Why three-point test cross?

 As the distance between genes increases, the possibility of double cross-over increases. When the two loci are farther apart on a chromosome, double crossovers occur between them and will tend to mask the recombinants. In a two-point test cross, some parental combinations result from double crossovers. These crossovers could not be identified as they do not produce recombinant gamete and progenies, all progenies will be of a parental type and the recombination frequency will become 0%. Hence it underestimates the actual distance between the genes.

This problem can be solved by including another marker (gene) between distantly located genes so that the recombinants can be identified. Hence three-point test cross is preferred for chromosome mapping. An important feature of all linkage maps is their linearity i.e., all genes in a given linkage group can be shown to map in a linear array. Let us presume that there are three genes A, B, and C present on the same chromosome (i.e., they are linked). There could be three possible linear orders in which these genes may be present on a chromosome. These are A-B-C, A-C-B, or BA- C. In one case, B is in the middle, and in the other two, C and A respectively are in the middle. Therefore, in finding out the linear order, the gene present in the center should be found out.

For this purpose, a three-point test- the cross is made, which involves crossing of a tri-hybrid ABC/ABC (obtained from across ABC/ABC X abc/abc) with triple homozygous recessive abc/abc. The progeny obtained will represent the gametes formed by the hybrid and is hypothetically considered in the following table.

Higher recombination frequency is observed between the genes placed farther apart in the chromosome. Farther the genes indicate the distance between them is more. Similarly, the lower the recombination frequency, the shorter is the distance. From the above example, the recombination frequency between A and C is the highest. So, B is present in between at 19.92 cM from A and 21.75 cM from C.