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

2. 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.
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

3. 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 N
meiosis 2N
parentals
recombinants N N N
different genotypes
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

4. 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
meiosis
meiosis
parental genotypes
meiosis A B a b
parental (P) genotypes
recombinant (R) genotypes
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

5. 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 B b a
A/a ; B/b
¼ A ; B P
¼ A ; b R
¼ a ; B R
¼ a ; b P
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

6. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

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

8. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

9. 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/A ; B/B
A/A ; B/b
A/a ; B/B
A/a ; B/b
A/A ; b/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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

10. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

11. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

12. 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/–
A/– ; b/b
a/a ; B/–
a/a ; b/b
The observed results are close to 1:1:1:1, allowing the deduction that the tested genotype was a dihybrid.
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

13. 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)
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

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

15. Cis dihybrid crossing-overA b a B P R
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%
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

16. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

17. 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)
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

18. 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)
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

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

20. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

21. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

22. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

23. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

24. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

25. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

26. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

27. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company

28. 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
Chapter 6: Eukaryote recombination © 2002 by W. H. Freeman and Company