Abstract
Chinese cabbage (Brassica rapa L. ssp. pekinensis), an important traditional vegetable indigenous to China, is a typical cross-pollinated Brassica crop exhibiting pronounced heterosis. However, its small flower organs make artificial pollination for hybrid seed production highly challenging. The use of male-sterile lines has emerged as a crucial approach in hybrid seed production. Therefore, understanding the genetic and molecular mechanisms underlying male sterility in Chinese cabbage holds profound theoretical and economic importance and is pivotal for advancing Chinese cabbage crossbreeding. Here, cytological comparative analysis of anthers from sterile line 366-2S and fertile line 366-2F revealed abnormalities in 366-2S during the late tetrad stage, including delayed tapetum degradation and the aggregation of tetrad microspores without separation, which prevented pollen production and caused male sterility. Construction of the F(2) segregating population, with 366-2S as the female parent and genetically diverse fertile material Y636-9 as the male parent, indicated that male sterility in 366-2S is controlled by a single recessive gene. Using bulked segregant analysis sequencing and kompetitive allele-specific polymerase chain reaction (KASP) technology, the sterile gene was mapped to 65 kb between the PA11 and PA13 markers, with 11 genes in the candidate region. Functional annotation, expression, and sequence variation analyses identified BraA09g012710.3C, encoding acyl-CoA synthetase 5, as a candidate gene for 366-2S male sterility. Quantitative real-time polymerase chain reaction analysis revealed minimal expression of BraA09g012710.3C in 366-2S but high expression in the flower buds of 366-2F. Further analysis of candidate gene DNA sequences identified a large deletion encompassing BraA09g012710.3C, BraA09g012720.3C, BraA09g012730.3C, and BraA09g012740.3C in sterile line 366-2S (A09: 7452347-7479709). Cloning and verification of the other three deleted genes in the F(2) population via agarose gel electrophoresis confirmed their presence in F(2) sterile individuals, indicating that their deletion was not associated with male sterility, underscoring BraA09g012710.3C as the key gene driving male sterility in 366-2S.