Yinghui Dong

Identification and comparative analysis of the Tegillarca granosa haemocytes microRNA transcriptome in response to Cd using a deep sequencing approach.

Background MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group. Results Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR. Conclusion Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.

Genetic analysis assessed by microsatellites for a diallel mating design of two geographical stocks of the blood clam Tegillarca granosa

To determine the potential for productive efficiency and genetic improvement in the blood clam Tegillarca granosa, four offspring populations (ZZ, ZK, KZ and KK) were produced from a diallel mating of two different geographical stocks (Z and K). The levels of genetic diversity and population structures of four populations were analyzed using 14 polymorphic microsatellites. The results showed that the mean observed heterozygosities (Ho) of reciprocal cross populations (ZK and KZ) was higher than those of pure populations(ZZ and KK). The largest values of genetic differentiation coefficient (Fst = 0.067) and Nei’s unbiased genetic distance (Dc = 0.263) were between ZK and KZ, and the smallest (Fst = 0.020, Dc = 0.116) were between ZZ and KK, which revealed that the largest genetic divergence was between the two reciprocal cross populations and the smallest was between two pure populations. This study demonstrated that the reciprocal cross populations of T. granosa had an extensive genetic difference and improvement, which may be advantageous for future breeding studies.

Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges

ABSTRACT Protease inhibitors play critical roles in numerous biological processes including host defense in all multicellular organisms. Eighty three evolutionary families of protease inhibitors are currently accommodated in the MEROPS database and the I84 family currently consists of 3 novel serine protease inhibitors from the eastern oyster Crassostrea virginica. In this study, we identified 2 new I84 family members from the Chinese razor clam Sinonovacula constricta, scSI‐1 and scSI‐2, using cDNA cloning and sequencing. The scSI‐1 cDNA consisted of 494 bp with a 282 bp ORF encoding a 93‐amino acid polypeptide that was predicted to have a 19‐amino acid signal peptide and a 74‐residue mature protein with a calculated molecular mass of 8248.5 Da. The scSI‐2 cDNA was 490 bp long with a 273 bp ORF encoding a 90‐amino acid polypeptide that was predicted to have an 18‐amino acid signal peptide and a 72‐residue nature protein with a calculated molecular mass of 7528.4 Da. ScSI‐1 and scSI‐2 shared high sequence similarity with the 3 known members of I84 family and both expressed primarily in the clam digestive glands. Protease inhibitory activity in the clam plasma also exhibited the signature kinetic characteristics of the I84 members from the oyster. In addition, levels of scSI‐1 and scSI‐2 gene expression in digestive glands and the protease inhibitory activity in plasma elevated significantly in clams challenged by bacterial injections and Vibrio harveyi was more effective than Staphylococcus epidermidis in inducing the gene expression and plasma protease inhibitory activity. Moreover, drastic changes of salinity and temperature also caused significant changes in the gene expression and plasma activity. These results indicated that scSI‐1 and scSI‐2 represented 2 new members of the I84 family and they likely play a role in clam host defense against infections and in reactions against physiochemical stressors. HighlightsTwo I84 family protease inhibitors were identified from Sinonovacula constricta.The new members shared multiple characteristics with previously known ones.Gene expression and activity elevated in response to various challenges.The inhibitors may be involved in razor clam host defense and stress responses.

Development and evaluation of a set of 135 EST-SNP markers in the transcriptome dataset of hard clam, Meretrix meretrix

The single nucleotide polymorphism (SNP) markers for the hard clam, Meretrix meretrix, are currently limited as the expressed sequence tags (ESTs), which are the main sequence resource, are insufficient. A massive amount of the ESTs generated from the transcriptome for this clam have made possible the rapid development of the high-throughput SNP markers. In the present study, we identified 135 SNPs from the transcriptome dataset of M. meretrix using high-resolution melting (HRM) analysis. The genetic parameter analysis of these markers showed that their observed heterozygosities ranged from 0.0000 to 0.7667 with an average of 0.3225, whereas their expected heterozygosities ranged from 0.3045 to 0.5085 with an average of 0.4615. The minor allele frequency of these markers varied from 0.1833 to 0.5000. Of the 135 SNPs, the nucleotides A/G (31.9%) and C/T (28.9%) accounted for a high percentage. 56 SNPs showed significant deviation from the Hardy–Weinberg equilibrium (P < 0.01) after Bonferroni correction. This study indicated that the transcriptome data provided an extremely invaluable resource for SNP discovery, and these novel SNPs could serve as useful markers for the genetic diversity assessment and molecular-assisted breeding of this species.