Beide Fu

Transcriptome Analysis of Silver Carp (Hypophthalmichthys molitrix) by Paired-End RNA Sequencing

The silver carp (Hypophthalmichthys molitrix) is among the most intensively pond-cultured fish species and is used in the wild to counteract water bloom in China. However, little genomic information is available for this species, especially regarding its ability to grow rapidly in water, even water contaminated with high concentrations of poisonous microcystin. In this study, we performed de novo transcriptome assembly and analysis of the 17.10 million short-read sequences produced by the Illumina paired-end sequencing technology. Using an improved multiple k-mer contig assembly method coupled with further scaffolding, 85 759 sequences were obtained. There were 23 044 sequences annotated with 3423 gene ontology terms for 104 196 term occurrences and the three corresponding organizing principles. A total of 38 200 assembled sequences were involved in 218 predicted Kyoto Encyclopedia of Genes and Genomes metabolic pathways. We also recovered 41 of 44 genes involved in the biosynthesis of glutathione. Of these, five genes were identified as experienced positive selection between silver carp and zebrafish, as determined by the likelihood ratio test. This report is the first annotated review of the silver carp transcriptome. These data will be of interest to researchers investigating the evolution and biological processes of the silver carp. This work also provides an archive for future studies of recent speciation and evolution of Cyprinidae fishes and can be used in comparative studies of other fishes.

A high-density genetic map and growth related QTL mapping in bighead carp (Hypophthalmichthys nobilis).

Growth related traits in fish are controlled by quantitative trait loci (QTL), but no QTL for growth have been detected in bighead carp (Hypophthalmichthys nobilis) due to the lack of high-density genetic map. In this study, an ultra-high density genetic map was constructed with 3,121 SNP markers by sequencing 117 individuals in a F1 family using 2b-RAD technology. The total length of the map was 2341.27 cM, with an average marker interval of 0.75 cM. A high level of genomic synteny between our map and zebrafish was detected. Based on this genetic map, one genome-wide significant and 37 suggestive QTL for five growth-related traits were identified in 6 linkage groups (i.e. LG3, LG11, LG15, LG18, LG19, LG22). The phenotypic variance explained (PVE) by these QTL varied from 15.4% to 38.2%. Marker within the significant QTL region was surrounded by CRP1 and CRP2, which played an important role in muscle cell division. These high-density map and QTL information provided a solid base for QTL fine mapping and comparative genomics in bighead carp.

A High-Density Genetic Linkage Map and QTL Fine Mapping for Body Weight in Crucian Carp (Carassius auratus) Using 2b-RAD Sequencing

A high-resolution genetic linkage map is essential for a wide range of genetics and genomics studies such as comparative genomics analysis and QTL fine mapping. Crucian carp (Carassius auratus) is widely distributed in Eurasia, and is an important aquaculture fish worldwide. In this study, a high-density genetic linkage map was constructed for crucian carp using 2b-RAD technology. The consensus map contains 8487 SNP markers, assigning to 50 linkage groups (LGs) and spanning 3762.88 cM, with an average marker interval of 0.44 cM and genome coverage of 98.8%. The female map had 4410 SNPs, and spanned 3500.42 cM (0.79 cM/marker), while the male map had 4625 SNPs and spanned 3346.33 cM (0.72 cM/marker). The average recombination ratio of female to male was 2.13:1, and significant male-biased recombination suppressions were observed in LG47 and LG49. Comparative genomics analysis revealed a clear 2:1 syntenic relationship between crucian carp LGs and chromosomes of zebrafish and grass carp, and a 1:1 correspondence, but extensive chromosomal rearrangement, between crucian carp and common carp, providing evidence that crucian carp has experienced a fourth round of whole genome duplication (4R-WGD). Eight chromosome-wide QTL for body weight at 2 months after hatch were detected on five LGs, explaining 10.1–13.2% of the phenotypic variations. Potential candidate growth-related genes, such as an EGF-like domain and TGF-β, were identified within the QTL intervals. This high-density genetic map and QTL analysis supplies a basis for genome evolutionary studies in cyprinid fishes, genome assembly, and QTL fine mapping for complex traits in crucian carp.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix)

Abstract Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4–5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

Development of 159 transcript-associated microsatellite markers in silver carp (Hypophthalmichthys molitrix)

The natural populations of silver carp (Hypophthalmichthys molitrix) have been declining due to habitat fragmentation, over-fishing and water pollution in China. In this study, 159 transcript-associated microsatellites were developed for silver carp by mining transcriptome sequences, and polymorphism of 27 randomly selected loci were further characterized in a wild population. The number of alleles per locus ranged from 4 to 9, the observed and expected heterozygosity ranged from 0.067 to 0.933 and 0.326 to 0.864, respectively. These novel microsatellite markers will be powerful tools to assess the population structure and genetic diversity in silver carp.

Quantitative trait loci mapping for feed conversion efficiency in crucian carp ( Carassius auratus )

QTL is a chromosomal region including single gene or gene clusters that determine a quantitative trait. While feed efficiency is highly important in aquaculture fish, little genetic and genomic progresses have been made for this trait. In this study, we constructed a high-resolution genetic linkage map in a full-sib F1 family of crucian carp (Carassius auratus) consisting of 113 progenies with 8,460 SNP markers assigning onto 50 linkage groups (LGs). This genetic map spanned 4,047.824 cM (0.478 cM/marker) and covered 98.76% of the crucian carp genome. 35 chromosome-wide QTL affecting feed conversion efficiency (FCE, 8 QTL), relative growth rate (RGR, 9 QTL), average daily gain (ADG, 13 QTL) and average daily feed intake (ADFI, 5 QTL) were detected on 14 LGs, explaining 14.0–20.9% of the phenotypic variations. In LGs of LG16, LG25, LG36 and LG49, several QTL affecting different traits clustered together at the identical or close regions of the same linkage group. Seven candidate genes, whose biological functions may involve in the energy metabolism, digestion, biosynthesis and signal transduction, were identified from these QTL intervals by comparative genomics analysis. These results provide a basis for elucidating genetic mechanism of feed efficiency and potential marker-assisted selection in crucian carp.

Comparative transcriptomic analyses of two bighead carp (Hypophthalmichthys nobilis) groups with different growth rates

Growth is one of the most important and desired economic traits for aquaculture species, and identification of loci controlling growth is a difficult task without genomic sequences. In this study, the liver transcriptomes of two groups (F and S) of bighead carp (Hypophthalmichthys nobilis) within a full-sib family with significant differences in growth rate were sequenced. Following de novo assembly of the combined reads from the two groups, a total of 410 differentially expressed genes were identified. Functional annotation and analysis of these genes indicated that some of these were involved in regulation of glucose levels and lipid metabolism, particularly fatty acid oxidation and transport. In addition to the differences on expression levels between the two groups, we also identified many non-synonymous coding single-nucleotide polymorphisms (SNPs) that were specific to each group, including SNPs from 4 genes involved in the lipid metabolism process (GO: 0006629). These differences in gene expression and DNA sequences may in part comprise the genetic background for the regulation of early growth rate in bighead carp.

Characterization and phylogenetic analysis of the complete mitochondrial genome from Rock Scallop (Crassadoma gigantea) using next-generation sequencing

Abstract In this study, the complete 18,495 bp mitochondrial genome was determined from Rock scallop (Crassadoma gigantea) using next-generation sequencing technology. The complete mitochondrial genome contained 12 protein-coding genes (PCGs), 2 ribosomal RNA genes, 23 transfer RNA genes, without ATP8 and D-loop, which was similar with most mitochondrial genomes of marine bivalve molluscs. Gene annotations, including gene order, genetic code, start and stop codons and codons bias, were identified. Phylogenetic tree was constructed using Neighbor-Joining (NJ) method based on the PCGs showed the present species clustered within the Pteriomorphia clade. This work should be of importance not only for the better understanding of the relationships within Pectinidae, but also for the development of useful genetic markers in Rock scallop aquaculture and restoration efforts.

Transcriptomic Profiles of Brain Provide Insights into Molecular Mechanism of Feed Conversion Efficiency in Crucian Carp (Carassius auratus)

Feed efficiency is an economically crucial trait for cultured animals, however, progress has been scarcely made in the genetic analyses of feed conversion efficiency (FCE) in fish because of the difficulties in measurement of trait phenotypes. In the present investigation, we present the first application of RNA sequencing (RNA-Seq) combined with differentially expressed genes (DEGs) analysis for identification of functional determinants related to FCE at the gene level in an aquaculture fish, crucian carp (Carassius auratus). Brain tissues of six crucian carp with extreme FCE performances were subjected to transcriptome analysis. A total of 544,612 unigenes with a mean size of 644.38 bp were obtained from Low- and High-FCE groups, and 246 DEGs that may be involved in FCE traits were identified in these two groups. qPCR confirmed that genes previously identified as up- or down-regulated by RNA-Seq were effectively up- or down-regulated under the studied conditions. Thirteen key genes, whose functions are associated with metabolism (Dgkk, Mgst3 and Guk1b), signal transduction (Vdnccsa1b, Tgfα, Nr4a1 and Tacr2) and growth (Endog, Crebrtc2, Myh7, Myh1, Myh14 and Igfbp7) were identified according to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations. Our novel findings provide useful pathway information and candidate genes for future studies of genetic mechanisms underlying FCE in crucian carp.

Molecular cloning, expression pattern of follistatin gene and association analysis with growth traits in bighead carp (Hypophthalmichthys nobilis)

Follistatin (FST) is a single-chain gonadal protein involving in various biological effects. FST plays important roles in not only ovary development but also body growth, whereas myostatin (MSTN) negatively regulates muscle growth. In this study, FST gene in bighead carp (HynFST) was cloned and characterized. A 5797 bp genomic sequence of HynFST, consisting six exons and five introns were cloned. The full-length cDNA of HynFST (2134 bp) has an open reading fragment encoding a polypeptide of 349 amino acids. Sequence comparison and phylogenetic analysis confirmed that FSTs are conserved throughout the vertebrates and HynFST belongs to FST-1 isoform. Nine single nucleotide polymorphisms (SNPs) of the HynFST were identified and three of them (g.2443 T > C, g.2852 T > C and g.5483A > G) were significantly associated with four growth-related traits. The average body weight of those fish with the combined genotype (CC CC GG) was 12.15-22.63% higher than that of triplotype (TT TT AA) in two bighead carp populations. HynFST was expressed in most of the development stages and various tissues with highest level in ovary. The co-expression results for FST and MSTN in brain and muscle of divergent weight groups showed that FST may inhibit MSTN expression, thus enhancing growth in bighead carp. Our results suggest that FST has significant genetic effects on the regulation of early growth in bighead carp. This study would facilitate the elucidation of multiple functions of FST gene in fish and exploration of the potentials as a gene marker in selective breeding programs for growth of bighead carp.