Identification of a Candidate Male-fertility Female-fertility Gene Tagged by an Endogenous Transposon in Soybean

In soybean the W4 gene controls the anthocyanin pigment biosynthesis in flowers. The mutant allele, w4-m, is characterized by variegated flowers. Loss of pigment production in the w4-m mutant was shown to be due to insertion of the Tgm9 transposon in the DFR2 gene. In the w4-m system, reversion of the unstable allele from variegated to normal purple color flower would indicate transposon’s excision, and its insertion at a second locus. We identified a male-sterile female-sterile mutant as a germinal revertant among the selfed progeny of w4-m. The objectives of our investigation were i) to map the sterility mutant; ii) to determine the association between sterility and presence of Tgm9 and; iii) to clone the male-sterility, female-sterility gene. We genetically mapped the sterility gene to 62 kb region on chromosome 16, flanked by   BARCSOYSSR_16_0428 and BARCSOYSSR_16_0430. Southern blot analysis confirmed that Tgm9 co-segregated with the fertility/sterility phenotype, suggesting that the sterility was caused by Tgm9. The pools of genomic DNA of homozygous fertile and sterile soybean plants were used to construct four adaptor-ligated genomic DNA libraries and were amplified using an adaptor primer and a transposon-specific primer. The band specific to the libraries of sterile plants were sequenced.  Comparison showed that the transposon insertion occurred in a DNA/RNA helicase gene, which is a DEAD-box superfamily member and is known to play a role in crossing over during meiosis. Characterization of the fertility gene will provide vital insight on structure and function of genes involved in reproductive biology of plants.