Association analysis for proline and fructan content in barley (Hordeum vulgare L.) under late spring cold stress using microsatellite markers

Introduction
Biotic and abiotic environmental stresses reduce the quantity and quality of agricultural products. Late spring cold stress, if it occurs, can cause significant damage to farmers and producers depending on the growth stage of the crop plants. Association analysis allows for the rapid and initial identification of quantitative trait loci based on linkage disequilibrium. This method is a powerful tool for explaining the genetics of complex agricultural traits and identifying alleles controlling these traits. The objective of this study was to evaluate the genetic diversity, population structure, linkage disequilibrium pattern and association analysis of some physiological traits of cultivated barley genotypes under late spring cold stress conditions using microsatellite (SSR) markers.

Materials and methods
The plant materials of this study were 60 cultivated barley genotypes including 20 commercial and 40 landraces, which were obtained from the Seed and Plant Improvement Research Institute, Karaj, Iran. The experiment was carried out as a factorial in a completely randomized design with three replications. The studied genotypes were placed under cold stress (+8 and -2 °C) at the reproductive stage (heading and flowering, Zadoks 68-50) and physiological data were measured and collected. Analysis of variance and comparison of means were performed by Tukey’s test at a probability level of 5% using SAS software version 9.4. software version 9.4. Also, genomic DNA was extracted using the CTAB method and its quality and quantity were determined using 1% agarose gel electrophoresis and spectrophotometer, respectively. To assess molecular diversity among the studied barley genotypes, DNA samples were amplified by 20 SSR markers using polymerase chain reaction (PCR) and the PCR products were loaded on metaphor-agarose gels. After electrophoresis, the bands in the gels were scored as co-dominant, and then the number of alleles, common allele frequency, marker discrimination power, polymorphic information content (PIC) and gene diversity were calculated using POWER MARKER software. Effective population structure analysis was also conducted using the Bayesian method in the Structure software. To identify genomic regions associated with the measured traits, association analysis based on mixed linear model (MLM) was conducted in TASSEL software, considering the population structure (Q) and the kinship (K) matrices as covariate variables.

Research findings
The results of the variance analysis for the physiological data indicated significant differences in all studied traits among barley genotypes under both temperature conditions of 8 °C and -2 °C. According to the mean comparison results, the highest amount of proline was observed in the local varieties Sahra and Jonoob at 8 °C, while in the -2 °C conditions, the highest levels were found in the genotypes Naik and Zahek. Conversely, the lowest proline levels at both temperature conditions were identified in the genotypes TN-02-6297 and TN-02-6400. Therefore, the two local varieties Naik and Zahek, with their higher proline levels at -2 °C, are introduced as cold-tolerant genotypes against late spring frost stress. Regarding fructan, this study also revealed that at 8 °C, the highest and lowest fructan levels were observed in the genotypes TN-02-6734 and TN-02-4975, respectively. Additionally, under -2 °C conditions, these values were recorded in the genotypes Bahman and TN-02-4952. Polymorphic information content varied from 0.59 for the marker Bmag0007 to 0.82 for the marker Bmag0032. The gene diversity ranged from 0.63 to 0.84, with an average of 0.74, and the frequency of common alleles varied from 0.21 to 0.52. Population structure analysis indicated the presence of two subpopulations among the studied barley genotypes. The results of association analysis based on the mixed linear model (MLM) identified five significant marker-trait associations (P < 0.05). Among these, three markers (Bmag0518, Bmag0211, and Ebmac0674) were significantly associated with proline-controlling genes under -2 °C conditions, one marker (Bmag0223) was associated with proline-controlling genes under +8 °C conditions, and one marker (Bmag0173) was linked to fructan-controlling genes under +8 °C conditions. Additionally, no marker was identified with a significant association for fructan levels under -2 °C conditions.

Conclusion
The results of the association analysis in this study showed that five markers, Bmag0518, Bmag0211, Ebmac0674, Bmag0223 and Bmag0173, had a significant association with the proline and fructan contents in barley under cold stress conditions. These markers, after validation with complementary tests, can be utilized in breeding programs by marker-assisted selection (MAS) to improve cold resistance in barley.