ISSR

Background : The main goal of the work was to analyse intraspecific variation in Elettaria cardamomum Maton (cardamom) using genome size, cytological studies and molecular marker data. Nuclear DNA content and molecular marker details furnish data on genome size and genetic diversity respectively among the studied accessions and both complement each other for evolutionary and taxonomic studies.
Results: The relative 2C genome size and total number of base pairs of cardamom was determined through flow cytometric analysis using propidium iodide staining. The nuclear DNA content was estimated in various sections of the species representing individuals from wild and cultivar genotypes following Zea mays L. CE-777 (2C = 5.43 pg) as internal reference standard. Chromosome number from growing root tip was examined following standard protocols. Twenty-six ISSR primers that generated polymorphic bands were used for genetic diversity analysis of the thirty accessions of cardamom. Estimated nuclear 2C DNA content ranged from 2.57 to 3.22 pg demonstrating 1.25-fold variation. The mean amount of 2C nuclear DNA of the cardamom was calculated as 2.87 pg which is equivalent of 2806 Mbp as the diploid genome size. The chromosome number was found to be 2n = 48. Among the thirty accessions of carda-mom studied using ISSR markers, C53 (feral from Bonacaud) showed a very prominent level of genetic diversity and was lowest for C96 (Avinash-I, a released variety from Indian Institute of Spices Research, Kozhikode).
Conclusion: These analyses revealed the existence of genetic variability within the studied cardamom accessions. The plant specimens also differed significantly in their genome size. However, the genetic variability parameters did not show any correlation with genome size.

A successful protocol has been developed for rapid clonal propagation of Aegle marmelos using nodal segments. The explants were cultured on MS medium augmented with different concentrations (0.5-5 mg/l) of BA, Kin and 2ip and combinations of BA (0.5 mg/l) with Kin/2ip (0.5-5 mg/l) and Kin (2 mg/l) with BA/2ip (0.5-5 mg/l). 0.5 mg/l BA induced the maximum number (8.40) of shoots. In vitro raised shoots were rooted on half strength MS medium supplemented with three auxins, nAA, iAA and iBA (0-2.5 mg/l). iBA (2.5 mg/l) was found to be better than iAA and nAA. The regenerated plantlets were acclimatized with 65% survival rate. inter Simple Sequence repeat (iSSr) marker assay was employed to validate the genetic fidelity of these regenerated plantlets. All banding profiles from micropropagated plants were monomorphic and comparable to those of the mother plant, thus ascertaining the true nature of the in vitro raised plants.

The family Orobanchaceae consists of parasitic species. One group of taxonomically problematic species is subsection Minores (Beck) Teryokhin genus Orobanche. The species in this subsection (except O. crenata) have been often assigned to aggregate O. minor because of their great morphological similarity. The aim of this study was to find useful molecular makers for characterization of the biodiversity of subsection Minores, Orobanche in Bulgaria and their phylogenetic relationships. Six species of subsection Minores were examined: O. minor Sm.; O. amethystea Thuill.; O. esulae Pančić var. bulgarica T.  Georgiev, O. pubescens d’Urv. O. loricata Rchb. and O. crenata Forssk. Genomic DNA was isolated from flowering stems and used as a template for ISSR PCR performed with ten different ISSR primers. The ISSR products were separated on agarose gel and visualized by UV-light. The molecular masses of the polymorphic bands were determined and used to fill Boolean matrices that were subjected to cluster analysis. The suitability of each primer for distinguishing each of the studied species was discussed. The resulting cladogram, based on the average Euclidean distances, displayed grouping by species and by geographic populations. This study confirmed that the assignment of the small-flowered species to agg. O. minor has only practical value and is not genetically justified. The study confirmed the independent taxonomic status of the Balkan endemic species O. esulae.

Sixteen Simple Sequence Repeats (SSR) and three Inter Simple Sequence Repeats (ISSR) primers were used to es-timate the genetic diversity and its distri-bution in twenty garlic clones. A high level of polymorphism amongst studied clones was found with both SSR and ISSR markers. The total number of bands that were detected by all used primers was 75 including 6 monomorphic, 5 unique and 64 polymorphic. The percentage of poly-morphism identified by SSR primers were varied between 33.3 and 100. However, all of the studied ISSR primers were pol-ymorphic conferring a 100% of polymor-phism. Results showed that each of the Asa14, Asa17, Asa18 and Asa59 primers generated one monomorphic band of 77, 120, 102 and 113 bp, respectively, in all of the studded garlic clones. Two monomor-phic bands of 104 and 177 bp were gener-ated by using Asa24 primer. Asa17 and Asa59 SSR primers produced only one unique band of 154 (Egaseed 2) and 646 bp (EGA 1), respectively. Two unique bands of 225 and 250 bp were detected for Egaseed 2 (ft) by using HB 13 ISSR pri-mer. The highest similarity value (0.969) was found between AZO 2 and AZO 3, while the lowest value (0.482) was found between AZO 4 and EGA 5 clones. Dendrogram of genetic distances amongst all tested clones showed two distinct ma-jor clusters with overlapping. In general, the present results reveal the importance of using molecular markers to assess ge-netic diversity among such closely related genotypes which were difficult to distin-guish with other markers.

The genetic variation and relationships among 16 pumpkin accessions of Cucurbita moschata & Cucurbita maxima were assessed based on variation in fruit shape, skin color, flesh and size of fruits. Polyacrylamide gel electrophoresis of seed protein and molecular markers revealed by SCoT and ISSR techniques. The SDS-PAGE electropherogram showed 12 bands; two (75 and 145 kDa) were found in accessions (1-8) and one (85 kDa) was found in accessions 9-16.  Other three bands were unique to accessions 1, 2 & 11. Five ISSR primers produced 79 markers ranging in molecular size from 130 to 2140 bp. Six SCoT primers produced 173 markers ranging from 135 to 2660 bp. The ISSR polymorphism among the examined accessions was 100% in the case of primers 49A, 44 B and HB-12, and was 92% for primer HB-15. Similarly, 100% polymorphism was scored for the primers SCoT8, SCoT11, SCoT12 and SCoT14. The lowest polymorphism was 93.94% in case of primer SCoT1. Our data based on protein, RAPD and ISSR data using the SYSTAT version 7.0 program clearly distinguished accessions from each other.

African Journal of Biotechnology Vol. 7 (23), pp. 4230-4243, 3 December, 2008 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2008 Academic Journals ... Analogy of ISSR and RAPD markers for comparative ... Shweta Gupta1, Mani Srivastava1, GP ...

Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegeta-tive propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars 'Aurea' and 'Elegantissima' were cultured on media enriched with six different sources of mac-ronutrients. Media were supplemented with either N 6-benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L −1 BA, 0.1 mg L −1 NAA, and 20–30 g L −1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L −1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for 'Aurea' and 90% for 'Elegantissima'. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymor-phic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197–199 and 184–187 distinct and reproducible band classes, respectively, in 'Aurea' and 'Elegantissima' plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

The purpose of this study was to document three Egyptian Moulokhya genotypes (namely; Fallahy, Saedy and Siewy), differentiate among them and identify their genetic relationships using Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR) and Sequence-Related Amplified Polymorphism (SRAP). The results of 15 RAPD, 15 ISSR primers and 40 SRAP primer combinations showed a total of 233, 239 and 574 reproducible bands, in which 66 (28.3%), 66 (27.6%) and 201 (35%), respectively were polymorphic and 37(15.9%), 65 (27.2%) and 257 (44.8%), respectively were specific markers. RAPD, ISSR and SRAP gave the highest percentages of polymorphism with Saedy genotype, which could be distinguished by one RAPD primer and two SRAP primer combinations. While four RAPD, three ISSR primers and 16 SRAP primer combinations distinguished Fallahy genotype, there were three RAPD, two ISSR primers and two SRAP primer combinations which distinguished Siewy genotype. The highest similarity indices were shown between Fallahy and Saedy genotypes (66%, 64%, 52% and 60%) using RAPD, ISSR, SRAP data and their combinations, respectively. The genetic relationships of the three genotypes as demonstrated by the phylogeny analysis, showed thatSiewy genotype was distinct from the other two genotypes, which were grouped together in one cluster. This implied that the genomic sequences of Siewy genotype varied at the genetic level and suggested that, it can be used as a genetic resource in the breeding programs to produce abiotic stress-tolerant genotypes. In conclusion, thesefindings not only provide a preliminary database for genetic biodiversity and can be useful in the breeding programs, but also are a necessity for production of Egyptian royalties of Moulokhyagenotypes.

Inter-simple sequence repeat (ISSR) analysis and aggressiveness assays were used to investigate genetic variability within a global collection of Fusarium culmorum isolates. A set of four ISSR primers were tested, of which three primers amplified a total of 37 bands out of which 30 (81%) were polymorphic. The intraspecific diversity was high, ranging from four to 28 different ISSR genotypes for F. culmorum depending on the primer. The combined analysis of ISSR data revealed 59 different genotypes clustered into seven distinct clades amongst 75 isolates of F. culmorum examined. All the isolates were assayed to test their aggressiveness on a winter wheat cv. ‘Armada’. A significant quantitative variation for aggressiveness was found among the isolates. The ISSR and aggressiveness variation existed on a macro- as well as micro-geographical scale. The data suggested a long-range dispersal of F. culmorum and indicated that this fungus may have been introduced into Canada from Europe. In addition to the high level of intraspecific diversity observed in F. culmorum, the index of multilocus association calculated using ISSR data indicated that reproduction in F. culmorum cannot be exclusively clonal and recombination is likely to occur.

Ten quantitative agromorpho-economic traits, six inter-simple sequence repeat (ISSR) primers, and three sequenced regions were employed to study intra-specific genetic diversity among twenty-eight accessions of West African and Asian okra (Abelmoschus spp L.) collected from eight geographical regions of Ghana. Pod yield per plant was analysed as dependent variable in relation to other agromorpho-economic traits, showing the correlation and contribution of each trait to crop yield. 50% germination and flowering were the most significant traits followed by plant height and average seeds per plant. Principal coordinate analysis defined three sets of traits, while Agglomerative Hierarchical Clustering (AHC) defined three clusters of the germplasms. ISSR detected very low level of polymorphism among the accessions. Testing the correlation between molecular data and morphological traits using Mantel test showed a significant positive correlation (r-value = 0.71, 0.90) with 50% flowering, fruiting and number of leaves per plant. Eclectic variation between Indiana and the rest of the accessions for both agromorpho-economic traits and molecular markers affirms its potential usefulness as a source of diverse genes for future breeding programmes. Sequencing of regions from all accessions, suggests that they are identical with a common ancestry. Outcomes of this study is timely for an ongoing okra hybridisation programme in Ghana.

Development of a robust set of 18 genomic microsatellite markers from turmeric (Curcuma longa L.) and its effective utilization in estimating the genetic diversity of 20 turmeric accessions are described. A total of 103 alleles were detected with an average of 5.7 alleles per locus. These markers displayed varied levels of polymorphism as evident from its discriminating power ranging from 0.19 to 0.70. The UPGMA cluster analysis of genetic distance values resolved the 20 turmeric accessions into five main groups. Three sets of genetically identical accessions were detected within the analyzed accessions, suggesting a revisit of the germplasm collection strategy based on vernacular identity. The entire grouping pattern of the entities was loose and independent of their geographical origins. These polymorphic SSR markers would be useful for the population genetic studies and germplasm management of turmeric.► Developed a robust set of 18 genomic microsatellite markers from turmeric (Curcuma longa L.). ► Genetic diversity analysis of 20 turmeric accessions collected from different geographical locations of India indicated a loose grouping pattern independent of geographical origins. ► Three sets of genetically identical accessions were detected within the analyzed accessions. ► This study confirms that collecting turmeric accessions based on vernacular identity could result in adding duplicates in germplasm collections. ► These polymorphic markers could be used for genetic diversity of turmeric for generating reliable molecular data for crop improvement of turmeric.

Costus pictus, belonging to the family Costaceae, is one of the valuable medicinal plants with its anti-diabetic property. Despite ever-increasing demand from the pharmaceutical industry, this species is being less exploited at molecular level. Hence, an effort has been made in the present study to characterize the 15 accessions of C. pictus collected from different geographical regions of India through random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. A total of 25 RAPD and 20 ISSR primers were used in the present study. The RAPD analysis generated 343 loci, of which 124 were polymorphic with an average of 4.96 loci per primer. While, ISSR primers produced 177 loci, of which 77 were polymorphic with an average of 3.85 loci per primer. The similarity coefficients ranged from 0.86-0.99, 0.84-0.95 and 0.86-0.96 for RAPD, ISSR and combined RAPD-ISSR, respectively. The UPGMA dendrogram generated using these data showed low level of divergence amon...

The progress technology of molecular markers to genetic diversity has enabled to an increase in our knowledge of rice genetics and our understanding of the structure and behavior of genomes. DNA markers technology becomes the markers of the study of the genetic analysis of rice species, cultivars and genomes, because they are able to assessment the genetic variation more precisely, cheaply and quickly. These techniques in special the use of molecular marker, have been utilized to study of DNA structure divergence in and during the create new sources of genetic diversity, genetic variation and species by introducing appropriate and new characteristic from related grass species and landraces. Development in molecular marker detection systems and in the techniques used to identify markers linked to useful traits has led large advances to be made in recent years. ISSR markers are highly polymorphic and are useful in researches on phylogeny, genome mapping, evolutionary biology, genetic diversity and gene tagging. This review investigator the use of this techniques for the inter simple sequence repeat (ISSR) fordetecting genetic analysis in rice

In agricultural systems, biodiversity includes diversity within species and among species and provides many benefits for production, resilience and conservation. This article aims to study the diversity of Trigonella foenum-graecum L., an annual herb of Leguminosae with a worldwide distributed culture, among thirty eight local populations collected from different Tunisian regions. The variability analysis was based on morphological characters, compositions of minerals in leaves and vitamins in seeds, and molecular profile. Morphological parameters consisted of vegetative and reproductive characters. Mineral analysis concerned sodium, potassium, iron, calcium and magnesium leaves contents. The analyzed vitamins contents in seeds were B1, B9 and C vitamins and molecular study was carried out by ISSR technique. The structure of the studied populations was established by the principal compound analysis (PCA) and by the unweighted pair group method using arithmetic means (UPGMA). Both an...

Aplochiton zebra (peladilla, zebra trout) is a galaxiid fish endemic to Patagonia and the Falkland Islands, where populations are threatened by salmonid introductions. Here we describe the isolation and characterization of 13 polymorphic microsatellite DNA markers that can be used for assessing the genetic variation and conservation status of this species. The variability of these markers was assessed in six rivers in Chilean Patagonia. The number of alleles ranged from 5 to 80 per locus and observed heterozygosities varied between 0.168 and 0.910. A protocol for multiplexing these markers is described.

The genetic variability and relationships among 14 date palm (Phoenix dactylifera L.) accessions representing six Egyptian cultivars were assayed using 27 RAPD and 10 ISSR primers. The level of polymorphism among the 14 accessions as revealed by RAPD and ISSR was 25.2% and 28.6%, respectively. These low levels of polymorphism reflect the narrow genetic background of these accessions. The genetic relationships among the 14 accessions were estimated in terms of similarity using Dice coefficients. The genetic similarity ranged from 96.1% to 99.5% and from 91.2% to 100% for RAPD and ISSR, respectively. The inter-cultivar relationships among the six date palm cultivars based on RAPD and ISSR revealed the highest genetic similarity between the cultivar Bertmoda and each of the cultivars Malkaby and Sakkoty. The RAPD and ISSR based dendrograms clustered the accessions belonging to each of the 3 cultivars Fraihy, Siwi and Gandila in separate groups. However, the reshuffling in the position ...

In order to identify QTLs controlling agronomic traits in 103 F3 families of Barley derived from cross Badia × Kavir was conducted based on randomized complete block design with three replications in 2014-2015 years, at the Faculty of Agriculture and Natural Resource of Gonbad Kavous University. For linkage map construction, 69 ISSR polymorphic alleles and 7 SSR markers were used  that covered 698 cM of the barley genome. 23 QTLs were identified. Among these mapped QTLs, qSW-3 (between ISSR29-7-ISSR31-5) and qSW-4 linked to (ISSR20-1), qSW-4 (between ISSR131-1-ISSR16-4), qSW-5 (between ISSR47-6-ISSR30-3), qSW-6 (between ISSR48-5-ISSR47-7), qSW-6 (between ISSR22-6-ISSR30-1), qSW-7 (ISSR48-1) for spike weight and qGL-4 (ISSR20-1) for grain length identified as major QTLs. It is suggested that Major QTLs should be considered in plant breeding programs and marker-assisted selection.