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Conservation Genetics Resources

, Volume 2, Supplement 1, pp 69–72 | Cite as

Mining single nucleotide polymorphisms from EST data of hard clam Meretrix meretrix

  • Hongjun Li
  • Dan Zhu
  • Xianggang Gao
  • Yun-feng Li
  • Jian Wang
  • Chongbo HeEmail author
Technical Note

Abstract

In this study, we describe the first set of single nucleotide polymorphism (SNP) genotyping assays for hard clam (Meretrix meretrix) that were discovered through expressed sequence tag (EST) database mining. A total of 3,129 ESTs were assembled and 228 putative SNPs were identified from 51 SNP-containing contigs, with an average of 0.64 SNPs per 100 bp. A/G (T/C) and A/C (T/G) types had a high frequency and accounted for 46.2% and 35.6%, respectively. Thirty-two SNP genotyping assays were designed for validation based on allele-specific polymerase chain reaction (PCR) and melting temperature (Tm)-shift analysis, and 15 (46.9%) of them showed polymorphisms with the minor allele frequency ranging from 0.075 to 0.475. Twelve (37.5%) assays failed due to amplification failure of one or both allele specific primers, and five (15.6%) assays lacked sufficient temperature discrimination for accurate genotype scoring. BLASTX gave significant hits for all 15 genotyped SNP-containing contigs but all were synonymous coding SNPs. These results indicate that EST data constitutes a valuable resource for SNP discovery and further maker development.

Keywords

Meretrix meretrix Single nucleotide polymorphism SNP AS-PCR Tm-shift 

Notes

Acknowledgements

This research was supported by grants from Program of Liaoning Province Science and Technology Commission (No. 2008203002) and Program of Liaoning Ocean and Fisheries Department (No. 200801).

References

  1. Barker G, Batley J, O’sullivan H, Edwards KJ, Edwards D (2003) Redundancy based detection of sequence polymorphisms in expressed sequence tag data using autoSNP. Bioinformatics 19:421−422CrossRefPubMedGoogle Scholar
  2. He CB, Cong LL, Ge LL, Liu WD, Zhou ZC, Gao XG (2008) AFLP analysis of cultured and wild hard clam (Meretrix meretrix) population. J Fish Sci China 15:215–221 (Chinese edition with English abstract)Google Scholar
  3. Kwok PY (2001) Methods for genotyping single nucleotide polymorphisms. Annu Rev Genomics Hum Genet 2:235–258CrossRefPubMedGoogle Scholar
  4. Li TW, Zhang AG, Su XR, Li CH, Liu BZ, Lin ZH, Chai XL (2006) The analysis of ITS2 in Meretrix meretrix with different stripes. Ocean Limn Sin 37:132–135 (Chinese edition with English abstract)Google Scholar
  5. Lin ZH, Lu RM, Zhou B, Dong YH (2009a) RAPD analysis of four populations of clam Meretrix meretrix. J Dalian Fish Univ 24:66–70 (Chinese edition with English abstract)Google Scholar
  6. Lin ZH, Huang XT, Dong YH, Bao ZM, Hu JJ (2009b) Analysis of Meretrix clams from Guangxi based on fAFLP markers and ITS sequences. Ocean Limn Sin 40:33–41 (Chinese edition with English abstract)Google Scholar
  7. Picoult-Newberg L, Ideker TE, Pohl MG, Taylor SL, Donaldson MA, Nickerson DA, Boyce-Jacino M (1999) Mining SNPs from EST databases. Genome Res 9:167–174PubMedGoogle Scholar
  8. Rozen S, Skaletsky HJ (2000) PRIMER 3 on the www for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
  9. Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S (2005) High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques 39:885–893CrossRefPubMedGoogle Scholar
  10. Wang SL, Sha ZX, Sonstegard TS, Liu H, Xu P, Somridhivej B, Peatman E, Kucuktas H, Liu ZJ (2008) Quality assessment parameters for EST-derived SNPs from catfish. BMC Genomics 9:450CrossRefPubMedGoogle Scholar
  11. Yeh FC, Yang R, Boyle TJ, Ye Z, Xiyan JM (2000) POPGENE 32, microsoft windows-based freeware for population genetic analysis. molecular biology and biotechnology centre. University of Alberta, EdmontonGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Hongjun Li
    • 1
  • Dan Zhu
    • 2
  • Xianggang Gao
    • 1
  • Yun-feng Li
    • 1
  • Jian Wang
    • 2
  • Chongbo He
    • 1
    Email author
  1. 1.Liaoning Key Laboratory of Marine Fishery Molecular BiologyLiaoning Ocean and Fishery Science InstituteDalianChina
  2. 2.College of Life SciencesLiaoning Normal UniversityDalianChina

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