Identification of class I LBD family members in upland cotton and function and haplotype analyses of GhLBD6 in regulating flowering period

Lateral organ boundaries domain (LBD) family genes play important roles in a wide range of plant biological processes. Previous studies have shown that class I LBD genes are involved in the regulation of flowering time; however, whether class I LBD genes have similar functions in upland cotton remains unclear. In this study, we identified and characterized class I LBD family members at the whole-genome level in upland cotton and screened flowering-related candidate genes through haplotype analysis. The expression patterns of candidate genes in different tissues and in early- and late-flowering varieties were examined using RNA-seq and RT-qPCR, respectively. The function of the target gene was validated by virus-induced gene silencing (VIGS), followed by analysis of its breeding-related evolutionary dynamics and the development of molecular markers. In total, 102 class I GhLBD genes were identified; they were unevenly distributed across 26 chromosomes and were classified into six groups based on evolutionary relationships. LBD members within the same group showed similar motif composition and arrangement. Integrating haplotype and expression analyses, GhLBD6 was identified as a candidate gene associated with flowering time. VIGS combined with paraffin sectioning and RT-qPCR showed that suppression of GhLBD6 accelerated floral bud differentiation and significantly advanced budding and flowering by 6.57 d and 6.86 d, respectively. Two haplotypes (GhLBD6-Hap 1 and GhLBD6-Hap 2) were identified in the GhLBD6 coding region; among them, GhLBD6-Hap 1 represents a favorable early-flowering allele and appears to have been under artificial selection during upland cotton breeding. Finally, we developed a kompetitive allele-specific PCR (KASP) molecular marker to distinguish the two GhLBD6 haplotypes. Together, these forward- and reverse-genetic results demonstrate that GhLBD6 contributes to flowering-time regulation in upland cotton, providing a basis for molecular breeding of early-maturing cotton.