Identification of cold-resistance genes in Vitis vinifera through integrated OcBSA and transcriptome analysis of an intraspecific hybrid population

Cold injury constrains the cultivation of Vitis vinifera, whereas conventional interspecific breeding can improve cold resistance at the expense of fruit quality. Efficient identification of cold-resistance loci within V. vinifera is therefore needed to support intraspecific breeding. We analyzed an F1 population derived from the cold-resistant, high-quality cultivar ‘Ecolly’ and ‘Marselan’. Outcross bulked-segregant analysis (OcBSA) was integrated with RNA sequencing (RNA-seq), and selected differentially expressed genes were validated by reverse-transcription quantitative PCR (RT-qPCR). Cold resistance in the hybrid population followed a normal distribution, confirming its suitability for gene mapping. OcBSA localized cold-resistance-associated regions to chromosomes 12, 18, and 25. Integration of OcBSA and RNA-seq identified 43 functionally annotated candidate genes enriched in 25 KEGG pathways involving amino acid, carbohydrate, secondary metabolite, energy, and signal transduction metabolism. Candidate genes included ASN1, MMT1, CYP73A4, GA2OX1, GA2OX6, SALR, GLO1, AGT1, MAN2, and SAG113, together with MYB, bHLH, and ERF transcription factors. RT-qPCR analysis of six differentially expressed genes was generally consistent with the RNA-seq results. CAB-151 was highly expressed in S_1, whereas PTI6 peaked in S_3 and decreased in the cold-resistant group. To our knowledge, this is the first use of integrated OcBSA and transcriptome analysis to identify cold-resistance genes in an intraspecific V. vinifera hybrid population. The identified candidate genes provide a foundation for functional validation and molecular-assisted breeding of high-quality, cold-resistant grapevine cultivars. An intraspecific Vitis vinifera hybrid population (‘Ecolly’ × ‘Marselan’) was used to dissect the genetic basis of cold resistance without compromising fruit quality. OcBSA combined with transcriptome (RNA-seq) analysis localized cold-resistance genes to chromosomes 12, 18, and 25. A total of 43 candidate genes were identified, enriched in 25 KEGG pathways spanning amino acid, carbohydrate, secondary metabolite, energy, and signal transduction metabolism. Key candidate genes (ASN1, CYP73A4, GA2OX1/6, SALR, GLO1, AGT1, MAN2, SAG113) and MYB, BHLH, and ERF transcription factors were associated with the cold-stress response. RT-qPCR validation was consistent with RNA-seq, supporting these genes as targets for molecular-assisted breeding of cold-resistant grapevine.