Abstract
Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivum L.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1–7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.
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References
Akhunov ED, et al (2003) The organization and rate of evolution of the wheat genome are correlated with recombination rates along chromosome arms. Genome Res (http://www.genome.org/cgi/doi/10.1101/gr.808603)
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic Local Alignment Search Tool. J Mol Biol 215:403–410
Bachem CWB, Van der Hoeven RS, de Brujin SM, Vreugdenhill D, Zabeau M, Visser RGF (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9:745–753
Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331
Brown GR, Kadel EE III, Bassoni DL, Kiehne KL, Temesgen B, van Buijtenen JP, Sewell MM, Marshall KA, Neale DB (2001) Anchor reference loci in loblolly pine ( Pinus taeda L.) for integrating pine genomics. Genetics 159:799–809
Burstin J, Deniot G, Potier J, Winachter C, Aubert G, Baranger A (2001) Microsatellite polymorphism in Pisum sativum. Plant Breed 120:311–317
Cardle L, Ramsay L, Milbourne D, Macaulay M, Marshall D, Waugh R (2000) Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics 156:847–854
Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR, Park WD, Ayres N, Cartinhour S (2000) Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice ( Oryza sativa L.). Theor Appl Genet 100:713–722
Cordeiro GM, Casu R, McIntyre CL, Manners JM, Henry RJ (2001) Microsatellite markers from sugarcane ( Saccharum spp.) ESTs cross transferable to Erianthus and sorghum. Plant Sci 160:1115–1123
Edwards KJ, Mogg R (2001) Plant genotyping by analysis of single nucleotide polymorphisms. In: Henry RJ (ed) Plant genotyping: the DNA fingerprinting of plants. CABI, Wallingford, UK, pp 1–13
Eujayl I, Sorrells ME, Baum M, Wolters P, Powell W (2002) Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theor Appl Genet 104:339–407
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Gupta PK, Varshney RK (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113:163–185
Gupta PK, et al (2002) Genetic mapping of 66 new microsatellite (SSR) loci in bread wheat. Theor Appl Genet 105:413–422
Holton TA, Christopher JT, McClure L, Harker N, Henry RJ (2002) Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat. Mol Breed 9:63–71
International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409:860–920
Jaccard P (1908) Nouvelles recherches sur la distribution florale. Bull Soc Vand Sci Nat 4:223–270
Kantety RV, La Rota M, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501–510
Maestri E, Malcevschi A, Massari A, Marmiroli N (2002) Genome analysis of cultivated barley ( Hordeum vulgare) using sequence-tagged molecular markers. Estimates based on divergence based on RFLP and PCR markers derived from stress-responsive genes, and simple sequence repeats (SSRs). Mol Genet Genomics 267:186–201
Metzgar D, Bytof J, Wills C (2000) Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res 10:72–80
Mogg R, Batley J, Hanley S, Edwards D, O'Sullivan H, Edwards KJ (2002) Characterization of the flanking regions of Zea mays microsatellites reveals a large number of useful sequence polymorphisms. Theor Appl Genet 105:532–543
Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with non-repetitive DNA in plant genomes. Nature Genet 30:194–200
Prasad M, Varshney RK, Kumar A, Balyan HS, Sharma PC, Edwards KJ, H-Singh, Dhaliwal HS, Roy JK, Gupta PK (1999) A microsatellite marker associated with a QTL for grain protein content on chromosome arm 2DL of bread wheat. Theor Appl Genet 99:341–345
Prasad M, Varshney RK, Roy JK, Balyan HS, Gupta PK (2000) The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat. Theor Appl Genet 100:584–592
Roy JK, Prasad M, Varshney RK, Balyan HS, Blake TK, Dhaliwal HS, Singh H, Edwards KJ, Gupta PK (1999) Identification of microsatellite on chromosome 6B and a STS on 7D of bread wheat showing an association with preharvest sprouting tolerance. Theor Appl Genet 99:336–340
Roy JK, Balyan HS, Prasad M, Gupta PK (2002) Use of SAMPL for a study of polymorphism, genetic diversity and possible gene tagging. Theor Appl Genet 104:465–472
Roy JK, Lakshmikumaran M, Balyan HS, Gupta PK (2004) AFLP-based genetic diversity and its comparison with diversity based on SSR, SAMPL and phenotypic traits in bread wheat. Biochem Genet, in press
Scott KD, Eggler P, Seaton G, Rossetto M, Ablett EM, Lee LS, Henry RJ (2000) Analysis of SSRs derived from grape ESTs. Theor Appl Genet 100:723–726
Sneath PHA, Sokol RR (1973) Numerical taxonomy. WH Freeman, San Francisco
Somers DJ, Kirkpatrick R, Moniwa M, Walsh A (2003) Mining single nucleotide polymorphisms from hexaploid wheat ESTs. Genome 49:431–437
Stack S, Campbell L, Henderson K, Eujayl I, Hanafey M, Powell W, Wolter P (2000) Development of EST-derived microsatellite markers for mapping and germplasm analysis in wheat. In: Abstracts of Plant and Animal Genome VIII, January 9–12, 2000, San Diego
Suarez MC, Bernal A, Gutierrez, Tohme J, Fregene M (2000) Developing expressed sequence tags (ESTs) from polymorphic transcript-derived fragments (TDFs) in cassava ( Manihot esculenta Crantz). Genome 43:62–67
Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST database for the development and characterization of gene derived SSR-markers in barley ( Hordeum vulgare L.). Theor Appl Genet 106:411–422
Varshney RK, Kumar A, Balyan HS, Roy JK, Prasad M, Gupta PK (2000) Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat. Plant Mol Biol Rep 18:5-16
Varshney RK, Thiel T, Stein N, Langridge P, Graner A (2002) In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cell Mol Biol Lett 7:537–546
Acknowledgements
This work was carried out in compliance with the current laws governing genetic experiments in India. The financial support received from the Department of Biotechnology, Government of India for carrying out this study, and from the University Grant Commission in the form of an Emeritus Fellowship to PKG is gratefully acknowledged. We also thank Dr. H. S. Dhaliwal for providing the seed material of the alien species, Dr. K. V. Bhatt for providing help in dendrogram preparation, and Dr. N. D. Young for supplying the macro used for the identification of EST-SSRs
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Gupta, P.K., Rustgi, S., Sharma, S. et al. Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Mol Genet Genomics 270, 315–323 (2003). https://doi.org/10.1007/s00438-003-0921-4
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DOI: https://doi.org/10.1007/s00438-003-0921-4