Polymorphic ISSR Research Articles

ISSR Fingerprinting to Ascertain the Genetic Relationship of <i>Curcuma sp.</i> of Tripura

Molecular fingerprints of four different species of Curcuma, viz., C. amada, C. caesia, C. longa and C. zedoaria, found in Tripura were developed using Inter Simple Sequence Repeats. Twenty ISSR primers generated 116 loci amplified in the range of 200 - 5000 bp with an average of 5.8 alleles and 1.6 effective alleles per locus. The percentage of polymorphic band was found to be 86.29 with an average of 5.15 per primer. Based on UPGMA algorithm these four species are placed in two different clusters that validate the classification based on external and internal morphological characters. The polymorphic ISSR markers generated from this study will be useful for understanding the genetic relationship of different species of the genus Curcuma.

Specific biological features and genetic variation of Chinese sleeper Perccottus glenii (Odontobutidae) in aquatic bodies of Tyumen oblast

Specific biological features, morphology, and genetic variation of an invasive species, the Chinese sleeper Perccottus glenii, in several lake and river ecosystems of the Tyumen oblast have been studied. This species displays relatively high rate of linear and weight growths. The main feed of the Chinese sleeper in both the river and lake ecosystems is the chironomid larvae. The diet is richer in the river ecosystem; predation plays an important role; and cannibalism is observed. As has been shown, the qualitative and quantitative characteristics of the Chinese sleeper infestations with local helminth species are by one order of magnitude lower as compared with the native fish species. A significant infestation rate is observed only for Nippotaenia mogurndae, a parasite specific for the Chinese sleeper. The population of this introduced species displays a high genetic variation for DNA markers. The rate of polymorphic ISSR bands is 73% and Nei’s genetic diversity is 0.26. The Chinese sleeper populations in the not directly connected river and lake (distance, 90 km) display a genetic identity of 99.5%, suggesting the absence of genetic differentiation of local populations, explainable by their recent origin from a single source of invasion. Morphological differences of the Chinese sleeper in different ecosystems appear on the same genetic basis and suggest a wide reaction norm characteristic of this species, underlying its ecological plasticity.

Additivity of ISSR Markers in Natural Hybrids of Related Forest Species Bromus benekenii and B. ramosus (Poaceae)

AbstractThe co-occurrence of hybrids and parental species in similar ecological niches poses a question on the role of traits additivity and overdispersion (emergence of new traits) in microevolutionary processes. We analysed genetic polymorphism ofBromus benekenii,B. ramosusand the spontaneous hybridB. benekenii×B. ramosusin sympatric and allopatric parts of the species distribution in Europe, based on non-coding regions of the taxon genomes (ISSR genetic fingerprinting). We tested 68 individuals in 7 populations, including a hybrid population in N France. Altogether 233 polymorphic ISSR bands (loci) were obtained. We found that the parent species were genetically distinct and the hybrids had an additive pattern of ISSR bands found in the putative parental species (NMDS, STRUCTURE); however, there was evidence of introgression towardsB. ramosus(NEWHYBRIDS, UPGMA classifications, Nei's D genetic distance).Bromus benekeniihad 72,B. ramosus21 and the hybrids 9 private bands (genetic overdispersion), probably resulting from the rearranged genomes. Based on its low genetic divergence index DW, the hybrid population seems to be at a young age. We argue that in the face of anthropogenic landscape transformations favouring secondary contacts, the hybrids may competitively replace the parental species in sympatric areas.

Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers

Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.

A comparative study of Inter Simple Sequence Repeat (ISSR), Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) loci in assessing genetic diversity inAmaranthus

Genetic diversity in a set of 31 Amaranthus accessions was analysed using RAPD, ISSR and SSR markers. Polymorphic Information Content (PIC) values of the polymorphic RAPD, SSR and ISSR markers ranged from 0.067 to 0.471, 0.178 to 0.452 and 0.11 to 0.362 with an average of 0.229, 0.371 and 0.441 per primer, respectively. Gene diversity for RAPD, SSR and ISSR markers respectively ranged from 0.032 to 0.487, 0.11 to 0.362 and 0.104 to 0.384.The similarity metrics values for the accessions ranged from 0.58 to 0.98 with RAPD, 0.70 to 1.00 with SSR and from 0.44 to 0.88 with ISSR markers. The dendrogram generated by the markers had five major clusters in case of RAPD markers, two distinct clusters in caseof SSR markers and sixclusters in caseof ISSR markers. Amongst all, ISSR markers provided greater confidence for the assessment of genetic diversity in comparison to SSR and RAPD markers. All the accessions studied were found genetically diverse which may be useful in breeding for improving the genotypes of Amaranthus.

A Strategy to Investigate the Intravarietal Genetic Variability in Vitis vinifera L. for Clones and Biotypes Identification and to Correlate Molecular Profiles with Morphological Traits or Geographic Origins

Grapevine is the most economically important and widely cultivated fruit crop in the world. Molecular markers have been used on Vitis vinifera to distinguish among both varieties and clones. Microsatellites are used to fingerprint varieties and several other techniques, reported in many papers, are used to analyze the differences among clones, but it is not available in the literature as a well defined strategy to screen a large number of Vitis cultivars. In fact, it is often necessary to use different techniques to investigate the genetic variability in different grapevine varieties and a proposed technique is used to study a cultivar, which is often not suitable for either the study of another cultivar or compare the genetic relationship among various cultivars. We describe here a strategy used for the analysis of several grapevine cultivars to describe a universal method to obtain DNA polymorphisms of Vitis vinifera genotypes from the same cultivar by using amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), microsatellites AFLP (M-AFLP), and ISSR molecular markers. The strategy here adopted permitted both to identify different biotypes (i.e., Primitivo), accessions (i.e., Garnacha tinta), and clones (i.e., Callet, Manto Negro, Moll) among the variability of same variety and to correlate the genetic differences to their geographical origins (i.e., Garnacha tinta; Malvasia nera di Brindisi/Lecce) or morphological traits (i.e., Malvasia of Candia). Here is also described the application of the protocol that allows to highlight the genetic variability accumulated during centuries of cultivations and selections of the same variety in different environments by vine growers.

A novel set of highly polymorphic chloroplast microsatellite and ISSR markers for the biofuel crop Jatropha curcas

A novel set of polymorphic ISSR and chloroplast microsatellite markers were identified to mark the genetic relationship among different accessions of Jatropha curcas. A total of 12 polymorphic ISSR primers used, amplified a total of 26 fragments among ten different accessions of J. curcas. The cross-species amplification of microsatellite loci is timesaving as well as a cost-effective approach for developing locus specific markers for new species. Further, in an attempt to reveal a high degree of polymorphism and genetic variation among different accessions of J. curcas chloroplast microsatellite primer pairs developed for Acorus calamus (Acoraceace) were examined for cross-species amplification and validation among them. Out of 18 cpSSRs, six (33.33%) were identified as resulting in good amplifications and polymorphism. The number of amplified alleles per cpSSR primer pair ranged from 2 to 4 with the total number of 18 size variants as whole. The 18 size variants at 6 loci among 10 accessions were combined in 4 different haplotypes. The total chloroplast DNA diversity (HT) and the differentiation (GST) were found as 0.700 and 0.560 respectively. Whereas, Shannon's information index (I), Nei's genetic diversity among different accessions of J. curcas using ISSR and cpSSR markers was estimated at 0.33±0.28; 0.22±0.19 and 0.94±0.35; 0.55±0.18 respectively. The genetic relationship among the accessions was established using UPGMA cluster analysis, which were grouped into two major clusters denoted as cluster-I and cluster-II. The implication of the results in devising the importance of a identified novel set of markers for the genetic relationship and genetic diversity analysis in J. curcas is discussed.

Life on the edge – High levels of genetic diversity in a cliff population of Bertya ingramii are attributed to B. rosmarinifolia (Euphorbiaceae)

Wind-facilitated migration of new genotypes into small, geographically disjunct populations should buffer them against local extinction. Bertya ingramii, a monoecious, wind-pollinated shrub, is restricted to three populations in a 4 km 2 area in eastern Australia. Populations are separated by deeply dissected gorges where it is unlikely that seeds are exchanged but where wind movement may facilitate pollen dispersal. Using 156 highly polymorphic ISSR markers, we found moderate genetic variation within and among populations of B. ingramii and less genetic diversity in a nearby and small population of the widespread Bertya rosmarinifolia. The smallest population of B. ingramii (<30 plants) had the highest genetic variation (65% polymorphic markers, Shannon Information Index = 0.30). AMOVA and a Bayesian analysis showed that molecular variance was equally distributed within and among populations suggesting that gene flow is as limited within as in among populations. Genetic distances between populations were only weakly explained by their relative geographic distances (mantel test, R 2 = 0.21, P = 0.001) but the distribution of private bands, the departure from Hardy–Weinberg equilibrium, and a UPGMA tree showed that the smallest population of B. ingramii was generally more similar to an upstream population of B. rosmarinifolia. Thirty-eight percent of bands in this small population of B. ingramii were exclusively shared with B. rosmarinifolia. This covert hybridisation may have been an ancient event but may be responsible for contemporary declines in germination and establishment in B. ingramii. The conservation implications are amplified by the endangered status of B. ingramii.

Processes affecting genetic structure and conservation: a case study of wild and cultivated Brassica rapa

When planning optimal conservation strategies for wild and cultivated types of a plant species, a number of influencing biological and environmental factors should be considered from the outset. In the present study Brassica rapa was used to illustrate this: to develop Scandinavian conservation strategies for wild and cultivated B. rapa, DNA-marker analysis was performed on 15 cultivated and 17 wild accessions of B. rapa plus 8 accessions of the cross compatible B. napus. The B. rapa cultivars were bred in Sweden and Finland in 1944-1997 and the wild B. rapa material was collected from Denmark, Sweden and United Kingdom. The B. napus accessions were bred within the last 20 years in the Scandinavian countries. Results were based on scoring of 131 polymorphic ISSR markers in the total plant material. A Bayesian Markov chain Monte Carlo (MCMC) approach implemented in NewHybrids demonstrated a clear distinction of B. rapa and B. napus individuals except for three individuals that seemed to be backcrosses. The backcrossed hybrids descended from two Swedish populations, one wild and one escaped. The overall pattern of genetic variation and structure in B. rapa showed that cultivated and wild B. rapa accessions formed two almost separated clusters. Geographical origin and breeding history of cultivars were reflected in these genetic relationships. In addition, wild populations from Denmark and Sweden seemed to be closely related, except for a Swedish population, which seemingly was an escaped cultivar. The study point to that many processes, e.g. spontaneous introgression, naturalisation, breeding and agricultural practise affected the genetic structure of wild and cultivated B. rapa populations.

Comparative analysis of genetic diversity in Indian bitter gourd ( Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd ( Momordica charantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high ( r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M. charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.

Inter and intra-population variability of Jatropha curcas (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers

Jatropha curcas (Euphorbiaceae) is an oil-bearing species with multiple uses and considerable potential as a bioenergy crop. The present investigation has been undertaken to assess the extent of genetic diversity in a representative set of 42 accessions of J. curcas encompassing different crop growing regions in India along with a non-toxic genotype from Mexico as a prelude for utilization of promising and genetically divergent materials in the breeding programmes. Molecular polymorphism was 42.0% with 400 RAPD primers and 33.5% with 100 ISSR primers between accessions indicating modest levels of genetic variation in the Indian germplasm. The within-population variation based on RAPD polymorphism was 64.0% and was on par with the inter-population variation. Polymorphic ISSR markers have been identified that could differentiate the Indian accessions from the Mexican genotype and two of them were converted to SCAR markers. The SCAR primer pair ISPJ1 amplified a 543 bp fragment in all the Indian populations, while ISPJ2 with a specific amplicon of 1,096 bp was specific to the Mexican genotype. Population-specific bands have been identified for the accession from Kerala (2 RAPD markers), Neemuch-1 from Rajasthan (1 each of RAPD and ISSR markers) and the non-toxic genotype from Mexico (17 RAPD and 4 ISSR markers), which serve as diagnostic markers in genotyping. The study indicates an immediate need for widening the genetic base of J. curcas germplasm through introduction of accessions with broader geographical background.

Population Structure and Evolutionary Dynamics of Wild–Weedy–Domesticated Complexes of Common Bean in a Mesoamerican Region

The diversity, structure, genetic flow, and evolutive relationships within and among three wild–weedy–domesticated complexes and a wild population isolated from crops of common bean (Phaseolus vulgaris L.) were analyzed under traditional agriculture, within a region of the Mesoamerican center of domestication. Their diversity was compared with the diversity of a local commercial variety and a breeding line. A morphological marker and 37 polymorphic ISSR loci were used. Total genetic diversity within the wild, weedy, and domesticated populations across the complexes was very similar (0.24, 0.22, and 0.26, respectively). Weedy populations within each complex were more closely related to the domesticated than to the wild populations, suggesting they originated from introgression of wild‐type alleles into the domesticated populations or predominant gene flow from domesticated to wild populations. The wild population in closest proximity to the crop within its complex was more similar to the domesticated and weedy populations of its complex than to the rest of the wild populations, suggesting displacement of the wild genetic diversity by gene flow from the domesticated population within its complex. The high values of differentiation among wild, weedy, and domesticated populations within each complex suggest high autogamy or genetic drift. However, the values of gene flow among populations within the complexes were close to one, theoretically sufficient to counteract genetic drift and/or autogamy. We therefore assume that human selection is the most important evolutionary mechanism for maintaining the high wild‐domesticated differentiation by negative farmer selection of cultivated plants with morphological characters that suggest introgression. Farmers may influence the magnitude and characteristics of gene flow among populations within each complex by the management of the distance between the crops and the wild populations, the diversity within the landraces sown, and the tolerance and harvesting of weedy populations. The high geographic differentiation of the wild populations, together with the local differences in human selection practices and agronomic management, could have generated multiple evolutionary lineages after domestication. Domesticated populations within complexes were between two and four times more diverse than the local commercial variety and four and nine times more diverse than the breeding line. New conservation and breeding strategies are suggested to maintain and use the gene pools from these complexes.

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.