Objective To screen and apply sterile lines with phenotypic marker traits, and enhanc the seed production purity in two-line hybrid rice.

Method In this study, the yellow-green leaf trait (YSL) obtained through natural mutation was introduced into two-line sterile line of rice (YOS-1) , resulting in a new two-line sterile line (YOS-2) carrying the yellow-green leaf marker. Combined with phenotype observation and data calculation of leaf color, determination of chlorophyll content, genetic analysis, transcriptome analysis, and candidate gene screening, the regulatory mechanism of the target genes on rice leaf color was clarified. The basic agronomic traits of YOS-2 were compared with its counterpart YOS-1. Furthermore, using each as the female parent, ten hybrid combinations were respectively constructed, and F₁ generations were subjected to comparative performance analysis.

Result In the F₃ population of YLS × R1173, the contents of chlorophyll a and chlorophyll b in the seedling and tillering stages of the yellow leaf and yellow glume materials were extremely significantly lower than those of the green leaf and green glume materials (P ≤ 0.01). The phenotypic characteristics of yellow leaf and yellow glume materials as well as green leaf and green glume materials were stable at each development stage. The yellow leaf and yellow glume traits were controlled by a pair of recessive nuclear genes. Compared to YOS-1, the three agronomic traits of YOS-2 (plant height, flag leaf length and flag leaf width) were predominantly governed by additive effects. Additionally, the average yield of hybrid combinations derived from YOS-2 was slightly higher than that of combinations derived from YOS-1. In the application of hybrid rice, YOS-2 could completely replace YOS-1. Three candidate genes (LOC_Os08g06220, LOC_Os08g06280, and LOC_Os08g07720) associated with the yellow leaf and yellow glume traits were initially screened out via BSA sequencing, transcriptome data analysis and gene annotation information in the molecular breeding database. Subcellular localization prediction analysis indicated that LOC_Os08g06220 was localized in chloroplasts, LOC_Os08g06280 in cell membranes, and LOC_Os08g07720 in cytoplasm.

Conclusion The breeding of YOS-2 (a male sterile line with yellow leaves and yellow glumes) has further enriched the germplasm pool of leaf-color marker-based male sterile lines, and simultaneously promoted the application of phenotypic marker traits in hybrid rice production. The exploration of genes controlling yellow leaf and yellow glume traits is conducive to further understanding the regulatory mechanisms of rice photosynthesis, chlorophyll synthesis and degradation, as well as its growth and development.