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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 F2:3 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.

Hull-less seed trait is preferred by nut and oil industries worldwide for snacking and oil extraction as it evades the expensive decorticating (dehulling) process. This seed trait is available in C. pepo only, which has small seed cavity, sensitive to various biotic and abiotic stresses, and restricted to temperate regions for cultivation. Contrarily, the related species C. moschata has wider adaptability, disease tolerance and high seed yield. Therefore, attempt was made to transfer this trait into C. moschata through conventional pollination and ovule culture using four parents of hull-less C. pepo and six of hulled C. moschata. Through conventional approach, few viable F1 seeds (12–23) were obtained by using C. pepo as female parent, but in three crosses (HLP36 × HM1343, HLP36 × HM1022 and HLP44 × HM1022) only, whereas, its use as male parent was not successful. This incompatibility issue of reciprocals was resolved through ovule culture of C. moschata genotypes HM1343 and HM6711 after 17 to 19 days of pollination with C. pepo genotypes HLP53 and HLP72, respectively. The hybridity of interspecific crosses was confirmed through SSR markers (alleles inherited from both the parents), morphological characters and micromorphological leaf traits (differed from both the parents). The successful transfer through interspecific hybridization was further established with the presence of hull-less seed in fruits of F2 populations. Outcome of this study would pave the way for enhancing the productivity and multi-season cultivation of snack-seeded pumpkin even in subtropical and tropical regions.