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Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers.

Quilang J, Wang S, Li P, Abernathy J, Peatman E, Wang Y, Wang L, Shi Y, Wallace R, Guo X, Liu Z - BMC Genomics (2007)

Bottom Line: A total of 5,542 ESTs were generated representing 4,688 unique sequences.Putative microsatellite and SNP markers were identified.These genome resources provide the material basis for future microarray development, marker validation, and genetic linkage and QTL analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849, USA. jpquilang@gmail.com

ABSTRACT

Background: The eastern oyster, Crassostrea virginica (Gmelin 1791), is an economically important species cultured in many areas in North America. It is also ecologically important because of the impact of its filter feeding behaviour on water quality. Populations of C. virginica have been threatened by overfishing, habitat degradation, and diseases. Through genome research, strategies are being developed to reverse its population decline. However, large-scale expressed sequence tag (EST) resources have been lacking for this species. Efficient generation of EST resources from this species has been hindered by a high redundancy of transcripts. The objectives of this study were to construct a normalized cDNA library for efficient EST analysis, to generate thousands of ESTs, and to analyze the ESTs for microsatellites and potential single nucleotide polymorphisms (SNPs).

Results: A normalized and subtracted C. virginica cDNA library was constructed from pooled RNA isolated from hemocytes, mantle, gill, gonad and digestive tract, muscle, and a whole juvenile oyster. A total of 6,528 clones were sequenced from this library generating 5,542 high-quality EST sequences. Cluster analysis indicated the presence of 635 contigs and 4,053 singletons, generating a total of 4,688 unique sequences. About 46% (2,174) of the unique ESTs had significant hits (E-value

Conclusion: A high quality normalized cDNA library was constructed. A total of 5,542 ESTs were generated representing 4,688 unique sequences. Putative microsatellite and SNP markers were identified. These genome resources provide the material basis for future microarray development, marker validation, and genetic linkage and QTL analysis.

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Low redundancy of C. virginica ESTs. Number of unique ESTs were plotted as a function of the total number of clones sequenced. Note that the relationship was nearly linear, suggesting a high rate of gene discovery.
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Figure 4: Low redundancy of C. virginica ESTs. Number of unique ESTs were plotted as a function of the total number of clones sequenced. Note that the relationship was nearly linear, suggesting a high rate of gene discovery.

Mentions: This work produced a total of 5,542 ESTs representing 4,688 unique sequences. This EST collection represents the largest number of unique sequences from the eastern oysters. Previous efforts in EST sequencing from this species revealed a high level of sequencing redundancy, largely due to very high proportion of mitochondrial genes [12,17]. In this work, the cDNA library was normalized, along with subtraction of the most abundantly expressed genes as determined from previous EST sequencing efforts. Cluster analysis indicated that only one contig was large representing mitochondrial sequences. For the most part, however, the normalized/subtracted cDNA library allowed efficient generation of unique EST sequences. This suggested that not only normalization, but also subtraction is required for the construction of cDNA libraries suitable for large-scale EST analysis in oysters. Our EST analysis here had a gene discovery rate of 84.6%, or a redundancy rate of 15.4%. Clearly, this is a reflection of the quality of the normalized library. As shown in Figure 4, after sequencing of 6,000 some clones, the rate of gene discovery was still in the linear phase in relation to the number of sequenced clones. This warrants the use of this normalized cDNA library for additional EST sequencing when funding situation permits.


Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers.

Quilang J, Wang S, Li P, Abernathy J, Peatman E, Wang Y, Wang L, Shi Y, Wallace R, Guo X, Liu Z - BMC Genomics (2007)

Low redundancy of C. virginica ESTs. Number of unique ESTs were plotted as a function of the total number of clones sequenced. Note that the relationship was nearly linear, suggesting a high rate of gene discovery.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919373&req=5

Figure 4: Low redundancy of C. virginica ESTs. Number of unique ESTs were plotted as a function of the total number of clones sequenced. Note that the relationship was nearly linear, suggesting a high rate of gene discovery.
Mentions: This work produced a total of 5,542 ESTs representing 4,688 unique sequences. This EST collection represents the largest number of unique sequences from the eastern oysters. Previous efforts in EST sequencing from this species revealed a high level of sequencing redundancy, largely due to very high proportion of mitochondrial genes [12,17]. In this work, the cDNA library was normalized, along with subtraction of the most abundantly expressed genes as determined from previous EST sequencing efforts. Cluster analysis indicated that only one contig was large representing mitochondrial sequences. For the most part, however, the normalized/subtracted cDNA library allowed efficient generation of unique EST sequences. This suggested that not only normalization, but also subtraction is required for the construction of cDNA libraries suitable for large-scale EST analysis in oysters. Our EST analysis here had a gene discovery rate of 84.6%, or a redundancy rate of 15.4%. Clearly, this is a reflection of the quality of the normalized library. As shown in Figure 4, after sequencing of 6,000 some clones, the rate of gene discovery was still in the linear phase in relation to the number of sequenced clones. This warrants the use of this normalized cDNA library for additional EST sequencing when funding situation permits.

Bottom Line: A total of 5,542 ESTs were generated representing 4,688 unique sequences.Putative microsatellite and SNP markers were identified.These genome resources provide the material basis for future microarray development, marker validation, and genetic linkage and QTL analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849, USA. jpquilang@gmail.com

ABSTRACT

Background: The eastern oyster, Crassostrea virginica (Gmelin 1791), is an economically important species cultured in many areas in North America. It is also ecologically important because of the impact of its filter feeding behaviour on water quality. Populations of C. virginica have been threatened by overfishing, habitat degradation, and diseases. Through genome research, strategies are being developed to reverse its population decline. However, large-scale expressed sequence tag (EST) resources have been lacking for this species. Efficient generation of EST resources from this species has been hindered by a high redundancy of transcripts. The objectives of this study were to construct a normalized cDNA library for efficient EST analysis, to generate thousands of ESTs, and to analyze the ESTs for microsatellites and potential single nucleotide polymorphisms (SNPs).

Results: A normalized and subtracted C. virginica cDNA library was constructed from pooled RNA isolated from hemocytes, mantle, gill, gonad and digestive tract, muscle, and a whole juvenile oyster. A total of 6,528 clones were sequenced from this library generating 5,542 high-quality EST sequences. Cluster analysis indicated the presence of 635 contigs and 4,053 singletons, generating a total of 4,688 unique sequences. About 46% (2,174) of the unique ESTs had significant hits (E-value

Conclusion: A high quality normalized cDNA library was constructed. A total of 5,542 ESTs were generated representing 4,688 unique sequences. Putative microsatellite and SNP markers were identified. These genome resources provide the material basis for future microarray development, marker validation, and genetic linkage and QTL analysis.

Show MeSH
Related in: MedlinePlus