Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (Hevea brasiliensis)

Expressed sequence tags derived simple sequence repeats (EST-SSRs), are different from traditional genomic SSR (gSSR) markers. They are more likely to be embedded in the functional gene sequences, less costly and time effective, and may provide abundant information. By analysis of 10,018 expressed sequence tags ( ESTs) out of 10,829 for rubber tree ( Hevea brasiliensis) available in public domain DNA databases, 799 SSR loci were found in the 643 non-redundant SSR-ESTs (SSR-containing ESTs), corresponding to one SSR in every 2.25 kb of the ESTs in rubber tree transcriptome. Of the total 799 SSRs in these ESTs, 673 (84.2%) contained simple repeat motifs while 126 (15.8%) represented compound motif types. Of the total EST-SSRs, 45.3% (362/799) were mononucleotide repeats (MNRs), 42.2% (337/799) were dinucleotide repeats (DNRs), 11.9% (95/799) were trinucleotide repeats (TNRs) and 0.6% (5/799) were tetranucleotide repeats (TTNRs) and hexanucleotide repeats (HNRs). The repeat motifs AAG and AG were the most abundant without regard to single nucleotide repeat. A total of 184 primer pairs were successful designed based on the non-redundant SSR-ESTs. Using 55 degrees C as annealing temperature, 110 primer pairs successfully amplified 12 H. brasiliensis cultivated varieties and four related species. Analysis on 74 alleles amplified by 30 randomly selected primer pairs indicated the medium polymorphism of the EST-SSRs developed. Based on 272 alleles detected by 87 EST-SSR markers, an assessment of genetic diversity was carried out on 12 H. brasiliensis cultivated varieties and four related species. In addition, investigation based on five selected EST-SSRs by cloning and sequencing cross some cultivated species and related species provided evidence for cross-species/genera transferability of the EST-SSR markers developed in this study.