Triticeae EST-SSRs

Welcome to

Triticeae EST-SSR Coordination!

Rajeev Varshney

Nils Stein

Last update 19 May 2005

Proposition

It would be useful if people doing research on Triticeae EST-SSRs knew each other, to help avoid redundant effort discovering, testing and characterizing the same microsatellites.

We're compiling a list of such people. If you'd like to see it, write matthews@greengenes.cit.cornell.edu and we'll send it to you. The assumption is that anyone who's interested in the list should be included in it, but let us know if this doesn't apply to you.

Coordination planning

Public EST-SSR data

GrainGenes Database
- 16000 microsatellite-containing ESTs from wheat, barley, rice, maize and sorghum, assembled into 2463 nonredundant "Uni-EST-SSR" contigs, with their consensus sequences. There are 530 superclusters containing Uni-EST-SSRs from more than one species. From Ju-Kyung Yu, Ramesh Kantety, Mauricio LaRota and Mark Sorrells.
  - Search the consensus sequences with BLAST.
  - Download the consensus sequences as FASTA (2 MBytes).
  - Find superclusters containing two desired species:
- 180 rye EST-SSRs, from Bernd Hackauf.
- 15 SSRs in known barley genes, from Manfred Heun. With results on the primers.
- 48 "cwem" EST-SSRs, from Junhua Peng. With wheat chromosome arm locations. New, May '05
- 301 "cfe" EST-SSRs, from Pierre Sourdille. With wheat chromosome arm locations. New, May '05
This Coordination site
- 80 additional unique EST-SSRs from wheat, checked for being different from anything in the Sorrells Uni-EST-SSRs. See "Consensus sequences + individual ESTs" in the BLAST database.
  From H.S. Balyan and P.K. Gupta.
  - 78 EST-SSR primers. Mostly from the same set of 80.
    From Gupta, Balyan, and Rustgi.
- EST-SSRs from Cornell (superclusters) and KSU (wheat ESTs), mapped in wheat and rice, and tested on barley, maize and sorghum. Updated, Feb '05
- 13,000 EST-SSR primers for wheat, barley and rice. Untested, for your exploration.
  From Mauricio La Rota.
- 266 wheat EST-SSR loci from 48 primer pairs, mapped to chromosome arm using Chinese Spring aneuploids.
  From Junhua Peng. Supplementary Material from FIG 5:80 (2005). New, May '05
CUGI PlantSSR Database
- 3900 barley SSR sequences from CUGI's EST project and 15,000 from dbEST.
Other? Do you have public data available?

Discussion and status reports

Chabane Kamel:
I already in collaboration with Dr. Gary Abblet from Lismore University (AUS) done some preliminary works using wild, cultivated and cultivars barley analysis. These results should be presented in the Next Eucarpia Symposium, November 2002, Italy.

Rajeev Varshney:
At IPK, Gatersleben, EST-derived microsatellite markers are being developed as part of the efforts in preparation of a high-density transcript map of barley. About 150 EST-based SSR-markers have been mapped so far to either one of the three populations 'IgrixFranka', 'SteptoexMorex', 'OWB RecxDom`. A manuscript on characterization and mapping of 76 of these EST-derived SSRs is in press (Thiel et al., 2002). Results on database mining and the integration of the markers to a genetic consensus map of barley have been discussed. Besides, a subset of 38 EST-derived SSR-markers comprising 114 alleles was also used to investigate genetic diversity among 54 barley cultivars. The markers are available for research on a MTA basis. Commercial applications would need a license agreement.

Further, in January 2002, SSR mining was performed for different cereal species in the then available set of ESTs (like, 87205 barley ESTs, 119158 maize ESTs, 101727 rice ESTs, 87216 sorghum ESTs, 75247 wheat ESTs, 8034 rye ESTs. etc.). SSRs were found to be present in 7- 10% of the total ESTs for the individual cereal species. Subsequently, non-redundant EST-SSRs were identified in each set and were used for primer design. Primer sequences (with annealing temp, product size, etc.) are available with us and may be obtained by contacting us (Varshney et al., 2002).

In the end, we propose to map a similar set of 'triticeae(/cereals) consensus EST-SSRs' (as computed by Mark Sorrells's laboratory) in different cereal species like barley, rice, wheat, rye, etc. Different laboratories having the interest (or engaged in mapping) in different crops like barley (our lab, any other lab ?), rice (Mark Sorrells), wheat (Mark Sorrells, Imad Eujayl, Pierre Sourdille, P K Gupta, Sukhwinder Singh), rye (Bernd Hackauf, our lab), etc. may collaborate on the same which will help for studying the syntenic relationships in different cereals. To achieve this goal, a set of putatively orthologous 'EST-SSRs' has to be defined (i.e. superclusters, Mark Sorrells) and mapped by independent labs!

Thiel T, Michalek W, Varshney R K and Graner A (2002). Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet (in press).

Varshney R K, Thiel T, Stein N, Langridge P and Graner A (2002) In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cell and Molecular Biology Lett. Vol. 7 (in press).

P.K. Gupta:
We would be prepared to share information and material with anybody interested in our wheat EST-SSRs.
We have screened over 15,000 bread wheat (Triticum aestivum L.) ESTs (expressed sequence tags) for the presence of possible mono- to hepta-nucleotide repeats (SSRs = simple sequence repeats). As many as 897 SSRs were identified. The SSRs scored, were defined by their minimum length, which ranged from 14 to 21 bp depending upon the length of the repeat unit itself (1-7 bp). The trinucleotide repeats were most abundant of all the SSRs detected and the average density of SSRs was one over every 9.29 kb EST sequences screened. As a representative sample, only 100 primer pairs were designed, which were also blasted against the dbEST entries of Hordeum vulgare and Triticum tauschii (D-genome donor of cultivated wheat) for E (expectation value) = 0.01. A proportion as high as 95% of the primer pairs exhibited 100% similarity between Hordeum and Triticum, indicating that the flanking sequences of SSRs are not only conserved over species but also over related genera within Poaceae.

We have already screened parental genotypes of four mapping populations (GPC, GW, PHST and ITMI) and 55 other wheat genotypes using 25 primer pairs from the above set of 100 primer pairs. Twenty of the above 25 primer pairs were functional. Four of these primers pairs showed presence/absence polymorphism in the parental genotypes, however, none of these primer pairs have detected length polymorphism in our material.

We have also developed a visual basic script, MSL (microsatellite locator) to harvest SSRs electronically from the available EST sequences.

References:

Rustgi S, Balyan H S and Gupta P K, 2002b. MSL and SNPL: Softwares for in silico mining of SSRs and SNPs. Indian J. Biotechnology (Communicated).

Rustgi S, Singh R, Kumar N, Balyan H S and Gupta P K, 2002a. EST-derived SSRs in bread wheat. (under preparation)).

Imad Eujayl:
I am fully interested in collaboration in EST-SSRs. We (with Sorrells and Powell) published recently an article in TAG (2002, Vol.104-2/4. EST-SSR for genotyping the A & B genomes of wheat. Further, I am interested in all SSR physical mapping and any work done in EST-SSR related to wheat end-product quality.

Nils Stein:
I think in the final stage it would be good to have a final set of SSR-containing EST clusters. People could then blast their set of ESTs against this est-unissr set to see what they have covered already. One could datamine directly for transferable SSRs between barley and wheat? If there are already big overlaps and/or if already the unissr set has been covered by the efforts of some labs I could imagine a joint publication between labs to report on the est-unissr set.

Trude Schwarzacher:
   We do not have an EST-SSR project as such, but have used published SSRs in wheat landraces to analyse the genetic variation and to possibly identify introgressed rye segments. This work is in collaboration with C. Ribeiro-Carvalho, H. Guedes-Pinto from the Department of Genetics and Biotechnology, ICETA, University of Tras-os-Montes and Alto Douro, 5001-911 Vila Real, Portugal.
   In parallel we have used SSR oligonucleotides as in situ hybridization probes and identified those that form large clusters in rye and wheat and are possibly embedded in highly repeated sequence families (in collaboration with A. Cuadrado, University of Alcala, Madrid, Spain)
   We are very interested in further SSR markers particularly those that go across species and that we could use for our triticeae diversity studies.
References:
   Ribeiro-Carvalho, C., Guedes-Pinto, H., Heslop-Harrison, J.S., Schwarzacher, T. (2002) Introgression of rye chromatin on chromosome 2D in the Portuguese wheat landrace 'Barbela'. Genome 44, 1122-1128.
   C. Ribeiro-Carvalho, H. Guedes-Pinto, G. Igrejas, P. Stephenson, T. Schwarzacher and J.S. Heslop-Harrison. Genetic diversity in the Portuguese wheat landrace 'Barbela' measured by microsatellite markers. In preparation.
  Cuadrado, A., Schwarzacher, T., Jouve, N. (2000) Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides. Theoretical and Applied Genetics 101, 711-717.
   Cuadrado, A. and Schwarzacher, T. (1998) The chromosomal organization of simple sequence repeats in wheat and rye genomes. Chromosoma 107, 587-594.

Jorge daSilva:
I published a paper recently on this issue (Da Silva, Jorge A. G. 2001. Genetics and Molecular Biology 24 (1-4), 155-159. PRELIMINARY ANALYSIS OF MICROSATELLITE MARKERS DERIVED FROM SUGARCANE EXPRESSED SEQUENCE TAGS (ESTs) and, currently, I am working on a proposal to submit to USDA applying EST-SSR for resistance gene tagging. Let me know if you would like more info on that.

Joerg Schondelmaier:
We are working on EST-SSRs especially in rye and to some extent in wheat (more) and barley (less). We are open to any exchange/collaboration.

Sukhwinder Singh:
I am working to develop and utilize EST-SSR markers in wheat. I have designed about 250 EST-SSR marker and 80% of them amplified good PCR products.

Robert Henry:
We analysed ITEC ESTs for SSRs when the database included just over 20000 ESTs. The results will be published in Molecular Breeding and I would be happy to supply those interested with further information.

Pierre Sourdille
Concerning our own project, a few months ago, we just made a quick look into ITEC ESTs and searched for SSRs. We found more than 1500 sequences bearing a microsatellite. We developed primer pairs for 112 and 84 revealed an amplification product, 34 showing poymorphism between our reference cultivars. Finally, 31 microsatellite loci were mapped on either the ITMI or Courtot x Chinese Spring populations. In addition, 28 monomorphic loci were physically assigned to chromosome arms using the Chinese Spring aneuploid lines. I think that these results can be made publicly available but I have to ask to the Genoplante committee.
May be a good thing would be to look for SSRs in the contigs that are now available and coordinate the efforts with the SNP group. May be we could set up a similar club (coordinated by Nils) where the participants would define primers and map or at least assign those polymorphic.

Gary Ablett:
Regarding EST - derived SSR's, we have made a few and have mapped some on some Australian barley and wheat populations. We may not spend time making more in the near future but we'd like to keep an eye on what's happening with regards to these markers. We hope to publish or at least post our findings soon.

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