Analysis of genetic diversity in barley (Hordeum vulgare) using restriction fragment length
polymorphism analysis
by Matthew Glenn Kramer
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Agronomy
Montana State University
© Copyright by Matthew Glenn Kramer (1987)
Abstract:
Exploitation of genetic diversity is the basis of plant breeding programs. Restriction fragment length
polymorphism analysis represents a new tool for the plant breeder in his efforts to detect genetic
variability. For this technology to be of use in barley breeding programs, it must be demonstrated that
heritable variation can be detected in barley using this technology. Examination of three barley
populations using two randomly selected genomic clones as probes across four restriction digests
showed a moderate amount of variation both within and between populations. This study reports the
detection of four polymorphic loci on at least four separate chromosomes, suggesting that this
technology holds promise for future genetics studies in this crop. ANALYSIS OF GENETIC DIVERSITY IN BARLEY (HORDEUM VULGARE)
USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS
by
Matthew Glenn Kramer
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Agronomy
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1987
UN Lift.
37?
ii
APPROVAL
of a thesis submitted by
Matthew Glenn Kramer
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Approved for the College of Graduate Studies
Date
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iii
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iv
TABLE OF CONTENTS
Page
LIST OF TABLES.... ............................. •
V
LIST OF FIGURES.....................
ABSTRACT.........
vii
REVIEW OF LITERATURE....................
I
PROBLEM STATEMENT.......................
I
8
MATERIALS AND METHODS...................
9
Genotype Selection...............................
Plant Tissue Preparation........................
DNA Extraction..........................
Restriction Digests....................
Electrophoresis..........................
Transfer of DNA......................
Probe Fragment Isolation.................
Nick Translation................................
Prehybridization/Hybridization..............
Washing........................................
Autoradiography........... .......,...........
Re-use of Membranes.....................
Data Analysis................................
9
10
10
11
11
12
13
14
14
15
15
16
16
RESULTS...................................
Characterization of Fragments...............
Evaluation of Polymorphism in Barley............
Allelism Among Probes Within Enzyme Treatments.
CONCLUSIONS.... ...............................
REFERENCES CITED..........................
17
17
18
20
45
49
V
LIST OF TABLES
Table
1.
2.
3.
4.
Page
VARIETIES AND RELATIONSHIPS OF
BARLEY CULTIVARS. ........................... .
10
TOTAL NUMBER AND SIZE OF RESTRICTION
FRAGMENTS OBSERVED USING PROBES LAMBDA 107
AND LAMBDA 167 ACROSS ENZYMES AND
GENOTYPES....... '.......... '. .............. ••
20
NUMBER AND SIZE OF LAMBDA 107
FRAGMENTS.... . .....................
22
NUMBER AND SIZE OF LAMBDA 167
FRAGMENTS........ ........................— •
22
LAMBDA 107 ALLELES
:HIND 3..................
24
6 . LAMBDA 107 ALLELES
: BAM Hl...'........
24
7.
LAMBDA 107 ALLELES
: ECO Rl... ...............
25
8.
LAMBDA 107 ALLELES
: ECO R5.............
26
9.
LAMBDA 167 FRAGMENTS :HIND 3.........
5.
35
10. LAMBDA 167 FRAGMENTS : BAM Hl...............
36
11. LAMBDA 167 FRAGMENTS : ECO Rl..........
36
12. LAMBDA 167 FRAGMENTS : ECO R5...............
37
13. DATA SUMMARY: BANDING PATTERN
DESIGNATIONS USED IN PHYLOGENETIC
ANALYSIS........................
vi
LIST OF FIGURES
Figure
1.
Page
HIND 3 RESTRICTION DIGEST
WESTERN 2-ROW TYPES.... .......................
2.
RESTRICTION DIGESTS OF LAMBDA 107
AND LAMBDA 167 USING BOTH HIND 3
AND ECO Rl ....................
3.
LAMBDA 107 ALLELES BETWEEN POPULATIONS :
BAM Hl....................................
4.
LAMBDA 107 ALLELES OF WESTERN 6-ROW TYPES :
HIND 3 .......................
5.
LAMBDA 107 ALLELES OF WESTERN 6-ROW TYPES :
BAM Hl... ................... .............
12
26
28
6.
LAMBDA 107 ALLELES MIDWEST 6-ROW TYPES :
HIND 3.......................
7.
LAMBDA 107 ALLELES MIDWEST 6-ROW TYPES :
ECO R5................. . ;.... ..............
30
BETWEEN POPULATION POLYMORPHISMS
REVEALED BY LAMBDA 167 : BAM Hl ............
37
LAMBDA 167 FRAGMENTS WESTERN 6-ROW TYPES :
HIND 3 ............ ..........................
38
8.
9.
10. LAMBDA 167 FRAGMENTS MID-WEST 6-ROW TYPES. :
HIND 3....... .............................. •
39
11. LAMBDA 167 FRAGMENTS MIDWEST 6-ROW TYPES :
BAM Hl................. ...... ...... ........
40
12. PHYLOGENETIC RECONSTRUCTION OF BARLEY
CULTIVARS.......................... .........
44
vii
ABSTRACT.
Exploitation of genetic diversity is the basis of plant
breeding programs. Restriction fragment length polymorphism
analysis represents a new tool for the plant breeder in his
efforts to detect genetic variability. For this technology
to be of use in barley breeding programs, it must be
demonstrated that heritable variation can be detected in
barley using this technology. Examination of three barley
populations using two randomly selected genomic clones as
probes across four restriction digests showed a moderate
amount of variation both within and between populations.
This study reports the detection of four polymorphic loci on
at least four separate chromosomes,
suggesting that this
technology holds promise for future genetics studies in this
crop.
I
REVIEW OF LITERATURE
All heritable variation can be attributed to
in sequence or
organization of
nucleic acids.
variation
The
recent
development and use of restriction endonucleases has allowed
the direct examination of
genomic DNA sequence
variation.
This direct examination of variation at the DNA level,
made
possible by the use of DNA fragments generated by the action
of restriction endonucleases, is called restriction fragment
length polymorphism
(RFLP) analysis.
viruses (Grodzicker, 1974), RFLPs
be both ubiquitous
markers (Burr
et
Discovered
first
in
have since been found
to
and numerous relative
al,
1983;
Seller
to other
and
genetic
Beckmann,
1983;
Helentjaris et al, 1985; Beckmann and Seller, 1986).
Restriction fragments themselves
specific
DNA
sequence
endonucleases, which
site
are generated by
recognition
of
catalyze endonucleolytic
the
restriction
cleavage
of
DNA resulting in fragments of a defined length (Nathans
and
Smith, 1975;
may
then
be
Nei and
Tajima, 1981).
electrophoretically
molecular size.
These
separated
on
fragments
the
basis
of
Differences between individuals and species
in the lengths of these fragments (i.e. RFLPs) may be due to
a variety
of genotypic
differences.
Specifically, one
more individual bases may differ (e.g. by point mutations)
or
2
resulting in the loss or gain of an enzyme recognition site.
The loss
or gain
of an
enzyme recognition
site by .point
mutation results in a DNA fragment of different size in
mutant relative
enzyme site
to its
which is
and/or deletions
isogenic normals ■for the
modified.
within
(Bottstein et al,
a
Alternatively,
fragment may
1980; Burr et
specific
insertions
alter
al, 1983).
the
it's
size
Alteration
of
fragment size leads to altered mobility which can
then be electrophoretically
complex
genomes,
visualized, or in
through
the
techniques (Southern, 1975).
agarose gel
such as
probe
is transferred
The
identification of allelic
of
DNA
suitable membrane
hybridized
resulting
with
an
matrix
radiolabelled
autoradiogram
variation of
of
hybridization
In this case, the DNA from
to a
nitrocellulose and
sequences.
use
the case
allows
the specific
probe
sequence.
The number of RFLPs available in a species is dependent
on the number of unique or low copy number sequences in
genome of
that
species.
single copy sequences may
In
barley, as
many
be available for RFLP
The number of potentially polymorphic loci is
unlimited
(Seller
Tanksley, 1986).
and Beckmann,
Genomic
RFLPs
Mendelian codominant markers
neutral
(Bottstein
Helentjaris
et
as
al,
are
1986;
analysis.
Bernatzky
inherited
Burr
500,000
theoretically
and are generally
1980;
et
the
as
and
simple
selectively
al,
et al, 1985; Seller and Beckman, 1983).
1983;
3
Linkage relationships
other genetic
progeny
among
markers can
of
a
between
be demonstrated
heterozygous
Helentjaris et al, 1985;
humans, RFLPs
RFLPs and
parent
RFLPs
and
by analysis
(Bottstein
of
1980;
Bernatzky and Tanksley, 1986).
have already
provided
a number
of
In
markers
which have been shown to be linked to disease loci
(Gusella
et al, 1984; Newmark, 1984).
The extent of RFLPs among eukaryotic genomes appears to
vary considerably.
In humans
a high
has been demonstrated (Bottstein et
1984; Helentjari's,
1985;
Jefferys,
1983; Jefferys
1985).
In
level of
variability
al, 1980; Baker et
et al,
1979;
plants, only
the
al,
Jefferys,
genomes
of
maize and tomato have been extensively studied (Burr, et al,
1983;
Johns,
et
al,
1983;
Bernatzky
and
Tanksley,
levels of
polymorphism have
Helentjaris,
1986).
been
found in
The level
tomato being is less
than that detected in
somewhat
of
barley
less
have
than
1985;
different
these
species
of variability detected
shown
maize
al,
Surprisingly
(Helentjaris, 1985).
studies
et
a
but
maize.
degree
of
greater
in
Limited
variability
than
tomato
would
be
(Saghai-Maroof et al, 1984).
The most
molecular
obvious
markers
initial
in
the
use of
RFLPs
construction
(Bottstein et al, 1980; Beckmann
of
genetic
as
maps
and Seller. 1983; Burr
et
al, 1983; Tanksley, 1983; Helentjaris et al, 1985; Bernatzky
and Tanksley, 1986).
The use of RFLPs as genetic markers
4
has been extensively applied in
the area of human
genetics
(Bottstein et al, 1980; Jefferys 1979; Jefferys et al,
a&b; Murray et al, 1984).
of RFLPs to a heritable
In
a number of studies,
disease locus and their
use as molecular markers
1985
linkage
subsequent
has been accomplished (Gusella
et
al, 1983; Phillips et al, 1983).
Seller and Beckmann identify three major areas in which
these new markers
may be used;
affecting quantitative genetic
the identification of
traits, genetic
loci
improvement
through evaluation of germplasm sources combined with marker
assisted introgression
of desirable
genes into
commercial
cultivars and varietal identification (Seller and
1983).
Linkage
technology
maps
will
produced
also
recombination rates,
improve our
allow
through
the
use
researchers
to
study unusual
knowledge
of plant
Beckmann,
of
RFLP
monitor
segregation ratios
evolution
(Bernatzky
and
and
Tanksley, 1986).
In the past, genetic
and
utilized
important
breeding
for
a
species.
linkage maps have been
limited
Recently,
technology
have
numerous biochemical markers
and zein proteins
This
advancement,
such as
the
agriculturally
advancements
in
localization
of
isozymes and
to morphological
limited
polymorphic biochemical markers
of
technical
permitted
with respect
though
number
developed
by
the
hordein
markers.
number
of
available to breeders,
has
nevertheless permitted the manipulation and utilization of a
5
number
of
economically
important
traits
such
as
male
sterility in tomato (Tanksley, Rick and Vallejos, 1984).
addition,
known
marker
loci
characterization of the
have
been
used
In
in
the
wheat-barley addition lines
(Islam
and Shepard, 1981).
Many economically important traits, such as yield,
quantitatively inherited.
these traits are due to
number of
small
loci.
or
at each
negative
Traits demonstrating
Some QTLs have
influencing
to
the
trait loci (QTLs)
with
morphological and biochemical markers
Beckmann and Seller, 1986).
trait
genetic variation
been mapped
of
(unlike
application of
in
this
respect to
known
(Soller et al,
1976;
It has been suggested that
phenotypic
and/or
QTL-Iinkage mapping
biochemical
markers)
techniques may
Chao and Blake,
due
within
enhance
efforts to map and manipulate economically significant
(Soller and Beckmann, 1986;
QTLs
unpublished).
Exciting as this may appear, it must be remembered that
number
of
offspring
RFLP-QTL
linkage
Beckmann,
1986)
which
appears
and
must
unavoidably
some
unreasonable (Ellis, 1986).
be
would
a
(Gelderman,
to the virtually unlimited number of RFLPs available
a genome
large
locus contributes
component
type are termed quantitative
1975).
differences
multiple allelic series at a
Each allele
positive
question.
Genetic
are
screened
large
argue
to
the
detect
(Seller
and
statistically
While controversy exists as
to
the possibility of producing a viable RFLP-QTL linkage map.
6
it is
clear that
by
using classical
methods it
will
possible to map the location of RFLP loci relative to
marker
loci
and
thus
produce
a
saturated
be
known
linkage
map
(Beckmann and Soller7 1986; Helentjaris7 1985; Bernatzky and
Tanksley7 1986; Chao and Blake7 unpublished).
Tanksley as well
saturated linkage
as others
map for
view the use
the introgression
of the
of
RFLP
desirable
genes from resource strains to cultivated varieties as their
most important potential use (Bernatzky and Tanksley7 1986).
It has been suggested
demonstrated that
(Seller and Plotkin-Hazan7 1977)
genes linked
to
marker alleles
and
can
be
effectively selected for and manipulated (Tanksley7 Rick and
Vallejos, 1984).
The use of RFLP technology to improve the efficiency of
backcrossing in
gene transfer
from exotic
germplasm
into
adapted lines could result in the reduction in the number of
generations of backcrossing required to adequately eliminate
the donor genotype while retaining the desired single
gene.
donor
This approach has been used successfully with
linked to
isozyme markers
wild type
species to
when transferring
a commercial
genes from
cultivar (Tanksley
Rick7 1980; Tanksley7 et al7 1981; Tanksley7 et al,
An additional advantage
ability to
differentiate
of RFLPs in
of
and
1982).
this context is
cultivars
a
their
the
same
species (Helentjaris7 1985; Beckmann and Soller7 1986;
Chao
and Blake, unpublished).
between
traits
This should allow the breeder not
7
only to use RFLP technology to introgress genes from
donor species,
but also
allow for
the movement
exotic
of
genes
between cultivars of the same species.
Perhaps the most
plant breeding
immediate use of
programs
will
be
RFLP technology
their
use
identification (Seller and Beckmann, 1983).
in
in
varietal
The development
of a number of RFLPs across a number of cultivars within a
I
species should yield a unique combination of fragments for
each cultivar.
This
unique
fragment
pattern
would
provide a solid basis for varietal identification.
et al (1985) have shown
in
humans
and
individuals .
In
Jefferys
that highly variable regions
exhibit
unique
plants
RFLP
there appears
patterns
to
be
then
exist
, between
sufficient
variability between cultivars that such a system of varietal
identification
Beckmann
and
is
indeed
Seller,
feasible
1986;
(Burr,
Helentjaris,
Bernatzky and Tanksley, 1986; Chao and Blake,
et
et
al,
al,
1983;
1985;
unpublished).
Furthermore it has been
calculated that depending upon
extent of
and
polymorphism
multiple
fragment exhibits, somewhere between
bands - should
be
sufficient
allelism
10 and 20
to
provide
that
the
each
polymorphic
positive
identification of cultivars (Seller and Beckmann, 1983).
If RFLPs are as abundant as hypothesized and their
distribution covers the entire genome of the organism being
studied, they will likely become the method of choice to
track a variety of genetic phenomena.
8
PROBLEM STATEMENT
One of the first major hurdles to overcome in
to apply
RFLP analysis
to a
plant species
attempting
is the
deter­
mination of the detectable levels of genetic variability
in
the species
to
to
be studied.
determine whether a sufficient
variability exists
both
This
attempted
level of detectable
within and
useful barley populations as
study has
between
agronomically
determined by RFLP
Two random genomic clones were used as probes.
analysis.
These probes
represent a single copy sequence and a multi-copy
sequence.
genetic
dispersed
9
MATERIALS AND METHODS
Genotype Selection
Barley genotypes
used in
this study
were drawn
from
three genetic backgrounds of spring barley in an attempt
assess the genetic diversity
of the major barley
found in the United
Varieties were selected
on pedigree and
following
States.
represent both parents
genetic
backgrounds:
("Manchuria" derivatives),* Western 6-row
derivatives)
Smyrna
and
Western
derivatives).
2-row
The
varieties used in this study
6-row
based
table
the
genotypes
genotypes
genotypes
following
varieties
and progeny of
Midwest
to
(Coast
(Hannchen
and
contains
the
and their relationship to
one
another.
Table I.
Varieties and Relationships of Barley Cultivars.
Midwest 6 Row
Genetic Background
Western 6 Row Western 2 Row
Parents: Darker
Traill
Unitan
Atlas
Coast
Betzes
Doman
Palliser
Progeny: Morex
Robust
Bedford
Hazen
Azure
Steptoe
Utah 1423
Or8408
Karla
Columbia
Karl
Clark
Lewis
Harrington
Gallatin
Bowman
Hector
Klages
10
Plant Tissue Preparation
Foliar tissue
obtained in
of
the above
mentioned
the following
manner:
were seeded with 30 g seed
per pot.
closed 24
hr. light
lyophilized
for
48
was
standard
pots
15.5 cm
Pots were placed in
environment and
days post planting, expanded
varieties
watered
daily.
Five
leaf tissue was harvested
hours
in
a
Virtis
a
and
large
scale
Total genomic DNA was obtained using a modified
method
lyophilizer.
DNA Extraction
of
Saghai-Maroof
(1984).
accomplished through
the use
4050 spectrophotometer.
was read at both
Quantification
of a
purity and concentration.
samples
LKB Biochrom
Ultraviolet
260 and 280 nm
of
was
Ultraspec
absorbance of
samples
in order to estimate
both
One O.D. was taken to equal 50 ug
/ml of nucleic acid at 260nm , while a ratio between the the
readings of
roughly
preparation of
samples were
2.0
nucleic
was
taken
to
acid (Maniatis,
subsequently adjusted
to a
indicate
et
a
pure
al,1979).
concentration
approximately I microgram DNA per microliter.
All
of
11
Restriction Digests
Restriction
digests
were
carried
different restriction endonucleases,
recognition site.
The
out
using
a six
base
restriction endonucleases used
were
as follows: Bam Hi, Hind
In
all
cases, enzymes were supplied by IBI, Inc. complete with
the
appropriate buffer.
3, Eco R I,
each with
four
and Eco R 5.
In all cases, 3Oug of total genomic DNA
was digested with 4OU of
the specified enzyme according
to
manufacturers instructions at 37 C for 16 hours.
Electrophoresis
The
using
resulting
0.8% agarose
approximately
buffers were 50
1979).
restriction
2
fragments
slab gels (resolution
volts/cm.
Both
the
gels were stained
separated
0.2-22.0 kb)
electrode
mM Tris-Borate/2mM EDTA
Upon completion of
were
and
at
gel
(Maniatis, et
al,
electrophoretic separation,
the
with ethidium
bromide at
0.4 ug/ml
and
visualized by transillumination using short wave ultraviolet
light.
type 57
A photograph
film
at
f8
of each
and
2
gel was
second
taken with
exposure.
restriction digest is shown in Figure I.
Polaroid
A
typical
12
Transfer of DNA
DNA restriction
fragments
were
transferred
from
the
agarose gel to a cationically charged support membrane using
a modified Southern blot technique (Gatti, et al 1984; Reed,
et al, 1985).
Figure I. Hind 3 Restriction Digest of Western 2-Row Types
Lanes:
I :Betzes,2:Doman,3:Palliser,4:Clark,5:Lewis,6:Harrington
7:Gallatin,8:Bowman,9:Hector,10:Klages
13
Probe Fragment Isolation
Two probes
were utilized
in
this study.
Both
were
barley genomic clones in the vector EMBL 4.
Lambda 107 is a
putative single copy
is a
multi copy
clone.
clone and
Specific
lambda 167
fragments of
dispersed
barley DNA
isolated from these phage after double digestion with
were
EcoRI
and Hind 3 utilizing the low melting point agarose isolation
technique discussed in Maniatis et al. (1982).
Figure 2. Restriction Digests of Lambda 107 and Lambda 167
Using Both Eco R I and Hind 3.
%
M i M i e MkIMH
- E M B L 4 DI GE ST S “
14
Nick Translation
Approximately
0.1 ug of cloned DNA fragment
32
radiolabelled with
P by nick translation (Rigby, et
1975).
was
al,
The reaction was carried out for two hours at 14
Labelled DNA was
separated from unincorporated
by filtration through
G-50 fine
(Maniatis
probes was measured
a 1.0
et al,
using a
ml "spun
1982).
nucleotides
column" of
Activity
C.'
of
Sephadex
labelled
Packard scintillation
counter
and those probes exhibiting a specific activity of at
least
8
1.0x10 cpm/ug DNA were utilized as probes.
denatured prior
to use
as follows:
All probes
to IOOul
of
were
labelled
probe, 500ul of sonicated salmon sperm DNA and IOOul of 0.4M
sodium hydroxide was added.
The mixture was centrifuged
to
consolidate the components and then denatured for 10 minutes
in a boiling water bath.
Prehybridization/Hybridization
Prehybridization of the nylon membrane was carried
in a 10 ml
out
volume of 1.5% SSPE,
1.0% SDS, 0.5% BLOTTO
and
0.5 mg/ml carrier DNA (sonicated
salmon sperm) at 60 C
for
18 hours. Hybridization was carried out in a 10 ml volume of
prehybridization mix to which was added the labeled
probe.
Hybridizations were carried out at 60 C for 2_4 hours.
15
Washing
Upon completion
removed
from
briefly in
2
their
X
of hybridization,
hybridization
SSC.
the membranes
solutions
After rinsing,
the
were
and
rinsed
membranes
were
washed successively by vigorous agitation for 15 minutes
in
approximately 200 ml of the following solutions:
2 X SSC/0.1% SDS
(room temperature)
0.5X SSC/ 0.1% SDS (room temperature)
0.1X SSC/ 0.1% SDS (room temperature)
The final
wash
was carried
out
for 30
minutes
in
0.1X
After the final wash the membranes were rinsed at
room
SSC/1.0%SDS at 55 C.A
Autoradiography
temperature in a
solution of
Whatman 3MM chromatography
wrapped in plastic wrap
0.IX SSC and
paper.
The membranes were
and taped to a
Whatman chromatography paper.
blotted dry
on
then
clean sheet of
The membrane was then
3MM
placed
in a Kodak ETO X-Ray exposure cassette with a piece of Kodak
X-Omat
X-Ray
film
and
intensification screen.
-70 C
freezer and
period of 3-10 days.
a
Du
Pont
The cassette was
the film
was allowed
Lightening
Plus
then placed in
a
to expose
a
for
16
Re-use of Membranes
Since two separate probes were
was necessary to probe first
used in this study,
with one clone and then
it
after,
exposure and subsequent stripping of the membrane, with
the
other.
was
Stripping
accomplished
as
the
old
follows:
probe
off
the
using
gentle
membranes
agitation,
the
membranes were washed for 15 minutes with a boiling solution
of 0.1X SSC/0.5% SDS.
This wash was then repeated a
time to insure removal of all residual probe.
was
then
prehybridized
as
described
The
second
membrane
previously
and
hybridization was carried out using the new probe.
Data Analysis
Data
program
analysis
of
was
the
carried
MSUSTAT
regressions were run
out
using
statistics
on standard
the
package.
molecular weight
run in conjunction with all restriction digests.
then a regression
equation was obtained
the determination of the approximate
Linear
markers
From
which allowed
size in base pairs
each restriction fragment.
From this data, it was
to compare
within and
cultivars both
MREGRESS
between
this
for
of
possible
populations
with respect to the loci represented by each of our probes.
17
RESULTS
Characterization of Fragments
Restriction fragments revealed through
to be homologous
to the
specific probes
autoradiography
utilized in
study were characterized in terms of their mobility
this
through
the agarose gel relative to known molecular size standards.
In this study, the molecular size standard used in all cases
was
phage
lambda
DNA
restriction endonuclease
digested
Hind 3.
restriction fragments of the
to
completion
This
with
digestion yields
following sizes: I:
125bp. The first
seven of these
2,028bp
fragments
are generally resolvable on a
0.8%
this study.
log linear regression on
By calculating a
molecular size
obtain
a
standard of
regression
for
specific
regression
Using the
each gel, it
was possible to
for
each
it was
equation
combination.
weight
agarose gel as used
each gel,
restriction
each
possible
autoradiograph of that specific gel.
equation
By taking the anti-log of
the
to
observed
for
molecular
on
the
The apparent molecular
weight is expressed as the log of the base pair size of
fragment.
in
standard/gel
derive an apparent
fragment
8
23,130bp,
2: 9,419bp, 3: 6,557bp, 4: 4,371bp, 5: 2,322 bp, 6:
7: 564bp and 8:
the
each value, a size
terms of base pairs was determined for each fragment.
the
in
18
Evaluation of Polymorphisms in Barley
The fundamental question to
be answered by this
study
is whether or not detectable genetic polymorphism exists
a
high
enough
development of
frequency
this
in
barley
technology
indeed variation can be
to
allow
for use
in
for
at
the
breeding.
demonstrated at a particular
If
locus
or set of loci, the alleles observed then have potential for
use as
genetic markers.
To resolve
this question,
barley populations were screened with two randomly
three
selected
genomic clones across four restriction enzyme digests.
The results of
this study indicate
that the level
variability detected by both
probes, lambda 107 and
167,
These
is
useful
moderately
for
molecular
high.
the detection
level.
The
of
probes
lambda
should
genetic variability
overall
analysis
of
of
prove
on
the
restriction
fragments characterized in this study revealed a total of 23
fragments using
probe lambda
enzyme combinations.
fragments
is
revealed a
from
all genotype . and
The size range in base pairs of
16,520
total of
107 across
to
1,968.
115 restriction
Probe
these
lambda
167
fragments across
all
genotype and enzyme combinations ranging in size from 16,945
to 1,291
base
pairs. Both
within and between
and size range of
probes
populations.
revealed
Table 2
polymorphisms
shows the
restriction fragments seen over
enzyme combinations using probes lambda 107 and 167.
number
genotype
19
Table 2. Total Number and Size of Restriction Fragments
Observed Using Probes Lambda 107 and 167 Across
Enzymes and Genotypes.
Probe
Lambda 107
Total Fragments
Lambda 167
Size (bp)
23
16,520-1,968
As can
be
generated
by
seen,
each
Total Fragments
• 115
the
probe
number
differs
Size (bp)
16,943-1,291
of
distinct
fragments
greatly. This
result
reflects the fundamental difference in the molecular
nature
of each probe. While both probes represent nuclear
sequences
mapping
as
and
shown
by
both
segregation
wheat-barley
analysis
(Chao
addition
and
line
Blake,
unpublished), they are quite different in terms of what they
represent on a molecular level.
lambda 107 is a single
7.
As previously stated, probe
copy sequence located on
chromosome
As such, it may be assumed that the restriction fragment
length polymorphisms observed through the use of this
probe
represent various alleles of a single locus.
Probe lambda 167 has been shown to be a multicopy
dispersed sequence located on at least four different
chromosomes (1,4,6 and 7).
As such
the
length polymorphisms
restriction
fragment
through the use Of this probe sequence
polymorphic forms of at least four loci.
it
may be assumed
that
observed
represent several
20
Allelism Among Probes Within Enzyme Treatments
Each enzyme
utilized in
this
study was
observed
generate a number of polymorphic fragments across
and
probes.
In
the
polymorphisms may be
case
lambda
probe
assumed to
forms of a single locus on
of. probe
of
167
lambda
genotypes
107
these
represent various
allelic
chromosome 7, while in the
it
may
be
assumed
to
that
case
these
polymorphisms represent various allelic forms of a number of
loci
dispersed
throughout
the
barley
genome.
Each
autoradiogram of every enzyme/probe/genotype combination was
examined
and
scored
for
resulting information is
the
presence
listed in Tables
of
RFLPs.
3 and
4.
The
Table
3shows both the number and size of alleles observed for each
restriction enzyme
using probe
lambda 107,
while Table
shows the same information for probe lambda 167.
Table 3. Number and Size of,Lambda 107 Fragments.
Enzyme
Hind 3
Frag.# Size Frag.#
I 16,520
I
2
8,318
2
7,775
3
3
4
3,048
4
5
2,818
5
6
2,432
6
7
Bam Hl
Eco Rl
Size
Frag.#
13,459
I
12,677
2
12,218
3
10,069
,4
8,830
■■ 5
6,194
1,968
ECO R 5
Size Frag. #
11,614
I
11,220
2
10,328
3
7,889
4
6,295 . 5
Size
13,243
12,474
12,035
11,041
10.069
4
21
Table 4. Number and Size of Lambda 167 Fragments.
Hind 3
Frag.#
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
39
40
41
42
Size Frag.#
16,943
I
16,406
2
15,996
3
4
14,859
14,015
5
13,050
6
12,775
7
11,912
8
9
11,561
11,027 10
10,839 11
10,375 12
10,116 13
9,908 14
9,725 15
9,370 16
9,162 17
8,820 18
7,870 19
7,568 20
7,295 21
6,823 22
6.628 23
6,412 24
5.998 25
5,584 26
5,376 27
5,130
4,920
4,677
4,540
4,159
3,926
3,793
3,508
3,133
2,723
2,355
2,094
1,614
1,291
Enzyme
Bam IHl
Size
Frag.#
15,241
I
14,521
2
13,964
3
13,213
4
12,350
5
11,455
6
11,041
7
10,839
8
10,666
9
10,280
10
9,921
11
9,280
12
13
8,179
7,728
14
6,966
15
6,223
16
5,808
17
5,572
18
5,012
19
4,831
20
4,571
21
3,873
22
2,974
2,793
2,716
2,523
1,435
ECO ]Rl
Size Frag.#
13,614
I
13,183
2
11,588
3
9,954
4
9,616
5
8,825
6
8,414
7
7,943
8
7,691
9
7,396 10
6,998 11
5,689 12
5,050 13
4,425 14
4,150 15
3,785 16
3,515 17
2,804 18
2,294 19
1,845 20
1,490 21
916 22
23
24
25
26
Eco :
Size
16,052
15,031
14,588
13,772
12,823
12,474
12,084
11,668
11,041
10,444
9,977
9,036
8,780
8,235
7,691
7,434
7,031
6,622
5,794
4,656
3,508
3,296
2,924
2,742
2,500
2,289
22
In this study genetic variation at the molecular
level
was demonstrated both within and between populations through
the use of
various restriction/enzyme/probe
combinations.
The following tables list the postulated alleles present
each population by restriction digest and variety for
probe
Lambda 107.
Table 5. Lambda 107 Alleles: Hind 3.
Population
Fragment #.
Postulated Allele #
Midwest 6 Row:
Larker
Traill
Morex
Robust
Bedford
Hazen
Azure
3
6
3
3
3
3
3
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT1423
OR8408
Karla
Columbia
Karl
3
3
3
3
3
5
n/d
3
5
I
I
I
I
I
3
n/d
I .
3
Western 2 Row:
Betzes
Palliser
Klages
Harrington
Gallatin
Bowman
6
2,5
5
5
1,5
6
2
4
3
3
5
2
I
2
I
I
I
I
I
in
23
Table 6. Lambda 107 Alleles: Bam HI.
Population
Midwest 6 Row: .
barker
Traill
Morex
Robust
Bedford
Hazen
Azure
Fragment #
Postulated Allele #
7
7
7
7
7
7
7
I
I
I
I
I
I
I
Western 6 Row
Unitan
Coast
Atlas
Steptoe
UT1423
OR8408
Karla
Columbia
Karl
3
I
I
2
2
5
2
2
2
2
3
3
4
4
5
4
4
4
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
3
4
4
4
4
3
4
3
4
6
2
6
6
6
6
2
6
2
6
7
24
Table 7. Lambda 107 Alleles: Eco Rl.
Population
Fragment #
Postulated Allele #
Midwest 6 Row:
Larker
Traill
Morex
Robust
Bedford
Hazen
5
3
3
3
3
3
Western 6 Row
Unitan
Coast
Atlas
Steptoe
UT1423
OR8408
Karla
Columbia
Karl
,2
2
2
2
2
I
2
2
2
3
3
3
3
3
4
3
3
3
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
. .
I
2
2
2
2
2
25
Table 8. Lambda 107 Alleles: Eco R5.
Population
Fragment #
Midwest 6 Row:
Darker
Traill
Morex
Robust
Bedford
Hazen
Azure
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT 1423
OR 8408
Karla
Columbia
Karl
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
4
5
3•
3
3
3
3
I
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
1,3
3
3
3
■3
1,3
3
3
As can be seen from
different alleles
3
4
. 5
3
3
3
3
5
3
3
the preceding Tables, probe
107 reveals substantial genetic
populations tested.
Postulated Allele #
lambda
variation between and among
This result indicates that a number
can be
detected at
example of this is shown in Figure 3.
the three populations studied was
probed with lambda 107.
a single
locus.
of
An
DNA of cultivars from
digested with Bam Hl
and
26
In
addition
to
the
variation
populations, this study reveals
observed
between
that a number of
different
alleles can also be detected at a single locus in
different
varieties within a
Western
6
population.
Figure 4 shows
DNA of
the
Row varieties digested with Hind 3 and probed with
Lambda 107.
While
there
are
similar
alleles
members of this group, two distinct alleles can be
among
observed
by this particular enzyme/genotype/probe combination.
Figure 3. Lambda 107 Alleles Between Populations: Bam HI.
Kb
Alleles
2319. 4-
#
I
_1
-2
-3
—4
654. 3—
2.3-
2.0-
I
2 3
4
5
6
7
8
9
10 11
the
12 13
Lanes:
I:Lambda Hind 3,2:Harrington,3:Clark,4:Lewis,5:Hector
6 :Klages,7: Betzes,8 :Larker,9 -.Morex, 10 :OR8408,11: Unitan,
I2:Steptoe,13:Coast.
27
Figure 4. Lambda 107 Alleles of Western 6 Row Varieties:
Hind 3.
Lanes:
I :Unitan,2:Coast,3:Atlas,4:Steptoe,5:UT1423,6: OR8408 ,
7:Karla,8 :Columbia,9:Horsford,10:Ridawn,11:Lambda Hind 3.
28
As well as the previous
variety of
examples. Figures 5-7 exhibit
enzyme/genotype combinations
which
demonstrate
several RFLPs using probe Lambda 107.
Figure 5. Lambda 107 Alleles Western
Bam HI.
6
a
Row Varieties:
Lanes:
I :Unitan,2:Coast,3:Atlas,4:Steptoe,5:UT1423,6:OR8408
7:Karla,8:Columbia,9:Horsford,10:Ridawn,11:Lambda Hind 3.
29
Figure 6. Lambda 107 Alleles Midwest 6 Row Varieties:
Hind 3.
A lle le s
Kb
2 3 .1 -
m
9 .4 -1
6 .5 43 —
2 .3 -
-2
2 .0 —
#
I
2
3 4
5
6
7
8
9 10
Lanes:
I :Lambda Hind 3,2:Prior,3:Clipper,4:Azure,5:Hazen,6:Bedford
7:Robust,8:Morex,9:Traill,10:Larker.
30
Figure 7. Lambda 107 Alleles Midwest 6 Row Varieties:
Eco R5.
Kb
-2 3 .1
A lle le s
r*
?2
“
_ 9.4
— 6.5
-4 .3
-2 .3
-
1 2
3
4
5
6
7
8
9
2.0
10
Lanes:
I:Lambda Hind 3,2:Prior,3:Clipper,4:Azure,5:Hazen,6:Bedford
7:Robust,8:Morex,9:Traill,10:Larker.
31
To summarize,
using
restriction enzyme
Midwest
6
-row
varieties show
Western
6
-row varieties show
Western 2-row
varieties
two alleles
4
alleles
Allele I is common to both the Midwest
row varieties.
6
3,
the
2),
the
and 3), and
the
(I and
2 alleles (I
show
Hind
(2,3,4,and
-row and Western
Allele 3 is common to the Western
Western 2-row varieties.
2-row and Midwest
Hind 3 alleles
6
5).
6
-row
6
-
and
Allele 2 is common to both Western
-row varieties.
using probe
This gives a total of
Lambda 107 three
of which
5
are
common to more than one population.
Using restriction
enzyme
Bam HI, the
varieties show I
allele (I).
The
Western
show
(2,3,4
5),
and
4
alleles
and
varieties show 2 alleles
(6
is common to
tions.
The
the Western
remaining
6
-row and
alleles
6
the
and 7). This
seven alleles using probe lambda 107.
Midwest
-row
6
-row
varieties
Western
2-row
gives a total
of
In this case allele 2
Western 2-row
appear
to
be
popula­
population
specific.
Using restriction
enzyme
Eco Rl, the
varieties show
two alleles
(I and 2).
varieties show
two alleles
as well
Western 2-row varieties show
total of five alleles
Midwest
6
-row
The Western
6
-row
(3 and
one allele (5).
for this enzyme
4), while
the
This gives
and once again
a
they
all appear to be population specific.
Using restriction
enzyme
Eco R5, the
varieties show three alleles (1,2 and 3).
Midwest
The Western
6
-row
6
^row
32
varieties show
one
allele
varieties show three
(3), while
alleles (3,4
total of five alleles
the
and 5
with one, number
Western
).
2-row
This gives
3, being common
a
to
each population.
An interesting note at this point is that although
varieties show
polymorphisms
with a
specific
most
restriction
enzyme, a number of varieties show polymorphisms across
two
or more enzymes (e.g. barker, Traill, OR8408 and Columbia).
This would seem to suggest that although most
polymorphisms
are due to
polymorphisms
point mutations, a
observed in
this study
number of the
appear to
be due
to insertion
or
deletion type events.
The
analysis
of
probe
straightforward.The molecular nature
data which is more complex.
detected both
within and
Lambda
107
of probe 167
was
provided
Again, genetic polymorphism was
between populations
although
this case these polymorphisms represent a number of
at differing loci.
alleles of one another
each
fragment
polymorphisms
which specific fragments
and so no attempt
represented in this
represent
has been made
to
It is possible however to characterize
in . terms
among
alleles
It was not possible within the bounds of
this study to determine
report them as such.
in
the
study.
of
molecular
size
enzyme/population
The following
and
observe
combinations
tables list
various specific restriction fragments observed using
Lambda 167 in specific enzyme/population combinations.
the
probe
33
Table 9. Lambda 167 Fragments: Hind 3.
Population
Fragment #
Pattern Designation
Midwest 6 Row:
barker
Traill
Morex
Robust
Bedford
Hazen
Azure
2,7,12,17,25,26,29,31
2,7,12,17,25,26,29,31,34,35
2,4,6,10,13,19,24,27,29,34,39,42
2,4,6,10,13,19,24,27,29,34,39,42
2,4,6,10,13,19,24,27,29,34,39,42
2,4,6,10,13,19,24,27,29,34,39,42,
2,9,14,19,24,27,29,39,42
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT 1423
OR 8408
Columbia
Karl
7,11,15,24,31,39,42
7,11,20,24,27,31,39,42
7,11,18,25,27,30,36,39,42
7,12,30,42
7,11,19,27,29,34,39,42
7,11,36,42
7,11,16,23,26,29,33,36,38,42
7,11,12,16,27,28,29,33,37,40,42
9
10
11
12
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
3,5,8,12,16,27,28,33,35,37,40,42
3,5,8,12,16,23,29,37,40,42
3,5,8,16,21,23,27,32,37,40,42
5,8,15,24,28,42
5,8,15,24,28,42
5,8,15,24,28,42
5,8,15,24,28,42
5,10,15,24,25,30,39,41,42
5,10,24,30,42
13
14
15
16
16
16
16
17
18
I
2
3
3
3
3
4
5
6
7
8
34
Table 10. Lambda 167 Fragments: Bam HI.
Population
Fragment #
Pattern Designation
Midwest 6 Row:
Darker
Traill
Morex
Robust
Bedford
Hazen
Azure
4,5,10,13,18,19,20,21,23,24,25,26
4,5,10,13,18,19,20,21,23,24,25,26
3,4,7,12,18,20,23,25,26
3,4,7,12,18,20,23,25,26
3,4,7,11,12,18,23,25,26
3,4,7,11,12,18,20,23,25,26
3,4,6,16,24,25
I
I
2
2
3
4
5
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT 1423
OR 8408
Karla
Columbia
Karl
2,5,8,12,13,14,17,21,22,23
2,5,6,7,8,12,13,18,21,22,23
n/d
n/d
2,5,7,12,13,19,21,22,23
2,5,7,12,13,14,17,21,22,23
2,5,7,12,13,14,17,18,21,22,23
2,5,7,12,13,14,17,18,19,21,22,23
2,5,8,12,13,14,17,18,21,22,23
6
9
10
11
10
Western 2 Row;
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
2,5,8,12,13,15,17,18,22,23,24,27
11
11
11
11
11
11
11
11
11
11
7
8
35
Table 11. Lambda 167 Fragments: Eco Rl.
Population
Fragment #
Pattern Designation
Midwest 6 row:
Darker
Traill
Morex
Robust
Bedford
Hazen
Azure
2,6,7,10,11,12,15,17,22,23,25
2,6,7,10,11,12,15,17,22,23,25
2,5,6,9,12,13,14,16,22,23,25
2,5,6,9,12,13,14,16,22,23,25
2,5,6,9,12,13,14,16,19,22,23,25
2,5,6,9,12,13,14,16,19,21,22,23,25
2,5,6,9,12,13,14,16,19,21,22,23,25
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT 1423
OR 8408
Columbia
Karl
6,11,12,14,16,20,22,25
6,8,12,14,15,16,20,22,25
6,11,12,14,16,20,22,25
6,11,12,14,16,20,22,25
6,11,12,14,16,20,22,25
6,11,12,14,16,20,22,25
6,11,12,14,16,20,22,25
6,11,12,14,16,20,22,25
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
1,6,11,12,14,16,18,20,21,22,24
I
I
2
2
3
4
4
5
6
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
7
36
Table 12. Lambda 167 Fragments: Eco R5.
Population
Fragment #
Midwest 6 Row:
Larker
Traill
Morex
Robust
Bedford
Hazen
Azure
Western 6 Row:
Unitan
Coast
Atlas
Steptoe
UT 1423
Or 8408
Karla
Columbia
Karl
Western 2 Row:
Betzes
Doman
Palliser
Hector
Klages
Lewis
Harrington
Gallatin
Pattern Designation
7,12,15,18,26
7,12,15,18,26
7,10,12,14,16,18,24,25
7,10,12,14,16,18,24,25
7,10,12,14,16,18,24,25
7,10,12,14,16,18,24,25
7,10,12,14,16,18,24,25
I
I
2
2
2
2
2
4,7,9,11,12,15,17,19,23,24
4,7,9,11,12,15,17,19,23,24
4,7,11,12,17,18,23,24
4,12,15,18,23,24
4,7,11,12,15,17,18,23,24
4,7,11,12,17,18,23,24,26
4,7,11,12,23,24
4,11,12,13,23,24
4,8,11,12,13,17,22,23,24
3
3
4
5
6
7
8
9
10
1,2,3,5,8,10,11,17,20,21,22
1,2,3,6,9,11,12,15,21,22
1,3,5,8,10,11,12,15,16,17,21,22
1,3,9,10,12,15,18,21,22
1,3,9,10,12,15,18,21,22
1,3,9,10,12,15,18,21,22
1,3,6,9,10,11,12,15,18,21,22
3,6,9,10,11,12,16,18,21,22
11
12
13
14
14
14
15
16
Results obtained through the use of this probe indicate
that several different alleles can be detected at several
loci
between
observation.
populations.
DNA from
populations examined
Lambda
167.
A
a
was
number
Figure
number of
digested
of
length polymorphisms
exist
populations.
result
This
varieties
with
distinct
both
demonstrates
8
and
indicates
from
three
probed
with
restriction
between
the
and
this
fragment
within
existence
various forms of several loci in cultivated barley.
the
of
37
Figure 8. Lambda 167 Between Population Polymorphisms:
Bam HI.
F ra g m e n ts
Kb
1
2
3
4
5
Lanes:
I :Lewis,2 :Klages,3:Robust,4:Hector,5:Morex,6 :Clark
7:Columbia,8 :Apex,9:Azure,1 0 :Teton,1 1 :Andre,1 2 :Bellona
13:Lambda Hind 3.
38
Figure 9. Lambda 167 Fragments of Western
Hind 3.
6
Row Varieties:
Kb
F ra g m en ts
-2 3 .1
715-
-9 .4
24-
-6 .5
31-
-4 .3
%
3439-
- 2 .3
-
2.0
42-
W
1 2 3 4
5 6 7 8
9
10
11
Lanes:
I :Unitan,2:Coast,3:Atlas,4:Steptoe,5:UTl423,6:OR8408,
7:Karla,8:Columbia,9:Horsford.10:Ridawn,11:Lambda Hind 3.
In addition
to the
variation between
populations
probe also reveals that a number of different
can be
detected
at
within a population.
is shown
6
in Figure
-row varieties
several loci
An example
9.
In this
was digested
in
polymorphisms
different
varieties
of this type of
variation
case
with the
Hind 3 and probed with Lambda 167.
this
DNA of
the
restriction
Western
enzyme
While similarities exist
39
in
the
banding
especially those
patterns
related
revealed
by
pedigree, there
number of
distinct polymorphisms
Figures 9
and
10 present
the
population/ enzyme combinations:
Midwest
6
-row
Bam Hl
between
varieties,
are
also
which can
be
observed.
same result
in
two
Midwest
respectively.
6
other
-row Hind 3
Note the
6
and
similarities
observed in varieties related by pedigree.
Figure 10. Lambda 167 Fragments Midwest
Hind 3.
a
Row Varieties:
Lanes:
I :Larker,2 :Traill,3:Morex,4:Robust,5:Bedford,6 :Hazen,
7:Azure,8 :Clipper,9:Prior,10:Lambda Hind 3.
40
Figure 11. Lambda 167 Fragments Midwest 6 Row Varieties:
Bam Hl.
1 2 3
4
5
6
7
8 9
10
Lanes:
I :Larker, 2 :Traill,3:Morex,4:Robust,5:Bedford,6 :Hazen,
7:Azure,8 :Clipper,9:Prior,10:Lambda Hind 3.
41
To summarize, restriction digests with Hind 3 yield the
largest number of polymorphisms between populations.
Of the
42 restriction fragments generated with this enzyme, 20
unique to
a specific
accounting
for
accounting for
nine
population; Western
of
seven of
them.
Midwest
them and
Western
accounting for the remaining four.
Bam
Hl
yielded
populations, out
13
total
generated by this enzyme.
are found in the Midwest
the Western 2-row
6
-row varieties.
total of
unique to
Eco
Rl
a specific
varieties
:-row
varieties
6
-row
varieties
fragments
of 27
unique
to
restriction
fragments
Eleven of these unique
fragments
-row varieties, two are found
types, and
restriction
2.2
6
2-row
Restriction digests with
restriction
of a
6
are
one is found
restriction
in the
digests
fragments, sixteen
population.
Midwest
Western
produced
of which
6
-row
6
varieties
-row varieties while the remaining four were
in Western 2-row varieties.
Finally, digestion with Eco
a specific population.
Seven
6
-row and
Midwest
6
are found
the Midwest
Hind 3
6
in
the
-row varieties respectively.
Variation among varieties within a population was
observed.
R5
which
the Western 2-row varieties while two each are found in
Western
in
found
yielded a total of 26 restriction fragments eleven of
were unique to
a
were
accounted for eight of these fragments, three were found
Western
in
digests yielded
17 polymorphisms
-row varieties,, 14 among the Western
6
varieties and 17 among the Western 2-row varieties.
also
within
-row
Bam Hl
42
yielded 13 polymorphisms among the Midwest
four among
Western
Western 2 row
6
-row
varieties.
6
-row
varieties
6
varieties and
Eco
R5 digests
6
and
^row
varieties,
none
among
yielded 12
-row varieties,
none among
the Midwest
varieties
and
Eco Rl digests
the Midwest
Finally, the
morphisms among
Western
-row
varieties.
morphisms among
Western
6
6
13 among
poly­
3 among
the Western
yielded
-row varieties,
the
the
2-row
7
9 among
poly­
the
the
Western
2-row
Lambda 167
and 107
probe
varieties.
The data derived
from both
screening was used in a reconstruction analysis to
evaluate
how well our results would resemble the known pedigree data.
This analysis would determine if it is possible to
estimate
genetic distance between genotypes using RFLP analysis.
data was
evaluated using
from the
Illinois
Natural
a program
History
determined relatedness between
called PAUP
Survey. . This
The
available
program
genotypes and constructed
a
phylogenetic tree which was compared to known pedigree data.
Table 13 presents the data.summary used for the phylogenetic
analysis. Figure 12 is a representation of the
tree derived from our data.
phylogenetic
43
Table 13. Data Summary: Banding Pattern Designations Used
in Phylogenetic Analysis.
Lambda 107
Lambda 167
Pattern Designation
H3 BHl Rl
Enzyme: H3 BHl Rl R5
Probe:
R5
Cultivar
Darker
Traill
Morex
Robust
Bedford
Hazen
Azure
Unitan
Coast
Atlas
Steptoe
UT1423
OR8408
Karla
Columbia
Karl
Betzes
Doman
Palliser
Hector
Klages
Lewis
Clark
Harrington
Gallatin
Bowman
I
2
I
I
I
I
I
I
I
I
I
I
3
3
3
3
2
I
I
I
I
I
I
I
2
3
3
4
4
5
4
4
6
6
6
4
6
6
3
6
6
6
3
5
6
2
7
6
I
I
I
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
5
3
3
3
3
3
3
3
3
3
3
3
4
5
2
2
2
2
3
3
3
3
3
4
3
3
5
5
5
5
5
5
5
5
5
5
5
6
I
I
I
I
I
I
2
2
2
2
2
2
2
3
4
5
3
4
4
5
6
6
2
2
2
3
3
4
5
11
10
12
9
7
7
7
7
7
7
7
11
8
11
11
8
12
11
8
11
8
13
14
14
14
7
8
9
7
10
8
9
13
14
15
16
16
16
16
17
18
11
8
11
8
11
8
11
8
11
8
11
8
6
7
8
9
10
15
16
44
Figure 12.
*
*
*
Phylogenetic Reconstruction of Barley Cultivars
I LARKER
**********
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
4*
****50
*
2
TRAILL
3
MOREX
4
ROBUST
****
6
*
*** *45
*
*
*
*
*
*
*
*
*******
*
*
*
*
*
*
*****45
*****49
*
*
*
*
*
*
*
*
*
*
*
48
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
HAZEN
*
*
*
AZURE
******
8
UNITAN
*
*
*******
COAST
*******27
*
* 1 0 ATLAS
*****44
*
*
*
*
43
*** 11 STEPTOE
*****28
**30
* 12 UTl423
* *
* * 15 COLUMBIA
****37
*
*
********* 23 OR8408
*
*
**29
*
*****32 * 14 KARLA
*
* 16 KARL
*
**** 17 BETZES
42
*
*
*
*
****18DOMEN
*
*
***32
*
*
*35
*19PALLISER
*
*
* *
*
*
*36 * 25 GALLATIN
* *
*******42
* **38 * 20 HECTOR
* * *
* * * 21 KLAGES
**39
33
* * * 23 CLARK
* *
40 * 22 LEWIS
* 34
* ****** 24 HARRINGTON
*
**** 26 BOWMAN
45
CONCLUSIONS
This
study
describes
the
evaluation
of
diversity in three barley populations using RFLP
genetic
analysis.
A moderate level of genetic diversity was observed using two
randomly selected
genomic clones
copy and one a dispersed
of barley,
one a
gene family sequence.
single
Due to: the
molecular nature of these probes it is possible to report at
least
four
polymorphic
separate chromosomes.
variation on
barley it
at
loci
on
at
least
Since only two clones have
least four
becomes
located
of
the seven
readily apparent
that
four
uncovered
chromosomes
this
in
technology
holds promise for future genetic studies within the species.
This study utilized randomly selected genomic
Earlier reports in the literature
cDNA probes
1985).
(Relentjaris
et
have described the use of
al, 1985;
These probes will evaluate
loci which
are
expressed as
clones.
Tanksley,
variation only at
translation
identify genes of interest,
coverage of
is impeded.
clones
In
contrast,
al,
those
products.
useful in attempting to
the genome
et
While
random
genomic
must be assumed to be random in their occurrence
and thus allow for
uniform fashion.
the evaluation of the
That
two genomic
genome in a
clones have
been
more
used
to demonstrate genetic variation represents a first step in
46
covering the
g e n o m e o f barley
with
randomly
distributed
molecular markers.
In comparing the two probes used in this study, several
points need
to be 'addressed.
probes were used in this
advantage.
Two fundamentally
study, each with its own
Probe lambda 107, a
easily interpretable data
this study.
different
distinct
single copy probe
in all
provides
combinations screened
Each polymorphism is assumed to be a
different
allelic form of a single locus on the basis of earlier
which was
done
to
characterize this
in
sequence
work
(Chao
and
Blake, unpublished).
In contrast, probe lambda 167 is a
probe.
dispersed
sequence
It reveals complex banding patterns which are
taken
to represent a number of polymorphic forms at several loci.
Initially it is
not possible to
determine which bands
are
allelic or how many loci are represented.
At first glance, the dispersed sequence probes
to
provide data unnecessarily complex.
of probe seems
loci are
likely to prove
available for
segregation
analysis
possible to
determine
However, this
advantageous since
evaluation with
of
F2
appear
one
individuals,
which polymorphisms
several
probe.
it
are
type
Using
should
allelic.
Helentjaris and others have demonstrated the feasibility
this approach
(Helentjaris,
et
Tanksley, 1985; Chao and Blake,
al,
1985;
unpublished).
be
Bernatzky
of
and
Furthermore,
since several loci are evaluated at once the liklihood of
47
finding useful polymorphisms is increased.
Initial
reports
in both maize and tomato indicate the advantage of multicopydispersed sequences
while
the
2 0
as
probes.
Helentjaris
reports
that
% of single copy probes reveal polymorphisms, 80% of
multicopy
dispersed
sequences
show
polymorphisms
(Helentjaris, et al, 1985).
As RFLP
linkage
markers are
map,
linkage
assigned positions
on the
barley
with
mapped
genes
controlling
agronomically
identified.
Selection
resistance to
easier.
multiple
(e.g.
dwarf
gene transfer
conferring
will
characters
virus)
genes with
genes
characters
useful
yellow
Marker associated
pyramiding of
effects
important
for
barley
previously
will
overlapping
resistance
(e.g.
be
may also
be
made
promote
phenotypic
to
powdery
mildew).
RFLPs
between
provide
a tool
populations.
for studying
The
virtually
genetic
unlimited
variation
number
of
RFLPs available within a species as well as their capability
to monitor
changes
extremely useful
in
in
untranslated
studies
phylogenetic relationships,
sequences
make
which
attempt
to
analyze
genetic
structure
them
determine
of
related populations and measure genetic diversity within and
between populations.
with pedigree
genetic
Our reconstruction analysis fits
analysis
distance
among
possible and useful.
and
suggests
genotypes
by
that
RFLP
well
estimation
of
analysis
is
In our case, evaluation of data by
48
PAUP
reconfirmed
the
relationships
determined
in ■ our
analysis.
To summarize then, this study indicates that a
substantial amount of detectable genetic variation exists
both within and between the barley populations screened
with two random genomic probes.
A saturated linkage map in
barley seems feasible and brings to light enormous potential
in
terms
of
genetic
improvement
and
manipulation.
Population improvement will benefit through improvements
selection efficiency for desirable traits.
in
Introgression of
desirable wild type alleles will be facilitated using marker
assisted"gene transfer. Screening of germplasm and isolation
of genes of
interest will be
RFLP saturated linkage map it
more random
genomic
dispersed type.
Care
yet
not
should
be taken
of
the
however
provide data which is
unnecessarily
analysis will need to be
To complete
a
will be necessary to, develop
clones, preferably
clones of this type which
complex
facilitated.
confusing.
multicopy
to
select
reasonably
Segregation
carried out on these clones
which they can be mapped to known marker loci.
That
after
barley
contains sufficient genetic diversity to lend itself to such
analysis has
been demonstrated
use of appropriate
means of
here.
The development
probe sequences should
applying a
basic molecular
achieve real agricultural improvement.
bring about
biology technique
and
the
to
49
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