Identification of a major QTL and candidate gene for flowering time in industrial hemp via BSA-seq and fine mapping

Industrial hemp (Cannabis sativa L.) is a photoperiod-sensitive short-day crop, yet the quantitative trait loci (QTLs) governing flowering time remain poorly characterized, limiting molecular breeding efforts. To identify genomic regions controlling photoperiodic flowering, we performed bulked segregant analysis sequencing (BSA-seq) coupled with fine linkage mapping using an F₂ population derived from a cross between day-neutral (Bubble Kush) and short-day (Aquawoman) accessions. Based on extreme flowering phenotypes, we pooled DNA from 50 early- and 50 late-flowering individuals. A major QTL, qHFX, was mapped to a 2.2 Mb region on chromosome X via ΔSNP-index and Euclidean distance algorithms. Using 15 Kompetitive allele-specific PCR (KASP) markers developed from parental polymorphisms, we refined qHFX to a 637-kb interval in 300 F₂ individuals. RNA-seq analysis of Aquawoman under short-day and long-day conditions identified five differentially expressed genes within this interval, with expression profiles validated by quantitative real-time PCR. Sequence analysis revealed a 1-bp indel in LOC115716363, which is homologous to Arabidopsis AHL20/22 and emerged as a strong candidate gene. Notably, heterologous overexpression of LOC115716363 in rice significantly delayed flowering, further supporting its role in flowering time regulation. Collectively, these findings elucidate the molecular basis of flowering time in industrial hemp and provide valuable genomic resources for breeding broadly adapted, high-yield varieties.