Integrative BSR-seq and association analysis identify GhLPA1 as a candidate gene associated with lint percentage in cotton

• A QTL hotspot ( qLP-D03-1 ) for lint percentage was identified via BSR-seq and GWAS approaches. • The candidate gene GhLPA1 was validated by VIGS to influence lint percentage. • A KASP marker based on high-LP GhLPA1 haplotype for marker-assisted breeding in cotton. Improving lint percentage (LP) through genetic breeding is crucial for stabilizing cotton fiber yield. To dissect the genetic basis of LP, a recombinant inbred line (RIL) population consisting of 300 lines were derived from a cross between the high-LP cultivar ‘CRI50’ and the low-LP standard line ‘TM-1’. Multi-environment phenotypic investigations revealed that LP showed a continuous distribution with high heritability ( H² =81.65%), indicating complex genetic control with strong genetic determination. Bulked segregant RNA-seq (BSR-seq) analysis of extreme LP pools identified a major quantitative trait locus (QTL), qLP-D03-1 , on chromosome D03. Further GWAS and QTL hotspot analysis narrowed the candidate region to approximately 250 kb on chromosome D03. Within this region, GhLPA1 ( Ghicr24_D03G124100 ), encoding a ribosomal protein with extraribosomal functions, was identified as the key candidate gene, and its function was confirmed by virus-induced gene silencing (VIGS) analysis. Haplotype analysis showed that the ‘CRI50’-type allele (Hap1) conferred significantly higher LP than the ‘TM-1’-type allele (Hap2). Furthermore, a kompetitive allele-specific PCR (KASP) marker developed from a functional SNP in the 3′ untranslated region (UTR) of GhLPA1 effectively differentiated genotypes with contrasting LP, which providing a valuable marker for marker-assisted selection (MAS) in cotton breeding.