Yaohua Shi

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

BackgroundThe 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).ResultsA 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 ≤ 1e-05) to the non-redundant protein database; 1,104 of which were annotated using Gene Ontology (GO) terms. A total of 35 microsatellites were identified from the ESTs, with 18 having sufficient flanking sequences for primer design. A total of 6,533 putative SNPs were also identified using all existing and the newly generated EST resources of the eastern oysters.ConclusionA 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.

Characterization of the Pearl Oyster (Pinctada martensii) Mantle Transcriptome Unravels Biomineralization Genes

Pearl oyster, Pinctada martensii, is a marine bivalve species widely distributed in tropic and subtropic marine coasts. Mantle is the special tissue of P. martensii that secretes biomineralization proteins inducing shell deposition as well as iridescent nacre both in the inner shell and artificial nucleus. The pearl oyster is very efficient for artificial pearl production and is therefore an ideal organism for studies into the processes of biomineralization. However, deficiency of transcriptome information limits the insight into biomineralization mechanisms and pearl formation. In this study, we sequenced and characterized the P. martensii mantle transcriptome using 454 pyrosequencing. A total of 25,723 unique transcripts were assembled from 220,824 quality reads, followed by annotation and Gene Ontology classification analysis. A total of 146 unique transcript segments homologous to 49 reference biomineralization genes were identified, including calcineurin-binding protein, amorphous calcium carbonate binding protein 1, calmodulin, calponin-like protein, carbonic anhydrase 1, glycine-rich shell matrix protein, lysine-rich matrix protein, mantle gene or protein, nacrein, pearlin, PIF, regucalcin, and shematrin. The sequence data enabled the identification of 10,285 potential single nucleotide polymorphism loci and 7,836 putative indels, providing a resource for molecular biomarker, population genetics, and functional genomic studies. A large number of candidate genes for biomineralization were identified, considerably enriching resources for the study of shell formation. These sequence data will notably advance biomineralization and transcriptome study in pearl oyster and other Pinctada species.

Development of Expressed Sequence Tags from the Pearl Oyster, Pinctada martensii Dunker

The pearl oyster, Pinctada martensii, is the primary species used for the aquaculture production of marine pearls in China and Japan. Genetic tools and resources are needed to study the genome of this species and to understand the molecular basis of development, growth, host defense, pearl formation, and other important traits. In this study, we developed a set of expressed sequence tags (ESTs) for P. martensii. We constructed cDNA libraries from adult tissues and sequenced 7,128 ESTs. Clustering analysis identified 788 contigs (covering 5,769 ESTs) and 1,351 singletons, yielding a total of 2,139 unique genes. Of these unique genes, only 935 had significant (E-value ≤ 0.005) hits in GenBank, and the remaining 1,204 (56.3%) were novel. Most of the known genes are related to cellular structure, protein binding, and metabolic processes. Putative host-defense genes (86) were identified including C-type lectin, ferritin, polyubiquitin, proteases, protease inhibitors, scavenger receptors, heat shock proteins, and RAS oncogenes. The EST sequences developed in this study provide a valuable resource for future efforts on gene identification, marker development, and studies on molecular mechanism of host defense in pearl oysters.

Characterization of 31 EST-derived microsatellite markers for the pearl oyster Pinctada martensii (Dunker).

We developed and characterized 31 microsatellite markers from expressed sequence tags of Pinctada martensii (Dunker). The number of alleles per locus ranged from 4 to 18 as determined in 44 individuals from a wild population. The expected heterozygosity ranged from 0.4121 to 0.9436, while the observed heterozygosity ranged from 0.4054 to 0.7273. Most of the loci are in Hardy–Weinberg equilibrium. These markers should be useful for population genetics studies, parentage and genome mapping in this species.

Identification and Characterization of 66 EST-SSR Markers in the Eastern Oyster Crassostrea virginica (Gmelin)

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.

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster, Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC50), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.