Alternative Splicing of NAC Transcription Factor Gene CmNST1 Is Associated with Naked Seed Mutation in Pumpkin, Cucurbita moschata

In pumpkin (Cucurbita moschata), the naked or hull-less seed phenotype has great benefits for breeding this crop for oil or snack use. We previously identified a naked seed mutant in this crop. In this study, we report genetic mapping, identification, and characterization of a candidate gene for this mutation. We showed that the naked seed phenotype is controlled by a single recessive gene (N). The bulked segregant analysis identified a 2.4 Mb region on Chromosome 17 with 15 predicted genes. Multiple lines of evidence suggested that CmoCh17G004790 is the most probable candidate gene for the N locus which encodes a NAC transcription factor WALL THICKENING PROMOTING FACTOR 1 (CmNST1). No nucleotide polymorphism or structural variation was found in the genomic DNA sequences of CmNST1 between the mutant and the wildtype inbred line (hulled seed). However, the cDNA sequence cloned from developing seed coat samples of the naked seed mutant was 112 bp shorter than that from the wildtype which is due to seed coat-specific alternative splicing in the second exon of the mutant CmNST1 transcript. The expression level of CmNST1 in the developing seed coat was higher in the mutant than in the wildtype during early seed coat development which was reversed later. Transcriptomic profiling with RNA-Seq at different stages of seed development in the mutant and wildtype revealed a critical role of CmNST1 as a master regulator for the lignin biosynthesis pathway during seed coat development while other NAC and MYB transcription factors were also involved in forming a regulatory network for the building of secondary cell walls. This work provides a novel mechanism for the well-characterized NST1 transcription factor gene in regulating secondary cell wall development. The cloned gene also provides a useful tool for marker-assisted breeding of hull-less C. moschata varieties.