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简单序列重复(ISSR)多态性及其在植物育种中的应用


Euphytica 128: 9–17, 2002. ? 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Inter simple sequence repeat (ISSR) polymorphism and its application in plant

breeding
M. Pradeep Reddy, N. Sarla? & E.A. Siddiq
Directorate of Rice Research, Rajendranagar, Hyderabad – 500 030, India; (? author for correspondence, e-mail: nsarla@hotmail.com)
Received 3 July 2001; accepted 6 March 2002

Key words: anchored primer, DNA marker, genome mapping, gene tagging, genetic diversity, ISSR-PCR

Summary Inter simple sequence repeat (ISSR)-PCR is a technique, which involves the use of microsatellite sequences as primers in a polymerase chain reaction to generate multilocus markers. It is a simple and quick method that combines most of the advantages of microsatellites (SSRs) and ampli?ed fragment length polymorphism (AFLP) to the universality of random ampli?ed polymorphic DNA (RAPD). ISSR markers are highly polymorphic and are useful in studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary biology. This review provides an overview of the details of the technique and its application in genetics and plant breeding in a wide range of crop plants.

Introduction DNA markers have proved valuable in crop breeding, especially in studies on genetic diversity and gene mapping. The commonly used polymerase chain reaction (PCR)-based DNA marker systems are random ampli?ed polymorphic DNA (RAPD), ampli?ed fragment length polymorphism (AFLP) and more recently simple sequence repeats (SSRs) or microsatellites (Staub et al., 1996; Gupta & Varshney, 2000). The major limitations of these methods are low reproducibility of RAPD, high cost of AFLP and the need to know the ?anking sequences to develop species speci?c primers for SSR polymorphism. ISSR-PCR is a technique that overcomes most of these limitations (Zietkiewicz et al., 1994; Gupta et al., 1994; Wu et al., 1994; Meyer et al., 1993). It is rapidly being used by the research community in various ?elds of plant improvement (Godwin et al., 1997). The technique is useful in areas of genetic diversity, phylogenetic studies, gene tagging, genome mapping and evolutionary biology in a wide range of crop species. In this method SSRs are used as primers to amplify mainly the interSSR regions. SSRs or microsatellites are short tandem repeats (STRs) or variable number of tandem repeats

(VNTRs) of 1–4 bases of DNA ubiquitously present in eukaryote genomes (Tautz & Renz, 1984). They are dispersed throughout the genome and vary in the number of repeat units. The details of the technique and its major applications are discussed in this review.

The technique Inter simple sequence repeat (ISSR) technique is a PCR based method, which involves ampli?cation of DNA segment present at an ampli?able distance in between two identical microsatellite repeat regions oriented in opposite direction. The technique uses microsatellites, usually 16–25 bp long, as primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly the inter- SSR sequences of different sizes. The microsatellite repeats used as primers can be di-nucleotide, tri-nucleotide, tetranucleotide or penta-nucleotide. The primers used can be 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 into the ?anking sequences (Zietkiewicz et al., 1994) (Figure 1). The technique combines most of the bene?ts of

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Figure 1. ISSR-PCR: A schematic representation of a single primer (AG)8 , unanchored (a), 3’-anchored (b) and 5’-anchored (c) targeting a (TC)n repeat used to amplify inter simple sequence repeat region ?anked by two inversely oriented (TC)n sequences. (a) Unanchored (AG)n primer can anneal anywhere in the (TC)n repeat region on the template DNA leading to slippage and ultimately smear formation (b) (AG)n primer anchored with 2 nucleotides (NN) at the 3’ end anneals at speci?c regions on the template DNA and produces clear bands (c) (AG)n primer anchored with 2 nucleotides (NN) at the 5’ end anneals at speci?c regions and ampli?es part of the repeat region also leading to larger bands.

11 AFLP and microsatellite analysis with the universality of RAPD. ISSRs have high reproducibility possibly due to the use of longer primers (16–25 mers) as compared to RAPD primers (10- mers) which permits the subsequent use of high annealing temperature (45– 60 ? C) leading to higher stringency. The studies on reproducibility show that it is only the faintest bands that are not reproducible. About 92–95% of the scored fragments could be repeated across DNA samples of the same cultivar and across separate PCR runs when detected using polyacrylamide (Fang & Roose, 1997; Moreno et al., 1998). 10 ng template DNA yielded the same ampli?cation products as did 25 or 50 ng per 20?l PCR reaction. The annealing temperature depends on the GC content of the primer used and usually ranges from 45 to 65 ? C. ISSRs segregate mostly as dominant markers following simple Mendelian inheritance (Gupta et al., 1994; Tsumura et al., 1996; Ratnaparkhe et al., 1998; Wang et al., 1998). However, they have also been shown to segregate as co-dominant markers in some cases thus enabling distinction between homozygotes and heterozygotes (Wu et al., 1994; Akagi et al., 1996; Wang et al., 1998; Sankar & Moore, 2001). Source of variability / polymorphism The evolutionary rate of change within microsatellites is considerably higher than most other types of DNA, so the likelihood of polymorphism in these sequences is greater. The source of variability in the ISSRs can be attributed to any one of the following reasons or any combination of these. (a) Template DNA Slippage of DNA polymerase during DNA replication and failure to repair mismatches is considered as a mechanism for creation and hypervariability of SSRs (Levinson & Gutman, 1987). Mutations at the priming site i.e. SSR could prevent ampli?cation of a fragment, as also in RAPD markers and thus give a presence/absence polymorphism. An insertion/deletion event within the SSR region or the ampli?ed region would result in the absence of a product or length polymorphism, depending on the ampli?ability of the resulting fragment size. Variability in number of nucleotides within a microsatellite repeat would result in length polymorphisms when using a 5’-anchored primer. (b) Nature of primer used The extent of polymorphism also varies with the nature (unachored, 3’-anchored, or 5’-anchored) and sequence of the repeats (motif) in the primer employed. When unanchored i.e only the SSRs are used as primers, the primer tends to slip within the repeat units during ampli?cation leading to smears instead of clear bands (Figure 1a). Extending the primer (anchoring) with 1 to 4 degenerate nucleotides at the 3’ end (Figure 1b) or 5’ end (Figure 1c) assures annealing only to the ends of a microsatellite in template DNA thus obviating internal priming and smear formation. Secondly, the anchor allows only a subset of the microsatellites to serve as priming sites. When 5’ anchored primers are used, the ampli?ed products include the microsatellite sequences and their length variations across a genome and therefore give more number of bands and a higher degree of polymorphism. Usually di-nucleotide repeats, anchored either at 3’ or 5’ end reveal high polymorphism (Blair et al., 1999; Joshi et al., 2000; Nagaoka & Ogihara, 1997). The primers anchored at 3’ end (Figure 1b) give clearer banding pattern as compared to those anchored at 5’ end (Tsumura et al., 1996; Blair et al., 1999; Nagaoka & Ogihara, 1997). Since the primer is a SSR motif the frequency and distribution of the microsatellite repeat motifs in different species also in?uence the generation of bands. There is a difference of abundance of SSRs between nuclear and organelle DNA sequences. Taking di- and tri-nucleotides together, one SSR was found every 33Kb in nuclear DNA compared to every 423-Kb of organelle DNA sequence (Wang et al., 1994). In general, primers with (AG), (GA), (CT), (TC), (AC), (CA) repeats show higher polymorphism than primers with other di-, tri- or tetra-nucleotide repeats. (AT) repeats are the most abundant di-nucleotides in plants but the primers based on (AT) would self- anneal and not amplify. Triand tetra-nucleotides are less frequent and their use in ISSRs is lesser than the di-nucleotides. The (AG) and (GA) based primers have been shown to amplify clear bands in rice (Blair et al., 1999; Joshi et al., 2000; Reddy et al., 2000; Sarla et al., 2000), trifoliate orange (Fang et al., 1997), Douglas ?r and sugi (Tsumura et al., 1996) and chickpea (Ratnaparkhe et al., 1998), whereas primers based on (AC) di-nucleotide repeats were found more useful in wheat (Nagaoka & Ogihara, 1997; Kojima et al., 1998) and potato (McGregor et al., 2000). Resolving power Rp is an index developed to compare the value of different primers in terms

12 of the informative bands obtained in a given set of germplasm (Prevost & Wilkinson, 1999). (c) Detection method The level of polymorphism detected has been shown to vary with the detection method used. Polyacrylamide gel electrophoresis (PAGE) in combination with radioactivity (labelled nucleotide in PCR reaction) was shown to be most sensitive, followed by PAGE with silver staining and then agarose-ethidium bromide system of detection. Markedly higher number of bands were resolved per primer when polyacrylamide was used compared to agarose (Moreno et al., 1998). In a study on trifoliate orange germplasm, silver staining using high quality chemicals could detect all the bands detected by autoradiography (Fang et al., 1997). However, high levels of polymorphism have been detected even when products of ISSR ampli?cation are resolved on agarose gels without radiolabelling (Tsumura et al., 1996; Arcade et al., 2000; Kojima et al., 1998; Wolff & Morgan-Richards, 1998; Sankar & Moore, 2001) Thus, the need for radioactivity can be avoided when many samples have to be screened as in germplasm characterization. ISSR-PCR is a simple, quick, and ef?cient technique. It has high reproducibility. The use of radioactivity is not essential.The primers are not proprietary (as in SSR-PCR) and can be synthesized by anyone. Variations in primer length, motif and anchor are possible. The primers are long (16–25 bp) resulting in higher stringency. The ampli?ed products (ISSR markers) are usually 200–2000 bp long and amenable to detection by both agarose and polyacrylamide gel electrophoresis. In the literature this technique and its variations have been referred to by different names (Table 1). based ISSR primers anchored at 5’ or 3’ end have been used in ?ngerprinting studies with high reproducibility for maintenance of cocoa collection (Charters & Wilkinson, 2000). ISSRs showed suf?cient polymorphism to distinguish between various cultivars of chrysanthemum (Wolff et al., 1995). Microspore derived plants could be distinguished from those derived from somatic tissues in anther culture of ?ax at an early seedling stage (Chen et al., 1998). Genetic diversity and phylogenetic analysis ISSRs have been successfully used to estimate the extent of genetic diversity at inter- and intra-speci?c level in a wide range of crop species which include rice (Joshi et al., 2000), wheat (Nagaoka & Ogihara, 1997), ?ngermillet (Salimath et al., 1995), Vigna (Ajibade et al., 2000), sweet potato (Huang & Sun, 2000) and Plantago (Wolff & Morgan-Richards, 1998). Superiority of ISSR-PCR over other marker techniques has been brought out in such investigations by various workers. Anchored SSR primers for instance, have been found to be more useful and reproducible than isozymes, RFLPs and RAPDs in the diversity analysis of trifoliate orange germplasm (Fang et al., 1997). ISSRs were more useful for the analysis of diversity in the genus Eleusine in terms of quality and quantity of data output as compared to RFLP and RAPD (Salimath et al., 1995). Signi?cantly, the ef?ciency of the technique was evident in characterization even at the varietal level of a species. For instance, three 5’ anchored primers together could distinguish 20 cultivars of Brassica napus (Charters et al., 1996). ISSR is the marker of choice for assessment of genetic diversity in cocoa (Charters & Wilkinson, 2000), gymnosperms such as Douglas ?r and sugi (Tsumura et al., 1996) and even fungi (Hantula et al., 1996). In a study on white lupin it has been demonstrated that among 10 primers used any two were suf?cient to distinguish all the 37 accessions studied (Gilbert et al., 1999). Similarly, 4 primers were suf?cient to distinguish 34 cultivars of potato (Prevost & Wilkinson, 1999) and 3 primers could distinguish 16 genotypes of redcurrant (Lanham & Brennan, 1998). The use of such highly informative primers lowers the cost, time and labour for diversity analysis. Various marker techniques have been used in phylogenetic investigations based on relative similarity. Inspite of their higher ef?ciency and reproducibility ISSR markers have as yet not been used extensively. It has however been found effective in

Application The potential for integrating ISSR-PCR into programs of plant improvement is enormous (Table 2). The major areas of the application of ISSR-PCR in different crops are discussed below. Genomic ?ngerprinting DNA ?ngerprinting is an important tool for characterization of germplasm and establishment of the identity of varieties/hybrids/parental sources etc. in plant breeding and germplasm management. Di-nucleotide

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Table 1. Synonyms of the ISSR-PCR technique and its variants S. No 1 2 3 4 5 6 7 Terms used MP-PCR, Microsatellite primed PCR (refers to unanchored primer) SSR-anchored PCR, Inter-SSR ampli?cation SPAR (single primer ampli?cation reaction) RAMPs (random ampli?ed microsatellite polymorphisms) RAMs (randomly ampli?ed microsatellites) AMP-PCR (anchored microsatellite primed PCR) ASSR (anchored simple sequence repeats) Reference Meyer et al. (1993) Zietkiewicz et al. (1994) Gupta et al. (1994) Wu et al. (1994) Hantula et al. (1996) Weising et al. (1998) Wang et al. (1998)

resolving problems relating to the phylogeny of Asian cultivated rice Oryza sativa (Joshi et al., 2000), wheat (Nagaoka & Ogihara, 1997), ?nger millet (Salimath et al., 1995), Vigna (Ajibade et al., 2000) and Diplotaxis species (Martin & Sanchez-Yelamo, 2000). There is immense scope to use this powerful technique in resolving species/inter-species status in many a genus and in deciding the distinctness of different genera within a family. Signi?cantly, genome/species speci?c ISSR markers have been reported in four genera Oryza (Joshi et al., 2000), Lolium and Festuca (Pasakinskiene et al., 2000) and Diplotaxis (Martin & Sanchez-Yelamo, 2000) which are useful in delineating species.

1998). CA polymorphisms had a biased distribution and GA polymorphisms were randomly dispersed. Gene tagging and marker assisted selection DNA markers closely linked to important agronomic traits greatly contribute to practical crop improvement programs. In rice, an ISSR marker generated by primer (AG)8YC was converted to a sequence tagged site (STS) marker to identify the fertility restoration gene, Rf-1 (Akagi et al., 1996). This co-dominant marker can be used in management of genetic purity of hybrid seed. In chickpea, ISSR markers UBC 855500 generated by primer (AG)8 YT and UBC 8251200 using primer (AG)8 T were linked to the gene conferring resistance to race 4 of Fusarium wilt (Ratnaparkhe et al., 1998). Markers closer to a given gene are generated by altering 5’ or 3’ anchors. Recently, ISSR-PCR was used in identifying two allelic dominant DNA markers, one linked in coupling and the other in repulsion phase to a major locus Fgr, which modulates fructose to glucose ratio in tomatoes (Levin et al., 2000). These PCR products were obtained from two ISSR-PCR reactions using (TC)8 CC and (TC)8 CG as primers. Another trait of value in hybrid seed production viz., temperature-sensitive genic male sterility has been tagged with an ISSR marker UBC 8551060 in rice (Hussain et al., 2000). ISSRs have also been used to generate species speci?c, gene speci?c and trait speci?c markers. While delineating the phylogenetic relationship among different species of the genus Oryza, 87 putative genome/species speci?c markers were identi?ed (Joshi et al., 2000). The 582 bp inter-SSR Festuca speci?c sequence and 1350 bp F. arundinacea speci?c sequence have potential as markers to con?rm presence of closely linked Festuca genes (Pasakinskiene et al., 2000). Likewise, race speci?c markers have been de-

Genome mapping ISSR markers are unmapped but can be used to saturate RFLP and SSR linkage maps. The RFLP map of barley was saturated with 60 ISSRs (referred as RAMPs in the study) which mapped to all chromosomes (Becker & Heun, 1995). Many of these markers are mapped in between clustered RFLPs, ?anking RFLP clusters, at the tips of chromosomes and more importantly in areas of low RFLP marker density. In Einkorn wheats, however, the nine ISSR markers mapped at or close to the RFLP marker positions (Kojima et al., 1998). ISSRs have also been used along with AFLP and RAPD markers in the mapping of Japanese and European larch genomes (Arcade et al., 2000). The genetic linkage map of Citrus was further saturated using 75 ISSR markers, which were dispersed among all the linkage groups (Sankar & Moore, 2001). Also it was shown that the level of segregation distortion of ISSRs is lower compared to RAPDs. In soybean, 58 ISSR markers were mapped onto 18 RAPD/RFLP linkage groups (Wang et al.,

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Table 2. Applications of ISSR-PCR technique S. No 1 Application Genomic ?ngerprinting Cocoa germplasm Potato cultivars Chrysanthemum cultivars Genetic diversity and phylogenetic analysis Rice cultivars Oryza granulata Wheat (Triticum sp.) Barley (Hordeum vulgare) Maize inbred lines (Zea mays) Fingermillet (Eleusine sp) Sorghum (Chinese) (Sorghum bicolor) White lupin germplasm (Lupinus albus) Vigna sp Pea germplasm (Pisum sativum) Soybean (Glycine max) Oilseed rape cultivars (Brassica napus) Sweet potato, wild relatives (Ipomoea sp) Potato cultivars (Solanum tuberosum) Redcurrant germplasm (Ribes sp) Grapevine germplasm (Vitis vinifera) Citrus cultivars (Citrus sp) Trifoliate orange germplasm (Poncirus trifoliata) Plantago major subspecies Gymnosperms, Douglas ?r and sugi Genome mapping Saturating RFLP linkage map in barley Construction of a genetic linkage map in Einkorn wheat Genetic mapping of Japanese and European types of larch Saturating genetic linkage map in citrus Saturating RFLP/RAPD linkage map in soybean Determining SSR motif frequency Recovery of microsatellite sequences in the mustard genome Distribution pattern of microsatellites across eukaryotic genomes Analysis of microsatellite frequency in rice cultivars Gene tagging and use in marker assisted selection Rf-1 gene for fertility restoration in rice Gene for resistance to Fusarium wilt Race 4 in chickpea Temperature sensitive genic male sterility in rice Fgr gene for modulating fructose to glucose ratio in tomato Genome/species speci?c markers in Lolium and Festuca Putative genome/species speci?c markers in Oryza. Race speci?c markers in fungi Evolutionary biology Diplotaxis species Diploid hybrid speciation in Penstemon Reference

Charters & Wilkinson, 2000 Prevost & Wilkinson, 1999 Wolff et al., 1995 Virk et al., 2000 Qian et al., 2001 Nagaoka & Ogihara, 1997 Sanchez et al., 1996 Kantety et al., 1995 Salimath et al., 1995 Yang et al., 1996 Gilbert et al., 1999 Ajibade et al., 2000 Lu et al., 1996 Wang et al., 1998 Charters et al., 1996 Huang & Sun, 2000 McGregor et al., 2000 Lanham & Brennan, 1998 Moreno et al., 1998 Fang & Roose, 1997 Fang et al., 1997 Wolff & Morgan- Richards, 1998 Tsumura et al., 1996 Becker & Heun, 1995 Kojima et al., 1998 Arcade et al., 2000 Sankar & Moore, 2001 Wang et al., 1998 Varghese et al., 2000 Gupta et al., 1994 Blair et al., 1999 Akagi et al., 1996 Ratnaparkhe et al., 1998 Hussain et al., 2000 Levin et al., 2000 Pasakinskiene et al., 2000 Joshi et al., 2000 Hantula et al., 1996 Martin & Sanchez-Yelamo, 2000 Wolfe et al., 1998

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15 veloped in various fungi groups using ISSRs (Hantula et al., 1996). Determining SSR motif frequency Perspectives ISSR analysis provides insights into the organization (clustered or not), frequency and levels of polymorphism of different simple sequence repeats in a genome. In rice and wheat, di-nucleotide simple sequence repeats used as primers gave the maximum number of bands and are, therefore, more common than any SSRs with larger units (Blair et al., 1999; Nagaoka & Ogihara, 1997). Poly(GA) based 3’-anchored primers produced 5 times as many bands as those with poly(GT) motif indicating low frequency or lack of clustering of (GT) motif (Blair et al., 1999). Using ISSRs it has been shown that tetra-nucleotide repeats were abundant across eukaryotic genomes (Gupta et al., 1994) and that tetramers of tetra-nucleotides AGAC and GACA are scattered within the genome of grasses (Pasakinskiene et al., 2000). It has been demonstrated in Brassica that enhanced recovery of microsatellite markers is possible using ISSR primers (Varghese et al., 2000). Studies on natural populations/ speciation The hypervariable nuclear ISSR markers have proved useful in testing hypotheses of speciation, introgression and systematics (Wolfe et al., 1998). The hybrid origin of Penstemon clevelandi was clearly brought out by the use of just 8 ISSR markers. Population of P. clevelandi has been found to have an additive pro?le of bands of the two proposed progenitor species viz. P. centranthifolius and P. spectabilis. On the other hand the population of P. spectabilis lacked the additive pro?le of bands of its proposed putative parents. The hybrid origin of P. spectabilis was thus negated and its origin was attributed instead to introgression of genes and not the genome of a related species. The utility of the technique has been demonstrated in a wide range of applications in molecular ecology in plant families which include Asteraceae, Brassicaceae, Hippocastanaceae, Orchidaceae, Poaceae, Scrophulariaceae and Violaceae (http://www.biosci.ohiostate.edu/ ?awolfe/issri.issr.html). Variation within and between populations can be compared using dispersed multilocus markers such as ISSR. It was shown that the amount of variation between O. granulata populations from different regions (49.2%) was higher than that between populations within a region (38%) As the need to protect proprietary germplasm is likely to increase in the future, ISSRs will have an important role in securing plant variety rights by virtue of its unique ef?ciency in distinguishing even closely related germplasm. To date, more polymorphism has been detected with the use of ISSRs than with any other assay procedure (Gupta et al., 1994; Salimath et al., 1995; Virk et al., 2000). In many of the studies for determining the extent of polymorphism or comparing marker systems only one family of SSRs, eg. tri-nucleotides or tetra-nucleotides had been used as primers. Such repeats are infrequent as compared to di-nucleotides and their use may not help arrive at precise classi?cation. As more data on the occurrence and distribution of SSR motifs becomes available, it should be possible to use primers that give more accurate span of the whole genome. Also, different combinations of the motif, anchor and length of primers can be used. Strategies to detect additonal polymorphism could include use of ISSRs in combination with RAPD (Joshi et al., 2000; Becker & Heun, 1995; Wu et al., 1994) or AFLP primers in the same reaction or restriction digestion of ISSR products (Becker & Heun, 1995). Unlimited combinations of motif and length of both primers and use of different restriction enzymes are thus possible. Well chosen primers can provide reasonably accurate ?ngerprinting and thereby quick estimate of genetic diversity especially in large sized accessions to identify core sets and the pattern of geographical distribution. The technique is not without limitations. For instance, there is the possibility as in RAPD, that fragments with the same mobility originate from non-homologous regions, which can contribute to some distortion in the estimates of genetic similarities (Sanchez et al., 1996). The molecular nature of the polymorphisms can be known only if the fragments extracted from the gel are sequenced. ISSR markers linked to the traits of agronomic importance have been sequenced and used as STS markers in marker aided selection. An attractive possibility is thus the use of ISSRs as probes for in-situ hybridization for physical mapping of homologous chromosome sites (Pasakinskiene et al., 2000). Another advantage in the use of ISSR markers lies in their linkage to SSR loci. or within a population (12%) using ISSR markers (Qian et al., 2001).

16 Although microsatellites themselves are probably nonfunctional and selectively neutral, they are known to be linked to coding regions, so that ISSRs are likely to mark gene rich regions (Kojima et al., 1998).
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