Isolation And Characterization Of Microsatellite Markers In Two Species Of Bivalve And Sea Urchin Strongylocentrotus Nudus

The microsatellite DNA markers of the xishishe clam Coelomactra antiquate, the ark shell Scapharca broughtonii and the sea urchin Strongylocentrotus nudus have been isolated by magnetic selective hybridization and data mining of expressed sequence tags (ESTs) in this study.The xishishe clam C. antiquata and the ark shell S. broughtonii are commercially important bivalve species in China, but has been suffering from severe population decline due to overexploitation and the deterioration of environmental conditions. To investigate the genetic variation and structures and promote conservation programs of the two species, polymorphic microsatellites of C. antiquate and S. broughtonii have been developed for the first time. Firstly, genomic DNA was extracted from the adductor muscle using a modified phenol-chloroform protocol and subsequently digested with Sau3AI. Size fractions of 300-1000 bp were isolated. Biotin-labeled dinucleotide repeat sequences [(CA)15] were hybridized to the DNA fragments and the hybridization complex was lifted out with streptavidin-coated magnetic spheres. After washing, the bound enriched DNA was eluted from the magnetic spheres, then cloned and sequenced. PCR primers for each microsatellite locus were designed using PRIMER 5, and tested on 40 individuals of wide C. antiquate and S. broughtonii. The allele number of the nine microsatellites of C. antiquata ranged from two to 28 per locus, with the observed heterozygosities ranging from 0.083 to 0.921; the allele number of the twelve microsatellites of S. broughtonii ranged from two to 22 per locus, with the expected heterozygosities ranging from 0.444 to 0.944.The exponentially increased expressed sequence tags (ESTs) in public databases provide a potential resource for genetic analysis. The first set of microsatellite markers for the endangered sea urchin S. nudus was developed from EST databases of S. purpuratus. A total of 2,000 S. purpuratus ESTs obtained from the GenBank (2007) database were screened using SSRHUNTER program, and 40 were selected for microsatellite marker optimization. But only nine pairs amplified the expected products. Amplification products were resolved via 6% denaturing polyacrylamide gel, and visualized by silver-staining. For the nine primer pairs, number of alleles per locus ranged from two to thirteen, while the observed and expected heterozygosities ranged from 0.000 to 0.645 and from 0.063 to 0.912, respectively.