Application of inter simple sequence repeat (ISSR) markers to plant genetics.
ABSTRACT Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)n, can be made with a degenerate 3'-anchor, such as (CA)8RG or (AGC)6TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with 32P or 33P via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.
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ABSTRACT: Green algae, Spirogyra (Chlorophyta), are found in a wide range of habitats including small stagnant water bodies, rivers, and streams. Species identification of Spirogyra based on morphological characteristics has proven to be a difficult process. An accurate identification method is required to evaluate genetic variations. This study is aimed at investigating the molecular profiling of 19 samples of Spirogyra from northern and northeastern Thailand. The morphological characteristics of each sample were recorded, viz. cell dimensions (width and length), along with the number and arrangement of chloroplast spirals/pyrenoids. With regard to a correlation of the biological and ecological parameters, conductivity was clearly significantly related to the number of pyrenoids. While DO is negatively related to the number of chloroplast spirals. Molecular studies with 10 ISSR primers were amplified to examine the DNA fingerprints. Morphological characters were determined to be significantly different by revealing 5 traits (P< 0.05) for all specimens. In addition, the DNA markers of all specimens were investigated using 10 ISSR primers. The results show that the PCR technique amplified 108 fragments. An analysis of the DNA fragments grouped all samples by ISRR-PCR, which were then separated into two groups according to their distribution.Saudi Journal of Biological Sciences 10/2014; · 0.74 Impact Factor
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ABSTRACT: Microdochium majus and Microdochium nivale are two of fungal pathogens that cause Fusarium head blight (FHB) in wheat, and have also caused pink snow mold in eastern Hokkaido, Japan. With the aim of assessing levels of genetic variation and population structure, 172 isolates of these Microdochium species obtained from five populations of infected wheat seeds were first classified into each species using polymerase chain reaction (PCR) amplification with specific primers. In total 165 (95.9 % of all isolates) and seven isolates (six of Tokachi populations and one of Abashiri populations) were identified as M. majus and M. nivale, respectively, indicating that M. majus was predominant and the main causal pathogen of FHB in this area. Inter-simple sequence repeat (ISSR) analysis showed that the total genetic diversity was 0.023 when estimated by Nei’s gene diversity index within the five populations dominated by M. majus. An AMOVA analysis also showed that 86.74 % of the total genetic variation was within populations and 13.26 % among populations. These results indicated that little genetic differentiation occurred among the five populations of M. majus. Based on the unweighted pair group method of cluster analysis using the ISSR data, all isolates were identified as one of eight haplotypes in M. majus or six haplotypes in M. nivale, allowing the construction of a dendrogram with two clades corresponding to each species. There was no correlation between the clustering of isolates and their geographic distribution on the tree. These findings show that migration is likely playing an important role in the population biology of M. majus, providing some support for the prediction of epidemics of fungicide resistant strains within populations of the FHB pathogen.European Journal of Plant Pathology 12/2014; 140(4). · 1.71 Impact Factor
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ABSTRACT: Fennel (Foeniculum vulgare Mill.) is an important medicinal plant with used for various purposes in different industries. In this study 25 different ecotypes of fennel from all over Iran were collected and their genetic diversity studied by seven ISSR primers. Seven ISSR primers generated 52 amplified fragments, of which 49 were polymorphic. The highest similarity coefficient among the ecotypes was between Chahestan and Haji abad whereas the minimum similarity coefficient observed between Fozveh and Moqan. In most cases, classifications were consistent with their geographical distribution for some ecotypes (like Givi and Khalkhal in close distance) and although with similarity in climate (like Damavand and Alamot with same climate). This study revealed that ISSR marker could properly separate these ecotypes based on geographical distribution and similarity in climates and showed the wide genetic diversity among Iranian fennels. In term of conservation program, it is highly recommended at least one conservation program for several accessions with near genetical distance should be conducted.Journal of Agricultural Science. 10/2012;