Transcriptome Analysis Reveals Molecular Underpinnings of Common Carp (Cyprinus carpio) Under Hypoxia Stress.

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp (Cyprinus carpio) is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp (C. carpio var wuyuanesis) and Songpu mirror carp (C. carpio var specularis). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet. This study tested the hypoxia tolerance of Songpu minor carp, Hebao red carp, and their hybrid F1 population by an acute hypoxia treatment. Muscle and liver tissues were used for transcriptome sequencing analysis to identify the key factors for hypoxia tolerance and explore the potential genetic mechanism for breeding high hypoxia tolerance in common carp. The comparative transcriptomic analysis revealed abundant hypoxia response-related genes and their differential regulation mechanism in these two tissues of different common carp strains under acute hypoxia, including immune response, cellular stress response, HIFs (hypoxia-inducible factors), MAP kinase, iron ion binding, and heme binding. Our findings will facilitate future investigation on the hypoxia response mechanism and provide a solid theoretical basis for breeding projects in common carp.

The genetic map of goldfish (Carassius auratus) provided insights to the divergent genome evolutions in the Cyprinidae family.

A high-density linkage map of goldfish (Carassius auratus) was constructed using RNA-sequencing. This map consists of 50 linkage groups with 8,521 SNP markers and an average resolution of 0.62 cM. Approximately 84% of markers are in protein-coding genes orthologous to zebrafish proteins. We performed comparative genome analysis between zebrafish and medaka, common carp, grass carp, and goldfish to study the genome evolution events in the Cyprinidae family. The comparison revealed large synteny blocks among Cyprinidae fish and we hypothesized that the Cyprinidae ancestor undergone many inter-chromosome rearrangements after speciation from teleost ancestor. The study also showed that goldfish genome had one more round of whole genome duplication (WGD) than zebrafish. Our results illustrated that most goldfish markers were orthologous to genes in common carp, which had four rounds of WGD. Growth-related regions and genes were identified by QTL analysis and association study. Function annotations of the associated genes suggested that they might regulate development and growth in goldfish. This first genetic map enables us to study the goldfish genome evolution and provides an important resource for selective breeding of goldfish.

The complete mitochondrial genome sequence of Silver Gudgeon (Gnathopogon argentatus).

The complete mitochondrial genome of Gnathopogon argentatus was determined to be 16,607 bp long circular molecule with a typical gene arrangement of vertebrate mitochondrial DNA. The complete mitochondrial genome of G. argentatus is 16,607 bp in length with 56.02% AT content, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. The complete mitochondrial genome of G. argentatus was obtained for the first time and would play an important role in population structure and conservation genetic studies.

Diversification of the duplicated Rab1a genes in a hypoxia-tolerant fish, common carp (Cyprinus carpio).

Common carp is a widely cultivated fish with longer than 2,000 years domestication history, due to its strong environmental adaptabilities, especially hypoxia tolerance. The common carp genome has experienced a very recent whole genome duplication (WGD) event. Among a large number of highly similar duplicated genes, a pair of Ras-associated binding-GTPase 1a (Rab1a) genes were found fast diverging. Four analogous Rab1a genes were identified in the common carp genome. Comparisons of gene structures and sequences indicated Rab1a-1 and Rab1a-2 was a pair of fast diverging duplicates, while Rab1a-3 and Rab1a-4 was a pair of less diverged duplicates. All putative Rab1a proteins shared conserved GTPase domain, which enabled the proteins serve as molecular switches for vesicular trafficking. Rab1a-1 and Rab1a-2 proteins varied in their C-terminal sequences, which were generally considered to encode the membrane localization signals. Differential expression patterns were observed between Rab1a-1 and Rab1a-2 genes. In blood, muscle, spleen, and heart, the mRNA level of Rab1a-1 was higher than that of Rab1a-2. In liver and intestine, the mRNA level of Rab1a-2 was higher. Expression of Rab1a-1 and Rab1a-2 showed distinct hypoxia responses. Under severe hypoxia, Rab1a-1 expression was down-regulated in blood, while Rab1a-2 expression was up-regulated in liver. Compared with the less diverged Rab1a-3/4 gene pair, common carp Rab1a-1/2 gene pair exhibited strong characteristics of sub-functionalization, which might contribute to a sophisticated and efficient Ras-dependent regulating network for the hypoxia-tolerant fish.

The complete mitochondrial genome sequence of Chinese lizard gudgeon (Saurogobio dabryi).

The complete mitochondrial genome of Saurogobio dabryi is 16,604 bp in length with 55.88% AT content, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. The termination-associated sequence and six conserved sequence blocks were also identified. Compared all protein-coding genes and whole genome sequence with four species, Saurogobio dabryi has higher similarity with Gobio gobio than others. The mitogenome sequence of Saurogobio dabryi would play an important role in population structure and conservation genetic studies.

The complete mitochondrial genome sequence of pike perch (Sander canadensis).

Pike perch (Sander canadensis) is a member of the largest order of Osteichthyes, Perciformes, and is an important ecological and economic freshwater species, which distributes in Ili River and Ergis River of Xinjiang Province, China. In this study, we sequenced the whole mitochondrial genome of pike perch, and analyzed the similarity with its related species. The mitochondrial genome of S. canadensis is 16,542 bp in length with 55.05% AT content, contained 13 protein coding genes, 22 tRNA genes, 2 ribosomal genes and an 892 bp non-coding region. In control region, 6 CSBs (CSB-1, CSB-2, CSB-3, CSB-D, CSB-E and CSB-F), one potential TAS and one poly-T region were identified. Comparing all protein-coding genes and whole genome sequence with 4 species of Perciformes (three species of Percidae, Perca flavescens. Percina macrolepida. Etheostoma radiosum and one outgroup Oreochromis sp. red tilapia), ND3 gene has the highest mutation rate, and S. canadensis has higher similarity with Perca flavescens than others. The mitochondrial genomic sequence will help us to study the conservation genetic and evolution of Percidae.

Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.

Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance.

[Screening and body correlation analysis of microsatellite markers related to intermuscular bone number in common carp (Cyprinus carpio)].

In this study, 149 polymorphic markers were screened from 200 microsatellite markers. From a family of mirror carp, which included 107 individuals. All samples were analyzed for body correlation, and intermuscular bone number was tested using the General Linear Model (GLM) single marker regression. Determination of the threshold values by 10,000 permutation tests showed that eight markers had significant correlation (P<0.05), in which HLJ3086, HLJ3642 and HLJ3515 had very significant correlation with intermuscular bone number (P<0.01). In addition, the genotypes of the captured correlative loci were determined by Duncan's test using SPSS17.0 software. Markers were used to screen the protein and nucleotide database in the National Center for Biotechnology Information (NCBI). Blasting results showed that HLJ2891 was highly correlated (92%) with latrophilin-2-like and HLJ3515 was highly correlated (81%) with serine/threonine-protein kinase 32B-like of zebrafish. These functional markers and genotypes may provide an efficient basis for marker-assisted selection of intermuscular bone number in mirror carp.

Rates and patterns of microsatellite mutations in common carp (Cyprinus carpio L.).

During genotyping 150 microsatellites in a F1 family of common carp, six mutations were found at five microsatellite loci. The overall mutation rate of common carp microsatellites was 2.53 X 10(-4) per locus per generation. At five loci, mutations increased the length of alleles by at least one repeat unit, suggesting mutations at microsatellite loci in common carp do not follow strict stepwise mutation model. The data on mutation rates and patterns can facilitate population genetics studies, and provide useful parameters for estimating a long-term effective population size of common carp.

[Studies on quantitative trait loci related to activity of lactate dehydrogenase in common carp (Cyprinus carpio)].

The reciprocal intergeneric hybrids between common wheat and Roegneria kamoji were successfully obtained by means of embryo culture. Morphology, chromosome pairing behavior at meiosis, fertility, and resistance to scab of the hybrid F1 and BC1 were studied. The results showed that the morphology of the reciprocal intergeneric hybrids F1 between R. kamoji and T. aestivum cv. Chinese Spring were intermediate type between the two parental species. The chromosome configuration at metaphase I (MI) of pollen mother cell (PMC) in reciprocal F1 was 40.33I + 0.78II + 0.03III and 40.40I + 0.79II, respectively. All of the F1 plants showed complete male sterility, and the seeds of BC1 were obtained by backcrossing with Chinese Spring pollen. The somatic chromosome numbers in BC1 plants of (R. kamoji x Chinese Spring) F1 x Chinese Spring ranged from 55 to 63. Many univalents were observed at MI of PMC, which resulted in the sterility of BC1 plants. Similarly, the chromosome numbers in BC1 plants of (Chinese Spring xR. kamoji) F1 x Chinese Spring also ranged from 55 to 63; however, many bivalents at MI of PMC and fertile pollen were observed resulting in partial fruitfulness in some BC1 plants by self-crossing. A plant (2n=63) with 42 wheat chromosomes and 21 R. kamoji chromosomes was obtained from R. kamoji x Chinese Spring cross, which had a chromosome configuration at MI of 26.40I + 18.30II. Because many univalents existed, this plant showed complete male sterility, and BC1 plants were obtained by back-crossing with Chinese Spring as the pollen parent. The chromosome numbers of BC1 ranged from 40 to 59, which contained less alien chromosomes. Although the morphology of the spike in BC1 plants was similar to that of Chinese Spring, these BC1 plants were still sterile. All F1 and most of the BC1 plants showed high resistance to Fusarium graminearum, which indicated that the resistance to scab from R. kmoji can be transferred into wheat.Microsatellite markers were used to make marker regression analysis on activity of lactate dehydrogenase based on double pseudo-testcross strategy using Windows Map Manager2.0 software. The parents that came from the cross between progenies of Hebao-cold tolerance red carp and Barbless carp and F2 progenies were used as segregating populations. For maker regression, a total of 12 markers associated with activity of lactate dehydrogenase were significant at P<0.05 and HLJE222 was significant at P<0.01. The variance explained by these loci, ranged from 4.00% to 10.00%. Locus HLJE222 was closely linked to the gene related to activity of lactate dehydrogenase of common carp. For further identification, EST-SSR markers were used to screen the protein and nucleotide database using bioinformatics tools in order to find the homologies. High sequence similarities of HLJE222 marker were observed with the nucleotide sequence of DAZ associated protein 1mRNA of zebrafish(94%), and protein sequence of DAZ associated protein 1(97%). DAZ protein is one of the short chain dehydrogenases, which is an important enzyme in the process of glucose metabolism in the organisms. This family contains a wide variety of dehydrogenases. This indicates that locus HLJE222 was closely linked to the gene associated with activity of lactate dehydrogenase of common carp.

[Correlation analysis of microsatellite DNA markers with body weight, length and height of common carp (Cyprinus carpio L.)].

Forty-seven microsatellite markers were selected to analyze the genomic DNA of 92 progenies derived from the recombinant inbred lines (RIL) of common carp, which came from the cross between Barbless carp and Hebao-cold tolerance red carp. The results showed that a total of 162 different alleles were found, and the number of alleles in each locus was 2 to 6. The DNA fragment length was 100 bp to 444 bp, and the number of mean valid alleles was 1.3069 to 4.2288. The value of heterozygosity was 0.2361 to 0.7677, and the mean polymorphism information content (PIC) was 0.5368. A GLM procedure was used to analyze the effects of these 47 microsatellites on body weight, length and height. Results uncovered HLJ695, HLJ716, HLJ739, HLJ759, HLJ774 and K16 had a significant impact on body weight, length and height, and HLJ776 had a significant impact on height. In addition, the genotypes of these correlative loci were determined.

[Mutation rate and pattern of microsatellites in gynogenetic silver crucian carp (Carassius autatus gibeblio)].

The natural gynogenetic triploid silver crucian carp (Carassius autatus gibeblio Bloch) provides a good system for studying evolutional genetics of the unisexual and polyploidy vertebrate. Microsatellites are abundant across genomes and show high levels of polymorphism and mutational rate, so they have been widely used for studying evolutional biology. In this study, the mutation rate and pattern at 33 microsatellite loci of silver crucian carp were investigated. As a result, it was found that the only one of 22 offspring had 18 mutant alleles at 15 microsatellite loci. The overall mutation rate of the 33 loci was 1.16x10(-2)/locus/generation (95% confidence interval 6.87x10(-3) and 1.83x10(-2)). The mutation rate in the gynogenetic triploid silver crucian carp was obviously higher than other fish, which was closely related to the transitional phase of parthenogenesis and gamogenesis in the natural gynogenetic fish. The repeat numbers had more than 10 times at 13 loci of the mutant alleles, and there was no obviously different in the mutant rate between the 11 compound microsatellite loci (1.31x10(-2) )and the 21 perfect microsatellite loci(1.00x10(-2)) (P = 0.67). The mutant rate had affinity with repeat numbers instead of repeat types and GC content in flanking sequences of microsatellite. The mutation pattern of silver crucian carp was very complexional, as well as some loci did not follow the Stepwise Mutation Model.

[Population genetic variation and structure analysis on five populations of mirror carp Cyprinus carpio L. using microsatellites].

In this paper, population genetic variability and genetic structure of five populations of an important cultivation species, mirror carp (Cyprinus carpio L.) were analyzed using 30 microsatellite loci. The observed (Ho) and expected (He) heterozygosity values, polymorphic information content (PIC) and number of effective alleles (Ae) were all determined. The genetic similarity coefficient and Nei's standard genetic distance were computed based on the allele frequencies. The Hardy-Weinberg equilibrium was checked by chi2 test. Genetic differentiation and hierarchical partition of genetic diversity were evaluated by FST and Nm. A dendrogram was constructed based on UPGMA methods using PHYLIP software package supported by a bootstrap value of 91.0%. Totally 7,083 fragments were procured. Their lengths were from 102 bp to 446 bp. For each locus, 1-16 alleles were amplified, adding up to 356 alleles in all the 5 populations. We found the genetic variability level was relatively high in all five populations, as shown by Ae = 1.07-2.30, He= 0.70-0.78 and PIC=0.69-0.75, respectively. The genetic similarity coefficients were all above 0.52, indicating their close genetic relationships. The UPGMA phylogenetic tree showed mirror carps sampled from Donggang, Fengcheng and Liaozhong were clustered into one group and the other two populations, both collected from Songpu, were grouped together. There were obvious relations between genetic distances and geographical distributions of the five populations. No fragments were amplified from some loci of EST-SSRs, which may suggest the loss of these loci in mirror carp genome or sequence divergence at the primer binding sites. These null alleles may result from selection because functional genes are under more selection pressure than non-encoding loci. Overall, population genetic variation is high for each of the five mirror carp, and the differentiations are also significant among populations.