Answer in brief: The standard approach is a complementation test, which determines whether two mutations affect the same gene or different genes based on the phenotype of the offspring from crossing the mutants. Explanation and guidance

• Core idea
  • If two mutants are crossed and the offspring display the wild-type phenotype, the mutations are in different genes (they complement each other).
  • If the offspring display the mutant phenotype, the mutations are in the same gene (they fail to complement).
• How the test works
  • Use recessive mutations to be informative. Each parent contributes a mutant allele for its own mutated gene.
  • In a cross between two homozygous recessive mutants:
    • If the organisms carry mutations in different genes (gene A and gene B), the F1 will be trans-heterozygous (A+/A and B+/B) and typically show wild-type because each gene still has a functional copy from the other parent.
    • If the mutations are alleles of the same gene, all F1s will be mutant since every individual lacks functional copies of that single gene.
• Practical considerations
  • Ensure both mutations are recessive; dominant mutations require different approaches.
  • Some cases may show more severe or novel phenotypes in trans-heterozygotes due to genetic interactions or dosage effects.
  • If a gene has multiple functions or if mutations affect different domains, the interpretation can be nuanced; additional crosses or molecular mapping may be needed.
• Alternatives and complements
  • Genetic mapping: locate each mutation on a chromosome; if they map to the same locus, they’re likely in the same gene.
  • Molecular approaches: sequencing the candidate genes can directly reveal whether the mutations are in the same gene.
  • Epistasis and complementation groups: in larger screens, grouping mutants by complementation tests defines “complementation groups” that correspond to distinct genes.

If you’d like, I can tailor this to a specific organism or provide a step-by- step experimental outline (cross design, expected phenotypes, controls) for your case.