Population Genetic Study of Fagopyrum tataricum from Western Himalaya Using ISSR Markers

Inter-simple sequence repeat (ISSR) markers were used to analyze genetic diversity and relatedness of 15 germplasms of Fagopyrum tataricum. Samples representing 75 individuals were collected from a range of altitudes in the Western Himalaya. The 13 ISSR primers revealed 98.1% polymorphism among populations, whereas average polymorphism was extremely low (2.18%) within populations. The coefficient of population differentiation was 0.9750, with limited gene flow (N _m) of 0.0128. The average PIC value of the ISSR markers was high (0.812), with a marker ratio of 0.65 and marker index of 6.66. The genetic diversity of F. tataricum significantly correlated with altitude and gene diversity, Shannon’s index, and the percentage of polymorphic bands. The genetic diversity among populations showed broad genetic base and provided a developmental strategy for crop improvement.     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Genetic variability and structure of Quercus brantii assessed by ISSR,IRAP and SCoT markers

Persian oak (Quercus brantii Lindl.) is one of the most important woody species of the Zagros forests in Iran. Three molecular marker techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) markers were compared for fingerprinting of 125 individuals of this species collected from different geographical locations of north-west of Iran. A total of 233 bands were amplified by 18 ISSR primers, of which 224 (96.10%) were polymorphic, and 126 polymorphic bands (97.65%) were observed in 129 bands amplified by 10 IRAP primers. Besides, 118 bands were observed for all 10 SCoT primers, of which 113 were polymorphic (95.71%). Average polymorphism information content (PIC) for ISSR, IRAP and SCoT markers was 0.30, 0.32 and 0.38, respectively, and this revealed that SCoT markers were more informative than IRAP and ISSR for the assessment of diversity among individuals. Based on the three different molecular types, cluster analysis revealed that 125 individuals taken for the analysis can be divided into three distinct clusters. The Jaccard's genetic similarity based on the combined data ranged from 0.23 to 0.76. These results suggest that efficiency of SCoT, IRAP and ISSR markers was relatively the same in fingerprinting of individuals. All molecular marker types revealed a low genetic differentiation among populations, indicating the possibility of gene flow between the studied populations. These results have an important implication for Persian oak (Q. brantii) germplasm characterization, improvement, and conservation.     

Inter-primer binding site retrotransposon and inter-simple sequence repeat diversity among wild Lens species

Even though lentil has been an important food legume for centuries, genetic studies in lentil are still in their infancy. Genetic diversity and relationships among wild Lens species from Turkey has seldom been investigated. Additionally, a limited number of simple sequence repeat (SSR) markers have been developed for use in breeding and genetic studies of lentil crop. In this study, molecular characterization of 50 accessions mostly from Turkey, belonging to 6 wild and 1 cultivated Lens species, was performed using newly developed inter-primer binding site (iPBS) retrotransposons and inter-SSR (ISSR) markers. The 10 iPBS primers generated a total of 151 scorable bands, of which 150 were polymorphic (99.3%) with an average of 15.0 polymorphic fragments per primer. The 10 ISSR primers detected 138 scorable bands showing 100% polymorphism, with an average of 13.5 bands per primer. The average polymorphism information content (PIC) value for ISSR markers (0.97) was higher than that for iPBS markers (0.90). Lens orientalis was found to be the most diverse species, raising the possibility of wide crosses with cultivated species Lens culinaris. Cultivated varieties also showed high level of polymorphism, at 82.92% and 51.92% with ISSR and iPBS markers, respectively. Lens lamottei and Lens tomentosus were found as the least polymorphic species using both marker systems. The grouping of accessions and species within clusters were almost similar when iPBS and ISSR graphs were compared. Our data also suggested the role of iPBS-retrotransposons as ‘a universal marker’ for molecular characterization of wild and cultivated Lens species.     

Genetic diversity in european and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs).

In this study, the use of inter-simple sequence repeats (ISSR) to assess genetic diversity between cultivated potatoes (Solanum tuberosum subsp. tuberosum) is reported. ISSR technology rapidly reveals high polymorphic fingerprints and thus determines the genetic diversity among potato cultivars. Nine primers were selected according to the number of amplified markers and the level of polymorphism detected. Three primers (GAG(CAA)5, CTG(AG)8, and (AG)8) were used to cluster the 28 potato accessions and 77 polymorphic markers were sufficient to identify all of the accessions. Among the 15 simple sequence repeat (SSR) motifs tested, the most abundant were CAA and AG. Argentinian- and European-grown potatoes were easily distinguished, with a higher level of genetic diversity among potatoes from Argentina. An ISSR study using a limited number of cultivars and very few primers clearly differentiated between all cultivars, thus ISSR was revealed to be a good tool for the genetic identification of potato and for future germplasm-management programs.     

Estimation of genetic diversity in some Iranian cornelian cherries (Cornus mas L.) accessions using ISSR markers

Iran is an important producer of cornelian cherries (Cornus mas L.). Seed propagation and long term human selection have given rise to a great diversity of trees. In this study, ISSR marker was used for genetic diversity evaluation of 40 accessions of cornelian cherries (C. mas L.). With 20 ISSR primers, 171 polymorphic bands were detected with polymorphism ratio range of 80–100%. The average polymorphism information content (PIC) was 0.46, which shows that the majority of primers are informative. The average values of Effective Multiplex Ratio (EMR), Marker Index (MI), effective number of alleles (Ne), Nei's gene diversity (He) and Shannon's information index (I) for all primers were 8.0, 3.75, 1.756, 0.416 and 0.595 respectively. Based on these results, ISSR analysis can be used for the characterization and grouping of cornelian cherry accessions. This is the first study demonstrating that ISSR analyses can be used to differentiate and classify cornelian cherry accessions.     

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.     

Assessment of genetic diversity in Salvadora persica L. based on inter simple sequence repeat (ISSR) genetic marker

Studies on the genetic variation in marginal populations and differentiation between them are essential for assessment of best gene conservation strategies and sampling schemes. In this study, ISSR markers were used to establish the level of genetic relationships and polymorphism 50 genotypes of Salvadora persica collected from 6 different regions of Hormozgan province. The ISSR analysis with 9 anchored primers also generated 105 scorable loci, of which 85 were polymorphic (80.95%). Parameters of genetic diversity and its partitioning were calculated. The genetic analysis demonstrated that S. persica maintain relatively high genetic diversity (PIC was 0.63, Na was 1.27 and Ho and He were 0.15 and 0.17 respectively). The coefficient of genetic differentiation among populations based on FST equaled 0.20. Genetic identities between population's pairs were high (mean I?=?0.88). These values are high as compared with other widespread congener species. Cluster analysis based on the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) revealed 3 main clusters for the ISSR data. The levels of genetic diversity maintained within populations of S. persica indicate that an appropriate sampling design for ex situ safeguarding should capture the majority of genetic diversity found within these taxa to help ensure the long term viability of this species. Furthermore, it could be inferred that ISSR markers are suitable tools for the evaluation of genetic diversity and relationships within the Salvadora persica.     

Genetic diversity in Vicia amoena (Fabaceae) germplasm resource in China using SRAP and ISSR markers

The genetic diversity in 17 germplasms of Vicia amoena L. from north China was analyzed using SRAP and ISSR markers. Three hundred and sixty-eight (94.11%) polymorphic bands (211 and 157 obtained from 20 pairs of SRAP and ISSR primers, respectively) were scored. Although SRAP was more effective than ISSR markers with higher PIC, RP and larger variation range of genetic distance, both the markers were useful for assessing V. amoena genetic diversity. Cluster analysis showed that the 17 germplasms were clustered into 5 groups. The results of principal coordinate analysis supported UPGMA clustering. The germplasms from source areas where the annual average temperature ranged from −1.0 to 5 °C exhibited the highest level of genetic diversity with the highest PPI, I and H. These results have important implications in genome mapping, breeding purposes, and germplasm conservation.     

Comparative Evaluation of SSR and ISSR Markers for Polymorphism in Landraces and Elite Varieties of Rice (Oryza sativa L)

The study aims at comparing SSR and ISSR markers for their ability to identify polymorphism in parents comprising three landraces and two elite varieties of rice. Both the SSR (0.51) and ISSR (0.46) primers showed almost similar values for Poly morphic Information Content (PIC). Maximum PIC values were observed with trinucleotide ISSR primers (0.67) followed by dinucleotide ISSR primers. In addition to the mapped SSR markers, ISSR markers used in the present study produced more polymorphic markers suggesting their utility in the survey of polymorphism between the parental lines belonging to the same sub-species of rice.     

Assessing Genetic Diversity in <Emphasis Type="Italic">Olea europaea</Emphasis> L. Using ISSR and SSR Markers

Olea europaea L. is one of the most economically important crops in the Mediterranean area, and known for having large genetic variability. In order to assess the genetic diversity, DNA from 41 olive cultivars, present in the protected denomination of origin (PDO) region of Trás-os-Montes, was screened using inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. Eleven ISSR primers amplified 135 reproducible bands of which 108 were polymorphic. The percentage of polymorphic bands detected by ISSR was 79%. The highest number of polymorphic bands was obtained by the use of primers UBC807 (15) and UBC809 (16). A total of 67 alleles were detected by six SSR primers, with an average of 11 alleles per primer. The number of alleles per locus ranged from five (ssrOeUaDCA05) to 15 (ssrOeUaDCA03). The observed heterozygosity ranged from 0.219 (ssrOeUaDCA05) to 0.900 (ssrOeUaDCA04), while the expected heterozygosity varied between 0.426 (ssrOeUaDCA05) and 0.887 (ssrOeUaDCA03). The polymorphism information content (PIC) values ranged from 0.392 (ssrOeUaDCA05) to 0.863 (ssrOeUaDCA03). The collection of primers selected gave a reasonable number of amplification products for the genetic diversity analysis. Based on the results, the genetic diversity among 41 olive cultivars is discussed. This study reveals the great importance of guaranteeing the differentiation of olive cultivars and their application for certification purposes.     

Analysis of genetic and virulence diversity of Cylindrocarpon liriodendri and C. macrodidymum associated with black foot disease of grapevine

Inter-simple sequence repeat (ISSR) analysis was used to investigate the genetic diversity of 87 Cylindrocarpon liriodendri and C. macrodidymum isolates, the causal agents of black foot disease of grapevine. The four ISSR primers (GT)₇, (CCA)₅, (CGA)₅ and (TCG)₅, were able to provide reproducible and polymorphic DNA fingerprint patterns and detected relevant genetic diversity in C. macrodidymum. The cluster analysis of ISSR data showed 21 different genotypes that were grouped in seven ISSR groups, from which two corresponded to C. liriodendri (G1 and G2) and five to C. macrodidymum (G3-G7). Nineteen isolates selected from the seven ISSR groups were inoculated in grapevine seedlings obtained from cv. ‘Tempranillo’. The pathogenicity tests detected virulence diversity in C. macrodidymum. The isolates belonging to ISSR groups G6 and G7 were significantly more virulent than the other C. macrodidymum and C. liriodendri isolates.     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

iPBS-Retrotransposons-based genetic diversity and relationship among wild annual Cicer species

Lack of requisite genetic variation in cultivated species has necessitated systematic collection, documentation and evaluation of wild Cicer species for use in chickpea variety improvement programs. Cicer arietinum has very narrow genetic variation, and the use of a wild relative in chickpea breeding could provide a good opportunity for increasing the available genetic variation of cultivated chickpea. Genetic diversity and the relationship of 71 accessions, from the core area of chickpea origin and domestication (Southeastern Turkey), belonging to five wild annual species and one cultivated species (Cicer arietinum) were analysed using iPBS-retrotransposon and ISSR markers. A total of 136 scorable bands were detected using 10 ISSR primers among 71 accessions belonging to 6 species, out of which 135 were polymorphic (99.3 %), with an average of 13.5 polymorphic fragments per primer, whereas iPBS detected 130 bands with 100 % polymorphism with an average of 13.0 bands per primer. C. echinospermum and C. pinnatifidum were the most diverse among species, whereas C. arietinum exhibited lower polymorphism. The average polymorphism information contents (PIC) value for both marker systems was 0.91. The clustering of the accessions and species within groups was almost similar, when iPBS and ISSR NeighborNet (NNet) planar graphs were compared. Further detailed studies are indispensable in order to collect Cicer germplasm, especially C. reticulatum, from southeastern Turkey particularly, from Karacada? Mountain for preservation, management of this species, and to study their genetic diversity at molecular level. This study also demonstrates the utility and role of iPBS-retrotransposons, a dominant and ubiquitous part of eukaryotic genomes, for diversity studies in wild chickpea and in cultivated chickpea.     

High efficiency and reliability of inter-simple sequence repeats (ISSR) markers for evaluation of genetic diversity in Brazilian cultivated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. accessions

Jatropha curcas L. is found in all tropical regions and has garnered lot of attention for its potential as a source of biodiesel. As J. curcas is a plant that is still in the process of being domesticated, interest in improving its agronomic traits has increased in an attempt to select more productive varieties, aiming at sustainable utilization of this plant for biodiesel production. Therefore, the study of genetic diversity in different accessions of J. curcas in Brazil constitutes a necessary first step in genetic programs designed to improve this species. In this study we have used ISSR markers to assess the genetic variability of 332 accessions from eight states in Brazil that produce J. curcas seeds for commercialization. Seven ISSR primers amplified a total of 21,253 bands, of which 19,472 bands (91%) showed polymorphism. Among the polymorphic bands 275 rare bands were identified (present in fewer than 15% of the accessions). Polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.26, 17.86 and 19.87 per primer, respectively, showing the high efficiency and reliability of the markers used. ISSR markers analyses as number of polymorphic loci, genetic diversity and accession relationships through UPGMA-phenogram and MDS showed that Brazilian accessions are closely related but have a higher level of genetic diversity than accessions from other countries, and the accessions from Natal (RN) are the most diverse, having high value as a source of genetic diversity for breeding programs of J. curcas in the world.     

Comparative analysis of ISSR marker polymorphism in populations of yak (Bos mutus) and in F1 hybrids between yak and cattle in the Sayan-Altai region

The genetic variability in seven yak populations from the Sayan-Altai region and in F₁ hybrids between yak and cattle (khainags) was investigated with the help of a technique that involves the use of inter simple sequence repeat (ISSR) markers generated with PCR primers (AG)⁹C and (GA)⁹C. Samples for the analysis were collected in Mongolia, Tuva, and Altai from 2008 through 2012. The examined yak populations differed in in the presence/absence of ISSR fragments, as well as in their frequency. In total, 46 ISSR fragments were identified using two marker systems; the proportion of polymorphic loci constituted 76% and 90% for the AG-ISSR and GA-ISSR markers, respectively. For the total sample of yaks, total genetic diversity (H _t), within-population diversity (H _s), and interpopulation diversity (G _st) constituted 0.081, 0.044, and 0.459 for the AG-ISSR and 0.137, 0.057, and 0.582 for the GA-ISSR markers, respectively. Based on ISSR finger-printing, species- and breed-specific DNA patterns were described for the three groups of animals (yaks, cattle, khainags). For the domestic yak, the species-specific profile was represented by eight ISSR fragments. Genetic relationships between the yak populations, cattle breeds, and khainags were examined with the help of four different approaches used in the analysis of population structure: estimation of phylogenetic similarity, multidimensional scaling, principal component analysis, and cluster analysis. Clear evidence on the differentiation of the populations examined at the interspecific, as well as at intraspecific, level were obtained. Similar (relative), as well as remote (isolated), yak populations were identified. Khainags occupy an intermediate position between yak and cattle. However, the data on the ISSR-PCR marker polymorphism (genome polymorphism, population structure) indicate that part of the analyzed khainag genome was more similar to the yak genome than to the cattle genome.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

Interspecific genetic analysis of orchids in Brazil using molecular markers

Several species of Orchidaceae, one of the largest plant families, are considered endangered throughout South America and legal protection policies are needed so they can be preserved. Inter simple sequence repeats (ISSRs) markers are a potential tool to be used in the phylogenetic reconstruction of closely related species. In this study, we evaluate the polymorphic information content (PIC) and optimum number of ISSR markers (ONM) for five Laeliinae orchids in order to assess genetic diversity. The phylogenetic relationships between Cattleya granulosa, an endangered Brazilian orchid, and four other native Brazilian species (Brassavola tuberculata, Cattleya bicolor, Cattleya labiata and Cattleya schofieldiana) were analyzed for genetic diversity and differentiation. The 11 selected primers generated 166 unambiguous loci (PIC = 0.354; ONM = 156). Of the five studied species, C. bicolor exhibited the highest level of genetic diversity (H _E  = 0.219), while C. labiata exhibited the lowest level (H _E  = 0.132). The percentage of genetic variation among species (analysis of molecular variance) was 23.26 %. The principal component analysis (PCA) of ISSR data showed that unifoliate and bifoliolate species are genetically divergent. Additionally, PCA indicated a close relation between C. granulosa and C. schofieldiana, a species considered to be a variety of C. granulosa by many researchers. Thus, we conclude that ISSR genetic markers are effective in detecting genetic differentiation among orchid species.     

Efficiency of Inter Simple Sequence Repeat (ISSR) markers for the assessment of genetic diversity of Moroccan pomegranate (Punica granatum L.) cultivars

Genetic diversity of 27 Moroccan pomegranate cultivars from three geographical regions of Morocco was screened using 61 Inter-Simple Sequence Repeat (ISSR) markers. Eight tested ISSR primers generated 70 bands of which 61 are polymorphic (87.14%). Nei's genetic distance ranged from (0.12 to 1) with a mean of 0.67. Nei genetic diversity parameters were 0.35 and 0.32 for H_t and H_s, respectively. Our results show considerable gene flow within and among pomegranate groups and a limited differentiation among groups as suggested by values of G_st (0.08) and N_m (5.80) parameters. The Unweighted Pair Group Method with Arithmetic Mean dendrogram and Principal Component Analysis clustered the pomegranate studied into two main clusters and did not show any relationships with the geographical origins of cultivars. Our results showed that ISSR markers can be a useful tool for detecting molecular polymorphism and studying the genetic diversity of pomegranate genotypes.