Investigation of genetic diversity of superior cocksfoot (Dactylis glomerata L.) genotypes using agronomic traits and ISSR markers for enhanced breeding strategies

Background and Objective: Cocksfoot (Dactylis glomerata L.) is a major cool-season perennial forage grass widely distributed in the rangelands of Iran and extensively used for livestock grazing and forage production. In grass breeding programs, knowledge of genetic variation is essential for achieving sustainable biomass production, broad adaptation, and long-term genetic improvement. Genetic diversity is the cornerstone of grass breeding because it enables effective parental selection, facilitates the exploitation of heterosis in polycross and synthetic varieties, and enhances yield stability and tolerance to biotic and abiotic stresses. Therefore, this study was conducted to investigate the genetic diversity of superior cocksfoot (Dactylis glomerata L.) genotypes using agronomic traits and ISSR molecular markers.
Materials and Methods: Twenty superior genotypes, previously selected based on forage yield, flowering date, and resistance to rust disease, were evaluated for both agronomic traits and ISSR molecular markers. The field experiment was conducted at the research farm of the Agricultural Biotechnology Research Institute, Tabriz, Iran, in 2020. Fifteen clonal plants from each genotype were established in compost-filled pots. After growth under greenhouse conditions, the seedlings were transplanted to the field. The experiment was arranged in a randomized complete block design with three replications. In each plot, five plants of each genotype were established at a spacing of 60 × 60 cm. No data were collected during the establishment year. Data on heading date, pollination date, plant height, panicle length, tiller number, crown diameter, seed yield, and forage dry matter yield at the first and second harvests were recorded in 2022. The data were subjected to analysis of variance, and mean comparisons were performed using the LSD test at the 5% probability level. Agronomic data were further classified using Ward’s cluster analysis. For molecular analysis, 29 ISSR primers were employed. Band presence or absence was scored, and genetic diversity parameters, including the number of effective alleles (Ne), Shannon’s information index (I), and expected heterozygosity (He), were estimated. Genetic relationships among genotypes were evaluated using the Neighbor-Joining clustering method and principal component analysis (PCA).
Results: The results of the field experiment revealed significant differences among genotypes for all measured traits. Genotypes Dg1, Dg6, Dg12, Dg13, and Dg18 exhibited superior forage dry matter and seed yields compared with the remaining genotypes. Cluster analysis based on agronomic traits classified the 20 cocksfoot genotypes into four distinct groups, containing 3, 8, 4, and 5 genotypes, respectively. Molecular analysis identified 346 alleles, of which 337 (97.4%) were polymorphic. The number of alleles per primer ranged from 7 to 20, with an average of 11.93 alleles. The mean values of Shannon’s information index, expected heterozygosity, and the number of effective alleles were 0.447, 0.315, and 1.533, respectively, indicating substantial genetic diversity among the evaluated genotypes. Cluster analysis based on ISSR markers also grouped the genotypes into four clusters, with cluster IV exhibiting the highest level of genetic divergence and diversity. The PCA results confirmed the clustering pattern obtained from the molecular data. The genetic similarity matrix, with coefficients ranging from 0.41 to 1.00, suggested that genetically distant clones could serve as suitable parental materials for polycross breeding to maximize heterosis. A comparison of the clustering patterns derived from ISSR markers and agronomic traits indicated that the grouping of genotypes was not fully consistent between the two analytical approaches.
Conclusion: Based on band clarity, reproducibility, and discriminatory ability, ISSR03, ISSR25, ISSR06, ISSR32, ISSR17, and ISSR15 were identified as the most informative markers for assessing genetic diversity in cocksfoot. Furthermore, the lowest Jaccard similarity coefficient (higher genetic distances) was observed between genotype Dg13 and genotypes Dg17, Dg18, and Dg19. Accordingly, genotypes Dg1, Dg6, Dg12, Dg13, and Dg18 demonstrated superior agronomic performance while also belonging to distinct groups with relatively greater genetic distances. These genotypes are therefore recommended as promising parental candidates for polycross or synthetic breeding programs aimed at maximizing heterosis and broadening the genetic base of cocksfoot cultivars.