UNIT 4 CYTOGENETICS OF ANEUPLOIDS AND
STRUCTURAL HETEROZYGOTES
Structure
4.1. Introduction
4.2. Objective
4.3. Aneuploidy
4.4. Monosomics
4.4.1. Methods of production of monosomics
4.4.2. Description and Identification of monosomics
4.4.3. –
4.4.4. Meiotic behaviour of monosomics
4.4.5. Transmission of monosomics
4.5. Trisomics
4.5.1. Trisomics in diploids
4.5.2. Trisomics are classified into 3 categories
4.5.3. Origin and source of trisomics
4.5.4. Use of monosomics and trisomics in chromosomal mapping of
diploid and polyploidy species
4.6. Euploidy
4.6.1. Haploidy
4.6.2. Polyploidy
4.6.2.1. Autopolyploidy
4.6.2.2. Allopolyploidy
4.1 INTRODUCTION
The expression and inheritance of the sum total of characters of an organism is
determined by the number and sequence of genes of its cells. Most of the plant and
animal species are diploid and in their somatic tissue chromosomes are found in
pairs. The number of chromosomes in a species normally remains constant
through successive generations and their results into the constancy of characters.
Gametes produced as a result of meiosis possess half as many chromosomes as the
somatic cells and in them each chromosome is represented only once. Fusion of
gametes brings together homologous chromosomes from the two parents and
restores the diploid number. Alternation of haploid and diploid generations occurs
in all sexual organisms. There is a great diversity in the number of chromosomes
in different species. But this number is fixed in a particular species. Chromosome
number is one of the characters that differentiate one species from another. In
plants the somatic chromosome number ranges from four (two pairs) in
Haplopappus to as many as 1260 in Ophioglossum. Constancy of the genetic
material through successive generations is essential for maintaining the identity of
a species. But variation is necessary if evolution has to occur. Sometimes such
[1]
variations involve whole chromosome or chromosome sets and can be seen
cytologically.
Variations that involve changes in number are of two types –
Euploidy- variations that involve entire sets of chromosome and,
Aneuploidy-variations that involve only single chromosome of a set.
Numerical changes in chromosomes or variations in chromosome number
means heteroploidy can be mainly of two types namely
(i)
Aneuploidy
(ii) Euploidy
Aneuploidy- means presence of chromosome number which is different than a
multiple of basic chromosome number, or variants that involve one or only a few
chromosomes but not the entire set.
Euploidy- on the other hand, means presence of chromosome number which is
similar multiple of basic chromosome number, or variation involve in entire sets
of chromosomes.
In above both alteration in chromosomes number, addition (hyperpoidy) or
loss of chromosomes, entire set or individually (hypoploidy) present.
Fig 4.1A diagrammatic representation of different types of numerical changes given
below –
Numerical alteration in chromosomes
Aneuploidy
Hypolpoidy
Euploidy
Hyperploidy
Hyperploidy
Haploidy
1x
Nullisomy
Monosomy
2n-(1x2)
2n-1
(pair)
Trisomy
2n+1
Hypolpoidy
Tetrasomy
2n+(1x2)
(pair)
Diploidy
2x
Triploidy
3x
Tetraploidy
4x
* Different types of numerical changes in chromosomes.
(x = basic chromosome number, 2n = somatic chromosome number, n = chromosome
number of a gamete)
[2]
4.2. OBJECTIVES
This unit aims:  To study the origin, breeding behavior and the phenotypic effects
resulting from the numeric alterations in chromosomes.
 To study the role of numeric alterations of chromosome in
agriculture and evolution.
4.3. ANEUPLOIDY
Aneuploidy can be either due to loss of any one or more chromosomes to
complete chromosome set (Hypoploidy). Hypoploidy is due to loss of single
chromosome is known as monosomy, and the individual that carries such number
is known as monosomics. The formula for monosomy is 2n-1. Another type of
hypo aneuploidy is nullisomy – where the loss of a pair chromosome from diploid
set. Both chromosome of a diploid set is absent. The general formula for
nullisomy is 2n-(1x2) or 2n-2.
Origin of aneuploidy
(i) Monosomy – When monosomy is present in diploid organism it can
not be tolerated because one complete chromosome is lacking.
Numbers of genes present in this chromosome were affected. This
unbalancing creates a problem, but when monosomy is present in
polyploid organism these could be easily produced and tolerable
because more than two chromosome of same combination is present.
Meiosis in monosomics behaves like haploids. In monosomics one of
the chromosomes will go to only one pole during division and other
pole will be devoid of it. Other chromosomes will asset normally. In
other words, monosomics behave like a haploid for one chromosome
and as a diploid for others.
(ii) Nullisomy – Another type of hypo-aneuploidy where a loss of pair of
homologous chromosome is seen. When it found in diploid organism
the organism doesn’t tolerate. The gametes produced by these
individual are devoid of one chromosome – means they are
aneuploids. The frequency of sterility is more in animals than in plants
because the plants are able to tolerate loses of chromosomes much
better than animals.
4.4 MONOSOMICS
[3]
Numbers of chromosomes in Nicotiana tabacum are 24, and all the 24
monosomics for that plant were reported by Clausenand Camerson (1944). Same
work was done by Sears (1954) for hexaploid bread wheat. A set of monosomic
for Gossypium hirsutum was reported by Endrizzi.
4.4.1 Methods of production of monosomics
(i)
From haploids – Sears (1939) got accidentally two haploids when he
crossed two Geneva – (Secale cereale and Chinese springwheat). That
haploid is used as female parent and crossed with normal hexaploid male.
The seeds produced by this method showing monosomics. When these
monosomics are selfed (inter crossing) nullisomic, trisomic and then later
on tetrasomics were found by selfing.
(ii) From backcrosses of inter specific hybrids – In N. tabacum
monosomics have been produced by crossing the polyploidy species with
one of its progenitors. The resultant F1 plant is backcrossed with
polyploidy crop by which monosomics are found.
Eg: In this eg. The haploid (n = 24) N. tabacum having 12 chromosomes
of N. Sylvestris (n = 12) and 12 chromosomes of N. tomentosa (n = 12).
This crop i.e. N. tabacum is crossed with N. sylvestris or (n = 24)
develops F1 plant. Obtained F1 plants has 12 pairs chromosomes of N.
sylvestris species and 12 single chromosomes of N. tomentosa. The
gametes of this crop contain n = 12 to 24 as they have 21 chromosomes
from N. sylvestris but the no. of single 12 chromosomes of N. tomentosa
varies from gametes to gametes resulting in n = 12 to 24. These are then
backcrossed with N. tabacum (n = 24) to develop monosomics for N.
tomentosa.
(a)
N. tabacum X N. sylvestris
(n = 24)
(n = 12)
F1 [12II (sylvestris) + 12I (tomentosa)] X N. tabacum
(n = 24)
(n = 12 to 24)
(b)
N. tabacum X N. tomentosa
(n = 24)
(n = 12)
23II + 1I (tomentosa)
(among other constitutions)
F1 [12II (tomentosa) + 12I (sylvestris)] X N. tabacum
(n = 24)
(n = 12 to 24)
[4]
23II + 1I (sylvestris)
(among other constitutions)
Fig.4.2 Production of monosomics through interspecific hybridization in
tobacco.
(iii) From partially asynaptic plants – In this type during meiotic metaphase
I, perfect bivalent formation is not observed, a variable number of
univalents are observed. These univalents will be distributed to the two
poles randomly during anaphase I.
(iv) Irradiation treatment – In case of Gossypium hirsutum (cotton) and
Avena sativa (Oats), irradiation of inflorescence to the production of
gametes with n+1 or n-1. It is a type of artificial treatment.
(v) Spontaneous production – Monosomics have been observed in wheat,
tobacco, cotton by natural occasional non-disjunction of a bivalent during
meiosis.
4.4.2 Description and Identification of monosomics
Sometimes monosomics are morphologically different or sometimes it may
not. It depends on genes present on that particular chromosome for which plant
is monosomic.
Monosomic condition is confirmed through chromosome count during mitotic
metaphase and by the use of a univalent at meiosis.
4.4.4 Meiotic behaviour of monosomics
Monosomics usually form bivalents in addition to a solitary univalent at end of
prophase I and metaphase I.
4.4.5 Transmission of monosomics
Breeding behaviour of monosomics has been studied by examining the
progeny obtained on selfing them and on crossing them separately as female
and male parents with the normal. On the basis of these studies, it is always
possible to calculate the frequency of functional deficient (n = 2x-1 or 3x-1)
gametes relative to normal (n = 2x or 3x) type of gametes. Although deficient
gametes are produced at a frequency higher than 75%, but they function in the
pollen at a very low frequency in wheat (4%), but at a variable frequency in
oats (1%-74%). In tobacco, although deficient pollen may function at a low
frequency, nullisomic zygotes, which normally do not survive. This situation
leads to the production of nullisomics in the progenies of selfed monosomics,
in variable frequency.
[5]
Eg, In case of wheat monosomics, (where normally n = 3x = 21), functional
male gametes are of 2 types,
i) n = 3x = 21,
ii) n = 3x-1 = 20.
The first category, male gametes are 96% while in second case only 4%
gametes are produced. But in female gametes 25% gametes carrying n = 3x =
21 while remaining 75% having n = 3x-1 = 20. When these types of gametes
crossed, percentage of nullisomics progeny is 3%, monosomics progeny is
73% and normal progeny is 24%.
4.5 TRISOMICS
4.5.1 Trisomics in Diploids
The first aneuploid chromosome number was reported in Oenothera hybrids by
Lutz (1909) and by Gates (1909) who recorded n = 15 while normal has n = 14.
Trisomy – Trisomics is an example of hyperaneuploidy where an organism has an
extra chromosome (2n+1). Since the extra chromosome may belong to anyone of
the different chromosomes of a haploid complement. The number of possible
trisomics in an organism will be equal to its haploid chromosome number.
Eg, Barley the haploid chromosome no. is 7 (n = 7) consequently, 7 trisomics are
possible.
4.5.2 Trisomics are classified into 3 categories
i)
ii)
iii)
Primary Trisomics – where an extra chromosome is identical to two
homologous chromosomes (diploid) in their gene sequence.
Secondary Trisomics – where the extra chromosome having same gene
in multiple copies (i.e. isochromosome). The extra chromosome having
duplicated and deficient gene.
Tertiary Trisomics – The extra chromosome should be product of
translocation i.e. this chromosome having genes of 2 or more other
chromosomes.
Trisomics were obtained for the first time in Datura stramonium by A. F.
Blakeslee and his coworkers. The genomic number of this species is n = 12.
Blakeslee and his coworkers found 12 primary trisomics, 24 secondary trisomics
and large no. of possible tertiary trisomics. In Datura stramonium most of the
trisomic were identified by the morphological features of fruit by shape and size.
[6]
Types of trisomy
2n + A
2n + B
2n + C
2n + D
2n + E
2n + F
2n + G
2n + H
2n + I
2n + J
2n + K
2n + L
Name
Rolled
Glossy
Buckling
Elongate
Echinus
Cockleber
Microcarpic
Reduced
Poinsettia
Spinach
Globe
Ilex
Figure 4.3
Different trisomics
of Datura
4.5.3 Table-Different
Origin and trisomics
sourceofofDatura
trisomics
Trisomics may originate spontaneously due to production of (n+1) type of
gametes; this gamete develops due to non-disjunction of a bivalent. Mostly
trisomics are produced by artificial methods.
Cytology of trisomics
A trisomic has an extra chromosome, which is homologous to one of the
chromosomes of the complement. During meiosis all of them synapse with each
other and at metaphase I they arrange themselves from a peculiar shape of
arrangement. (Fig 4.4)
Somatic Chromosome
Metaphase I configuration
[7]
Primary trisomics
Secondary trisomics
Tertiary trisomics
Fig 4.4 Different types of trisomics and their meiotic configurations at
metaphase I
4.5.4 Use of monosomics and trisomics in chromosomal
mapping of diploid and polyploidy species
The result of trisomic ratios can be utilized for locating genes on specific
chromosome or for finding out distances of these genes from centromere, this
technique is called chromosome mapping. If linkage groups are already
established in an organism, trisomics can be effectively used for assigning these
linkage groups to specific chromosomes. Since the segregation ratio for genes
located on the chromosome involved in trisomic condition differs from that, genes
located on other chromosomes, through these ratios one can found out on which
chromosome a particular gene is located. This technique has been successfully
applied in maize, tomato, barley, datura and Arabidopsis.
[8]
4.6 EUPLOIDY
It means presence of chromosome number which is similar multiple of basic
chromosome number, or variation involve in entire sets of chromosomes.
Each set of chromosomes is designated by the letter n and the numerical prefix
refers to the number of sets or the level of ploidy.
4.6.1 Haploidy
Haploid number of chromosomes is normally found in gametes of diploid (2n)
organisms, but sometimes the whole organism may be haploid. Lower organism
like bacteria and fungi are haploid for most of their life and become diploid or
partially diploid for only a brief period.
Monoploidy and haploidy
A distinction should be made between monoploidy and haploidy. Monoploidy
have a single basic set of chromosomes e.g. 2n = x = 7 in barley or 2n = x = 10 in
corn. Haploids, on the other hand represent individuals having half the somatic
chromosome number found in normal individual. Therefore, individuals having 2n
= 3x = 21 in wheat would also be haploids. These latter kind of haploids obtained
from polyploids are often called polyhaploids in order to distinguish them from
monoploids.
While reviewing the work on haploids in flowering plants in 1963, G. Kimber
and R. Riley (now, John Inner Centre or JRC, Norwich, UK), gave a classification
for haploids. They classified haploids in euhaploids and aneuhaploids which as
the term indicate are derived from euploids and aneuploids respectively. A
modified classification given by K. J. Kasha is presented. Euhaploids will
include monohaploids as well as polyhaploids.
Euhaploids
Monohaploids
Polyhaploids
Allopolyhaploids
Autopolyhaploids
Aneuhaploids
Disomic Haploids (n+1)
Addition Haploids (n+1.etc)
Nullisomic Haploids (n-1)
Substitution Haploids (n-1+1)
Misdivision Haploids
Haploids
[9]
Origin, Occurence and Production of Haploids
The haploids may originate at different points during the life cycle. Their
spontaneous production may be facilitated by environmental factors and these may
also be produced in the laboratory by artificial methods.
Based on the point in the life cycle involved, haploid production systems can be
broadly classified in three main categories namely:
i) parthenogenesis and apogamy, ii) chromosome elimination and iii) culture
methods
1. Production of haploids through parthenogenesis and apogamy
The development of an embryo from the egg cell without the participation of a
male gamete is parthenogenesis, and when the embryo develops from any other
gametophytic cell other than egg cell without fertilization, it is apogamy.
There are varieties of methods, which bring about parthenogenetic production of
haploids like:
a. Spontaneous- .It is largely due to parthenogenetic development of egg or any
other cell of the embryo sac.
b. Artificial treatments- The induction of haploids can be facilitated due to
different treatments like irradiation treatment including X-rays and UV rays, hot
and cold temperature shocks, wounds or injury, and chemical treatment
c. Delayed pollination- A delay in pollination has also been found to induce
parthenogenetic development of egg into a haploid embryo.
d. Wide hybridization- In this method, when a suitable pollinator is used, pollen
stimulates the development of embryo without fertilization.
2. Production of haploids through chromosome elimination
Cases are known where either spontaneously or due to specific treatments, the
chromosome number was reduced to half in the somatic tissues, a phenomenon
described as somatic reduction or reductional mitosis. Swaminathan and Singh
(1958) induced a haploid branch of watermelon by irradiation of the seed used.
3. Production of haploids through culture methods
a. Through anther and pollen culture. It is one of the popular methods for
production of haploids using artificial culture medium. This leads to the growth of
microspores into sporophytes.
[10]
b. Through ovule culture. Haploids have also been successfully produced from
cultured female gametophytes.
Haploidy is common in plants but rare in animals except some diploid species of
insects, rotifers, mites, etc., which produce haploid males parthenogenetically. In
haploids each chromosome represented only once due to which there is no
zygotene pairing and all the chromosomes appear as univalents on a metaphase
plate at the time of meiosis. During anaphase each chromosome moves
independently of the other and goes to either of the poles. According to the law of
probability the chance that a particular chromosome will go to a particular pole is
half and the chance that all the chromosomes of a haploid set will go to the same is
½ x ½ x ½…n times, where n = number of chromosomes in the haploid set.
Therefore, the frequency of gametes with the haploid set or n number of
chromosomes will be (½)n. This indicates that higher the number of chromosomes
in a haploid set, lesser will be the frequency of all of them being included in the
same gamete. Gametes with less than the haploid number of chromosomes are
normally not viable. Haploid organism, therefore, are highly sterile. Male bees and
such haploid animals are exceptions and in them there is no meiosis at the time of
gamete formation. Cells arising as a result of mitosis get differentiated into sperms
and are, therefore, haploid and viable. Haploid individuals usually develop
abnormally in many diploid animals. Amphibian haploids have been induced by
various means, but the embryos rarely reach the adult stage. In many plants, such
as Nicotiana, Datura, Oiyza, Secale etc., fully viable haploids can be formed, but
they turn out to be sterile because meiosis produces inviable gametes.
In all organisms’ haploid and diploid generations alternate but in evolved
organisms, like higher plants and animals, the diploid generation predominates and
the haploid generation is transitory and short lived. As we go down the
evolutionary scale, the balance shifts in favour of the haploid generation, so much
so that in micro-organisms major part of the life span is haploid. During haploid
state there is only one copy of each chromosome and only one allele of each gene.
Consequently, each gene is expressed whether it is dominant or recessive. This
facilitates genetic experiments and this is the reason why micro-organisms have
been helpful in genetic studies. For the same reason scientists are trying to develop
haploid strains of flowering plants. Success has been achieved in developing
haploid strains of Nicotiana, Datura, Triticum, etc., but the problem of their poor
growth and sterility has yet to be overcome.
Haploid strains of crop are useful in another way. Present day diploid and
polyploidy species are the result of evolution through ages and inconceivable
[11]
diversity and heterozygosity has been generated due to mutation and
recombination.
Heterozygosity is a hindrance in plant breeding because the recessive genes keep
on appearing during successive generations. If the chromosome number of a
haploid strain is duplicated, a diploid will arise which will be homozygous for all
the characters and genes. Such a strain will be an ideal starting material for crop
breeding. This is the reason why biologists are synthesizing haploid strains of
crops like rice, wheat, etc. It was Maheshwari and Guha (1966) of Delhi
University who, for the first time, were successful in obtaining haploid plants of
tobacco by culturing anthers on suitable media. Since then this technique has been
used for developing haploids in a number of plants. In the same department Sinha
and Bhojwani have been able to induce haploidy in somatic cells of onion by
growing the roots in a medium containing para-fluorophenylalanine. Chemical
induction of haploidy in higher plants, if successful on a large scale, will be a very
useful tool in the hands of plant breeders.
4.6.2 Polyploidy
In polyploidy cells and organisms the number of chromosome sets exceeds that of
the diploid. Depending on the number of chromosome sets, polyploidy can be
classified as triploids, tetrapoloids, pentaploids, etc. diploid organisms possess
polyploidy cells and tissue. Diploid human cells have 46 chromosomes each, but
in cancer cells this number may reach 100 or more due to abnormal divisions. In
the liver of mice about 40 percent of the cells are tetraploid and about five percent
are octaploid. Polyploidy nuclei, cells and tissue are very common even in plants.
Endosperm nuclei are generally triploid because of triple fusion. Sometimes they
may be tetra-, penta-, hexa-, or polyploids of even higher order.
Origin and Induction of Polyploidy
Failure of normal mitotic divisions results into nuclei with increased sets of
chromosomes. During mitosis in a diploid somatic cell, if the chromosomes
duplicate and divide but cytokinesis fails to occur, a tetraploid cell arises. Normal
mitotic division of this cell gives rise to a tetraploid tissue. Organs or branches that
develop from such a tissue are tetraploid and flower borne on such branches
produce diploid instead of haploid gametes. A doubling of the number of somatic
chromosomes in a sex organ results into a doubling of the number of gametic
chromosomes. A fusion of two diploid gametes produces a tetraploid individual. A
fusion of a diploid and a haploid gamete results into a triploid individual. Even
abnormal meiosis may result into a diploid gamete instead of a haploid in a diploid
[12]
organism. Polyploidy can also occur if an egg is fertilized by more than one male
gamete.
Polyploidy can be induced with the help of chemicals, by giving temperature
shocks or by causing mechanical injuries to tissues. In tomato plants disorganized
cell division occurs at cut ends of stem and a callus tissue develops. Most of the
cells in such a tissue are tetraploid rather than diploid. About seven percent of the
branches developed from such a tissue are polyploid.
Similarly, octaploid branches can be developed on tetraploid tissues. In plants, like
maize, polyploidy can be induced by temperature shocks which disorganize
cytokinesis. Tissues developed from a polyploid cell are polyploid. In maize short
pulses of high temperature are necessary for inducing polyploidy. In other
organisms short pulses of low temperatures or alternating high and low
temperatures are required for this purpose. Divided nuclei can be prevented from
separating in Spirogyra by refrigeration or by treatment with ether or chloroform.
If nuclei do not separate and cytokinesis occurs, the result is a cell with two nuclei
and another cell without any nucleus. A cell with two nuclei divides to give rise to
polyploidy cells.
Chemicals, like indole acetic acid induce cell division in non-meristematic cells,
thereby inducing polyploidy. If the rate of nuclear division is increased,
polyploidy cells arise because cell wall synthesis, which is responsible for
cytokinesis, is not able to cope with the increased division rate of the nucleus.
When roots of leguminous plants are infected with nitrogen-fixing bacteria like
Rhizobium, the level of indoleacetic acid increases in cortical cells and this causes
polyploidy. It is a matter of dispute whether indoleacetic acid induces polyploidy
or simply helps in division and multiplication of a polyploidy cell.
Let us imagine that A, B1, B2, and C are four different haploid sets of
chromosomes and that genomes B1 and B2 are related. (Fig 4.5)
[13]
AAAAB1B1B1B1
Autoallopolyploid
2n = 56
AB1B1B2
Hybrid
2n = 28
AAB1B1CC
Allohexaploid
2n = 42
AAAA
Autotetraploid
2n = 28
AAB1B1
Allotetraploid
2n = 28
B1B1B2B2
Segmental
allotetraploid
2n = 28
B2B2B2B2
Autotetraploid
2n = 28
AB1C
Allotriploid
hybrid
2n = 21
AA
Diploid
2n = 14
AB1
Hybrid
2n = 14
B1B1
Diploid
2n = 14
B1B2
Hybrid
2n = 14
B2B2
Diploid
2n = 14
CC
Diploid
2n = 14
Figure 4.5 Different kinds of polyploids and their derivation from one or more basic
diploid species
[14]
Chemicals like colchicines and acenapthene are believed to interfere with spindle
formation and thus induce polyploidy. Colchicine is an alkaloid which is extracted
from corms of Colchicum autumnale or Colchicum luteum of the family Liliaceae.
The corm is used as a carminative, laxative, aphrodisiac and is useful in gout,
rheumatism and disease of liver and spleen and also as an external application to
lessen inflammation and pain. Eigsti (1937) noticed that if onion bulbs are allowed
to root in a 0.05 percent aqueous solution of colchicines, some cells show
polyploidy and if colchicines treatment is continued as many as 1000
chromosomes can be found in a cell.
As colchgicine prevents the formation and organization of spindle fibre, the
metaphase chromosomes of affected cells (C-metaphase or colchicines metaphase)
do not move to a metaphase plate and remain scattered in the cytoplasm. Even
cytokinesis is prevented and with duplications of chromosomes their number goes
on increasing.
Soon after Eigsti discovered the polyploidy inducing potentiality of colchicines,
Blakeslee, Avery and Nebel (1937) used it for getting a polyploidy plant.
Now-a-days colchicine is one of the most widely used chemicals for induction of
polyploidy and for causing related effects. As colchicine interferes with spindle
formation, its effects are limited to dividing or meristematic cells. Once a
polyploidy cell is formed, its normal division gives rise to a polyploidy tissue,
branch, flowers, fruits, seeds, etc., and a polyploidy variety is established.
Polyploidy
There are mainly three different kinds of polyploids, namely
i)
Autopolyploid,
ii)
Allopolyploids, and
iii)
Auto allopolyploids.
[15]
Species A
2n = AA
Species B
2n = BB
Species C
2n = CC
F1
2n = AB
Autotriploid
3n = AA
Autotetraploid
4n = AAAA
Triploid
3n = ABB
Allotetraploid
4n = AABB
Triploid
3n = ABC
Autohexaploid
6n = AAAAAA
Autoallohexaploid
6n = AABBBB
Autopentaploid
5n = AAAAA
Allohexaploid
6n = AABBCC
Fig. 4.6. Interrelationships of auto- and allopolyploids. (After Stebbins, 1950).
[16]
4.6.2.1 Autopolyploids
Autopolyploids are those polyploids, which has the same basic set of
chromosomes multiplied. For instance, if a diploid species has two similar sets of
chromosomes or genomes (AA), an autotriploid will have three similar genomes
(AAA) and an autotetraploid will have four such genomes (AAAA) as shown in
figure.
Origin and production of autopolyploids.
The autopolyploids may occur in nature or may be artificially produced. When
they are found in nature, their autopolyploid nature is inferred mainly by their
meiotic behaviour. One of the very common examples of natural autopolyploidy
relevant to northern India pertains to common ‘doob’ grass (Cynodon dactylon). In
U.P. and Bihar, common ‘doob’ grass was found to be an autotriploid as inferred
from its meiotic behaviour (Gupta and Srivastava, 1970). It is perhaps successful
due to efficient vegetative reproduction, because, as will be seen, autopolyploids
are normally triploids and set no seeds. Autotriploids are also known in
watermelons, sugarbeet, tomato, grapes and banana; although in several of these
cases the polyploids have been artificially produced. Similarly autopolyploids are
known in rye (Secale cereale), corn (Zea mays), red clover (trifolium prantese),
berseem (trifolium alexandrium), marigolds (Tagetes), snapdragons (Antirrhinum),
Phlox, grapes, apples, etc. In Oenothera lamarckiana, an American plant, a giant
mutant described by Hugo de Vries was later discovered to be an autotetraploid.
Induced autopolyploids.
In many cases listed above, autopolyploidy is artificially induced. Since
polyploids are normally larger and more vigorous, their role in crop improvement
has been realized and techniques developed for artificial induction of polyploidy.
Polyploidy is mainly induced by treatment with aqueous solution of a drug called
colchicine. This drug has the property of arresting and breaking the spindle so that
a cell division without cell wall formation may be affected leading to doubling of
chromosome number. The concentration of aqueous solution of colchicines may
vary from 0.01% to 0.50% and the treatment may be given in one of the following
manners.
(I)
Seed treatment may be mainly given by soaking seeds for different
durations in aqueous solution of colchicines.
(II)
Injections of colchicines solution may also be given at seedling stage so as
to inject solution into cortex tissue with the help of a hypodermic needle.
(III) Axillary bud treatment is also effective. Since bud is meristematically
active, placing cotton soaked in colchicine on the bud and continuous
dropping of solution on cotton leads to induction of polyploidy in the
branch arising from the treated bud.
[17]
(IV)
Shoot apex treatment is brought about just like bud treatment and is fairly
effective, but the shoot apex should come in direct contact of the solution.
In order to facilitate this, young leaves covering the shoot apex may be
removed.
Colchicine is an alkaloid extracted from seed and corm of Colchicine autumnale.
The action of colchicines and its use in inducing polyploidy was first studied in
1930’s. The successful doubling of chromosome number was described for the
first time by Levan (1938) and by Eigsti (1938 - 40). It was surprising however,
that colchicine does not affect colchicum, the plant from which it is extracted. This
was attributed to the presence of an ‘anticolchicine’. The success of colchicine
treatment depend on three prerequisites, namely
(i)
direct contact of growing tissue with solution,
(ii)
effective concentration of colchicine and
(iii) The optimal stage of development.
These three conditions are met with in the methods of treatment described above.
Effects of chromosome doubling.
One of the important effects of polyploidy is to produce ‘gigantism’. The
autopolyploids may be normally larger in size. Sometimes plants may be smaller
than diploids. But leaves, flowers, and the cells themselves may be bigger in size.
Some of the important effects of polyploidy are as follows:
With increase in cell size, water content increases leading to decrease in osmotic
pressure. This results into loss of resistance against frost, etc.
Growth rate decreases due to slower rate of cell division; this leads to a decrease
in auxin supply and a decrease in respiration.
In autopolyploids, time of blooming is delayed and prolonged due to slow growth
rate.
At higher ploidy level (autooctoploids or higher), the adverse effects are highly
pronounced and lead to death of plants.
Cytology of autopolyploid.
In an autopolyploid, there will be more than two sets of homologous
chromosomes. This leads to formation of multivalents instead of bivalent as found
in diploids. An important difference exists even between autotriploids and
autotetraploids, because while in the latter normal disjunction is possible giving
rise to diploid gametes, in triploids it is not possible. In an autotriploid, there are
three sets of homologous chromosomes. If these three sets are normally paired,
trivalents can not disjoin normally and will either disjoin 2:1 chromosomes to two
poles or will disjoin 1:1 leaving one chromosome as a laggard. The number of
chromosomes in gametes of triploid organism, therefore, will vary from n to 2n.
Most of these gametes are unbalanced leading to high degree of sterility.
[18]
In autotetraploids, since there are four sets of chromosomes, quadrivalents are
formed, which disjoin in a normal 2:2 manner giving diploid gametes. Rarely
however, a quadrivalent may disjoin 3:1 or may leave a chromosome as a laggard
at anaphase I. Therefore autotetraploids also have a certain degree of sterility,
although it will not be as high as on autotriploids.
4.6.2.2 Allopolyploids.
Polyploidy may also result from doubling of chromosome number in a F1 hybrid
which is derived from two distinctly different species. This will bring two
different sets of chromosomes in F1 hybrid. The number of chromosomes in each
of these two sets may differ. Let A represent a set of chromosomes (genome) in
species X, and let B represent another genome in a species Y.The F1 will then
have one A genome and another B genome. The doubling of chromosomes in this
F1 hybrid (AB) will give rise to a tetraploid with two A and two B genomes. Such
a polyploidy is called an allopolyploid or amphidiploid.
Raphanobrassica is a classical example of allopolyploidy. In 1927, G. D.
Karpechenko, a Russian geneticist, reported a cross between Raphanus sativus
(2n = 18) and Brassica oleracea (2n = 8) to produce F1 hybrid which was
completely sterile. This sterility was due to lack of chromosome pairing, since
there is no homology between genomes from Raphanus sativus and Brassica
oleracea. Among these sterile F1 hybrids, Karpechenko found certain fertile
plants. On cytological examination these fertile plants were found to have 2n = 36
chromosomes, which showed normal pairing into 18 bivalents.
Radish
(9R + 9R)
Gametes :
F1 :
Gametes :
F1 :
Cabbage
(9C + 9C)
9R
9C
(9R + 9C)
Diploid hybrid
(9R 9C)
(9R 9C)
(18R + 18C)
Raphanobrassica
[19]
Fig 4.7 Example of an allopolyploidy.
(a)
Evolution of Wheat. Common cultivated wheat is another important
example of allopolyploidy, although its allopolyploid nature has now been
questioned. There are three different chromosomes numbers in the genus
Triticum, namely 2n = 14, 2n = 28, 2n = 42. The common wheat is hexaploid
with 2n = 42 and is derived from three diploid speices: (i) AA = Triticum
aegilopoides (2n = 14), (ii) BB = Aegilops speltoides ? (2n = 14)(in the past
evidence was made available, showing that Ae. Speltoides may not be the
donor of E genome; it is also believed that the donor of E genome may never
be discovered) and (iii) DD = Aegilops tauschii, earlier known as Ae.squarrosa
(2n = 14). The hexaploid wheat, therefore, is designated as AABBDD, the
tetraploid (2n = 28) as AABB and diploid (2n = 14) as AA. There is, however,
evidence available now which suggests that A, B and D genomes from three
diploid species mentioned above are not much different from one another, so
that it is believed that the three diploid progenitors of common hexaploid
wheat were derived from a common ancestor. For this reason, the common
hexaploid wheat is now considered an autopolyploid rather than an
allopolyploid. At best, it may be a segmental allopolyploid. (Fig.4.8)
(b)
Triticale (x Triticosecale), a new man made cereal. During the last
decades, considerable emphasis has been laid on the possibility of utilizing a
new man made cereal known as triticale, on a commercial scale. It is already
grown in an estimated area of more than three million hectares and research at
several places all over the world is in progress to improve this man made crop.
Triticale is the first man made crop, in so far as it resulted as an artificial
allopolyploid derived by crossing wheat (Triticum) and rye (Secale).
Depending upon whether tetraploid (2n = 4x = 28) or hexaploid (2n = 6x = 42)
wheat is utilized for the synthesis, one would get hexaploid triticale (2n = 6x
= 42) or octoploid triticale (2n = 8x = 56) respectively. In each case, only
diploid rye (2n = 2x = 14) was used.
(a)
Triticum durum X Secale cereale
2n = 28
2n = 14
F1 hybrid (sterile)
2n = 21
Chromosome
doubling
2n = 42
Hexaploid triticale
[20]
(b)
Triticum aestivum X Secale cereale
2n = 42
2n = 14
Hybrid
2n = 14
AB
Chromosome doubling
Aegilops speltoides
X
2n = 14
Triticum dicoccoides
BB
4n = 28
Wild
AABB
Triticum boeoticum
2n = 14
AA
Wild
Triticum monococcum
2n = 14
AA
Cultivated
Wild
F1 hybrid (sterile)
2n = 28
Chromosome
doubling
2n = 56
Octoploid triticale
Fig.4.9 Artificial synthesis of a) Hexaploid triticale b) Octoploid triticale
[21]
Fig.4.8 Diagrammatic representation of the origin of tetraplooid and hexaploid
cultivated wheat from their wild ancestors.
Triticum durum
Triticum dicoccum
Aegilops sqarrosa
X
4n = 28
4n = 28
2n = 14
AABB
AABB
DD
Cultivated
Cultivated
Wild
Check Your Progress
Note:
(a) Write your answer in the
(b) Compare your answer with
of the unit.
Q.1 What is
allopolyploidy?Describe the
Raphanobrassica was produced.
Hybrid
3n = 21
ABD
space given below,
key given at the end
Chromosome doubling
Triticum spelta, Triticum
vulgare. Etc.
6n = 42
AABBDD
Cultivated
experiment by which
………………………………………………………………………………………
………………………………………………………………………………………
………………
Q.2 How many trisomics were found in Datura?
[22]
………………………………………………………………………………………
………
Q.3 What are secondary and tertiary trisomics?
………………………………………………………………………………………
………
Q.4 How did triticum aestivum be produced artificially?
………………………………………………………………………………………
………………………………………………………………………………………
……………...
Q.5 Differentiate between Aneuploid and euploid.
………………………………………………………………………………………
………………………………………………………………………………………
………………………………………………………………………………………
……………………....
Let’s Sum Up
 Change in the number and structure of chromosome may occur
spontaneously or experimentally by the action of radiation or chemicals.
 Chromosomal changes in number are of two main kinds: in euploids, the
set is kept balanced. In aneuploids there is a loss or gain of one or more
chromosomes.
 In plants, polyploids (triploid, tetraploiid etc) are rather common. They
originate by reduplication without cytokinesis.
 Polyploidy can be induced by colchicines, this substance is used in
agriculture to improve certain plant species.
 Allopolyploidy consists of the formation of a hybrid with different sets of
chromosomes.
 Among the aneuploid organisms there are the trisomic (i.e. with three
similar chromosomes instead of normal pair) and the monosomic (missing
one member of a pair of homologous chromosomes).
Assignment –
a)
b)
c)
d)
Role of aneuploidy and euploidy in agriculture.
Meiotic behavior of autopolyploid and allopolyploids.
Aneuploid abnormalities in Drosohila, Datura and Human.
Behavior of metaphase configuration in different types of trisomics.
[23]
Check Your Progress: Key
Q.1 A polyploidy containing two or more different genomes.By crossing radish
and cabbage and in F1 hybrid the chromosome number is doubled to produce
raphanobrassica.
Q.2 12 primary trisomics, 24 secondary trisomics and a large number of tertiary
trisomics.
Q.3 Refer section 4.5.2
Q.4 Refer section 4.6.2.2
Q.5An aneuploid organism has a chromosome number that is not an exact multiple
of the basic chromosome number while euploid has exact multiple of the basic
number.
References
Cell and molecular biology-P.K.Gupta
Cytogenetics, Plant Breeding and evolution-U Sinha and Sunita Sinha
Cytogenetics- P.K.Gupta
Cell Biology-P.S.Verma
Chromosomes-M.S.Clark and W.J.Wall
Cell and molecular biology-Ajay Paul
Cell and molecular biology -De Robertis
[24]
UNIT-5 MOLECULAR CYTOGENETICS AND
ALIEN
GENE
TRANSFER
THROUGH
CHROMOSME MANIPULATION
Structure
5.0 Introduction
5.1 Objectives
5.2 Molecular Cytogenetics:
5.2.1 Nuclear DNA Content
5.2.2 C-value Paradox
5.2.3 Cot Curve and its Significance
5.2.4 Restriction Techniques
5.2.5 Multigene Families and their Evolution
5.2.6 In-Situ Hybridization – Concept and Techniques
5.2.7 Physical Mapping of Genes on Chromosomes
5.2.8 Computer Assisted Chromosome Analysis
5.2.9 Chromosome Microdissection and Microcloning
5.2.10 Flow Cytometry and Confocal Microscopy in Karyotype Analysis
5.3 Alien Gene Transfer Through Chromosome Manipulation:
5.3.1 Transfer of Whole Genome – Examples from Wheat, Archis & Brassica
5.3.2 Transfer of Individual Chromosomes and Chromosome Segments
5.3.3 Methods for Detecting Alien Chromatin
5.3.4 Production, Characterization and Utility of Alien Addition &
Subtraction Lines RNA Splicing
5.3.5 Genetic Basis of Inbreeding and Heterosis
5.3.6 Exploitation of Hybrid Vigour
5.4 Let Us Sum Up
5.5 Check Your Progress
5.6 Check Your Progress: The Key
5.7 Assignment
5.8 References
5.0 INTRODUCTION
Molecular cytogenetics is a branch of biology in which nucleic acid and other cytological
molecules which being involve in genetic inheritance are studied. In eukaryotes nucleus
consist DNA as genetic material which have different components like chromatin,
nucleosomes, histone etc. Nucleolus also have important role. Various techniques are
now available to measure the amount of genome, number and position of genes and many
other such type of work for the research and applicability purposes.
[25]
Breeding programmes aiming at transferring desirable genes from one species to another
through interspecific hybridization and backcrossing often produce monosomic and
disomic addition as intermediate crossing products. Such aneuploids contain alien
chromosomes added to the complements of the recipient parent and can be used for
further introgression programmes, but lack of homoeologous recombination and
inevitable segregation of the alien chromosome at meiosis make them often less ideal for
producing stable introgression lines. Monosomic and disomic additions can have specific
morphological characteristics, but more often do they need additional confirmation by
molecular marker analyses and assessment by fluorescence in situ hybridization with
genomic and chromosome specific DNA as probes. Their specific genetic and
cytogenetic properties make them powerful tools for fundamental research elucidating
regulation of homoeologous recombination, distribution of chromosome specific markers
and repetitive DNA sequences, and regulation of heterologous gene expression. In above
chapter it has been tried to present the major characteristics of such interspecific
aneuploids highlighting their advantages and drawback for breeding and fundamental
research. Therefore present chapter is being prepared to enlighten the role of nucleus in
genetics along with techniques of genetic engineering which are also helpful to improve
the quality of plants of agricultural, horticultural importance.
5.1 OBJECTIVE
Purpose of this unit is to cover the following points to describe the utility of molecular
cytogenetics:
C-value genomic content in a haploid cell which is related to genome size and
varies organism to organism,
The Cot curve (an S-shaped curve) is obtained by plotting the fraction of singlestranded DNA remaining (C/C0) as a function of the logarithm of the product of
the initial concentration and the elapsed time,
Eukaryotic DNA consists of at least three types of sequences nonrepetitive
(unique-sequence DNA), moderately repetitive DNA, highly repetitive DNA,
Restriction techniques involve methods for locating specific DNA sequences,
cutting DNA at precise locations, amplifying a particular DNA sequence,
mutating and joining DNA fragments to produce desired sequences and
procedures for transferring DNA sequences into recipient cells,
A multigene family involves members of a family of related proteins encoded by
a set of similar genes. Multigene families are believed to have arisen by
duplication and variation of a single ancestral gene,
In situ hybridization techniques locate specific nucleic acid sequences in cells or
on chromosomes,
The ultimate goal of gene mapping is to clone genes, especially disease genes.
Once a gene is cloned, we can determine its DNA sequence and study its protein
product,
Computer programmes are very helpful to analyse the karyotype and
chromosomal information,
[26]
Chromosome microdissection is a specialized way of isolating specific regions by
removing the DNA from the band and making that DNA available for further
study (microcloning),
Flow cytometry is very fast and informative, quantitative and qualitative analysis
method which objects include chromosomes and nuclei,
Alien gene transfer is aiming to improve the crop quality by using the different
properties of plant like heterosis, hybrid vigour etc.
5.2 MOLECULAR CYTOGENETICS
5.2.1 Nuclear DNA Content
The nucleus mainly have chromosome and nucleoplasm. The molecule of DNA in a
single human chromosome ranges in size from 50 x 106 nucleotide pairs in the smallest
chromosome (stretched full-length this
molecule would extend 1.7 cm) up to
250 x 106 nucleotide pairs in the
largest (which would extend 8.5 cm).
Stretched end-to-end, the DNA in a
single human diploid cell would extend
over 2 meters. In the intact
chromosome, however, this molecule
is packed into a much more compact
structure. The packing reaches its
extreme during mitosis when a typical
chromosome is condensed into a
structure about 5 µm long (a 10,000fold reduction in length).
Chromatin - The nucleus contains
Figure 5.1: Electron micrograph showing chromatin
from the nucleus of a chicken red blood cell (birds,
unlike most mammals, retain the nucleus in their
mature red blood cells). The arrows point to the
nucleosomes. You can see why the arrangement
of nucleosomes has been likened to "beads on a
string"(courtesy of David E. Olins and Ada L.
Olins).
the chromosomes of the cell. Each
chromosome consists of a single
molecule of DNA complex with an
equal mass of proteins. Collectively,
the DNA of the nucleus with its
associated proteins is called chromatin
(Figure 5.1). Most of the protein consists of multiple copies of 5 kinds of histones. These
are basic proteins, bristling with positively charged arginine and lysine residues. (Both
Arg and Lys have a free amino group on their R group, which attracts protons (H+)
giving them a positive charge.) Just the choice of amino acids you would make to bind
tightly to the negatively-charged phosphate groups of DNA.
Chromatin also contains small amounts of a wide variety of nonhistone proteins. Most of
these are transcription factors (e.g., the steroid receptors) and their association with the
DNA is more transient.
[27]
Nucleosomes - Two copies of each of four kinds of histones
H2A
H2B
H3 and
H4
form a core of protein,
the nucleosome core
(Figure 5.2). Around this
is wrapped about 147
base pairs of DNA. From
20–60 bp of DNA link
Figure 5.2:Nuclosome, Histone and Linker DNA
one nucleosome to the
next. Each linker region is occupied by a single molecule of histone 1 (H1).
The binding of histones to DNA does not depend on particular nucleotide sequences in
the DNA but does depend critically on the amino acid sequence of the histone. Histones
are some of the most conserved molecules during the course of evolution. Histone H4 in
the calf differs from H4 in the pea plant at only 2 amino acids residues in the chain of
102.
The formation of nucleosomes helps somewhat, but not nearly enough, to make the DNA
sufficiently compact to fit in the nucleus. In order to fit 46 DNA molecules (in humans),
totaling over 2 meters in length, into a nucleus that may be only 10 µm across requires
more extensive folding and compaction.
 interactions between the exposed "tails" of the core histones cause nucleosomes to
associate into a compact fiber 30 nm in diameter.
 these fibers are then folded into more complex structures whose precise configuration
is uncertain and which probably changes with the level of activity of the genes in the
region.
Histone Modifications - Although their amino acid sequence (primary structure)
is unvarying, individual histone molecules do vary in structure as a result of chemical
modifications that occur later to individual amino acids. These include adding:
 acetyl groups (CH3CO-) to lysines
 phosphate groups to serines and threonines
 methyl groups to lysines and arginines
Although 75–80% of the histone molecule is incorporated in the core, the remainder - at
the N-terminal - dangles out from the core as a "tail" (not shown in the Figure 5.2).
The chemical modifications occur on these tails, especially of H3 and H4. Most of theses
changes are reversible. For example, acetyl groups are
 added by enzymes called histone acetyltransferases (HATs)(not to be confused
with the "HAT" medium used to make monoclonal antibodies!) and
 removed by histone deacetylases (HDACs).
More often than not, acetylation of histone tails occurs in regions of chromatin that
become active in gene transcription. This makes a kind of intuitive sense as adding acetyl
groups neutralizes the positive charges on Lys thus reducing the strength of the
association between the highly-negative DNA and the highly-positive histones. But there
is surely more to the story.
[28]

Acetylation of Lys-16 on H4 prevents the interaction of their "tails" needed to
form the compact 30-nm structure of inactive chromatin. But this case involves
interrupting protein-protein not protein-DNA interactions.
 Methylation, which also neutralizes the charge on lysines (and arginines), can
either stimulate or inhibit gene transcription in that region.
o methylation of lysine-4 in H3 is associated with active genes while
o methylation of lysine-9 in H3 is associated with inactive genes. (These
include those imprinted genes that have been permanently inactivated in
somatic cells.) and
 Adding phosphates causes the chromosomes to become more - not less - compact
as they get ready for mitosis and meiosis.
In any case, it is now clear that histones are a dynamic component of chromatin and not
simply inert DNA-packing material.
Histone Variants - We have genes for 8 different varieties of histone 1(H1). Which
variety is found at a particular linker depends on such factors as
o the type of cell,
o where it is in the cell cycle, and
o its stage of differentiation.
In some cases, at least, a particular variant of H1 associates with certain transcription
factors to bind to the enhancer of specific genes turning off expression of those genes.
Some other examples of histone variants:
o H3 is replaced by CENP-A ("centromere protein A") at the
nucleosomes near centromeres. Failure to
substitute CENP-A for H3 in this regions
blocks centromere structure and function.
o H2A may be replaced by the variant H2A.Z
at the boundaries between euchromatin and
heterochromatin.
o All the "standard" histones are replaced by variants
as sperm develop.
In general, the "standard" histones are incorporated into the
nucleosomes as new DNA is synthesized during S phase of the
cell cycle. Later, some are replaced by variant histones as
conditions in the cell dictate.
Euchromatin versus Heterochromatin - The
density of the chromatin that makes up each chromosome
(that is, how tightly it is packed) varies along the length of
the chromosome.
 dense regions are called heterochromatin,
 less dense regions are called euchromatin (Figure 5.3).
Figure 5.3: The diagram represents
a hypothetical model of how
euchromatin and heterochromatin
may be organized during interphase
in a vertebrate cell.
Heterochromatin
 is found in parts of the chromosome where there are few or no genes, such as
[29]
o
o
centromeres and
telomeres
 is densely-packed;
 is greatly enriched with transposons and other "junk" DNA;
 is replicated late in S phase of the cell cycle;
 has reduced crossing over in meiosis.
 Those genes present in heterochromatin are generally inactive; that is, not
transcribed and show
o increased methylation of the cytosines in CpG islands of the DNA;
o decreased acetylation of histones and
o increased methylation of lysine-9 in histone H3, which now provides a
binding site for heterochromatin protein 1 (HP1), which blocks access by
the transcription factors needed for gene transcription.
Euchromatin
 is found in parts of the chromosome that contain many genes;
 is loosely-packed in loops of 30-nm fibers.
 These are separated from adjacent heterochromatin by insulators.
 In yeast, the loops are often found near the nuclear pore complexes. This would
seem to make sense making it easier for the gene transcripts to get to the cytosol.
However, in animal cells, gene transcription appears to be repressed near the inner
surface of the nuclear envelope.
 The genes in euchromatin are active and thus show
o decreased methylation of the cytosines in CpG islands of the DNA
o increased acetylation of histones and
o decreased methylation of lysine-9 in histone H3.
Nucleosomes and Transcription - Transcription factors cannot bind to their
promoter if the promoter is blocked by a nucleosome. One of the first functions of the
assembling transcription factors is to either expel the nucleosome from the site where
transcription begins or at least to slide the nucleosomes along the DNA molecule. Either
action exposes the gene's promoter so that the transcription factors can then bind to it.
The actual transcription of protein-coding genes is done by RNA polymerase II (RNAP
II). In order for it to travel along the DNA to be transcribed, a complex of proteins
removes the nucleosomes in front of it and then replaces them after RNAP II has
transcribed that portion of DNA and moved on.
The Nucleolus - During the period between cell divisions, when the chromosomes
are in their extended state, 1 or more of them (10 in human cells) have loops extending
into a spherical mass called the nucleolus. Here are synthesized three (of the four) kinds
of RNA molecules (28S, 18S, and 5.8S) used in the assembly of the large and small
subunits of ribosome.
28S, 18S, and 5.8S ribosomal RNA is transcribed (by RNA polymerase I) from hundreds
to thousands of tandemly-arranged rDNA genes distributed (in humans) on 10 different
chromosomes. The rDNA-containing regions of these 10 chromosomes cluster together
in the nucleolus. (In yeast, the 5S rRNA molecules - as well as transfer RNA molecules are also synthesized (by RNA polymerase III) in the nucleolus. Once formed, rRNA
[30]
molecules associate with the dozens of different ribosomal proteins used in the assembly
of the large and small subunits of the ribosome.
But all proteins are synthesized in the cytosol - and all the ribosomes are needed in the
cytosol to do their work - so there must be a mechanism for the transport of these large
structures in and out of the nucleus. This is one of the functions of the nuclear pore
complexes.
Nucleoplasm - The term "nucleoplasm" is still used to describe the contents of the
nucleus. However, the term disguises the structural complexity and order that seems to
exist within the nucleus. For example, there is evidence that DNA replication and
transcription occur at discrete sites within the nucleus. C-value is the mass of DNA
(expressed, for example, in picograms per cell) in the haploid genome of a species.
5.2.2 C-value Paradox / Engima
C-value - The term C-value refers to the amount of DNA contained within a haploid
nucleus (e.g., in a gamete or one half the amount in a diploid somatic cell) of a eukaryotic
organism. In some cases (notably among diploid organisms), the terms C-value and
genome size are used interchangeably, however in polyploids the C-value may represent
two genomes contained within the same nucleus. Greilhuber et al. (2005) have suggested
some new layers of terminology and associated abbreviations to clarify this issue, but
these somewhat complex additions have yet to be used by other authors. C-values are
reported in picograms.
Origin of the term - Many authors have incorrectly assumed that the "C" in "Cvalue" refers to "characteristic", "content", or "complement". Even among authors who
have attempted to trace the origin of the term, there had been some confusion because
Hewson Swift did not define it explicitly when he coined it in 1950. In his original paper,
Swift appeared to use the designation "1C value", "2C value", etc., in reference to
"classes" of DNA content (e.g., Gregory 2001, 2002); however, Swift explained in
personal correspondence to Prof. Michael D. Bennett in 1975 that "I am afraid the letter
C stood for nothing more glamorous than 'constant', i.e., the amount of DNA that was
characteristic of a particular genotype" (quoted in Bennett and Leitch 2005). This is in
reference to the report in 1948 by Vendrely and Vendrely of a "remarkable constancy in
the nuclear DNA content of all the cells in all the individuals within a given animal
species" (translated from the original French). Swift's study of this topic related
specifically to variation (or lack thereof) among chromosome sets in different cell types
within individuals, but his notation evolved into "C-value" in reference to the haploid
DNA content of individual species and retains this usage today.
C-value paradox history - In 1948, Roger and Colette Vendrely reported a
"remarkable constancy in the nuclear DNA content of all the cells in all the individuals
within a given animal species", which they took as evidence that DNA, rather than
protein, was the substance of which genes are composed. The term C-value reflects this
observed constancy. However, it was soon found that C-values (genome sizes) vary
[31]
enormously among species and that this bears no relationship to the presumed number of
genes (as reflected by the complexity of the organism). For example, the cells of some
salamanders may contain 40 times more DNA than those of humans. Given that C-values
were assumed to be constant because DNA is the stuff of genes, and yet bore no
relationship to presumed gene number, this was understandably considered paradoxical;
the term C-value paradox was used to describe this situation by C.A. Thomas, Jr. in 1971.
The discovery of non-coding DNA in the early 1970s resolved the C-value paradox. It is
no longer a mystery why genome size does not reflect gene number in eukaryotes: most
eukaryotic (but not prokaryotic) DNA is non-coding and therefore does not consist of
genes, and as such total DNA content is not determined by gene number in eukaryotes.
The human genome, for example, comprises only about 1.5% protein-coding genes, with
the other 98.5% being various types of non-coding DNA (especially transposable
elements) (International Human Genome Sequencing Consortium 2001). It is unclear
why some species have a remarkably higher amount of non-coding sequences than others
of the same level of complexity. Non-coding DNA may have many functions yet to be
discovered. Though now it is known that only a fraction of the genome consists of genes,
the paradox remains unsolved.
The term "C-value enigma" represents an update of the more common but outdated term
"C-value paradox" (Thomas 1971), being ultimately derived from the term "C-value"
(Swift 1950) in reference to haploid nuclear DNA contents. The term was coined by
Canadian biologist Dr. T. Ryan Gregory of the University of Guelph in 2000/2001. In
general terms, the C-value enigma relates to the issue of variation in the amount of noncoding DNA found within the genomes of different eukaryotes.
The C-value enigma, unlike the older C-value paradox, is explicitly defined as a series of
independent but equally important component questions, including:
a) What types of non-coding DNA are found in different eukaryotic genomes, and in
what proportions?
b) From where does this non-coding DNA come, and how is it spread and/or lost
from genomes over time?
c) What effects, or perhaps even functions, does this non-coding DNA have for
chromosomes, nuclei, cells, and organisms?
d) Why do some species exhibit remarkably streamlined chromosomes, while others
possess massive amounts of non-coding DNA?
Variation among species - C-values vary enormously among species. In
animals they range more than 3,300-fold, and in land plants they differ by a factor of
about 1,000 (Bennett and Leitch 2005; Gregory 2005). Protist genomes have been
reported to vary more than 300,000-fold in size, but the high end of this range (Amoeba)
has been called into question. Variation in C-values bears no relationship to the
complexity of the organism or the number of genes contained in its genome, an
observation that was deemed wholly counterintuitive before the discovery of non-coding
DNA and which became known as the C-value paradox as a result. However, although
there is no longer any paradoxical aspect to the discrepancy between C-value and gene
number, this term remains in common usage. For reasons of conceptual clarification, the
various puzzles that remain with regard to genome size variation instead have been
suggested to more accurately comprise a complex but clearly defined puzzle known as
[32]
the C-value enigma. C-values correlate with a range of features at the cell and organism
levels, including cell size, cell division rate, and, depending on the taxon, body size,
metabolic rate, developmental rate, organ complexity, geographical distribution, and/or
extinction risk.
Calculating C-values - By using the data in Table 5.1, relative weights of
nucleotide
pairs can be
Relative
calculated as
Nucleotide
Chemical formula
molecular
follows: AT =
weight
615.3830 and
2′-deoxyadenosine 5′-monophosphate C10H14N5O6P
331.2213
GC
=
2′-deoxythymidine 5′-monophosphate C10H15N2O8P
322.2079
616.3711.
2′-deoxyguanosine 5′-monophosphate C10H14N5O7P
347.2207
Provided the
2′-deoxycytidine 5′-monophosphate
C9H14N3O7P
307.1966
ratio of AT to
*Source of table: Doležel et al., 2003*
GC pairs is 1:1, the mean relative weight of
one nucleotide pair is 615.8771 (±1%). The
relative molecular weight may be converted to an absolute value by multiplying it by the
atomic mass unit (1 u), which equals one-twelfth of a mass of 12C, i.e., 1.660539 × 10-27
kg. Consequently, the mean weight of one nucleotide pair would be 1.023 × 10-9 pg, and
1 pg of DNA would represent 0.978 × 109 base pairs.
Table 5.1: Relative Molecular Weights of Nucleotides
The formulas for converting the number of nucleotide pairs (or base pairs) to picograms
of DNA and vice-versa are:
Genome size (bp) = (0.978 x 109) x DNA content (pg)
DNA content (pg) = genome size (bp) / (0.978 x 109)
1 pg = 978 Mb
The current estimates for human female and male diploid genome sizes are 6.406 × 109
bp and 6.294 × 109 bp, respectively. By using the conversion formulas given above,
diploid human female and male nuclei in G1 phase of the cell cycle should contain 6.550
and 6.436 pg of DNA, respectively.
The phenomenon that, frequently, C values do not correlate with the evolutionary
complexity of species; they are large in some small organisms. This is presumably due to
the fact that sizeable portions of the DNA do not code for proteins and either have other
regulatory functions or are functionless.
5.2.3 C0t Curve and its Significance
C0t Curve - The curve obtained by plotting the data of a reassociation kinetics
experiment. Since the reassociation of DNA is a bimolecular, second-order reaction, it
follows that C/C0=17 (1 + A:C00 where ^_ is the second-order rate constant (L molds'"1),
t is the time (s), C0 is the initial concentration of single-stranded DNA (moles of
nucleotide per liter), and C is the concentration of single stranded DNA remaining in the
reaction mixture at time / (moles of nucleotide per liter). The cot curve is obtained by
plotting the fraction of single-stranded DNA remaining (C/C0) as a function of log (C0Oi
that is, the logarithm of the product of the initial concentration and the elapsed time. The
cot curve is an S-shaped curve. See also reassociation kinetics.
[33]
Variation in Eukaryotic DNA Sequences - Prokaryotic and eukaryotic cells
differ dramatically in the amount of DNA per cell, a quantity termed an organism’s C
value (Table 5.2). Each cell of a fruit fly, for example, contains 35 times the amount of
DNA found in a cell of the bacterium E.
Table 5.2: Genome sizes of various
coli. In general, eukaryotic cells contain
organisms
more DNA than that of prokaryotes, but
Approximate
variability in the C values of different
Organism
Genome Size
eukaryotes is huge. Human cells contain
(bp)
more than 10 times the amount of DNA
λ -bacteriophage
50,000
found in Drosophila cells, whereas some
E. coli (bacterium)
4,600,000
salamander cells contain 20 times as
Saccharomyces
cerevisiae 13,500,000
much DNA as that of human cells.
(yeast)
Clearly, these differences in C value
Arabidopsis thaliana (plant) 100,000,000
cannot be explained simply by
Drosophila
melanogaster 140,000,000
(insect)
differences in organismal complexity.
Homo sapiens (human)
3,000,000,000
So what is all this extra DNA in
Zea mays (corn)
4,500,000,000
eukaryotic cells doing? We do not yet
Amphiuma (salamander)
765,000,000,000
have a complete answer to this question,
but examination of DNA sequences has revealed that eukaryotic DNA has complexity
that is absent from prokaryotic DNA.
Denaturation and Renaturation of DNA - The first clue that the DNA of
eukaryotes contains several
types of sequences came from
the results of studies in which
double-stranded DNA was
separated and then allowed to
reassociate. When doublestranded DNA in solution is
heated, the hydrogen bonds that
Figure 5.4: The slow heating of DNA causes the two
hold the two strands together are
strands to separate (denature).
weakened and, with enough heat,
the two nucleotide strands
separate completely, a process called denaturation or melting (Figure 5.4). DNA is
typically denatured within a narrow temperature range. The midpoint of this range, the
melting temperature (Tm), depends on the base sequence of a particular sample of DNA:
G–C base pairs have three hydrogen bonds, whereas A–T base pairs only have two; so
the separation of G–C pairs requires more energy than does the separation of A–T pairs.
A DNA molecule with a higher percentage of G–C pairs will therefore have a higher Tm
than that of DNA with more A–T pairs.
The denaturation of DNA by heating is reversible; if single-stranded DNA is slowly
cooled, single strands will collide and hydrogen bonds will again form between
complementary base pairs, producing double-stranded DNA (Figure 5.4). This reaction,
called renaturation or reannealing, takes place in two steps. First, single strands in
solution collide randomly with their complementary strands. Second, hydrogen bonds
[34]
form between complementary bases. Two single-stranded molecules of DNA from
different sources will anneal if they are complementary, a process termed hybridization.
For hybridization to take place, the two strands do not have to be complementary at all
their bases - just at enough bases to hold the two strands together. The extent of
hybridization can be used to measure the similarity of nucleic acids from two different
sources and is a common tool for assessing evolutionary relationships. The rate at which
hybridization takes place also provides information about the sequence complexity of
DNA.
Renaturation Reactions and C0t Curves - In a typical renaturation
reaction, DNA molecules are first sheared into fragments several hundred base pairs in
length. Next, the fragments are heated to about 100° C, which causes the DNA to
denature. The solution is then cooled slowly, and the amount of renaturation is measured
by observing optical absorbance.
Double-stranded DNA absorbs less
UV light than does single-stranded
DNA; so the amount of
renaturation can be monitored by
shining a UV light through the
solution and measuring the amount
of the light absorbed.
The amount of renaturation
depends on two critical factors: (1)
initial concentration of singlestranded DNA (C0) and (2) amount
of time allowed for renaturation
(t). Other things being equal, there
will be more renaturation at higher
Figure 5.5: A C0t curve represents the fraction of
DNA remaining single stranded in a renaturation
concentrations of DNA, because
reaction, plotted as a function of DNA concentration _
high concentrations increase the
time (C0t). This graph is a typical C0t curve for a
likelihood
that
the
two
prokaryotic organism.
complementary
strands
will
collide. There will also be more renaturation with increasing time, because there are more
opportunities for two complementary sequences to collide. These two factors together
form a parameter called Cot, which equals the initial concentration multiplied by the
renaturation time (Co x t = Cot).
A plot of the fraction of single-stranded DNA as a function of Cot during a
renaturation reaction is called a C0t curve. A typical Cot curve for a prokaryotic organism
is shown in figure 5.5. The upper left-hand side of the curve represents the start of the
renaturation reaction, when all of the DNA is single stranded, and so the proportion of
single-stranded DNA is 1. As the reaction proceeds, single stranded DNA pairs to form
double-stranded DNA, represented by the decreasing fraction of single-stranded DNA. At
the end of the reaction, the proportion of single stranded DNA is 0, because all of the
DNA is now double stranded. The value at which half of the DNA is reannealed is called
Cot ½.
[35]
The rate of renaturation also depends on the size and complexity of the DNA molecules
used. Consider the following analogy. Suppose we distribute 100 cards equally among
the students in a class. We ask each student to write his or her name on the cards, and we
put all the cards in a hat. We then randomly draw two cards from the hat and see if the
names on the two cards match. If they don’t match, we put them back in the hat; if they
do match, we remove them, and we continue drawing until all the cards have been
removed. If there are only four students in the class, each student will receive 25 cards.
Because each student’s name is on 25 cards, the chance of drawing two cards that match
is high, and we will quickly empty the hat. If we do the same exercise in another class
with 50 students, again using 100 cards, each student’s name will appear on only two
cards, and the chance of removing two cards with the same name
is much lower. Thus, it will take longer to empty the hat. This exercise resembles what
occurs in the renaturation reaction. If we start with the same total amount of DNA, but
there are only a few different
sequences in the DNA, a chance
collision
between
two
complementary fragments is more
likely to occur than if there were
many different sequences. Therefore
DNA from organisms with larger
genomes will have a larger C0t
value. Thus far, we have considered
renaturation reactions in which each
DNA sequence is present only once
in each molecule. If some sequences
are present in multiple copies, these
sequences will be more likely to
collide with a complementary copy,
and renaturation of these sequences Figure 5.6: A typical C0t curve for a eukaryotic
will be rapid. Think about our organism contains several steps. The first step in the
analogy of drawing names from a curve represents DNA renaturing at very low C 0t
hat. Imagine that we have 50 values, because these sequences are present in many
copies (highly repetitive). The second step represents
students and 100 cards; each student DNA renaturing at intermediate C0t values; these
gets two cards. This time, the sequences are present in an intermediate number of
students write only their first names copies (moderately repetitive). The last step represents
on the cards. Again, we place the DNA that renatures slowly; these sequences are
cards in the hat and draw out two present singly or in few copies (unique).
cards at random.
If there are five students in the class named Scott, this name will appear on ten cards; so
the chance of drawing out two cards at random bearing the name Scott is fairly high. On
the other hand, if there is only one Susan in the class, this name will appear on only two
cards, and the chance of drawing out two cards with the name Susan is low. The cards
with Scott match up more quickly than the cards with Susan, because there are more
copies with the name Scott. Similarly, in a renaturation reaction, if some sequences of
DNA are present in multiple copies, they will renature more quickly.
[36]
Types of DNA Sequences in Eukaryotes - For most eukaryotic organisms,
C0t curves similar to the one presented in figure 5.6 are produced and indicate that
eukaryotic DNA consists of at least three types of sequences. Slowly renaturing DNA
consists of sequences that are present only once or at most a few times, in the genome.
This nonrepetitive, unique-sequence DNA includes sequences that code for proteins, as
well as a great deal of DNA whose function is unknown. The more rapidly renaturing
DNA represents two kinds of repetitive DNA - DNA sequences that exist in multiple
copies. Although not identical, these copies are similar enough to reanneal.
Moderately repetitive DNA typically consists of sequences from 150 to 300 bp in length
(although they may be longer) that are repeated many thousands of times. Some of these
sequences perform important functions for the cell; for example, the genes for ribosomal
RNAs (rRNAs) and transfer RNAs (tRNAs) make up a part of the moderately repetitive
DNA. However, much of the moderately repetitive DNA has no known function in the
cell. Moderately repetitive DNA itself is of two types of repeats. Tandem repeat
sequences appear one after another and tend to be clustered at a few locations on the
chromosomes. Interspersed repeat sequences are scattered throughout the genome.
An example of an interspersed repeat is the Alu sequence, each of which consists of
about 200 bp. The Alu sequence is present more than a million times in the human
genome and makes up about 11% of each person’s DNA. Short repeats, such as the Alu
sequences, are called SINEs (short interspersed elements).
Longer interspersed repeats consisting of several thousand base pairs are called LINEs
(long interspersed elements). Most interspersed repeats are transposable genetic elements,
sequences that can multiply and move.
The other major class of repetitive DNA is highly repetitive DNA. These short
sequences, often less than 10 bp in length, are present in hundreds of thousands to
millions of copies that are repeated in tandem and clustered in certain regions of the
chromosome, especially at centromeres and telomeres. Highly repetitive DNA is
sometimes called satellite DNA, because it has a different base composition from those of
the other DNA sequences and separates as a satellite fraction when centrifuged at high
speeds. Highly repetitive DNA is rarely transcribed into RNA. Although these sequences
may contribute to centromere and telomere function, most highly repetitive DNA has no
known function.
5.2.4 Restriction Techniques
Restriction (Recombinant DNA) Techniques - In the sections that follow,
we will examine some of the following techniques of recombinant DNA technology and
see how they are used to create recombinant DNA molecules: (1.) Methods for locating
specific DNA sequences, (2.) Techniques for cutting DNA at precise locations, (3.)
Procedures for amplifying a particular DNA sequence billions of times, producing
enough copies of a DNA sequence to carry out further manipulations, (4.) Methods for
mutating and joining DNA fragments to produce desired sequences and (5.) Procedures
for transferring DNA sequences into recipient cells
[37]
Cutting and Joining DNA Fragments - The key development that made
recombinant DNA technology possible was the discovery in the late 1960s of restriction
enzymes (also called restriction endonucleases) that recognizes and make doublestranded cuts in the sugar–phosphate backbone of DNA molecules at specific nucleotide
sequences. These enzymes are produced naturally by bacteria, where they are used in
defense against viruses. In bacteria, restriction enzymes recognize particular sequences in
viral DNA and then cut it up. A bacterium protects its own DNA from a restriction
enzyme by modifying the recognition sequence, usually by adding methyl groups to its
DNA.
Three types of restriction enzymes have been isolated from bacteria (Table 5.3). Type I
restriction enzymes recognize specific sequences in the DNA but cut the DNA at random
sites that may be some
Table 5.3: Types of restriction enzymes
distance (1000 bp or
Activity of
ATP
Type
Cleavage Site
more)
from
the
Enzyme
Required
I
Cleavage
and
Yes
Random sites distant recognition
sequence.
methylation
from recognition site
Type
III
restriction
II
Cleavage only
No
Within recognition site
enzymes
recognize
III
Cleavage
and
Yes
Random
sites
near specific sequences and
methylation
recognition site
cut the DNA at nearby
sites, usually about 25 bp away. Type II restriction enzymes
recognize specific sequences and cut the DNA within the
recognition sequence. Virtually all work on recombinant DNA is
done with type II restriction enzymes; discussions of restriction
enzymes throughout this book, refers to type II enzymes. More than
800 different restriction enzymes that recognize and cut DNA
at more than 100 different sequences have been isolated from
bacteria. Many of these enzymes are commercially available;
examples of some commonly used restriction enzymes are
Figure 5.7: Action of
given in Table 5.3. Each restriction enzyme is referred to by a
HindIII producing sticky /
short abbreviation that signifies its bacterial origin. The
cohesive / staggered
sequences recognized by restriction enzymes are usually from
end DNA fragments.
4 to 8 bp long; most enzymes recognize a sequence of 4 or 6
bp. Most recognition sequences are palindromic - sequences that
read the same forward and backward.
Notice in Table 5.3 that the sequence on the bottom strand is the
same as the sequence on the top strand, only reversed. All type II
restriction enzymes recognize palindromic sequences. Some of
the enzymes make staggered cuts in the DNA. For example,
HindIII recognizes the sequence as given in figure 5.7, HindIII
cuts the sugar-phosphate backbone of each strand at the point
indicated by the arrow, generating fragments with short, singlestranded overhanging ends: Such ends are called cohesive ends
Figure 5.8: Action of
or sticky ends, because they are complementary to each other
PvuII
producing
blunt
end
DNA
and can spontaneously pair to connect the fragments. Thus DNA
fragments.
fragments can be “glued” together: any two fragments cleaved
by the same enzyme will have complementary ends and will pair (Figure 5.8). When their
[38]
cohesive ends have paired, two DNA fragments can be joined together permanently by
the enzyme DNA ligase, which seals nicks between the sugar–phosphate groups of the
fragments. Not all restriction enzymes produce staggered cuts and sticky ends. PvuII cuts
in the middle of its recognition site, producing blunt-ended fragments: Fragments with
blunt ends must be joined together in other ways.
Figure 5.8: Restriction enzymes make doublestranded cuts in the sugar–phosphate backbone of
DNA, producing cohesive, or sticky, ends.
The sequences recognized by a restriction enzyme occur randomly within genomic DNA.
Consequently, there is a relation between the length of the recognition sequence and its
frequency of occurrence: there are fewer long recognition sequences than short sequences
because the probability of all the bases being in the required order is less. Restriction
enzymes are the workhorses of recombinant DNA technology and are used whenever
DNA fragments must be cut or joined. In a typical restriction reaction, a concentrated
solution of purified DNA is placed in a small tube with a buffer solution and a small
amount of restriction enzyme. The reaction mixture is then heated at the optimal
temperature for the enzyme, usually 37o C. Within a few hours, the enzyme cuts all the
restriction sites in the DNA, producing a set of DNA fragments (Figure 5.9). Restriction
enzymes can cut linear as well as circular both type of DNA (Figure 5.10). DNA
fragments obtain by the action of restriction enzyme can further use for gel
electrophoresis and cloning purpose (Figure 5.11). DNA fragments can be inserted into
the plasmid by 3 methods (a) restriction cloning (b) cloning by tailing and (c) cloning by
using linkers, in each method restriction enzyme again play important role (Figure 5.11).
Thus restriction enzymes are major components of restriction techniques.
[39]
Table 5.4: Characteristics of some common type II restriction enzymes used
in recombinant DNA technology
Note: The first three letters of the abbreviation for each restriction enzyme refer to the bacterial species from
which the enzyme was isolated (e.g., Eco refers to E. coli). A fourth letter may refer to the strain of bacteria
from which the enzyme was isolated (the “R” in EcoRI indicates that this enzyme was isolated from the RY13
strain of E. coli). Roman numerals that follow the letters allow different enzymes from the same species to be
identified. For convenience, molecular geneticists have come up with idiosyncratic pronunciations of the
names: EcoRI is pronounced “echo-R-one,” HindIII is “hin-D-three,” and HaeIII is “hay-three.” These
common pronunciations obey no formal rules and simply have to be learned.
[40]
Figure 5.9: The number of restriction sites is related to the number of fragments produced when DNA is cut by a
restriction enzyme.
A
B
C
D
A
Figure 5.10: Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge.
(Photo courtesy of Carol Eng.)
[41]
Figure 5.11 : A foreign
DNA fragment can be
inserted into a plasmid
with the use of (a)
restriction cloning, (b)
tailing, or (c) linkers.
[42]
5.2.5 Multigene Families and their Evolution
The term “Multigene Families” is used to include groups of genes from the same
organism that encode proteins with similar sequences either over their full lengths or
limited to a specific domain. DNA duplications can generate gene pairs. If both copies
are maintained in subsequent generations then a multigene family will exist. A
multigene family is a member of a family of related proteins encoded by a set of similar
genes. Multigene families are believed to have arisen by duplication and variation of a
single ancestral gene. Examples of multigene families include those that encode the
actins, hemoglobins, immunoglobulins, tubulins, interferons, histones etc.
DNA duplications that involve one or more genes generate gene pairs. If both copies are
maintained in subsequent generations then a multigene family will exist in the genome.
Because most duplications occur adjacent to the original copy, a subsequent duplication
encompassing both paralogs may generate a family of four chromosomal rearrangements
disperse the multigene families throughout the genome. Dispersed members of the
multigene family can still be recognized by sequence comparison.
The significance of recognizing multigene families is that the members may have related
functions. Genes that are identical or nearly identical in sequence and regulation can be
considered to encode isoforms rather than members of a multigene family. In addition,
genes that were derived from a common ancestral gene but have diverged extensively
may not be recognized as related.
The term “Super-Family” is used to describe a group of proteins with significant
sequence similarity to each other but with clearly defined multigene families. The
individual multigene families are likely to have distinct functions that select for shared
sequences that vary from the global consensus sequence seen in the whole super-family.
The term "clan" is used for related protein families that share some properties but display
no clear phylogenetic relationship with each other. It covers cases of convergent
evolution of proteins with similar functions but no convincing evidence of a common
origin.
Comparative genomics has increasingly shown that most eukaryotic genes are derived
from genes that were present in one form or another in the eukaryotic ancestor.
Subsequent gene loss or amplification led to quantitative and qualitative differences
observed in distant phyla.
(1) Origins and Evolution of the Formin Multigene Family that Is
Involved in the Formation of Actin Filaments - In eukaryotes, the
assembly and elongation of unbranched actin filaments is controlled by formins, which
are long, multidomain proteins. These proteins are important for dynamic cellular
processes such as determination of cell shape, cell division, and cellular interaction. Yet,
no comprehensive study has been done about the origins and evolution of this gene
family. We therefore performed extensive phylogenetic and motif analyses of the formin
genes by examining 597 prokaryotic and 53 eukaryotic genomes. Additionally, we used
three-dimensional protein structure data in an effort to uncover distantly related
sequences. Our results suggest that the formin homology 2 (FH2) domain, which
promotes the formation of actin filaments, is a eukaryotic innovation and apparently
originated only once in eukaryotic evolution. Despite the high degree of FH2 domain
[43]
sequence divergence, the FH2 domains of most eukaryotic formins are predicted to
assume the same fold and thus have similar functions. The formin genes have
experienced multiple taxon-specific duplications and followed the birth-and-death model
of evolution. Additionally, the formin genes experienced taxon-specific genomic
rearrangements that led to the acquisition of unrelated protein domains. The evolutionary
diversification of formin genes apparently increased the number of formin's interacting
molecules and consequently contributed to the development of a complex and precise
actin assembly mechanism. The diversity of formin types is probably related to the range
of actin-based cellular processes that different cells or organisms require. Our results
indicate the importance of gene duplication and domain acquisition in the evolution of
the eukaryotic cell and offer insights into how a complex system, such as the
cytoskeleton, evolved.
(2) Molecular evolution of a multigene family in group A
Streptococci - The emm genes are members of a gene family in group ‘A’
streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or
immunoglobulin-binding proteins. Previously sequenced genes in this family have been
named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as
the "emm gene family." The genes in the emm family are located in a cluster occupying 36 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes.
Most GAS strains contain one to three tandemly arranged copies of emm-family genes in
the cluster, but the alleles within the cluster vary among different strains. Phylogenetic
analysis of the conserved sequences at the 3' end of these genes differentiates all known
members of this family into four evolutionarily distinct emm subfamilies. As a starting
point to analyze how the different subfamilies are related evolutionarily, the structure of
the emm chromosomal region was mapped in a number of diverse GAS strains by using
subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal
patterns of the genes in the emm gene cluster were found. These nine chromosomal
patterns support a model for the evolution of the emm gene family in which gene
duplication followed by sequence divergence resulted in the generation of four major
gene subfamilies in this locus.
(3) Molecular Evolution of the Cecropin Multigene Family in
Drosophila: Functional Genes vs. Pseudogenes - Multigene families
are formed by genes originated by gene duplication that have retained a certain degree of
similarity. The different members are often arranged in a compact cluster although they
might be more or less dispersed in the genome, mostly due to chromosomal
rearrangements subsequent to the gene duplications. Members of a family can be
functional or nonfunctional (pseudogenes). Functional members can be very similar as
the copies might have retained the same function and be redundant. However, one of the
copies may have acquired a new function and suffered a certain degree of differentiation,
which would be best explained by the action of Darwinian selection (Ohta 1994). On the
other hand, pseudogenes can accumulate substitutions due to the lack of functional
constraints. Concerted evolution of the different copies of a gene, which is facilitated by
their compact clustering, can restrict the functional differentiation as well as the loss of
function of the copies (Walsh 1987). Otherwise, members of a family where concerted
[44]
evolution is weak or absent have a higher probability to become pseudogenes (Walsh
1995).
The cecropin multigene family of Drosophila melanogaster is a family with both
functional genes and pseudogenes. The functional genes of this family code for
cecropins, which are antibacterial peptides involved in the insect humoral immune
response (Kylsten et al. 1990; Tryselius et al. 1992). In Drosophila this response is
mediated by at least another eight different kinds of peptides: defensin, attacin, diptericin,
drosocin, metnikowin, drosomycin, andropin, and lysozyme (Engstrom 1997; Hetru et al.
1997; Meister et al. 1997). The humoral response constitutes together with the cellular
response the immune system in insects (Hultmark 1993).
In D. melanogaster the Cecropin region was cloned and sequenced by Kylsten et al. 1990
and by Tryselius et al. 1992. In an ~7-kb region these authors detected four functional
Cecropin genes (CecA1, CecA2, CecB and CecC) and two pseudogenes (Cec1 and
Cec2). All functional genes are expressed upon bacterial infection, mainly in the fat body,
although at different times during development: CecA1 and CecA2 are essentially
expressed in larvae and adults while CecB and CecC are mainly expressed during the
pupal stage (Hultmark 1993).
5.2.6 In-Situ Hybridization – Concept and Techniques
In Situ Hybridization - In situ hybridization is another method for determining the
chromosomal location of a particular gene. This method requires a DNA copy of the gene
or its RNA product, which is used to make a molecule (called a probe) that is
complementary to the gene of interest. The probe is made radioactive or is attached to a
special molecule that fluoresces under ultraviolet (UV) light and is added to
chromosomes from specially treated cells that have been spread on a microscope slide.
The probe binds to the complementary DNA sequence of the gene on the chromosome.
The presence of radioactivity or fluorescence from the bound probe reveals the location
Figure 5.12 : In situ hybridization determine the chromosomal location of a gene. (a) FISH
technique: in this case, the bound probe reveals sequences associated with the centromere.
(b) SKY technique: 24 different probes, each specific for a different human chromosome and
producing a different color, identify the different human chromosomes. (Courtesy of Dr. Hesed
Padilla-Nash and Dr. Thomas Ried, NIH.)
[45]
of the gene on a particular chromosome (Figure 5.12a). The use of fluorescence in situ
hybridization (FISH) has been widely used to identify the chromosomal location of
human genes. In Spectral Karyotyping (SKY) (Figure 5.12b), a set of 24 FISH probes,
each specific to a different human chromosome and attached to a molecule that fluoresces
a different color, allows each chromosome in a karyotype to be identified.
Figure 5.13 : In situ hybridization to locate specific genes on
chromosomes. Here, six different DNA probes have been used to mark
the location of their respective nucleotide sequences on human
chromosome 5 at metaphase. The probes have been chemically labeled
and detected with fluorescent antibodies. Both copies of chromosome 5
are shown, aligned side by side. Each probe produces two dots on each
chromosome, since a metaphase chromosome has replicated its DNA
and therefore contains two identical DNA helices. (Courtesy of David C.
Ward.)
Figure 5.14 : In situ hybridization for RNA localization in tissues.
Autoradiograph of a section of a very young Drosophila embryo that
has been subjected to in situ hybridization using a radioactive DNA
probe complementary to a gene involved in segment development. The
probe has hybridized to RNA in the embryo, and the pattern of
autoradiographic silver grains reveals that the RNA made by the gene
(called ftz) is localized in alternating stripes across the embryo that are
three or four cells wide. At this stage of development (cellular
blastoderm), the embryo contains about 6000 cells. (From E. Hafen, A.
Kuriowa, and W.J. Gehring, Cell 37:833-841, 1984. © Cell Press.)
[46]
In Situ Hybridization Techniques Locate Specific Nucleic Acid
Sequences in Cells or on Chromosomes - Nucleic acids, no less than
other macromolecules, occupy precise positions in cells and tissues, and a great deal of
potential information is lost when these molecules are extracted by homogenization. For
this reason, techniques have been developed in which nucleic acid probes are used in
much the same way as labeled antibodies to locate specific nucleic acid sequences in situ,
a procedure called in situ hybridization. This can now be done both for DNA in
chromosomes and for RNA in cells. Labeled nucleic acid probes can be hybridized to
chromosomes that have been exposed briefly to a very high pH to disrupt their DNA base
pairs. The chromosomal regions that bind the probe during the hybridization step are then
visualized. Originally, this technique was developed using highly radioactive DNA
probes, which were detected by autoradiography. The spatial resolution of the technique,
however, can be greatly improved by labeling the DNA probes chemically instead of
radioactively. For this purpose the probes are synthesized with special nucleotides that
contain a modified side chain, and the hybridized probes are detected with an antibody
(or other ligand) that specifically recognizes this side chain (Figure 5.13).
In situ hybridization methods have also been developed that reveal the distribution of
specific RNA molecules in cells in tissues. In this case the tissues are not exposed to a
high pH, so the chromosomal DNA remains double-stranded and cannot bind the probe.
Instead the tissue is gently fixed so that its RNA is retained in an exposed form that will
hybridize when the tissue is incubated with a complementary DNA or RNA probe. In this
way the patterns of differential gene expression can be observed in tissues. In the
Drosophila embryo, for example, such patterns have provided new insights into the
mechanisms that create distinctions between cells in different positions during
development (Figure 5.14).
5.2.7 Physical Mapping of Genes on Chromosomes
"Gene mapping" refers to the mapping of genes to specific locations on chromosomes. It
is a critical step in the understanding of genetic diseases. There are two types of gene
mapping:
Genetic Mapping - using linkage analysis to determine the relative position between two
genes on a chromosome.
Physical Mapping - using all available techniques or information to determine the
absolute position of a gene on a chromosome.
The ultimate goal of gene mapping is to clone genes, especially disease genes. Once a
gene is cloned, we can determine its DNA sequence and study its protein product. For
example, cystic fibrosis (CF) is the most common lethal inherited disease in the United
States. As many as 1 in 2500 Americans of Northern European descent carries a gene
with CF. In 1985, the gene was mapped to chromosome 7q31-q32 by linkage analysis.
Four years later, it was cloned by Francis Collins and his co-workers. We now know that
the disease is caused by the defect of a chloride channel - the protein product of this
disease gene. Physical mapping techniques which are most common are following two:-
[47]
1) Somatic cell hybridization and
2) Fluorescent In situ Hybridization (FISH)
The resolution of FISH is much higher than somatic cell hybridization.
Mapping by DNA Sequencing - Another means of physically mapping genes
is to determine the sequence of nucleotides in the DNA. With this technique, physical
distances between genes are measured in numbers of base pairs. Continuous sequences
can be determined for only relatively small fragments of DNA; so, after sequencing,
some method is still required to map the individual fragments. This mapping is often
done by using the traditional gene mapping that examines rates of crossing over between
molecular markers located on the fragments. It can also be accomplished by generating a
set of overlapping fragments, sequencing each fragment, and then aligning the fragments
by using a computer program that identifies the overlap in the sequence of adjacent
fragments. With these methods, complete physical maps of entire genomes have been
produced.
Physical Chromosome Mapping - Genetic maps reveal the relative positions
of genes on a chromosome on the basis of frequencies of crossing over, but they do not
provide information that can allow us to place groups of linked genes on particular
chromosomes. Furthermore, the units of a genetic map do not always precisely
correspond to physical distances on the chromosome, because a number of factors other
than physical distances between genes (such as the type and sex of the organism) can
influence rates of crossing over. Because of these limitations, physical-mapping methods
that do not rely on rates of crossing over have been developed.
Deletion Mapping - One method for determining the chromosomal location of a
Figure 5.15 : Deletion mapping can be used to determine the chromosomal location of a gene. An individual
homozygous for a recessive mutation in the gene of interest (aa) is crossed with an individual heterozygous for a
deletion.
[48]
gene is deletion mapping. Special staining methods have been developed that make it
possible to detect chromosome deletions, mutations in which a part of a chromosome is
missing. Genes are assigned to regions of particular chromosomes by studying the
association of a gene’s phenotype or product and particular chromosome deletions.
In deletion mapping, an individual
that is homozygous for a recessive
mutation in the gene of interest is
crossed with an individual that is
heterozygous for a deletion (Figure
5.15). If the gene of interest is in the
region
of
the
chromosome
represented by the deletion (the red
part of chromosome in Figure 5.15),
approximately half of the progeny
will display the mutant phenotype
(see Figure 5.15a). If the gene is not
within the deleted region, all of the
progeny will be wild type (see Figure
5.15b). Deletion mapping has been
used to reveal the chromosomal
locations of a number of human
genes. For example, Duchenne
Muscular Dystrophy is a disease that
causes progressive weakening and
degeneration of the muscles.
From its X-linked pattern of
inheritance, the mutated allele
causing this disorder was known to
be on the X chromosome, but its
precise location was uncertain.
Examination of a number of patients
having
Duchenne
Muscular
Dystrophy, who also possessed small
deletions, allowed researchers to
position the gene to a small segment
of the short arm of the X
chromosome.
Somatic-Cell Hybridization
- Another method used for
positioning genes on chromosomes
is somatic cell hybridization, which
Figure 5.16 : Somatic-cell hybridization can be used to
requires the fusion of different types
determine which chromosome contains a gene of interest.
of cells. Most mature somatic (non
sex) cells can undergo only a limited
number of divisions and therefore cannot be grown continuously. However, cells that
[49]
have been altered by viruses or derived from tumors that have lost the normal constraints
on cell division will divide indefinitely; these
types of cells can be cultured in the laboratory
and are referred to as a cell line. Cells from
two different cell lines can be fused by treating
them with polyethylene glycol or other agents
that alter their plasma membranes. After
fusion, the cell possesses two nuclei and is
called a heterokaryon. The two nuclei of a
heterokaryon eventually also fuse, generating a
hybrid cell that contains chromosomes from
both cell lines. If human and mouse cells are
mixed in the presence of polyethylene glycol,
fusion results in human–mouse somatic-cell
hybrids (Figure 5.16). The hybrid cells tend to
lose chromosomes as they divide and, for
reasons that are not understood, chromosomes
from one of the species are lost preferentially.
In human–mouse somatic-cell hybrids, the
human chromosomes tend to be lost, whereas
the mouse chromosomes are retained.
Eventually, the chromosome number stabilizes
when all but a few of the human chromosomes
have been lost. Chromosome loss is random
and differs among cell lines. The presence of
these extra” human chromosomes in the mouse
genome makes it possible to assign human
genes to specific chromosomes. In the first step
of this procedure, hybrid cells must be
separated from original parental cells that have
not undergone hybridization. This separation is
accomplished by using a selection method that
allows hybrid cells to grow while suppressing
the growth of parental cells. The most
Figure 5.17 : HAT medium can be
commonly used method is called HAT
used to separate human–mouse hybrid
selection (Figure 5.17), which stands for
cells from the original hybridized cells.
hypoxanthine, aminopterin, and thymidine,
three chemicals that are used to select for hybrid cells.
In the presence of HAT medium, a cell must possess two enzymes to synthesize DNA:
thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT).
Cells that are tk - or hprt - cannot synthesize DNA and will not grow on HAT medium.
The mouse cells used in the hybridization procedure are deficient in TK, but can produce
HPRT (the cells are tk – hprt +); the human cells can produce TK but are deficient for
HPRT (they are tk + hprt - ). On HAT medium, the mouse cells do not survive, because
they are tk - ; the human cells do not survive, because they are hprt - . Hybrid cells, on the
other hand, inherit the ability to make HPRT from the mouse cell and the ability to make
[50]
Figure 5.18 : Somatic-cell hybridization is used to assign a gene to a particular human chromosome. A panel
of six cell lines, each line containing a different subset of human chromosomes, is examined for the presence
of the gene product (such as an enzyme). A plus sign means that the gene product is present; a minus sign
means that the gene product is missing. Four of the cell lines (A, B, D, and F) have the gene product,
indicating that the gene is present on one of the chromosomes found in these cell lines. The only chromosome
common to all four of these cell lines is chromosome 4, indicating that the gene is located on this
chromosome.
TK from the human cell; thus, they produce both enzymes (the cells are tk + hprt +) and
will grow on HAT medium.
To map genes using somatic-cell hybridization requires the use of a panel of different
hybrid cell lines. The cell lines of the panel differ in the human chromosomes that they
have retained. For example, one cell line might possess human chromosomes 2, 4, 7, and
8, whereas another might possess chromosomes 4, 19, and 20. Each cell line in the panel
is examined for evidence of a particular human gene.
The human gene can be detected either by looking for the protein that it produces or by
looking for the gene itself with the use of molecular probes. Correlation of the presence
of the gene with the presence of specific human chromosomes often allows the gene to be
assigned to the correct chromosome. For example, if a gene was detected in both of the
aforementioned cell lines, the gene must be on chromosome 4, because it is the only
human chromosome common to both cell lines (Figure 5.18).
Two
genes
determined to be
on
the
same
chromosome with
the
use
of
somatic-cell
hybridization are
said
to
be
syntenic genes.
This term is used
Figure 5.19: 7.21 Genes can be localized to a specific part of a
chromosome by using somatic-cell hybridization.
Note: If the gene
product is present in a
cell line with an intact
chromosome
but
missing from a line
with a chromosome
deletion, the gene for
that product must be
located in the deleted
region.
[51]
because syntenic genes may or may not exhibit linkage in the traditional genetic senseremember that two genes can be located on the same chromosome but may be so far apart
that they assort independently. Synthenic refers to genes that are physically linked,
regardless of whether they exhibit genetic linkage. (Synteny is sometimes also used to
refer to gene loci in different organisms located on a chromosome region of common
evolutionary origin.) Sometimes somatic-cell hybridization can be used to position a gene
on a specific part of a chromosome. Some hybrid cell lines carry a human chromosome
with a chromosome mutation such as a deletion or a translocation. If the gene is present
in a cell line with the intact chromosome but missing from a line with a chromosome
deletion, the gene must be located in the deleted region (Figure 5.19) Similarly, if a gene
is usually absent from a chromosome but consistently appears whenever a translocation
(a piece of another chromosome that has broken off and attached itself to the
chromosome in question) is present, it must be present on the translocated part of the
chromosome.
Fluorescent In situ Hybridization (FISH) - This technique has been
described in section 5.2.6.
5.2.8 Computer Assisted Chromosome Analysis
Chromosome Analysis - Comparing the DNA of different organisms can show
how closely related they are. Since for each species the DNA information is organized in
a characteristic number of chromosomes, the number of chromosomes is a reasonable
indicator of the relatedness of two similar species.
Sometimes the DNA information on a chromosome is reorganized. Chromosomes can
sometimes fuse with each other or can exchange chromosome "arms". When this
happens, DNA information is not always lost, but it can become mixed up. This sort of
rearrangement may not cause problems for the individual who carries the change --- as
long as all the DNA is still present.
A mule is the product of two
different species (a horse and a
donkey) mating with each other.
The fact that these two different
types of animals can mate and
produce viable offspring tells
scientists that horses and donkeys
are closely related. However,
mules are always sterile. Why is
this? Horses and donkeys have
different chromosome numbers.
The fact that horses and donkeys
have
different
chromosome
numbers tells scientists that these
two are different species.
Figure 5.20: One Utah species originally assigned to
For the mule, having parents with Notholaena has 27 chromosomes.
different chromosome numbers
[52]
isn't a problem. During mitotic cell division, each of the chromosomes copies itself and
then distributes these two copies to the two
daughter cells. In contrast, when the mule is
producing sperm or egg cells during meiosis,
each pair of chromosomes (one from Mom
and one from Dad) need to pair up with each
other. Since the mule doesn't have an even
number of homologous pairs (his parents had
different chromosome numbers), meiosis is
disrupted and viable sperm and eggs are not
formed.
Using Chromosomes To Classify
Plant Species - Variations in
Figure 5.21: Species of Notholaena from
chromosome number are even more common other states have multiples of 30
in the plant kingdom. In plants, chromosome chromosomes
number is an important indicator for
determining relationships between
plant species. Scientists at the Utah
Museum of Natural History recently
used studies of chromosome number to
show that a Utah fern was not the same
species as a very similar fern found in
other states. Studies of the Utah Jones
Cloak Fern (originally thought to be a
Notholaena species) showed that the
cells of this plant have 27
chromosomes (Figure 5.20).
Other species of Notholaena found in
other states have 30 chromosomes
(Figure 5.21). When combined with
the results of other studies, the
difference in chromosome number
helped to prove that the Utah species
actually belonged in the genus
Argyrochosma, a very distant relative
of Notholaena .
This sort of information is important
because
it
helps
conservation
biologists understand the distribution
of each different species of plant. With
this sort of information, scientists are
better able to decide which plants are
Figure 5.22 : A logical tree structure of the dynamic encoding
rare and require protection by means
process. (Note: When a branch terminates with "finish," a
such as the endangered species list.
correct decision can probably be made. When a branch
terminates with a numbered "return," the data must be
augmented, or modified if possible, then reconsidered
starting at the corresponding auxiliary entry point of the
decision tree.)
[53]
The purpose of the computer program is twofold: (i) to illuminate the basic mechanisms
by which a human recognizes an object, such as a chromosome, and distinguishes it from
other entities; and (ii) the employment of these mechanisms is an automatic and precise
extraction of chromosome features.
The computer program must locate a single entity- chromosome or nonchromosome-then
identify as many of its characteristics as possible and employ them to make a decision. If
the characteristics noted are sufficient to satisfy the observer that his decision will be
correct, he will make a decision. Otherwise, he will investigate alternative ways of
looking at the object. On a computer, this takes the form of a decision tree (Figure 5.22).
The chromosome is defined and recognized by the presence or absence of "arms" and of
their shape and size.
Computer chromosome recognition has an extensive literature. For example : Ledley et al. describe syntax-directed methods using a special scanner for computer
input of pictures.
Hilditch and Rutovitz present a comprehensive discussion of shape-oriented
chromosome recognition experiments using the same scanner.
Rutovitz employs two of our concepts: "a series of increasingly complex alternatives"
and "a polar coordinate representation of the boundary."
Hilditch describes the separation of touching chromosomes,
While Gallus and Neurath improve earlier programs by incorporating boundary
analysis.
Here is an example of a work which presents methods for data reduction and isolation of
visual entities (chromosomes, blobs, touching and overlapping chromosomes) as well as
boundary encoding via a scaled polar plot which enables analysis for overlap resolution.
It has an experimental
program which exemplifies
feature
encoding
and
decision
making
in
hierarchical fashion. Few
steps of this program are as
follows which is explained
here only by program
generated pictures.
(1) Data Reduction
and
Isolation
of
Separate Entities The first phase of the
computer program modifies
the data representing the
picture so that it becomes as
small a data base as possible.
The scanned picture is input
in the form of numbers
representing gray levels, or
Figure 5.23 : Points are assigned to an object as
the boundary is traced.
[54]
relative darkness, corresponding to a particular small area of the original photograph. A
picture of 1 000000 points is converted into a different form and reduced to some 10 000
pairs of coordinates (Figure 5.23).
(2) Dynamic Encoding of Information for Feature Extraction - The
actual encoding of information is the major task of the computer program. Two
techniques are incorporated in the program. These are :(a) Integral projections or chord profiles (Figure 5.24) and
(b) The production of a polar plot (Figure 5.25) of the suspected chromosome.
These two methods are not parallel. The integral projection technique is used as a pretest
to determine which branches of the tree should be followed. At a later time the polar plot
is used for the more explicit decision making.
Figure 5.24
detached part.
:
Chromosome
with
[55]
(3) Analysis and Decision
Making - Objects encountered by the
decision making apparatus are being
evaluated for possible membership in
four categories: normal X chromosome,
abnormal
X
chromosome,
Y
chromosome, and non-chromosome.
(4) Classification - Figure 5.26 is
an example of what is obviously a
complete
and
probably
normal
chromosome, although it is not an
excellent image.
Figure 5.25 : Polar plot of chromosome with
detached arm.
Figure 5.26 : An example of a complete and probably normal chromosome.
[56]
(5) Overlap Resolution –
Result could indicate an object of
the type as shown in figure 5.27
and could be interpreted as two
X chromosomes, one on top of
the other. The polar plot does not
make it immediately obvious that
this is the case, but it does
indicate that the possibility
exists.
Once the coordinates have been
translated as shown in figure
5.28, the polar plot can be
reexamined in a new light. It is
assumed that the largest peaks in
this profile are not part of the
chromosome.
The
experimental
pattern
recognition program described
here employs several feature
extraction techniques to enable
computer
analysis
of
chromosome patterns. The program
-performs data reduction to isolate
objects from the scene in an
efficient manner based on the line at
a time input of scanned picture data
from magnetic tape, and retaining
the useful boundary information in
this process. One test-a convexity
indication eliminating blobs-is based
on the easily computed integral
projections/chord profiles used
elsewhere in pattern recognition by
Pavlidis and Ball. Boundary data is
encoded and features obtained
(peaks in a polar plot of the
boundary indicating chromosome
arms) as the program continues past
this test through its decision tree.
Features are summarized in a pattern
vector which consists of the relative
maxima and minima in the final
polar plot of the chromosome, while
the record of translations and
deformation which led to this plot is
Figure 5.27 : Two overlapping chromosomes.
Figure 5.28 : Overlapping chromosomes reconsidered
using a new origin.
[57]
retained. Methods for using the data to classify the chromosomes (normal X
chromosome, abnormal X chromosome, Y chromosome, and nonchromosome) are
described which could easily be incorporated in the existing program, to make it an
operational program.
Finally, a method for resolving cases of overlapping chromosomes by selecting subsets of
the polar plot peaks was presented, as was a method for assigning a confidence level to
each decision (Figure 5.29).
The key concept presented here is a two-level logical structure of the pattern recognition
program which allows it to select the procedures and criteria most likely to lead to a
correct decision. In particular, the program uses a changing organization of the features
of the object discovered as the decision-making task changes. The resulting pattern
recognition concept we term dynamic encoding for feature extraction, while the overall
task this program is intended to perform is that of flexible decision making needed for
picture processing.
5.2.9 Chromosome Microdissection and Microcloning
Chromosome microdissection is a
technique that physically removes a
large section of DNA from a
complete chromosome (Figure 5.30).
The smallest portion of DNA that
can be isolated using this method
comprises 10 million base pairs hundreds or thousands of individual
genes.
Scientists who study chromosomes
are known as cytogeneticists. They
are
able
to
identify
each
chromosome based on its unique
pattern of dark and light bands.
Certain abnormalities, however,
cause chromosomes to have unusual
banding patterns. For example, one
chromosome may have a piece of
another chromosome inserted within
it, creating extra bands. Or, a portion
of a chromosome may be repeated
over and over again, resulting in an
unusually wide, dark band (known
as a homogeneously staining region).
Some chromosomal aberrations have
been linked to cancer and inherited
genetic
disorders,
and
the
chromosomes of many tumor cells
exhibit
irregular
bands.
To
Figure 5.29 : Decomposition of the plot in order to distinguish
two chromosomes.
[58]
understand more about what causes these conditions, scientists hope to determine which
genes and DNA sequences are located near
these
unusual
bands.
Chromosome
microdissection is a specialized way of isolating
these regions by removing the DNA from the
band and making that DNA available for further
studies.
To
prepare
cells
for
chromosome
microdissection, a scientist first treats them with
a chemical that forces them into metaphase: a
phase of the cell's life-cycle where the
chromosomes are tightly coiled and highly
visible. Next, the cells are dropped onto a
microscope slide so that the nucleus, which
holds all of the genetic material together, breaks
apart and releases the chromosomes onto the
slide. Then, under a microscope, the scientist Figure 5.30 : Procedure of microdissection
locates the specific band of interest, and, using a of a chromosome
very fine needle, tears that band away from the
rest of the chromosome. The researcher next produces multiple copies of the isolated
DNA using a procedure called PCR (polymerase chain reaction). The scientist uses these
copies to study the DNA from the unusual region of the chromosome in question.
Microcloning is a process in which very small amount of chromosome/genome is cloned
into a host. A little discussion about the microcloning process in different host is present
here.
Genome require isolation of large numbers of DNA probes from defined regions of the
chromosomes. In addition, the effective use of genetic linkage analysis with polymorphic
DNA probes from the human genome has localized more and more inherited diseases of
unknown etiology, as well as specific forms of cancer, to refined regions of various
chromosomes. Thus, the next major task is to isolate the genes underlying these diseases
for better understanding, prevention, diagnosis, and treatment of the disease.
A direct approach to this objective, as proposed by Edstrom and his colleagues, is to use
the chromosome microdissection technique to remove physically the chromosomal region
of interest and to clone the minute quantities of the dissected chromosomal DNA by a
microcloning procedure. This approach has been successfully applied first with the
Drosophila polytene chromosome and later with the mammalian metaphase chromosome
(Figure 5.31).
Recently, a modification of this method was described by Ludecke et al. in which
significantly increased cloning efficiency was achieved by ligation of the dissected
chromosomal DNA to a vector, followed by polymerase chain reaction (PCR) to amplify
the dissected sequences. The PCR products were cloned into the plasmid vector pUC13.
Using this method, these investigators obtained between 5000 and 20,000 clones from
each of the dissected chromosome regions. The advantages of this method over the
original microcloning procedure include (i) banded and stained chromosome preparations
can be used, (ii) fewer dissected chromosome fragments are needed, and (iii) large
numbers of microclones can be obtained.
[59]
Another method of PCR microcloning was described by Saunders et al. and by Johnson
for a universal amplification of microdissected Drosophila polytene chromosome
fragments. In this method, a polytene chromosome band was dissected, cleaved with
either Sau3A or MboI, and ligated to a linker adaptor. The linker adaptor was constructed
from oligonucleotides of 24-mer and 20-mer annealed to yield a 5' protruding Sau3A or
MboI sequence. The 20-mer of the linker adaptor was used as a primer for PCR.
The amplified products were digested again with Sau3A or Mbo I and cloned into the
compatible BamHI site of a plasmid vector. Large numbers of clones were obtained by
FIG. 5.31 : (a) Unstained normal human metaphase spread; the arrow indicates the location of a
chromosome 2. (b) Same spread, showing the chromosome 2 after the removal of the distal half
of the short arm with a fine glass needle.
this method. More importantly, Saunders et al. also compared the PCR microcloning
method with the conventional microcloning method and showed that the PCR method
generated probes representing >90% of the dissected genomic region, whereas the probes
from the conventional method covered only 3-4% of the same dissected region.
Moreover, the PCR microcloning by the linker-adaptor method appears to offer
additional useful features over the PCR microcloning method of Ludecke et al. : (i)
several different linker sequences and restriction enzymes can be used, and (ii) a more
efficient sticky-end ligation, instead of blunt-end ligation, is used.
Several approaches have been used to obtain a number of clones specific to a
subchromosomal region for use in mapping. The phenol-enhanced reassociation
technique, as used for Duchenne muscular dystrophy, relies on the presence of a deletion
which spans the region of interest. Hybrids containing only specific chromosomal
fragments can be constructed by chromosome-mediated gene transfer, from which
libraries can be prepared. However, this method depends on the natural presence or
chance integration of a selectable marker.
[60]
An alternative and more generally applicable technique clones picogram amounts of
DNA isolated by the direct microdissection of chromosomes. This method was first
applied to DNA from individual bands of Drosophila melanogaster polytene
chromosomes and was extended to mammalian chromosomes with the isolation of clones
mapping to the mouse t complex and to a proximal region of the mouse X chromosome.
In mouse genetics, the technique has fulfilled the promise of fine genetic and molecular
mapping of individual chromosome regions and allows the isolation and characterization
of previously unclonable genetic loci. With the use of Robertsonian translocations for
chromosomal identification, every chromosomal region of mice is now available for
dissection.
Microdissection and microcloning of the euchromatin-heterochromatin transition region
of the Drosophila melanogaster polytene X chromosome and part of the euchromatin of
chromosome 4 reveals that they share certain features characteristic of (3heterochromatin, which is morphologically defined as the loosely textured material at the
bases of some polytene chromosome arms. Both are mosaics of many different middlerepetitive DNA sequences interspersed with single-copy DNA sequences. Sixty percent
of cloned inserts derived from division 20 and about 40 percent from subdivisions l9EF
of the X chromosome harbor at least one repetitive DNA sequence in an average insert of
4.5 kilobases. No repeats have significant cross-hybridization to any of the eleven
satellite DNAs, or to the clustered-scrambled sequences present in pDml. The repetitive
elements are, in general, confined to the g3-heterochromatic regions of polytene
chromosomes, but some are adjacent to nomadic elements. Chromosome 4, however, has
some repeats spread throughout its entire euchromatin. These data have implications for
the structure of transition zones between euchromatin and heterochromatin of mitotic
chromosomes and also provide a molecular basis for reexamining some of the unusual
classical properties of chromosome 4.
5.2.10 Flow Cytometry and Confocal Microscopy in Karyotype
Analysis
Flow Cytometry - Often the experimenter is not interested in keeping the cells but
simply in analyzing the distribution of cell size and/or surface molecules on the cells in
the suspension. The signals from the detectors can generate these sorts of data, a
procedure known as flow cytometry.
The data are analyzed by computer and can be plotted in several ways. The more
elaborate flow cytometers can use two different lasers to detect as many as three different
colors on the cells in the suspension. Here are some examples (all courtesy of Becton
Dickinson Immunocytometry Systems).
Counting T and B cells by flow cytometry - A sample of normal
human blood was
treated with a
fluorescent
monoclonal
antibody specific
for the T cell
Figure 5.32: Flow Cytometry for counting T and B Cells
(a) Different Peaks of T and B cell (b) Separation of T and B cells.
[61]
surface antigen CD3 and a monoclonal antibody conjugated to a different fluorescent dye
and specific for the B cell surface antigen designated CD19. Fluorescence intensity
(logarithmic) is plotted on the x axis; cell number on the y axis. Note that the number of
B cells is substantially less than that of T cells. The right-hand panel shows another way
of plotting the data.
Counting T-cell subsets in normal
human blood - Fluorescent monoclonal
antibodies were directed against the
CD4 molecule (left) and CD8 molecule
(right). CD4+ T cells are responsible for
several cell-mediated immune responses
and giving help to B cells. CD8+ T cells
are cytotoxic T lymphocytes (CTLs). Figure 5.33: Two subset of T cell : CD4
molecules (left) and CD8 molecules (right).
The preponderance of CD4+ over CD8+
cells shown here is typical of healthy humans. In AIDS patients, this ratio becomes
reversed and the CD4+ subset may eventually disappear.
Flow Cytometry and Genome Analysis - Flow cytometry allows for fast
and informative, quantitative and qualitative analysis of objects including chromosomes
and nuclei, normally by measuring the fluorescence of molecules that are specifically
bound to structures of interest (Figure 5.34; Melamed et al., 1990). The molecules
measured are often fluorochrome-conjugated antibodies or fluorescent dyes binding
specifically to DNA or proteins.
Figure 5.34 : The optical and mechanical
arrangement of a flow cytometer capable of
measuring the fluorescence of stained particles,
such as chromosomes of nuclei, in a fluid stream.
After arrangement, the particles, by now
contained in droplets, are sorted by charged
plates depending on their fluorescence.
Flow cytogenetics underpins much of the
human genome project: the Department of
Energy Human Genome Program reports that
"among the resources most crucial to mapping
progress are the libraries of clones
representing each of the human chromosomes.
This chromosome-specific clone library
production
from
physically
purified
chromosomes depends on the unique
chromosome-sorting facilities" at Los Alamos
and
Lawrence
Livermore
National
Laboratories (Anon, 1993). Flow technology
is also recognized as being critical to pig and
bovine genome projects (Miller et al., 1992; Dixon et. al., 1992).
[62]
Joe Gray, from the University of California, San Francisco, and Scott Cram, Life Science
Division Leader at Los Alamos National Laboratory, described some of the advantages of
flow cytometry for plant molecular cytogenetics and genome analysis. "The analysis and
sorting of plant chromosomes is of considerable economic interest. As is the case for
mammalian chromosomes, flow karyotyping and chromosome sorting provide the
opportunity for gene mapping and the construction of chromosome-specific libraries"
(Gray and Cram, 1990).
Since they wrote this, there have been successful applications of the methods in plants for
genome size measurements (including the specific AT and GC base-pair content), cell
cycle analysis, flow karyotyping (by measuring the DNA content of chromosomes),
chromosome sorting and production of chromosome-enriched DNA libraries, although
these analyses are not as yet extensively exploited.
In this short review, I aim to highlight potential and recent applications of flow cytometry
to plant genomes; the literature on chromosome analysis has been reviewed recently by a
group of European collaborators from the Czech Republic, Italy and Germany (Dolezel et
al. 1994), while many techniques for flow analysis of plants are discussed in the same
paper and elsewhere (e.g., Heslop-Harrison and Schwarzacher, 1995).
Genome size analysis - Changes between species, during
differentiation and during the cell cycle - Analytical information about the
physical size of plant genomes and their state of replication is easily obtainable from flow
cytometry. Knowing the number of base pairs in a genome is valuable for studies of new
species, and an extensive list based on flow cytometric estimates was published by
Arumuganathan, now at the University of Lincoln, Nebraska, and Lisa Earle, from
Cornell University, in 1991.
Flow cytometry provides a fast and accurate way to look at changes in genome size
during evolution and differentiation. Establishment of ploidy and aneuploidy changes
during tissue culture, and examination of intra- and inter-specific variation of DNA
content can all be important in plant hybridization, breeding, and genetic manipulation
programs (Dolezel et al. 1994; Leitch et al. 1992). Perhaps, in contrast to animals,
polyploidy often accompanies differentiation, and is an important part of plant
development, with different cell types having characteristic ploidies (Galbraith et al.
1991; Bino et al. 1993). Such differentiation by polyploidization is important for
understanding the regulation of gene expression in differentiated tissues, and for
understanding the nature of tissues used for plant regeneration and transformation.
Flow cytometry provides an accurate method for determining the proportions of cells in
G1, S and G2/M stages of the cell cycle. These data can be used to calculate cell cycle
times, which are needed in studies of the genetics and control of this process, and are
useful for analysis of aspects of crop growth and development.
Flow karyotyping - Flow karyotypes, giving the average sizes of chromosomes
from mitotic cells, are quick, accurate and quantitative. To make a flow karyotype, a
suspension of many thousands of chromosomes is made and stained with a fluorochrome
which binds quantitatively to DNA. The fluorescence of chromosomes is measured as
they pass individually through a cytometer (Figure 5.35), giving a histogram where each
peak represents one or a group of chromosomes.
[63]
Flow methods enable differences as small as 1.5 to 4 Mb to be analyzed in humans, and
both aneuploidy and many chromosome deletions can be detected easily. In plants, eight
species have been flow karyotyped so far, including Haplopappus gracilis, a plant with
only two pairs of chromosomes, four solanaceous species and Melandrium album (with
sex chromosomes), wheat (Triticum aestivum), and field or broad bean (Vicia faba).
Chromosome sorting for gene mapping and library construction
- After identification of a
Figure 5.35 : A "flow karyotype" of a wheat cell line. The continuous
trace is a frequency distribution histogram showing the number of
chromosomes with each particular fluorescence. The five sharp
peaks correspond to single chromosomes or pairs of chromosomes,
while the smoother curves are formed where many individual
chromosome types are not resolved (right), or from broken
chromosomes, nuclei, or organelles (left). This graph is derived from
a two-wavelength analysis, shown as the diagonal line where density
of dots is proportional to number of chromosomes detected of each
fluorescence. Some peaks on the continuous line are now resolved
as two peaks (arrow). The karyotype is derived from analysis of 50,
000 chromosomes, taking about 5 minutes. I thank Nigel Miller at the
Babraham Institute, Cambridge, for assistance in operating the flow
cylinder.
polymerase chain reaction (PCR) can be mapped by using
chromosome
by
its
fluorescence,
particular
chromosomes can be sorted
from others in a flow
cytometer
by
either
electrical (as in Figure
5.34)
or
mechanical
deflection. Chromosome
sorting has been achieved
from
important
crop
species including wheat,
tomato
(Lycopersicon
esculentum), and field
bean. Sorted chromosomes
serve as a source of DNA
for
production
of
chromosome-specific and
chromosome recombinant
DNA
libraries.
A
chromosome-specific
recombinant DNA library
from chromosome 4A
showed 20-fold enrichment
of clones as compared to
a random genomic library
(Wang et al. 1992).
Partitioning the genome
before cloning can reduce
the effort required for
screening a library.
Dot blots, in which
chromosomes are sorted
onto membranes, have
been used for gene
mapping by probing with
cDNA and other clones.
Primers
for
the
sorted chromosomes as the
[64]
DNA source (Macas et al. 1993), and subchromosomal localization is possible by probing
sorted chromosomes from karyotypes with translocations. Sorted chromosomes also can
be used for microinjection, bombardment, or electroporation into regenerable tissues to
make, perhaps, chromosome-specific transfectants.
Quantification - Flow cytometry can be used to measure small DNA molecules down to 20 kb, below the threshold of separation by normal electrophoresis. The ability
to quantify accurately large (from 20 kb to chromosomal size) DNA molecules may be
useful in analysis of large genomic features - the distribution of Notl or M1u1 islands, for
example. Work at National Flow Cytometry Resource Center, Los Alamos, and
elsewhere, is pushing the limits to base-pair resolution: DNA sequencing at rates of tens
of base pairs per second may be feasible by Ba/31 exonuclease digestion of a single DNA
molecular and cytometric identification of the bases as they are cut off. In situ
hybridization methods in suspension may enable accurate quantification of numbers of
copies of repetitive DNA sequences on different chromosomes in a species -- something
often difficult to do by other methods.
Conclusions - Applications of flow cytogenetics range from detection of aberrant
cell cycles and changes in nuclear DNA amounts to sorting of chromosomes for gene
mapping and library construction. The methods are used extensively in the human
genome project, and are applicable to plants. The fast and quantitative results make the
method an important analytical tool which has the potential to advance many aspects of
plant genome analysis.
CONFOCAL MICROSCOPY - Confocal microscopy is an optical imaging
technique used to increase micrograph contrast and/or to reconstruct three-dimensional
images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that
are thicker than the focal plane. This technique has been gaining popularity in the
scientific and industrial communities. Typical applications include life sciences and
semiconductor
Basic concept - The principle of confocal imaging was patented by Marvin Minsky
in 1957. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen
is flooded in light from a light source. Due to the conservation of light intensity
transportation, all parts of the specimen throughout the optical path will be excited and
the fluorescence detected by a photodetector or a camera. In contrast, a confocal
microscope uses point illumination and a pinhole in an optically conjugate plane in front
of the detector to eliminate out-of-focus information. Only the light within the focal plane
can be detected, so the image quality is much better than that of wide-field images. As
only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging
requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning
lines) in the specimen. The thickness of the focal plane is defined mostly by the square of
the numerical aperture of the objective lens, and also by the optical properties of the
specimen and the ambient index of refraction.
[65]
Types - Three types of confocal microscopes are commercially available: Confocal
laser scanning microscopes (Figure 5.36), spinning-disk (Nipkow disk) confocal
microscopes and Programmable Array Microscopes (PAM). Confocal laser scanning
microscopy
yields
better image quality
than Nipkow and PAM,
but the imaging frame
rate was very slow (less
than 3 frames/second)
until recently; spinningdisk
confocal
microscopes
can
achieve video rate
imaging - a desirable
feature for dynamic
observations such as
live
cell
imaging.
Confocal laser scanning
microscopy has now
been
improved
to
provide better than
video
rate
(60
frames/second) imaging
by using MEMS based
scanning mirrors.
Biologists
quickly
realized that confocal
microscopy offers one
big advantage: the
ability
to
perform
Figure 5.36 : "Confocal Microscope" - Path for Light
optical sectioning at the
cellular
level
in
a
noninvasive way. It eliminates the need to embed material and cut serial sections.
The confocal microscope offers biologists the chance to investigate the microscopic
world in 3-dimensions. A second advantage is the ability to quantify the visual data that
greatly enhanced the use of confocal microscopy. The scientific literature contains many
confocal microscope studies ranging from the cellular to the macroscopic. Molecular
biology in particular is exploiting the technology. It is being used to unravel the 3dimensional functioning of genes in nuclei, for example to show when they become
active (Rowland and Nickless 1999). In fact, confocal microscopy is now applied
worldwide not only in all avenues of the biological sciences but also the physical sciences
The Confocal Microscope Facility at Massey University is a multi-user facility used by
plant biologists, veterinary researchers, food technologists, dairy product researchers,
microbiologists, cytogeneticists, molecular biologists and even technologists examining
bitumen (asphalt like products). The potential of the machine is limited only by the type
of questions one wishes to ask. Unquestionably, it has created a sense of inquiry where
[66]
features relating to growth and development, injury and disease, mode of infection of
pathogens, chromosomal behavior, milk processing and product development all require
some quantitative understanding of events that unfold.
5.3 ALIEN GENE TRANSFER THROUGH CHROMOSME
MANIPULATION
Genetic improvement of crops has traditionally been achieved through sexual
hybridization between related species, which has resulted in numerous cultivars with high
yields and superior agronomic performance. Conventional plant breeding, sometimes
combined with classical cytogenetic techniques, continues to be the main method of
cereal crop improvement. More recently, through the introduction of new tools of
biotechnology, crossing barriers have been overcome, and genes from unrelated sources
have become available to be introduced asexually into plants. Cereal crops were initially
difficult to genetically engineer, mainly due to their recalcitrance to in vitro regeneration
and their resistance to Agrobacterium infection. Systematic screening of cultivars and
explant tissues for regeneration potential, development of various DNA delivery methods
and optimization of gene expression cassettes have produced transformation protocols for
the major cereals, although some elite cultivars still remain recalcitrant to transformation.
Most of the transgenic cereals developed for commercial purpose exhibit herbicide and/or
insect resistance; traits that are often controlled by a single gene. In recent years, more
complex traits, such as dough functionality in wheat and nutritional quality of rice have
been improved by the use of biotechnology. The current challenges for genetic
engineering of plants will be to understand and control factors causing transgene
silencing, instability and rearrangement, which are often seen in transgenic plants and
highly undesirable in lines to be used for crop development. Further improvement of
current cereal cultivars is expected to benefit greatly from information emerging from the
areas of genomics, proteomics and bioinformatics.
5.3.1 Transfer of Whole Genome - Examples from Wheat, Archis
& Brassica
In recent years, with advent of the development of efficient plant regeneration systems in
cereal crops, the field of recombinant DNA technology has opened up new avenues for
genetic transformation of crop plants. Monocots particularly cereals were initially
considered difficult to genetically engineer, primarily due to their recalcitrance to in vitro
regeneration and their resistance to Agrobacterium. Continuous efforts and systematic
screening of cultivars and tissues for regeneration potential, development of various DNA
delivery methods and optimization of gene expression cassettes have led the development
of reliable transformation protocols for the major cereals including wheat. Since the
production of first fertile transgenic wheat plants in 1992s, microprojectile-mediated gene
transfer has proved the most successful method for genetic transformation of wheat not
only for the introduction of marker genes but also agronomically important genes for
improving quality of wheat flour, transposons tagging, building resistance against fungal
[67]
pathogen and insects, engineering male sterility, and resistance to drought stress. Despite
tremendous successes in producing fertile transgenic wheat plants using various methods
and approaches, elite cultivars of wheat still remain recalcitrant to transformation.
Moreover, in comparison with other major cereals like rice and maize, the development
of a high throughput wheat transformation system has been slowed and severely affected
by genotype effects on plant regeneration, low transformation efficiencies and problems
with transgene inheritance and stability of expression. Majority of the researcher
worldwide have used genetic engineering to tailor wheat for specific end-use by using
immature embryos as the primary target tissue for the delivery of desired foreign genes.
Hence, we have focused our attention to the work done on stable gene expression and
transformation of wheat by employing microprojectile bombardment and Agrobacterium
as a source of foreign DNA delivery into immature embryos. Recent advances in wheat
transformation especially successes in genetic transformation of wheat with
agronomically important genes and novel and innovative approaches for wheat
transformation based on different selection schemes are also discussed.
Wide hybrids in the Triticeae tribe have been attempted and studied for over a 100 years.
The first such hybrid was between wheat and rye (Wilson, 1876). Rimpau (1891)
described 12 plants recovered from seed of a wheat-rye hybrid that represented the first
Figures 5.37 : 3-D anaglyphs of
pollen grains stained with acridine
orange. Bar = 10μm. (3) a single
Hoheria sp. pollen grain; (4)
Myosotis monroi pollen grains on the
surface of a stigma; (5) Agathis
australis (kauri) pollen grain obtained
from a swamp profile, estimated age
10,000 years. (6) and (7) 3-D
anaglyphs of dividing somatic cells of
Vicia faba, broad bean, root-tips
stained with Feulgen. (6) mostly
interphase cells, note the bright
heterochromatic knobs. Also note
the early prophase at left, late
prophase in middle, and metaphase
top right. Bar = 5μm; (7) a prominent
anaphase in the center and a
prophase cell at top left. Bar =3μm.
(8) 3-D anaglyph of germinating
yeast spores stained with acridine
orange. Bar = 5μm. Color images of
these anaglyphs are found on the
cover of this issue. Anaglyph 3-D
glasses are included with this issue.
View the color image using the
glasses: Left eye = blue, right eye =
red.
triticale. Farrer (1904) reported
studies
on
wheat-barley
[68]
hybridization; however, Shepherd and Islam (1981) considered it improbable that these
were true intergeneric hybrids. Several perennial grasses were hybridized with wheat in
the early 1930s with the objectives of transferring disease resistance and perenniality into
annual crops. Many hybrids involving Triticum and several Aegilops species were also
made during the 1920s and 1930s from which the genomic relationships of the two
genera were derived. The large-scale practical use of the hybrids, however, was delayed
until the advent of colchicine treatment in the late 1930s. The ability to double the
chromosome number of hybrids using colchicine had both practical and theoretical
consequences. The production of fertile amphiploids provided the way to develop X
Triticosecale Wittmack as a new cereal crop. Also advanced were evolutionary studies
when McFadden and Sears (1946) resynthesized T. aestivum, discovering that Ae.
tauschii (syn. T. tauschii) was the D-genome donor to bread wheat.
With the advancement of hybridization techniques (Kruse, 1973) and embryo culture
(Murashige, 1974), wide hybridization became a more common practice involving more
perennial species. In reviews of the progress of wide hybridization, intense interest was
expressed among breeding programmes in utilizing the genetic resources available in the
perennial Triticeae for cereal improvement (Dewey, 1984; Sharma and Gill, 1983a;
Mujeeb-Kazi and Kimber, 1985; Sharma, 1995; Wang, 1989).
Of the approximately 325 species in the tribe Triticeae, about 250 are perennials and 75
are annuals (Dewey, 1984). Relatively few perennials have been hybridized with wheat
essentially because of the complexity of doing so and due to embryo rescue/regeneration
constraints. The perennials, which include many important forage grasses, have the
potential to serve as a vital genetic reservoir for the improvement of annual grasses.
These include the major cereals: bread wheat, durum wheat, triticale, barley and rye.
Perennials successfully utilized for improving wheat are predominantly in the
Thinopyrum group.
This section is focus on achieving agricultural production targets with emphasis on bread
and durum wheat. Such production targets are to be achieved by enforcing crop
improvement protocols based upon the utilization of genetic diversity, crucial for
durability of stress resistances and tolerances and for ensuring sustainability. Major
emphasis is devoted to consideration of the exploitation of ‘alien’ genetic diversity,
encompassing interspecific and intergeneric hybridization categories.
5.3.2 Transfer of Individual Chromosomes and Chromosome
Segments
Chromosome 5B mechanism - There seems to be no parallel to the
chromosome 5B-like manipulative approach that encompasses mono-5B, phph or nullitetrasomic stocks as the maternal wheat sources in wide-crosses. These stocks enhance
wheat/alien recombinations in the F1 hybrids and all involve the ph system (Sharma and
Gill, 1983a, 1983b, 1983c; Darvey, 1984; Mujeeb-Kazi et al., 1984; Forster and Miller,
1985; Sharma and Bäenziger, 1986). The resultant F1 hybrids exhibit a high meiotic
chromosome pairing frequency, but obtaining back-cross derivatives was considered to
be a major problem. Sharma and Gill (1986) encountered similar constraints when T.
aestivum x Aegilops species hybrids were produced. Subsequently, Ter-Kuile et al.
(1988) reported success with the ph maternal system using T. aestivum x Ae. variabilis as
[69]
the test cross. Since then, numerous ph manipulative high pairing F1 hybrids have been
routinely produced and advanced to BCI or BCII (Rosas et al., 1988). However, as an
alternative, since a general constraint prevails, it may be appropriate to produce the F1
hybrid with a highly crossable wheat (PhPh) and either back- or top-cross it with the
phph stock (Sharma and Gill, 1986). Additional options for influencing the PhPh locus
are associated with this locus being suppressed by Ae. Mutica or Ae. speltoides; a
procedure that could be incorporated at the F1 stage with a low recombination hybrid or
on desired alien disomic addition lines. Achieving high recombination is emphasized
primarily because the T. aestivum crop species with its phenomenal cytogenetic
flexibility via Ph manipulation offers remarkable opportunities for alien gene transfers
and incorporation of homoeologous segments introduced in the best location in the
recipient wheat chromosomes.
Some other novel systems for genetic manipulation in intergeneric hybridization have
experimental priority and are targetted to yield a tremendous agricultural impact via
germplasm developed by this procedure. The following are strategies that the authors feel
must be pursued vigorously in the quest to keep product development at a pace ahead of
the population surge anticipated during the next two decades. Exploiting these strategies
is anticipated to render available diverse genes from alien sources that have been
insufficiently utilized and that the authors feel shall provide a durable and sustainable
practical focus through gene pyramiding. The need to match or exceed the impact of the
T1BL.1RS spontaneous translocation (Rajaram et al., 1983) and the induced T1AL.1RS
germplasm (Islam-Faridi and Mujeeb-Kazi, 1995; Villareal et al., 1996) ranks high. With
the relative ease of hybridization (Sharma, 1995) and the range of hybrids and alien
chromatin exchange stocks available (Jiang et al., 1994; Friebe et al., 1996), some
diversification seems appropriate as the next century begins. Can intergeneric
hybridization protocols be modified to yield faster returns of quality end products?
Though closely related genomes hold a priority for wheat improvement, additional genes
from ‘diverse’ gene pools also offer unique resistance durability and are anticipated to
contribute to sustainable cropping systems. The various gene sources contributing to C.
sativus resistance elucidates this concept. These genes, when pyramided, have the
potential to ensure resistance durability across several locations where C. sativus is a
wheat production constraint. From the earlier BH1146 cultivar resistance, the
improvement present in the Th. curvifolium plus Chinese wheat cultivars has been
significantly dramatic (cultivars Chirya and Mayoor) and has remained durable across
several countries for approximately 12 years. However, complacency in not introgressing
more diverse genes must not prevail for C. sativus or for any other biotic stress. In
essence, the progress from BH1146 to Th. curvifolium and/or usage of Chinese wheat
cultivar derivatives (Chirya and Mayoor), coupled with the extensive variation identified
in the A-, B- and D-genome accessions, is seen as a guarantee for stability over years to
come. It is a path being followed for C. sativus and can be extended to address other
stress objectives. The above approach, as the next century begins, is totally removed from
some wide hybridization views. Fedak et al. (1994) expressed that wide-crossing for
purposes of gene transfer be done as a last resort when the variability for a particular trait
is exhausted or is non-existent in the primary gene pool. The authors, however, view
wide-crossing as a complementary approach, conducted simultaneously and integrated
within conventional breeding programmes. This approach contributes multiple diverse
[70]
novel genes from all gene pools by pyramiding them with the conventional genetic
resource present in the primary pool commonly used by breeders. Even if these are
‘major’ alien genes, their multiplicity and diversity shall provide an advantage when
pyramided with the conventionally available ‘minor’ genes that recognizably contribute
to durable resistance.
Distantly related species (e.g. tertiary gene pool) are complex to exploit, but their
potential use in crop improvement is very high. To exemplify, the contributions of S.
cereal in wheat improvement cannot be overlooked (Islam-Faridi and Mujeeb-Kazi,
1995; Mujeeb-Kazi et al., 1996), and a major role of Thinopyrum species for BYDV
resistance further attests to the use of this distant diversity (Henry et al., 1996).
Involvement of other tertiary gene pool species in wheat germplasm has been the subject
of a few recent reviews (Jiang et al., 1994; Sharma, 1995; Friebe et al., 1996). A modified
tertiary gene pool transfer strategy, which should receive greater emphasis in the future,
is aimed at providing maximum recombination between wheat and alien species
chromosomes in the early hybrid
generation stages. The enhanced
recombination will be a consequence
of cytogenetic Ph locus manipulation,
irradiation, callus induction, etc. Ph
manipulations will involve the use of
chromosome 5B genetic stocks and
use of the relatively newer PhI
germplasm option (Chen et al., 1994).
The latter warrants more exploitation.
The
sooner
the
wheat/alien
chromosomal exchanges occur in an
intergeneric hybridization programme
involving tertiary gene pool species,
the sooner appropriate breeding
protocols will be incorporated, stress
screening coupled with homozygosity
will find its place and, with current
molecular diagnostic strength, alien
introgression(s) identification will
occur and be exploited. Researchers'
vision is to shorten the tertiary gene
pool conventional genetic transfer
protocols to a short-term productoriented programme akin to the
Figure 5.38 : Molecular marker - assisted genetic
interspecific approach that capitalizes
manipulation involving the ph locus on chromosome
upon the primary and secondary gene
5B mediated by maize induced doubled haploidy.
pool species. The vision projected
above will better address the
incorporation of polygenically controlled traits, hopefully embolic, into wheat of which
the alien Th. elongatum chromosomal control (3E, 4E and 7E) for salinity tolerance is
one example (Dvorak et al., 1988).
[71]
Immediate priority has been assigned to a doubled haploid wheat/maize-based
manipulation protocol that utilizes
the Ph F1 wheat/alien hybrids
maintained at the International
Maize and Wheat Improvement
Center (CIMMYT) as a living
herbarium. The protocol is
applicable to amphiploids and
fertile BCI combinations.
The double haploidy role in
salvaging Ph-based F1 hybrids has
become an option to enable phmediated alien introgression(s)
without having to remake complex
F1 hybrids using the ph genetic
stock (Sears, 1977) as the maternal
parent. Because of CIMMYT’s
living F1 herbarium involving
wheat and several alien species (Ph
locus present), BCI derivatives can
be produced by pollinating these
Ph F1 wheat/alien hybrids with the
Chinese Spring Ph ph wheat
genetic stock. The BCI progenies
(Ph ph) are crossed with maize to
yield poly-haploids that possess the
Ph or ph locus. The entire wheat
and
alien
chromosomal
complement is represented.
The haploids derived from the Ph
BCII=back-cross II
ph BCI derivatives possessing the
ph recessive gene get identified at
Figure 5.39 : Schematic
showing the crossing scheme
the seedling stage by a polymerase
of Triticum aestivum /Aegilops
chain reaction (PCR) based
tauschii direct hybridization
diagnostic analysis (Gill and Gill,
and back-cross advance
1996; Qu et al., 1998), which
enhances programme efficiency
(Figure 5.38) and sets a crop improvement programme in place using an integration of
the breeding methodologies. The ph locus facilitates wheat/alien chromosomal exchanges
and generates translocation stocks that are between homoeologous and nonhomoeologous chromosomes. They are Robertsonian translocations generally, but
smaller alien exchanges are also produced. The diagnostic protocols are FISH followed
by Giemsa C-banding. Derivatives of interest possess the critical translocation
chromosome(s), but also have entire alien chromosomes that are eliminated via backcrossing (Mujeeb-Kazi, 2001b).
[72]
Current emphasis has shifted for delivering farm crop products at a fast pace. Such
impacts shall be a consequence of gene
transfers from the closely related diploid
species and their accessional diversity.
In some cases, even swifter results can
be obtained as exemplified by the Dgenome direct crossing procedures
(Figure 5.39) (Alonso and Kimber,
1984; Gill and Raupp, 1987). Greater
efficiency emerges by mediating direct
transfers with sexually induced double
haploidy (DH) for achieving rapid
homozygosity (Mujeeb-Kazi and RieraLizarazu, 1996).Identifying genes in
different resistant accessions and
pyramiding these prior to crossing with
wheat also appears challenging (Figure
5.40, Figure 5.41). One approach is to
intercross resistant synthetic hexaploids
for a stress, advance the F1, select
resistant F2/F3 derivatives that combine
the effect of the two divergent Ae.
tauschii accessions, incorporate double
haploidy and end up with a homozygous
SH=synthetic hexaploid
stock.
Figure 5.39 : Schematic showing steps
involved in pyramiding accessional
diversity of Aegilops tauschii
Figure 5.40 : Schematic indicating a novel
gene pyramiding strategy associating stressresistant diverse genomes (AA of Triticum
monococcum and DD of Aegilops tauschii)
SH = Synthetic Hexaploid
*Seven such double haploids result
Use of this stock in wheat improvement
will simultaneously permit incorporating
two different genes from the Ae. tauschii
accessions (Figure 5.40). Another
approach
is
intercrossing
two
genomically divergent resistant diploid
accessions and developing tetraploid
stocks. Use of resistant A and D-genome
accessions is an example (Figure 5.41).
In order to hasten such gene
identifications, the- DH approach has
provided further usage and is now being
advantageously utilized for modified
complete or partial monosomic analyses.
The partial analysis is conducted when
[73]
resistance is associated with the D genome of SH wheats (Figure 5.42). The F1
monosomics of 1D to 7D chromosomes (2n=6x=40 + 1D to 40 + 7D) when crossed with
maize yield 21 chromosome polyhaploids with the 1D to 7D contributions coming from
resistant SH wheats. Doubling these n=3x=21 polyhaploid plants with colchicines results
in stable 42 chromosome double haploids. Each DH now possesses the homozygous 1D
to 7D chromosomes of the resistant SH parent being analysed for the chromosomal
location(s) of the resistant gene(s). Upon screening, the non-segregating resistant DHs are
attributed with having the gene(s) in them. The stable monosomic derived DH
germplasm, apart from simplifying the conventional monosomic analyses, also facilitates
global distribution of the developed germplasm. The germplasm enables experimental
repetition without have necessary when the conventional monosomic analytical
procedure is followed.
Alien Transfers for Durum Wheat Improvement - Progress in durum
wheat/alien species hybridization has not been as exhaustive as bread wheat. However,
the need to diversify the durum genetic base is crucial and can be achieved by
incorporating the diversity of the primary, secondary and tertiary gene pools. The
intergeneric recombination constraints can presumably be overcome by using the ph1c
Capelli genetic stock that requires greater investigating to fit agricultural goals.
For interspecific durum wheat improvement,
the A and the B genome diversity through
their AAAABB/AABBBB amphiploid routes
allows for cross combinations to be made
between the resistant amphiploids and elite
durum cultivars. This facilitates introgression
and exploitation of resistant traits in breeding
programmes by utilizing appropriate breeding
protocols. This alien diversity based durum
improvement programme is currently in its
infancy, but the authors do anticipate
contributions for resistant transfers to be
achieved for durums.
Challenging is the exploitation of D-genome
resistances for durum wheat improvement,
and at a high priority would be the transfer of
scab (F. graminearum) resistance genes.
Additionally, it must be mentioned the
potential been assigned to these diseases of Dgenome resistance transfers to durum wheat of
genes associated with salinity tolerance,
Figure 5.42 : Steps
drought tolerance, S. tritici, C. sativus and associated in conducting a
BYDV resistance, with quality being an partial
monosomic
integral part in all A-, B- and D-genome analysis, where resistance
accessional transfers. These genomic transfers is located within the
hexaploid
would be a consequence of recombinational synthetic
(2n=6x=42)
D-genome
events due to the preferential A- and D- chromosomes
[74]
genomic chromosome pairing represented as seven bivalents in the presence of the ph
locus. The bivalents are generally of the A- and D-genome chromosomes and univalents
of the B genome, as inferred separately from meiotic C-banding data (unpublished). The
authors’ current tester system to demonstrate the D- to A-genome genetic exchange
efficacy is for C. sativus and S. tritici from some D-genome resistant accessions. Durum
wheat cultivars are highly susceptible for both of these biotic stresses, and since
CIMMYT has ideal screening protocols with reliable screening locations in Mexico,
priority has from the secondary gene pool, the potential of using Ae. speltoides resistance
diversity for several stresses does also exist for durum wheat improvement.
Whole-Arm Translocation - One possibility of introducing into a cultivated
background less than an entire alien chromosome is to substitute one arm of the latter for
a corresponding wheat arm. In, wheat, such transfers are made possible by the strong
tendency of univalent chromosomes to misdivide at meiosis and give rise to one-armed
(telocentric) chromosomes. If both an alien chromosome and its wheat homoeologs are
present as univalents and misdivide in the same sporocyte, the resulting telocentrics can
occasionally fuse at the centromere and produce a bibrachial chromosome, with one alien
and one wheat arm. One major limit of such a method for introducing alien variation
resides in the low frequency with which the correct, balanced combination can be
recovered, whether from spontaneous or from radiation-induced events. Moreover, a
whole arm often possesses too much unwanted alien chromatin along with the target
gene(s). however, before the extensive use of induced homoeologous pairing as the
method of choice and, in some instances, to make possible use of desired introgressions
involving A or B genome chromosomes already obtained at the 6s level, a number of
alien transfers had been attempted into durum wheat involving entire chromosomal arms.
Among the few that met with relatively good success in a tetraploid background is the
1BL.1RS translocation. This translocation, together with the corresponding one involving
wheat chromosome 1A (1A.1RS), represents the most successful wheat-alien transfer
effectively employed in common wheat breeding worldwide. The short arm of Rye
chromosome 1R is known to carry many important genes for resistance to wheat
pathogens, including the yellow rust resistance gene Yr9, the leaf rust resistance gene
Lr26, the stem rust resistance gene Sr31, the powdery mildew resistance gene Pm8 and
Pm17 and the green bug resistance genes Gb2 and Gb6.
Transfer of Chromosomal Segments - The general outcome of the research
described above demonstrates that a whole alien arm, particularly if it originates from
relatively distant alien species, finds little acceptance by durum wheat. Among the
possible strategies of chromosome engineering that enable controlled introductions of
chromosome segments from related Triticeae into cultivated wheat, the one based on
manipulations of the wheat chromosome pairing control system, particularly the use of
mutations of Ph1, is by far the most effective.
Radiation-induced transfers have the inherent drawback of producing essentially random
translocation. Although many such translocations in wheat tend to involve homoeologous
chromosomal segments, these are greatly outnumbered by those involving non
homoeologous. Moreover, even for compensating transfers, attainment of intercalary
translocations remains an extremely rare event. The only documented case of an
[75]
intercalary wheat-alien transfer produced by radiation treatment appears to be that of a
non compensating translocation of a relatively short 6RL segment containing the H25
gene for resistance to Hessian Fly proximally inserted into the wheat 4AL arm. This
introduction, originally obtained in common whe3at, was recently incorporated via
homologous recombination into durum. Gametic transmission of translocated
chromosome, showing disturbance on the male side in the BC1 generation, appeared
normal in BC2 and BC3 derivatives. Moreover, the resultant translocation stock was
vigorous and had a seed set similar to the durum parent Cando thus demonst6rating its
usefulness in breeding.
Apart from this exceptional case, the past and more recent experience on wheat
chromosome engineering clearly highlights that the Ph1-mediated approach offers the
greatest promise for obtaining alien transfers. This is because disturbance of the recipient
chromosome and genotype balance, if any, is minimal, and the benefits of the alien
genetic contribution(s) are more readily available for practical utilization. One reason for
this is the cytogenetic affinity of the recipient chromosome to the donor chromosome,
because homoeologous are almost exclusively involved in Ph1-promoted pairing. When
the critical alien chromosome or chromosome arm is as an addition or substitution into
wheat genome, the potential for pairing between the alien and the wheat homeologs is the
highest if the two are present as univalents. Another important advantage of the Ph1mediated approach resides in the possibility to progressively shorten the amount of alien
chromatin flanking the desired gene(s) during successive steps of the transfer procedure.
This can be accomplished in different ways, depending on the material available as well
as on the position of the target gene(s) on the chromosome. If the alien products with
short, terminal alien segments. If, however, the alien gene has a median or more proximal
location, additional manipulations are needed to further shorten the alien segment due to
the expected low frequency of double crossover, especially between homoeologous
chromosomes. One possibility consists of allowing the donor and recipient chromosomes
to undergo repeated rounds of Ph1-induced homoeologous recombination. This, however,
can cause an excessive accumulation of unwanted background translocations, leading to
considerable gametic and zygotic instability and eventually to loss of potentially
desirable types. An alternative and perhaps better strategy was originally suggested and
successfully applied by Sears. By combining two complementary transfer chromosomes,
which resulted from single exchanges on the proximal and distal sides of the target gene,
respectively, crossing-over can occur in the homologous region shared by them. This will
give rise to a product equivalent be determined by applying FISH with a highly repeated
(pSc119.2) and low-copy RFLP sequence (PSR907) as probe. These physical markers
allowed the alien segment to be precisely located distal to the 3BS Xprs907 locus, in the
adjacent subtelomic interval separating the two most distal pSC119.2 sites of 3BS, is
known to be genetically positioned at less than 25 centimorgans (cM) from the 3B
centromere.
The above example highlights how molecular cytogenetic techniques, such as FISH
using specific DNA sequences or total genomic DNA (fl-GISH) of the alien species as
probe, allow a precise assessment of the physical amount of exchanged material. This
represents a critical parameter to estimate the potential impact of an alien transfer on the
recipient genotype, particularly when operating at the less tolerant 4x ploidy level.
[76]
5.3.3 Methods for Detecting Alien Chromatin
Hybrid Validation: Diagnostics which also Include Detection of
Alien Chromatin - Initial hybrid identification is based upon mitotic counts in root
tips collected at various hybrid development stages (Mujeeb-Kazi and Miranda, 1985). A
Figure 5.43 :
Figure no. 1, 2
and 3 showing
somatic
chromosomes
of
Triticale
“Badger”, a pair
of
1B/1R
chromosomes
and
an
interphase cell
in “Ning 8026”
respectively
probed
with
labelled
rye
DNA
and
blocked
with
unlabelled
wheat DNA, the
rye
chromosomes
and chromatin
(brown)
could
be
followed.
Figure
4,
5
showing
the
somatic
chromosomes
of
Triticale
Badger and the
1R(1D)
substitution line
“84056-1-36-1”
respectively
probed
with
PTA 71, six
signals
in
Badger
and
eight in “840561-36-1” could be
observed
(arrows).
[77]
normal intergeneric F1 hybrid possesses half the chromosome number of each parent
involved in the combination. For hybrids of different polyploidy levels, a mere number
count is adequate initial verification. There are, however, cases where the alien species
are hexaploid, such as wheat, and hybrids would then have 42 chromosomes. These may
be difficult to classify categorically as hybrids, but with superb primary and secondary
constriction resolution of wheat 1B, 6B and 5D chromosomes (Mujeeb-Kazi and
Miranda, 1985), identification of hexaploid hybrids is simplistic. Additional identification
can be made by employing chromosome-banding techniques. Karyotypic differences play
a part, but positive claim to hybridity must be accompanied by clear meiotic analyses.
In some situations, the alien genome may be totally or partially eliminated, resulting in
the production of polyhaploid/haploid or aneuploid F1 hybrids. The two aspects are
classified under: (i) genome elimination; and (ii) aneuploid F1 hybrids.
A modified hybrid phenotype is also good proof of hybridity, but often a hybrid may not
express the features of one parent as in the case of wheat x barley or its reciprocal
combination (Islam et al., 1978). In general, however, a majority of the F1 wheat x alien
(reciprocal also) hybrids involving all the A-B- and D-genome diploids, annual and
perennial, Triticeae genera and species exhibit a co-dominant phenotype (Mujeeb-Kazi et
al., 1987, 1989). Similar is the phenotype modification of their amphiploids. More recent
diagnostic procedures utilized for the detection of hybrids and/or their advanced
derivatives are in situ hybridization (FISH, GISH, etc.), electrophoretic analyses
(biochemical) and molecular detection of the presence of alien chromatin. Some
examples are as follows:-
(1) Fluorescence In-Situ Hybridization Techniques for Brassica:
Methodological Development and Practical Applications Fluorescence In Situ Hybridization (FISH) methods were developed for Brassica which
allow the physical localization of labeled DNA sequences at chromosomal sub-arm level.
In addition, Genomic In Situ Hybridization (GISH) enabled all Brassica diploid genome
components apart from the highly similar A and C genomes to be distinguished, and R
genome chromatin was reliably detected in Brassica napus x Raphanus sativus hybrids.
Hybrids among the Brassicaceae can be relatively easily produced and are an ideal
method for generating new lines containing agronomically important pest and disease
resistance genes. Efficient monitoring of alien chromatin is however critical for
successful transfer of alien chromosomes or chromosome segments containing useful
genes. The small size of chromosomes in Brassica and its close relatives is particularly
problematic for the identification of alien chromosomes in Brassica hybrids, and
translocations cannot be reliably identified by conventional cytogenetic methods. GISH
was shown to be an effective practical method for detecting amounts and locations of
foreign chromatin in Brassica hybrids, while other FISH applications were useful for
chromosome identification and investigations of Brassica genome evolution.
(2) Identification of Alien Chromatin and Ribosomal DNA in
Wheat by In-Situ Hybridization - In situ hybridization has used to detect
somatic cells of hexaploid Triticale “Badger” 1B/1R translocation line “Nine 8026” and
IR (1D) substitution line “84056-1-36” using biotin labelled total rye genomic DNA and
wheat rDNA as probe (Figure 5.43).
[78]
(3) FISH and RFLP Marker-Assisted Introgression of Festuca
mairei Chromosomes into Lolium perenne - Plant breeders and
geneticists have attempted for several decades to combine perennial ryegrass (Lolium
Figure 5.44 : Southern hybridization of the HindIII - or EcoRI-digested genomic DNA of Lolium
perenne (Lp), Festuca mairei (Fm), and their hybrid derivatives with (A) TF109, identifying the
presence of F. mairei genome in F1, F2, and BC1 plants; (B) TF515, a Festuca genomespecific probe, showing strong hybridization only in F1, BC1, and some BC1G1 plants (M =
labeled DNA/HindIII fragments)
perenne L.) with fescue (Festuca spp.) to create novel
forage grasses containing both high forage quality and
good drought tolerance (Figure 5.44). Difficulty in
selecting true hybrids with alien chromatin or chromosome
addition–substitution has been a major barrier in Festuca x
Lolium breeding programs. In this investigation,
fluorescence in situ hybridization (FISH) and restriction
fragment length polymorphism (RFLP) markers were used
to monitor transfer of Festuca mairei, St. Yves
chromosomes into L. perenne through intergeneric hybrids.
Among 64 hybrid plants of the BC1G1 generation
(intercrossed progeny of first backcross to L. perenne),
chromosome addition and substitution of F. mairei were
identified by FISH using total genomic DNA of F. mairei
as a probe (Figure 5.45). Forty-two clones from a PstIgenomic DNA library of F. arundinacea Schreb, were used
to screen for the presence of F. mairei DNA in the hybrid
plants. These RFLP probes rapidly identified presence of
the F. mairei genome in F1, F2, BC1, but not in BC2
plants. In contrast, genomic FISH on meiotic cells
effectively detected any F. mairei chromosomes as well as
chromosomal pairing relationships in any hybrid. By
Figure 5.45 : Genomic
FISH using labeled F.
mairei DNA as a probe
effectively
detected
substitution of a pair of
Festuca
mairei
chromosomes
in
a
Lolium-like hybrid plant
[79]
monitoring and selectively introducing F. mairei chromosomes into ryegrass, these
molecular markers may accelerate the Festuca x Lolium breeding for improvement of
ryegrass.
Figure 5.46 : Detection of rye chromatin using genomic in situ hybridization. Yellow-orange to yellow
fluorescence shows hybridization of the total genomic DNA rye probe. A: Metaphase chromosome
spread of the disomic wheat-rye addition line 1R showing two rye chromosomes identified by yelloworange arms and bright yellow telomeres (see arrows). B: Metaphase chromosome spread of Atlas,
showing the absence of rye DNA probe hybridization. C: Metaphase spread of Century, showing the
detection of two rye chromosome arms by the yellow-orange fluorescence and bright yellow telomeres
(see arrows). D: The number of alien chromosome arms can also be counted in interphase nuclei of
Century where they form two distinct fluorescent yellow domains (see arrows).
[Normal View 101K | Magnified View 298K]
[80]
(4) Use of fluorescence genomic in situ hybridization (GISH) to
detect the presence of alien chromatin in wheat lines differing in
nuclear DNA content - Genomic in situ hybridization was used to identify the
number and size of alien chromosomes or chromosome arms. Probe hybridization sites
were detected by FITC-linked antibodies that fluoresced yellow under blue light
excitation and allowed visualization of rye chromatin material. TA3601, a disomic
wheat-rye addition line involving the addition of two complete rye chromosomes, is
pictured in Figure 5.46A. Forty-four chromosomes were observed with two chromosomes
appearing yellow-orange with each end of the chromosome stained bright yellow. The
bright yellow-stained chromosome telomeric ends represent heterochromatic regions of
the chromosome. In addition, nuclei were also seen to have four distinct signals
representing the heterochromatic regions of genetic material. These results were observed
in all four of the disomic wheat-rye addition line. Figure 5.46B is a metaphase spread of
Atlas, with an absence of the FITC signal, indicating the absence of rye chromatin.
Century (Figure 5.46C) had 42 chromosomes with two chromosomes that exhibited one
red arm and one yellow-orange arm with a bright yellow signal on the telomere. The
nuclei of Century (Figure 5.46D) exhibited two labeled domains in the nuclei at
interphase. The near-isolines of Century also displayed two FITC-stained chromosome
arms and two yellow domains in the nucleus. All of the Century-derived plants analyzed
were observed to have the positive signal. Chisholm metaphase spreads had an absence of
FITC signal. Nuclei were absent of any labeled domains. Near isolines derived from
Chisholm did not exhibit FITC signals in either metaphase chromosome spreads or
nuclei. At no time was a positive signal observed in the Atlas or Chisholm lines or the
isolines of Chisholm.
5.3.4 Produciton, Characterization and Utility of Alien Addition &
Subtraction Lines RNA Splicing
Production of alien chromosome addition / substitutions and
their utility in plant genetics - Plant geneticists and breeders have gained great
interest in extending genetic variation of crop plant using exotic germplasm from related
species. In a long term crossing programme, known as introgressive hybridization,
economically or otherwise important genes were incorporated into the recipient parent by
sexual or somatic hybridization between the crop and a related species or genera,
followed by consecutive backcrossing with the recipient parent while selecting for the
favourable trait(s). In the offspring families aneuploidy individuals could thus be isolated
containing only a single alien chromosome added to the full cell complement of the
recipient parent. Monosomic addition were first described by Leighty and Taylor (1924)
but use and potential were better demonstrated in the comprehensive study of O’Mara
(1940). Khus (1973), Gupta (1995) and Sybega (1992) gave a full overviews of alien
addition (The commonly used term monosomic addition lines is incorrect because lines
assume homozygosity for a particular gene or chromosome variant, and so will not
segregate in the next generation) in relation to other aneuploids in plant genetics.
Although the number of papers on alien addition are almost countless, reports on full sets
with all alien chromosomes appeared only for monosomic additions with chromosomes
[81]
of Beta webbiana, B. patellaris and B. procumbens to beet, Solanum lycoperiscoides to
tomato, tomato to potato, Oryza officinalis to rice, a number of Triticeae species,
including rye and barley, to Triticum aestivum, maize to oat, and onion to Allium
fistulosm. Table 5.5 present an overview of complete sets, together with their parental
species, marker selections, cytogenetics and references.
Production of additions and substitutions - Three major barriers can be
distinguished in introgressive hybridization : i) incompatibility or incongruence between
the parental species, and hence the difficulties in producing viable interspecific or
intergeneric hybrids; ii) sterility of the F1 and sometimes also of the BC1, thus
hampering transfer of alien chromosomes through backcrossing and ii) rare or negligible
meiotic recombination between the alien chromosomes and one of its homoeologous
counterparts, preventing incorporation of alien chromatin into the recipient genome.
Methods have been developed to overcome these natural crossing barriers. One of them
makes use of chromosome doubling to produce allopolyploids from which fertile
allotriploids can be derived. A second method makes use of pollen mixes, containing
pollen for syngamy and “mentor” pollen from the maternal species to facilitate
fertilization by foreign pollen. Alternatively, desirable traits can be transferred indirectly
using bridge cross with a related (wild) species or cultivar that is compatible with either
parental species. Although time consuming, such interim crosses have successfully been
applied to various crop species including alfalfa, beet, lettuce and onion species.
Melchers et al. (1978) was the first to make somatic hybrids, later followed by many
others.
Table 5.5:
The development of cell culture technology for protoplast fusion and regeneration, for the
production of somatic hybrids from fused protoplasts of related species that are
incompatible in a sexual cross was a further step to bridge the gap between non-crossable
A highly advanced example of somatic hybridization technology was developed by Sree
Ramulu et al. who isolated micronuclei containing one or few chromosome s from the
donor species fused them with complete euploid protoplasts from the recipient crop. This
microprotoplast fusion technique directly produced asymmetric somatic hybrids with the
cell complement of a monosomic addition, thus skipping the problems of interspecific
incongruity and BC1 sterility. Although potentially very promising, this advanced
strategy never received large-scale practical application.
Hybrid embryo abortion after fertilization is an important problem generally occurring in
(somatic) hybrids from wide crosses and reflects disharmony between the genomes of the
parental species that result into embryo mortality, endosperm break down, seed
inviability and hybrid sterility. Different in vitro embryo rescue techniques were
developed to overcome this problem of hybrid embryo degeneration and have been
applied to a large variety of crops and ornamental species. Once the backcross progenies
could be recovered, aneuploid individuals with alien chromosomes, including monosomic
additions could be isolated. As maintenance of monosomic additions was difficult due to
sterility, inferior viability and low chromosome transmission through the germline ,
additions of potato, onion and beet background were often kept in vitro or retained by
[82]
vegetative propagation. Transmission of the alien chromosome sand viability of the
additions can difficult to obtain. Production of disomics was only possible when the alien
chromosome was transmitted also through the male germ line, or they can be obtained by
meiotic non-disjunction of the alien chromosome in the monosomic addition parent. Use
Figure 5.47 : Genomic painting (genomic in situ hybridization) shows the number and
behaviour of alien chromosomes in backcross individuals. Here we show the monosomic
addition carrying chromosome 3 of tomato added to tetraploid potato (2n=4x=+1=49)
hybridized with total genomic tomato DNA as probe and 50x unlabeled potato DNA as
competitor. (a) Mitotic metabphase complement. (b) Pachytene complement. Bars equal 10
μm.
of derived ditelosomic additions, in which single individual alien chromosomes were,
replaced by their telocentrics, often improved chromosomal transmission considerably.
The final step in introgressive hybridization programmes involves meiotic recombination
between alien chromosome and one of its homoeologous counterparts in order to stably
incorporate its chromatin into the recipient parental genome. Generally, additions derived
from wide interspecific or intergeneric hybrids display little or no homoeologous
recombination at all, and hence leave the alien chromosome s as univalents at metaphaseI. In the corresponding substitution lines, association of homoeologous chromosomes
may be far higher due to the absence of homologous partners. Transfer of desired
chromosome regions to wheat is generally most successful in the Ph1 background of
wheat allowing high levels of homoeologous recombination. Other means of alien
chromosome introgression can be achieved by breakage and fusion of chromosome
fragments, either spontaneously by radiation-induction or by gametocidal factor in
Aegilops cylindrical, which have especially been applied in the cereal species.
Characterization of the alien chromosome(s) - Monosomic additions
can be selected on the basis of specific alien traits, like disease resistance, aberrant plant
phenotype, species-specific molecular markers and karyotype analysis to demonstrate the
presence of an extra chromosome. Morphological traits can be qualitative, like the
[83]
characteristic liguleless leaves of the maize chromosome 3 monosomic addition in oat
and the monogenic dominant resistance genes, or inherit quantitatively, such as plant size
and spike morphology. The morphological traits in the monosomic addition set of Beta
vulgaris carrying an extra chromosome from B. procumbens or B. patellaris appear only
partly chromosome-specific, and hence are not adequate for the identification of all alien
chromosomes without additional markers. Monosomic additions derived from
hybridization between genetically related parental species can have phenotypes often
resembling its corresponding primary trisomics.
Alien chromosomes in the additions may sometimes be morphologically distinguishable,
as was shown in a karyotype analysis of four nematode resistant monosomic addition of
beet containing different alien chromosomes from Beta procumbens or B. patellaris.
Chromosome morphology was for a long time described in terms of centromere position,
arm lengths, C- and N-banding profiles, and heterochromatin pattern in cell complements
at pachytene. Flow cytometry can be helpful when chromosome size and GC/AT ratio are
sufficient to identify the alien chromosome in the flow karyogram and can even be
applied to isolate large numbers of alien chromosomes, as was demonstrated for a
monosomic addition with maize chromosome 9 to oat.
The by far most important tool to visualize alien chromosomes in genomic in situ
hybridization (GISH) or genomic painting, a fluorescence in situ hybridization protocol
using total genomic donor DNA as probe and non-labelled DNA from the recipient
parent as competitor. An example of GISH of a monosomic addition of tomato
chromosome 3 in potato is shown in the Figures 5.47a and 5.47b. Reports on establishing
the
number
of
alien
chromosomes in intergeneric
backcross
families
are
numerous, like tomato to
potato, maize to oat, Beta
corolliflora in beet, Solanum
brevidens to potato and Sgenome chromosomes in
wheat. The technique is also
considered
effective
in
painting
and
studying
individual chromosomes in
plants. GISH has also the
advantage of demonstrating
interspecific and intercenteric
translocations, and shows
recombinant chromosomes
resulting from homoeologous
recombination.
For the identification of the
alien chromosome in the
Figure 5.48 : RFLP molecular markers for the
addition stocks, additional
characterization and selection of monosomic
additions with a specific alien chromosome. Southern
analysis is required using
hybridization of Dral/HindIII markers, T=tomato,
chromosome specific “wild”
P=potato and 1-11, a series of offspring plants from a
monosomic addition 3 backcross.
[84]
morphological traits, isozyme markers including RFLPs, RAPDs, microsatellites and
repetitive sequence DNA fingerprints (Figure 5.48).
The integrity and identification of the alien chromosome in a monosomic addition
carrying a Solanum brevidens chromosome in potato background were obtained by
sequential genomic painting and FISH with BACs diagnostic for each chromosome. The
advantage of this 2-step procedure is that both genomic origin and genetic identity of the
alien chromosome can be established in a single experiment.
Properties of additions - The potential of alien additions for breeding
programmes largely depends on the genetic distance of the parental species and hence, on
the possibility of the alien chromosome to recombine with one of its homoeologous
counterparts by crossing over. When parent can be combined in a sexual cross, such as
Wheat and Rye, Maize and Pennisetum, Festuca and Lolium, crossovers between
homoeologous chromosomes are not rare, which may reveal recombinant chromosomes,
even in the first backcross generations. In the case of distant parental species when
somatic hybridization and embryo rescue are required for the production of monosomic
additions, the alien chromosome generally fails to synapse and / or recombine at meiotic
prophase I, remains lagged behind in the equatorial plane at anaphase-I and may get lost
at later stages. Its univalence may result in centromere breakage and thus producing
monotelosomic and isotelosomic additions in the progeny. In the derived disomic
additions, the two alien chromosomes will pair and form chiasmata, resulting in the
formation of univalents and hence, in unbalanced gametes.
The extent of crossover recombination between the homoeologous in the monosomic
additions depends primarily on the genetic relation between the parental species, but can
also vary between the different alien chromosomes in the monosomic additions and in
different genetic backgrounds. When homoeologous recombination in the interspecific
hybrids is rare, it will be even more seldom or entirely absent in the derived backcross
generations due to high preferential pairing. Additional causes for suppression of
crossover and recombination are the heterochromatic pericentromeric chromosome
regions and heterozygosity for (small) chromosomal rearrangements like duplications,
inversions and translocations or pericnetromeric regions.
Geneticists have undertaken several strategies to improve homoeologous recombination.
When genotypes lacking the Ph1 locus that controls suppression of homoeologous
recombination are often used in breeding programmes for transferring desirable genes
from rye, and other related cereal species to wheat. More recent studies on the mode of
Ph1 have shown that the locus not directly control homoeologous recombination
suggesting the need for an entirely new approach to the introgression of alien genetic
variation into wheat. Very little is known about other genetic systems controlling
synapsis and recombination between homoeologous chromosomes in plant polyploids. In
Lolium amphidiploids diploidizing genes carried by A-chromosomes and supernumerary
B-chromosomes controlled bivalent formation. This system has so far not been applied to
introgressive hybridization programmes.
Diploid plants with one chromosome replaced by its homoeologous can be easily
obtained in backcross offspring families of interspecific hybrids and monosomic
additions. The heteromorphic (homoeologous) bivalents in such monosomic substitutions
generally demonstrate higher levels of crossover recombination between the alien
[85]
chromosomes and its homoeologous counterpart than in the corresponding monosomic
addition, and are therefore more appropriate for producing recombination chromosomes.
A large series of substitution lines have been produced from Festuca pratensis x Lolium
perenne hybrids and used GISH to establish the sites of homoeologous crossover events
in the recombinant chromosomes. The range of substitution lines each with different
recombinant chromosomes provided excellent material for physical mapping the
introgressed F. pratensis chromosomes segments and comparing genetic and physical
maps for the molecular markers on these chromosomes.
Gamma-ray irradiation of monosomic oat-maize additions was used by produce radiation
hybrid lines containing centric maize chromosome fragments or oat-maize translocation
chromosome. These lines, which lack one or more parts of the original maize
chromosome, allow mapping of molecular markers on a sub-chromosome level of the
maize genome.
Transmission rates are generally far higher through the female than the male line, and
greatly vary between and among the addition sets, as was found for Oryza australiensis
to rice, Solanum lycopersicoides to tomato and onion to A. fistulosum. In the monosomic
additions of Solanum lycopersicoides chromosomes to tomato, transmission for
chromosome 10 was 24%, whereas for chromosome 6 no transmission at all could be
recorded. In an extensive study transmission values amounted 0-32% for chromosome 9,
whereas that of chromosome 6 varied from 14-88% between the different families.
Transmission rate can vary considerable for the different alien chromosomes and is
generally higher for the larger chromosomes. A recent study encompassing three
consecutive generations in the Brassica rapa monosomic additions in Raphanus sativs,
revealed average transmission rates ranging from 26% to 44%.
Applications and Scientific impact of additions and substitutions
These are as follows : 1. Introgressive hybridization
2. Gene/markers localization
3. Construction of chromosome specific libraries
4. Heterologous gene expression
5. Organization of the alien chromosomes
6. Studies on chromosome pairing of alien chromosome pairs using GISH
7. Use in genomics and physical mapping
5.3.5 Genetic Basis of Inbreeding and Heterosis
Heterosis is a term used in genetics and selective breeding. The term heterosis, also
known as hybrid vigour or outbreeding enhancement, describes the increased strength of
different characteristics in hybrids; the possibility to obtain a genetically superior
individual by combining the virtues of its parents.
Heterosis is the opposite of inbreeding depression, which occurs with increasing
homozygosity. The term often causes controversy, particularly in terms of the selective
breeding of domestic animals, because it is sometimes believed that all crossbred plants
or animals are genetically superior to their parents; this is true only in certain
circumstances: when a hybrid is seen to be superior to its parents, this is known as hybrid
[86]
vigor. When the opposite happens, and a hybrid inherits traits from their parents that
makes them unfit for survival, the result is referred to as outbreeding depression. Typical
examples of this are crosses between wild and hatchery fish that have incompatible
adaptations.
Genetic basis of heterosis - When a population is small or inbred, it tends to
lose genetic diversity. Inbreeding depression is the loss of fitness due to loss of genetic
diversity. Inbred strains tend to be homozygous for recessive alleles that are mildly
harmful (or produce a trait that is undesirable from the standpoint of the breeder).
Heterosis or hybrid vigor is the tendency of outbreed strains to exceed both inbred
parents in fitness.
Selective breeding of plants and animals, including hybridization, began long before
there was an understanding of underlying scientific principles. In the early 20th century,
after Mendel's laws came to be understood and accepted, geneticists undertook to explain
the superior vigor of many plant hybrids. Two competing hypotheses, which are not
mutually exclusive, were developed:


Dominance hypothesis. The dominance hypothesis attributes the superiority of
hybrids to the suppression of undesirable recessive alleles from one parent by
dominant alleles from the other. It attributes the poor performance of inbred
strains to loss of genetic diversity, with the strains becoming purely homozygous
at many loci. The dominance hypothesis was first expressed in 1908 by the
geneticist Charles Davenport (Figure 5.49).
Overdominance hypothesis. Certain combinations of alleles that can be obtained
by crossing two inbred strains are advantageous in the heterozygote. The
overdominance hypothesis attributes to heterozygote advantage the survival of
many alleles that are recessive and harmful in homozygotes. It attributes the poor
performance of inbred strains to a high percentage of these harmful recessives.
The overdominance hypothesis was developed independently by Edward M. East
(1908) and George Shull (1908).
Figure 5.49 Figure- Genetic basis of
heterosis. Dominance hypothesis.
(Scenario A) - Fewer genes are
under-expressed in the homozygous
individual. Gene expression in the
offspring is equal to the expression
of the fittest parent. Overdominance
hypothesis (Scenario B) - Overexpression of certain genes in the
heterozygous offspring. (The size of
the circle depicts the expression
level of gene A)
Dominance and overdominance have
different consequences for the gene
expression profile of the individuals. If over-dominance is the main cause for the fitness
advantages of heterosis, then there should be an over-expression of certain genes in the
[87]
heterozygous offspring compared to the homozygous parents. On the other hand, if
dominance is the cause, fewer genes should be under-expressed in the heterozygous
offspring compared to the parents. Furthermore, for any given gene, the expression
should be comparable to the one observed in the fittest of the two parents.
Historical retrospective - Population geneticist James Crow, who in his younger
days believed that overdominance was a major contributor to hybrid vigor, has
undertaken a retrospective review of the developing science. According to Crow, the
demonstration of several cases of heterozygote advantage in Drosophila and other
organisms first caused great enthusiasm for the overdominance theory among scientists
studying plant hybridization. But overdominance implies that yields on an inbred strain
should decrease as inbred strains are selected for the performance of their hybrid crosses,
as the proportion of harmful recessives in the inbred population rises. Over the years,
experimentation in plant genetics has proven that the reverse occurs, that yields increase
in both the inbred strains and the hybrids, suggesting that dominance alone may be
adequate to explain the superior yield of hybrids. Only a few conclusive cases of
overdominance have been reported in all of genetics. Since the 1980s, as experimental
evidence has mounted, the dominance theory has made a comeback.
Crow writes, "The current view ... is that the dominance hypothesis is the major
explanation of inbreeding decline and the high yield of hybrids. There is little statistical
evidence for contributions from overdominance and epistasis. But whether the best
hybrids are getting an extra boost from overdominance or favorable epistatic
contributions remains an open question."
Inbreeding depression is usually defined as the lowered fitness or vigour of inbred
individuals compared with their non-inbred counterparts, observed in many (but by no
means all) organisms. Its converse is heterosis, the 'hybrid vigour' manifested in
increased size, growth rate or other parameters resulting from the increase in
heterozygosity in F1 generation crosses between inbred lines.
Problems arise in defining fitness and vigour. So long as we are discussing populations of
wild plants, we have the concept of Darwinian fitness: if individuals of one genotype
survive to breed more than others, then that genotype confers greater fitness. Fitness is an
observed quantity that integrates the effect of all characters that influence the ability of
the organism to live and reproduce. As natural selection can only act to increase the
frequency of an allele in proportion to the extent to which that allele increases fitness, it
was predicted (Falconer, 1989) that the amount of heritable variance for a trait would be
inversely proportional to its effect on the organism's fitness. In other words, there would
be more inheritable variance in relatively 'neutral' characters than in ones that increased
the organism's viability or fecundity. Studies on three very different animal species
tended to confirm this prediction regarding individual characters (Kruuk et al., 2000), but
the measurement of overall fitness in populations remains problematical.
Vigour is another vague concept: but to most growers it is synonymous with rate of
growth or biomass accumulation. In an annual, this is well correlated with production of
grain, flowers or fruit since these plants maximise their investment in seed production.
But a perennial may increase its growth rate only to reinvest these resources in further
vegetative reproduction; examples are bulbil watsonia and crow garlic that have become
weeds by the strategy of vegetative reproduction with little or no seeding.
[88]
Vigour has sometimes been used as a measure (or even a near-synonym) of fitness in
discussions of heterosis. But a plant can be too vigorous for its own good: it can become
structurally unstable and vulnerable to destruction by mechanical stress or dependent on a
higher and more reliable supply of water and nutrients than the environment can
guarantee. And an individual plant whose accelerated growth rate causes it to flower long
before the rest of its population may - if it can self-pollinate - have increased its fitness.
But its fitness will be reduced to zero if it's a self-incompatible annual.
The
relation
between
fitness and vigour in a wild
population is not a straight
line, but a curve with a
maximum:
In the language of games
theory, natural selection
might be said to favour
strategies that lead to a
minimax outcome - the
optimum attainable in the
real world rather than the
theoretical maximum.
Darwinian fitness is not a
single character, but the
outcome of every trait in an individual that influences the contribution it makes to the
next generation. In populations of wild plants, heterosis for a range of quantities
presumed to have a strong influence on fitness is more meaningful than heterosis for
vigour alone. For example, Buza et al. (2000) measured germination rate, seedling
survivorship and vegetative growth rate in their attempt to quantify inbreeding depression
in relict Swainsona populations. Balanced polymorphisms in populations are often due to
the heterozygotes having a higher fitness than either homozygote (Ford, 1964).
Possible explanations of this heterosis for overall fitness include:
1. The formation of homozygotes in which alleles with more or less recessive
deleterious effects become fully expressed, but had been masked by the
dominants in heterozygotes. Recessive and mildly deleterious mutations are
common, and if located close to a beneficial allele they will increase in frequency
as it does, being "sheltered" from selection in the heterozygotes. By the time a
beneficial mutant is common enough for its homozygotes to occur in significant
numbers in the population, it may be linked with enough harmful recessives to
make these homozygotes less fit than the heterozygote (Fisher, 1927; Ford, 1965).
In this case, heterosis is said to be caused by dominance complementation. This
may also be described loosely as epistasis of the harmful genes over the beneficial
ones - but only if that term is used in the broadest sense.
2. Overdominance - the fitness of the heterozygote per se is higher than either
homozygote. This could arise because genes normally have pleiotropic effects,
contributing too many measurable traits of the plant. If a mutation is selected for a
positive effect on fitness or some desired trait, its other effects are likely to be
deleterious. Because a mutant allele at first spreads through a population via
[89]
heterozygotes, selection will tend to modify and optimise its action by making its
beneficial effects dominant and its deleterious effects recessive (Gustafsson,
1950; Sheppard, 1953). This hypothesis has been used to explain balanced
polymorphisms, for example in the case of Dactylis glomerata (Apirion &
Zohary, 1961).
In both explanations, a beneficial dominant effect is associated with a deleterious
recessive effect. Whether these correlated effects are due to the same gene or to closely
linked but separate genes may be a difficult point to decide. Both overdominance and
dominance complementation may contribute in varying degrees to observed cases of
heterosis. But evidence for actual instances of overdominance remains scarce.
Three predictions of the dominance complementation hypothesis suggest how it might be
tested:
1. Inbreeding depression due to genes with deleterious recessive effects that are
linked to genes with beneficial dominant effects may be removed by selection; but
if it is due to overdominance it cannot.
2. A period of inbreeding can 'purge' deleterious recessive alleles from a population
since these will have an increased chance of appearing in homozygotes than can
be eliminated by selection (Barrett & Charlesworth, 1991).
3. If two strains of a plant are each selected for heterosis in the F1 produced by
crossing them with a third strain, heterosis will also increase in the F1 between
these selected strains because they are being purged of deleterious alleles. But if
heterosis was due to overdominance, the two strains would converge, since each
would be selected for alleles that differed from those of the tester strain at each
locus, and their hybrid will show inbreeding depression instead of heterosis.
Wild plants that are normally inbreeders, or have a mixed inbreeder/outbreeder strategy,
might be expected to have a genetic architecture that minimises inbreeding depression. In
one experiment confirming this, the inbred seeds from cleistogamous flowers of Viola
species had only slightly lower fitness than the outcrossed seeds from chasmogamous
flowers on the same plants (Berg & Redbo-Torstensson, 1999).
Inbreeding depression is caused mainly by deleterious recessive alleles in both Mimulus
guttatus (an outbreeder) and the closely related inbreeder M. micranthus (Dudash & Carr,
1998). Natural selection in experimentally inbred lines of M. guttatus purged the few
alleles with major deleterious effects, but numerous other alleles, each with a small
effect, accounted for almost all the depression (Willis, 1999).
Cultivated plants - But most of the practical interest in heterosis centers on
cultivated plants, and when plant breeders say 'fitness' they really mean 'crop yields'. It is
more useful to speak of heterosis for a particular statistic of the crop: for example, leaf
area or seed size.
Heterosis is also modified by the interactions between genotype and environment in
cultivation. Hybrid sorghum can show heterosis for yield; but this effect varies widely
between trials conducted at sites differing in seasonal water supply (Chapman et al.,
2000), so that it is more meaningful to characterise a particular hybrid line as showing
heterosis for yield at a specific locality or under certain environmental conditions.
In practice, yield and other measurable traits desired by breeders are often correlated with
traits that increase the fitness of the plant: increased efficiency of metabolism,
[90]
photosynthesis etc. As a convenient approximation, we can speak loosely of 'beneficial'
and 'deleterious' effects.
Heterosis for size, vigour or yield is most evident in outbreeding crops. The classic case
is maize; in which heterosis has long been exploited in the production of uniformly highyielding F1 seed in commercial quantities.
The definitive experiment by Sprague (1983) showed that this instance of heterosis is due
wholly or mainly to dominance complementation. Over many years, two maize
populations were each selected for increased yield in the hybrids produced by crossing
them with another inbred 'tester' strain. F1 hybrids between the two selected strains
showed increased yield, as predicted by the dominance complementation hypothesis.
They also produced increased yields in hybrids with other testers.
If dominance complementation is the only factor involved in heterosis for yield, the high
productivity of hybrid maize may have been matched by open-pollinated cultivars if
enough work had been invested in their development. However, Fu & Dooner (2002)
have reported the surprising discovery that maize cultivars differ in the set of genes they
carry: some loci in one cultivar lack corresponding alleles in another line. In this case,
any component of the heterosis effect due to complementation between different loci
could never be fixed in an open-pollinated line, since these loci would remain on separate
chromosomes. Maize may be a special case among crop plants - both because its genome
has been doubled in size by retrotransposon activity that produced multiple paralogs of
some genes, and also because it has been more radically changed by the domestication
process than have other crops such as rice.
Xiao et al. (1995) demonstrated that overdominance is not a major cause of heterosis for
yield in a cross between the two subspecies of rice, because there was no correlation
between most traits and overall genome heterozygosity, heterozygotes were never
superior to both homozygotes in analysis of quantitative trait loci, and some F8 inbred
lines were actually superior to the F1 for all traits evaluated.
Trials support the hypothesis that heterosis in wheat is due to dominance
complementation, with linkage and interaction of alleles (Pickett & Galwey, 1997) - to
the loss of those who would like to exploit hybrid wheat as they have done hybrid maize.
In some other outbreeding crop plants, such as cucumbers (Cramer & Wehner, 1999),
crossing inbred lines rarely leads to heterosis.
The hypothesis of dominance complementation is also supported by evidence that
polyploids are to some extent buffered against inbreeding depression because they have
additional copies of each gene (Bingham et al., 1994; Husband & Schemske, 1997).
A third explanation of heterosis - An alternative theory was proposed by
Milborrow (1998). He suggested that growth of a plant may be limited by the genes that
regulate certain metabolic pathways down to lower levels than the maximum possible.
Heterozygotes may partially escape this regulation because they have two slightly
different alleles for these genes, allowing greater flow on these pathways. This is not
overdominance; but, like the overdominance hypothesis, it predicts that heterozygotes
have an inherent advantage in vigour that cannot be duplicated by any amount of
selection in open-pollinated homozygous lines.
The most interesting part of Milborrow's theory is the implication as to why there are so
many "weak" alleles in a population to start with. These are not sublethal mutants that
[91]
have accumulated under the protection of closely linked genes that are strongly selected,
but a necessary part of the genetic adaptation of the population.
Natural selection will maintain weak alleles because an individual with all the strong
alleles will be vigorous but of lower fitness. Therefore, selection maintains the average
individual in the population at that level of vigour which maximises fitness. This may
explain observations that the level of heterosis in wild populations depends on their
habitat, sometimes termed habitat-dependent heterosis (Lloyd, 1980). The fitness
advantage of heterozygotes is often greater under more severe conditions (e.g.- Dudash,
1990).
Genetic variation allows more vigorous and less vigorous individuals to exist and take
advantage of changing conditions under which they may have the advantage. This is a
mixed strategy; and as Von Neumann & Morgenstern (1953) showed, mixed strategies
are necessary to achieve minimax outcomes.
But artificial selection of plants in cultivation aims to maximise desired parameters yield, size etc - within an artificial, optimum environment. It may therefore be possible to
purge the alleles that cause inbreeding depression from cultivated lines.
(1) Genetic basis of heterosis and inbreeding depression in rice
(Oryza sativa L.) - The genetic basis of heterosis was studied through mid-parent,
standard variety and better parent for 11 quantitative traits in 17 parental lines and their
10 selected hybrids in rice (Oryza sativa L.). The characters were plant height. days to
flag leaf initiation, days to first panicle initiation, days to 100% flowering, panicle length,
flag leaf length, days to maturity, number of fertile spikelet/panicle, number of effective
tillers/hill, grain yield/10-hill, and 1000-grain weight. In general the hybrids performed
significantly better than the respective parents. Significant heterosis was observed for
most of the studied characters. Among the 10 hybrids, four hybrids viz., 17A×45R,
25A×37R, 27A×39R, 31A×47R, and 35A×47R showed highest heterosis in
10-hill grain yield/10-hill. Inbreeding depression of F2 progeny was also studied for 11
characters of 10 hybrids. Both positive and negative inbreeding depressions were found
in many crosses for the studied characters, but none was found significant. Selection of
good parents was found to be the most important for developing high yielding hybrid rice
varieties.
(2) Overdominant epistatic loci are the primary genetic basis of
inbreeding depression and heterosis in rice. I. Biomass and
grain yield - To understand the genetic basis of inbreeding depression and heterosis
in rice, main-effect and epistatic QTL associated with inbreeding depression and
heterosis for grain yield and biomass in five related rice mapping populations were
investigated using a complete RFLP linkage map of 182 markers, replicated phenotyping
experiments, and the mixed model approach. The mapping populations included 254
F(10) recombinant inbred lines derived from a cross between Lemont (japonica) and
Teqing (indica) and two BC and two testcross hybrid populations derived from crosses
between the RILs and their parents plus two testers (Zhong 413 and IR64). For both BY
and GY, there was significant inbreeding depression detected in the RI population and a
high level of heterosis in each of the BC and testcross hybrid populations. The mean
performance of the BC or testcross hybrids was largely determined by their heterosis
[92]
measurements. The hybrid breakdown (part of inbreeding depression) values of
individual RILs were negatively associated with the heterosis measurements of their BC
or testcross hybrids, indicating the partial genetic overlap of genes causing hybrid
breakdown and heterosis in rice. A large number of epistatic QTL pairs and a few maineffect QTL were identified, which were responsible for >65% of the phenotypic variation
of BY and GY in each of the populations with the former explaining a much greater
portion of the variation. Two conclusions concerning the loci associated with inbreeding
depression and heterosis in rice were reached from our results. First, most QTL
associated with inbreeding depression and heterosis in rice appeared to be involved in
epistasis. Second, most (approximately 90%) QTL contributing to heterosis appeared to
be overdominant. These observations tend to implicate epistasis and overdominance,
rather than dominance, as the major genetic basis of heterosis in rice.
5.3.6 Exploitation of Hybrid Vigour
Hybrid vigour, which overcomes “inbreeding depression”, and which is a way to increase
genetic diversity and stress resilience in a crop, is an important breeding concept. It is
especially relevant to canola in Australia. Theoretical and experimental understanding of
hybrid vigour in several crops is an extremely sound reason to be confident that GM
canola hybrids are more profitable than conventional varieties.
There are many silly untrue assumptions floating around the community about
commercial seed hybrid varieties - eg that commercial seeds are sterile or do go grow
without fertilizer, that it is worth going back to explain what hybrids are. The story starts
with maize (Indian corn) in America.
In 1908 the U.S. plant breeder G. H. Shull reported the results of crossing maize plants
from two different inbred lines. The cross between two highly homozygous lines
produced heterozygous offspring, because the lines were homozygous for different sets of
genes. The result was astonishing. The two inbred lines had each produced about 20
bushels of maize per acre in the last crop. Their outbreed offspring quadrupled this yield,
to 80 bushels per acre! This unanticipated strength in the heterozygous outcross was
called hybrid vigor; and such hybrids have played an important role in increasing maize
yield.
Hybrid vigour can be fully exploited when plants are cross pollinators rather than selfpollinators. Thus exploitation of hybrid vigour is not possible with self-pollinating wheat
and barley, but is possible with maize, sunflower and sorghum. Although the commercial
breeding of hybrid maize has focussed on yield improvements, there are interesting
complexities in the actual traits that allow this achievement. Modern hybrids are highly
stress tolerant plants, and they can be seeded at high plant densities per acre. Scientific
publications of Duvick and Cassman (1999) and Tollenaar et al. (1994) show that modern
maize hybrids have increased stress tolerance rather than high yield potential.
Because of the spectacular potential of hybrid vigour demonstrated with maize, plant
breeders have tried to develop artificial methods to allow practical cross-breeding with
other crops. The Chinese have developed non-GM artificial methods that generate rice
hybrids, and Bayer market GM canola hybrids.
The phenomenon of heterosis and its importance in crop improvement have been known
since decades. Recognizing that commercial exploitation of hybrid vigour would,
[93]
however, depend on economic viability of hybrid seed production. Varied genetic and
non-genetic approaches for selective emasculation of one of the parents have been
evolved and used. Cytoplasmic-genetic male sterility is the worldwide used system
among them Yet, its cumbersome nature, labour expansive seed production method and
restircted parental choice warranting an alternative has led to the discovery in some of the
cereal crops. Physiological characterization of various sources of photoperiod and
temperature sensitive genic male sterility (TGMS/PGMS) in a model crop like rice has
helped group them into 3-4 categories on the basis of their critical sterility and fertility
points, while genetic analysis has revealed it to follow simple mendelian mode of
inheritance and 2-3 putative genes (pms1, pms2, tms1, tms2, tms3) to govern them.
Development of stable and agronomically superior PGMS and TGMS lines using
promising gene sources and identification of ideal locations/seasons for hybrid seed
production and multiplication of parental TGMS or PGMS line has facilitated evolution
of two-line hybrids of rice for commercial planting. It is hoped that more precise
understanding of the physiology, biochemistry, genetics and molecular bases of these
environment sensitive male sterility sources would greatly help extent this innovative
system of hybrid breeding to a wide range of crop plants in the coming year.
Exploitation of hybrid vigour on a commercial scale involves extra expenditure in the
production of hybrid seeds. In a study for developing the hybrid of brinjals an increase in
yield of hybrids was in the order of 50 to 100 percent. Over the parental means and this
compensate for the extra cost of production. This is a sufficient reason to advocate the
use of hybrid seeds of brinjals for commercial growing in India.
In Tamilandu region of India it is recently proof that exploitation of hybrid vigour is very
successful which was done for developing rice hybrid (Oryza sativa L.) through green
manure and leaf colour chart (LCC) based nitrogen application.
5.4 LET US SUM UP
Type II restriction enzymes cut DNA at specific base sequences. Some restriction
enzymes make staggered cuts, producing DNA fragments with cohesive ends;
others cut both strands straight across, producing blunt-ended fragments. There
are fewer long recognition sequences in DNA than short sequences.
Eukaryotic DNA comprises three major classes: unique-sequence DNA,
moderately repetitive DNA, and highly repetitive DNA. Unique-sequence DNA
consists of sequences that exist in one or only a few copies; moderately repetitive
DNA consists of sequences that may be several hundred base pairs in length and
is present in thousands to hundreds of thousands of copies. Highly repetitive DNA
consists of very short sequences repeated in tandem and present in hundreds of
thousands to millions of copies.
When double-stranded DNA is heated, it denatures, separating into singlestranded molecules. On cooling, these single-stranded molecules pair and re-form
double-stranded DNA, a process called renaturation. A C0t curve is a plot of a
renaturation reaction.
[94]
Physical-mapping methods determine the physical locations of genes on
chromosomes and include deletion mapping, somatic-cell hybridization, in situ
hybridization, and direct DNA sequencing.
Additions can basically be subdivided into two classes, each with their own
properties and applications: (i) Class 1 in which homoeologous recombination
between the alien chromosome and one of its homoeologous counterparts occurs
at reasonable frequency. These additions produce various recombinants and so are
particularly suitable for introgressive hybridization, for comparing physical and
genetic maps, and for studying the effect of individual chromosomes on crossover
(chiasma) distribution and frequencies. More knowledge is required especially
about the genes controlling homoeologous recombination, chiasma distribution
and unreduced gamete formation, and for genetic programmes aiming at
producing monosomic substituions in order to force homoeologous chromosomes
to pair and synapse. (ii) In class 2 homoeologous recombination does not occur in
a normal genetic background, i.e. not disturb in genes controlling meiotic
recombination, homoeologous recombination or unreduced forms of meiosis (like
first and second division restitution). This group of interspecific aneuploids are
less attractive3 for breeders as long as introgressive hybridization cannot be
achieved by homoeologous recombination, but can provide significant
information for various cytogenetic and genomic studies including chromosome
disposition, molecular organization of repetitive and single copy sequences,
heterologous gene expression and starting material for flow sorted or
microdissected alien chromosome samples for chromosome specific DNA
sequences.
By virtue of its several advantages over the traditional cytoplasmic-genetic male
sterility system, the environment sensitive genic male sterility would be an ideal
alternative for effective exploitation of hybrid vigour in rice and other crops.
Nevertheless, sustainable use of this system on a commercial scale would require
further refinement of the system, keeping in view the impending global warming,
the effect of temperature-light interaction and the influence of not very well
understood Fd and other minor gene complexes on the stability of fertility-sterility
reversion. The following are some of the areas, warranting more in-depth study:
o Streamlining of breeding methodology for development of PGMS and
TGMS lines.
o Precise characterization of all the usable PGMS and TGMS lines derived
from different sources for the benefit of users in the seed industry.
Development and use of molecular markers for improving
breeding/selection efficiency.
o Development of EGMS system for developing more productive intersubspecific hybrids.
[95]
5.5 CHECK YOUR PROGRESS
NOTE: (1) Write your answer in the space given below.
(2) Compare your answer with the one given at the end of this unit.
‫( א‬1) Fill in the blanks :
(a) The DNA of the nucleus with its associated proteins is called ……………..
(b) The cells of some …………… may contain 40 times more DNA than those of
humans.
(c) ………. restriction enzymes recognize specific sequences and cut the DNA at
nearby sites, usually about 25 bp away.
(d) …………………… technique was developed using highly radioactive DNA
probes, which were detected by radiography.
(e) HAT selection, which stands for ……………., ……………, and ………….,
three chemicals that are used to select for hybrid cells.
(f) Scientists who study chromosomes are known as …………………….
‫( א‬2) Write the answer of following questions :
(a) Describe the multigene family in bacteria and insect.
(b) Explain how production of alien addition/substitution is useful.
5.6 CHECK YOUR PROGRESS : THE KEY
‫( א‬1) (a) Chromatin (b) Salamanders (c) Type III (d) In situ hybridization
(e) Hypoxanthine, aminopterin and thymidine (f) Cytogeneticists
‫( א‬2) (a) see section 5.2.5 (b) see section 5.3.4
[96]
5.7 ASSIGNMENT
Collect the information about Indian plants in which alien gene transfer
methods have been applied successfully.
5.8 REFERENCES
Our courteous thanks to following two authors/publishers for preparing the
various section of this chapter:B. Alberts et al., ‘Molecular Biology of the Cell’: 4th Ed. (2002). Garland.
Benzamin A. Pierce, ‘Genetics : A Coneptual Approach’
Other helping resources are as follows:Scalenghe F, Turco E, Edstrom JE, Pirrotta V, Melli M. 1981. “Microdissection
and cloning of DNA from a specific region of Drosophila melanogaster polytene
chromosomes”. In Chromosoma. 82(2):205-16.
Mapping Genomes - From Genomes by T. A. Brown, 2002.
Linkage disequilibrium maps and association mapping - J. Clin. Invest., 2005.
Linkage Disequilibrium in Humans: Models and Data - Am. J. Hum. Genet.,
2001.
Cytogenetic and Molecular Studies on Tomato Chromosomes using Diploid
Tomato and Tomato Monosomi Additions in Tetraploid Potato From Song-Bin
Chang.
Linkage Disequilibrium and the Search for Complex Disease Genes - Genome
Research, 2000.
Gray JW, Cram LS. 1990. Flow karyotyping and chromosome sorting. In: Flow
Cytometry and Sorting. New York: Wiley Liss, 503-529.
Heslop-Harrison JS, Schwarzacher T. 1995. Flow cytometry and chromosome
sorting. In: Fukui K, ed. Plant Chromosomes: Laboratory Methods. CRC, Baton
Rouge. In press.
Dewey, D.R. 1984. The genomic system of classification as a guide to
intergeneric hybridization with the perennial Triticeae. In J.P. Gustafson, ed.
Gene manipulation in plant improvement, p. 209-279. New York, NY, USA,
Plenum Press.
Allen Klinger, member, IEEE, Arnold kochman, and Nikitas Alexandridis. 1971.
“Computer Analysis of Chromosome Patterns: Feature Encoding for Flexible
Decision Making”. In IEEE Transactions on Computers, Vol. C-20, No. 9
Pawley JB (editor) (2006). Handbook of Biological Confocal Microscopy, 3rd
ed., Berlin: Springer. ISBN 038725921x.
Hybrid Vigor inn Plants and its Relationship to Insect Pollination - a section from
Insect Pollination Of Cultivated Crop Plants by S.E. McGregor, USDA
******
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