Inter-simple Sequence Repeat Technique Research Articles

Somaclonal Variation in Callus Cultures of Rose Periwinkle, <i>Catharanthus Roseus L</i>. Under Induced Salt and Osmotic Stresses

The experiment was carried out at the laboratories of the National Commission for Biotechnology in Syria during the period 2020-2022 to detect the somaclonal genetic variation occurring in callus cultures at different ages and to compare them with the <em>In vitro</em> growing plants<em> </em>and<em> </em>introduced species of Rose Periwinkle that grew up in the Syrian environment, in addition, exploring the effect of abiotic stresses in causing genetic variations in callus, by the Inter Simple Sequence Repeats technique (ISSR) using 21 primers. Seeds were germinated on an MS nutrient medium devoid of growth regulators; explants were transferred to a propagation medium fortified with Naphthalene Acetic Acid (NAA) (1 mg L<sup>-1</sup>) and 6-Benzyladenine (BA) (2 mg L<sup>-1</sup>). Callus was developed from Rose periwinkle <em>Catharanthus roseus L</em>. leaves using 5C01 medium fortified with NAA and Kinetin (Kin). After that, callus was exposed to gradually increasing levels of PEG-6000-induced osmotic stress and salt stress (NaCl) with a higher level every 30 days. The molecular study results proved the effectiveness of all the used primers (21 primers) in presenting polymorphism between the studied samples at a rate of 92.16%. The cluster analysis showed separating the analyzed samples into two main clusters. The first cluster contained the osmotic stressed callus, while the second cluster separated into two sub-clusters that had the rest of the studied samples. It was found that there were genetic variations between <em>In vitro</em> plants and the callus. In addition, the stressed callus (salt and osmotic) was compared to the unstressed callus, and this confirmed that callus cultures were an essential source of genetic variations, and the ISSR technique was an effective tool for detecting these variations.

Association study between some cultivated species and their wild relatives from Apiaceae, Asteraceae and Brassicaceae families based on molecular and DNA barcoding in Egypt

Concerns about food security worldwide have been raised due to the adverse impacts of climate change on agricultural productivity. Utilizing wild relatives of cultivated plants in breeding schemes is a potential solution to address the abovementioned challenges. Wild relatives refer to indigenous plant species that share genetic ancestry with recently cultivated plants. The capacity of plants to withstand abiotic stresses and enhance crop yield stability in cold and arid environments renders them a valuable prospective resource for breeding programs. Molecular markers are employed to assess the genetic variability of cultivated plants and their wild relatives under investigation. This study employed a set of five primers derived from ISSR (Inter Simple Sequence Repeats) and SCoT (Start Codon Targeted) to assess comparative research between cultivated species and their wild relatives from three families: Apiaceae (Apium gravolens L., A. leptophyllum (Pers.) F.Muell., Daucus carota L.& D. litoralis Sm.), Asteraceae (Lactuca sativa L. & L. serriola L.) and Brassicaceae (B. oleracea L., Brassica tourneforttii Gouan., Raphanus sativus L. & R. raphanistrum L.). The ISSR technique yields approximately 206 bands, whereas the SCoT primers generate approximately 207 bands. The ISSR primers yielded 169 polymorphic bands, exhibiting a polymorphism rate of 82.04%, whereas the SCoT primers produced 169 polymorphic bands with a polymorphism rate of 81.64%. The Brassicaceae has been found to exhibit the highest resolving power (RP) of 48.25, as determined by ISSR. The primary objective of DNA barcoding is to create a communal database of DNA sequences that can be utilized to identify organisms and clarify their taxonomic classification. The findings indicate that the internal transcribed spacer (ITS) regions exhibit high efficacy as a DNA barcoding tool. Overall, the results obtained from two molecular markers and DNA barcoding analyses suggest a significant degree of genetic diversity and relatedness among the cultivated taxa and their wild relatives examined in this study.

Comparative assessment of ISSR, DAMD and RAPD markers for evaluation of genetic diversity of gerbera (Gerbera jamesonii Bolus ex Hooker f.) cultivars

Genetic diversity is the best way to improve available genetic resources for breeding programs in gerbera. In present study, genetic diversity of 22 gerbera cultivars was investigated using inter-simple sequence repeat (ISSR), directly amplified minisatellite DNA (DAMD), and Random Amplified Polymorphic DNA (RAPD) markers. Average polymorphism information content (PIC) for ISSR, DAMD and RAPD markers was 0.40, 0.41 and 0.40, respectively. Cluster analysis for ISSR, DAMD and RAPD divided the cultivars into three distinct clusters. The comparative analysis of the three markers (ISSR, DAMD and RAPD) showed that DAMD had superiority over RAPD and ISSR in characterization of genetic diversity in Gerbera. To our knowledge, this is the first report of a comparison of performance among DAMD, ISSR and RAPD techniques on a set of gerbera genotypes. Overall, our results showed that DAMD markers well represented different genotypes of gerbera diversity.

POPULATION GENETIC DIVERSITY OF CAMELLIA DILINHENSIS ON THE DI LINH PLATEAU OF VIETNAM REVEALED BY ISSR AND SCOT MARKERS

Genetic diversity of naturally distributed Camellia dilinhensis populations on the Di Linh plateau of Vietnam was assessed by the inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) techniques separately and then by combining data from both techniques for satisfactory results. The genetic diversity parameters and genetic distances among individuals found with the ISSR technique (HeI = 0.1420, II = 0.2092, PPBI = 35.22%, GSCsI = 0.765–0.988, and AGSCI = 0.915) are lower than those found with the SCoT technique (HeS = 0.2100, IS = 0.381, PPBS = 52.27%, GSCsS = 0.644–0.985, and AGSCS = 0.866). Based on the combined data from both techniques, the level of genetic diversity of the investigated population is PPB = 43.77%, He = 0.1720, I = 0.2582, and the genetic similarities among individuals are GSCs = 0.764–0.973 with an average of AGSC = 0.894. The SCoT technique differentiated between individuals better and reflected a higher level of genetic diversity in the population than the ISSR technique, but the ISSR technique revealed more loci in Camellia dilinhensis plants than did the SCoT technique.

Diversity of Colletotrichum Species causing Anthracnose Disease from Mango cv. Nam Dork Mai See Tong based on ISSR-PCR

Background: Anthracnose disease caused by the genus Colletotrichum is one of the crucial problems occurring in the field, along with postharvest diseases and affects mango quality in Thailand. In particular, the Nam Dork Mai See Tong cultivar, which is highly susceptible to the disease, is an important product for exportation. Methods: In this research, thirty-seven Colletotrichum species isolate were obtained from anthracnose disease in mango cv. Nam Dork Mai See Tong in three provinces in Thailand. Morphological studies and molecular techniques using species-specific primers were investigated; moreover, the diversity of pathogens was analyzed using PCR amplification of inter simple sequence repeats (ISSRs) with 6 primers, including pathogenicity tests. Result: Morphological studies and molecular detection with species-specific primers revealed that 32 isolates belonged to the C. gloeosporioides species complex and 5 isolates to the C. acutatum species complex. The genetic diversity of pathogens was analyzed. PCR amplification using 6 ISSR primers produced 35 polymorphic bands. These bands were used to construct UPGMA, in which cluster analysis divided the 37 isolates into 3 main groups and 8 subgroups at 61-73% Jaccard similarity coefficient with cophenetic correlation (r) = 0.6781. The ISSR technique showed the greatest genetic variation among isolates collected from different locations. Hence, a study based on ISSR markers was profitable to investigate the phylogenetic relationship of the genus Colletotrichum. Pathogenicity tests revealed that PC006 (Ca) and CS005 (Cg) showed the highest aggressiveness, with disease incidences of 84.74 and 80.90%, respectively. This study indicates that the diversity of pathogenic Colletotrichum species related to mango plantations in Thailand is increasing.

Isolation, identification, and study of the genetic diversity between three entomopathogenic nematodes belonging to Heterorhabditis sp. using ISSR technique

BackgroundEntomopathogenic nematodes (EPNs) are widely used in biological control for soil-dwelling stages of many insect pests that are characterized by their safety to most non-target organisms and to the environment.ResultsThe objectives of the present study were isolation of EPNs from agricultural soil in Egypt for further use in biological control programs and study the genetic variation among them using the molecular marker inter-simple sequence repeats (ISSR). Three out of 25 soil samples collected from fields cultivated with strawberry, tangerine, and pumpkin were positive for the presence of EPNs, using the Galleria baiting technique. Sequencing of the internal transcribed spacer (ITS) region indicated that the isolates obtained belong to Heterorhabditis sp. The ITS sequences were submitted to the National Center for Biotechnology Information (NCBI) and registered under accession nos. MH553165, MH553168, and MH553169. Six ISSR primers were used. The numbers of polymorphic bands were 42 out of 56, and the polymorphism percentage was 75%. The highest number of bands was 12 bands generated by primer ISSR8 followed by UBC-809 (11 bands) while recorded the lowest band number (4 bands), the percentage of polymorphism ranged from 40% (ISSR1) to 100% (ISSR6).ConclusionISSR marker can be considered a good marker to study genetic diversity and detecting the genetic polymorphism among the nematodes species.

ISSR and RAPD primers selection for assessing genetic diversity of Enhalusacoroides (L.f.) Royle

Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) are common PCR-based molecular markers used to study genetic diversity both between and within species. One of important steps in ISSR and RAPD analyses is primer selection to obtain bright PCR products. The aim of this study was to select ISSR and RAPD primers that produced scorable fragments of PCR product for genetic diversity evaluation of seagrass Enhalusacoroides (L.f.) Royle. The study also aimed to compare ISSR and RAPD technique in producing informative fragments. Enhalusacoroides is one seagrass species widely distributed in Indonesia. Seagrass samples were collected from Sanur Coastal Water and from Nusa Dua Coastal Water. DNA was extracted using CTAB method. Ten ISSR primers and eight RAPD primers were tested. Results showed that RAPD was more informative than ISSR. Among ten ISSR primers, only four primers resulted in scorable fragments in all samples, and only one among those primers gave polymorphic pattern. As many as 15 fragments have resulted from three ISSR primers and only 1 fragment was polymorphic. All RAPD primers produced scorable products, and three primers resulted in polymorphic fragments. A total of 42 RAPD fragments were produced and 4 from them were polymorphic fragments

Field Performance and Genetic Stability of Micropropagated Gooseberry Plants (Ribes grossularia L.)

Gooseberry (Ribes grossularia L.) is a small fruit crop producing valuable fruits, which is constantly gaining importance. In vitro propagation of this species can significantly support the production of virus-free planting material and accelerate the introduction of new cultivars to the market. The aim of presented study was to assess field performance and genetic stability of micropropagated plants (MPs) of four gooseberry cultivars, “Captivator”, “Hinnonmaki Rot”, “Invicta”, and “Resika”. The growth vigor and yield of MPs and plants propagated by standard methods from softwood cuttings (ST) were evaluated in a field experiment. Microscopic observations of the number and length of the stomata of MP and ST plants were carried out. Two DNA-based techniques, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR), were used to assess genetic stability of MP plants. For analysis of genetic stability of ST plants, the ISSR technique was applied. For three cultivars, Captivator, Hinnonmaki Rot, and Invicta, the plants’ growth vigor and fruit yield were greater in MP plants than in ST plants. In the case of Resika, most of these parameters were higher in ST plants. Microscopic observations of the stomata indicated a lack of differences in the length between MP and ST plants, while the stomata frequency on leaves of MP plants was higher than that of ST plants. The genetic variability of MP plants, assessed by AFLP, ranged from 0.35% for Hinnonmaki Rot to 2.12% for Resika. The results of ISSR analysis of MP plants showed variability from 0% in the case of Hinnonmaki Rot and Resika to 4% and 8.69% for Captivator and Invicta, respectively. No polymorphism was detected among ST plants of all analyzed gooseberry cultivars.

Assessment of genetic diversity and phylogenetic relationship among grouper species Epinephelus spp. from the Saudi waters of the Arabian Gulf.

Understanding of fish genetic characterization plays a vital role in the conservation and utilization of fish genetic resources of grouper species. The present study was carried out to assess the genetic diversity and phylogenetic relationships in five grouper species, Epinephelus spp. from eastern Saudi Arabian coast using two molecular marker systems, inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. In total, 219 individuals grouper specimens (Epinephelus tauvina, E. coioides, E. bleekeri, E. malabaricus, and E. areolatus) were genotyped with 10 ISSR and 11 SSR selected primers. The ISSR produced 94 DNA fragments, of which 44 were polymorphic with an average of 2.13 fragment per primer. While SSR primers generated 107 alleles, all of them were polymorphic with an average 9.72 per primer. ISSR and SSR techniques demonstrated a high level of gene diversity and genetic distances illustrated by UPGMA dendrograms among the grouper species. The results proved that the SSR markers were highly informative and efficient in detecting genetic variability and relationships of the Epinephelus spp.

Analysis of Genetic Diversity Between Eriobotrya japonica (Thunb.) Lindl. (Rosaceae) Populations Growing in the Aegean Region of Turkey

In this study, we performed a genetic diversity using RAPD (Random amplified polymorphic DNA) and ISSR (Inter-simple sequence repeats) markers for some Eriobotrya japonica populations grown in the Aegean region of Turkey. PCR was performed with all DNA samples and primers with ability of scoring band. Phylogenetic analysis and pairwise genetic distance between the populations were calculated by using the PAUP analysis program. According to the RAPD data, a total of 33 bands were obtained and 21 of them were polymorphic. The closest genetic distance (0.09677) was found between Aydın and Nazilli populations, while the most distant value was found 0.44000 between Salihli and Nazilli populations. According to the ISSR data, a total of 43 bands were obtained and 18 of them were polymorphic. The closest genetic distance (0.09302) was found between İzmir and Aydın populations, while the most distant values (0.48276) were found between Denizli and Nazilli populations. As a result of this study, the rate of polymorphism obtained in ISSR technique was found to be lower than the RAPD technique.

Genetic polymorphism in populations of voles and shrews from the Kronotsky Reserve (Kamchatka Peninsula, Russia)

We studied genetic polymorphism of four mammal species Myodes rutilus, Myodes rufocanus, Sorex isodon, from four localities, the Valley of Geysers, Uzon volcanic caldera, the Death Valley, and the Shore of Kuril Lake. In total, 172 individuals were genotyped using the inter-simple sequence repeat technique. We observed the lowest polymorphism in shrews S. caecutiens. In this species, 68.8 % of bands were polymorphic, and Nei’s genetic diversity (h) was 0.27, while these values in S. isodon were 81.3 % and 0.29, respectively. Populations of M. rufocanus were the most polymorphic among the studied species (P = 91.4, h = 0.34). Polymorphism in M. rutilus from Kamchatka (P = 87.2, h = 0.29) was similar to that from Western Siberia. In addition, we found a high genetic differentiation of rodent populations. The interpopulation component of genetic variability was about 30–40 % (GST = 0.31 in M. rutilus and 0.39 in M. rufocanus). Gene flow among populations of M. rutilus from Kamchatka was two times lower than that of populations of the species from taiga ecosystems in Siberia. Key words: Myodes, Sorex, Differentiation of populations, Genetic variability, ISSR markers, Kamchatka Peninsula

Genetic polymorphism in populations of voles and shrews from the Kronotsky Reserve (Kamchatka Peninsula, Russia)

We studied genetic polymorphism of four mammal species Myodes rutilus, Myodes rufocanus, Sorex isodon, from four localities, the Valley of Geysers, Uzon volcanic caldera, the Death Valley, and the Shore of Kuril Lake. In total, 172 individuals were genotyped using the inter-simple sequence repeat technique. We observed the lowest polymorphism in shrews S. caecutiens. In this species, 68.8 % of bands were polymorphic, and Nei’s genetic diversity (h) was 0.27, while these values in S. isodon were 81.3 % and 0.29, respectively. Populations of M. rufocanus were the most polymorphic among the studied species (P = 91.4, h = 0.34). Polymorphism in M. rutilus from Kamchatka (P = 87.2, h = 0.29) was similar to that from Western Siberia. In addition, we found a high genetic differentiation of rodent populations. The interpopulation component of genetic variability was about 30–40 % (GST = 0.31 in M. rutilus and 0.39 in M. rufocanus). Gene flow among populations of M. rutilus from Kamchatka was two times lower than that of populations of the species from taiga ecosystems in Siberia. Key words: Myodes, Sorex, Differentiation of populations, Genetic variability, ISSR markers, Kamchatka Peninsula

In vitro regeneration and molecular characterization of Jatropha curcas plant

Background and objectiveA simple, rapid, efficient, and reproducible protocol for callus induction and regeneration of plantlets from callus of Jatropha curcas plant was established.Materials and methodsRandomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses were used to determine the genetic variation between the regenerated, micropropagated, and mother plants.ResultsThe highest callus induction percentage from leaf explant was recorded with MS medium containing 2.5 mg/l BA (6-benzylaminopurine) + 1.0 mg/l NAA (1-naphthaleneacetic acid). Leaf-derived callus was grown on medium containing 2.0 mg/l BA + 0.2 mg/l IBA (indole-3-butyric acid) for adventitious shoot regeneration. In addition, using five random RAPD primers with the tested samples produced 117 amplified products out of which 25 were polymorphic bands resulting in 21.37% polymorphism, whereas the five ISSR primers used yielded 116 scorable bands out of which 22 were polymorphic bands producing a polymorphism pecentage of 18.96.ConclusionAn optimized protocol for large-scale production of J. curcas plants using plant biotechnology tools was achieved. RAPD and ISSR techniques would introduce an alternative system for large-scale production and establishment of genetically stable plants.

Assessment of genetic fidelity in microclones of curry leaf plants [Murrayakoenigii (L.) Spreng.]using ISSR markers

Murrayakoenigii (L.) Spreng., commonly called Indian curry leaf, is an aromatic shrub highly valued for its medicinal potentialviz. anticonvulsant, antitumor, anti-inflammatory, antidiabetic, antiviral and diuretic.Its leaves, locally known as curry patta, are used widely for culinary purpose, particularly in India. However, M. koenigii,due to poor rate of fruit set and seeds with short viability period and poor rate of germination, are sparsely distributedin wild, and has attractedresearchers for propagation through biotechnological approaches. In this context, maintaining genetic fidelity is a vital for assessing genetic uniformity in micropropagated plantlets as variations within the progeny can result in serious losses to the end users.therefore, it is necessary to screen them for their genetic makeup whether they are true-to-type or not. Molecular techniques like Inter Simple Sequence Repeat (ISSR) that are not influenced by environmental factors are appropriate tools to analyse genetic fidelity of in vitro propagated plantsas, and generate reliable and reproducible results. In this study, we tried to evaluate genetic fidelity of micro-clones of tissue culture raised M. koenigii using ISSR technique. Twenty five ISSR primers were used to amplify genomic DNA from in vitro raised field grown plants and mother plant. Out of 25 primers screened, a total 465 amplified products were obtained from 10 ISSR primers. Out of which, 55 were monomorphic across the mother plant and its micropropagated progenies of 10 ISSR primers 05 showed profiles identical to mother plant. Similarity matrix based on Jaccard’s coefficient and pair-wise values between mother plant and tissue cultured plant ranged from 0.91 to 1.00, indicating a high degree of genetic fidelity.

Intra- and inter-cultivar genetic variability in Bulgarian tobacco

PURPOSE: The genus Nicotiana is a well-defined group of species of which tobacco (Nicotiana tabacum L.) is the most important crop plant and plays a significant role in the economies of many countries. Recent advances in molecular genetics of the crop allowed the identification of many important genes and their location on chromosomes. However, the genetic analysis of this inbreeding, highly homozygous plant that serves as a model in many other studies is still very much work in progress. METHODS: A large proportion of the tobacco genome is represented by highly repeated DNA sequences, which makes molecular markers, based on them, an obvious first choice in diversity studies. Here we present an assessment with Inter-Simple Sequence Repeat (ISSR) markers of the intra- and inter-cultivar variability in a set of Bulgarian tobacco varieties. RESULTS: The screening of the 4 primers revealed a varying number of bands generated by different primers. The total number of bands varied between 4 and 13. No correlation between the total number of bands and the number of polymorphic bands was observed. Testing with ISSR primers revealed that plants with different DNA profiles can be found in varieties used. The highly similar profiles when E7 primer was used in two of the tested varieties from different production types indicate that the DNA fingerprinting technique may present a particular challenge in this crop. CONCLUSIONS: The present study demonstrates the power of the ISSR technique as a tool for diversity identification in tobacco.

Assessment of Genetic Relationships among Cultivated and Wild Pistachios (Pistacia vera L.) using Molecular Markers

Abstract Iran is one of the main diversity centers and origins of pistachios in the world. Pistachio cultivation spread first within the ancient Persian Empire and then moved gradually westward. Knowledge of the genetic relationships among wild and cultivated varieties of pistachio is important for the efficient utilization of the available germplasm resources. Three molecular marker strategies, namely, inter-simple sequence repeat (ISSR), inter-retrotransposon amplified polymorphism (IRAP), and retrotransposon microsatellite amplified polymorphism (REMAP), were used to study the genetic relationships among 35 pistachio accessions including 15 wild-type genotypes of Pistacia vera and 20 important cultivars from Iran. According to the results, high levels of polymorphism were observed for all three marker systems. REMAP and IRAP techniques had the higher mean values of genetic relationships parameters than ISSR technique. The results from this study showed that the 5′LTR2, Sukkula, Sukkula + UBC855, and 5′LTR2 + UBC811 primers were the most informative and could be used to evaluate the genetic relationships of pistachios accessions. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) properly separated the accessions and divided them into four main groups. The presence of most cultivated genotypes in a group indicates genetic erosion of cultivated pistachio in Iran. Wild-type genotypes of P. vera are located in different clusters indicating the high diversity of the genotypes. The results provide useful genetic information about wild pistachios in northeastern of Iran and indicate that the use of wild pistachios in breeding programs could be useful for generating new genotypes with interesting characters.

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels.

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR-RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.

GENETIC ASSESSMENT IN SOME MEDICINAL PLANTS UTILIZING MOLECULAR MARKERS

The interest in medicinal plants is not only due to the fact that it is a source of food additives, but it is also a major source of medicines. Medical plants contain many important vital ingredients used in the treatment of many diseases. Therefore, medicinal plants are considered a safe source of medical drugs. Many medicinal plants have a significant economic importance to increase their demand. The plant families include many families, such as solanaceae family, which is one of the largest plant families with more than 3000 plant species And the plants of this family contains many important biological components and because of its importance, it was necessary to study and identify these plants by making fingerprinting, detect the molecular genetic markers for this family, study the genetic diversity of these species and determine the relationships between species of this family by using genetic techniques such as the technique of Inter simple sequence repeat (ISSR). The results obtained in the five solanaceae species (Lycium shwaii, Hyoscyamus muticus, Solanum nigrum from Northwestern coast, Solanum nigrun from Saint Katherine, and Nicotiana gluca) indicated that, 6 primers were applied. The HB15 primer which gave 12 bands, showed the highest polymorphism 58.33%, and the 49A primer, which gave 6 bands gave a lowest polymrphism 16.66% between the used plants populations, these results mean that ISSR technique is an powerful tool to make genetic diversity assessment for species.

Genetic diversity analysis of cultivated Korarima [Aframomum corrorima (Braun) P.C.M. Jansen] populations from southwestern Ethiopia using inter simple sequence repeats (ISSR) marker

BackgroundKorarima (Aframomum corrorima) is a perennial and aromatic herb native and widely distributed in southwestern Ethiopia. It is known for its fine flavor as a spice in various Ethiopian traditional dishes. Few molecular studies have been performed on this species so far. In the present paper, the ISSR technique was employed to study the genetic diversity in populations of cultivated A. corrorima.ResultsSeven ISSR primers produced a total of 86 clearly scorable DNA bands. High levels of genetic diversity were detected in cultivated A. corrorima (percentage of polymorphic bands = 97.67%, gene diversity = 0.35, Shannon’s information index = 0.52). Analysis of molecular variance (AMOVA) showed that 27.47% of the variation is attributed to the variation among populations and 72.53% to the variation within populations. The Fst (0.28) value showed a significant (p < 0.0001) genetic differentiation among populations. This was supported by the high coefficient of gene differentiation (Gst = 0.32) and low estimated gene flow (Nm = 1.08). A neighbor-joining dendrogram showed that the thirteen cultivated populations were separated into three clusters, which was in good accordance with the results provided by the two dimensional and three dimensional coordinate analyses. However, the clusters did not reveal clear pattern of populations clustering according to their geographic origin. This could be due to human mediated transfer of genetic material among different localities.ConclusionThe genetic diversity in populations of A. corrorima from the southwestern part of Ethiopia was relatively high. This finding should be taken into account when conservation actions, management policies for the species and site identification for in situ and ex situ conservation strategies are developed. Mizan Teferi II population displayed the highest genetic diversity; this population should be considered as the key site in designing conservation strategies for this crop. In addition, Jimma I and Jimma II populations with lowest genetic diversity, should also be considered due to the putative risk of extinction that they face because of the low genetic diversity.

Research Article Parapatric speciation found in Limnonectes (Dicroglossidae) species in Thailand

Biodiversity stems from speciation, and species identification allows scientists to measure species biodiversity. However, some closely related species, particularly amphibians that choose to inhabit the same environment, are difficult to identify. Juvenile specimens of Limnonectes gyldenstolpei (Lg) and L. taylori (Lt) were used as models to study the speciation process in populations with a parapatric distribution. Samples of the two species were collected from six provinces in the north and northeast of Thailand. Inter-simple sequence repeat (ISSR) data of five loci showed 96 polymorphic bands and species-specific bands of the primers UBC811 (Lg) and UBC824 (Lt). There was limited genetic variation within the same population, 8% in Lg and 2% in Lt, whereas a high genetic variation was observed between populations from different provinces 90% and 97% in Lg and Lt, respectively. An unweighted pair-group method using arithmetic averages (UPGMA) tree was constructed for each population using Jaccard’s similarity efficient. The tree demonstrated genetic differentiation between the two species. Principal coordinates analysis also supported the UPGMA diagram by showing no clear clusters with close genetic distances. The results of the molecular variance analysis within the population were high, indicating that they were genetically similar, and that microhabitat is an important factor influencing genetic distribution. Overall, the present study proves the hypothesis that the distribution of the two species is in a Parapatric form and that the ISSR technique is inappropriate tool to study the differentiation of a species complex.