Genetic Diversity in Barley as Revealed by Microsatellite Markers and Association Analysis of These Markers by Traits Related to Freezing Tolerance

Genetic diversity of 35 barley genotypes was evaluated by microsatellite markers and the association of these markers with several characters related to freezing tolerance were studied. DNA was extracted from young barley leaves using the CTAB method and then multiplied with 44 microsatellite primer pairs from which 36 primers produced clear banding pattern. Amplified products were separated using 6% polyacrylamide denaturing gel electrophoresis. Zero-one binary data were recorded for the presence (one) or absence (zero) of the bands. Furthermore, for each marker alleles were named alphabetically as a, b, c, etc. From the data obtained, polymorphic information content (PIC) and average gene diversity were calculated for each microsatellite primer pair. Then, the barley genotypes were grouped based on neighbor-joining algorithm and the distance coefficient of Kimura 2-parameters. Furthermore, the fresh and dry weight of leaves, crowns and seedlings, percent of tissue water content, amount of tissue water loss, frost survival rate at -6, -10, -14, -16 and -18 oC and LT50 of the barley genotypes were measured and association analysis of the microsatellite markers with the phenotypic values of these traits was carried out. Numbers of polymorphic alleles were 268, with an average of 7.44 alleles per locus. Marker Hvm74 with 21 alleles and Hvm2a with three alleles had the maximum and minimum number of alleles, respectively. The lowest and highest major allele frequency of 0.13 and 0.53 belonged to Hvm20 and Hvm14 markers, respectively. Average major allele frequency was 0.31. PIC ranged from 0.42 (for Hvm2) to 0.94 (for Hvm74) with an average of 0.74. The gene diversity ranged from 0.53 to 0.93 with an average of 0.78. Highest amount of heterozygosity (0.25) was related to Hvm74 markers. The results of cluster analysis showed that microsatellite markers were highly capable of identifying genetic differences among barley genotypes. Association analysis revealed significant association of 62 alleles belonging to 38 microsatellite markers with 12 out of 13 characters evaluated on the barely genotypes under study. Maximum and minimum number of alleles were observed in relation to crown moisture (with 10 alleles) and relative water loss (with one alleles), respectively. Five alleles were associated with LT50, which may be used in barley breeding programs if they contribute to freezing tolerance consistently in other experiments.