1- Yasouj University
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: (155 Views)
Extended Abstract
Background: Lentil (Lens culinaris Medic) is one of the most important plants of the legume family, which play an important role in providing human food and are known as an important source of protein. There is 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 marker has shown high efficiency as a dominant marker in studying genetic diversity and genetic relationships between different genotypes of plants, especially lentil. So, 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. Leaves DNA was extracted using CTAB method and their quality on agarose gel and its quantity was examined using spectrophotometer in the central laboratory of the Faculty of Agriculture, Yasouj University. DNA samples with suitable quality were selected for 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. Statistical analyses such as the percentage of polymorphic bands, polymorphic information content, and Shannon index were calculated and the efficiency of the ISSR markers was examined. Then, the genotypes were grouped by cluster analysis (Jaccard similarity coefficient and UPGMA method) and principal component analysis, and the genotypes with the greatest genetic distance were introduced for further breeding programs. Excel, NTSYS PC 2.02, and Popgene 1.32 softwares were used.
Results: A total of 190 bands were produced, of which 186 were polymorphic. UBC840 marker produced the highest number of bands (17 bands), all of which showed polymorphism. Among the markers, the highest polymorphic information content (PIC) belonged to UBC830 marker (0.49) and the lowest belonged to UBC814 marker (0.13). Nine markers had PICs greater than 0.4, which were considered suitable for genetic differentiation of genotypes. The average Shannon index was 0.845. The highest Shannon index, which indicates intera-population variation, was related to UBC853 marker (1.4) and the lowest was related to UBC814 primer (0.3). 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 in the first group (C1) two genotypes, in the second group (C2) most of the genotypes (55.6%), in the third group (C3) two genotypes; in the fourth group (C4) 11 genotypes, and in the fifth group (C5) only one genotype. 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 in the grouping obtained from the first two components, only 42.09% of the total variance contributed. 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 compared to other markers. Considering 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, in order to take a fundamental step towards 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, which play an important role in providing human food and are known as an important source of protein. There is 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 marker has shown high efficiency as a dominant marker in studying genetic diversity and genetic relationships between different genotypes of plants, especially lentil. So, 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. Leaves DNA was extracted using CTAB method and their quality on agarose gel and its quantity was examined using spectrophotometer in the central laboratory of the Faculty of Agriculture, Yasouj University. DNA samples with suitable quality were selected for 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. Statistical analyses such as the percentage of polymorphic bands, polymorphic information content, and Shannon index were calculated and the efficiency of the ISSR markers was examined. Then, the genotypes were grouped by cluster analysis (Jaccard similarity coefficient and UPGMA method) and principal component analysis, and the genotypes with the greatest genetic distance were introduced for further breeding programs. Excel, NTSYS PC 2.02, and Popgene 1.32 softwares were used.
Results: A total of 190 bands were produced, of which 186 were polymorphic. UBC840 marker produced the highest number of bands (17 bands), all of which showed polymorphism. Among the markers, the highest polymorphic information content (PIC) belonged to UBC830 marker (0.49) and the lowest belonged to UBC814 marker (0.13). Nine markers had PICs greater than 0.4, which were considered suitable for genetic differentiation of genotypes. The average Shannon index was 0.845. The highest Shannon index, which indicates intera-population variation, was related to UBC853 marker (1.4) and the lowest was related to UBC814 primer (0.3). 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 in the first group (C1) two genotypes, in the second group (C2) most of the genotypes (55.6%), in the third group (C3) two genotypes; in the fourth group (C4) 11 genotypes, and in the fifth group (C5) only one genotype. 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 in the grouping obtained from the first two components, only 42.09% of the total variance contributed. 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 compared to other markers. Considering 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, in order to take a fundamental step towards 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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