Integrated BSA-seq and RNA-seq analyses reveal CmRABE1c as a negative regulator of drought tolerance and enable the development of a promoter SNP marker in Chinese chestnut

Abstract

Drought stress severely limits both yield and nut quality of Chinese chestnut (Castanea mollissima Blume), an economically and ecologically important woody grain species native to China. However, the genetic basis of drought tolerance in Chinese chestnut remains a research gap. Our study fills this gap by identifying novel drought-responsive genes, which represents the first comprehensive molecular characterization of drought adaptation in this species. To dissect the genetic basis of drought tolerance and develop functional markers, we investigated 316 F1 seedlings derived from the cross between the drought-tolerant ‘Yanlong’ cultivar and the drought-sensitive ‘Shuofeng’. Bulked segregant analysis coupled with next-generation sequencing (BSA-seq) identified 80 drought-associated quantitative trait loci (QTLs) across 12 chromosomes. Integration of BSA−seq, RNA-seq, and parental resequencing resulted in the identification of a core candidate gene, CmRABE1c, encoding a ras-related protein. CmRABE1c overexpression in tobacco and poplar confirmed its negative regulatory role in drought tolerance. Notably, a GG-to-AA substitution at -312/-313 bp in the CmRABE1c promoter disrupted the GT1-motif and was significantly associated with drought tolerance across 60 chestnut accessions. Furthermore, transgenic tobacco and poplar assays proved this SNP may be diagnostic by showing that the AA allele confers superior drought tolerance compared to the GG allele. This diagnostic marker likely can be directly applied in marker-assisted selection (MAS) to screen and develop drought-tolerant chestnut cultivars, thereby accelerating breeding efficiency. In summary, this study provides valuable genetic resources and a robust functional SNP marker for drought resistance breeding in Chinese chestnut.