Amplified Fragment Length Polymorphism Primer Combinations Research Articles

English

At present, due to the increasing popularity of Jatropha curcas as a source of biodiesel and the generation of nontoxic and high yielding varieties of the plant, there is an immediate need for genetic improvement. However, conclusions on the genetic basis of J. curcas are still inconsistent. The objective of this study is to use amplified fragment length polymorphisms (AFLP) to survey the genetic diversity ofJ. curcas in Southeast Asian countries and China to aid in genetic improvement, and to preliminarily get the message on the genetic relationships of J. curcasbetween China and Southeast Asian. We collected a total of 240 samples from three Asian countries, two African countries and different geographical regions in China. In this study, six AFLP primer combinations were used to amplify, and they yielded a total of 352 scorable loci, 14.78% of which were polymorphic. The number of loci scored per primer combination ranged from 53 (E-ACT/M-CAA and E-ACG/M-CAT) to 68 (E-ACC/M-CAA), with an average of 58.67 loci per primer combination. The rate of polymorphisms found for different primer combinations ranged from 12.90 (E-ACA/M-CAT) to 16.98% (E-ACG/M-CAT). The AMOVA revealed that 36.18% of the variance occurred among populations and 63.82% occurred within populations. The above indicators suggest that the genetic germplasms of J. curcas have a narrow genetic diversity in China and Southeast Asia. The clustering of genotypes based on the AFLP markers shows that the genetic relationship between cluster II and III is close, indicating that the origin of J. curcas in China may be from Southeast Asian. Our next work is to find the bands linkage with specific populations or specific properties and convert it into a sequence-characterized amplified region marker to aid in the genetic improvement of this energy plant.   Key words: Jatropha curcas, genetic diversity, amplified fragment length polymorphism, genetic relationship.

Development of SCAR and CAPS markers linked to a recessive male sterility gene in lettuce (Lactuca sativa L.)

Genetic male sterility (GMS) has been a useful system for the production of hybrid varieties in self-pollinated plants. We obtained a GMS line developed from a spontaneous mutation in lettuce (Lactuca sativa L.). Genetic analysis in our previous study revealed that the sterility was controlled by a recessive gene which was named ms-S. For simple and quick screening of individuals showing male sterility, we attempted molecular mapping of the ms-S locus using an amplified fragment length polymorphism (AFLP) technique. From the examination of 4,096 AFLP primer combinations, 63 AFLP markers were found to be linked to the gene and nine of them were successfully converted into sequence characterized amplified region (SCAR) markers and cleaved amplified polymorphic sequence (CAPS) markers. Linkage analysis indicated that these nine markers were closely linked to the ms-S gene and all were located on the same side of the gene. The minimum genetic distance between the ms-S gene and a marker was 3.1 cM. These results provide additional information for map-based cloning of the ms-S gene and will be of great help for lettuce breeding using GMS to produce F1 hybrids.

AFLP assessment of genetic diversity among Indian Mucuna accessions

Amplified fragment length polymorphism (AFLP) marker was used to assess diversity in germplasm collection of Mucuna species which has gained tremendous attention in the recent past due to its promising nutritional, agronomic and medicinal attributes. Twenty five accessions comprising five species, collected from seven states of India were evaluated with twelve AFLP primer combinations that generated a total of 1,612 fragments with an average of 134 fragments per primer combination. The values of polymorphic information content (PIC), marker index (MI) and the resolving power (Rp) demonstrated the utility of the primer combinations used in the present study for discriminating the Mucuna accessions. UPGMA and Principal coordinate analysis (PCoA) of the genotypic data revealed clustering of accessions as per phenetic and genetic relationships. The Jaccard's similarity coefficient values suggested good variability among the M. pruriens accessions indicating their utility in breeding programs. Molecular diversity presented in this study combined with the datasets on other morphological/agronomic traits will be highly useful for selecting appropriate accessions for plant improvement through conventional as well as molecular breeding approaches and for evolving suitable conservation strategies.

Separation of the genera in the subtribe Cassiinae (Leguminosae: Caesalpinioidae) using molecular markers

Random amplified polymorphic DNA (RAPD), Inter simple sequence repeat (ISSR) and Amplified fragment length polymorphism (AFLP) markers were used to verify the segregation of the genus Cassia L. senso lato into three distinct genera namely Chamaecrista Moench., Senna P. Mill. and Cassia L. sensostricto Eighteen representatives of the three taxa were characterized using the molecular markers. 25 RAPD, six ISSR primers and six AFLP primer combinations resulted in the amplification of 612, 115 and 622 bands (loci) respectively. Most of the loci are found to be polymorphic, showing high degrees of genetic diversity among the different taxa studied. The dendrogram constructed on the basis of the RAPD, ISSR and AFLP data using SHAN clustering, divided Cassia L. senso lato. into three different clusters as Chamaecrista Moench. Senna P. Mill. and Cassia L. senso stricto High bootstrap value revealed that all the clusters were stable and robust. It was observed from the present investigation that these genera have their identity at molecular level, which supports the elevation of the genus Cassia L. senso lato to the level of subtribe Cassiinae and segregation into three distinct genera instead of intrageneric categories.

Genetic Relationships among Native Species and Hybrid Cultivars of Asian Dendrobium (Orchidaceae) Using Amplified Fragment Length Polymorphism Markers

This study addresses the phylogenetic relationships among native species and hybrid cultivars of Asian Dendrobium by amplified fragment length polymorphism (AFLP). The plant materials of this study are composed of 37 accessions belonging to native species in China and 63 accessions proposed to be hybrid cultivars originating from Japan and Korea. Eight AFLP primer combinations produced a total of 1658 fragments with an average of 207 fragments per primer pair, of which 1655 bands were polymorphic. Specific AFLP markers were identified in 29 of 100 tested Dendrobium accessions. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Dice's similarity coefficient matrix and also average similarity of each species and cultivar. The tested 100 Asian Dendrobium accessions were grouped into seven clusters with the similarity coefficient of 0.49. A first cluster consisted of 63 hybrid cultivars, 17 species of section Dendrobium, one species of section Formosae, and one species of section Callista. A second, fourth, and seventh cluster included five, three, and two species of section Dendrobium, respectively. A third group comprised five species of section Formosae. A fifth and sixth cluster contained three and two species of section Callista, respectively. These results indicated that the genetic relationships among tested Asian Dendrobium accessions were related to their origins, morphological classification, flower color, and pedigree, to some extent.

Identification of a Male-Specific Amplified Fragment Length Polymorphism (AFLP) and a Sequence Characterized Amplified Region (SCAR) Marker in Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. is a dioecious species. Currently, there is no method to identify the sex during the juvenile stage that lasts a relatively long time. This study aimed to develop a sex specific Amplified Fragment Length Polymorphism (AFLP) marker for Eucommia ulmoides Oliv. Of a total of 64 AFLP primer combinations screened, primer combination E-ACA/M-CTT produced a 350 bp male-specific marker. This 350 bp AFLP marker was converted into a 247 bp Sequence Characterized Amplified Region (SCAR) marker. Results suggest that the SCAR marker can be utilized for early sexual identification in Eucommia ulmoides Oliv., and it will greatly facilitate future breeding programs.

Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers

Helleborus is a genus of herbaceous perennials belonging to the family Ranunculaceae. Within this genus six sections with a total of 22 species are found. The largest section Helleborastrum contains 16 species for which genetic relationships are still unclear. This study represents the first genetic analysis in the genus Helleborus, including the two newly described species H. liguricus and H. abruzzicus based on multilocus amplified fragment length polymorphism (AFLP) markers with a genome-wide distribution in combination with nuclear DNA content data. Chromosome analyses of roots tips revealed a number of 2 n = 32 for the selected species, which was congruent with previous observations. The nuclear DNA content of Helleborus was estimated by flow cytometry applying propidium iodide staining and varied between 18 and 33 pg/2C, depending on the species. For AFLP analyses, 19 out of the 22 Helleborus species were studied using 10 AFLP primer combinations, resulting in a total of 1109 polymorphic bands among all species including the outgroup. The genetic distances between species varied between 0.034 and 0.330. Based on genetic distances a phenogram using the Neighbor-joining cluster method with bootstrap analysis was calculated. The results support the previously suggested division of the genus into six sections and thereby approve AFLP data to be applicable for phenetic analyses. Moreover, this genetic information is significant for the development of future Helleborus breeding strategies.

Genetic diversity in tetraploid switchgrass revealed by AFLP marker polymorphisms

Switchgrass (Panicum virgatum) is a perennial warm-season grass native to North America that has been identified as a dedicated cellulosic biofuel crop. We quantified genetic diversity in tetraploid switchgrass germplasm collected at Oklahoma State University and characterized genetic relatedness among the collections from distinct regions. Fifty-six tetraploid accessions, including seven upland and 49 lowland genotypes from throughout the US, were examined. The amplified fragment length polymorphism (AFLP) procedure was utilized to generate DNA profiling patterns that were scored visually. Sixteen selective AFLP primer combinations were used to amplify 452 polymorphic bands. The accessions' genetic similarity coefficients, UPGMA (unweighted pair-group method with arithmetic averaging) cluster analysis and principle coordinate analysis, were performed. The upland and lowland accessions clustered according to ecotypes, with one exception (TN104). Genetic similarity coefficients among the accessions ranged from 0.73 to 0.95. Analysis of molecular variance (AMOVA) was performed, showing significant differences between the upland and lowland genotypes. The trnL marker confirmed that TN104 was a lowland genotype, but the trnL marker identification of upland and lowland genotypes was not consistent with the AFLP analysis in two germplasms (Miami and AR4).

Genetic diversity of Aquilegia (Ranunculaceae) species and cultivars assessed by AFLPs

Species of the genus Aquilegia are exceptionally diverse in their floral morphology and color, commonly known as columbine. They are widely planted ornamentals and are highly attractive for hummingbirds. However, little is known about their genetic diversity. We examined the genetic diversity of the species and cultivars using amplified fragment length polymorphism (AFLP) markers. Sixteen EcoRI/MseI AFLP primer combinations produced 327 informative polymorphic bands, with a mean of 20.4 bands scored per primer. Jaccard's coefficient of similarity varied from 0.61 to 0.93, indicative of high levels of genetic variation. Cluster analysis using the unweighted pair group method with arithmetic mean algorithm placed the 64 accessions into two main clusters, each divided into two sub-clusters. The AFLP variability was significantly associated with the geographic origins, as the Asian species and the North American species grouped into two distinct clusters. The genetic diversity found among Aquilegia demonstrated the potential value of Chinese germplasm for cultivar improvement and for widening the genetic basis of breeding programs and breeding material selection. We concluded that AFLPs are informative and can provide significant insights for genetic diversity research in columbine species.

English

‘Waan chak modlook’ (Curcuma comosa Roxb.) is an important medicinal plant of Thailand, but the high similarity in morphological characteristics observed amongCurcuma spp. may cause confusion in its utilization. The amplified fragment length polymorphism (AFLP) marker was used to identify and elucidate the phylogenetic relationships among 97 accessions of ‘Waan chak modlook’ collected throughout Thailand. Nine AFLP primer combinations generated a total of 202 bands, of which, 158 bands were polymorphic, with an average of 17.56 bands per primer pair. Pairwise similarity estimated between all samples ranged from 0.39 to 1.00 with an average of 0.67. The phylogenetic tree derived from AFLP data showed that the ‘Waan chak modlook’ accessions were divided into five clusters. Based on morphological characterizations, all samples could be assigned to four species: Curcuma sp.; Curcuma latifolia Rosc.;Curcuma elata Roxb.; and C. comosa. The results indicated that there were otherCurcuma species that were misused as C. comosa. The DNA fingerprint data along with the morphological data provided the keys for the accurate identification of C. comosa from the other three related species.   Key words: Curcuma comosa, AFLP, DNA fingerprinting, Waan chak modlook, genetic diversity.

Improvement of the recessive genic male sterile lines with a subgenomic background in Brassica napus by molecular marker-assisted selection

Both the pollination control system and genetic distance are major factors in the utilization of crop heterosis. The recessive genic male sterile line (RGMS) 7-7365A (Bnms3ms3ms4ms4) has been widely applied to hybrid seed production because it can generate a completely male sterile population by crossing with the 7-7365C temporary line (Bnms3ms3rfrf). In this study, the sterile genes of 7-7365A were transferred to the new Brassica napus lines 7-749 and 7-750 with a high content of subgenomes by backcross breeding. We used the amplified fragment length polymorphism (AFLP) technique combined with bulk segregant analysis (BSA) to identify markers linked to the BnMs4 gene. Twelve AFLP markers linked to the BnMs4 gene were identified. Of them, SA06MG09 and P08MG16 were the closest makers, which were on either side of the gene at a distance of 0.9 and 0.8 cM, respectively. Twenty AFLP primer combinations were used to screen the F2, BC1F3, and BC2F4 populations from the breeding program, and the markers linked to the BnMs3 and BnMs4 genes were used to screen the BC2F4 populations. As a result, we obtained two types of improved sterile lines, 7-749A and 7-750A, and their indexes of subgenomic components (ISG) were 44.2–49.8 and 20.2–26.6%, respectively. The combining ability analyses of seed yield character were conducted in the crosses from the three sterile lines and ten restorers within a random block design in three environments for two successive years. The general combining ability (GCA) of the two improved sterile lines were significantly higher than the GCA of 7-7365A in every environment tested. The two improved sterile lines had stability in seed yield, and they will be used in the future for hybrid seed production.

Detection of genomic changes in transgenic Bt rice populations through genetic fingerprinting using amplified fragment length polymorphism (AFLP)

A highly precise molecular marker technique, Amplified Fragment Length Polymorphism (AFLP) was used to characterize the changes in genomic structure occurred due to introduction of foreign gene and/ or due to the induced tissue culture stress during the development of various transgenic Bt rice populations. The transgenic rice plants having complex to simple Bt gene and selectable marker gene organizations were selected from the six insect resistant Bt rice lines. Our results clearly demonstrate that integration pattern, extent of rearrangement of foreign DNA and method of transformation may influence the genomic changes in transgenic rice populations. Eleven of fourteen AFLP primer combinations tested, generated distinct scorable banding pattern, which were further used in this study. Three primer combinations E-TA / M-CTT (IRRI-NPT), E-AG / M-CAC (Tulasi) and E-AA / M-CAG (IR 68899B) produced only monomorphic bands in all the transgenic and control rice plants .A percentage of 0.61% of 430 million bases of haploid rice genome were examined by the use of 11 AFLP primer combinations employed in this study. The DNA fingerprints generated by AFLP analysis, of each Bt rice population was compared with their respective non-transgenic control and was found to be > 97% similar. This indicated that the introduction of Bt gene into the genome of six rice varieties showed few genomic changes. The comparison shows that fewer changes were observed among the transgenic plants developed by Agrobacterium infection than that of developed through particle bombardment. Transgene integration pattern and their copy number were associated with the extent of genomic changes observed in the transgenic Bt rice varieties. AFLP fingerprints of the six transgenic rice varieties evidenced few, but consistent polymorphic bands between the transgenic individuals with different PI values. Relationships among the transgenic populations with their control rice plants were expressed in the form of dendrograms.

A genetic linkage map of the Japanese eel ( Anguilla japonica) based on AFLP and microsatellite markers

The first genetic linkage map of the Japanese eel ( Anguilla japonica) was constructed based upon Amplified Fragment Length Polymorphism (AFLP) and microsatellite (STR) markers with an F 1 pseudo-testcross strategy. The segregation of 106 microsatellite loci, which included 74 newly developed, and 463 polymorphic loci identified from 56 AFLP primer combinations was studied in 46 F 1 individuals derived from a single female and a male. A total of 319 markers (99 STRs + 220 AFLP) are placed on the female map and 314 markers (91 STRs + 223 AFLP) on the male map. The female map spans 1732.4 cM (Kosambi) in 22 linkage groups with an average spacing of 7.2 cM; the male map spans 964.9 cM in 22 linkage groups with an average spacing of 6.3 cM. The average ratio of male:female recombination rates was 1:2.11. The estimated coverage of the genome was 82.4% for the female map and 74.0% for the male map. This map is the first linkage map in the order Anguilliformes and provides a basis for mapping quantitative trait loci (QTL) and for breeding applications.

Allogyogenetic Progeny are Produced from a Hybrid Abalone Cross of FemaleHaliotis diversicolorand MaleHaliotis Discus Discus

ABSTRACT Interspecific hybrid families of female Haliotis diversicolor × male H. discus discus were produced and analyzed using amplified fragment length polymorphism (AFLP) technology to reveal the genetic makeup of F1 progenies. The survival rates of the hybrid F1 were very low, ranging from 0–0.13%. Twenty hybrid F1 from 3 families along with 3 different female parents and their common male parent were analyzed with 3 AFLP primer combinations. In total, 266 markers were detected. Genetic relationships among the progenies and the parents were evaluated by generating a similarity and genetic distance matrix. The genetic divergence between Haliotis diversicolor and Haliotis discus was at a high level, with genetic distance ranging from 1.471–1.492. The AFLP band patterns of hybrid F1 progeny were similar to those of the female parents, but were quite different from that of the male parent. The mean genetic distance between hybrid F1 and their female parents were 0.024–0.039, slightly less than that among ...

Molecular identification of interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin using amplified fragment-length polymorphism and microsatellite markers

Interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin were produced in this study. The hybridity of the interspecific hybrids was confirmed by using the methods of amplified fragment-length polymorphism (AFLP) and microsatellite [simple sequence repeats (SSR)] markers. Five AFLP primer combinations were used to develop the AFLP profiles of H. discus hannai, H. gigantea and their reciprocal hybrids. AFLP analysis revealed that genetic variations of H. discus hannai and H. gigantea were relatively diverse and each species holds species-specific bands. The AFLP profiles of reciprocal hybrids showed that all of the hybrids inherited bands specific to H. discus hannai and H. gigantea. Of a total of 20 microsatellite loci, which were selected from H. discus hannai microsatellite markers evaluated, eight loci were polymorphic in H. gigantea samples, with an average of 3.375 alleles per locus. Preliminary screening showed that, two of these eight microsatellite loci (Awb002 and Awb022) could be used as species-specific markers to distinguish the hybrids and their parental species. Simple sequence repeats analysis showed that the reciprocal hybrids inherited one allele from each parent for both of the two SSR loci investigated. These data strongly suggest that the induced interspecific hybrid is a true hybrid between H. discus hannai and H. gigantea.

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.

Musa Genetic Diversity Revealed by SRAP and AFLP

The sequence-related amplified polymorphism (SRAP) technique, aimed for the amplification of open reading frames (ORFs), vis-â-vis that of the amplified fragment length polymorphisms (AFLP) were used to analyze the genetic variation and relationships among forty Musa accessions; which include commercial cultivars and wild species of interest for the genetic enhancement of Musa. A total of 403 SRAP and 837 AFLP amplicons were generated by 10 SRAP and 15 AFLP primer combinations, of which 353 and 787 bands were polymorphic, respectively. Both cluster analysis of unweighted pair-grouping method with arithmetic averages (UPGMA) and principal coordinate (PCO) analysis separated the forty accessions into their recognized sections (Eumusa, Australimusa, Callimusa and Rhodochlamys) and species. The percentage of polymorphism amongst sections and species and the relationships within Eumusa species and subspecies varied between the two marker systems. In addition to its practical simplicity, SRAP exhibited approximately threefold more specific and unique bands than AFLP, 37 and 13%, respectively. SRAP markers are demonstrated here to be proficient tools for discriminating amongst M. acuminata, M. balbisiana and M. schizocarpa in the Eumusa section, as well as between plantains and cooking bananas within triploid cultivars.

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS AND AGRONOMIC TRAITS ANALYSIS PROVIDE STRATEGIES FOR IMPROVEMENT OF BITTER GOURD (MOMORDICA CHARANTIA L.)

Some reports on genetic diversity based on morphological characterization on bitter gourd (Momordica charantia L.) are available; no work has been conducted to estimate genetic diversity using amplified fragment length polymorphism (AFLP) markers in accessions of south-China type. In the present study, 36 genotypes of M. charantia including few commercially cultivars, collected and separated from different regions accorded with the target of the breeding of bitter gourd of south-China type, were analyzed for diversity both at morphology and markers levels. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was not in consonance with the relative agronomic traits. Among 64 random decamer primers screened 8 were polymorphic and informative enough to analyze all the 36 genotypes, which yielded a total of 1142 bands, of which 992 (87%) bands were polymorphic. The polymorphic bands per AFLP primer combination are 82-89%. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.47 to 0.87, indicated the genetic diversity of the examined 36 accessions. Moreover, putative AFLP loci defined genetic relationships that allowed for partitioning of traits into two distinct groups after cluster analysis. The cluster patterns based on agronomic traits and molecular variation was different, which provided for the development of strategies for genetic analysis and crop improvement in the species.

Genetic Diversity of Common Carpetgrass Revealed by Amplified Fragment Length Polymorphism Markers

Common carpetgrass [Axonopus fissifolius (Raddi) Kuhlm.] is a perennial, warm‐season grass. Its low maintenance attributes make it a desirable turfgrass species for use in the southeastern United States. Information on the extent of genetic diversity at the molecular level for common carpetgrass germplasm is limited. Accordingly, the objectives of this study were to assess the genetic diversity and relatedness among 59 common carpetgrass accessions, including 48 accessions collected from the southeastern United States, one from Hawaii, one from the NPGS, and another nine derived from commercial seed (seed‐derived) using fluorescence‐labeled amplified fragment length polymorphism (AFLP) markers. Twenty selected AFLP primer combinations generated a total of 1046 amplification bands, of which 469 (44.1%) were polymorphic. Genetic similarity coefficients among accessions ranged from 0.807 to 0.980. A low level of genetic diversity (Shannon's information index = 0.24, gene diversity = 0.16) was observed for this collection. Naturalized accessions had relatively higher genetic diversity than seed‐derived accessions. Accessions from both sources were randomly distributed throughout the dendrogram with no definite geographic or source patterns. More comprehensive sampling throughout its center of origin is needed to increase the genetic diversity of common carpetgrass for breeding.

Analysis of Genetic Diversity in Pongamia [Pongamia pinnata (L) Pierrre] using AFLP Markers

In recent years, Pongamia has been considered as important renewable source of biodiesel, however not much molecular information is available in this species. Molecular characterization of this legume tree will enhance our understanding in improving the optimal yields of oil through breeding and enable us to meet the future demands for biodiesel. To assess the molecular genetic diversity in 46 Pongamia pinnata accessions collected from six different states of India, amplified fragment length polymorphism (AFLP) marker system was employed. Five AFLP primer combinations produced 520 discernible fragments, of which 502 (96.5%) were polymorphic. AFLP primer informativeness was estimated evaluating four parameters namely polymorphism information content (PIC), effective multiplex ratio (EMR), marker index (MI) and resolving power (RP). In total, 51 unique fragments were detected of which 19 unique fragments were observed with primer combination E-ACG / M-CTA. Although neighbour joining (NJ) method did not group accessions strictly according to their region of collection, a good level of genetic diversity was observed in examined germplasm. However, accessions collected from Karnataka showed comparatively higher diversity than accessions from other states. The diverse accessions identified in this study may be useful in Pongamia pinnata improvement to meet the future demands of biodiesel.