Inter-simple-sequence-repeat (ISSR) polymorphisms are ...

1 See discussions, stats, and author profiles for this publication at: (ISSR) polymorphisms are useful for findingmarkers associated with disease resistance gene clustersArticle in Theoretical and Applied Genetics September 1998 DOI: authors:Some of the authors of this publication are also working on these related projects:Soybean genetics View projectPermaculture View projectMilind RatnaparkheICAR-Indian Institute of Soybean Research, Indore, India60 PUBLICATIONS 1,619 CITATIONS SEE PROFILEM. Tekeoglu stanbul20 PUBLICATIONS 1,331 CITATIONS SEE PROFILEF rederick Joseph MuehlbauerWashington State University257 PUBLICATIONS 8,518 CITATIONS SEE PROFILEAll content following this page was uploaded by Milind Ratnaparkhe on 29 April user has requested enhancement of the downloaded by P. M. A. TigerstedtM. B. Ratnaparkhe M. Tekeoglu F. J. Muehlbauer ()USDA-ARS, Grain Legume Genetics and Physiology Unit,and Department of Crop and Soil Sciences,Washington State University, Pullman WA 99164, USAFax:#1 509-335-7692 Theor Appl Genet (1998) 97 : 515 519(Springer-Verlag 1998M.)

2 B. Ratnaparkhe M. Tekeoglu F. J. MuehlbauerInter- simple - sequence -repeat (ISSR) polymorphisms are useful for finding markersassociated with disease resistance gene clustersReceived: 20 January 1998 / Accepted: 19 March 1998 AbstractWe describe a simple and new approach,based on inter - simple sequence repeats (ISSRs), forfinding markers linked to clusters of disease resistancegenes. In this approach, simple sequence repeats (SSR)are used directly in PCR reactions, and markers foundto be linked to disease resistance genes provide impor-tant information for the selection of other sequenceswhich can be used with PCR to find other linkedmarkers. Based on an ISSR marker linked to a gene ofinterest, many new markers can be identified in thesame region. We previously demonstrated that ISSR markers are useful in gene tagging and identifieda marker, UBC-855500, linked to the gene for resistanceto fusarium wilt race 4 in chickpea.

3 This ISSR markerprovided the information used in the present study forselecting other primers which amplified a region linkedto the gene for resistance to fusarium wilt race 4. Theprimers were based on homology with the (AC)/se-quence and were used for PCR amplifications. Changesin the sequence were at the anchor region of theprimers. The repeat (AC)8T amplified a marker, UBC-8251200, which was located cM from the gene forresistance to fusarium wilt race 4 and was closer thanother markers. These results indicated that ISSR markers can provide important information for thedesign of other primers and that by making changes atthe 3@and 5@anchors close linkage to the desired genecan be found. The approach allows rapid scanning ofthe targeted region and may provide important in-formation for genome analysis of plant wordsMicrosatellite Disease resistance Mapping sequence -directed approachIntroductionInter- simple sequence repeats (ISSRs) are a new type ofDNA marker which involves the use of microsatellitesequences directly in the polymerase chain reaction(PCR) for DNA amplifications (Gupta et al.)

4 1994; Ziet-kiewicz et al. 1994; Sanchez et al. 1996). This techniqueenables amplification of genomic DNA and providesinformation about many loci simultaneously. Recentreports have shown Mendelian inheritance of simple - sequence -repeat (SSR) fragments scored as dominantmarkers (Tsumura et al. 1996) or as codominantmarkers (Wu et al. 1994). simple sequence repeats(SSRs) are short tandem repetitive DNA sequenceswith a repeat length of few base pairs (1-5) (Litt andLuty 1989). The sequences are abundant, dispersedthroughout the genome and highly polymorphic incomparison with other molecular markers (Akkaya etal. 1992; Morgante and Olivieri 1993; Wang et al. 1994).SSRs have been used successfully in the genome map-ping of a variety of crop species including maize, rice,barley and wheat (Senior and Heun 1993; Wu andTanksley 1993; Saghai-Maroof et al. 1994; Roder et ).

5 However, due to the technical difficulties in de-veloping SSRs, these markers have not been commonlyused for gene tagging in plants. ISSR has been pro-posed as a new source of genetic markers which over-comes the technical limitations of restriction fragmentlength polymorphism (RFLP) and random amplifiedpolymorphic DNA (RAPD).Ratnaparkhe et al. (1998) were the first to demon-strate that ISSR markers are useful in gene tagging andcan be used for finding markers linked to the gene ofinterest. Previous reports indicated that SSRs arenot randomly distributed in the genome but are oftenclustered (Arens et al. 1995; Broun and Tanksley 1996).The clustering of SSRs has also been observed in hu-mans (Erickson et al. 1988) and on sex chromosomes ofdifferent organisms (Epplan 1988; Nanda et al. 1990).Similarly, disease resistance genes have also been foundto form clusters (Sheperd and Mayo 1972; Islam et ; Kesseli et al.)

6 1993). Studies on disease resistancegenes have indicated a high level of polymorphism andthe presence of SSRs at certain loci (Yu et al. 1996). Inthe study presented here we exploited the combinationof ISSR markers to find markers at the fusarium wiltdisease resistance gene cluster. The approach is basedon the association of a cluster of SSRs with the diseaseresistance gene cluster. We also investigated the poten-tial of an ISSR-directed approach for selecting othersequences which can be used to find linked markersand for marker enrichment in the desired region. Thetechnique is based on the use of SSR primers withvariations at 5@and 3@anchors, which provides markerslinked to the desired gene. We studied the inheritanceof ISSR polymorphisms using 96 recombinant inbredlines (RILs) derived from the cross (ICC-4958), a cultivated chickpea germplasm linewith resistance to fusarium wilt, (PI 489777), the closest wild relative of the cultivatedspecies, and identified markers linked to the gene forresistance to fusarium wilt races 4 and and methodsPlant materialA set of 96 F6-derived F7recombinant inbred lines (RILs) obtainedfrom a cross (ICC-4958) (PI 489777) was used in this study.

7 The RILs were developed by thesingle-seed descent procedure. Scoring for resistance to fusarium wiltin the two parents and 96 RILs was done in the extractionDNA was isolated from vegetative buds and leaf tissues of the parentsand RILs using the microprep method of Doyle and Doyle (1987).One gram of each sample was submerged in liquid nitrogen and thenground to a fine powder. The powder was quickly transferred toa tube containing ml of ice cold extraction buffer ( , , 5 mMEDTA, pH ). The tube was briefly shaken, ml of nuclei lysis buffer (2 MNaCl, , 50 mMEDTA,2% CTAB, pH ) was then quickly added, followed by 3 ml of 5%sarkosyl solution. Sample sets were incubated in a 65 C waterbath for20 min. After incubation, the tubes were allowed to cool for a fewminutes and then 18 ml of chloroform/isoamyl alchohol (24 : 1) wasadded to each tube. The tubes were then centrifuged at 500gfor15 min.

8 The aqueous layer was removed and extracted again with15 ml chloroform mixture. Finally DNA was precipitated with chilledethanol and suspended in 1 ml of TE preparation and inoculation proceduresInoculum was prepared from a single-spored fungal isolate grownon sterile filter paper placed on potato-dextrose-agar (PDA) asdescribed by Tullu (1996). When the filter paper was completelycolonized by fungus, only those colonies representative of the wildtype were aseptically removed and placed in a fresh petri dish to dryfor 5 days in a laminar flow hood. The dried filter paper wasaseptically cut into pieces using a pair of sterile scissors, and thesepieces were then used to prepare the primary inoculum. The conidiaconcentration was adjusted to 1]106spores per milliliter witha hemacytometer. Twelve to twenty seeds of each RIL were grown inthe greenhouse (21 26 C) in single rows in plastic trays filled withsterile coarse perlite.

9 When the seedlings reached the three- tofour-nodal stage, they were carefully removed from the perlite,pruned while submerged in the spore suspension and, after about5 min in the spore suspension, replanted into the perlite. Plants werethen scored as susceptible or resistant over the next 2 primersOne hundred primers of 15 23 nucleotides in length (UBC setK9)were obtained from the Biotechnology Laboratory, University ofBritish Columbia, Vancouver, British Columbia, Canada and usedfor polymerase chain reaction (PCR) amplification and electrophoresisPCR amplification was performed in 10 mMTRIS-HCl pH ,50 mMKCl, Triton]100, mMMgCl2, , ,of primer, 30 ng of genomic DNA per 25ll of reactionvolume and 1 unit of aqpolymerase. The amplifications werecarried out on a Perkin Elmer Cetus 9600 programmed for 35 cyclesof de-naturation at 94 C for 30 s, annealing at 50 C for 30 s andextension at 72 C for 2 min, with a 10-min final extension at 72 products were separated on 2% agarose gels, then stained withethidium bromide and scored for presence or absence of ISSR markers are dominant, a locus was considered to bepolymorphic if the band was present in one parent and not in theother.

10 Linkage analysis was performed using the MAPMAKER program (Lander et al. 1987)ResultsWe previously demonstrated that when the simple se-quence repeat (AC)8YT is used directly in a PCR reac-tion it amplifies a marker, UBC-855500, which is linkedto the gene for resistance to fusarium wilt race 4 (Rat-naparkhe et al. 1988). The aim of our present study wasto determine the suitability of the (AC)/sequences asguidelines for selecting new primers for use in markerenrichment at the desired region. For this we studiedthe inheritance of ISSR polymorphism in a cross ofcultivated chickpea ( ) and the closely re-lated wild species ( ). Based on the(AC)8YT sequence which amplifies a marker linked tothe disease resistance gene, we selected other sequencescontaining AC repeats, but ones that varied at the3@and We found that other SSRs with ACrepeats also amplified fragments that were linked to thefusarium wilt resistance genes.