The Genetic Loci Associated with Fiber Development in Upland Cotton (Gossypium hirsutum L.) Were Mapped by the BSA-Seq Technique

Cotton fiber quality improvement remains a fundamental challenge in breeding programs due to the complex genetic architecture underlying fiber development. The narrow genetic base of upland cotton (Gossypium hirsutum L. ) and the quantitative nature of fiber quality traits necessitate innovative approaches for identifying and incorporating superior alleles from related species. We developed a BC6F2 population by introgressing chromosome segments from the sea island cotton variety Xinhai 36 (G. barbadense) into the upland cotton variety Xinluzhong 60 (G. hirsutum). Based on fiber strength phenotyping, we constructed two DNA bulks representing extreme phenotypes (20 superior and 12 inferior individuals) for bulked segregant analysis sequencing (BSA-Seq). High-throughput sequencing generated 225. 13 Gb of raw data with average depths of 20× for parents and 30× for bulks. SNP calling and annotation were performed using GATK and ANNOVAR against the upland cotton reference genome (TM-1). BSA-Seq analysis identified 13 QTLs primarily clustered within a 1. 6 Mb region (20. 6–22. 2 Mb) on chromosome A10. Within this region, we detected nonsynonymous mutation genes involving a total of six genes. GO and KEGG enrichment analyses revealed significant enrichment for carbohydrate metabolic processes, protein modification, and secondary metabolite biosynthesis pathways. Integration with transcriptome data prioritized GHA10G1043, encoding a β-amylase family protein, as the key candidate gene. Functional validation through overexpression and RNAi knockdown in Arabidopsis thaliana demonstrated that GHA10G1043 significantly regulates starch content and β-amylase activity, though without visible morphological alterations. This study successfully identified potential genomic regions and candidate genes associated with cotton fiber strength using chromosome segment substitution lines combined with BSA-Seq. The key candidate gene GHA10G1043 provides a valuable target for marker-assisted selection in cotton breeding programs. Our findings establish a foundation for understanding the molecular mechanisms of fiber quality formation and offer genetic resources for developing superior cotton varieties with enhanced fiber strength.