Genetics Chapter 7 Part 3

3. Determining the Gene Sequence during Mapping Requires the Analysis of Multiple Crossovers

Study of single crossovers between 2 linked genes

o Provides basis of determining distance between the genes
o When linked genes are studied, chromosome sequence is difficult to determine
Multiple exchanges occur between chromatids of a tetrad
o Facilitates process of producing extensive chromosome maps
3 or more linked genes are investigated simultaneously
o Determines the following:
1. Sequence of genes
2. Distances between the genes

Multiple Crossovers
-

In a single tetrad
o Two, or more exchanges may occur between nonsister chromatids
o Exchanges are a result of crossover events
Double Crossovers (DCOs)
o Double exchanges of genetic material
o For this to be possible there is a need of 3 gene pairs
Each heterozygous, for 2 alleles
o 2 separate and independent events or exchanges must occur simultaneously
Probability of a single exchange between A and B or B and C genes relate directly to distance between the
loci (will make sense in the figure that will be shown below)
o The closer the distance, the less likely the single exchange will occur between either of the 2 sets of
loci
Product Law
o Probability of 2 independent events occurring simultaneously is equal to the product of the
individual probabilities

Frequency of Double crossover gametes is ALWAYS LOWER than the single crossover class of gametes
o Given:
Crossover gametes resulting from single exchanges are recovered 20% of the time between
A and B (p = 0.20)
Crossover gametes recovered between B and C is 30% (p = 0.30)

What youre solving for:

The probability of recovering a double crossover gamete arising from 2 exchanges
(Between A and B and between B and C)
o Solution using Product Law
(0.20)(0.3) = 0. 06
o Statement
Frequency of double crossover gametes is 6%, it is lower than that of the single-crossover
class of gametes
Scenario:
o 3 genes are close together along 1 chromosome it is expected that the frequency of double
crossover gametes is extremely low
o Example:
Given
A B distance is 3 mu
B C distance is 2 mu
Solution
(0.03)(0.02) = 0.0006 or 0.06%
Statement
6 events in 10,000
What does this mean if there are only 6 double frequency events in 10,000?
o In a mapping experiment with closely linked genes:
Large number of offsprings are required to detect double crossover
events
it is unlikely that a double crossover will be observed even if 1000
offspring are examined
If 4 or 5 genes are mapped, even fewer triple and quadruple
crossovers are expected to occur
o

Three Point Mapping in Drosophila

-

Mapping 3 or more linked genes in a single cross

3 criteria for a successful mapping cross
1. Genotype of organism producing crossover gametes must be heterozygous at all loci
2. Cross constructed should determine all genotypes by observing phenotypes of resulting offspring
Problem: Not all genotypes can be observed directly
Solution: Each phenotypic class must reflect genotype of the gametes of the parents producing it
3. Sufficient number of offspring must be produced in mapping experiment to recover representative
sample of all crossover classes

3, X linked recessive mutant genes were used in the example in figure 8 namely:
1. Yellow body color (y)
2. White eye color (w)
3. Echinus eye shape (ec)
In diagramming the cross the 1st step is:
-

Assume theoretical sequence even though we do not know if it is correct

Seen in figure 8, we initially assume the sequence of the genes to be y w ec
If assumption is incorrect
o Analysis will demonstrate and reveal correct sequence

In P1 generation example:
-

Hemizygous males are crossed to all homozygous females for all recessive mutant alleles
Hemizygous, P1 males are wild type with all 3 traits
o Said to have a wild-type phenotype
Females, exhibit mutant traits
o Yellow body color, white eyes, and echinus eye shape

In the F1 generation example: (Fulfillment of 1st criterion)

-

Consists of females that are heterozygous in all 3 loci, making them wild type
Males that are hemizygous to all 3 mutant alleles (because of the Y chromosome)
Genotype of F1 females fulfills first criterion for mapping:
o Heterozygous at 3 loci
o Can serve as a source of recombinant gametes generated by crossing over
NOTE: Other arrangements are possible that could produce a heterozygous genotype
o Heterozygous female could have y and ec mutant alleles on 1 homolog and w allele on the other
o This could occur if in P1 cruss, one parent was yellow, echinus and the other parent was white

Fulfillment of 2nd Criterion

-

2nd criterion is met by the gametes formed by the F1 males

o Every gamete of the F1 male contains an X chromosome with 3 mutant alleles or a Y chromosome
that is inert for the 3 loci
o Whichever type participates in fertilization, genotype of gamete by F1 female will be expressed
phenotypically in F2 male and female
o All F1 noncrossover and crossover gametes can be detected by observing the F2 phenotypes

Chromosome map can now be constructed from the crosses:

1. Determine which F2 phenotypes correspond to noncrossover and crossover categories
a. To determine noncrossover F2
i. Identify individuals derived from parental gametes formed by F1 female
ii. Each gamete contains X chromosome unaffected by crossing over
iii. Laws of segregation dictates an approximate equal proportion of 2 types of gametes;
producing F2 phenotypes
iv. Because they derive from a heterozygote
Genotypes of the 2 parental gametes and the resultant F2 phenotypes complement
one another
Example: one is wild type; other is completely mutant
Reciprocal classes of gametes and phenotypes
One chromosome shows 1 mutant allele while another shows the other 2
mutant alleles

2 noncrossover phenotypes easily recognized because they exist in greatest proportions

-

In figure 8, gametes 1 and 2 present in greatest numbers

Flies that express yellow, white and echinus phenotypes and flies that are normal for all 3 characteristics
constitute the noncrossover category (94.44%) of F2

Double Crossover Phenotypes

-

Low probability in occurrence

They must be present in the least numbers
This group represents 2independent but simultaneous single crossover events
Reciprocal phenotypes present in gamete 7
o Shows mutant traits yellow echinus but normal eye
Reciprocal to gamete 8
o Mutant white but normal body and eye
Constitute only 0.06% of offspring for F2

Remaining 4 phenotypic classes represent 2 categories from Single Crossovers

-

Gametes 3 and 4 reciprocal phenotypes produced by single crossover events

o Occurs between yellow and white loci
o Equal to 1.50% of F2 offspring
Gametes 5 and 6
o 4% of F2 offspring
o Represent reciprocal phenotypes resulting from single crossover events between white and
echinus loci

Map distances separating 3 loci can be calculated

-

Distance between y and w or between w and ec is equal to the percentage of all detectable exchanges
between them
o Includes all single crossovers and double crossovers
Double crossovers are included because they represent simultaneous single crossovers
Gametes 3, 4, 7 and 8 for the y and w genes total 1.50% + 0.06% or 1.56 mu
Distance between w and ec is equal to the percentage of offspring from exchange between the 2 loci
o gametes 5, 6, 7, 8 totaling 4.00 % + 0.06% = 4.06 mu
map of 3 loci shown at the bottom of the figure

DETERMINING THE GENE SEQUENCE

-

in most mapping experiments, the gene sequence is not known

o this constitutes another variable in the analysis
o more straightforward method:
this method is based on the fact that there are only 3 possible arrangements, each
containing one of the 3 genes between the other two
1. w y ec (y in the middle)
2. y ec w (ec in the middle)

3. y w ec (w in the middle)
-

Steps during analysis to determine gene order

1. Assume any of the 3 orders above
a. First determine arrangement of alleles along each homolog of heterozygous parent, giving rise
to noncrossover and crossover gametes
b. Example, F1 female
2. Determine whether a double crossover event occurring within the arrangement will produce
a. Observable double cross-over phenotypes
i. These phenotypes occur least frequently and easily identified
3. If the order does not produce the predicted phenotypes, then try again until u get it hahaha (trial and
error daw seryoso)

Analysis of Figure 9
1. Assuming that y is between w and ec, arrangement I of alleles in F1 heterozygote is:
w y ec
w+ y+ec+
a. We know this because of how the P1 generation was crossed
b. P1 female contributes an X chromosome with w, y and ec alleles
c. P1 male contributes X chromosome bearing w+, y+ , ec+ alleles
2. A double crossover within arrangement yields the following:
w y+ ec
and
w+ y ec+
a. Following fertilization, if y is in the middle, F2 double crossover phenotypes correspond to
gametic genotypes yielding:
i. offspring that express white, echinus phenotype
ii. offspring that expresses yellow phenotype
b. determination of actual double crossover phenotypes reveal them to be yellow, echinus flies and
white flies
i. therefore, our assumed order is incorrect
ii. awtsu beh

3. if we consider arrangement II with the ec/ ec+ alleles in the middle or arrangement III with the w/w+ alleles
in the middle
(II)

y ec w
y+ ec+ w+

or

(III)

y w ec
y+ w+ ec+

a. arrangement II provides predicted double crossover phenotypes

i. do not correspond to actual (observed) double crossover phenotypes
ii. predicted phenotypes are yellow, white flies and echinus flies in the F2 generation
iii. this order is also incorrect
b. arrangement III produces observed phenotypes
i. observed phenotyeps yellow, echinus flies and white flies
ii. this arrangement, with the w gene in the middle is correct
SUMMARY:
-

first determine arrangement of alleles on homologues of heterozygote yielding the crossover gametes
o determine by locating the reciprocal noncrossover phenotypes
test each of 3 possible orders to determine which yields the observed double crossover phenotypes
o the one that represents the correct order