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Coilia nasus is an important economic fish species in China due to its excellent meat quality and wonderful flavor. The germplasm resources of C. nasus have recently been seriously degraded by human activities. It is urgent to develop the genetic markers of C. nasus in order to apply to conservation of this species. In this study, we used C. nasus transcriptomic data from GENBANK to identify single nucleotide polymorphism (SNP) markers for this species. From 148,156 unigenes with functional annotations, 85,454 putative SNPs were identified. Forty-one of the 61 SNP loci (including 31 loci that were associated with osmoregulation-related genes and 10 loci associated with other functions) were successfully validated and showed bi-allelic polymorphism in three rivers. Nine, six and eleven of these SNP loci deviated significantly from Hardy–Weinberg equilibrium (P

Bighead carp (Aristichthys nobilis) is one of the most economically and ecologically important fish species widely stocked in lakes or cultured in ponds throughout China. The natural populations are facing the stringent challenges of germplasm resources reduction because of pollution and culture. Thus, developing sufficient molecular markers for the conservation and molecular-aided selection of bighead carp is urgent. In this study, we used SNaPshot to develop single nucleotide polymorphism (SNP) markers in bighead carp. A total of 38861 putative SNP markers were identified from the RAD sequencing of bighead carp. Among the markers, 28 SNPs were randomly selected and 25 loci exhibited significant polymorphisms in genotyping by SNaPshot. To the best of our knowledge, this is the first report about SNP marker development in bighead carp based on SNaPshot. These SNP markers can provide an effective tool for the resource conservation and genetic improvement of bighead carp.

Global warming significantly affects fish, particularly cold-water fish, because increased temperature adversely impacts their abilities to grow or reproduce, and eventually influences their fitness or even causes death. To survive, fish may alter their distribution or behavior to avoid the stress, and perhaps acclimate or evolve resistance to the elevated temperature. Brachymystax lenok tsinlingensis is an endangered cold-water species in China, and it has been found to alter the altitudinal distribution, decrease swimming efficiency and develop resistance under heat exposure, which badly impact the continuing conservation work. To better protect them, it is essential to understand how they respond to thermal stress behaviorally and physiologically. Therefore, the fish were exposed to 24.5 °C and based on the time taken for them to lose equilibrium, they were separately sampled as sensitive and tolerant groups. Both gill and liver tissues were collected from both groups for transcriptome sequencing. Sequencing results demonstrated that control and tolerant groups were similar in transcriptomic patterns and sensitive groups differentially expressed more genes than tolerant ones, suggesting the gene expression of tolerant groups may return to base levels as exposure time increased. Tissue differences were the major factor affecting gene expression, and they also displayed different physiological responses to heat stress. Consistent with other studies, heat shock response, immune response, metabolic adjustment and ion transport were found to be triggered after exposed to elevated temperature. The findings would contribute to a better understanding of responding mechanisms of fish to thermal stress and provide guidance for future conservation programs.

In order to understand the influence of human activities such as habitat fragmentation on freshwater fish population evolution, we investigated and compared the genetic diversity and phylogeography of Sinibrama macrops populations in the Oujiang River and Ling River. Mitochondrial control region sequences (D-loop region) of 131 specimens from six populations were obtained and analyzed. The diversity of main stream in the Ou River was lower than that in Ling River. Changtan population showed the lowest diversity (H = 0.646 ± 0.077; π = 0.00060 ± 0.00820). Pairwise F

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

We performed annealing control primer (ACP)-based differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to isolate differentially expressed genes (DEGs) from the stage IV ovary and ovotestis of the rice field eel, Monopterus albus. Using 20 arbitrary ACP primers, 14 DEG expressed-sequence tags were identified and sequenced. The transcriptional expression of one DEG, G2, was significantly greater in the ovotestis than the stage IV ovary. To understand the role of G2 in sex inversion, G2 cDNA was cloned and semi-RT-PCR, real time PCR were performed during gonad development. The full-length G2 cDNA was 650 base pairs (bp) and it comprised a 5′-untranslated region (UTR) of 82 bp, a 3′-UTR of 121 bp and an open reading frame of 444 bp that encoded a 148-amino acid protein. The expression of G2 was weak during early ovarian development until the stage IV ovary, but expression increased significantly with gonad development. We speculate that G2 may play an important function during sex inversion and testis development in the rice field eel, but the full details of the function of this gene requires further research.

Eleven polymorphic microsatellite DNA markers from topmouth culter (Culter alburnus Basilewsky) were developed by magnetic beads and their amplification characteristics were described. The number of alleles ranged from 5 to 10 with an average of 6.7 per locus in a sample of 24 individuals. The mean observed and expected heterozygosity were 0.6288 and 0.7946, respectively. The average polymorphism information content (PIC) was 0.7435. No loci that deviated significantly from Hardy–Weinberg equilibrium (P = 0.05) were found except loci EI19 and EI24.

In this paper, the complete mitochondrial DNA (mtDNA) sequence of Thryssa kammalensis was determined. The mitochondrial genome is 16,884 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region as those found in other vertebrates, with the gene identical to that of typical vertebrates. The overall base composition of the heavy strand is 31.07% A, 24.82% T, 28.33% C, and 15.78% G, with an AT bias of 55.89%. Phylogeny of T. kammalensis suggested more close relationship with Lycothrissa crocodilus and Setipinna taty. The complete mitogenome of this species can provide a basic data for the studies on population history, molecular systematics, phylogeography, stock evaluation, and conservation genetics.

The objective of this study was to investigate gene transcription profiles of the stage IV ovary and the ovotestis of the rice field eel (Monopterus albus) in an attempt to uncover genes involved in sex reversal and gonad development. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from the stage IV ovary and the ovotestis. In total 100 positive clones from the libraries were selected at random and sequenced, and then expressed sequence tags (ESTs) were used to search against sequences in the GenBank database using the BLASTn and BLASTx search algorithms. High quality SSH cDNA libraries and 90 ESTs were obtained. Of these ESTs, 43 showed high homology with genes of known function and these are associated with energy metabolism, signal transduction, transcription regulation and so on. The remaining 47 ESTs shared no homology with any genes in GenBank and are thus considered to be hypothetical genes. Furthermore, the four genes F11, F63, R11, and R47 from the forward and reverse libraries were analyzed in gonad, brain, heart, spleen, liver, kidney and muscle tissues. The results showed that the transcription of the F11 and F63 genes was significantly increased while the expression of the R11 and R47 genes was significantly decreased from IV or V ovary. In addition, the results also indicated that the four genes' expression was not gonad-tissue specific. This results strongly suggested that they may be involved in the rice field eel gonad development and/or sex reversal.

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptapeptide hepatotoxin produced by cyanobacteria. The toxin accumulates rapidly in the liver where it exerts most of its damage, but the molecular mechanisms behind its toxicity remain unclear. Here, suppression subtractive hybridization (SSH) was used to identify alterations in gene transcription of the silver carp (Hypophthalmichthys molitrix) after exposure to MC-LR. After hybridization and cloning, the forward and reverse subtractive cDNA libraries were obtained. At random, 150 positive clones (70 forward and 80 reverse) were selected and sequenced from the subtractive libraries, which gave a total of 88 gene fragment sequences (48 forward and 40 reverse). Sequencing analysis and homology searches showed that these ESTs represented 75 unique genes and 13 duplicates. Of the 75 unique genes, 38 shared high homology with fish genes of known functions, including immune-related genes, transporters and some involved in cell metabolism. Four sequenced genes (Fs59, Fs70, Rs2 and Rs15) were analyzed further using semi-quantitative RT-PCR. The genes from the forward library (Fs59 and Fs70) were found to be transcriptionally upregulated, while the genes from the reverse library (Rs2 and Rs15) were found to be transcriptionally downregulated. These results confirmed the successful construction of the subtractive cDNA library that was enriched for genes that were differentially transcribed in the silver carp liver challenged with MC-LR.

Giant freshwater prawn (GFP; Macrobrachium rosenbergii) is an exotic species that was introduced into China in 1976 and thereafter it became a major species in freshwater aquaculture. However the gene discovery in this species has been limited to small-scale data collection in China. We used the next generation sequencing technology for the experiment; the transcriptome was sequenced of samples of hepatopancreas organ in individuals from 4 GFP groups (A1, A2, B1 and B2). De novo transcriptome sequencing generated 66,953 isogenes. Using BLASTX to search the Non-redundant (NR), Search Tool for the Retrieval of Interacting Genes (STRING), and Kyoto Encyclopedia of Genes and Genome (KEGG) databases; 21,224 unigenes were annotated, 9,552 matched unigenes with the Gene Ontology (GO) classification; 5,782 matched unigenes in 25 categories of Clusters of Orthologous Groups of proteins (COG) and 20,859 unigenes were consequently assigned to 312 KEGG pathways. Between the A and B groups 147 differentially expressed genes (DEGs) were identified; between the A1 and A2 groups 6,860 DEGs were identified and between the B1 and B2 groups 5,229 DEGs were identified. After enrichment, the A and B groups identified 38 DEGs, but none of them were significantly enriched. The A1 and A2 groups identified 21,856 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function and the KEGG pathway defined 2,459 genes had a KEGG Ortholog - ID (KO-ID) and could be categorized into 251 pathways, of those, 9 pathways were significantly enriched. The B1 and B2 groups identified 5,940 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function, and the KEGG pathway defined 1,543 genes had a KO-ID and could be categorized into 240 pathways, of those, 2 pathways were significantly enriched. We investigated 99 queries (GO) which related to growth of GFP in 4 groups. After enrichment we identified 23 DEGs and 1 KEGG PATHWAY ‘ko04711’ relation with GFP growth.