chapter 6: eukaryote recombination© 2002 by w. h. freeman and company chapter 6 genetic...

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

Chapter 6

Genetic Recombination in Eukaryotes

Linkage and genetic diversity


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.


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

N2N

N

N

N

N

different genotypes

parentals recombinantsmeiosis


Recombination (2)• Bringing together of two or more pairs of

alleles into new combinations

A/aB/b

a/ab/b

A/AB/B

AB

ab

AB

ab

Ab

aB

parental (P) genotypes recombinant (R) genotypes

parental genotypes

meiosis meiosis

meiosis


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


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


Dihybrid testcross (1)• Determines genotype of dihybrid by

crossing to homozygous recessive testerA/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


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


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


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


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


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.


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)


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

Bcis trans

A

Ba

b a

BA

b

meiotic crossing-over

AB/ab aB/Ab Ab/aB AB/ab


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

BP

R

R

P

A b

a B


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


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)


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)


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.


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


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


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


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. =


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


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


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 22 )(

χ


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


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 chapter 6 genetic...

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