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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
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
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)
Figure 4.1 Gametes produced through meiosis
Figure 4.2 The frequency of recombination between two mutant alleles
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.
Figure 4.3 The frequency of recombination between two genes depends on
the genes
Genetic Map
Figure 04.05: The frequency of recombination is used to construct a genetic map.
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.
Genetic Mapping
• Recombination changes the allelic arrangement on
homologous chromosomes
Figure 04.04: Crossing-over between two genes.
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
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
Figure 04.06: Diagram of chromosomal configurations in 50 meiotic cells.
Figure 4.7 Crossing-over outside the region between two genes is not
detectable through recombination
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
Gene Mapping: Double Crossing Over
• Two exchanges taking place between genes, and
both involving the same pair of chromatids, result
in nonrecombinant chromosomes
Figure 04.08: Crossovers between marker genes A and B.
Figure 4.9 Possible genetic maps
Figure 4.10 Genetic map of chromosome 10 of corn, Zea mays
Adapted from an illustration by E.H. Coe.
Figure 04.12: The result of two crossovers in the interval between two genes 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
Figure 04.11: Chromosome 2 in Drosophila as it appears in metaphase of mitosis
and in the genetic map.
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
Table 04.01 Interpreting in a Three-Point Cross.
Figure 4.14 The order of genes in a three-point testcross
Figure 4.15 Result of single crossovers in a triple heterozygote
Figure 4.16 Result of double crossovers in a triple heterozygote
Table 4.2 Comparing Reciprocal Products in a Three-Point Cross
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 04.17: Mapping functions.
Genetic Mapping
• Coefficient of coincidence = observed number of
double recombinants divided by the expected
number
• Interference = 1-coefficient of coincidence
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.
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.
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)
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.
Figure 04.18: SNP genotype of an individual.
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
Figure 04.19: The restriction enzyme EcoRI cleaves double-stranded DNA
wherever the sequence 5-GAATTC-3 is present.
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 04.20: A minor difference in the DNA sequence of two molecules can be
detected if the difference eliminates a restriction site.
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
Figure 04.21B: A type of genetic variation that is widespread in most natural
populations of animals and plants.
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
Figure 04.22: Human pedigree showing segregation of SSR alleles.
Copy-number variations (CNVs)
• 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
variation (CNV).
• The extra or missing copies of the genome in
CNVs can be detected by means of hybridization
with oligonucleotides in DNA microarrays.
Tetrad Analysis
• In some species of fungi, each
meiotic tetrad is contained in a saclike 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 04.24: Formation of an
ascus containing all of the four
products of a single meiosis.
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
Figure 4.24 Life cycle of the yeast Saccharomyces cerevisiae
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
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 04.26: Types of
unordered asci produced
with two genes in different
chromosomes.
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
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
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
Figure 04.28: The life cycle of Neurospora crassa.
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
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
Figure 04.29 (top): First- and second-division segregation in Neurospora.
Figure 04.28 (bottom): First- and second-division segregation in Neurospora.
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
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
Figure 4.29 Mismatch repair
Figure 04.31: Mismatch repair resulting in gene conversion.
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
Figure 04.32: Double-strand break in a duplex DNA molecule.
Adapted from D. K. Bishop and D. Zickler,
Cell 117 (2004): 9-15
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
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
Figure 04.33: 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