Identification and characterization of 66 EST-SSR markers in the eastern oyster Crassostrea virginica (Gmelin).(Report)

ABSTRACT Large numbers of genetic markers are needed for genomic analyses in the eastern oyster (Crassostrea 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. Thirty-four 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.

KEY WORDS: expressed sequence tags, simple sequence repeats, linkage mapping, population genetics, oyster, Crassostrea virginica

INTRODUCTION

The eastern oyster (Crassostrea virginica Gmelin, 1791) is an economically important mollusc that has supported important fishery industries in the United States. However, the eastern oyster populations and fishery in much of the Mid-Atlantic region have been devastated by overfishing, habitat destruction, and diseases (MacKenzie 1996). Aquaculture production of the eastern oyster is on the rise and increasingly demands superior stocks. Genetic improvement of oyster stocks can greatly benefit from a better understanding of the oyster genome and genes that control economically important traits such growth and disease resistance. An important step in genomic research is the development of a large set of genetic markers for genetic and QTL (quantitative trait loci) mapping.

For a long time, there were few genetic markers for the eastern oyster. It is only recently that DNA-based genetic markers became available. Genetic linkage maps have been constructed for the eastern oyster using primarily amplified fragment length polymorphisms (AFLPs) (Yu & Guo 2003). Although AFLPs are efficient markers and widely used in aquaculture species, they are dominant and less informative markers, and not readily transferable among populations. AFLP-based genetic maps have limited applications unless codominant markers are added. Codominant markers such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) are better suited for genome mapping because they are more informative and easily transferable. Whereas codominant markers are ideal, they are also more difficult and expensive to develop. At the present time, there are only about 95 SSRs available in C. virginica including seven from Brown et al. (2000), four from Yu and Guo (2003, 2006), 31 from Reece's laboratory (Reece et al. 2004, Carlsson et al. 2006, Carlsson & Reece 2007) and 53 from Wang & Guo (2007). Whereas the number of SSRs markers is sufficient for population genetics studies, it is inadequate for genome mapping. Large numbers of SSR markers (hundreds) are needed for genome mapping and population-wide association studies.

SSR markers are typically developed from genomic libraries enriched for SSR. Recently, expressed sequences tags (ESTs) have been shown to be good sources of SSR markers (Zhan et al. 2005, Wang et al. 2007, Wang & Guo 2007). ESTs are part of expressed genes, and the EST-derived SSRs can be considered as type I markers and used to map genes of known functions. We have previously identified and characterized 53 EST-SSRs by screening a database of 9,101 C. virginica ESTs with stringent criteria of having at least eight di-nucleotide and five other repeats (Wang & Guo 2007). These 53 EST-SSRs are highly polymorphic and useful for mapping and population studies. In this study, we mined the same EST database again using a lower threshold (>5 di-nucleotide and four other repeats) in an attempt to obtain more SSR markers. Here we report the development and characterization of 66 new polymorphic EST-SSR markers in selected individuals from three populations and a full-sib family.

MATERIALS AND METHODS