Spore Genotype Phenotype
A GEN I wild-type
B GEN1 wild-type
C genl-62 mutant
Since genl-62 is a single alteration in the GEN1 gene, the only possible tetrad that can result from this cross is one containing two GEN1 spores and two genl-62 spores. If a thousand tetrads were dissected all would be 2 wild-type: 2 mutants because only a single mutant gene is segregating in this cross. Thus, the 2:2 segregation pattern is consistent with the fact that a single genetic difference exists between the parent strains in the cross.
But what if one does not know whether a mutant strain contains a single mutant alteration? Perhaps two or more mutations in different genes are needed to produce the mutant phenotype observed. Perhaps the mutant strain has a complex phenotype and exhibits several abnormalities. Are all these abnormal phenotypes associated with a single gene mutation or are there several mutations in the strain each causing a specific phenotype? These questions can be answered by crossing the mutant strain to a wild-type strain (one that has not been exposed to mutagenesis and selection). If tetrad analysis of many tetrads derived from this heterozygous diploid gives only two mutant spores and two wild-type spores, then this is strong evidence that one is working with a mutant strain carrying a mutation in a single gene. If all the mutant phenotypes are exhibited by all the mutant spores, then one can conclude that the single gene mutation has several phenotypic effects, i.e. it is pleiotropic. If one finds some other segregation pattern, such as one mutant spore to three wild-type spores, or if the phenotypes segregate from one another, then one must consider the possibility that the mutant strain contains mutations in two or more genes (see Cross 3 below). There are other interpretations of a 1:3 ratio, such as a high rate of gene conversion or aneuploidy, but these are less likely particularly if the mutations were induced by a mutagen and are not spontaneous.
The 'single gene cross' has other uses. If one crosses two mutant strains believed to contain different mutant alleles of the same gene, such as a deletion and a single base change, then these alleles should always segregate to different spores. All of the haploid spores resulting from the heterozygous diploid should contain either one or the other mutant allele. Given this segregation pattern, only tetrads containing four mutant and no wild-type will result, except for rare recombinants between the alleles if recombination is possible. Thus, a 4:0 result in all tetrads is strong evidence that the mutations are in the same gene, or extremely closely linked genes. This is shown in Cross 2 below.
Cross 2: genl-33 x genl-62 (genotypes of parental strains) (mutant) (mutant) (phenotype of parental strains)
(genotype of diploid)
(phenotype of diploid)
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