Three point test cross in Drosophila:

• Wild-type Male Drosophila was crossed with female Drosophila homozygous for three recessive X-linked mutations—scute (sc) bristles, echinus (ec) eyes, and crossveinless (cv) wings to obtain F1 progeny.
• Wild Male Drosophila= (sc+, ec+, cv+)
• Mutated female Drosophila = (sc, ec, cv)
• Then F1 progeny were intercrossed to produce F2 flies, which are then classified and counted.
• The F1 males carried the three recessive mutations on their single X chromosome. Thus, this intercross was equivalent to a testcross with all three genes in the F1 females present in the homozygous form.
• The F2 progeny flies from the intercross comprised eight phenotypically distinct classes, two of them are parental and six recombinant.

Gene order:

• The parental classes were by far the most numerous (1158+1455=2613). The less numerous recombinant classes each represented a different kind of crossover chromosome.
• To figure out which crossovers were involved in producing each type of recombinant, we must first determine how the genes are ordered on the chromosome.
• There are three possible gene orders :

1. sc—ec—cv
2. ec—sc—cv
3. ec—cv—sc

• Four of the recombinant must have come from a single crossover in one of the two regions of the genes. The other two recombinant must have come from double crossing over—one exchange in each of the two regions. Because a double crossover switches the gene in the middle with respect to the genetic markers on either side of it, it is used for determining the gene order.
• Again, intuitively, double crossover occur much less frequently than a single crossover. Therefore, among the six recombinant classes, the two rare ones must represent the double crossover chromosomes
• From the given example, the double crossover must have occurred in class 7 (sc ec+cv) and class 8 (sc+ec cv+), each containing a single recombinant F2 progeny.
• Comparing these rare recombinant to parental class 1 (sc ec cv) and class 2 (sc+ec+Cv+), the echinus allele has been switched with respect to scute and crossveinless.
• Consequently, the echinus gene must be located between the other two.
• Therefore the correct gene order is sc–ec–cv.

Map distance:

• It is the distance between each pair of gene and it is obtained by estimating the average number of crossovers.
• Total map distance between these three genes is map distance between sc and ec plus map distance between ec and

i. Map distance between sc and ec:

• We can obtain the length of the region between sc and ec by identifying the recombinant classes that involved a crossover between these genes.
• There are four such classes: class 3 (sc ec+cv+), class 4 (sc+ec cv), class 7 (sc ec+cv), and class 8 (sc+ec cv+).
• Classes 3 and 4 involved a single crossover between sc and ec, and classes 7 and 8 involved two crossovers, one between sc and ec and the other between ec and
• We can therefore use the frequencies of these four classes to estimate the average number of crossovers between sc and ec:
• Average crossover between sc and ec =(163+130+1+1) /3248

=0.091 Morgan

=9.1 centiMorgan or Map unit

• Thus, in every 100 chromosomes coming from meiosis in the F1 females, 9.1 had a crossover between sc and ec.
• The distance between these genes is therefore 9.1 map units.

ii. Map distance between ec and cv:

• In a similar way, we can obtain the distance between ec and cv.
• Four recombinant classes involved a crossover in this region: class 5 (sc ec cv+), class 6 (sc+ec+cv), class 7 and class 8.
• The double recombinants are also included here because one of their two crossovers was between ec and cv.
• The average cross between ec and cv =(192+148+1+1)/3248

=0.105 morgan

= 10.5 centiMorgans or map unit

Total map distance:

• Combining the data for the two regions, the map is sc—9.1— ec—10.5— cv
• Thus map distances between sc and cv= 9.1 cM +10.5 cM =19.6 cM

Alternative way of calculating map distance:

• Directly calculating the average number of crossovers between these genes:
• Recombination frequency (RF)= Non–crossover + Single crossover + Double crossover

= (0)*(1158+1455)/3248 + 1 (163+130+192+148)/3248  + 2 (1+1)/3248

= 0 + 0.195 + 0.0006

= 0.196 Morgan

= 19.6 CentiMorgan

Inference and coefficient of coincidence:

• Inference is the phenomenon of inhibition of crossover of by another crossover nearby.
• For example, the crossover frequency between sc and ec in region I was (163 +130 +1+1)/3248 =0.091, and crossover frequency between ec and cv in region II was (192+148 +1 +1)/3248 =0.105.
• If we assume both crossover are independence of each other, the expected frequency of double crossovers in the interval between sc and cv would be 0.091 *0.105 = 0.0095.
• But actual observed frequency of double crossover is (1+1)/3248 = 0006
• Double crossovers between sc and cv were much less frequent than expected.
• The result suggest one crossover inhibited the occurrence of another nearby, a phenomenon called interference
• The extent of the interference is measured by the coefficient of coincidence (C).
• Coefficient of coincidence is the ratio of observed frequency to double cross to expected frequency to double cross.
• C= (observed frequency of double crossovers)/(expected frequency of double crossovers)

=0.0006/0.0095

C=0.063

Level of inference (1-C):

• Level of inference = 1-C
• =1-0.063
• =0.937
• Because in this example the coefficient of coincidence is close to zero, its lowest possible value, interference was very strong (I is close to 1).
• cases:
• if a coefficient of coincidence equal to 1; no interference between crossover at all which means the crossovers occurred independently of each other.
• If a coefficient of coincidence is equal to 0; very strong inference between crossover therefore double cross do not occur.
• ** map distance less than 20cM has very strong inference. Thus, double crossovers seldom occur in short chromosomal regions.
• The strength of interference is therefore a function of map distance

Three point test cross: gene order, map distance, inference and coefficient of coincidence and level of inference

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