Combining genome-wide association study and transcriptome analysis to identify molecular markers and genetic basis of population-asynchronous ovarian development in Coilia nasus.

Coilia nasus, a migratory fish species found in the middle and lower reaches of the Yangtze River and along offshore areas of China, possesses considerable aquacultural and economic potential. However, the species faces challenges due to significant variation in the gonadal development rate among females, resulting in inconsistent ovarian maturation times at the population level, an extended reproductive period, and limitations on fish growth rate due to ovarian prematurity. In the present study, we combined genome-wide association study (GWAS) and comparative transcriptome analysis to investigate the potential single nucleotide polymorphisms (SNPs) and candidate genes associated with population-asynchronous ovarian development in C. nasus. Genotyping of the female population based on whole-genome resequencing yielded 2 120 695 high-quality SNPs, 39 of which were suggestively associated with ovarian development. Of note, a significant SNP peak on LG21 containing 30 suggestively associated SNPs was identified, with cpne5a determined as the causal gene of the peak. Therefore, single-marker and haplotype association analyses were performed on cpne5a, revealing four genetic markers ( P<0.05) and seven haplotypes (r 2>0.9) significantly associated with the phenotype. Comparative transcriptome analysis of precociously and normally maturing individuals screened out 29 and 426 overlapping differentially expressed genes in the brain and ovary, respectively, between individuals of different body sizes. Integrating the GWAS and transcriptome analysis results, this study identified genes and pathways related to hypothalamic-pituitary-gonadal axis hormone secretion, extracellular matrix, angiogenesis, and gap junctions involved in population-asynchronous ovarian development. The insights gained from this study provide a basis for a deeper understanding of the molecular mechanisms underlying ovarian development in fish and may facilitate the genetic breeding of C. nasus strains exhibiting population-synchronous ovarian development in the future.

Chromosome-level genome assembly of the yellow-cheek carp Elopichthys bambusa.

Yellow-cheek carp (Elopichthys bambusa) is a typical large and ferocious carnivorous fish endemic to East Asia, with high growth rate, nutritional value and economic value. In this study, a chromosome-level genome of yellow-cheek carp was generated by combining PacBio reads, Illumina reads and Hi-C data. The genome size is 827.63 Mb with a scaffold N50 size of 33.65 Mb, and 99.51% (823.61 Mb) of the assembled sequences were anchored to 24 pseudo-chromosomes. The genome is predicted to contain 24,153 protein-coding genes, with 95.54% having functional annotations. Repeat elements account for approximately 55.17% of the genomic landscape. The completeness of yellow-cheek carp genome assembly is highlighted by a BUSCO score of 98.4%. This genome will help us understand the genetic diversity of yellow-cheek carp and facilitate its conservation planning.

The genetic basis of the leafy seadragon's unique camouflage morphology and avenues for its efficient conservation derived from habitat modeling.

The leafy seadragon certainly is among evolution's most "beautiful and wonderful" species aptly named for its extraordinary camouflage mimicking its coastal seaweed habitat. However, limited information is known about the genetic basis of its phenotypes and conspicuous camouflage. Here, we revealed genomic signatures of rapid evolution and positive selection in core genes related to its camouflage, which allowed us to predict population dynamics for this species. Comparative genomic analysis revealed that seadragons have the smallest olfactory repertoires among all ray-finned fishes, suggesting adaptations to the highly specialized habitat. Other positively selected and rapidly evolving genes that serve in bone development and coloration are highly expressed in the leaf-like appendages, supporting a recent adaptive shift in camouflage appendage formation. Knock-out of bmp6 results in dysplastic intermuscular bones with a significantly reduced number in zebrafish, implying its important function in bone formation. Global climate change-induced loss of seagrass beds now severely threatens the continued existence of this enigmatic species. The leafy seadragon has a historically small population size likely due to its specific habitat requirements that further exacerbate its vulnerability to climate change. Therefore, taking climate change-induced range shifts into account while developing future protection strategies.

Genetic Diversity and Population Structure of Coilia nasus Revealed by 2b-RAD Sequencing.

Coilia nasus is a threatened migratory species in the Yangtze River Basin. To reveal the genetic diversity of natural and farmed populations of C. nasus and the status of germplasm resources in the Yangtze River, the genetic diversity and structure of two wild populations (Yezhi Lake: YZ; Poyang Lake: PY) and two farmed populations (Zhenjiang: ZJ; Wuhan: WH) of C. nasus were analyzed using 44,718 SNPs obtained via 2b-RAD sequencing. The results indicate that both the wild and farmed populations had low genetic diversity, and germplasm resources have undergone varying degrees of degradation. Population genetic structure analyses indicated that the four populations may have come from two ancestral groups. Different amounts of gene flow were identified among WH, ZJ, and PY populations, but gene flow among YZ and other populations was low. It is speculated that the river-lake isolation of Yezhi Lake is the main cause of this phenomenon. In conclusion, this study revealed that genetic diversity reduction and germplasm resource degradation had occurred in both wild and farmed C. nasus, suggesting that conservation of its resources is of great urgency. This study provides a theoretical basis for the conservation and rational exploitation of germplasm resources for C. nasus.

Enhanced weathering input from South Asia to the Indian Ocean since the late Eocene.

Enhanced silicate weathering induced by the uplift of the Himalayan-Tibetan Plateau (HTP) has been considered as the major cause of pCO2 decline and Cenozoic cooling. However, this hypothesis remains to be validated, largely due to the lack of a reliable reconstruction of the HTP weathering flux. Here, we present a 37-million-year record of the difference in the seawater radiogenic neodymium isotopic composition (ΔεNd) of Ocean Drilling Program (ODP) sites and Fe-Mn crusts between the northern and central Indian Ocean, which indicates the contribution of regional weathering input from the South Asian continent to the Indian Ocean. The results show a long-term increase in ΔεNd and thus provide the first critical evidence of enhanced South Asian weathering input since the late Eocene. The evolution coincided well with major pulses of surface uplift in the HTP and global climatic transitions. Our foraminiferal εNd record suggests that tectonic uplift and silicate weathering in South Asia, especially in the Himalayas, might have played a significant role in the late Cenozoic cooling.

Single-cell transcriptomes and runx2b-/- mutants reveal the genetic signatures of intermuscular bone formation in zebrafish.

Intermuscular bones (IBs) are mineralized spicules, present in the myosepta of many, but not all, teleost species. IBs are often small and sharp, and they consequently limit how the fish can be processed; the IBs may cause injury or trauma if lodged in consumers' throats or mouths, and therefore affect the appeal of the fish to many consumers. The development of IBs in teleosts is still not fully understood and the molecular basis of IB development remains to be established. Here, the characteristics of IB tissue are evaluated based on single-cell transcriptomics in wild-type zebrafish. The analysis defined 18 distinct cell types. Differentiation trajectories showed that IBs are derived from tendons and that a core tendon-osteoblast cell lineage is related to IB formation. In particular, the functions of 10 candidate genes were evaluated via CRISPR-Cas9 mutants. Among those, runx2b-/- mutants completely lost IBs, while swimming performance, growth and bone mineral density were not significantly different from runx2b+/+ zebrafish. Comparative single-cell RNA sequencing (scRNA-seq) analysis in runx2b-/- and runx2b+/+ zebrafish revealed the role of osteoblasts in IB formation. In addition, differentially expressed genes were enriched in the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathway after runx2b deletion. This study provides evidence for the crucial role of runx2b regulation in IB formation. Genetic breeding can target runx2b regulation and generate strains of commercial fish species without IBs, which can improve the safe consumption and economic value of many farmed fish species.

Functional differentiation of bmp2a and bmp2b genes in zebrafish.

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b-/- embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a-/- zebrafish. The results showed that the expression of runx2b in bmp2a-/- was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.

Functional Differentiation of BMP7 Genes in Zebrafish: bmp7a for Dorsal-Ventral Pattern and bmp7b for Melanin Synthesis and Eye Development.

Bone morphogenetic protein 7 (BMP7) belongs to the transforming growth factor ß (TGF-ß) family, which not only induces cartilage and bone formation, but also regulates eye development and melanoma tumorigenesis in mammals. In teleosts, BMP7 differentiates into two subtypes, bmp7a and bmp7b, which have clearly differentiated structures. To fully understand the functional differentiation of bmp7a and bmp7b in fish species, we successfully constructed bmp7a and bmp7b gene deletion mutants in zebrafish using CRISPR/Cas9-mediated gene editing technology. Our results showed that bmp7a mutation caused abnormal development of the embryo's dorsal-ventral pattern that led to death; bmp7b mutation induced growth inhibition and increased melanin production in the skin and eye of mutants. Histological analysis revealed that melanin in the retina of the eyes in bmp7b mutants increased, and behavioral observation showed that the vision and sensitivity to food of the mutants were reduced. Transcriptome analysis of the skin and eye tissues showed that the expression changes of wnt7ba and gna14 in bmp7b mutants might promote the increase of melanin. Additionally, the eye transcriptome analysis indicated that changes in the structure of the eyes in bmp7b mutants led to defects in phototransduction, and seven DEGs (rgs9a, rgs9b, rcvrn2, guca1d, grk1b, opn1mw4, and gc2) were identified as key candidate genes that affected the photonic response of the eyes. The study revealed the functional differentiation of bmp7a and bmp7b in teleosts and the first report about the inhibitory effect of bmp7b on melanogenesis may provide useful information for the future research on human melanoma-related diseases.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Genome sequences reveal global dispersal routes and suggest convergent genetic adaptations in seahorse evolution.

Seahorses have a circum-global distribution in tropical to temperate coastal waters. Yet, seahorses show many adaptations for a sedentary, cryptic lifestyle: they require specific habitats, such as seagrass, kelp or coral reefs, lack pelvic and caudal fins, and give birth to directly developed offspring without pronounced pelagic larval stage, rendering long-range dispersal by conventional means inefficient. Here we investigate seahorses' worldwide dispersal and biogeographic patterns based on a de novo genome assembly of Hippocampus erectus as well as 358 re-sequenced genomes from 21 species. Seahorses evolved in the late Oligocene and subsequent circum-global colonization routes are identified and linked to changing dynamics in ocean currents and paleo-temporal seaway openings. Furthermore, the genetic basis of the recurring "bony spines" adaptive phenotype is linked to independent substitutions in a key developmental gene. Analyses thus suggest that rafting via ocean currents compensates for poor dispersal and rapid adaptation facilitates colonizing new habitats.

A new species of Pegasus (Syngnathiformes: Pegasidae) from the South China Sea.

A new fish species from the South China Sea, Pegasus nanhaiensis sp. nov., is described herein. The new species is readily distinguished from all other species of the genus Pegasus with the following characteristics: A rounded hump-like tubercle on each of dorsal plates I, II, and III. Noticeable hexagonal patterns on the dorsal carapace with a clear boundary, and two paired caudolateral plates overlapping the junctions between tail rings II and III, and between tail rings IV and V. The integrated evidence from both morphological and genetic studies indicates that P. nanhaiensis represents an independent lineage within the genus Pegasus.

Genome-Wide Integrated Analysis Revealed Functions of lncRNA-miRNA-mRNA Interaction in Growth of Intermuscular Bones in Megalobrama amblycephala.

Intermuscular bone (IB) occurs in the myosepta of teleosts. Its existence has an adverse influence on the edible and economic value of fish, especially for aquaculture species belonging to Cypriniformes. The growth mechanism of IBs is quite lacking. In this study, we firstly used single molecular real-time sequencing (SMRT) technology to improve the draft genome annotation and full characterization of the transcriptome for one typical aquaculture species, blunt snout bream (Megalobrama amblycephala). The long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression profiles in two IB growth stages (1 and 3 years old) were compared through transcriptome and degradome analyses. A total of 126 miRNAs, 403 mRNAs, and 353 lncRNAs were found to be differentially expressed between the two stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the significantly upregulated map2k6 and cytc in the MAPK/p53 signaling pathway and the significantly downregulated lama3 and thbs4b in the extracellular matrix (ECM)-receptor pathway may play a key regulatory role in IB growth. Bioinformatics analysis subsequently revealed 14 competing endogenous RNA (ceRNA) pairs related to the growth of IBs, consisting of 10 lncRNAs, 7 miRNAs, and 10 mRNAs. Of these, dre-miR-24b-3p and dre-miR-193b-3p are core regulatory factors interacting with four lncRNAs and three mRNAs, the interaction mechanism of which was also revealed by subsequent experiments at the cellular level. In conclusion, our data showed that IBs had higher activity of cell apoptosis and lower mineralization activity in IB_III compared to IB_I via interaction of MAPK/p53 and ECM-receptor signaling pathways. The downregulated zip1 interacted with miR-24a-3p and lnc017705, decreased osteoblast differentiation and Ca2+ deposition in the IB_III stage. Our identified functional mRNAs, lncRNAs, and miRNAs provide a data basis for in-depth elucidation of the growth mechanism of teleost IB.

Development of Teleost Intermuscular Bones Undergoing Intramembranous Ossification Based on Histological-Transcriptomic-Proteomic Data.

Intermuscular bones (IBs) specially exist in lower teleost fish and the molecular mechanism for its development remains to be clarified. In this study, different staining methods and comparative proteomics were conducted to investigate the histological structure and proteome of IB development in Megalobrama amblycephala, including four key IB developmental stages (S1-IBs have not emerged in the tail part; S2-several small IBs started to ossify in the tail part; S3-IBs appeared rapidly; S4-all the IBs appeared with mature morphology). Alcian blue and alizarin red S stained results indicated that IBs were gradually formed from S2 to S4, undergoing intramembranous ossification without a cartilaginous phase. A total of 3368 proteins were identified by using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. Functional annotation showed that proteins which were differentially expressed among stages were involved in calcium, MAPK, Wnt, TGF-ß, and osteoclast pathways which played a critical role in bone formation and differentiation. Three proteins (collagen9α1, stat1, tnc) associated with chondrocytes did not exhibit significant changes through S2 to S4; however, proteins (entpd5, casq1a, pvalb, anxa2a, anxa5) which associated with osteoblasts and bone formation and differentiation showed significantly a higher expression level from S1 to S2, as well as to S3 and S4. These further demonstrated that development of IBs did not go through a cartilaginous phase. The inhibitors of TGF-ß and Wnt pathways were tested on zebrafish (sp7/eGFP) and the results indicated that both inhibitors significantly delayed IB development. This study provides a comprehensive understanding of the IB ossification pattern, which will help further elucidate the molecular mechanisms for IB development in teleosts.

End-member modeling of the grain-size record of Sikouzi fine sediments in Ningxia (China) and implications for temperature control of Neogene evolution of East Asian winter monsoon.

The Late Cenozoic East Asian winter monsoon (EAWM) enhancement has been attributed to several factors, such as uplift of the Tibetan Plateau, retreat of the Paratethys Sea, and global cooling related to polar ice volume increment. However, the fundamental forcing factors remain enigmatic due to the absence of long and continuous climate records and sensitive indicators. Here we reanalyzed the published grain-size record of Sikouzi fine sediments in the western Chinese Loess Plateau through end-member (EM) modeling. The results indicate that EM 2 with grain-size peaks between 10-100 µm decreased in content from 20.1 to 17 Ma and stepwise increased from 17 to 0.07 Ma during the following six stages (17-15 Ma, 15-12 Ma, 12-8 Ma, 8-6 Ma, 6-4 Ma and 4-0 Ma). Such varying trends can be successively correlated in seven stages with the integrated benthic δ18O record, implying that global warming weakened the EAWM from 20.1 to 17 Ma and global cooling has stepwise strengthened the EAWM since 17 Ma. Therefore, we conclude that global temperature change played a major role on the evolution of EAWM during the Neogene period. By contrast, Late Cenozoic palaeogeographic reorganization caused by uplift of the Tibetan Plateau and retreat of the Paratethys Sea contributed less to the evolutionary evolution of EAWM. Spectral analysis of the EM 2 data first provided direct evidence of orbitally influenced deposition in the study area and thus the EAWM variations during the Neogene period. The 100-kyr period became weak since ~10 Ma, possibly due to the decrease in sensitivity of a more stable, continental-scale ice sheet in Antarctica to local insolation forcing, deserving further investigation.

Construction of a high-density linkage map and fine mapping of QTLs for growth and gonad related traits in blunt snout bream.

High-density genetic maps based on SNPs are essential for fine mapping loci controlling specific traits for fish species. Using restriction-site associated DNA tag sequencing (RAD-Seq) technology, we identified 42,784 SNPs evenly distributed across the Megalobrama amblycephala genome. Based on 2 parents and 187 intra-specific hybridization progenies, a total of 14,648 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs) of maternal and paternal map. The total length of the integrated map was 3,258.38 cM with an average distance of 0.57 cM among 5676 effective loci, thereby representing the first high-density genetic map reported for M. amblycephala. A total of eight positive quantitative trait loci (QTLs) were detected in QTL analysis. Of that, five QTL explained ≥35% of phenotypic variation for growth traits and three QTL explained ≥16% phenotypic variation for gonad related traits. A total of 176 mapped markers had significant hits in the zebrafish genome and almost all of the 24 putative-chromosomes of M. amblycephala were in relatively conserved synteny with chromosomes of zebrafish. Almost all M. amblycephala and zebrafish chromosomes had a 1:1 correspondence except for putative-chromosome 4, which mapped to two chromosomes of zebrafish caused by the difference in chromosome numbers between two species.

Evolution of Fish Let-7 MicroRNAs and Their Expression Correlated to Growth Development in Blunt Snout Bream.

The lethal-7 (let-7) miRNA, known as one of the first founding miRNAs, is present in multiple copies in a genome and has diverse functions in animals. In this study, comparative genomic analysis of let-7 miRNAs members in fish species indicated that let-7 miRNA is a sequence conserved family in fish, while different species have the variable gene copy numbers. Among the ten members including let-7a/b/c/d/e/f/g/h/i/j, the let-7a precursor sequence was more similar to ancestral sequences, whereas other let-7 miRNA members were separate from the late differentiation of let-7a. The mostly predicted target genes of let-7 miRNAs are involved in biological process, especially developmental process and growth through Gene Ontology (GO) enrichment analysis. In order to identify the possible different functions of these ten miRNAs in fish growth development, their expression levels were quantified in adult males and females of Megalobrama amblycephala, as well as in 3-, 6-, and 12-months-old individuals with relatively slow- and fast-growth rates. These ten miRNAs had similar tissue expression patterns between males and females, with higher expression levels in the brain and pituitary than that in other tissues (p < 0.05). Among these miRNAs, the relative expression level of let-7a was the highest among almost all the tested tissues, followed by let-7b, let-7d and let-7c/e/f/g/h/i/j. As to the groups with different growth rates, the expression levels of let-7 miRNAs in pituitary and brain from the slow-growth group were always significantly higher than that in the fast-growth group (p < 0.05). These results suggest that let-7 miRNA members could play an important role in the regulation of growth development in M. amblycephala through negatively regulating expression of their target genes.

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development.

BACKGROUND: Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. RESULTS: The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P < 0.05), which was in accordance with the results from histological structures. In comparison groups of 1-IB-vs-2-IB and 1-Rib-vs-2-Rib, 33 and 51 differently expressed proteins were identified and the function annotation results showed that these proteins were involved in regulating bone development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. CONCLUSIONS: Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.

Dynamic mRNA and miRNA expression analysis in response to intermuscular bone development of blunt snout bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of lower teleosts, is attracting more attention because they are difficult to remove and make the fish unpleasant to eat. By gaining a better understanding of the genetic regulation of IB development, an integrated analysis of miRNAs and mRNAs expression profiling was performed on Megalobrama amblycephala. Four key development stages were selected for transcriptome and small RNA sequencing. A number of significantly differentially expressed miRNAs/genes associated with bone formation and differentiation were identified and the functional characteristics of these miRNAs/genes were revealed by GO function and KEGG pathway analysis. These were involved in TGF-ß, ERK and osteoclast differentiation pathways known in the literature to affect bone formation and differentiation. MiRNA-mRNA interaction pairs were detected from comparison of expression between different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating bone development and differentiation. A negative regulation effect of two miRNAs was verified through dual luciferase reporter assay. As a unique public resource for gene expression and regulation during the IB development, this study is expected to provide forwards ideas and resources for further biological researches to understand the IBs' development.

Mitochondrial Genome Variation after Hybridization and Differences in the First and Second Generation Hybrids of Bream Fishes.

Hybridization plays an important role in fish breeding. Bream fishes contribute a lot to aquaculture in China due to their economically valuable characteristics and the present study included five bream species, Megalobrama amblycephala, Megalobrama skolkovii, Megalobrama pellegrini, Megalobrama terminalis and Parabramis pekinensis. As maternal inheritance of mitochondrial genome (mitogenome) involves species specific regulation, we aimed to investigate in which way the inheritance of mitogenome is affected by hybridization in these fish species. With complete mitogenomes of 7 hybrid groups of bream species being firstly reported in the present study, a comparative analysis of 17 mitogenomes was conducted, including representatives of these 5 bream species, 6 first generation hybrids and 6 second generation hybrids. The results showed that these 17 mitogenomes shared the same gene arrangement, and had similar gene size and base composition. According to the phylogenetic analyses, all mitogenomes of the hybrids were consistent with a maternal inheritance. However, a certain number of variable sites were detected in all F1 hybrid groups compared to their female parents, especially in the group of M. terminalis (♀) × M. amblycephala (♂) (MT×MA), with a total of 86 variable sites between MT×MA and its female parent. Among the mitogenomes genes, the protein-coding gene nd5 displayed the highest variability. The number of variation sites was found to be related to phylogenetic relationship of the parents: the closer they are, the lower amount of variation sites their hybrids have. The second generation hybrids showed less mitogenome variation than that of first generation hybrids. The non-synonymous and synonymous substitution rates (dN/dS) were calculated between all the hybrids with their own female parents and the results indicated that most PCGs were under negative selection.

De Novo Transcriptome Analysis of Two Seahorse Species (Hippocampus erectus and H. mohnikei) and the Development of Molecular Markers for Population Genetics.

Seahorse conservation has been performed utilizing various strategies for many decades, and the deeper understanding of genomic information is necessary to more efficiently protect the germplasm resources of seahorse species. However, little genetic information about seahorses currently exists in the public databases. In this study, high-throughput RNA sequencing for two seahorse species, Hippocampus erectus and H. mohnikei, was carried out, and de novo assembly generated 37,506 unigenes for H. erectus and 36,113 unigenes for H. mohnikei. Among them, 17,338 (46.23%) unigenes for H. erectus and 17,900 (49.57%) for H. mohnikei were successfully annotated based on the information available from the public databases. Through comparing the unigenes of two seahorse species, 7,802 candidate orthologous genes were identified and 5,268 genes among them could be annotated. In addition, gene ontology analysis of two species was similarly performed on biological processes, cellular components, and molecular functions. Twenty-four and twenty-one unigenes in H. erectus and H. mohnikei were annotated in the biosynthesis of unsaturated fatty acids pathways, and both seahorses lacked the Δ12 and Δ15 desaturases. Total of 8,992 and 9,116 SSR loci were obtained from H. erectus and H. mohnikei unigenes, respectively. Dozens of SSR were developed and then applied to assess the population genetic diversity, as well as cross-amplified in a related species, H. trimaculatus. The HO and HE values of the tested populations for H. erectus, H. mohnikei, and H. trimaculatus were medium. These resources would facilitate the conservation of the species through a better understanding of the genomics and comparative genome analysis within the Hippocampus genus.