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Analogy of ISSR and RAPD markers for comparative analysis of genetic diversity among different <i>Jatropha curcas</i> genotypes
Abstract
The phylogenetic relationships of 13 Jatropha genotypes from different parts of the India were analysed using 34 polymerase chain reaction (PCR) markers (20 random amplified polymorphic DNAs (RAPDs) and
14 inter simple sequence repeats (ISSRs)). Amplification of genomic DNA of the 13 genotypes, using RAPD analysis, yielded 107 fragments that could be scored, of which 91 were polymorphic, with an average of
4.55 polymorphic fragments per primer. Number of amplified fragments ranged from one (OPA20, OPB19, OPD13) to nine (OPA18) and which varied in size from 200 to 2,500 bp. Percentage of polymorphism
ranged from 40% (OPB18) to a maximum of 100% (14 primers). Resolution power ranged from a minimum of 0.153 (OPA20, OPB19) to a maximum of 11.23 (OPB15). Out of 25 ISSR primers used, 14 were able to amplify. These primers produced 81 bands across 13 genotypes, of which 62 were polymorphic. The number of amplified fragment ranging from two (ISSR 7, ISSR 8, ISSR 16) to nine (ISSR 12) and which varied in size from 200 to 2,500 bp. Of the 81 fragments amplified bands, 62 were polymorphic, with an average of 4.42 polymorphic fragments per primer. Percentage of polymorphism ranged from 37.5% (ISSR 2, ISSR10) to a maximum of 100% (seven primers). The primers based on poly (GA) produced maximum number of bands (nine) while, poly (AT) and many other motifs gave no amplification at all with any of these thirteen genotypes. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection, as they detected 84.26% as compared to 76.54% for ISSR markers. But, resolving power (Rp), average bands per primer, Nei’s genetic diversity (h), Shannon’s Information Index (I), total genotype diversity among population (Ht), within population diversity (Hs) and gene flow (Nm) estimates were more for ISSR (7.098, 5.79, 0.245, 0.374, 0.244, 0.137 and 0.635, respectively) as compared to RAPD markers (5.669, 5.35, 0.225, 0.359, 0.225, 0.115 and 0.518, respectively). The regression test between the two Nei’s genetic diversity indexes gave r2 = 0.3318, showing low regression between RAPD and ISSR
based similarities. Regression value for ISSR and ISSR + RAPD combined data is moderate (0.6027), while it is maximum for RAPD and ISSR+RAPD based similarities (0.9125). Thus both the markers are equally important for genetic diversity analysis in Jatropha curcas. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR. Principal Coordinates Analysis (PCA) analysis was also employed to evaluate the resolving power of the markers to differentiate between the genotypes. These analyses, carried out for both (ISSR and RAPD) markers, allowed us to identify four main groups partially corresponding to the four J. curcas collection sites. The results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of these genotypes.
14 inter simple sequence repeats (ISSRs)). Amplification of genomic DNA of the 13 genotypes, using RAPD analysis, yielded 107 fragments that could be scored, of which 91 were polymorphic, with an average of
4.55 polymorphic fragments per primer. Number of amplified fragments ranged from one (OPA20, OPB19, OPD13) to nine (OPA18) and which varied in size from 200 to 2,500 bp. Percentage of polymorphism
ranged from 40% (OPB18) to a maximum of 100% (14 primers). Resolution power ranged from a minimum of 0.153 (OPA20, OPB19) to a maximum of 11.23 (OPB15). Out of 25 ISSR primers used, 14 were able to amplify. These primers produced 81 bands across 13 genotypes, of which 62 were polymorphic. The number of amplified fragment ranging from two (ISSR 7, ISSR 8, ISSR 16) to nine (ISSR 12) and which varied in size from 200 to 2,500 bp. Of the 81 fragments amplified bands, 62 were polymorphic, with an average of 4.42 polymorphic fragments per primer. Percentage of polymorphism ranged from 37.5% (ISSR 2, ISSR10) to a maximum of 100% (seven primers). The primers based on poly (GA) produced maximum number of bands (nine) while, poly (AT) and many other motifs gave no amplification at all with any of these thirteen genotypes. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection, as they detected 84.26% as compared to 76.54% for ISSR markers. But, resolving power (Rp), average bands per primer, Nei’s genetic diversity (h), Shannon’s Information Index (I), total genotype diversity among population (Ht), within population diversity (Hs) and gene flow (Nm) estimates were more for ISSR (7.098, 5.79, 0.245, 0.374, 0.244, 0.137 and 0.635, respectively) as compared to RAPD markers (5.669, 5.35, 0.225, 0.359, 0.225, 0.115 and 0.518, respectively). The regression test between the two Nei’s genetic diversity indexes gave r2 = 0.3318, showing low regression between RAPD and ISSR
based similarities. Regression value for ISSR and ISSR + RAPD combined data is moderate (0.6027), while it is maximum for RAPD and ISSR+RAPD based similarities (0.9125). Thus both the markers are equally important for genetic diversity analysis in Jatropha curcas. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR. Principal Coordinates Analysis (PCA) analysis was also employed to evaluate the resolving power of the markers to differentiate between the genotypes. These analyses, carried out for both (ISSR and RAPD) markers, allowed us to identify four main groups partially corresponding to the four J. curcas collection sites. The results of the present study can be seen as a starting point for future researches on the population and evolutionary genetics of these genotypes.
