Development and Evaluation of New Non-Redundant EST-SSR Markers from Gossypium.

A software Clustal X was used to analyse the redundancy of 393 753 ESTs of Gossypium available in public database. By mining 349 815 non-redundant ESTs, a total of 11 372 SSR loci derived from 10 507 ESTs using a software SSRmine developed by ourselves were observed. The frequency of ESTs containing SSRs was 3%, with an average of one SSR in every 21 kb of EST sequence. Besides, trinucleotide and hexanucleotide repeats were found to be the most abundant among 2–6-nucleotide repeat types, accounting for 34.1% and 40.6% respectively. In dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, pentanucleotide repeats and hexanucleotide repeats, AG/CT, AAG/CTT, AAAT/ATTT, AAAAG/CTTTT, AAAAAG/CTTTTT motifs accounted for the highest proportion respectively. Two hundred pairs of new non-redundant EST-SSR primers were developed based on 410 EST sequences removed the redundancy which have not been developed so far in Gossypium arboreum L.,G. hirsutum,and G. barbadense. Andwe used a software SSRmine developed by ourselvesto obtain non-similarity primers, designated CRI (Cotton Research Institute) XXX through six steps, including SSR primer sequences download, pretreatment, Blastn, extraction of primer numbers of similarity score more than 81%, extraction of redundant primers pairs and making redundant primers in a line, to remove homologous sequences from themselves and similar primers released in CMD from different cotton species. Among them, 100 primers were evaluated in polymorphism information content (PIC) and transferability using twelve cotton species including seven representative diploids species and five tetraploid species. The results showed that a total of 56 from the 100 pairs of SSR primers could be amplified the stable and clear polymorphic bands in the 12 accessions mentioned above, moreover, 35 out of 56 pairs of primers were polymorphic, with the primer polymorphism ratio of 35%. PIC of these primers ranged from 0.097 to 0.888, with the average of 0.482. Totally, the transferability among twelve cotton species was 100% for a pair of EST-SSR primers from Gossypium barbadense L., 81% for 25 primers from G. arboreum and 80.1% for 74 primers from G. hirsutum, respectively.