SSR mining in coffee tree EST databases: potential use of EST–SSRs as markers for the Coffea genus
- 605 Downloads
Expressed sequence tags (ESTs) from Coffea canephora leaves and fruits were used to search for types and frequencies of simple sequence repeats (EST–SSRs) with a motif length of 1–6 bp. From a non-redundant (NR) EST set of 5,534 potential unigenes, 6.8% SSR-containing sequences were identified, with an average density of one SSR every 7.73 kb of EST sequences. Trinucleotide repeats were found to be the most abundant (34.34%), followed by di- (25.75%) and hexa-nucleotide (22.04%) motifs. The development of unique genic SSR markers was optimized by a computational approach which allowed us to eliminate redundancy in the original EST set and also to test the specificity of each pair of designed primers. Twenty-five EST–SSRs were developed and used to evaluate cross-species transferability in the Coffea genus. The orthology was supported by the amplicon sequence similarity and the amplification patterns. The >94% identity of flanking sequences revealed high sequence conservation across the Coffea genus. A high level of polymorphic loci was obtained regardless of the species considered (from 75% for C. liberica to 86% for C. canephora). Moreover, the polymorphism revealed by EST–SSR was similar to that exposed by genomic SSR. It is concluded that Coffea ESTs are a valuable resource for microsatellite mining. EST-SSR markers developed from C. canephora sequences can be easily transferred to other Coffea species for which very little molecular information is available. They constitute a set of conserved orthologous markers, which would be ideal for assessing genetic diversity in coffee trees as well as for cross-referencing transcribed sequences in comparative genomics studies.
KeywordsSSR mining EST–SSR Coffea Transferability Genetic variation
This work was partly supported by EU grant No. ICA4-CT-2001-10068. The authors wish to thank I. Mougenot, C. Fizames, B. Piegu, A. Wissocq, F. Lechauve, F. Moreews, X. Argout, F. Chevalier, and many Genetrop researchers for their involvement in the development of ESTdb, and M. Lorieux for his help in developing the SSR script (http://www.mpl.ird.fr/bioinfo/). Thank to Dr. Santiago C.González-Martínez for his valuable comments on the manuscript.
- Bhat PR, Krishnakumar V, Hendre PS, Rajendrakumar P, Varshney RK, Aggarwal RK (2005) Identification and characterization of expressed sequence tags-derived simple sequence repeats, markers from robusta coffee variety ‘CxR’ (an interspecific hybrid of Coffea canephora × Coffea congensis). Mol Ecol Notes 5:80–83CrossRefGoogle Scholar
- Dirlewanger E, Cosson P, Tavaud M, Aranzana J, Poizat C, Zanetto A, Arus P, Laigret F (2002) Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor Appl Genet 105:127–138PubMedCrossRefGoogle Scholar
- Dufour M, Hamon P, Noirot M, Ristrerucci AM, Brottier P, Vico V, Leroy T (2001) Potential use of SSR markers for Coffea spp. genetic mapping. In: ASIC (ed) 19th international science colloquium on coffee, Trieste, ItalyGoogle Scholar
- N’Diaye A, Noirot M, Hamon S, Poncet V (2006) Genetic basis of species differentiation between Coffea liberica Hiern and C. canephora Pierre: analysis of an interspecific cross. Genet Resour Crop Evol (in press)Google Scholar
- Park YH, Alabady MS, Ulloa M, Sickler B, Wilkins TA, Yu J, Stelly DM, Kohel RJ, el-Shihy OM, Cantrell RG (2005) Genetic mapping of new cotton fiber loci using EST-derived microsatellites in an interspecific recombinant inbred line cotton population. Mol Genet Genomics 274:428–441PubMedCrossRefGoogle Scholar
- Rovelli P, Mettulio R, Anthony F, Anzueto F, Lashermes P, Graziosi G (2000) Microsatellites in Coffea arabica L. In: Sera T, Soccol CR, Pandey A, Roussos S (eds) Coffee biotechnology and quality. Kluwer, Netherlands, pp 123–133Google Scholar
- Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
- Sambrook J, Fristch EF, Maniatis T (1989) Molecular cloning–a laboratory manual. Cold Spring Harbor Laboratory edn. Cold Spring HarborGoogle Scholar