The order of the genes has been arbitrarily assigned because at this point we do not know which is the middle gene.
Additionally, the alleles in these heterozygotes are in coupling configuration (because all the wild-type dominant alleles were inherited from one parent and all the recessive mutations from the other parent), although the testcross can also be done with genes in repulsion.
In the three-point testcross, we cross the F1 heterozygotes with flies that are homozygous for all three recessive mutations. In many organisms, it makes no difference whether the heterozygous parent in the testcross is male or female (provided that the genes are autosomal) but, in Drosophila, no crossing over takes place in males. Because crossing over in the heterozygous parent is essential for determining recombination frequencies, the heterozygous flies in our testcross must be female. So we mate female F1 flies that are heterozygous for all three traits with male flies that are homozygous for all the recessive traits:
st female X
st st male
The progeny produced from this cross are listed in 4 FIGURE 7.13. For each locus, two classes of progeny are produced: progeny that are heterozygous, displaying the dominant trait, and progeny that are homozygous, displaying the recessive trait. With two classes of progeny possible for each of the three loci, there will be 23 = 8 classes of phenotypes possible in the progeny. In this example, all eight phenotypic classes are present but, in some three-point crosses, one or more of the phenotypes may be missing if the number of progeny is limited. Nevertheless, the absence of a particular class can provide important information about which combination of traits is least frequent and ultimately the order of the genes, as we will see.
To map the genes, we need information about where and how often crossing over has occurred. In the ho-mozygous recessive parent, the two alleles at each locus are the same; and so crossing over will have no effect on the types of gametes produced; with or without crossing over, all gametes from this parent have a chromosome with three recessive alleles ( st e ss ). In contrast, the heterozygous parent has different alleles on its two chromosomes; so crossing over can be detected. The information that we need for mapping, therefore, comes entirely from the gametes produced by the heterozygous parent. Because chromosomes contributed by the ho-mozygous parent carry only recessive alleles, whatever al-leles are present on the chromosome contributed by the heterozygous parent will be expressed in the progeny.
As a shortcut, we usually do not write out the complete genotypes of the testcross progeny, listing instead only the alleles expressed in the phenotype (as shown in Figure 7.13), which are the alleles inherited from the heterozygous parent.
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