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.