Chapter 4
Gene Linkage and
Genetic Mapping
Important Definitions
• Locus = physical location of a gene on a chromosome
• Homologous pairs of chromosomes often contain
alternative forms of a given gene = alleles
• Different alleles of the same gene segregate at Meiosis
I
• Alleles of different genes assort independently in
gametes
• Genes on the same chromosome exhibit linkage:
inherited together
2
Genetic Mapping
• Gene mapping determines the order of genes and
the relative distances between them in map units
• 1 map unit = 1 cM (centimorgan)
In double heterozyote:
• Cis configuration = mutant alleles of both genes are
on the same chromosome = ab/AB
• Trans configuration = mutant alleles are on different
homologues of the same chromosome = Ab/aB
3
Genetic Mapping
• Gene mapping methods use recombination
frequencies between alleles in order to determine
the relative distances between them
• Recombination frequencies between genes are
inversely proportional to their distance apart
• Distance measurement: 1 map unit = 1 percent
recombination (true for short distances)
4
Genetic Mapping
• Recombination results from crossing-over between
linked alleles
• The linkage of the genes can be represented as a
genetic map, which shows the linear order of the
genes along the chromosome spaced so that the
distances between genes is proportional to the
frequency of recombination between them.
5
Genetic Map
Figure 4.5: The frequency of recombination is used to construct a genetic map6
Genetic Mapping
• Genes with recombination frequencies less than 50
percent are on the same chromosome = linked
• Linkage group = all known genes on a chromosome
• Two genes that undergo independent assortment
have recombination frequency of 50 percent and
are located on nonhomologous chromosomes or
far apart on the same chromosome = unlinked
7
Recombination
• Recombination between linked genes occurs at
the same frequency whether alleles are in cis or
trans configuration
• Recombination frequency is specific for a
particular pair of genes
• Recombination frequency increases with
increasing distances between genes
• No matter how far apart two genes may be, the
maximum frequency of recombination between
any two genes is 50 percent.
8
Genetic Mapping
• Recombination changes the allelic
arrangement on homologous chromosomes
Figure 4.4: Crossing-over between two genes
9
Genetic Mapping
• The map distance (cM) between two genes equals
one half the average number of crossovers in that
region per meiotic cell
• The recombination frequency between two genes
indicates how much recombination is actually
observed in a particular experiment; it is a measure
of recombination
10
Figure 4.6: Diagram of chromosomal configurations in 50 meiotic cells
11
Genetic Mapping
• Over an interval so short that multiple crossovers
are precluded (~ 10 percent recombination or
less), the map distance equals the recombination
frequency because all crossovers result in
recombinant gametes.
• Over the short interval genetic map = linkage map
= chromosome map
12
Genetic Mapping
• The frequency of recombination between two
mutant alleles is independent of whether they
are present in the same chromosome or in
homologous chromosomes.
13
Figure 4.2: The frequency of recombination between two mutant alleles
14
Gene Mapping: Double Crossing Over
• Two exchanges taking place between genes, and
both involving the same pair of chromatids, result
in nonrecombinant chromosomes
Figure 4.8: Crossovers between marker genes A and B
15
Figure 4.12: The result of two crossovers in the interval between two genes
16
is indistinguishable from independent assortment of the genes
Genetic vs. Physical Distance
• Map distances based on recombination frequencies
are not a direct measurement of physical distance
along a chromosome
• Recombination “hot spots” overestimate physical
length
• Low rates in heterochromatin and centromeres
underestimate actual physical length
17
Figure 4.11: Chromosome 2 in Drosophila
as it appears in metaphase of mitosis and
in the genetic map
18
Genetic Mapping: Three-Point Cross
• In any genetic cross, the two most frequent types
of gametes are nonrecombinant; these provide
the linkage phase (cis versus trans) of the alleles
in the multiply heterozygous parent.
• The two rarest classes identify the doublerecombinant gametes.
• The effect of double crossing-over is exchange of
members of the middle pair of alleles between the
chromosomes
19
Table 4.1 Interpreting in a Three-Point Cross
20
Genetic Mapping
• Mapping function: the relation between genetic
map distance and the frequency of recombination
• Chromosome interference: crossovers in one
region decrease the probability of a second
crossover close by
Figure 4.17: Mapping functions
21
Genetic Mapping
• Coefficient of coincidence = observed
number of double recombinants divided
by the expected number
• Interference = 1-coefficient of coincidence
22
Mapping Genes in Human Pedigrees
• Methods of recombinant DNA technology are used
to map human chromosomes and locate genes
• Genes can then be cloned to determine structure
and function
• Human pedigrees and DNA mapping are used to
identify dominant and recessive disease genes
• Polymorphic DNA sequences are used in human
genetic mapping.
23
Mapping Genes in Human Pedigrees
• Most genes that cause genetic diseases are rare, so
they are observed in only a small number of families.
• Many mutant genes of interest in human genetics are
recessive, so they are not detected in heterozygous
genotypes.
• The number of offspring per human family is
relatively small, so segregation cannot usually be
detected in single sibships.
• The human geneticist cannot perform testcrosses or
backcrosses, because human matings are not
dictated by an experimenter.
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Genetic Polymorphisms
• The presence in a population of two or more relatively
common forms of a gene or a chromosome is called
polymorphism
• A prevalent type of polymorphism is a single base pair
difference, simple-nucleotide polymorphism (SNP)
• SNPs in restriction sites yield restriction fragment
length polymorphism (RFLP)
• Polymorphism resulting from a tandemly repeated short
DNA sequence is called a simple sequence repeat (SSR)
25
SNPs
•
SNPs are abundant in the human genome.
• The density of SNPs in the human genome
averages about one per 1300 bp
• Identifying the particular nucleotide present at
each of a million SNPs is made possible through
the use of DNA microarrays composed of about
20 million infinitesimal spots on a glass slide the
size of a postage stamp.
26
Figure 4.18: SNP genotype of an individual
27
RFLPs
• Restriction endonucleases are used to map genes as
they produce a unique set of fragments for a gene
• There are more than 200 restriction endonucleases in
use, and each recognizes a specific sequence of DNA
bases
• EcoR1 cuts double-stranded DNA at the sequence
5'-GAATTC-3' wherever it occurs
28
Figure 4.19: The restriction enzyme EcoRI cleaves double-stranded DNA
wherever the sequence 5-GAATTC-3 is present
29
RFLPs
• Differences in DNA sequence generate different DNA
cleavage sites for specific restriction enzymes
• Two different alleles will produce different fragment
patterns when cut with the same restriction enzyme
due to differences in DNA sequence
Figure 4.20: A minor difference in the DNA sequence of two molecules can be
detected if the difference eliminates a restriction site
30
SSRs
• A third type of DNA polymorphism results from
differences in the number of copies of a short DNA
sequence that may be repeated many times in tandem
at a particular site in a chromosome
• When a DNA molecule is cleaved with a restriction
endonuclease that cleaves at sites flanking the
tandem repeat, the size of the DNA fragment
produced is determined by the number of repeats
present in the molecule
• There is an average of one SSR per 2 kb of human
DNA
31
Figure 4.21B: A type of genetic variation that is widespread in most natural
populations of animals and plants
32
Mapping Genes in Human Pedigrees
• Human pedigrees can be analyzed for the inheritance
pattern of different alleles of a gene based on
differences in SSRs and SNPs
• Restriction enzyme cleavage of polymorphic alleles
that are different in RFLP pattern produces different
size fragments by gel electrophoresis
33
Figure 4.22: Human pedigree showing segregation of SSR alleles
34
Copy-number polymorphisms (CNPs)
• A substantial portion of the human genome can
be duplicated or deleted in much larger but still
submicroscopic chunks ranging from 1 kb to 1
Mb.
• This type of variation is known as copy-number
polymorphism (CNP).
• The extra or missing copies of the genome in
CNPs can be detected by means of hybridization
with oligonucleotides in DNA microarrays.
35
Tetrad Analysis
• In some species of fungi, each
meiotic tetrad is contained in a
sac-like structure, called an
ascus
• Each product of meiosis is an
ascospore, and all of the
ascospores formed from one
meiotic cell remain together in
the ascus
Figure 4.23: Formation of an ascus containing
all of the four products of a single meiosis
36
Tetrad Analysis
• Several features of ascus-producing organisms are
especially useful for genetic analysis:
 They are haploid, so the genotype is expressed
directly in the phenotype
 They produce very large numbers of progeny
 Their life cycles tend to be short
37
Ordered and Unordered Tetrads
• Organisms like Saccharomyces cerevisiae, produce
unordered tetrads: the meiotic products are not
arranged in any particular order in the ascus
• Unordered tetrads have no relation to the geometry
of meiosis.
• Bread molds of the genus Neurospora have the
meiotic products arranged in a definite order
directly related to the planes of the meiotic
divisions—ordered tetrads
• The geometry of meiosis is revealed in ordered
tetrads
38
Tetrad Analysis:
Unordered Tetrads
• In tetrads when two pairs
of alleles are segregating,
three patterns of
segregation are possible
• Parental ditype (PD) = two
parental genotypes
• Nonparental ditype (NPD)
= only recombinant
combinations
• Tetratype (TT) = all four
genotypes observed
Figure 4.25: Types of unordered asci produced
with two genes in different chromosomes
39
Tetrad Analysis: Unordered Tetrads
• The existence of TT for linked genes
demonstrates two important features of
crossing-over:
– The exchange of segments between parental
chromatids takes place in prophase I, after the
chromosomes have duplicated
– The exchange process consists of the breaking
and rejoining of the two chromatids, resulting in
the reciprocal exchange of equal and
corresponding segments
40
Tetrad Analysis
• When genes are unlinked, the parental ditype
tetrads and the nonparental ditype tetrads are
expected in equal frequencies: PD = NPD
• Linkage is indicated when nonparental ditype
tetrads appear with a much lower frequency than
parental ditype tetrads: PD » NPD
• Map distance between two genes that are
sufficiently close that double and higher levels of
crossing-over can be neglected, equals
1/2 x (number TT / total number of tetrads) x 100
41
Tetrad Analysis: Ordered Tetrads
• Ordered asci also can be classified as PD, NPD, or
TT with respect to two pairs of alleles, which
makes it possible to assess the degree of linkage
between the genes
• The fact that the arrangement of meiotic products
is ordered also makes it possible to determine
the recombination frequency between any
particular gene and its centromere
42
Figure 4.27: The life cycle of Neurospora crassa
43
Tetrad Analysis: Ordered Tetrads
• Homologous centromeres of parental chromosomes
separate at the first meiotic division
• The centromeres of sister chromatids separate at the
second meiotic division
• When there is no crossover between the gene and
centromere, the alleles segregate in meiosis I
• A crossover between the gene and the centromere
delays segregation alleles until meiosis II
44
Tetrad Analysis: Ordered Tetrads
• The map distance between the gene and
its centromere equals:
1/2 x (number of asci with second division
segregation/total number of asci) x 100
• This formula is valid when the gene is
close enough to the centromere and there
are no multiple crossovers
45
Figure 4.28 (top): First- and second-division segregation in Neurospora
46
Figure 4.28 (bottom): First- and second-division segregation in Neurospora
47
Gene Conversion
• Most asci from heterozygous Aa diploids demonstrate
normal Mendelian segregation and contain ratios of
2A : 2a in four-spored asci, or 4A : 4a in eight-spored
asci.
• Occasionally, aberrant ratios are also found, such as
3A : 1a or 1A : 3a and 5A : 3a or 3A : 5a.
• The aberrant asci are said to result from gene
conversion because it appears as if one allele has
“converted” the other allele into a form like itself
48
Gene Conversion
• Gene conversion is frequently accompanied by
recombination between genetic markers on either
side of the conversion event, even when the flanking
markers are tightly linked
• Gene conversion results from a normal DNA repair
process in the cell known as mismatch repair
• Gene conversion suggests a molecular mechanism
of recombination
49
Figure 4.30: Mismatch repair resulting in gene conversion
50
Recombination
• Recombination is initiated by a double-stranded
break in DNA
• The size of the gap is increased by nuclease
digestion of the broken ends
• These gaps are repaired using the unbroken
homologous DNA molecule as a template
• The repair process can result in crossovers that
yield chiasmata between nonsister chromatids
51
Figure 4.31: Double-strand break in a duplex DNA molecule
Adapted from D. K. Bishop and D. Zickler, Cell 117 (2004): 9-15
52
Recombination: Holliday Model
• The nicked strands unwind, switch partners,
forming a short heteroduplex region with one
strand and a looped-out region of the other
strand called a D loop
• The juxtaposed free ends are joined together,
further unwinding and exchange of pairing
partners increase the length of heteroduplex
region—process of branch migration
53
Recombination: Holliday Model
• One of two ways to resolve the resulting structure,
known as a Holliday junction, leads to recombination,
the other does not
• The breakage and rejoining is an enzymatic function
carried out by an enzyme called the Holliday
junction-resolving enzyme
54
Figure 4.32: EM of a Holliday structure
Illustration modified from B. Alberts. Essential Cell Biology. Garland
Science, 1997. Illustration reproduced with permission of Huntington
Potter, Johnnie B. Byrd Sr., Alzheimer’s Center & Research Institute
55