Isolation Of Microsatellite Markers From Chinese Shrimp(Fenneropenaeus Chinensis)

Recombinant positive clones containing microsatellite sequences of Chinese shrimp(Fenneropeneaus chinensis) were obtained through rapid screening small-size fractionated genomic libraries with PCR technique. The primers used in PCR were T₇/Sp₆ promotor primers located at both sides of multiple clone site of pGEM-3zf(+)and STR primers:STR1:5'-ATATATATATATAT-3' and STR2:5'-TCTCTCTCTCTCTC-3' designed according to core repeats motifs of microsatellites. Thirty-one microsatellites of Chinese shrimp F. chinensis were obtained after sequencing for the first time. Among the 31 microsatellites in 18 positive recombinant positive clones are 23 perfect ones(74%), 2 imperfect(7%), 1 compound perfect (3%), 5 compound imperfect (16%). By using the on-line bioinformatics software Repeatmasker on internet, the ESTs database of Chinese shrimp was screened. 229 microsatellite sequences were obtained from 10446 ESTs sequences, about 2.19% in whole database. A total 146 dinucleotide repeats and 58 trinucleotide repeats were found in these 229 microsatellite sequences and most were perfect repeats, with (AT)n in dinucleotide repeats and (TAA)_n in trinucleotide repeats being the most common, about 28.82% and 17.03% in the total microsatellite sequences respectively.Nineteen primer pairs were designed from microsatellites using computer program Primer Premier (Version 5.00). Of the 16 primers that produced product, Eleven primers were polymorphic in about 20 Chinese shrimp samples and Eight primers were suitable for microsatellite analysis.In these eight microsatellite loci, the alleles numbers distributed from 5 to 15, and the range of these alleles were 165 to 305bp and similar with their predicted size. The expected heterozygosity and the PIC values of them distributed from 0.59 to 0.89 and0.56 to 0.88, respectively. These values showed that these 8 microsatellite loci were suitable for genetic analysis.