Application of inter simple sequence (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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- " Polymorphic markers based on short DNA sequences reveal genome variations among expressed and non-expressed regions and are quick, reliable and reproducible. Among the different molecular markers, inter simple sequence repeat (ISSR) markers are rapid, cost effective and do not require any sequence information of the genome under study  or any radioactive labelling based assay. ISSR-polymerase chain reaction (PCR) analysis involves gene amplification of a region between two inversely oriented microsatellites placed at an amplifiable distance. ISSR markers have been used to resolve polymorphisms among plant accessions by generating a large number of markers that target multiple microsatellite loci distributed across the genome  and also among highly related species. "
ABSTRACT: Genetic diversity fosters the quintessence of speciation and species acclimatization of plants in their in situ environment. Members of Cucurbitaceae, an edible and economically vital crop family, have spread across the world, dominating the tropical regions. Thus, a study of the genetic relationships among cucurbit cultivars would throw light onto the extent of diversification among these vegetal crops. The present study endeavours to understand the phylogenetic patterns and relatedness among selected species of cucurbits, using inter simple sequence repeat (ISSR) markers, which are quick, reliable and produce sufficient polymorphisms for large-scale DNA fingerprinting purposes. A total of 117 bands, of which 57 were polymorphic, were amplified by five primers. The phylogram generated on the basis of Jacquards’ similarity coefficient revealed a close genetic relationship between C. maderaspatanus and C. melo, while C. sativus, a member of the same genus, was placed as a distant relative from both species, thereby demonstrating remarkable diversification among members of the same genus.Biotechnology & Biotechnological Equipment 07/2015; DOI:10.1080/13102818.2015.1052760 · 0.38 Impact Factor
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- "Inter-simple sequence repeat (ISSR) is a PCR-based technique that involves the amplification of DNA sequences between simple sequence repeats (SSR) by means of anchored or non-anchored SSR homologous primers (Zietkiewicz et al. 1994). ISSR does not require information about genome sequence in advance and can detect a greater number of polymorphisms than RFLP or RAPD (Godwin et al. 1997). ISSR is therefore an efficient tool for analysing genetic diversity within closely related species (Yu et al. 2008), and for studying genetic populations of fungi (Menzies et al. 2003; Chadha and Gopalakrishna 2007). "
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). DOI:10.1007/s10658-014-0509-3 · 1.71 Impact Factor
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- "Therefore, molecular approaches using the PCR method have been used to resolve and support taxonomic evidence related to various organisms including algae. A number of molecular markers, such as amplified fragment length polymorphism (AFLP) (Vos et al., 1955), rDNA sequences (Wu et al., 2001), and inter-simple sequence repeats (ISSR) (Godvin et al., 1997; Wolfe and Randle, 2001) including microsatellite markers (Widmer et al., 2010), have been applied widely in the identification of the genetic diversity of many living organisms, including green algae (Shen, 2008), such as Entomophthora fungus (Lihme et al., 2009; Alaniz et al., 2009) and Gerbera spp. (Bhatia et al., 2009). "
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; 113(4). DOI:10.1016/j.sjbs.2014.10.004 · 0.74 Impact Factor