Identification and characterization of 66 EST-SSR markers in the Eastern oyster Crassostrea Virginica (Gmelin)

Large numbers of genetic markers are needed for genomic analyses in the eastern oyster (Crassoslrea virginica). We previously identified 53 simple sequence repeat (SSR) markers from an expressed sequence tag (EST) database using a high selection standard. We mined the same EST database again using a lower threshold (>5 di-nucleotide and 4 other repeats) and identified 330 new SSR-containing ESTs. Primers were designed for 201 suitable sequences, and PCR was successful for 137. The screening of 113 primer pairs that produced fragments shorter than 800 bp produced 66 polymorphic SSR markers, which were characterized in 30 oysters from three populations and a full-sib family. The SSRs had an average of 5.4 alleles per locus, ranging from 2-12. Thirtyfour loci segregated in the family, with seven showing significant deviation from Mendelian ratios after Bonferroni correction. Nullalleles were observed at 17 loci. The EST-derived SSRs are part of expressed genes, and most of them should be useful for gene and genome mapping. This study shows that more SSR markers can be developed from ESTs using lower selection standards.