Q.61 A drought tolerant rice genotype was found to be associated with a missense
mutation in the gene A. Which one or more of the following experiments is/are
appropriate to validate whether the mutation in A is the causal factor for drought
tolerance?
(A) Introduce the same mutation in a drought sensitive rice genotype and test if it
becomes drought tolerant.
(B) Delete the wild–type A in drought sensitive plant and test if it becomes drought
tolerant.
(C) Determine the stability of the protein encoded by the wild–type and the mutant
forms of A.
(D) Repair the mutation in the drought tolerant rice genotype and test if it becomes
drought sensitive.

Options (A), (C), and (D) are appropriate experiments to validate the missense mutation in gene A as the causal factor for drought tolerance in rice.​

Option Analysis

Option A: Introduce mutation in sensitive genotype
Introducing the same missense mutation into a drought-sensitive rice genotype using CRISPR-Cas9 and observing if it gains drought tolerance directly tests gain-of-function causality. This recreates the mutant allele in a clean genetic background, confirming the mutation alone confers the phenotype.​

Option B: Delete wild-type A in sensitive plant
Deleting the wild-type gene A in a drought-sensitive plant tests loss-of-function, which would likely make it more sensitive or unchanged, not tolerant. This does not validate the specific missense mutation’s gain-of-function role in tolerance.​

Option C: Protein stability comparison
Missense mutations often alter protein structure or stability; comparing wild-type and mutant protein stability under stress explains the mechanism if the mutant form is more stable or functional. This supports causality by linking genotype to phenotype biochemically.​

Option D: Repair mutation in tolerant genotype
Repairing the mutation (complementation with wild-type A) in the drought-tolerant genotype and testing if it becomes sensitive is a classic rescue experiment. It proves the mutation is necessary and sufficient for the tolerant phenotype.​

A drought tolerant rice genotype linked to a missense mutation in gene A requires rigorous validation to confirm causality for drought tolerance. Key experiments like CRISPR-based mutation introduction and complementation provide definitive proof, essential for rice breeding and CSIR NET life sciences preparation.​

Why Validate Causal Mutations?

Missense mutations change a single amino acid, potentially enhancing protein function for stress tolerance. Standard genetic validation uses forward and reverse genetics to isolate the mutation’s effect from genetic background noise.​

Recommended Experiments

• CRISPR Mutation Recreation (Option A): Edit sensitive rice to match the mutant allele; tolerance gain confirms causality.​

• Protein Stability Assay (Option C): Mutant proteins often show altered folding/stability under drought; assays like thermal shift validate mechanism.​

• Complementation Repair (Option D): Restore wild-type A in tolerant plants; sensitivity reversion proves necessity.​

Invalid Approaches

Gene knockout (Option B) tests null alleles, irrelevant for gain-of-function missense variants.​

This framework aligns with rice drought tolerance breeding using CRISPR for precise edits.