Genetic dissection of cold stress tolerance and yield potential under cold stress in nested synthetic wheat (Triticum aestivum L.) introgression libraries using multi-locus genome-wide association and haplotype analysis

Cold stress is a major abiotic constraint to wheat production in temperate regions, particularly in the Kashmir Valley of north-western Himalayas, India, where prolonged low temperatures delay growth, reduce yield, and disrupt the rice-wheat cropping system. To address this challenge, we evaluated a panel of 340 nested synthetic hexaploid wheat introgression lines, developed by introgressing allelic diversity from Aegilops tauschii and Triticum durum into bread wheat. The panel was phenotyped for cold stress tolerance, electrolyte leakage index, and yield-related traits across three temperate environments and genotyped using the 35 K Axiom® Wheat Breeder's Array. Genome-wide association study using FarmCPU and BLINK models identified 152 stable marker-trait associations (MTAs), with - log₁₀(P) values ranging from 3.00-14.64. Several stable MTAs surpassed the Bonferroni-adjusted significance threshold and were designated as high-confidence loci. A number of stable MTAs co-localized with previously reported genomic regions, whereas others represent potentially novel loci. Multiple pleiotropic loci were detected, indicating potential for simultaneous improvement of multiple traits. Haplotype analysis identified 32 major LD blocks significantly associated with target traits, with favorable haplotypes conferring enhanced stress tolerance and productivity. Several putative candidate genes were identified within chromosome-specific LD intervals of high-confidence MTAs. Literature mining, in silico expression profiling and network analyses further supported their functional relevance in cold stress adaptation and yield regulation. The identified genomic regions, haplotypes, and candidate genes provide valuable resources for marker-assisted and haplotype-based breeding to develop cold-tolerant, high-yielding wheat cultivars suited to temperate regions, thereby supporting the sustainability of rice-wheat cropping systems.