Jianxin Feng

Genome-Wide SNP Discovery from Transcriptome of Four Common Carp Strains

Background Single nucleotide polymorphisms (SNPs) have been used as genetic marker for genome-wide association studies in many species. Gene-associated SNPs could offer sufficient coverage in trait related research and further more could themselves be causative SNPs for traits. Common carp (Cyprinus carpio) is one of the most important aquaculture species in the world accounting for nearly 14% of freshwater aquaculture production. There are various strains of common carp with different economic traits, however, the genetic mechanism underlying the different traits have not been elucidated yet. In this project, we identified a large number of gene-associated SNPs from four strains of common carp using next-generation sequencing. Results Transcriptome sequencing of four strains of common carp (mirror carp, purse red carp, Xingguo red carp, Yellow River carp) was performed with Solexa HiSeq2000 platform. De novo assembled transcriptome was used as reference for alignments, and SNP calling was done through BWA and SAMtools. A total of 712,042 Intra-strain SNPs were discovered in four strains, of which 483,276 SNPs for mirror carp, 486,629 SNPs for purse red carp, 478,028 SNPs for Xingguo red carp and 488,281 SNPs for Yellow River carp were discovered, respectively. Besides, 53,893 inter-SNPs were identified. Strain-specific SNPs of four strains were 53,938, 53,866, 48,701, 40,131 in mirror carp, purse red carp, Xingguo red carp and Yellow River carp, respectively. GO and KEGG pathway analysis were done to reveal strain-specific genes affected by strain-specific non-synonymous SNPs. Validation of selected SNPs revealed that 48% percent of SNPs (12 of 25) were tested to be true SNPs. Conclusions Transcriptome analysis of common carp using RNA-Seq is a cost-effective way of generating numerous reads for SNP discovery. After validation of identified SNPs, these data will provide a solid base for SNP array designing and genome-wide association studies.

Comparative Transcriptome Analysis Reveals the Genetic Basis of Skin Color Variation in Common Carp

Background The common carp is an important aquaculture species that is widely distributed across the world. During the long history of carp domestication, numerous carp strains with diverse skin colors have been established. Skin color is used as a visual criterion to determine the market value of carp. However, the genetic basis of common carp skin color has not been extensively studied. Methodology/Principal Findings In this study, we performed Illumina sequencing on two common carp strains: the reddish Xingguo red carp and the brownish-black Yellow River carp. A total of 435,348,868 reads were generated, resulting in 198,781 assembled contigs that were used as reference sequences. Comparisons of skin transcriptome files revealed 2,012 unigenes with significantly different expression in the two common carp strains, including 874 genes that were up-regulated in Xingguo red carp and 1,138 genes that were up-regulated in Yellow River carp. The expression patterns of 20 randomly selected differentially expressed genes were validated using quantitative RT-PCR. Gene pathway analysis of the differentially expressed genes indicated that melanin biosynthesis, along with the Wnt and MAPK signaling pathways, is highly likely to affect the skin pigmentation process. Several key genes involved in the skin pigmentation process, including TYRP1, SILV, ASIP and xCT, showed significant differences in their expression patterns between the two strains. Conclusions In this study, we conducted a comparative transcriptome analysis of Xingguo red carp and Yellow River carp skins, and we detected key genes involved in the common carp skin pigmentation process. We propose that common carp skin pigmentation depends upon at least three pathways. Understanding fish skin color genetics will facilitate future molecular selection of the fish skin colors with high market values.

An ultra-high density linkage map and QTL mapping for sex and growth-related traits of common carp (Cyprinus carpio)

High density genetic linkage maps are essential for QTL fine mapping, comparative genomics and high quality genome sequence assembly. In this study, we constructed a high-density and high-resolution genetic linkage map with 28,194 SNP markers on 14,146 distinct loci for common carp based on high-throughput genotyping with the carp 250 K single nucleotide polymorphism (SNP) array in a mapping family. The genetic length of the consensus map was 10,595.94 cM with an average locus interval of 0.75 cM and an average marker interval of 0.38 cM. Comparative genomic analysis revealed high level of conserved syntenies between common carp and the closely related model species zebrafish and medaka. The genome scaffolds were anchored to the high-density linkage map, spanning 1,357 Mb of common carp reference genome. QTL mapping and association analysis identified 22 QTLs for growth-related traits and 7 QTLs for sex dimorphism. Candidate genes underlying growth-related traits were identified, including important regulators such as KISS2, IGF1, SMTLB, NPFFR1 and CPE. Candidate genes associated with sex dimorphism were also identified including 3KSR and DMRT2b. The high-density and high-resolution genetic linkage map provides an important tool for QTL fine mapping and positional cloning of economically important traits, and improving common carp genome assembly.

Phylogeny and Evolution of Multiple Common Carp (Cyprinus carpio L.) Populations Clarified by Phylogenetic Analysis Based on Complete Mitochondrial Genomes

Common carp (Cyprinus carpio L.) is one of the oldest, most widely farmed commercially important freshwater fish in the world. However, many undetermined phylogenetic relationships and origins of common carp lineages remain, which are obstacles to conservation and genetic breeding of this species. Phylogenetic analyses based on molecular tools are helpful to distinguish the origin of species, understand and clarify their evolutionary history, and provide a genetic basis for selective breeding. In this study, we demonstrated a method to extract complete mitochondrial genome sequences from whole-genome resequencing data using the Illumina platform. The complete mitochondrial genome sequences of 26 individuals representing nine strains were obtained and subjected to a phylogenetic analysis. We reconstructed the phylogenetic topologies of the nine strains and analyzed the haplotypes. Results from both analyses suggested that the genome sequences belonged to two distinct subspecies from Europe and East Asia. We also estimated the time of divergence of the nine strains, which was up to 100 KYA. The phylogenetic results clarified the breeding history of Songpu mirror carp and suggest that this species may be hybrid of paternal European mirror carp and maternal Xingguo red carp. The results also support a previous hypothesis that koi may have originated from or have close ancestry with Oujiang color carp in China.

Intraspecific mitochondrial variations between Rhinogobio typus from the Yellow River and Yangtze River.

Abstract The complete mitochondrial genome of the first individual Rhinogobio typus collected from the Yellow River were sequenced and compared with the previously reported complete mitochondrial sequence of Rhinogobio typus from the Yangtze River. The length of their circular mitochondrial genome was determined to be 16 599 and 16 608 bp respectively. The comparison of two mitochondrial genomes revealed 237 base pair substitutions and 17 insertions or deletions (indels), including 182 base pair substitutions and 2 indels in protein-coding region. Phylogenetic tree was constructed based on complete mitogenomes of the two populations and closely related 13 teleost species to assess their phylogenic relationship and evolution.

Dynamic regulation of mRNA and miRNA associated with the developmental stages of skin pigmentation in Japanese ornamental carp.

The Japanese ornamental carp (Cyprinus carpio var. Koi) is famous for multifarious colors and patterns, making it commonly culture and trade across the world. Although functional genes and inheritance of color traits have been commonly studied, seldom attentions were focused on the genetic regulation during the developmental process of pigmentation. To better understand the mechanism of skin color development, we observed the morphogenesis of pigment cells during the post-embryonic stages and analysed the temporal expression pattern of mRNAs/miRNAs profiles in four distinct developmental stages. 59 and 103 differentially expressed genes/miRNAs (DEGs/DEMs) associated with pigmentation and skin were identified, including pax7, mitf, tyr, tyrp1, etc., and the highest DEGs were detected at 11 days post hatching (dph). In addition, the functional characteristics of mRNAs/miRNAs associated with pteridine and carotenoid pathway were also examined. Furthermore, 65 miRNA-mRNA interaction pairs related to pigmentation, pteridines and carotenoids metabolism were detected between different stages. Interestingly, the largest pairs appeared in the transition from 11 dph to 48 dph, which had the similar trend with DEGs further manifesting the importance of 11 dph. This study produced a comprehensive programme of DEGs/DEMs during color development, which will provide resources to understand the regulation mechanism in color formation. The understanding of genetic basis in color formation might promote the production and breeding of the Koi carp.

Genetic Mapping of Head Size Related Traits in Common Carp (Cyprinus carpio)

Head size is important economic trait for many aquaculture fish which is directly linked to their carcass yield. The genetic basis of head size trait remains unclear in many widely cultured fish species. Common carp (Cyprinus carpio) is one of the most widely studied fish due to its importance on both economic and environmental aspects. In this study, we performed genome-wide association study using 433 Yellow River carp individuals from multiple families to identify loci and genes potentially associated with head size related traits including head length (HL), head length/body length ratio (HBR), eye diameter (ED), and eye cross (EC). QTL mapping was utilized to filter the effects of population stratification and improve power for the candidates identification in the largest surveyed family with a published genetic linkage map. Twelve SNPs showed significant for head size traits in GWAS and 18 QTLs were identified in QTL mapping. Our study combining both GWAS and QTL mapping could compensate the deficiency from each other and advance our understanding of head size traits in common carp. To acquire a better understanding of the correlation between head size and body growth, we also performed comparisons between QTLs of head size traits and growth-related traits. Candidate genes underlying head size traits were identified surrounding the significant SNPs, including parvalbumin, srpk2, fsrp5, igf1, igf3, grb10, igf1r, notch2, sfrp2. Many of these genes have been identified with potential functions on bone formation and growth. Igf1 was a putative gene associated with both head size and body growth in Yellow River carp. The teleost-specific igf3 was a candidate head size related gene, related to both HL and HBR. Our study also indicated the importance of Igf signaling pathway for both growth and head size determination in common carp, which could be potentially used in future selective breeding in common carp as well as other species.

Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio).

The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.

Complete mitochondrial genomes of two ornamental fishes

Abstract The complete mitochondrial genomes of two ornamental fishes, black molly (Poecilia sphenops) and blue gourami (Trichogaster trichopterus), were obtained by the traditional polymerase chain reaction (PCR)-based sequencing approach. The mitogenomes of P. sphenops and T. trichopterus are determined as 16,533 bp and 16,456 bp in length, respectively. Both the genomes include 22 transfer RNA genes, 13 protein-coding genes and 2 ribosomal RNA genes. Phylogenetic tree was constructed based on the complete mitogenomes of these two species and closely related 20 teleost species to assess their phylogenic relationship and evolution.