Association Analysis for Photosynthetic Pigments in Bread Wheat (Triticum aestivum L.) under Late Cold stress

Bread wheat (Triticum aestivum L.) is the most widely cultivated cereal and a globally significant crop. Understanding physiological trait responses to late spring cold stress in susceptible and tolerant cultivars provides insights into cold tolerance mechanisms. One critical physiological trait affected by cold stress is leaf chlorophyll content. This study aimed to investigate the changes of photosynthetic pigments and their association with SSR markers in bread wheat subjected to late spring cold stress.

Changes in the concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were evaluated in 70 bread wheat varieties under two temperature treatments: +8°C (control) and -2°C (cold stress). The cultivars were grown in a greenhouse under a completely randomized design with three replications. During the reproductive stage (Zadoks growth stages 48–60), temperature treatments were applied by gradually reducing the temperature from 24°C to the target temperature at a rate of -2°C per hour. Plants were maintained at the target temperature for two hours before being returned to 24°C. Sampling for trait measurements and molecular experiments was conducted 24 hours after the stress treatment.

Mean comparisons revealed significant variations among cultivars across temperature treatments. For chlorophyll a, Moghan2 exhibited the highest change (20.70), while Karaj3 showed the lowest (4.30). For chlorophyll b, Chamran2 showed the highest change (7.78 mg.g⁻¹), whereas Baharan showed the lowest (0.16). Total chlorophyll changes were highest in Moghan2 (20.70) and lowest in Karaj3 (4.30). For carotenoids, Ehsan exhibited the highest change (1.75), while Neyshabour displayed the lowest (0.009). Cultivars with minimal pigment changes were identified as potentially tolerant to late spring cold stress. Marker-trait association analysis identified specific SSR markers linked to pigment traits under control and stress conditions. Four SSR markers were identified under control conditions (+8°C) and four under cold stress (-2°C) for chlorophyll a. For chlorophyll b, three SSR markers were linked under control conditions and five under cold stress conditions. Total chlorophyll was associated with five SSR markers under both environments. Four SSR markers were identified under control conditions for carotenoids, while only one was detected under cold stress.

Structure analysis revealed that most genotypes were not clustered based on origin or growth type. Due to significant genotype × environment interactions, marker-trait associations were analyzed separately for control and stress conditions. More quantitative trait loci (QTL) were identified at 8°C than at -2°C. Several markers showed consistent associations across environments, demonstrating high stability and utility for marker-assisted selection. These results highlight the efficacy of SSR markers in evaluating genetic diversity and population structure among wheat genotypes, providing valuable tools for breeding programs aimed at improving cold tolerance.