Volume 18, Issue 2 (4-2026)
J Crop Breed 2026, 18(2): 79-91 |
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Rahmani A, Dehdari M, Amiri Fahliani R, Karimizadeh R. (2026). Assessment of the Genetic Diversity of Lentil (Lens culinaris Medic) Genotypes using ISSR Markers. J Crop Breed. 18(2), 79-91. doi:10.61882/jcb.2026.1634
URL: http://jcb.sanru.ac.ir/article-1-1634-en.html
URL: http://jcb.sanru.ac.ir/article-1-1634-en.html
1- Department of Agronomy and Plant Breeding, Faculty of Agriculture, Yasouj University, Yasouj, Iran
2- Dryland Agricultural Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Gachsaran, Iran
2- Dryland Agricultural Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Gachsaran, Iran
Abstract: (413 Views)
Extended Abstract
Background: Lentil (Lens culinaris Medic) is one of the most important plants of the legume family, playing an important role in providing human food, known as an important source of protein. There is a rich germplasm of this plant in Iran and the world. Given that a large number of varieties and breeding lines are produced and introduced by internal and exotic breeders every year, it is essential to study their genetic diversity for use in breeding programs. It is possible to study genetic diversity through morphological, biochemical, and DNA markers. The use of molecular markers to study genetic diversity is more reliable than other markers due to the lack of influence of environmental factors and extensive polymorphism. ISSR markers have shown high efficiency as a dominant marker in studying genetic diversity and genetic relationships between different genotypes of plants, especially lentil. Therefore, this study was designed and implemented to study the genetic diversity of different lines and several common varieties of lentil using the ISSR marker.
Methods: In this experiment, fresh leaf samples of 36 lentil genotypes (including 33 new breeding lines and three common cultivars), which were obtained from the Gachsaran Rainfed Research Station, were collected and frozen at -40 ˚C. Leaf DNA was extracted using the CTAB method, and its quality and quantity were examined on agarose gel and using a spectrophotometer, respectively, in the central laboratory of the Faculty of Agriculture, Yasouj University. DNA samples with suitable quality were selected for the further step, and then PCR amplification was performed using 20 ISSR primers that had shown high polymorphism in other plants in previous studies. In the desired samples, the presence and absence of bands were scored as one and zero, respectively. The percentage of polymorphic bands, polymorphic information content, and the Shannon index were calculated with statistical analyses, followed by examining the efficiency of the ISSR markers. Then, the genotypes were grouped by cluster analysis (Jaccard’s similarity coefficient and the UPGMA method) and principal component analysis. The genotypes with the greatest genetic distance were introduced for further breeding programs. Excel, NTSYS PC 2.02, and Popgene 1.32 software were used for the analyses.
Results: In total, 186 out of 190 produced bands were polymorphic. UBC840 marker produced the highest number of bands (17 bands), all of which showed polymorphism. Among the markers, the highest and the lowest polymorphic information content (PIC) belonged to UBC830 (0.49) and UBC814 (0.13) markers, respectively. Nine markers had PICs greater than 0.4, which were considered suitable for the genetic differentiation of genotypes. The average Shannon index was 0.845. The highest and the lowest Shannon index, which indicates intra-population variation, were recorded for UBC853 (1.4) and UBC814 (0.3) primers, respectively. In general, considering the measured characteristics, UBC855 and UBC853 primers are recommended for genetic studies in lentil. Cluster analysis classified 36 lentil genotypes into five groups. There were two genotypes in the first group (C1), most of the genotypes (55.6%) were in the second group (C2), two genotypes were in the third group (C3), 11 genotypes were in the fourth group (C4), and only one genotype was in the fifth group (C5). The greatest genetic distance was observed between the fifth and second groups (for example, the Jaccard similarity coefficient was 0.08 between genotype 33 and genotype 19). Therefore, it is expected that the crossing between genotype 33 (group 5) and the genotypes of the second group would witness more transgressive segregation. The Sepehr and Kimiya cultivars were placed in one group, which probably indicates their common origin, while the Gachsaran cultivar was placed in a different group. Most genotypes with the same origin were placed in the same group, although some differences were also observed. Principal component analysis showed that many components played a role in explaining the total variance; for example, the first five components explained 51.82% of the total variance. This result indicates a suitable distribution of the markers in the lentil genome. The grouping of genotypes based on the first two components partially confirmed the grouping obtained from cluster analysis, although it also had some differences. In cluster analysis, all the variance contributed to the grouping, while only 42.09% of the total variance contributed to the grouping obtained from the first two components. Therefore, the existence of differences in the grouping results is not far from expected.
Conclusion: Similar to some studies, the ISSR markers used in this study showed high efficiency for examining genetic diversity and distinguishing lentil genotypes. Among them, two primers, including UBC855 and UBC853, were superior to the other markers. Given the extensive polymorphism shown by these markers, it is suggested that they can be used to study the association analysis with important lentil traits, including drought tolerance, grain yield, and protein percentage, to take a fundamental step toward improving these traits. On the other hand, a wide diversity was observed among the studied genotypes in terms of ISSR markers. The grouping of genotypes showed that genotypes with high genetic distance can be selected and introduced for continuing lentil breeding programs. Among them, the highest genetic distance was observed between only the fifth group genotype and the second group genotypes, which is suggested to be used in future studies.
Background: Lentil (Lens culinaris Medic) is one of the most important plants of the legume family, playing an important role in providing human food, known as an important source of protein. There is a rich germplasm of this plant in Iran and the world. Given that a large number of varieties and breeding lines are produced and introduced by internal and exotic breeders every year, it is essential to study their genetic diversity for use in breeding programs. It is possible to study genetic diversity through morphological, biochemical, and DNA markers. The use of molecular markers to study genetic diversity is more reliable than other markers due to the lack of influence of environmental factors and extensive polymorphism. ISSR markers have shown high efficiency as a dominant marker in studying genetic diversity and genetic relationships between different genotypes of plants, especially lentil. Therefore, this study was designed and implemented to study the genetic diversity of different lines and several common varieties of lentil using the ISSR marker.
Methods: In this experiment, fresh leaf samples of 36 lentil genotypes (including 33 new breeding lines and three common cultivars), which were obtained from the Gachsaran Rainfed Research Station, were collected and frozen at -40 ˚C. Leaf DNA was extracted using the CTAB method, and its quality and quantity were examined on agarose gel and using a spectrophotometer, respectively, in the central laboratory of the Faculty of Agriculture, Yasouj University. DNA samples with suitable quality were selected for the further step, and then PCR amplification was performed using 20 ISSR primers that had shown high polymorphism in other plants in previous studies. In the desired samples, the presence and absence of bands were scored as one and zero, respectively. The percentage of polymorphic bands, polymorphic information content, and the Shannon index were calculated with statistical analyses, followed by examining the efficiency of the ISSR markers. Then, the genotypes were grouped by cluster analysis (Jaccard’s similarity coefficient and the UPGMA method) and principal component analysis. The genotypes with the greatest genetic distance were introduced for further breeding programs. Excel, NTSYS PC 2.02, and Popgene 1.32 software were used for the analyses.
Results: In total, 186 out of 190 produced bands were polymorphic. UBC840 marker produced the highest number of bands (17 bands), all of which showed polymorphism. Among the markers, the highest and the lowest polymorphic information content (PIC) belonged to UBC830 (0.49) and UBC814 (0.13) markers, respectively. Nine markers had PICs greater than 0.4, which were considered suitable for the genetic differentiation of genotypes. The average Shannon index was 0.845. The highest and the lowest Shannon index, which indicates intra-population variation, were recorded for UBC853 (1.4) and UBC814 (0.3) primers, respectively. In general, considering the measured characteristics, UBC855 and UBC853 primers are recommended for genetic studies in lentil. Cluster analysis classified 36 lentil genotypes into five groups. There were two genotypes in the first group (C1), most of the genotypes (55.6%) were in the second group (C2), two genotypes were in the third group (C3), 11 genotypes were in the fourth group (C4), and only one genotype was in the fifth group (C5). The greatest genetic distance was observed between the fifth and second groups (for example, the Jaccard similarity coefficient was 0.08 between genotype 33 and genotype 19). Therefore, it is expected that the crossing between genotype 33 (group 5) and the genotypes of the second group would witness more transgressive segregation. The Sepehr and Kimiya cultivars were placed in one group, which probably indicates their common origin, while the Gachsaran cultivar was placed in a different group. Most genotypes with the same origin were placed in the same group, although some differences were also observed. Principal component analysis showed that many components played a role in explaining the total variance; for example, the first five components explained 51.82% of the total variance. This result indicates a suitable distribution of the markers in the lentil genome. The grouping of genotypes based on the first two components partially confirmed the grouping obtained from cluster analysis, although it also had some differences. In cluster analysis, all the variance contributed to the grouping, while only 42.09% of the total variance contributed to the grouping obtained from the first two components. Therefore, the existence of differences in the grouping results is not far from expected.
Conclusion: Similar to some studies, the ISSR markers used in this study showed high efficiency for examining genetic diversity and distinguishing lentil genotypes. Among them, two primers, including UBC855 and UBC853, were superior to the other markers. Given the extensive polymorphism shown by these markers, it is suggested that they can be used to study the association analysis with important lentil traits, including drought tolerance, grain yield, and protein percentage, to take a fundamental step toward improving these traits. On the other hand, a wide diversity was observed among the studied genotypes in terms of ISSR markers. The grouping of genotypes showed that genotypes with high genetic distance can be selected and introduced for continuing lentil breeding programs. Among them, the highest genetic distance was observed between only the fifth group genotype and the second group genotypes, which is suggested to be used in future studies.
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