Gene mapping and development of molecular markers for thousand-grain weight in rye based on bulked segregant analysis

Xinjiang wild rye (<i>Secale cereale</i> subsp. <i>segetale</i>) is a wild rye species that was discovered in Xinjiang Province of China in the 20th century. It is a relatively understudied cereal crop within the <i>Poaceae</i> family, and there is currently a lack of research on its yield-related traits. Mapping genes controlling thousand-grain weight (TGW) is crucial for developing high-yielding rye cultivars. In this study, bulk segregant analysis sequencing (BSA-seq) was performed on a cross-pollinated (CP) hybrid population derived from cultivated rye (Z837) and Xinjiang wild rye (90R13) to identify genomic regions associated with TGW. Molecular markers were then developed in the region of the initially mapped genes to further localize the TGW gene. BSA-seq analysis identified 10 candidate regions containing 68 single nucleotide polymorphism (SNP) loci across the seven chromosomes and unassembled chromosomal segments of rye. Molecular markers were designed for these loci and PCR-based validation was performed, yielding six high-quality Kompetitive allele-specific PCR (KASP) markers and one simple sequence repeat (SSR) marker. After evaluating amplification efficiency, a single KASP marker, <i>TGW-16</i>, proved highly effective for selecting germplasm with superior TGW. Functional annotation of the candidate gene <i>ScWN7R01G304400</i> (linked to <i>TGW-16</i>) revealed that it encodes a protein containing an RNA recognition motif (RRM1) domain. These findings establish both genetic resources and methodological frameworks for marker-assisted breeding in rye, laying a solid foundation for cultivating elite varieties with optimized TGW performance.