If two mutant organisms have the same phenotype and the mutant sequence has not been identified, how do you know whether the two organisms have mutations in the same gene or in two different genes that both are required for the same process?
The answer can be determined by a special test cross known as a complementation test. For this test, two homozygous recessive organisms are crossed. If the mutations are in the same gene then both copies of the gene will be mutant in the F1 offspring and they will exhibit the same phenotype as their parents. In this case, the exact mutations may be different (for example, a missense in one mutant and a nonsense in the other mutant), but both alleles of a single gene are mutant and therefore the recessive phenotype will be observed. In contrast, if the two mutants have homozygous recessive mutant alleles for two different genes, gene1-/- and gene2-/- for example, then the offspring will inherit a good copy of gene1+ from the gene2-/- mutant and a wild-type copy of gene2 from the gene1-/- mutant parent.
Summary: Interpreting a complementation test
Cross Mutant 1 x Mutant 2
If the F1s all have the wild-type phenotype, the two mutants complement each other and the mutations are in two different genes.
If the F1s all have the mutant phenotype, the two mutants fail to complement each other and the mutations are likely in the same gene.