Identification of single major QTL and candidate gene(s) governing hull-less seed trait in pumpkin

The hull-less pumpkin (Cucurbita pepo) seed does not require de-hulling before use for human consumption, as a result highly preferred by the oil, nut, and baking industries. In hull-less seeds, a single recessive gene is responsible for the absence of outer thick seed coat layers; however, the genomic region and gene(s) controlling the trait are unclear to date. In this study, four crosses attempted to derive F₂ and backcross populations confirmed the single recessive gene inheritance of hull-less seed trait in populations adapted to the sub-tropical climate. The candidate genomic region for hull-less seed trait was identified through the BSA-QTLseq approach using bulks of F_2:3 progenies from a cross of HP111 (hulled) and HLP36 (hull-less). A novel genomic region on chromosome 12 ranging from 1.80 to 3.86 Mb was associated with the hull-less seed trait. The re-sequencing data identified a total of 396 SNPs within this region and eight were successfully converted into polymorphic KASP assays. The genotyping of segregating F₂ (n = 160) with polymorphic KASP assays resulted in a 40.3 cM partial linkage map and identified Cp_3430407 (10 cM) and Cp_3498687 (16.1 cM) as flanking markers for hull-less locus (Cphl-1). These flanking markers correspond to the 68.28 kb region in the reference genome, and the marker, Cp_3430407 successfully predicted the genotype in 93.33% of the C. pepo hull-less germplasm lines, thus can be used for marker-assisted selection in parents polymorphic for the hull-less seed trait. The Cphl-1-linked genomic region (2.06 Mb) encompasses a total of 182 genes, including secondary cell wall and lignin biosynthesis-related transcriptional factors viz., "NAC" (Cp4.1LG12g04350) and "MYB" (Cp4.1LG12g03120). These genes were differentially expressed in the seeds of hulled and hull-less genotypes, and therefore could be the potential candidate genes governing the hull-less seed trait in pumpkin.