Xinhui Zhang

Genome Sequencing of the Perciform Fish Larimichthys crocea Provides Insights into Molecular and Genetic Mechanisms of Stress Adaptation

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.

The Sinocyclocheilus cavefish genome provides insights into cave adaptation

BackgroundAn emerging cavefish model, the cyprinid genus Sinocyclocheilus, is endemic to the massive southwestern karst area adjacent to the Qinghai-Tibetan Plateau of China. In order to understand whether orogeny influenced the evolution of these species, and how genomes change under isolation, especially in subterranean habitats, we performed whole-genome sequencing and comparative analyses of three species in this genus, S. grahami, S. rhinocerous and S. anshuiensis. These species are surface-dwelling, semi-cave-dwelling and cave-restricted, respectively.ResultsThe assembled genome sizes of S. grahami, S. rhinocerous and S. anshuiensis are 1.75 Gb, 1.73 Gb and 1.68 Gb, respectively. Divergence time and population history analyses of these species reveal that their speciation and population dynamics are correlated with the different stages of uplifting of the Qinghai-Tibetan Plateau. We carried out comparative analyses of these genomes and found that many genetic changes, such as gene loss (e.g. opsin genes), pseudogenes (e.g. crystallin genes), mutations (e.g. melanogenesis-related genes), deletions (e.g. scale-related genes) and down-regulation (e.g. circadian rhythm pathway genes), are possibly associated with the regressive features (such as eye degeneration, albinism, rudimentary scales and lack of circadian rhythms), and that some gene expansion (e.g. taste-related transcription factor gene) may point to the constructive features (such as enhanced taste buds) which evolved in these cave fishes.ConclusionAs the first report on cavefish genomes among distinct species in Sinocyclocheilus, our work provides not only insights into genetic mechanisms of cave adaptation, but also represents a fundamental resource for a better understanding of cavefish biology.

Mudskipper genomes provide insights into the terrestrial adaptation of amphibious fishes

Mudskippers are amphibious fishes that have developed morphological and physiological adaptations to match their unique lifestyles. Here we perform whole-genome sequencing of four representative mudskippers to elucidate the molecular mechanisms underlying these adaptations. We discover an expansion of innate immune system genes in the mudskippers that may provide defence against terrestrial pathogens. Several genes of the ammonia excretion pathway in the gills have experienced positive selection, suggesting their important roles in mudskippers’ tolerance to environmental ammonia. Some vision-related genes are differentially lost or mutated, illustrating genomic changes associated with aerial vision. Transcriptomic analyses of mudskippers exposed to air highlight regulatory pathways that are up- or down-regulated in response to hypoxia. The present study provides a valuable resource for understanding the molecular mechanisms underlying water-to-land transition of vertebrates.

The seahorse genome and the evolution of its specialized morphology

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a ‘pelvic fin-loss’ phenotype similar to that of seahorses.

Transcriptome analysis reveals the molecular mechanisms underlying growth superiority in a novel grouper hybrid (Epinephelus fuscogutatus♀× E. lanceolatus♂)

BackgroundGroupers (Epinephelus spp.) have been widely cultivated in China and South-East Asian countries. As a novel hybrid offspring crossed between E. fuscogutatus♀ and E. lanceolatus♂, Hulong grouper exhibits significant growth superiority over its female parent, which made it a promising farmed species in grouper aquaculture industry in China. Hulong grouper present a good combination of beneficial traits from both parent species, but the molecular mechanisms of its heterosis still remain poorly understood.ResultsBased on RNA sequencing and gene expression profiling, we conducted comparative transcriptome analyses between Hulong grouper and its parents E. fuscoguttatus & E. lanceolatus. Six hundred sixty-two and 5239 differentially expressed genes (DEGs) were identified in the brains and livers, respectively. GO enrichment analysis of these DEGs revealed that metabolic process and catalytic activity were the most enriched GO terms. Further analysis showed the expressions of GnRH1and GnRH3 in the brain, and GH/IGF axis related genes such as IGF-1, IGF-2b, IGFBP-1, IGFBP-2, IGFBP-4 and IGFBP-5a in the liver of the hybrid F1 were significantly up-regulated, which is in accordance with the growth superiority of hybrid grouper. Meanwhile, expressions of genes related to the protein and glycogen synthesis pathway, such as PI3KC, PI3KR, Raptor, EIF4E3, and PP1 were up-regulated, while PYG expression was down-regulated. These changes might contribute to increased protein and glycogen synthesis in the hybrid grouper.ConclusionsWe identified a number of differentially expressed genes such as GnRH1 and GnRH3, and genes involved in GH/IGF axis and its downstream signaling pathways for protein and glycogen synthesis in Hulong Grouper. These findings provided molecular basis underlying growth superiority of hybrid grouper, and comprehensive insights into better understanding the molecular mechanisms and regulative pathways regulating heterosis in fish.

High-throughput identification of novel conotoxins from the Chinese tubular cone snail (Conus betulinus) by multi-transcriptome sequencing

BackgroundThe venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides or conotoxins. These peptides have proven to be valuable pharmacological probes and potential drugs because of their high specificity and affinity to important ion channels, receptors and transporters of the nervous system. Most previous studies have focused specifically on the conopeptides from piscivorous and molluscivorous cone snails, but little attention has been devoted to the dominant vermivorous species.ResultsThe vermivorous Chinese tubular cone snail, Conus betulinus, is the dominant Conus species inhabiting the South China Sea. The transcriptomes of venom ducts and venom bulbs from a variety of specimens of this species were sequenced using both next-generation sequencing and traditional Sanger sequencing technologies, resulting in the identification of a total of 215 distinct conopeptides. Among these, 183 were novel conopeptides, including nine new superfamilies. It appeared that most of the identified conopeptides were synthesized in the venom duct, while a handful of conopeptides were identified only in the venom bulb and at very low levels.ConclusionsWe identified 215 unique putative conopeptide transcripts from the combination of five transcriptomes and one EST sequencing dataset. Variation in conopeptides from different specimens of C. betulinus was observed, which suggested the presence of intraspecific variability in toxin production at the genetic level. These novel conopeptides provide a potentially fertile resource for the development of new pharmaceuticals, and a pathway for the discovery of new conotoxins.

A chromosome-level genome assembly of the Asian arowana, Scleropages formosus

Asian arowana (Scleropages formosus), an ancient teleost belonging to the Order Osteoglossomorpha, has been a valuable ornamental fish with some varieties. However, its biological studies and breeding germplasm have been remarkably limited by the lack of a reference genome. To solve these problems, here we report high-quality genome sequences of three common varieties of Asian arowana (the golden, red and green arowana). We firstly generated a chromosome-level genome assembly of the golden arowana, on basis of the genetic linkage map constructed with the restriction site-associated DNA sequencing (RAD-seq). In addition, we obtained draft genome assemblies of the red and green varieties. Finally, we annotated 22,016, 21,256 and 21,524 protein-coding genes in the genome assemblies of golden, red and green varieties respectively. Our data were deposited in publicly accessible repositories to promote biological research and molecular breeding of Asian arowana.

Comparative Transcriptomic Study of Muscle Provides New Insights into the Growth Superiority of a Novel Grouper Hybrid

Grouper (Epinephelus spp.) is a group of fish species with great economic importance in Asian countries. A novel hybrid grouper, generated by us and called the Hulong grouper (Hyb), has better growth performance than its parents, E. fuscoguttatus (Efu, ♀) and E. lanceolatus (Ela, ♂). We previously reported that the GH/IGF (growth hormone/insulin-like growth factor) system in the brain and liver contributed to the superior growth of the Hyb. In this study, using transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR), we analyzed RNA expression levels of comprehensive genes in the muscle of the hybrid and its parents. Our data showed that genes involved in glycolysis and calcium signaling in addition to troponins are up-regulated in the Hyb. The results suggested that the activity of the upstream GH/IGF system in the brain and liver, along with the up-regulated glycolytic genes as well as ryanodine receptors (RyRs) and troponins related to the calcium signaling pathway in muscle, led to enhanced growth in the hybrid grouper. Muscle contraction inducing growth could be the major contributor to the growth superiority in our novel hybrid grouper, which may be a common mechanism for hybrid superiority in fishes.

De novo assembly and comparative transcriptome analysis of the foot from Chinese green mussel (Perna viridis) in response to cadmium stimulation

The Chinese green mussel, Perna viridis, is a marine bivalve with important economic values as well as biomonitoring roles for aquatic pollution. Byssus, secreted by the foot gland, has been proved to bind heavy metals effectively. In this study, using the RNA sequencing technology, we performed comparative transcriptomic analysis on the mussel feet with or without inducing by cadmium (Cd). Our current work is aiming at providing insights into the molecular mechanisms of byssus binding to heavy metal ions. The transcriptome sequencing generated a total of 26.13-Gb raw data. After a careful assembly of clean data, we obtained a primary set of 105,127 unigenes, in which 32,268 unigenes were annotated. Based on the expression profiles, we identified 9,048 differentially expressed genes (DEGs) between Cd treatment (50 or 100 μg/L) at 48 h and the control, suggesting an extensive transcriptome response of the mussels during the Cd stimulation. Moreover, we observed that the expression levels of 54 byssus protein coding genes increased significantly after the 48-h Cd stimulation. In addition, 16 critical byssus protein coding genes were picked for profiling by quantitative real-time PCR (qRT-PCR). Finally, we reached a primary conclusion that high content of tyrosine (Tyr), cysteine (Cys), histidine (His) residues or the special motif plays an important role in the accumulation of heavy metals in byssus. We also proposed an interesting model for the confirmed byssal Cd accumulation, in which biosynthesis of byssus proteins may play simultaneously critical roles since their transcription levels were significantly elevated.

A genome-wide association study on growth traits in orange-spotted grouper (Epinephelus coioides) with RAD-seq genotyping

The orange-spotted grouper, Epinephelus coioides, is one of the most popular fish in China and Southeast Asian countries because of its important economic value. However, molecular mechanism underlying the growth of orange-spotted grouper has never been fully understood. Herein, we performed a genome-wide association study (GWAS) on a natural population of 198 individuals aiming to screen the whole genome of orange-spotted grouper for identification of growth-related loci by restrictionsite associated DNA sequencing. In this research, 261,366 single nucleotide polymorphisms (SNPs) were developed, in which 110 SNPs were identified to be correlated with growth and 20 SNPs were further confirmed to be associated with both body weight and total length. From these identified SNPs, we annotated a total of 34 genes, including adgrb2, csnkza1, cers5, col22a1, creb5, dnd1, dzank1, dnai1, npy2r, fat3, lrrk2, lrp5, map3k9, and so on. Among these candidate genes, npy2r (neuropeptide Y receptor Y2) was reported to play a critical role in growth of the orange-spotted grouper. In addition, population structure, principal component analysis, kinship matrix and linkage disequilibrium were examined to verify the accuracy and reliability of our GWAS results. Our data will also provide a valuable genetic resource for further marker-assisted selection program to improve growth quality in groupers.