Inter simple sequence repeat (ISSR) polymorphism and its ...

1 See discussions, stats, and author profiles for this publication at: simple sequence repeat (ISSR) polymorphism and its application inplant breedingArticle in Euphytica January 2002 DOI: :1020691618797 CITATIONS944 READS5,2553 authors, including:Some of the authors of this publication are also working on these related projects:Wide hybridization in Brassicas View projectMolecular breeding to introgress physiological traits for improving rice productivitry under water limiting conditions View projectPradeep Reddy MarriDow AgroSciences108 PUBLICATIONS 2,881 CITATIONS SEE PROFILES arla NeelamrajuIndian Institute of Rice Research (previously Directorate of Rice Research)225 PUBLICATIONS 3,108 CITATIONS SEE PROFILEAll content following this page was uploaded by Pradeep Reddy Marri on 10 November user has requested enhancement of the downloaded :9 17, 2002.

2 2002 Kluwer Academic Publishers. Printed in the simple sequence repeat (ISSR) polymorphism and its application inplant breedingM. Pradeep Reddy, N. Sarla & SiddiqDirectorate of Rice Research, Rajendranagar, Hyderabad 500 030, India; ( author for correspondence, 3 July 2001; accepted 6 March 2002 Key words:anchored primer, DNA marker, genome mapping, gene tagging, genetic diversity, ISSR-PCRS ummaryInter simple sequence repeat (ISSR)-PCR is a technique, which involves the use of microsatellite sequences asprimers in a polymerase chain reaction to generate multilocus markers. It is a simple and quick method thatcombines most of the advantages of microsatellites (SSRs) and amplified fragment length polymorphism (AFLP)to the universality of random amplified polymorphic DNA (RAPD).)

3 ISSR markers are highly polymorphic and areuseful in studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary biology. Thisreview provides an overview of the details of the technique and its application in genetics and plant breeding in awide range of crop markers have proved valuable in crop breed-ing, especially in studies on genetic diversity andgene mapping. The commonly used polymerase chainreaction (PCR)-based DNA marker systems are ran-dom amplified polymorphic DNA (RAPD), amplifiedfragment length polymorphism (AFLP) and more re-cently simple sequence repeats (SSRs) or microsatel-lites (Staub et al.)

4 , 1996; Gupta & Varshney, 2000). Themajor limitations of these methods are low reprodu-cibility of RAPD, high cost of AFLP and the need toknow the flanking sequences to develop species spe-cific primers for SSR polymorphism . ISSR-PCR isa technique that overcomes most of these limitations(Zietkiewicz et al., 1994; Gupta et al., 1994; Wu etal., 1994; Meyer et al., 1993). It is rapidly being usedby the research community in various fields of plantimprovement (Godwin et al., 1997). The technique isuseful in areas of genetic diversity, phylogenetic stud-ies, gene tagging, genome mapping and evolutionarybiology in a wide range of crop species.

5 In this methodSSRs are used as primers to amplify mainly the Inter -SSR regions. SSRs or microsatellites are short tandemrepeats (STRs) or variable number of tandem repeats(VNTRs) of 1 4 bases of DNA ubiquitously presentin eukaryote genomes (Tautz & Renz, 1984). Theyare dispersed throughout the genome and vary in thenumber of repeat units. The details of the techniqueand its major applications are discussed in this techniqueInter simple sequence repeat (ISSR) technique is aPCR based method, which involves amplification ofDNA segment present at an amplifiable distance inbetween two identical microsatellite repeat regionsoriented in opposite direction.

6 The technique uses mi-crosatellites, usually 16 25 bp long, as primers in asingle primer PCR reaction targeting multiple gen-omic loci to amplify mainly the Inter - SSR sequencesof different sizes. The microsatellite repeats used asprimers can be di-nucleotide, tri-nucleotide, tetra-nucleotide or penta-nucleotide. The primers used canbe either unanchored (Gupta et al., 1994; Meyer et al.,1993; Wu et al., 1994) or more usually anchored at 3 or 5 end with 1 to 4 degenerate bases extended intothe flanking sequences (Zietkiewicz et al., 1994) (Fig-ure 1). The technique combines most of the benefits of10 Figure : A schematic representation of a single primer (AG)8, unanchored (a), 3 -anchored (b) and 5 -anchored (c) targeting a(TC)nrepeat used to amplify Inter simple sequence repeat region flanked by two inversely oriented (TC)nsequences.

7 (a) Unanchored (AG)nprimer can anneal anywhere in the (TC)nrepeat region on the template DNA leading to slippage and ultimately smear formation (b) (AG)nprimer anchored with 2 nucleotides (NN) at the 3 end anneals at specific regions on the template DNA and produces clear bands (c) (AG)nprimer anchored with 2 nucleotides (NN) at the 5 end anneals at specific regions and amplifies part of the repeat region also leading to and microsatellite analysis with the universalityof RAPD. ISSRs have high reproducibility possiblydue to the use of longer primers (16 25 mers) as com-pared to RAPD primers (10- mers) which permits thesubsequent use of high annealing temperature (45 60 C) leading to higher stringency.

8 The studies onreproducibility show that it is only the faintest bandsthat are not reproducible. About 92 95% of the scoredfragments could be repeated across DNA samples ofthe same cultivar and across separate PCR runs whendetected using polyacrylamide (Fang & Roose, 1997;Moreno et al., 1998). 10 ng template DNA yieldedthe same amplification products as did 25 or 50 ngper 20 l PCR reaction. The annealing temperaturedepends on the GC content of the primer used andusually ranges from 45 to 65 segregate mostly as dominant markers fol-lowing simple Mendelian inheritance (Gupta et al.,1994; Tsumura et al.)

9 , 1996; Ratnaparkhe et al., 1998;Wang et al., 1998). However, they have also beenshown to segregate as co-dominant markers in somecases thus enabling distinction between homozygotesand heterozygotes (Wu et al., 1994; Akagi et al., 1996;Wang et al., 1998; Sankar & Moore, 2001).Source of variability / polymorphismThe evolutionary rate of change within microsatellitesis considerably higher than most other types of DNA,so the likelihood of polymorphism in these sequencesis greater. The source of variability in the ISSRs canbe attributed to any one of the following reasons orany combination of these.(a) Template DNAS lippage of DNA polymerase during DNA replica-tion and failure to repair mismatches is consideredas a mechanism for creation and hypervariability ofSSRs (Levinson & Gutman, 1987).

10 Mutations atthe priming site SSR could prevent amplifica-tion of a fragment, as also in RAPD markers andthus give a presence/absence polymorphism . An in-sertion/deletion event within the SSR region or theamplified region would result in the absence of aproduct or length polymorphism , depending on theamplifiability of the resulting fragment size. Variab-ility in number of nucleotides within a microsatelliterepeat would result in length polymorphisms whenusing a 5 -anchored primer.(b) Nature of primer usedThe extent of polymorphism also varies with thenature (unachored, 3 -anchored, or 5 -anchored) andsequence of the repeats (motif) in the primer em-ployed.