Chapter6_GeneticRecombinationEukaryotes.ppt

Genetic Recombination in Eukaryotes

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
meiocyteN
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) genotypesrecombinant (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
Bb B
a aA
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/btestcross
Parental
F
1
F
1 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 ; BA/A ; B/BA/A ; B/bA/a ; B/BA/a ; B/b
A ; bA/A ; B/bA/A ; b/bA/a ; B/bA/a ; b/b
a ; BA/a ; B/BA/a ; B/ba/a ; B/Ba/a ; B/b
a ; bA/a ; B/bA/a ; b/ba/a ; B/ba/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/b3/16 A/– ; b/b1/16 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
P
1 A/a ; B/b ; C/c ; D/d  P
2 a/a ; B/b ; C/c ; D/D
# gametes P
1
2 x 2 x 2 x 2 = 16
# gametes P
2
1 x 2 x 2 x 1 = 4
# genotypes in F
12 x 3 x 3 x 2 = 36
# phenotypes in F
1
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
ba
B a
bA
B
cis
trans
A
Ba
b
a
BA
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
ba
B
A
ba
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
tsrecombinan double expected #
tsrecombinan 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
EO
2
2 )(
χ

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
a
1
/a
2
 a
+
and a
1,2