Designing Pinus Massoniana SSR Primers From Pinus EST Database

By designing Pinus massoniana SSR primers from Pinus EST database,the main results were as follows:1.359521 ESTs of Pinus in the database of NCBI were downloaded and analyzed,resulting in 56776 non-redundant ESTs with total length about 51,061,225 kb. Totally 2315 SSRs loci distributed in 2057 ESTs were detected,accounting for 4.08% of the non-redundant ESTs. The average distance of the EST–SSRs were about 22.06 kb. Mononucleotide,dinucleotide and trinucleotide repeats were the main types,accounting for 94.94% of all the SSRs. 1631 SSRs of dinucleotide and trinucleotide repeats , 117 SSRs of tetranucelotide , pentanucleotide and hexanucleotide were found. AT/AT and AAG/CTT were the most frequent motifs,accounting for 72.92% and 19.02% in dinucleotide and trinucleotide repeats,respectively.2.The EST-SSRs decreased obviously while the number of repeat bases and SSRs repeats were increasing. It showed base bias of EST-SSRs. During all the SSRs except mononucleotide repeats,there were 488 SSRs longer than 20 bp(including 20 bp),1260 between 12 bp and 20 bp,that meaned the SSRs which had the highest polymorphism of potential take 27.92%,the SSRs which polymorphism of potential were higher take 72.08%. The potential of the EST-SSRs for designing primers specific to flanking sequences was assessed. These EST-SSRs designed 135 primer pairs,eventually 51 pairs to amplify the effective rate of 37.78% amplified,there were 39 pairs of polymorphisms,polymorphism rate of 28.89%. In 51 primer pairs corresponding to the type of SSRs,P-type accounted for 74.51%,I-type accounted for 3.92%,C-type accounted for 21.57%.3.Using BioXM 2.0 software to check 51 pairs of primers with 104 kinds of the common restriction enzyme site,14 pairs of primers detected 19 kinds of restriction sites,for:AatII,AluI,BfaI,BspKT6I,BstUI,ChaI,Csp6I,CviRI,DpnI,HaeIII,MaeII,MboI,MseI,PstI,RsaI,SelI,TaiI,TaqI,Tsp509I. The existence of these sites were 27.45%.