Funct Integr Genomics (2005) 5: 260–263DOI 10.1007/s10142-005-0146-1

SHORT COMMUNICATION

Aakash Goyal . Rajib Bandopadhyay .Pierre Sourdille . Takashi R. Endo .Harindra S. Balyan . Pushpendra K. Gupta

Physical molecular maps of wheat chromosomes

Received: 22 February 2005 / Revised: 10 April 2005 / Accepted: 10 April 2005 / Published online: 19 May 2005# Springer-Verlag 2005

Abstract In bread wheat, a set of 527 simple sequencerepeats (SSRs) were tried on 164 deletion lines, leading toa successful mapping of 270 SSRs on 313 loci covering all21 chromosomes. A maximum of 119 loci (38%) werelocated on B subgenome, and a minimum of 90 loci (29%)mapped on D subgenome. Similarly, homoeologous group7 carried a maximum of 61 loci (19%), and group 4 carrieda minimum of 22 loci (7%). Of the cited 270 SSRs, 39 hadmultiple loci, but only eight of these detected homoe-ologous loci. Linear order of loci in physical maps largelycorresponded with those in the genetic maps. Apparently,distances between each of only 26 pairs of loci significantlydiffered from the corresponding distances on genetic maps.Some loci, which were genetically mapped close to thecentromere, were physically located distally, while otherloci that were mapped distally in the genetic maps werelocated in the proximal bins in the physical maps. Thissuggested that although the linear order of the loci waslargely conserved, variation does exist between genetic andphysical distances.

Keywords Simple sequence repeats (SSRs) .Genetic maps . Deletion lines . Physical maps

Introduction

Bread wheat (Triticum aestivum L. em Thell, 2n=42,AABBDD) has a large genome (∼16 million kb/hap-loid cell), which is ∼35 times the size of rice genome and∼110 times the size of Arabidopsis genome (Bennett andSmith 1976). During the last decade, molecular markers—mainly including restriction fragment length polymor-phism (RFLPs) and SSRs (including EST-SSRs)—wereextensively used for genetic and physical mapping in wheat(for references, see Varshney et al. 2004; Somers et al.2004; Sourdille et al. 2004; Song et al. 2005; Peng andLapitan 2005). Among the different classes of markersused for the construction of molecular maps in the past,SSRs were the markers of choice, because they belong to aclass of markers that are codominant, locus-specific andsuitable for detecting a high level of polymorphism (Röderet al. 1995; Plaschke et al. 1995; Gupta et al. 1996; Guptaet al. 2002). In bread wheat, as many as ∼2,150 SSRs havealready been genetically mapped, of which only ∼1,050SSRs have been physically mapped (Röder et al. 1998;Varshney et al. 2001; Sourdille et al. 2004; Song et al.2005). This leaves a large number of genetically mappedSSRs that are yet to be physically assigned. The presentstudy marks an effort to prepare physical maps of the re-maining wheat SSR markers and to study the relationshipbetween distribution of markers and recombination bycomparing the physical map with available genetic maps(Sourdille et al. 2004; Somers et al. 2004). As many as 527SSR markers were tried, of which 270 markers were suc-cessfully mapped on 313 loci.

Materials and methods

Plant material

A set of 21 nulli-tetrasomic (NT) lines and 24 ditelosomic(DT) lines of Chinese Spring (kindly provided by Dr. B. S.Gill, Kansas State University, USA) were used for local-isation of markers on individual chromosomes and their

Electronic Supplementary Material Supplementary material isavailable for this article at http://dx.doi.org/10.1007/s10142-005-0146-1.

A. Goyal . R. Bandopadhyay . H. S. Balyan . P. K. Gupta (*)Molecular Biology Laboratory, Department of Genetics &Plant Breeding, Ch. Charan Singh University,Meerut, Uttar Pradesh, 250 004, Indiae-mail: pkgupta36@vsnl.comTel.: +91-121-2768195Fax: +91-121-2768195

P. SourdilleUMR INRA–UBP Amélioration et Santé des Plantes,234, Avenue du Brézet,63039 Clermont-Ferrand, France

T. R. EndoGraduate School of Agriculture, Kyoto University,Kyoto, 606-8502, Japan

arms. A total of 164 homozygous overlapping deletionlines from a total of 436 available deletion lines were usedfor bin localisation (for details, see Table 1, Fig. 1). Thedetails of these deletion lines are available elsewhere(Endo and Gill 1996). The number of deletion lines used inthe present study ranged from 2 to 7 per arm of a chro-mosome and 6 to 11 for an individual chromosome. De-letion lines of 4BS were not available to us. Three SSRgenetic maps, including two prepared by Sourdille et al.(2004) and one by Somers et al. (2004), were used forcomparison with physical map constructed during thepresent study.

SSR markers

We used 527 SSR primer pairs (167 wmc, 192 gwm, 34cfa, 74 cfd and 60 psp) for physical mapping. Primersequences for wmc, cfa and cfd markers were availableto us and those for gwm markers were supplied byM. Röder. Primers were mainly synthesised either byIllumina Inc. (USA) or MWG (Germany) except for pspmarkers, for which primer aliquots were kindly suppliedby P. Stephenson (John Innes Centre, UK). (For DNA iso-lation and PCR analyses, see Prasad et al. 2000.)

Results and discussion

Physical maps of SSRs prepared duringthe present study

In the present study, using 270 SSRs, we prepared aphysical maps of 313 SSR loci (114 Xwmc, 153 Xgwm, 17Xcfa, 25 Xcfd and 4 Xpsp), which represented nearly onethird of the ∼1,100 SSRs loci that were genetically mappedearlier but were not yet placed on the physical maps. These313 SSR loci were distributed on all 21 chromosomes, al-though the distribution was not random (ESM Fig. 1a–u).

A set of additional 257 SSR markers (71 wmc, 62 gwm, 18cfa, 50 cfd and 56 psp), which were also used for physicalmapping, could not be unambiguously mapped, becausein each case, the relevant band of expected size wasalso available in at least one of the two corresponding null-

Table 1 A summary of the number of terminal deletion lines used and the number of SSR loci physically mapped to chromosomes of all theseven homoeologous groups of the three (A, B and D) subgenomes of bread wheat

Homoeologousgroup

A subgenome B subgenome D subgenome Total no.

Deletionlinesused

Locimapped

Region(D, C, I)

Deletionlinesused

Locimapped

Region(D, C, I)

Deletionlinesused

Locimapped

Region(D, C, I)

Deletionlinesused

Locimapped

Region(D, C, I)

1 6 10 (4, 4, 2) 10 14 (5, 9, 0) 8 14 (4, 10, 0) 24 38 (13, 23, 2)2 6 16 (6, 8, 2) 9 18 (7, 9, 2) 9 19 (6, 10, 3) 24 53 (19, 27, 7)3 7 19 (10, 6, 3) 9 18 (8, 8, 2) 6 10 (5, 1, 4) 22 47 (23, 15, 9)4 7 09 (7, 2, 0) 7 05 (2, 3, 0) 8 08 (4, 2, 2) 22 22 (13, 7, 2)5 11 15 (3, 7, 5) 9 24 (6, 13, 5) 6 14 (7, 5, 2) 26 53 (16, 25, 12)6 6 12 (5, 7, 0) 8 22 (4, 13, 5) 8 05 (3, 2, 0) 22 39 (12, 22, 5)7 10 23 (8, 14, 1) 7 18 (8, 8, 2) 7 20 (6, 14, 0) 24 61 (22, 36, 3)Total 53 104 (43, 48, 13) 59 119 (40, 63, 16) 52 90 (35, 44, 11) 164 313 (118, 155, 40)

D Number of loci mapped in distal region, C number of loci mapped in centromeric region, I number of loci mapped in interstitial region

Fig. 1 PCR amplification profiles used for physical mapping ofSSR primers: a wmc216 mapped in the proximal region of 1DL;Lane M 100-bp ladder, 1 CS, 2 N1DT1A, 3 N1DT1B, 4 DT1DS, 5DT1DL, 6 1DL-4, 7 1DL-1, 8 1DL-6, 9 1DL-2. b cfa2134 mappedin interstitial region of 3AL; Lane M 100-bp ladder, 1 CS, 2N3AT3B, 3 N3AT3D, 4 DT3AS, 5 DT3AL, 6 3AL-2, 7 3AL-1, 83AL-3, 9 3AL-8

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isomic tetrasomics. The plausible explanation for this un-expected result is that the concerned SSR in each case wasperhaps either present on more than one homoeologues,exhibiting no polymorphism or the concerned region wasinvolved in a translocation. These 257 SSRs markers remainthe subject of future research.

The information about the physical map involving all the21 chromosomes belonging to three subgenomes and sevenhomoeologous groups is summarised in Table 1. Thedistribution of SSR loci in the three subgenomes and theseven homoeologous groups did not differ from theirdistribution on the genetic maps. In the present study, amaximum of 119 loci were assigned to the B subgenome(with the highest DNA content) and a minimum of 90 locito the D subgenome (with the lowest DNA content). Sim-ilarly, a maximum of 61 loci were assigned to homoeolo-gous group 7 and a minimum of 22 loci to homoeologousgroup 4. The distribution pattern of EST loci in chromo-some bin map of bread wheat in a recent study was nodifferent, with B subgenome having significantly highernumber of mapped EST loci than the A and D subgenomes(Qi et al. 2004). While the DNA contents in the three sub-genomes were proportionate to the relative frequenciesof mapped loci, there is also evidence of higher level ofpolymorphism in the B subgenome than in the A and Dsubgenomes. This is attributed to higher level of gene du-plications preexisting in ancestral diploid donor speciesclosely related to the open pollinated species Aegilopsspeltoides (Huang et al. 2002; Li and Gill 2002). This ex-plains the relatively higher frequency of loci mapped on theB subgenome.

Comparison of the physical maps with available SSRgenetic maps

The physical maps prepared during the present study werecompared with three available SSR genetic maps, includ-ing the integrated SSR genetic map prepared by Somerset al. (2004), and the two other maps, i.e. CtCS (Courtot×Chinese Spring) and ITMI (International Triticeae Map-ping Initiative) maps prepared by Sourdille et al. (2004).A high degree of collinearity of SSRs was observed be-tween the present physical map and the three genetic maps,which was not unexpected. Similar collinearity betweenphysical and genetic maps of SSRs was recently reportedby Sourdille et al. (2004) and Song et al. (2005), althoughthe collinearity of markers mapped within the same bin cannot be examined in the physical maps of bread wheatprepared using the available deletion lines.

A small fraction of SSRs (12 SSR loci, 3.77%) werephysically mapped in regions other than those on the cor-responding genetic maps. These 12 SSRs include the fol-lowing: gwm124, 1BL; gwm425, 2AS; gwm356 andwmc261, 2AL; gwm374, 2BS; gwm5, 3AS; wmc206 andwmc326, 3BL; wmc446 and gwm397, 4AL; wmc417,6BL; and wmc435, 7BL (also see ESM Fig. 1a–u). Foreight of these 12 markers, located on nine different chro-mosome arms, this may be attributed to the occurrence of

paralogues or chromosome rearrangements, as was alsoinferred by Weng et al. (2000) for group 6 chromosomes ofbread wheat. Of the remaining four loci, two loci (wmc326and wmc206) were genetically mapped together at a dis-tance of 7 cM in the interstitial region on the 3BL; in aphysical map, these are placed together in a 3BL bin justadjacent to the centromere. The other two loci (wmc446and gwm397) were genetically mapped together at a dis-tance of 9 cM near the centromere on 4AL, but were placedtogether in the telomeric bin in the physical map of 4AL.These two pairs of two markers each may represent casesof the subtle chromosomal rearrangement. Alternatively,these may be separated from other genetically mapped loci,through long, low recombination regions.

Redundancy of SSR loci on physical maps

Another interesting feature of the present study involvedphysical mapping of multiple loci for individual SSRs.There were 39 (14.44%) such SSRs, which together al-lowed mapping of 82 loci representing 36 duplicate, twotriplicate and one quadruplicate loci. Of these multilocusSSRs, only eight SSRs (each detecting two loci) detectedtwo homoeoloci each; the remaining multilocus SSRs per-haps did not represent homoeoloci, even though some ofthem were present on homoeologous chromosomes ataltered positions. It is thus obvious that although SSRs areknown to be locus-specific, rarely homoeoloci and para-logues (nonhomoeologous duplicates) are also available, asalso reported in a number of earlier studies (Gupta et al.2002). Also, in an earlier study, 19% of EST loci involvingredundancy mapped physically to the nonhomoeologoussets of chromosomes in bread wheat (Qi et al. 2004). Sim-ilarly, 30% RFLP loci were reported to be duplicated onthe genetic map of Am genome of T. monococcum byDubcovsky et al. (1996). Analysis of wheat EST unigenesrevealed that among the total duplication events, a largemajority occurred even at the diploid level (Akhunov et al.2003). On the basis of the results of the present study,however, it is not possible to establish an ancestral rela-tionship between these redundant loci, although we feeltempted to speculate that these duplications could alsooccur due to the movement of SSRs containing transposon-like sequences, from chromosome to chromosome, as pro-posed by Qi et al. (2004) for EST loci. This hypothesis issupported by the fact that SSRs are indeed present insequences of transposable elements available in TREPdatabase (our unpublished results).

Possible future fine mapping of loci within bins

Both in the present study and in some earlier studies, dele-tion mapping was used to place SSRs in individual binscircumscribed by the breakpoints of deletion lines. How-ever, a disadvantage of the chromosome bin map is that theloci mapped within individual chromosome bins cannot beordered within a bin. To overcome this problem in case of

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ESTs mapped to wheat chromosome bins, in silico orderingof sequenced EST loci using the rice genome sequenceswas recently reported in bread wheat (Sorrells et al. 2003;Conley et al. 2004; Hossain et al. 2004; Linkiewicz et al.2004; Miftahudin Ross et al. 2004; Munkvold et al. 2004;Peng et al. 2004; Randhawa et al. 2004). However, a sim-ilar approach for ordering of SSRs mapped during thepresent study may not be successful, as the SSRs usedduring the present study were derived from genomic librar-ies and sequences homologous to the primers of these SSRsare generally not available in the rice genome sequences(our unpublished results). An approach that may poten-tially help in ordering the SSRs within the bins is thedevelopment and use of new deletion lines that may furthersubdivide the chromosome bins identifiable by the avail-able deletion lines. Using gamma irradiation of ChineseSpring monosomics for chromosome 1A, 2A and 3A ofbread wheat, we are currently developing such deletionlines for use in future studies on physical mapping of thesethree chromosomes. The SSRs assigned to specific chro-mosome bins during the present study may also be used asanchor points to the physical BAC contig global map pro-posed under the auspices of International Genome Re-search on Wheat (IGROW) (Gill et al. 2004).

Acknowledgements Thanks are due to Dr. P. Stephenson for pro-viding aliquots of 53 psp SSR primers, to Dr. B. S. Gill for providingthe Nulli–Tetrasomic and Ditelosomic lines and to Dr. R. Appels foruseful suggestions on the original manuscript. Financial support forthis study was provided by the Department of Atomic Energy-Boardof Research in Nuclear Science (DAE-BRNS), Government of India,Mumbai, India.

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