SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus.
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.
Poncet
Valérie
V
UMR 1097 Diversité et Génomes des Plantes Cultivées (DGPC), IRD, Institut de Recherche pour le Développement, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France. poncet@mpl.ird.fr
Rondeau
Myriam
M
Tranchant
Christine
C
Cayrel
Anne
A
Hamon
Serge
S
de Kochko
Alexandre
A
Hamon
Perla
P
D003001:Cloning, Molecular; D040503:Coffea / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D005638:Fruit / Q000235:genetics; D005819:Genetic Markers; D018895:Microsatellite Repeats; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014197:Trees / Q000235:genetics
D018744:DNA, Plant; D005819:Genetic Markers