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Development and Characterization of EST-SSR Markers in the Eastern Oyster Crassostrea virginica

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Abstract

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.

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Acknowledgments

We thank Dr. Arnaud Tanguy for assistance with primer design for some of the loci and Dr. Patrick Gaffney for helpful comments. This study was supported by grants from New Jersey Commission on Science and Technology (02-2042-007-11) and NOAA Sea Grant Oyster Disease Research Program (R/OD-2003, NJMSC-6520–0011 and 12) and the Gulf Oyster Industry Program. This is publication no. 2007–50 of IMCS and NJSG–07–667.

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  1. Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue, Port Norris, NJ, 08349, USA

    Yongping Wang & Ximing Guo

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  1. Yongping Wang
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  2. Ximing Guo
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Correspondence to Ximing Guo.

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Wang, Y., Guo, X. Development and Characterization of EST-SSR Markers in the Eastern Oyster Crassostrea virginica . Mar Biotechnol 9, 500–511 (2007). https://doi.org/10.1007/s10126-007-9011-7

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  • DOI: https://doi.org/10.1007/s10126-007-9011-7

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