Maize Genetics Cooperation Newsletter vol 85 2011
Which seed color gene of r1 responds to inhibitors (Inr) and enhancers (Enr) of aleurone color?
Stinard and Sachs (2002. J Hered 93:421-428) and Stinard et al. (2009. J Hered 100:217-228) recently reported on r1 haplotype-specific inhibitors and enhancers of aleurone color. Genetic analysis of the R1-r(sd2) (spotted dilute) haplotype led Stinard et al. (2009) to conclude that one of the seed color genes (S1 or S2) of the seed color (S) subcomplex of R1-r(sd2) contains a dSpm insert, and the other seed color gene responds to dominant alleles at inhibitory (Inr) or enhancing (Enr) loci (see Figure 4 of Stinard et al. 2009 for a proposed model). However, it was unclear which seed color gene carries the dSpm insert and which gene responds to Inr and Enr. Intragenic recombination experiments were designed in order to answer this question.
Intragenic recombination has been used a means of dissecting the structure and function of the genic components of the compound r1 locus (see Kermicle, JL. 1988. pp. 81-89 in Nelson, OE Jr (ed). Plant transposable elements. Plenum Press, NY for a review). The S subcomplex of R1-r(Std), the presumed progenitor of R1-r(sd2), carries duplicate genes, S1 and S2 arranged as inverted repeats flanking a promoter region (Walker et al. 1995. EMBO J 14:2350-2363). Both S1 and S2 are expressed in the aleurone, so a mutation in one of the S genes of R1-r(Std) would not be expected to give a visible phenotype. However, intragenic recombination knocking out the S2 gene followed by genetic analysis of the derivatives can help answer the question of which gene in R1-r(sd2) carries a dSpm insert and which one responds to inhibitors and enhancers. For these experiments, the r1-sc:m3 allele was chosen to recombine with S2. r1-sc:m3 carries a Ds insertion in the simplex Sc (aleurone Self-color) gene, which is homologous to and in direct orientation with respect to S2 on chromosome 10 (Kermicle, JL. 1980. Science 208:1457-1459; in combination with conclusions of Walker et al. 1995). Recombination between S2 and r1-sc:m3 proximal to the Ds insertion point would result in the transfer of the Ds element to S2, knocking it out and at the same time placing it under control of Ac should Ac be present in the genome. Experiments were devised to discriminate between the following two possibilities:
(1) Possibility 1: S1 responds to Inr and Enr alleles. r1-sc:m3 was crossed to R1-r(Std) (genotype S1 S2). The F1 was crossed by r1-g Enr1-Fcu Inr1. If Ds is transferred to S2 by intragenic recombination, and if S1 responds to the mutable enhancer allele Enr1-Fcu and the inhibitory Inr1 allele, then the derivatives carrying s2::Ds with S1 would be expected to show aleurone color sectoring on a pale background in the presence of Enr1-Fcu and Inr1. Nonrecombinants would have full aleurone color due to the presence of the uninhibited, nonmutant S2 gene. This cross was performed in 2008, and out of a total of 10,288 scorable kernels (with aleurone color), 11 mottled exceptions were obtained. Subsequent testing of the exceptions revealed that the mottling was not heritable in 10 of the exceptions—they were actually parental R1-r(Std) types. One exception did not germinate. Therefore, from an effective population of 9,353 kernels, no visible recombinants were obtained. These negative results could mean that either the population size was not sufficient to isolate the desired recombinants, or that S1 does not respond to Enr and Inr alleles.
(2) Possibility 2: S2 responds to Inr and Enr alleles. r1-sc:m3 was crossed to R1-r(sd2). The F1 was crossed by r1 inr1 Spm. If Ds is transferred to S2 by intragenic recombination, and if S1 of R1-r(sd2) carries a dSpm insert, then the derivatives carrying s2::Ds with s1::dSpm would be expected to show typical small revertant sectors (S1 s2::Ds) on a colorless (s1::dSpm s2::Ds) background (no Ac present). Nonrecombinants (s1::dSpm S2) would have full aleurone color since an inhibitory Inr1 allele was absent from this cross. This cross was performed in 2009, and out of a total of 8,740 scorable kernels (with aleurone color), 3 mutable exceptions were obtained, for a frequency of 3.4 X 10-4. Subsequent testing of the exceptions revealed that all three were heritable. Two of the exceptions also responded to Ac, which is what would be expected if Ds was transferred to S2 by intragenic recombination (s1::dSpm s2::Ds); one exception did not respond to Ac. The non-responder to Ac could represent a spontaneous mutation in S2 (s1::dSpm s2) rather than a recombination event. The frequency of Spm Ac double responders was 2.3 X 10-4.
The recovery of the expected and heritable events from the latter intragenic recombination experiment demonstrates that it is the S2 gene that responds to Inr and Enr alleles, and the S1 gene of R1-r(sd2) carries a dSpm insert. The frequency of recombination between Sc and S2 (2.3 X 10-4) is comparable with that observed between other components of the r1 gene complex (e. g. 4.49 X 10-4 between the P (Plant color) gene and r1-sc:m3 restoring kernel color, Dooner, HK and Kermicle, JL. 1986. Genetics 113:135-143; and 3.93 X 10-4 and 3.68 X 10-4 for recombination between P and S2 in R1-r(Std) resulting in deletion of sequences between P and S2, Dooner, HK and Kermicle, JL. 1971. Genetics 67:427-436.).
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