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: Safflower (Carthamus tinctorious L.) is valued as a source of high quality vegetable oil. 20 ISSR primers were used to assess the genetic diversity of 18 accessions of safflower collected from different geographical regions of Iran. The ISSR primers combinations revealed 57.6 % polymorphism, among 338 genetic loci amplified from the accessions. The sum of effective number of alleles and observed number of alleles were 29.76 and 36.77, respectively. To understand genetic relationships among these cultivars, Jacquards’ similarity coefficient and UPGMA clustering algorithm were applied to the ISSR marker data set. ISSR markers grouped accessions into two main clusters and four sub clusters. Also, the principal coordinate analysis (PCoA) supported the cluster analysis results. The results showed these genotypes have high genetic diversity, and can be used for alternative safflower breeding program.Physiology and Molecular Biology of Plants 05/2013;
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ABSTRACT: A comparison of the efficiency of RAPD and ISSR primers using different genetic parameters like polymorphism percentage, effective alleles per locus (Aep), genetic diversity per locus (Hep), Shannon diversity index (I), polymorphic information content (PIC) and marker index (MI) has been performed in 8 Indian grown Corchorus spp. (2n = 14) of the family Tiliaceae (C. capsularis L., C. olitorius L., C. aestuans L., C. fascicularis Lamk., C. pseudocapsularis L., C. pseudoolitorius I. and Z., C. tridens L. and C. trilocularis L.). The objective of the work is to develop a simple, efficient and cost effective primer based system for quick genetic evaluation of Corchorus germplasms. Result indicated that OPA 03, OPA 05, OPA 06, OPB 03, OPB 06, OPC 05 and OPC 10 among the employed RAPD markers and (GA)12, (CA)8GC and (GATA)4 among ISSR primers are efficient and effective. Further, relatedness between the species has also been noted using principal component analysis (PCA) and cluster analysis by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) with an objective for efficient breeding and crop improvement. Unrooted phylogenetic tree constructed from molecular data of the studied species revealed possible divaricated mode of origin of Corchorus.The Nucleus. 03/2014; 56(1):23-30.
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ABSTRACT: Genetic variation in three populations of Parkia timoriana (DC.) Merr. grown in the Manipur state of northeast India was analysed using inter-simple sequence repeat (ISSR) markers. A total of 30 individual trees representing three populations were sampled and studied using 22 University of British Columbia (UBC set no. 9) primers in the present study. Of the total 22 primers, 19 primers produced distinct, reproducible and well-resolved fragments. Overall, a total number of 111 fragments were generated by the 19 primers and of which, 51 were polymorphic (45.94 %). The average number of loci and polymorphic loci generated per primer were 5.84 and 2.68, respectively. The genetic variation generated by ISSR markers within the three populations studied ranges from 33.33 to 18.92 %. The overall genetic differentiation (Gst) among populations was estimated to be 0.29, and the number of gene flow (Nm) was estimated to be 1.23 per generation between populations. Of the total genetic variance, 70.04 % was attributed to within-population diversity while 4.72 % differences to the among-populations. The genetic similarity across the individuals belonging to the three populations was represented by the dendrogram showing the grouping of the individuals into three major groups which is also supported by the principle component analysis. The present finding asserts the effectiveness of ISSR procedure for assessing genetic variations of P. timoriana populations and provides valuable genetic information that can be utilized for breeding and conservation strategies.Applied biochemistry and biotechnology 11/2013; · 1.94 Impact Factor