Question 1 asks you to suggest 3 experiments that could help demonstrate that a pea mutant resembling the ‘r’ mutant of pea that segregates as a single recessive nuclear locus is a new allele of the R locus. You know already that the ‘R’ gene (SBE1) is cloned and recessive alleles (r) are available. Answer (any three of the answers below): a. Do a classical complementation test by crossing the new mutant and an ‘r’ mutant (homozygous known recessive allele of R). If the new mutant is homozygous for a recessive allele of ‘R’ allele then it should fail to complement the ‘r’ mutant and the F1 seeds will be wrinkled. b. Clone (PCR) and sequence the SBE1 gene from the new mutant. If the new mutant is homozygous for an ‘r’ allele then you should find a mutation in its SBE1 gene. c. Transform the new pea mutant with a genomic clone of the SBE1 gene. If the new mutant is homozygous for a recessive allele of ‘R’ then the wild type SBE1 gene should complement the mutant phenotype (transgene complementation). d. Assuming a DNA-based marker for the R gene (that does not affect the phenotype) is available and wild type and the new mutant have different alleles you could cross a wild type pea plant to the new mutant and show that the DNA based R allele in the new mutant and new phenotype co-segregate. If the new mutation is an ‘R’ allele then it will co-segregate with a DNA-based marker for ‘R’ (this experiment assumes that a DNA-based polymorphism for ‘R’ between wild type and the new mutant can be found). Technically, you could use the new mutant phenotype to clone the defective gene by map-based cloning. If the gene turns out to be ‘R’ (SBE1) then you know that new mutation is in fact R. However in practice no one would do this because it would take a very long time compared to any on the approached listed above. Question 2. Answer: The technician failed to establish whether the new mutant phenotypes were due to a single nuclear recessive mutation. Thus no conclusion could be drawn from the complementation experiment performed.