Identification of candidate gene controlling TSSL via BSA-seq and fine mapping and germplasm innovation in rice

Stigma exsertion ratio (SER) of rice male sterile line directly affects the hybrid rice F₁ seed production, while SER was directly affected by the total of stigma and style length (TSSL). To improve the F₁ seed production, it is necessary to identify the genes or quantitative trait loci (QTLs) controlling TSSL. In this study, we used the 7001 S with short TSSL, Zitai S with long TSSL and F₂ to preliminarily map the QTL controlling TSSL to a 2.71 Mb interval using BSA-seq method. Based on the known SSR marker and newly designed InDel markers, the target region was further narrowed down to a 58.2 kb interval between TSL25 and TSL128 on chromosome 3. Gene knockout experiment was used to confirm Os03g0407400 (GS3) as a candidate gene controlling TSSL. Furthermore, according to the sequence alignment difference in the coding region of GS3 between 7001 S and Zitai S, the marker M1 was developed and revealed to be closely linked to TSSL. Combining fertility identification and molecular marker-assisted selection, a fertility-stable near-isogenic line (NIL) of C815S with long TSSL was created. It was found through small-scale F₁ seed production combination trials that the outcrossing seed setting ratio of combination NIL-C815S × R919 was 39.31%, and the yield of the F₁ seeds harvested from the female parents in the unit area was 237.50 g, which were both higher than that of the combination C815S × R919. These results laid a foundation for further research on the TSSL trait in rice and enriched its molecular regulatory network.