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1. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Chapter 6
Genetic Recombination in
Eukaryotes
Linkage and genetic diversity

2. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Overview
• In meiosis, recombinant products with new
combinations of parental alleles are generated by:
– independent assortment (segregation) of alleles on
nonhomologous chromosomes.
– crossing-over in premeiotic S between nonsister
homologs.
• In dihybrid meiosis, 50% recombinants indicates
either that genes are on different chromosomes or
that they are far apart on the same chromosome.
• Recombination frequencies can be used to map gene
loci to relative positions; such maps are linear.
• Crossing-over involves formation of DNA
heteroduplex.

3. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Recombination (1)
• A fundamental consequence of meiosis
– independent assortment (independent
segregation)
– crossing-over between homologous chromatids
• Yields haploid products with genotypes
different from both of the haploid genotypes
that originally formed the diploid meiocyte
N
N
2N
N
N
N
N
different genotypes
parentals recombinants
meiosis

4. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Recombination (2)
• Bringing together of two or more pairs of
alleles into new combinations
A/a
B/b
a/a
b/b
A/A
B/B
A
B
a
b
A
B
a
b
A
b
a
B
parental (P) genotypes recombinant (R) genotypes
parental genotypes
meiosis meiosis
meiosis

5. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Independent assortment (1)
• Also known as independent segregation
• Consequence of independent alignment of
chromosomes in meiotic bivalents
A/A ; B/B  a/a ; b/b
A/a ; B/b
¼ A ; B P
¼ A ; b R
¼ a ; B R
¼ a ; b P
OR
Alternate bivalants
A
B
b B
a a
A
b

6. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Independent assortment (2)
• For genes on different (nonhomologous) pairs of
chromosomes, recombinant frequency is always 50%
A/A ; B/B  a/a ; b/b
A/a ; B/b
¼ A ; B P
¼ A ; b R
¼ a ; B R
¼ a ; b P
A/A ; b/b  a/a ; B/B
A/a ; B/b
¼ A ; B R
¼ A ; b P
¼ a ; B P
¼ a ; b R
50%
recombinants

7. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Dihybrid testcross (1)
• Determines genotype of dihybrid by
crossing to homozygous recessive tester
A/A ; b/b  a/a ; B/B
A/a ; B/b  a/a ; b/b testcross
Parental
F1
F1 gametes
tester gametes
a ; b
progeny
proportions
progeny
phenotypes
¼ A ; B A/a ; B/b ¼ A B
¼ A ; b A/a ; b/b ¼ A b
¼ a ; B a/a ; B/b ¼ a B
¼ a ; b a/a ; b/b ¼ a b
1:1:1:1
ratio

8. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Dihybrid testcross (2)
• Best way to study recombination is in a
dihybrid testcross
– only dihybrid produces recombinant genotypes
– all homozygous recessive tester gametes alike
• Typical 1:1:1:1 ratio a result of independent
assortment in dihybrid
• Each genotype in progeny has unique
phenotype
• Observed by Mendel in testcrosses with two
pairs of traits

9. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Dihybrid selfing
• Cross between two A/a ; B/b dihybrids
– recombination occurs in both members of cross
– recombination frequency is 50%
A ; B A ; b a ; B a ; b
A ; B A/A ; B/B A/A ; B/b A/a ; B/B A/a ; B/b
A ; b A/A ; B/b A/A ; b/b A/a ; B/b A/a ; b/b
a ; B A/a ; B/B A/a ; B/b a/a ; B/B a/a ; B/b
a ; b A/a ; B/b A/a ; b/b a/a ; B/b a/a ; b/b
Ratio: 9 A/– ; B/– 3 A/– ; b/b 3 a/a ; B/– 1 a/a ; b/b

10. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Product rule
• Multiply probabilities of independent occurrences
to obtain probability of joint occurrence
• E.g. branched tree or grid methods
• For mating A/a ; B/b  A/a ; B/b
– Segregation at A, gives ¾ A/– and ¼ a/a in progeny
– Segregation at B, gives ¾ B/– and ¼ b/b in progeny
¾ A/– ¼ a/a
¾ B/– 9/16 A/– ; B/– 3/16 a/a ; B/–
¼ b/b 3/16 A/– ; b/b 1/16 a/a ; b/b

11. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Independent assortment: multiple loci
• Calculations can be made for any gene
combination using predicted outcomes at
single loci and the product rule
P1 A/a ; B/b ; C/c ; D/d  P2 a/a ; B/b ; C/c ; D/D
# gametes P1 2 x 2 x 2 x 2 = 16
# gametes P2 1 x 2 x 2 x 1 = 4
# genotypes in F1 2 x 3 x 3 x 2 = 36
# phenotypes in F1 2 x 2 x 2 x 1 = 8
Frequency of
A/– ; B/– ; C/– ; D/–
½ x ¾ x ¾ x 1 = 9/32

12. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Deducing genotypes from ratios
• Genetic analysis works in two directions
– predict genotypes in offspring
– determine genotypes of parents in cross
• Specific expectations, e.g., 1:1:1:1 and 9:3:3:1
can be used to deduce genotypes
• Testcross example:
Phenotype # observed
A/– ; B/– 310
A/– ; b/b 295
a/a ; B/– 305
a/a ; b/b 290
The observed results are
close to 1:1:1:1, allowing
the deduction that the
tested genotype was a
dihybrid.

13. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Crossing-over (CO)
• Breakage and rejoining of homologous
DNA double helices
• Occurs only between nonsister chromatids
at the same precise place
• Visible in diplotene as chiasmata
• Occurs between linked loci on same
chromosome
– cis: recessive alleles on same homolog (AB/ab)
– trans: recessive alleles on different homologs
(Ab/aB)

14. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Cis – trans crossing-over
• Drawing shows only chromatids engaged in crossing-over
• Effect is to switch between cis and trans
A
b
a
B a
b
A
B
cis trans
A
B
a
b a
B
A
b
meiotic crossing-over
AB/ab  aB/Ab Ab/aB  AB/ab

15. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Cis dihybrid crossing-over
• Parental (P) and recombinant (R) classes each have both
alleles at each locus (reciprocal)
• Each crossover meiosis yields two P chromosomes and
two R chromosomes
• Because CO does not occur in each meiocyte, frequency of
recombinants (R) must be <50%
A
b
a
B
A
b
a
B
P
R
R
P
A b
a B

16. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Crossing-over
• No loss of genetic material, just formation
of new chromatids
• Parental chromatids are noncrossover
products
• Recombinant chromatids are always
products of crossing-over
• All four genes (A, B, a and b) are present
between both parental chromatids and
between both recombinant chromatids

17. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Consequences of crossing-over
• Frequency of recombinant gametes is 0-
50%, depending on frequency of meiocytes
with crossing-over
• Results in deviation from 1:1:1:1 in
testcrosses
– parental combination is most frequent
– recombinant combination is rarest
• Allows drawing of linkage maps based on
recombination frequencies (RF)

18. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Recombination frequency (RF)
• Experimentally determined from frequency
of recombinant phenotypes in testcrosses
• Roughly proportional to physical length of
DNA between loci
• Greater physical distance between two loci,
greater chance of recombination by
crossing-over
• 1% recombinants = 1 map unit (m.u.)
• 1 m.u. = 1 centiMorgan (cM)

19. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Linkage maps
• RF is (60+50)/400=27.5%, clearly less than 50%
• Map is given by:
# observed
140
50
60
150
A B
27.5 m.u.

20. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Mapping
• RF analysis determines relative gene order
• RF between same two loci may be different
in different strains or sexes
• RF values are roughly additive up to 50%
– multiple crossovers essentially uncouple loci,
mimicking independent assortment
• Maps based on RF can be combined with
molecular and cytological analyses to
provide more precise locations of genes

21. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Trihybrid testcross
• Sometimes called three-point testcross
• Determines gene order as well as relative
gene distances
• 8 categories of offspring
– for linked genes, significant departure from
1:1:1:1:1:1:1:1
• Works best with large numbers of offspring,
as in fungi, Drosophila

22. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Analysis of trihybrid testcross data
• Identify pairs of parental and recombinant offspring
– parental (noncrossover); most abundant
– double crossovers; least abundant
– single crossovers; intermediate abundance
• identify on the basis of reciprocal combinations of alleles
• Determine gene order by inspection (the parental
gene order yields double crossovers by switching
middle genes)
• Calculate RF for single crossovers, adding double
crossovers each time
• Draw map

23. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Interference
• Crossing-over in one region of chromosome
sometimes influences crossing-over in an
adjacent region
• Interference = 1 – (coefficient of coincidence)
• Usually, I varies from 0 to 1, but sometimes it
is negative, meaning double crossing-over is
enhanced
ts
recombinan
double
expected
#
ts
recombinan
double
observed
#
c.o.c. 

24. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Genetic maps
• Useful in understanding and experimenting
with the genome of organisms
• Available for many organisms in the
literature and at Web sites
• Maps based on RF are supplemented with
maps based on molecular markers,
segments of chromosomes with different
nucleotide sequences

25. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Chi-square test
• Statistical analysis of goodness of fit
between observed data and expected
outcome (null hypothesis)
• Calculates the probability of chance
deviations from expectation if hypothesis is
true
• 5% cutoff for rejecting hypothesis
– may therefore reject true hypothesis
– statistical tests never provide certainty, merely
probability

26. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Chi-square application to linkage
• Null hypothesis for linkage analysis
– based on independent assortment, i.e., no
linkage
– no precise prediction for linked genes in
absence of map
• for all classes
• Calculated from actual observed (O) and
expected (E) numbers, not percentages



E
E
O 2
2 )
(


27. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Mechanism of meiotic crossing-over
• Exact mechanism with no gain or loss of
genetic material
• Current model: heteroduplex DNA
– hybrid DNA molecule of single strand from
each of two nonsister chromatids
– heteroduplex resolved by DNA repair
mechanisms
• May result in aberrant ratios in systems that
allow their detection

28. Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company
Recombination within a gene
• Recombination between alleles at a single
locus
• In diploid heterozygous for mutant alleles of
the same gene, recombination can generate
wild-type and double mutant alleles
• Rare event, 10-3 to 10-6, but in systems with
large number of offspring, recombination can
be used to map mutations within a gene
a1/a2  a+ and a1,2