Identification of a Novel Candidate Gene for Chilling Tolerance in Pumpkin (Cucurbita moschata) Using Whole-Genome Resequencing

Chilling stress is one of the major abiotic constraints limiting cucurbit crop winter cultivation globally. Pumpkins (Cucurbita moschata) have been frequently used as a bloomless rootstock to enhance the fruit vigor and glossiness of cucumber (Cucumis sativus). Nonetheless, pumpkins are less tolerant to chilling stress than fig-leaf gourd (Cucurbita ficifolia), limiting their winter cultivation usage. Hence, it necessitates identifying chilling-tolerant pumpkin germplasm to identify the stress-responsive genes and to ease rootstock breeding in the face of unpredictable future climates. Our study considered the root fresh weight (RFW) and shoot fresh weight (SFW) growth under chilling stress as a tolerance index to assess the low-temperature tolerance to screen 407 pumpkin germplasm collection. Based on the preliminary characterization, we isolated the chilling-tolerant (IT 278592 [CT]) and the chilling-susceptible (IT 274039 [CS]) lines. The bulked-segregant analysis-assisted next-generation sequencing uncovered the existence of single nucleotide polymorphism (SNP) variations in three genes, including CmoCh06G003170 (encodes for F4P12_330), CmoCh16G010790 (Nucleoredoxin 1 [CmoNRX1]), and CmoCh15G013730 (DNA binding with one finger (Dof) protein 4.6 [CmoDof4.6]), while comparing the allelic variations between the CT and CS lines. Accordingly, Sanger sequencing and polymorphic marker analysis also asserted the presence of missense mutations in the three candidate genes. Despite SNP variation in three genes, the qRT-PCR analysis uncovered the chilling stress-responsive expression only in the CmoDof4.6 gene. The CmoDof4.6 gene was downregulated in the CT line compared to the CS line in response to the chilling stress implying that it could be a potential candidate gene and needed further experimental attention. Altogether, we identified a novel chilling stress-responsive gene in pumpkin germplasm that could facilitate the identification and breeding of chilling-tolerant pumpkin rootstock.