Detection of QTLs and QTL–environment interactions for fruit branch number in Upland cotton (Gossypium hirsutum L.) using a restricted two-stage multi-locus genome-wide association study

Fruit branch number (FBN) is an important architectural trait of Upland cotton (Gossypium hirsutum L.). In this study, FBN was investigated in 340 Upland cotton accessions across five environments. The restricted two-stage multi-locus genome-wide association study was employed for the first time to dissect the genetic architecture of FBN. A total of 37,010 high-quality single-nucleotide polymorphism markers (SNPs) were used to generate 6,749 single-SNP and 6,151 multi-SNP linkage disequilibrium block markers. A total of 78 quantitative trait loci (QTLs) for FBN were detected, with four QTLs consistently detected across different environments. Three QTLs, qFBN-A13, qFBN-D03-2, and qFBN-D06, were further validated through single-marker/haplotype analysis. Gene Ontology (GO) enrichment analysis for genes near the three QTLs revealed that 22 significantly enriched terms (34 underlying genes), including “regulation of DNA-templated transcription,” “oxygen binding,” and “heme binding.” RNA-seq analysis further revealed that seven genes (GH_A13G1575, GH_D03G1306, GH_D03G1319, GH_D06G0408, GH_D06G0424, GH_D06G0436, and GH_D06G0442) exhibited high expression levels in stem tissue, suggesting their potential roles in fruit branch formation. Additionally, 18 QTL-by-environment interactions (QEIs) were detected, with five QEIs validated by analyzing phenotypic differences among genotypes across various environments. The GO enrichment analysis of genes near these five QEIs identified 22 significantly enriched terms (28 underlying genes), including “oxidoreductase activity, acting on metal ions,” “copper ion binding,” and “protein neddylation.” Five genes (GH_A06G1857, GH_A06G1859, GH_A06G1884, GH_D07G0658, and GH_D09G1041) showed high expression levels in the stem, indicating their potential involvement in fruit branch formation through gene–environment interactions. In total, 12,905 SNPLDB markers were constructed from 37,010 high-quality SNPs. A total of 78 QTLs and 18 QEIs were detected. Three stable QTLs for FBN were validated through single-marker/haplotype analysis. Five QEIs for FBN were validated through phenotypic difference analysis. Seven QTL-related genes and five QEI-related genes may contribute to FBN.