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Abstract In hexaploid bread wheat ( Triticum aestivum L. em. Thell), ten members of the IWMMN ( International Wheat Microsatellites Mapping Network ) collaborated in extending the microsatellite (SSR = simple sequence re-peat) genetic map. Among a much larger number of mi-crosatellite primer pairs developed as a part of the WMC(Wheat Microsatellite Consortium ), 58 out of 176 primer pairs tested were found to be polymorphic between theparents of the ITMI ( International Triticeae Mapping Initiative) mapping population W7984 ×Opata 85 (ITMIpop). This population was used earlier for the con-struction of RFLP ( Restriction Fragment Length Polymor-phism) maps in bread wheat (ITMI map). Using the ITMIpopand a framework map (having 266 anchor mark-ers) prepared for this purpose, a total of 66 microsatelliteloci were mapped, which were distributed on 20 of the 21chromosomes (no marker on chromosome 6D). These 66mapped microsatellite (SSR) loci add to the existing 384microsatellite loci earlier mapped in bread wheat. Keywords Triticum aestivum · Bread wheat · Molecular genetic maps · Microsatellites · SSRs Introduction Bread wheat is one of the most important world foodcrops. It is a hexaploid, having three closely related geno-mes (A, B, D), each with seven chromosomes. The 21wheat chromosomes are arranged in a two-way classifica-tion with seven homoeologous groups, each group havingthree chromosomes, one from each of the three genomes(for a review see Gupta 1991). It also has a large genomeof 16 ×10 9bp (Gupta et al. 1991; Bennett and Leitch 1995), of which more than 80% is repetitive DNA. Thismakes bread wheat a difficult material for genome-widestudies. Despite this, detailed RFLP genetic maps withmore than 1,500 mapped loci spanning a genetic distanceof over 3,700 c M (a density of 4. 4 Mb per c M) are nowavailable for bread wheat, due to concerted efforts madeby several laboratories, coordinated by the ITMI (see Communicated by P. Langridge P. K. Gupta (✉) · H. S. Balyan CCS University, Meerut, Indiae-mail: pkgupta@ndf. vsnl. net. in K. J. Edwards Department of Biological Sciences, University of Bristol, UK P. Isaac Agrogene, Moissy-Cramayel, France V. Korzun Lochow-Petkus Gmb H, Grimsehlstr. 31, 37574 Einbeck, Germany M. Röder Institute for Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany M.-F. Gautier · P. Joudrier INRA, Montpellier, France A. R. Schlatter IRB-INTA, 1712 Castelar, Buenos Aires, Argentina J. Dubcovsky University of California, Davis, USA R. C. De la Pena · P. Jack · G. Penner Monsanto, USA M. Khairallah CIMMYT, Mexico M. J. Hayden · P. Sharp University of Sydney, Quality Wheat CRC and Plant Breeding Institute, Camden NSW 2570, Australia B. Keller Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland R. C. C. Wang USDA-ARS-FRRL, 695 N 1100 E, Logan, Utah 84322-6300, USA J. P. Hardouin Benoist Ets, France P. Leroy UMR INRA-UBP ASP, 234 Avenue du Brézet, 63 039 Clermont-Ferrand Cedex 2., France Theor Appl Genet (2002) 105:413-422 DOI 10. 1007/s00122-002-0865-9 P. K. Gupta · H. S. Balyan · K. J. Edwards · P. Isaac V. Korzun · M. Röder · M.-F. Gautier · P. Joudrier A. R. Schlatter · J. Dubcovsky · R. C. De la Pena M. Khairallah · G. Penner · M. J. Hayden · P. Sharp B. Keller · R. C. C. Wang · J. P. Hardouin · P. Jack P. Leroy Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat Received: 2 July 2001 / Accepted: 8 October 2001 / Published online: 19 June 2002 © Springer-Verlag 2002

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Gupta et al. 1999 for a review). Physical maps for all 21 chromosomes involving a sizable proportion of the genet-ically mapped loci are also available (Gill et al. 1993;Kota et al. 1993; Hohmann et al. 1994; Ogihara et al. 1994; Delaney et al. 1995a, b; Mickelson-Young et al. 1995; Varshney et al. 2001). These genetic and physicalmaps, however, are still far from the ultimate objective ofgetting a map as saturated as those available for the toma-to and rice genomes. Therefore, the addition of moremarkers on these maps is a valuable objective for thewheat research community. The preparation of microsat-ellite or SSR (simple sequence repeat) maps was onesuch attempt, where a total of 279 and 55 microsatelliteloci were mapped in Germany by Röder et al. (1998) and Pestsova et al. (2000) respectively, and 50 microsatelliteloci were mapped in UK by Stephenson et al. (1998). There are other published or unpublished reports of addi-tional mapped microsatellite loci, which include 144 loci(beloning to 137 microsatellites) mapped in Australia(Harker et al. 2001), 205 loci mapped in Canada (person-al communication, D. Somers) and 337 loci mapped in France (Sourdille et al. 2001). However, only the recentmaps from Australia and Canada include some wmcloci from a total of 396 microsatellites developed through theefforts of the WMC, which was started in the year 1996. The WMC was an international effort to develop and usethese second-generation microsatellite markers, since de-velopment of these markers by individual laboratorieswas considered time-consuming and expensive. Microsat-ellite markers were considered superior to markersmapped earlier, the majority of which were RFLP mark-ers. The microsatellite markers are locus-specific and PCR-based, thus making them attractive. Although theinitial development of microsatellite markers may becostly, once developed they are cost effective and conve-nient for a variety of purposes including QTL analysisand marker-assisted selection. To aid this, an Internation-al Wheat Microsatellites Mapping Network (IWMMN)was created in February 1997, with the main objective tomap additional microsatellite loci (particularly the WMCprimers) on the bread wheat ITMI map. For this purpose the ITMI population W7984 ×Opata 85 consisting of ap-proximately 130 recombinant inbred lines (RILs) wasavailable, of which 70 RILs were considered adequate forsubsequent mapping work (Röder et al. 1998). IWMMNis a world-wide goodwill network that depends essential-ly on collaboration among all its members and providesfor a mechanism, whereby researchers at the internationallevel can release and exchange among themselves valu-able data concerning the mapping of wheat microsatel-lites. The results of the first mapping efforts of the IWMMN are reported here. Materials and methods Genomic clones and development of microsatellites Several genomic libraries enriched for a number of microsatellites were prepared by K. Edwards (Edwards et al. 1996) and 48 cloneswere supplied by Agrogene (coordinator of the WMC) to each member of the WMC for the purpose of sequencing. The sequencedata was collated into a database by Agrogene who designed prim-er sets using the software Primer Pick (in-house software of Agro-gene). Primers were synthesized by each member of WMC fromtheir corresponding sequence data and were then pooled by Agro-gene, which distributed primer aliquots to all members of WMC,for characterization and genetic mapping. Chromosome assignment In some cases, the markers were assigned to specific chromo-somes using Chinese Spring nullisomic-tetrasomic lines and tospecific arms of chromosomes using Chinese Spring ditelocentriclines. DNA extraction and distribution The ITMI population W7984 ×Opata 85 (ITMI pop), used earlier (Nelson et al. 1995a) for the preparation of RFLP and microsatel-lite maps, was utilized for mapping in the present study. At the UMR INRA-UBP ASP of Clermont-Ferrand (Institut National dela Recherche Agronomique), France, total DNA was extracted and purified as described by Lu et al. (1994). After lyophilization,the same samples of total DNA extracted and purified from the 70 RILs were distributed to the ten members of the IWMMN formapping analysis. These 70 RILs are a subset (following datafrom RFLP and cytogenetic analysis, data not shown) of 115 RILs(F7 progeny) of the ITMI popused previously to build the ITMImap(Nelson et al. 1995a, b, c; Marino et al. 1996; Röder et al. 1998; Pestsova et al. 2000). Segregating data were collated intoa database at the INRA of Clermont-Ferrand for mapping analysis. Genetic mapping A reference ITMI mapconsisting of 266 anchor markers (mainly RFLPs; Leroy et al. 1997) was prepared using MAPMAKER/Exp Version 3. 0b (Lander et al. 1987) and marker data from 70 RILswere used for mapping each of the new microsatellite markers. Linkage groups were established by using a maximum recombina-tion fraction of 0. 35 and a minimum LOD score of 3. 0. Markerswith minimal missing data or segregation distortion were selectedto build a skeleton map for each chromosome. The order was thenrefined using the MAPMAKER 'ripple' command. Other markerswere assigned to intervals between the anchor markers using the MAPMAKER 'assign' command at LOD 3. 0 and recombina-tion fraction 0. 35. The markers were also mapped using the MAPMAKER 'map' command. The loci on the skeleton mapwere checked using the MAPMAKER 'links any' command. Seg-regation distortion was calculated using an in-house S+ program. Map distance (c M) values were calculated using Haldane mappingfunction (Haldane1919). In some cases, the “assigned values” dif-fered from the “mapped values”. Only mapped values were usedfor integrating the mapped microsatellite markers into the skeletonmap. Results Functional and polymorphic primer pairs A total of 396 microsatellite primer pairs, the '' wmcse-ries”, were developed by the WMC, which had 38 mem-bers in November 1998. Of the 396 wmcprimer pairs thus designed by WMC, during the present study a totalof 176 primer pairs were used by ten members of the IWMMN, to detect polymorphism between synthetic414

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W7984 and the wheat variety Opata 85, the parents of the ITMI pop. Fifty eight (58) primer pairs (33%) were found to identify polymorphism, and were therefore usedfor genotyping the set of 70 RILs from the ITMI pop. These 70 RILs have been found to be adequate for adding new molecular markers on the existing map andare the same which were earlier used successfully for ge-netic mapping of 279 microsatellite loci (Röder et al. 1998). Microsatellites used for genotyping by differentmembers of the IWMMN during the present study arelisted in Table 1 and the primer sequences of these mi-crosatellites are given in Table 2. Mapping of microsatellite loci A reference skeleton framework ITMI map, consisting of 266 anchor loci, was prepared for mapping purposes,and the genotyping data of the ITMI popwere used for gene-tic mapping on this framework map. Fifty eight(58) primer pairs amplified 66 mappable loci. For 20 ofthe 58 primer pairs, the loci amplified were also as-signed to specific chromosomes or their specific arms,utilising Chinese Spring nullisomic-tetrasomic lines andthe ditelocentric lines (Table 2). The genetic map show-ing the loci mapped during the present study along withthe reference loci is shown in Fig. 1. In this figure, thechromosomal centromeric positions on the genetic mapare approximate and are based on earlier mapping ana-lyses conducted using the ITMI pop(Nelson et al. 1995a, b, c; Marino et al. 1996; Röder et al. 1998; Pestsova et al. 2000) and recent comparative mappingconducted using deletion maps (Cadalen, personal com-munication; Langridge, personal communication). Thelinear order of marker loci for each chromosome skeletonwas also verified from the reference ITMI mapprepared in these earlier studies. The results of genetic mappingwere in agreement with the results of the chromo-some assignment undertaken using nullisomic-tetra-somic lines and ditelocentric lines in 15 of the 20 cases(Table 2). Distribution of mapped loci on genomes and homoeologous groups The B genome had a maximum of 27 from the 66 loci mapped, while A and D genomes had 20 and 19 loci re-spectively (Fig. 1). Such a predominance of mapped mi-crosatellites on the B genome was also observed in themap constructed earlier by Röder et al. (1998). Amongthe seven homoeologous groups also, the microsatelliteswere not uniformly distributed. A maximum of 18 lociwere mapped on the three chromosomes of homoeolog-ous group 2, a minimum of six loci each were availableon chromosomes of groups 6 and 7, and no locus wasmapped on chromosome 6D. There was no consistentpattern in the distribution of loci in individual linkagegroups; in some cases the loci were restricted to the dis-tal regions, while in other cases loci mapped near thecentromere or were evenly distributed. Microsatellites having multiple loci (homoeologous vs non-homoeologous) Out of the 58 primer pairs used, only 12 amplified more than one mappable locus, so that the majority of markersmapped in this study were chromosome specific. This istrue for the majority of microsatellites in general, sincein an earlier study, out of 15 functional microsatelliteprimer pairs examined in this manner, only five ampli-fied more than one locus suggesting that only about20-30% microsatellites have homoeoloci (Varshney etal. 2000). These results are also in agreement with thoseobtained with the 'gwm' set of microsatellites, where on-ly 20% of the markers detected more than one locus(Röder et al. 1998). It is possible that homoeoloci for in-dividual microsatellite loci in two other genomes carrynull alleles thus allowing no amplification. Only se-quencing of the corresponding regions in homoeologouschromosomes could resolve this question. In the presentstudy, however, homoeoloci were detected by only 6 outof the 58 pairs of primers, of which six primer pairs415 Table 1List of microsatellite markers analyzed by individual members of the IWMMN Laboratory wmc microsatellites Number of microsatellitesanalyzed UMR INRA-UBP ASP, France wmc163, wmc166, wmc167, wmc168, wmc169, wmc173, wmc175, 10 wmc177, wmc181, wmc182 Monsanto, USA wmc147, wmc149, wmc153, wmc154, wmc156, wmc157, wmc161, 8 wmc213 CCS University, India wmc254, wmc256, wmc257, wmc261, wmc262, wmc264, wmc265 7University of California, USA wmc41, wmc43, wmc44, wmc47, wmc48, wmc49, wmc51, wmc52 8CIMMYT, Mexico wmc322, wmc326, wmc327, wmc329, wmc331 5Agriculture and Agri-Food, Canada wmc94, wmc95, wmc96, wmc97, wmc104, wmc105 6University of Sydney, Australia wmc232, wmc233, wmc238, wmc243, wmc245 5University of Zurich, Switzerland wmc272, wmc273, wmc276 3USDA-ARS, Utah, USA wmc24, wmc25, wmc27 3Benoist Ets, France wmc215, wmc216, wmc219 3 Total 58

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416 Table 2Primer sequences of 58 microsatellites used for mapping Microsatellites Forward primer (5 ′3′) Reverse primer (5 ′3′) Chromosome/arma wmc24 GTGAGCAATTTTGATTATACTG TACCCTGATGCTGTAATATGTG 2A wmc25 TCTGGCCAGGATCAATATTACT TAAGATACATAGATCCAACACC 2AS, 2BS, 2DSwmc27 AATAGAAACAGGTCACCATCCG TAGAGCTGGAGTAGGGCCAAAG-wmc41 TCCCTCTTCCAAGCGCGGATAG GGAGGAAGATCTCCCGGAGCAG-wmc43 TAGCTCAACCACCACCCTACTG ACTTCAACATCCAAACTGACCG-wmc44 GGTCTTCTGGGCTTTGATCCTG TGTTGCTAGGGACCCGTAGTGG 1Bwmc47 GAAACAGGGTTAACCATGCCAA ATGGTGCTGCCAACAACATACA 7Awmc48 GAGGGTTCTGAAATGTTTTGCC ACGTGCTAGGGAGGTATCTTGC-wmc49 CTCATGAGTATATCACCGCACA GACGCGAAACGAATATTCAAGT-wmc51 TTATCTTGGTGTCTCATGTCAG TCGCAAGATCATCAGAACAGTA-wmc52 TCCAATCAATCAGGGAGGAGTA GAACGCATCAAGGCATGAAGTA-wmc94 TTCTAAAATGTTTGAAACGCTC GCATTTCGATATGTTGAAGTAA-wmc95 GTTTTTGTGATCCCGGGTTT CATGCGTCAGTTCAAGTTTT-wmc96 TAGCAGCCATGCTTAGCATCAA GTTTCAGTCTTTCACGAACACG-wmc97 GTCCATATATGCAAGGAGTC GTACTCTATCGCAAAACACA-wmc104 TCTCCCTCATTAGAGTTGTCCA ATGCAAGTTTAGAGCAACACCA 6BSwmc105 AATGTCATGCGTGTAGTAGCCA AAGCGCACTTAACAGAAGAGGG-wmc147 AGAACGAAAGAAGCGCGCTGAG ATGTGTTTCTTATCCTGCGGGC-wmc149 ACAGACTTGGTTGGTGCCGAGC ATGGGCGGGGGTGTAGAGTTTG 2Bwmc153 ATGAGGACTCGAAGCTTGGC CTGAGCTTTTGCGCGTTGAC-wmc154 ATGCTCGTCAGTGTCATGTTTG AAACGGAACCTACCTCACTCTT-wmc156 GCCTCTAGGGAGAAAACTAACA TCAAGATCATATCCTCCCCAAC-wmc157 CTTGATCCAAGTGGTTCTTTCC TCCAAATGTTTGCGAAACCTGA-wmc161 ACCTTCTTTGGGATGGAAGTAA GTACTGAACCACTTGTAACGCA-wmc163 TTACACCCATCAGGGTGGTCTT GTCTATCCATACGACAAA-wmc166 ATAAAGCTGTCTCTTTAGTTCG GTTTTAACACATATGCATACCT-wmc167 AGTGGTAATGAGGTGAAAGAAG TCGGTCGTATATGCATGTAAAG 2Dwmc168 AACACAAAAGATCCAACGACAC CAGTATAGAAGGATTTTGAGAG-wmc169 TACCCGAATCTGGAAAATCAAT TGGAAGCTTGCTAACTTTGGAG 3Awmc173 TGCAGTTGCGGATCCTTGA TAACCAAGCAGCACGTATT-wmc175 GCTCAGTCAAACCGCTACTTCT CACTACTCCAATCTATCGCCGT-wmc177 AGGGCTCTCTTTAATTCTTGCT GGTCTATCGTAATCCACCTGTA-wmc181 TCCTTGACCCCTTGCACTAACT ATGGTTGGGAGCACTAGCTTGG-wmc182 GTATCTCACGAGCATAACACAA GAAAGTGTATGGATCATTAGGC-wmc213 ATTTTCTCAAACACACCCCG TAGCAGATGTTGACAATGGA-wmc215 CATGCATGGTTGCAAGCAAAAG CATCCCGGTGCAACATCTGAAA-wmc216 ACGTATCCAGACACTGTGGTAA TAATGGTGGATCCATGATAGCC 7Bwmc219 TGCTAGTTTGTCATCCGGGCGA CAATCCCGTTCTACAAGTTCCA-wmc232 GAGATTTGTTCATTTCATCTTCGCA TATATTAAAGGTTAGAGGTAGTCAG 4ALwmc233 GACGTCAAGAATCTTCGTCGGA ATCTGCTGAGCAGATCGTGGTT 5DSwmc238 TCTTCCTGCTTACCCAAACACA TACTGGGGGATCGTGGATGACA-wmc243 CGTCATTTCCTCAAACACACCT ACCGGCAGATGTTGACAATAGT 2BLwmc245 GCTCAGATCATCCACCAACTTC AGATGCTCTGGGAGAGTCCTTA 2AS, 2BS, 2DSwmc254 AGTAATCTGGTCCTCTCTTCTTCT AGGTAATCTCCGAGTGCACTTCAT 1Awmc256 CCAAATCTTCGAACAAGAACCC ACCGATCGATGGTGTATACTGA 6A, 6Dwmc257 GGCTACACATGCATACCTCT CGTAGTGGGTGAATTTCGGA 2Bwmc261 GATGTGCATGTGAATCTCAAAAGTA AAAGAGGGTCACAGAATAACCTAAA 2Awmc262 GCTTTAACAAAGATCCAAGTGGCAT GTAAACATCCAAACAAAGTCGAACG 4A, 5Bwmc264 CTCCATCTATTGAGCGAAGGTT CAAGATGAAGCTCATGCAAGTG 3Awmc265 GTGGATAACATCATGGTCAAC TACTTCGCACTAGATGAGCCT 2Bwmc272 TCAGGCCATGTATTATGCAGTA ACGACCAGGATAGCCAATTCAA-wmc273 AGTTATGTATTCTCTCGAGCCTG GGTAACCACTAGAGTATGTCCTT-wmc276 GACATGTGCACCAGAATAGC AGAAGAACTATTCGACTCCT-wmc322 CGCCCCACTATGCTTTG CCCAGTCCAGCTAGCCTCC-wmc326 GGAGCATCGCAGGACAGA GGACGAGGACGCCTGAAT-wmc327 TGCGGTACAGGCAAGGCT TAGAACGCCCTCGTCGGA-wmc329 ACAAAGGTGCATTCGTAGA AACACGCATCAGTTTCAGT-wmc331 CCTGTTGCATACTTGACCTTTTT GGAGTTCAATCTTTCATCACCAT-a Assigned using nullisomic-tetrasomics and ditelocentrics Fig. 1Molecular linkage map of bread wheat showing the posi-tions of 66 new microsatellite loci. For each chromosome, markersare shown on the right and genetic distances in c M (centimorgans)are shown on the left. The new wmc ( wheat microsatellite con-sortium) microsatellite loci that were mapped during the presentstudy are shown in bold italics. Primer pairs that amplify morethan one locus on homoeologous chromosomes are marked *, and markers with more than one locus on non-homoeologouschromosomes are marked **. The approximate positionof the centromere on each chromosome is shown by a box and the approximate length in c M (Haldane) is given at the bottomof each chromosome L

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417

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418 Fig. 1(continued)

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419 Fig. 1(continued)

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(wmc25, wmc43, wmc48, wmc175, wmc181, wmc329) each detected two homoeoloci (Table 3). In at least twocases (wmc96, wmc166), the multiple loci amplified bythe same primer pair belonged to non-homoeologouschromosomes; in four other cases (wmc169, wmc173,wmc322, wmc323) multiple loci were available butcould not be assigned to specific chromosomes. Discussion The present study adds to the repertoire of molecularmarkers, so far used for the construction of molecularmaps in bread wheat. These maps were extensively usedfor comparative genomics, although all classes ofmapped markers were not found to be equally useful forthis purpose (Devos and Gale 1997). It is also knownthat the resolution available in the existing molecular ge-netic maps in bread wheat is not satisfactory either formap-based cloning or for gene tagging that is requiredfor marker-aided selection. Therefore, there is a need toadd more molecular marker loci to the available maps. The present study is an effort in this direction. The results of genetic mapping in the present study were largely in agreement with those of chromosome as-signment done using nullisomic-tetrasomic and ditelo-centric lines, although this exercise of chromosome as-signment could not be undertaken for all the microsatel-lite primer pairs used in the present study (only 20 primerpairs could be used for chromosome assignment; see Table 2). For some of the microsatellite markers, themapping results of the present study involving the ITMIpopwere also confirmed either by using another mapping population (at CIMMYT by M. Khairallah, per-sonal communication) or in another independent studyusing ITMI pop(at Agriculture Canada, in Winnipeg by D. Somers, personal communication). For two markers,namely wmc41 (associated with grain protein content)and wmc104 (associated with pre-harvest sprouting), theresults of physical mapping obtained using deletionstocks (Endo and Gill 1996) were also in agreement withthe present results of genetic mapping (Varshney et al. 2001). Hopefully, physical mapping for all other SSR loci mapped during the present study will certainly beundertaken in the future with the help of available dele-tion lines. In bread wheat, molecular markers that have been used for mapping can be broadly classified into threegroups: (1) those having triplicate homoeoloci, one locus each on three chromosomes of a homoeologous group,(2) those having multiple loci, but not on homoeologouschromosomes, and (3) those which are chromosome spe-cific, each with a single locus. RFLPs can also be of twotypes, those derived from c DNA probes (based on con-served expressed sequences) generally having triplicatehomoeoloci, and others derived from genomic DNA,which may or may not have triplicate homoeoloci. Insharp contrast to these RFLPs, microsatellite primersusually amplify a specific locus each, and therefore theygenerally belong to the third category. In the presentstudy, only a small proportion of microsatellite primerpairs (12 out of 58) amplified more than one locus, andonly 50% of these multilocus microsatellite primers am-plified homoeoloci (Table 3). Other multilocus microsat-ellites amplified loci on non-homoeologous or unknownchromosomes. The microsatellite wmc96 amplified twoloci, on non-homoeologous chromosome arms 4AS and5AS; similarly wmc166 amplified two loci on non-homoeologous chromosome arms 2DS and 7BL. If it isassumed that multiple loci in bread wheat should be either homoeoloci or duplicated loci, then the loci for thesame microsatellite on non-homoeologous chromosomesmay be either due to translocations or to duplications between non-homoeologous chromosomes. Many suchtranslocations and duplications were actually detected inplants, using the approach of comparative genomics in-volving use of heterologous probes for molecular map-ping. In wheat also, a locus-containing gene encoding areceptor-like kinase was shown to be duplicated on chro-mosomes 3S and 1S. The duplication on 1S was found tobe specifically occurring in the whole Triticeae (Feuilletand Keller 1999). In any case, the microsatellite markersmay not prove very useful for comparative genomics toresolve the conservation of colinearity. However, theyare very useful for gene tagging and QTL analysis. Efforts are being made worldwide to develop and map additional microsatellite primers, so that within a fewyears at least 1,000 microsatellite loci will be availableon the map. For instance, recently, M. Röder and her col-leagues at Gatersleben (Germany) have developed a setof 55gdmmicrosatellite markers for the D genome of bread wheat (Pestsova et al. 2000), and in France an ad-ditional set of 337 microsatellite loci were mapped on allthe three genomes (Sourdille et al. 2001). Other sets ofmicrosatellites including some wmcloci are being mapped in Australia (Harker et al. 2001) and Canada 420 Table 3Homoeoloci among 66 microsatellite loci assignedon a skeleton map usingthe ITMIpop Homoeologous group Genome and chromosome arm (S = short arm; L = long arm) AS AL BS BL DS DL 1--wmc329-wmc329-2-wmc181 wmc25-wmc25 wmc181---wmc175-wmc175 3--wmc43-wmc43-4--wmc48-wmc48-

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(personal communication, D. Somers; no details were made available). Thus the remaining wmc and other ad-ditional microsatellite markers will be mapped in duecourse of time. The WMC has already initiated the development of another set of wmcmarkers in its second phase with fewer members, and it may also undertake designing aset of anchored wmc primers from sequences that wereearlier considered unsuitable for primer designing (P. Isaac, personal communication). In future, microsatel-lites will also be developed through searches of genomicand EST (expressed sequence tag) sequence resources,that are becoming freely available from the ITEC (Inter-national Triticeae EST Consortium, http://wheat. pw. usda. gov/genome/index. html), the USDA/ARS wheat endo-sperm EST project, and its joint NSF project “The Struc-ture and Function of the Expressed Portion of the Wheat Genomes” (http://wheat. pw. usda. gov/NSF/htmlversion. html). In addition, microsatellites will be searched for ina large number of proprietary ESTs available from theprivate sector. The new microsatellite markers, whichwill thus become available, will also be eventuallymapped giving a fairly saturated map to be used for map-based cloning and gene tagging in bread wheat. The ITMI is also making new coordinating efforts towardsthe development of functional microsatellites and SNPs(Single Nucleotide Polymorphisms), and it is proposedthat a set of 150 core microsatellites spanning the wholegenome be developed and a core germplasm for charac-terization of these microsatellites may be assembled forfuture users (ITMI meeting at San Diego, California,January 13, 2001, during the Plant and Animal Genome IX meeting). Molecular genetic maps in bread wheat, in the past, as well as in the present study, have been prepared with thejoint effort of several laboratories. In such an exercise, itis necessary to ensure that different laboratories use thesame mapping population. Further, while adding newmarkers to an existing map jointly by different laborato-ries, as being done in the present study, it is important toensure that not only the same mapping population beused by different laboratories, but also that this popula-tion does not differ from the one used for preparing theoriginal map that is being extended. In bread wheat, amapping population (ITMI pop) consisting of 130 RILs, was initially prepared and a set of 115 RILs was used bydifferent laboratories for preparing the genetic maps. However, subsequently it was realized that a smallersubset of 70 RILs along with a framework map may ac-tually be adequate for adding new markers to the exist-ing map. This subset of 70 RILs was also earlier utilizedsuccessfully by Röder et al. (1998) in their study involv-ing the mapping of 279 SSR loci. In the present study,the same sample of DNA from such a single set of 70 RILs of the ITMI popwas used by each of the ten laboratories for mapping 66 microsatellite loci. However,when different subsets of the ITMI popare used, or seeds of the ITMI popare obtained from different sources, no major differences were actually observed (Mark Sorrells,personal communication), thus indirectly validating the mapping of 66 microsatellite loci in the present study. Despite this, since there may be additional problemswith the identity of RILs due to chromosome rearrange-ments, and other errors due to handling, it is recom-mended that in future mapping efforts, fresh seed shouldalways be obtained from the same source. It is also sug-gested that the subset of the ITMI popbeing used for mapping be validated by genotyping them using a subsetof mapped representative markers from all the 21 chro-mosomes, to ensure that no alterations of any kind thatmay interfere with mapping have occurred over time. One should, however, also realize that even if differentsubsets are used by different laboratories engaged inmapping, eventually rare mistakes will be sorted out andstandard high-density integrated maps with a variety ofmolecular markers should become available to be usedfor a variety of purposes including marker-aided selec-tion and map-based cloning of genes. Wheat workersworldwide look forward to further developments for thesaturation of genetic maps with new molecular markersincluding SSRs and SNPs. Acknowledgements Thanks are due to B. Charef (INRA at Clermont-Ferrand) and Joy K. Roy (Meerut, India) for their helpin the preparation of the manuscript, and to Dr. D. Somers (AAFC,Winnipeg) and Dr. M. Sorrells (Cornell University) for useful dis-cussion and advice. References Bennett MD, Leitch IJ (1995) Nuclear DNA amounts in angio-sperms. 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