Microsatellite markers from tea plant expressed sequence tags (ESTs) and their applicability for cross-species/genera amplification and genetic mapping

Bioinformatic mining of EST datasets is an efficient and economical method for developing functional microsatellite markers. The aim of this work is to generate new tea plant EST-SSR markers and to evaluate their potentials for cross-species/genera amplification and in particular genetic linkage mapping. A total collection of 8008 untapped tea plant ESTs were retrieved from GenBank and then clustered into 2982 non-redundant unique sequences, in which 561 microsatellites were identified. Of these SSRs, the di-nucleotide repeats were the most common repeat types (255, 45.5%), then followed by tri- (18.9%), hexa- (14.3%), penta- (13.5%), tetra- (5.3%), hepta- (2.1%), octa- (0.2%), and deca-nucleotide repeats (0.2%). AG/CT and AGA/TCT predominated in dimeric and trimeric repeat motifs, respectively. BLASTX homology searches found that 319 SSR-containing ESTs had significant Arabidopsis protein hits (E-value < 1.00E−05). In total, a set of 97 primer pairs was successfully designed for marker development and validation. Finally, fifty-four primers yielded repeatable and distinct amplification products among 21 tea plant genotypes, however, only 30 loci were polymorphic with an average allele number of 3.4/locus (range: 2–6 alleles/locus). Cross-amplification showed that 87% (26/30) primers could yield scorable products in at least one of nineteen Theaceae species. The transferability ranged from 7 to 77% with a mean of 30%. Furthermore, twenty-five SSR loci were polymorphic for the parents of a pseudo-testcross mapping population of tea plant, indicating they could be utilized for genetic linkage mapping. These novel informative EST-SSR markers will be a useful addition to genetic analysis and molecular breeding programs in tea plant and its related species.

► Novel EST-SSR markers were developed for tea plant.
► The mean polymorphism information content (PIC) value was 0.42, indicating these EST-SSRs had moderate allelic diversity.
► Twenty-six (87%) of the EST-SSRs showed robust inter-specific/generic transferability among nineteen Theaceae species.
► Twenty-five (83%) of the EST-SSRs were genetic linkage mappable.