Portland State University Bi 341

Chapter 4: Gene Linkage and Genetic Mapping

Kim H. Brown, PhD
 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)
 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 Map

Figure 04.05: The frequency of recombination is used to construct a genetic map.

 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
 Recombination
• Recombination between linked genes occurs at the
same frequency whether alleles are in cis or trans
configuration
A B
• Recombination frequency Cis
is specific for a particular a b
pair of genes
• Recombination frequency A b
increases with increasing Trans
a B
distances between genes
• No matter how far apart two genes may be, the
maximum frequency of recombination between any
two genes is 50 percent.
 Genetic Mapping
• Recombination changes the allelic arrangement on
homologous chromosomes

Figure 04.04: Crossing-over between two genes.

 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.
 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
 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 04.02: The frequency of recombination between two mutant alleles.
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 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
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 double-
recombinant 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.
 What is the Gene Order?
Lz Gl su
lz gl Su
 How Far Apart?
Order: Lz Su Gl

Lz su gl 40
lz Su Gl 33
Lz su Gl 4
lz Su gl 2
79/740 = 0.107 = 10.7%

Lz Su gl 59
lz su Gl 44
Lz su Gl 4
lz Su gl 2
109/740 = 0.147 = 14.7%

lz su gl

| 10.7 map units | 14.7 map units |

 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

• 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
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
 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.
What are Structural Variants (i.e., CNVs)?
 Structural Variants

 on20SNPs,
Based YRBtheand 20 CEU
difference between the genomes of two humans was thought to be 0.1%

Integrating CNVs,
 42M probes this
- 450 bp. number goes up to 0.8%
Resolution
 11700
with ~3.5%CNVof the Reference Genome Variable.
regions
How Many Copies?
Structural Variant Implications
Identified Diseases with Related CNVs
 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
 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

 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
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
 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
 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
 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
 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
• 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