Identification and fine mapping of a stigma exsertion mutant gene (Bolsem) in ornamental kale (Brassica oleracea var. acephala)

Stigma exsertion is a plant trait wherein stigmas emerge from flower buds before anthesis. It is a key determinant of the ornamental kale mating system, greatly increases the efficiency of hybrid seed production, and represents a technique to promote crossing pollination. The present study investigated a stigma exsertion mutant (‘Y009-10-5’) in ornamental kale and determined that the trait was determined by a recessive loss-of-function allele at the Bolsem (B. oleracea stigma exsertion mutant) locus. Then, using linkage analysis, with 350 F2 progeny and 98 SSR markers, the Bolsem locus was mapped to a 65.64-kb region of Chromosome 7, between the markers SSRCWS5 and SSRLS3. Sequence analysis and Brassica Database analysis identified two genes in this region: Bol005242, a gene of unknown function, and Bol005241, an ortholog of the Arabidopsis gene ABAP1, which controls cell proliferation and was, therefore, considered a potential candidate for Bolsem. Further sequence analysis identified two single-nucleotide polymorphisms (SNPs) in Bol005241. One SNP was found in the third exon, with the ‘Y009-10-5’ sequence yielding a TAA stop codon; therefore, it is predicted as the significant effects on stigma exsertion. Bol005241 also fully co-segregated with the exon SNP, and quantitative real-time PCR indicated that Bol005241 exhibited pistil-specific expression. Pollination experiments were performed to determine the effect of pollination timing on hybrid seed production. The results of this study provide a basis for elucidating the molecular mechanism underlying stigma exsertion, which could significantly improve hybrid seed production in ornamental kale.