Analysis of genetic differences underlying chilling stress tolerance using whole genome Re-Sequencing in walnut (Juglans regia L.)

Abstract Walnut (Juglans regia L.) is prized worldwide for both its nutritional value and economic importance, yet it remains vulnerable to cold stress, with significant differences in tolerance among varieties. This study combined physiological analyses with whole-genome resequencing (WGS) to evaluate the cold stress responses of two varieties, ‘Qingxiang’ and ‘Liaoning No.8’. Under chilling stress (0 °C), we measured electrolyte leakage and antioxidant enzyme activity, applying both exogenous methyl jasmonate (MeJA) and the jasmonate inhibitor DIECA. Genomic variations were analyzed using WGS. Results showed that ‘Liaoning No.8’ exhibited superior cold tolerance. Application of MeJA reduced electrolyte leakage by 37% and MDA accumulation by 52% on average, whereas DIECA exacerbated stress-related damage. WGS achieved 16.24–16.26× coverage and identified 2.73–2.78 million SNPs, 378–382k InDels, 25–26k SVs, and 7.2–7.9k CNVs. Twenty genes containing sequence variants showed transcriptional responses under cold stress that were significantly correlated with mutation density (r = 0.62, P < 0.01). One gene, XM_018985465.2, which lacked SNPs in ‘Liaoning No.8’, was expressed 4.2 times more in this variety, suggesting cis-regulatory influence. These findings highlight the role of jasmonic acid signaling in enhancing cold tolerance in walnut and offer genomic insights into its underlying adaptive mechanisms.