Genome-wide identification, phylogeny and expressional profile of the Dmrt gene family in Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon Dmrt gene family was identified and characterized for the first time. A total of 5 putative Dmrt genes were identified. The gene structure, conserved protein domain and the phylogenetic relationship of Dmrt gene family were systematically analyzed. The expressed profile of Chinese sturgeon Dmrt genes in gonad, pituitary and hypothalamus in the male and female were investigated. The results indicated that the accumulation of Dmrt genes was involved in different tissues, and the expression profile also differed among each Dmrt genes. ASDmrt1A, ASDmrt2, ASDmrt3, and ASDmrtA1 were highly expressed in the testis in comparison with other tissue. This result showed that ASDmrt1A, ASDmrt2, ASDmrt3, and ASDmrtA1 played an important role in the development of testicle, and may be useful tool in distinguishing between male and female of Chinese sturgeon. Our study will provide a basis for additional analyses of Chinese sturgeon Dmrt genes. This systematic analysis provided a foundation for further functional characterization of Dmrt genes with an aim of study of Chinese sturgeon Dmrt gene family.

Developing microsatellite duplex PCR reactions for sterlet (Acipenser ruthenus) and their application in parentage identification.

The sterlet (Acipenser ruthenus) is one of the 27 sturgeon species and is well-known for its wide distribution and small body size in comparison to other sturgeons. For assessing the population genetics and parentage identification of sterlet, ten microsatellites developed for Chinese sturgeon and cross-amplified in sterlet were tested by 40 individuals of sterlet. The ten microsatellites were developed using transcriptome sequencing of Chinese sturgeon. The expected heterozygosity (HE), observed heterozygosity (HO), Shannon-Weiner diversity indices (H') and polymorphic information content (PIC) of the 10 microsatellites ranged from 0.466 to 0.751, from 0.438 to 0.938, from 0.66 to 1.51 and from 0.368 to 0.716, respectively. Combined exclusion probability based on the genotype of pair parent known (CE-PP), one parent known (CE-2P), and no parent known (CE-1P) of the 10 microsatellites were 99.99%, 99.96%, and 99.49%, respectively. These result showed that the 10 microsatellites should be helpful for assessing the population genetics and parentage identification of sterlet.

Effects of feeding frequency on juvenile Chinese sturgeon Acipenser sinensis.

In this study, the effects of different feeding frequencies on the growth and the expression of genes in the GH/IGF axis were assessed in juvenile Chinese sturgeon. The newly hatched Chinese sturgeons were bred for 38 days at three different feeding frequencies groups (feeding frequency of two times a day, TWD; three times a day, THD; and four times a day, FOD), and the expression levels of the GH/IGF axis responses to feeding frequency were determined by quantitative real-time PCR. In addition, the full-length of the Coding Sequences of IGF I and IGF II genes (489-bp and 660-bp, respectively), were cloned and analyzed from Chinese sturgeon the first time. Multiple sequence alignments of IGFs revealed that Chinese sturgeon are high sequence identity to IGFs from other species. The phylogenetic relationships based on the IGF I and IGF II amino acid sequences were consistent with the traditional classification. After 38 days of growth, the three different feeding frequencies groups of Chinese sturgeon had no significant difference of body length, body weight, specific growth rate, the survival rate, the rate of weight gain and the condition factor. However, the relative expression of Chinese sturgeon GH in the pituitary decreased with increasing feeding frequency. The relative expression of Chinese sturgeon GHR in liver and skeletal muscle was deceased with increasing feeding frequency, while the relative expression of GHR in stomach and intestines at THD group was significantly higher than that of at TWD group and FOD group (p < 0.05). The relative expression of Chinese sturgeon IGF I in liver increased significantly with increasing feeding frequency (p < 0.05). The relative expression of IGF I in stomach and skeletal muscle was similar at the three groups. The relative expression of IGF I in intestines was significantly higher at FOD group than at TWD group and THD group (p < 0.05). The relative expression of Chinese sturgeon IGF II in liver at TWD group was significantly higher than that at THD group and FOD group (p < 0.05). However, the relative expression of IGF II in stomach, intestines and skeletal muscle at THD group was higher than that at TWD group and FOD group. Based on these previous studies that liver IGF I is regarded as a biomarker of growth performance, this result suggested that the juvenile Chinese sturgeon is better for growth when feeding four times daily compared to twice and thrice daily.

Development and characterization of a duplex PCR assay in Chinese sturgeon (Acipenser sinensis) for genetic analysis.

Chinese sturgeon (Acipenser sinensis) has been listed as a critically endangered species on the IUCN Red List and is an endemic fish of China. Five sets of duplex polymerase chain reactions (PCR) assays were developed with 10 tetranucleotide microsatellites for Chinese sturgeon. The size of CS57, ZHX43, ZHX69, AS105, ZHX51, AS074, ZHX2, AS078, AS026 and AS073 products in 184 Chinese sturgeon individuals ranged from 257-305, 191-241, 251-285, 172-244, 236-260, 169-209, 194-234, 92-176, 165-257 and 120-164, respectively. The observed allele number of the 10 microsatellites ranged from 7 to 16, and the total number of alleles was 106. The number of alleles per individual in CS57, ZHX43, AS105, AS074, AS078 and AS026 was 1-4. The number of alleles per individual in ZHX69, ZHX51, ZHX2 and AS073 was 2-4. The mean number of alleles per locus per individual ranged from 2.01-3.76. The expected heterozygosity (HE), observed heterozygosity (HO), polymorphic information content (PIC) and Shannon-Weiner diversity index (H') ranged from 0.582 to 0.899, from 0.676 to 1, from 0.518 to 0.886 and from 1.034 to 2.34, respectively. Despite many advantages, the use of microsatellites as genetic analysis tools can be limited by the cost of the associated experiment. To solve this problem, this set of five duplex PCRs will provide tools that are more helpful, less expensive and less time consuming than others used for genetic analyses in Chinese sturgeon.

Genome-wide analysis of the Chinese sturgeon sox gene family: identification, characterisation and expression profiles of different tissues.

The sox family is assumed to be responsible for a number of developmental systems. Genome sequencing technology makes it possible to scan sox genes and conduct characteristic analyses of different species. In fish, full characterisation of sox genes at the genome-wide level has been reported for pufferfish Takifugu rubripes, medaka Oryzias latipes, tilapia Oreochromis niloticus and channel catfish Ictalurus punctatus. However, no systematic investigation of the sox family in sturgeons (Acipenseridae) has been reported to date. This study conducted genome-wide identification of the sox genes in the Chinese sturgeon Acipenser sinensis and profiled their tissue distribution between male and female individuals. In total, 19 sox genes were identified, including soxb1, b2, c, d, e, f and h, in the Chinese sturgeon. Genomic structure analysis indicated relatively conserved exon-intron structures in each sox group and phylogenetic analysis supported the previous classification of the sox family. Most of the sox genes showed a tissue-specific expression pattern, indicating the possible involvement of Chinese sturgeon sox genes at different developmental processes such as cardiac and gonadal development. This study provides a comprehensive resource of Chinese sturgeon sox genes and enables a better understanding of the evolution and function of the sox family.

Transcriptome profiling reveals candidate cleft palate-related genes in cultured Chinese sturgeons (Acipenser sinensis).

The Chinese sturgeon (Acipenser sinensis) is an anadromous fish distributed in the Yangtze River and the East China Sea. In this study, we report the novel finding of cleft palates in Chinese sturgeons derived from artificial fertilization. To explore the genetic basis of palate malformation in A. sinensis, Illumina RNA-seq technology was used to analyze the transcriptome data of farmed Chinese sturgeons with normal palates and cleft-palates. Raw reads were obtained and assembled into 808,612 unigenes, with an average length of 509.33 bp and an N50 of 574 bp. Sequence similarity analyses against four public databases (Nr, UniProt, KEGG, and COGs) found 158,642 unigenes that could be annotated. GABAergic synapses and TGF-ß signal pathways were the two most enriched pathways with high Rich Factors in the analyses of differentially expressed genes. In these two signal pathways, six genes (GABRA4, GS, GNS, S6K, PITX2, and BMP8) were found as candidate cleft-palate genes in Chinese sturgeon. These findings contribute to our understanding of cleft palate genetics in sturgeon, while simultaneously adding to our knowledge about craniofacial development.

The complete mitochondrial genome of the hybrid sturgeon of Huso dauricus (♀) × Acipenser schrenckii (♂).

The complete mitochondrial genome sequence of the hybrid of Huso dauricus (♀) × Acipenser schrenckii (♂) is described in this study. The 16,693bp long circular molecule consisted of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region, showed a typical vertebrate pattern. All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. Base composition of the heavy strain was A(30.40%), T(24.19%), C(29.26%), G(16.16%), and with A + T bias of 54.59%. Comparing with the complete mitochondrial genome of its parents, the hybrid sturgeon of Huso dauricus (♀) × Acipenser schrenckii (♂) was consistent with a maternal inheritance. The complete mitogenome of the hybrid sturgeon of Huso dauricus (♀) × Acipenser schrenckii (♂) provides an important dataset for the exploration of mitochondrial inheritance mechanism.

Near-Full-Length Genome Sequence of a Novel Reovirus from the Chinese Mitten Crab, Eriocheir sinensis.

A novel Eriocheir sinensis reovirus (EsRV) was identified using deep-sequencing techniques in crabs afflicted with trembling disease (TD). Near-full-length genome sequences of 12 segments of EsRV were obtained. The genome of EsRV will facilitate further studies on the causative agent of TD.

In-depth transcriptome analysis of Coilia ectenes, an important fish resource in the Yangtze River: de novo assembly, gene annotation.

Coilia ectenes is an important teleost species in the Yangtze River and a model organism that can be used to study the protection of fish resources. In this report, we performed de novo transcriptome sequencing of ten cDNA libraries from the brain, gill, heart, intestine, kidney, liver, muscle, stomach, ovary, and testis tissues. A total of 352 million raw reads of 100 base pairs were generated, and 130,113 transcripts, corresponding to 65,350 non-redundant transcripts, with a mean length of 1520 bp, were assembled. BLASTx-based gene annotation (E-value<1 × 10(-5)) allowed the identification of 73,900 transcripts against at least one of four databases, including the NCBI non-redundant database, the GO database, the COG database, and the KEGG database. Our study provides a valuable resource for C. ectenes genomic and transcriptomic data that will facilitate future functional studies of C. ectenes.

In-depth transcriptome analysis of the red swamp crayfish Procambarus clarkii.

The red swamp crayfish Procambarus clarkii is a highly adaptable, tolerant, and fecund freshwater crayfish that inhabits a wide range of aquatic environments. It is an important crustacean model organism that is used in many research fields, including animal behavior, environmental stress and toxicity, and studies of viral infection. Despite its widespread use, knowledge of the crayfish genome is very limited and insufficient for meaningful research. This is the use of next-generation sequencing techniques to analyze the crayfish transcriptome. A total of 324.97 million raw reads of 100 base pairs were generated, and a total of 88,463 transcripts were assembled de novo using Trinity software, producing 55,278 non-redundant transcripts. Comparison of digital gene expression between four different tissues revealed differentially expressed genes, in which more overexpressed genes were found in the hepatopancreas than in other tissues, and more underexpressed genes were found in the testis and the ovary than in other tissues. Gene ontology (GO) and KEGG enrichment analysis of differentially expressed genes revealed that metabolite- and immune-related pathway genes were enriched in the hepatopancreas, and DNA replication-related pathway genes were enriched in the ovary and the testis, which is consistent with the important role of the hepatopancreas in metabolism, immunity, and the stress response, and with that of the ovary and the testis in reproduction. It was also found that 14 vitellogenin transcripts were highly expressed specifically in the hepatopancreas, and 6 transcripts were highly expressed specifically in the ovary, but no vitellogenin transcripts were highly expressed in both the hepatopancreas and the ovary. These results provide new insight into the role of vitellogenin in crustaceans. In addition, 243,764 SNP sites and 43,205 microsatellite sequences were identified in the sequencing data. We believe that our results provide an important genome resource for the crayfish.

Sex-lethal gene of the Chinese mitten crab Eriocheir sinensis: cDNA cloning, induction by eyestalk ablation, and expression of two splice variants in males and females.

Sex-lethal (Sxl) plays an important role in sex determination in insects. In this study, the full-length complementary DNA (cDNA) of Sxl of the Chinese mitten crab Eriocheir sinensis (EsSxl) was cloned. EsSxl is expressed during different developmental stages of embryos, and its expression level in the cleavage stage is lower than that in other stages of embryonic development, such as the original zoea stage when it reaches the highest level. The expression level of EsSxl in eight different tissues was investigated. EsSxl was expressed in seven examined tissues, excluding eyestalk, and the highest expression levels were observed in testis and hepatopancreas. EsSxl expression in testis was 13-fold higher than that in ovary. After induction by eyestalk ablation, changes in EsSxl expression were also tissue-specific, with EsSxl expression increasing by 2.6-fold and 11.5-fold in ovary and muscle, respectively, between 0 and 7 days after eyestalk ablation and decreasing by 2.0-fold in testis between 0 and 3 days after eyestalk ablation and by 265-fold in hepatopancreas between 0 and 7 days after eyestalk ablation. Two splice variants of EsSxl were identified, and the only difference between them was a 77-bp alternatively spliced intron that is transcripted in EsSxl1 and skipped in EsSxl2. Both variants were expressed in males and females, demonstrating the lack of a sex-specific expression pattern for Sxl in Chinese mitten crab.

Calcium-calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting.

Calcium-calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium-calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3,262 bp in length and has an open reading frame (ORF) of 1,038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.