Comparative Transcriptomic Analysis of Gonad Development and Renewal of Ovoviviparous Black Rockfish (Sebastes Schlegelii)

Comparative Transcriptomic Analysis of Gonad Development and Renewal of Ovoviviparous Black Rockfish (Sebastes Schlegelii) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Comparative Transcriptomic Analysis of Gonad Development and Renewal of Ovoviviparous Black Rockfish (Sebastes Schlegelii) Jianshaung Li, Haishen Wen, Likang Lyu, Yun Li, Xiaojie Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-70073/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Black rockfish ( Sebastes schlegelii ) has an ovoviviparous reproductive pattern and long-term sperm storage, which resulting in asynchronous gonadal development between the sexes. While the comprehensive understanding of gonad development of black rockfish has not been well studied. Here, we study the gonad development and germ cell renewal by histology and RNA-seq. Results: In this study, RNA-seq was performed on both testis and ovary to characterize key pathways and genes during development and gamete maturation in black rockfish. Different expression genes (DEGs) were identified and annotated in 4 comparisons (F_III vs F_IV, F_IV vs F_V, M_III vs M_IV and M_IV vs M_V). Based on enriched analysis of DEGs in testis, 11 and 14 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mapped in M_III vs M_IV group and M_IV vs M_V group, respectively. And DEGs in ovary development periods were also classified into 10 biological function group. The results of the q-PCR expression pattern of the selected genes was significantly correlated with the RNA-Seq results, implying the reliability and accuracy of the RNA-Seq analysis. Conclusion: The categories intercellular interaction and cytoskeleton, molecule amplification and repairment in cell cycle were revealed to be crucial in testis development and spermatogenesis along with a series of metabolite biosynthesis. Our results provided a comprehensive insight into the black rockfish gonad development for further study of reproductive physiology and molecular biology in ovoviviparity. Epigenetics & Genomics Black rockfish Transcriptomic Gonad development RNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Spermatozoa and oocyte in fish species are highly specialized cells fusing into an embryo which will develop into a mature organism producing gametes again ( 1 ) . Germ cell development and renewal are required by gonad development during the entire reproductive lifespan of teleost. Spermatogenesis and oogenesis are both highly organized processes and crucial for transmitting the genetic information to next generation ( 2 , 3 ) . Many aspects of gametogenesis unfold their difference between female and male despite the development of spermatozoa and oocyte follow common principles ( 2 ) . Teleost represent the most diverse and numerous group in vertebrates and the researches on spermatogenesis in teleost are mainly focused on a few species such as Atlantic cod ( Gadus morhua ) ( 4 ) , tilapia ( Oreochromis niloticus ) ( 5 ) , rainbow trout ( Oncorhynchus mykiss ) ( 6 , 7 ) , zebrafish ( Danio rerio ) ( 8 ) and so on. The molecular mechanism of fish spermatogenesis has been reviewed as well ( 9 ) . Besides, the studies of oogenesis in teleost fish have been reported in recent decades, including the related gene in tilapia ( 5 ) , rainbow trout ( 10 ) , Least killifish ( Heterandria formosa ) ( 11 ) and pejerrey ( Odontesthes bonariensis ) ( 12 ) . In recent years, some molecular techniques and tools are rising their importance in identification of key pathways and genes in some biological process. RNA-seq platform based on the next-generation sequencing technology (NGS) is considered to be a revolutionary and efficient tool for investigating the key pathways and genes in gonad development. It has been already applied in study of reproduction system in many species including pig ( Susscrofa domestica ) ( 13 ) , American alligator ( Alligator mississippiensis ) ( 14 ) , swimming crab ( Portunus trituberculatus ) ( 15 ) . A lot of researches also focused on gonad development and germ cell renewal in teleost revealed the potential mechanism in germ cell and gonad development ( 16 , 17 ) , sex determination and differentiation ( 18 , 19 ) , sex-related genes in both sexes ( 20 , 21 ) . Fishes take different reproductive strategies including oviparity which is common in most teleost for laying eggs before fertilization, viviparity in some Chondrichthyes species whose embryos develop inside ovary with direct nutrition connect with maternal ( 22 ) , and the recent well-studied ovoviviparity which eggs fertilized in ovary with development of lecithotrophic larva in some of teleost including the black rockfish ( Sebastes schlegelii ) ( 23 – 25 ) in the present study. Previous studies on black rockfish were mainly focused on environmental toxicology and immune ( 26 – 28 ) , response to stress ( 24 , 29 ) , whole genomic data analysis ( 30 , 31 ) and reproductive physiology ( 32 – 35 ) . While the reproductive studies of black rockfish were most on single gene identification analysis, ( 32 – 34 , 36 , 37 ) and function ( 38 , 39 ) . While little is known about the comprehensive understanding of gonad development of black rockfish. In this study, RNA-seq was performed on both testis and ovary to characterize key pathways and genes during development and gamete maturation in black rockfish. The transcripts were de novo assembled and annotated greatly enriched the black rockfish gene database. The identified pathways and genes in this study also provided a novel insight into the reproductive biology of ovoviviparious teleost. Results 1. Basic physiological data and histology analysis identified different stages of ovary and testis H&E stain was processed to identified the different stages of gonad development from October to March of the next year. As shown in Fig. 1 , testis was characteristic as stage III which spermatogenesis was not observed yet in October until early November when spermatogenesis was completed as a mark for stage IV. After mating in January of the next year, sperm was totally emptied from testis, showed the testis was in stage V. Stage III oocyte was observed in early November with lipid drop and yolk accumulation initiate. Lipid drop and yolk were showed to fuse together in oocyte at stage IV. Completion of the first meiotic division and first polar body discharge were observed at stage V oocyte. According to the identification by H & E stain, 3 different stages of both gonads were distinguished and collected and named as F_III, F_IV and F_V for stage III, IV and stage V ovary, while M_III, M_IV and M_V for stage III, IV and stage V testis. Average bodyweight and gonad weight were showed in Additional file 1. In testis, both high body weight (795.09g ± 58.63 g) and gonadosomatic index (GSI) (1.26) were conventionally observed in winter which mating behavior occurs in ovoviviparous black rockfish. While high body weight (880.68g ± 99.48 g) and GSI (7.57) of ovary were showed in middle March of the next year when the oocytes mature and fertilization was coming. 2. de novo assembly and annotation of black rockfish gonad transcripts RNA-Seq was performed on both ovary and testis samples from 3 different stages. A total of 1,029,619,820 raw reads (150 bp) were obtained from 18 gonad samples on the Illumina HiSeq X ten platform. After preprocessing and filtration of low-quality sequences, clean read count was 998,950,272 ( Additional file 2 ). The de novo assembled transcriptome included 517,848 genes with the N50 of 1,660 indicated a high-quality assembly. The genes of black rockfish gonads were annotated in 7 public databases including Nr, Nt, KO, SwissProt, PFAM, gene ontology (GO)and KOG with 61.63% of genes annotated in at least 1 database. The Nonredundant (NR) annotation showed that 72.6% of genes were annotated in 5 fish species with the highest sequence similarity compared to large yellow croaker ( Larimichthys crocea ) (Fig. 2 a). The functional classification of transcriptome data is the basic requirement for the application of functional genomic approaches in fishery research. Analysis based on GO and KEGG database are the common methods in functional classification of transcriptomic sequence. Result showed Blast2Go assigned 123,181 genes into 56 functional GO terms (Fig. 2 b). Regarding the 3 primary ontology categories, Biological Process (BP) represents the majority (26 terms) of annotations, followed by Cell Component (CC) (20 terms) and Molecular Function (MF) (10 terms). Based on the analysis of level 2 GO terms, cellular process (GO:0009987) showed the most annotations genes in BP. And for CC, cell (GO:0005623) and cell part (GO:0044464) contained the highest numbers of annotations. The GO terms related to MF with the highest number of annotations were binding (GO:0005488), catalytic activity (GO:0003824). KEGG analysis was performed to understand the higher order functional information of biological system ( 49 ) . Based on the analysis, totally 70,174 genes were annotated into 5 categories on 32 significantly enriched KEGG pathways (Fig. 2 c). 3. Analysis different expression genes in gonad development stage A total of 33,393 DEGs were obtained from 4 different gonad development libraries (adjusted p-value 2) (Fig. 3 ). Among them, 464 DEGs (151 up-regulated and 313 down-regulated) were significantly expressed in F_III vs F_IV, and 329 DEGs (109 up-regulated and 220 down-regulated) were significantly expressed in F_IV vs F_V. While male showed less time in reproductive period revealed much more DEGs than female, which 3,858 DEGs (1,611 up-regulated and 2,247 down-regulated) were significantly expressed in M_III vs M_IV and 30,160 DEGs (24,446 up-regulated and 5,714 down-regulated) were significantly expressed in M_IV vs M_V. 3.1 Identification of DEGs in testis development of black rockfish Based on these DEGs mentioned above, 3,858 and 30,160 annotated DEGs were obtained from M_III vs M_IV group and M_IV vs M_V group, respectively (Fig. 4 a). Heatmap analysis of these 32,772 DEGs expressed in all 3 stages of testis revealed that most of these genes in both stage III and IV testis showed similar expression pattern, which up-regulated genes in stage III (M_III) and stage IV testis (M_IV) showed down-stream in stage V (M_V) testis, suggested that the expression profile of these DEGs and male reproductive process including gamete mature were directly proportional (Fig. 4 b). GO analysis of these DEGs showed most of them were mapped onto MF term ( p v alue < 0.01), especially molecule binding GO terms (anion binding, GO:0043168, small molecule binding, GO:0036094 and so on) (Fig. 4 c). 11 and 14 significantly enriched KEGG pathways were mapped between M_III vs M_IV group and M_IV vs M_V group ( p-value < 0.01), respectively (Table 1 ). In M_III vs M_IV group, 11 KEGG pathways were classified into 3 categories, including intercellular interaction and cytoskeleton , molecule amplification and repairment in cell cycle , and other (Fig. 5 ). The DEGs in category intercellular interaction and cytoskeleton , including extracellular matrix (ECM)-receptor interaction, focal adhesion and regulation of actin cytoskeleton, were basically up-regulated in stage IV compared with stage III. DEGs in category molecule amplification and repairment in cell cycle , including cell cycle, ubiquitin mediated proteolysis, DNA replication, Fanconi anemia pathway, RNA transport and mRNA surveillance pathway, were significantly enhanced in stage III, when the spermatogenesis start, cell division and protein biosynthesis are processing, compared with stage IV. In addition, some other pathways such as steroid hormone biosynthesis was up-regulated in stage IV testis. As shown in Fig. 6 , 14 KEGG pathways were classified into 5 categories, including progesterone induced gamete mature , molecule amplification and repairment in cell cycle , endoplasmic reticulum-related protein processing and exocytosis in nervous system , infection and immune related and other in M_IV vs M_V group. In category progesterone induced gamete mature , most DEGs showed up-regulated in stage IV testis due to the importance of steroid hormone to germ cell division. Category molecule amplification and repairment in cell cycle , which also showed up-regulated in both stage III and stage IV, implied the drastic changes in cell metabolism continue the whole reproductive process. The protein processing in endoplasmic reticulum, synaptic vesicle cycle and retrograde endocannabinoid signaling in categories endoplasmic reticulum-related protein processing and exocytosis in nervous system showed the most interest and abundant DEGs. DEGs, such as sar1b , sec13 , sec24c and slc18a2 are involved in transport vesicles, stx2 is related to epithelial morphogenesis due to exocytosis, cacna1b regulates hormone or neurotransmitter release, while gria1 and grm1 for receipting glutamate neurotransmitter message. These results suggested these genes expressing variated significantly between stage III and IV testis were inseparable with the intense reproductive stages. As shown in Additional file 3 , 3 KEGG pathways cell cycle, mRNA surveillance pathway and RNA transport, which were both up-regulated in M_III vs M_IV and M_IV vs M_V, indicated that the activity process in the whole reproductive cycle. Interestingly, Ubiquitin mediated proteolysis, which was highly expressed in M_III vs M_IV and M_IV vs M_V, presented totally different DEGs, suggested that the pathway may play different roles in different development stages. In Oocyte meiosis pathway, sgo1 , stag3 , smc1b and smc3 were up-regulated in stage IV, suggested the exit of meiosis of gamete in final reproductive stage. Besides, DEGs in p53 signaling pathway, ccne1 , ccnd2 , cdk2 with up-regulated and srsf10 with down-regulated expressed profile, which srsf10 repressed cell cycle G1 and G2 phase arrested by inhibition of ccne1 , ccnd2 , cdk2 , resulting cell cycle was active in stage III and IV testis. Table 1 significant enriched KEGG pathways in M_IIIvsM_IV group and M_IVvsM_V group. Stage KEGG pathway ID Input gene Regulation M_IIIvsM_IV Intercellular interaction and cytoskeleton ECM-receptor interaction ko04512 39 DOWN Focal adhesion ko04510 64 DOWN Regulation of actin cytoskeleton ko04810 59 DOWN Molecule amplification and repairment in cell cycle Cell cycle ko04110 39 UP Ubiquitin mediated proteolysis ko04120 27 UP DNA replication ko03030 10 UP Fanconi anemia pathway ko03460 12 UP mRNA surveillance pathway ko03015 23 UP Steroid hormone biosynthesis ko00140 21 DOWN Glycosylphosphatidylinositol (GPI)-anchor biosynthesis ko00563 7 UP M_IVvsM_V Progesterone induced gamete mature Progesterone-mediated oocyte maturation ko04914 117 UP Oocyte meiosis ko04114 129 UP Molecule amplification and repairment in cell cycle mRNA surveillance pathway ko03015 97 UP Cell cycle ko04110 140 UP Ubiquitin mediated proteolysis ko04120 143 UP RNA transport ko03013 139 UP p53 signaling pathway ko04115 69 UP Apoptosis - multiple species ko04215 34 UP endoplasmic reticulum-related protein processing and exocytosis in nervous system Protein processing in endoplasmic reticulum ko04141 159 UP Synaptic vesicle cycle ko04721 102 UP Retrograde endocannabinoid signaling ko04723 179 UP Infection and immune related Staphylococcus aureus infection ko05150 49 DOWN Transcriptional misregulation in cancer ko05202 174 DOWN Pertussis ko05133 73 DOWN RNA transport ko03013 30 DOWN Huntington's disease ko05016 205 UP 3.2 Identification of DEGs in ovary development of black rockfish Due to the special reproductive strategy in female black rockfish, the transcriptomic level changes in ovary seemed not as much drastic as testis. Annotation and enrichment analysis only achieved 464 and 329 DEGs (adjusted p-value 2) in F_III vs F_IV group and F_IV vs F_V group, respectively (Fig. 7 a). The expression pattern of all these 765 DEGs were different from testis, which up-regulated in stage IV (F_IV) and V (F_V) ovary, suggested the delayed development in ovary compared with testis (Fig. 7 b). Interestingly, GO analysis of these 765 DEGs with a total of 205 DEGs were annotated in membrane (GO:0016020) in CC (Fig. 7 c). Additional file 4 showed the detail of these 205 DEGs, most of which participated in membrane-anchored enzymatic reaction or molecular transport. 10 accurately biological function classifications and several subclassifications were accomplished with cell cycle , cell junction , cell structure , metabolism , DNA binding and transcription regulated , immune system , nervous system , molecular transport , protein modification , RNA binding , and signal transduction including. Among these classifications, Molecular transport showed the most abundant (40 DEGs) of all, indicated the importance of matter transport process during ovary development stage of black rockfish. 4. Validation of RNA-Seq results by qRT-PCR 10 DEGs were randomly selected for q-PCR analysis to validate the present RNA-Seq data. The results showed that the q-PCR expression pattern of the selected genes was significantly correlated with the RNA-Seq results (R 2 : 0.933–0.9559). Totally, the RNA-Seq data were confirmed by the q-PCR results, implying the reliability and accuracy of the RNA-Seq analysis (Fig. 8 ). Discussion The black rockfish, as an economically crucial fish species, has been processed a lot of works, especially in reproductive and toxic physiology and molecular biology due to the unusual ovoviviparous strategy ( 24 , 27 , 30 ) . While the transcriptomics profile of the black rockfish gonad development was less clear. In order to investigate underlying mechanisms, a series of sex-related genes and biological pathways should be determined for further exploration. The present study mainly focused on identification of the key genes and pathways in both ovary and testis development and germ cell renewal by histology and RNA-seq. Identification and terminology of gonad development of the black rockfish Reproduction of most teleost is an annual and cyclic event, while gonads present different development stages for germ cells renewal. Critical phases of fish reproductive cycle were determined by a conceptual model based on specific histological and physiological markers ( 50 ) . Black rockfish testis go through 4 phases during development, including the developing phase when no spermatozoa present in lumen of lobules or sperm ducts (described as stage III in the present study), spawning phase when spermatogenesis active (described as stage IV), and regressing phase with few or no sperm (described as stage V) ( 50 , 51 ) . Different from the oviparity strategy, black rockfish ovulated eggs fertilized and retained in ovary during the gestation phase ( 50 ) . In the present study, the description of the developmental stage of ovary as stage III, stage IV and stage V ovary indicated the terminology of developing phase, early developing subphase and actively spawning subphase, respectively ( 50 , 52 ) . Key pathways in testis development and gametogenesis of male black rockfish Testis of male black rockfish showed drastic variation from stage III to stage V which reflected in both histology results and transcriptomic profile. During the development from stage III to stage IV, one category of pathways intercellular interaction and cytoskeleton showed upregulation significantly. Similar to mammals, cellular interactions among sertoli cells and germ cells of fish in the seminiferous epithelium play important structural and functional roles as well. Testis structure undergo dramatic morphofunctional changes during reproductive season to activate spermatogenic arrangement ( 53 , 54 ) . It suggested that actin-related adhesion among sertoli cells and sertoli cells to germ cells are associated with multiple events crucial for spermatogenesis and normal fertility, especially for spermatid elongation in stage IV testis ( 54 , 55 ) . In the transcriptomics analysis of spotted knifejaw ( Oplegnathus punctatus ), similar intercellular interaction pathway (cell adhesion molecules) was significantly enriched in testis as well ( 20 ) . In fish, cytoplasmic extensions of sertoli cells form a envelop of a single synchronously developing group of germ cells derived from a single spermatogonium. The form of the group of germ cells, namely spermatogenesis is a highly organized and coordinated process in which diploid spermatogonia proliferate and differentiate to mature spermatozoa ( 2 ) . Both processes required a large amount of molecule, including DNA, RNA, protein, lipid and steroid, for cell amplification. In the present study, category Molecule amplification and repairment in cell cycle , including cell cycle, ubiquitin mediated proteolysis, DNA replication, fanconi anemia pathway, mRNA surveillance pathway, RNA transport, p53 signaling pathway and apoptosis, showed significant up-regulated in both stage III and IV testis. Different from mammals, fish show a cystic type of spermatogenesis and go through different stages by an accompanying group of sertoli cells. The shape shifting of a cystic which is enveloped by sertoli cells is accompanied by strong proliferation of sertoli cells ( 56 , 57 ) . In African catfish and Nile tilapia, sertoli cell proliferation occurred primarily during spermatogonial proliferation ( 57 ) . In addition, in Leporinus taeniatus spermatogenesis is also processed as which diploid spermatogonia proliferate and differentiate to haploid spermatozoa ( 58 ) . On the other hand, fanconi anemia pathway was confirmed as an efficient DNA repairment pathway ( 59 , 60 ) . In fanconi anemia disease causing genes mutant zebrafish, the DNA damage repairment failed and caused a series of abnormal development. Testis development and spermatogenesis required a mass of metabolites, especially for androgens. In fish, transduction of the gonadotropin signal stimulates the production of 11-ketotestosterone (11-KT), a major androgen for fish ( 61 ) , functionally activity the androgen receptors ( 62 , 63 ) . To complete the process, enzymes and other proteins are required for biosynthesis ( 64 ) . In the present study, the steroid hormone biosynthesis pathway and the key genes 3b-hsd and cyp11a1 were significant up-regulated in stage IV testis, indicating the importance of steroidogenesis for spermatogenesis ( 62 , 65 ) . A few pathways related to RNA transcription and translation transportation, namely RNA transport, mRNA surveillance pathway and ubiquitin mediated proteolysis, were also significantly enriched in stage IV testis, suggested the metabolism and synthesis under gonadotropin and steroid hormone have been crucial for maintenance of spermatogenesis ( 62 , 66 ) . Interestingly, these pathways (progesterone-mediated oocyte maturation and oocyte meiosis) were both enriched in stage IV testis, implying the importance of the role that progestogen plays in spermatogenesis. Serum 17α, 20β-dihydroxy-4-pregnen-3-one (DHP), as fish specific progestin, exhibited higher in stage IV concomitant with testicular development in male turbot ( Scophthalmus maximus ) ( 67 ) . Knock out of the nuclear receptor of DHP resulted a smaller testis and a lower GSI compared with normal testis in Nile tilapia ( Oreochromis niloticus ) ( 68 ) . Key genes in ovary development and gametogenesis of female black rockfish Due to the special reproductive strategy in female black rockfish, the transcriptomic level changes in ovary seemed not as much drastic as testis. A total of 765 DEGs were enriched significantly which classified into 10 classifications including cell cycle, cell junction, cell structure, metabolism, DNA binding and transcription regulated, immune system, nervous system, molecular transport, protein modification, RNA binding, signal transduction and unclassified genes. Similar to the testis of black rockfish, ovary development presented significantly enriched DEGs in cell cycle and junction and cytoskeleton as well. The matrix metalloproteinases (MMPs) are a family of extracellular proteinases which plays a role in the ECM remodeling associated with many physiological and pathological processes ( 69 ) . Previous studies in mammal have shown increased MMP19 expression in preovulatory follicles ( 70 ) , and estrogen receptor knockout lead to down-regulation of MMP19 and failure in release of mature oocyte ( 71 ) . In the present study, mmp19 was also up-regulated in stage IV ovary, which may be due to follicles mature and rupture afterward in black rockfish. Black rockfish vipr gene showed a significant increase in stage IV ovary implied its critical role in folliculogenesis. Both neuropeptides vasoactive intestinal polypeptide (vip) and pituitary adenylate cyclase-activating polypeptide (pacap) are considered to stimulate the ovarian functions such as steroidogenesis, cAMP accumulation in rat granulosa cells ( 72 ) through the PACAP/VIP receptors. VIPR has been found equal affinity with both PACAP and VIP ( 73 ) . In zebrafish, the vipr expression maintained high during follicle until full-grown stage ( 74 ) . The classification molecular transport showed the most DEGs in ovary development of black rockfish with 20 genes of solute carrier (SLC) gene superfamily enriched. The SLC gene superfamily encodes series of membrane transporters ( 75 ) including passive transporters, cotransporters and exchangers in various cellular membranes ( 76 ) . The members of slc6 gene family ( slc6a11 , slc6a15 , slc6a6 , slc6a8 ) which performs Na + and Cl − dependent neurotransmitter transport for γ-aminobutyric acid (GABA), creatine and taurine were differentially expressed along ovary development. The result was coincident with the transcriptomic analysis of hapuku ( Polyprion oxygeneio s) ( 77 ) . However, these transport proteins were more commonly mentioned in central nervous system of vertebrates ( 78 ) , which imply the SLCs may also functional in ovary. Zinc is necessary for meiotic in zebrafish and mammals ( 79 , 80 ) , the zinc transport proteins slc30 gene family was found expressed in oocyte and cumulus cells during maturation in mouse ( Mus musculus ) ( 81 ) , which may be a proper explanation that slc30a9 showed up-regulated in early oocyte mature for zinc transport. Conclusions The present study is the first report of the transcriptomic information of the ovoviviparous black rockfish gonad development. Several important candidate pathways and genes in both testis and ovary development have been identified. Among these pathways and genes, the categories intercellular interaction and cytoskeleton , molecule amplification and repairment in cell cycle were revealed to be crucial in testis development and spermatogenesis along with a series of metabolite biosynthesis. Some key genes emerged in ovary development such as mmp19 and neuropeptide receptor vipr in follicles mature and rupture and the membrane transporter family slc6 and slc30 in various ways. These data provided a comprehensive insight into the black rockfish gonad development for further study of reproductive physiology and molecular biology in ovoviviparity. Materials And Methods 1. Animal and sample collection Together, 27 adult male and 27 adult female black rockfish cultured in northern Yellow Sea were obtained from October to March of the next year. Nine individuals were sampled for each development stage in all 3 development stages in both sexes. Fish were acclimatized at a density of 10 individuals per tank (diameter 1 m, height 1.5 m) under laboratory conditions for 2 days without feeding. After acclimation, individuals were anesthetized with MS222 (200 mg /L). Body weight and gonad weight were measured and GSI was calculated. Gonad were also collected immediately for both histology and RNA isolation. 2. Histology analysis and RNA isolation Testis and ovary of different development stages were fixed in Bouin’s solution, dehydrated and embedded in paraffin. Tissue sections were cut into 6 µm by a microtome (Leica, Wetzler, Germany) and stained with hematoxylin-eosin. All section photos were taken by Olympus bright field light microscope (Olympus, Tokyo, Japan). Gonads were collected and frozen in liquid nitrogen for further total RNA isolation with TRIzol reagent (Invitrogen, USA). The quality and concentration of the total RNA were assessed by agarose gel electrophoresis and Agilent 2100 Bioanalyzer system (Agilent Technologies, USA), respectively. 3. Library construct and transcriptome sequencing In order to mask the difference among sample repetitions, equal amount of total RNA from 3 individual ovaries or testis in same development were pooled together. 18 sequencing libraries were generated NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s instructions and index codes were added to attribute sequences to each sample. Samples were sequenced on an Illumina Hiseq X ten platform and 150 bp paired-end reads were generated. Raw sequences were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) with accession numbers of PRJNA573572. 4. De novo assembly and annotation of sequencing reads De novo assembly was performed on gonad clean reads using the Trinity assembly software suite ( 40 ) without a reference genome. Transcripts (both contigs and singletons) were annotated by BLASTx searches ( 41 ) using NCBI non-redundant (Nr), NCBI nucleotide sequences (Nt) and Swiss-Prot databases with a cutoff “e-value” of < 1e − 5 . Domain-based comparisons with Protein family (Pfam) and KOG (a eukaryote-specific version of the Clusters of eukaryotic Ortholog Groups) databases were performed by RPS-BLAST tool from locally installed NCBI BLAST + v2.2.28 and HMMER 3.0 program, respectively. Annotated transcripts were analyzed to GO classification with the aid of Blast2Go program ( 42 ) . These gene terms were then enriched on the three GO categories (Biological Process, Cellular Component and Molecular Function at level 2) using the GOseq R package ( 43 ) . KEGG, which is a database of biological systems, maps were retrieved by online KEGG Automatic Annotation Server for the overview of metabolic pathway analysis ( 44 , 45 ) . 5. Differential gene expression analysis The reads of each library were mapped to the de novo assembled transcripts with the bowtie 2 program for mismatch check ( 46 ) . Count numbers of mapped reads and FPKM (expected number of Fragment Per Kilobase of transcript sequence per Millions base pairs sequenced) were achieved and normalized by RSEM V1.2.15 ( 47 ) . Differential expression statistical analysis of different development stage of gonad was conducted by the DEGSeq R package ( 48 ) with a cutoff “q-value” of 0.01 and |log 2 (fold change)|>2. Transcripts with absolute fold change values over 2.0 were marked as significantly differential expressed genes. 6. Experimental validation by quantitative real-time PCR Expression analysis of 10 selected DEGs were performed by quantitative real-time PCR (qPCR) with specific primers validate our Illumina sequencing data. Primers were listed in Additional file 5 . Samples were generated from F_III, F_IV, F_V ovary and M_III, M_IV, M_V testis in the preceding experiment. After RNA extraction and reverse transcription, all the cDNA products were diluted to 500 ng/µL. The 20µL qPCR reaction mixture consisted of 2µL cDNA template, 0.4µL of both primers, 10µL of KAPA SYBR®FAST qPCR Master Mix (2X), 0.4µL of ROX and 6.8µL of RNAase-free water. PCR amplification was performed as that incubated in a 96-well optical plate at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and a final extension at 72 °C for 2 min. qPCR was performed using the StepOne Plus Real-Time PCR system (Applied Biosystems) and 2 −ΔΔCT method was used to analysis the expression level of genes. Abbreviations DEGs: Different expression genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; NGS: The next-generation sequencing technology; GSI: Gonadosomatic index; GO: Gene ontology; NR: Nonredundant; BP: Biological Process; CC: Cell Component; MF: Molecular Function; ECM: Extracellular matrix; 11-KT: 11-ketotestosterone; DHP: 17α, 20β-dihydroxy-4-pregnen-3-one; MMPs: Matrix metalloproteinases; vip: vasoactive intestinal polypeptide; pacap: pituitary adenylate cyclase-activating polypeptide; SLC: Solute carrier; GABA: γ-aminobutyric acid; NCBI: the National Center for Biotechnology Information; Nr: non-redundant; Nt: nucleotide sequences; Pfam: Protein family; KOG: a eukaryote-specific version of the Clusters of eukaryotic Ortholog Groups; qPCR: quantitative real-time PCR; Declarations Ethics approval and consent to participate All procedures involved in dealing of fish in this study were approved by Animal Research and Ethics Committees of Ocean University of China (Permit Number: 20141201) prior to the initiate of the study. The studies did not involve endangered or protected species. And all experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. Consent for publication Not applicable Availability of data and materials The datasets generated and analysed during this study were deposited in the Short Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra) of the National Center for Biotechnology Information (NCBI) with accession numbers of PRJNA573572. And other data supporting the conclusion of this article is included within the article, and can be found in the additional files. Competing interests The authors declare that they have no competing interests Funding This research was supported by The National Natural Science Funds (41676126). Our funding agencies did not play a role in the study design, data collection, analysis, interpretation of the data, or preparation of the manuscript. Authors' contributions WHS, LJF and QX designed the study; LJS performed the transcriptome and qRT-PCR experiment; LLK, WXJ, YYJ and LJS performed in samples collection; LJS wrote the manuscript and QX provided manuscript editing and feedback; All authors read and approved the final manuscript. Acknowledgements Not applicable. References Labbé C, Robles V, Herraez MP. Epigenetics in fish gametes and early embryo. 2017; 472: 93-106. Schulz RW, de França LR, Lareyre J-J, LeGac F, Chiarini-Garcia H, Nobrega RH , et al. Spermatogenesis in fish. Gen Comp Endocrinol. 2010; 165: 390-411. Kagawa H. Oogenesis in teleost fish. Aqua-bioscience Monograghs, 2013; 6: 99-127. Almeida FF, Kristoffersen C, Taranger GL, Schulz RW. Spermatogenesis in Atlantic cod (Gadus morhua): a novel model of cystic germ cell development. Biol Reprod. 2008; 78: 27-34. Kobayashi T, Kajiura-Kobayashi H, Nagahama Y. 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Supplementary Files SupplementaryFile.zip Cite Share Download PDF Status: Under Review Version 1 posted Review # 2 received at journal 04 Apr, 2021 Editorial decision: Major revision 04 Apr, 2021 Reviewer # 2 agreed at journal 16 Jan, 2021 Review # 1 received at journal 25 Oct, 2020 Reviewer # 1 agreed at journal 03 Oct, 2020 Reviewers invited by journal 22 Sep, 2020 Editor assigned by journal 01 Sep, 2020 Submission checks completed at journal 31 Aug, 2020 Editor invited by journal 31 Aug, 2020 First submitted to journal 30 Aug, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-70073","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":2061496,"identity":"f48b62ea-ea73-44a9-8649-59de5c62fd9f","order_by":0,"name":"Jianshaung Li","email":"","orcid":"https://orcid.org/0000-0003-4100-4326","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianshaung","middleName":"","lastName":"Li","suffix":""},{"id":2061497,"identity":"0e2d516d-a8a3-4d81-b983-d0026bceb797","order_by":1,"name":"Haishen Wen","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haishen","middleName":"","lastName":"Wen","suffix":""},{"id":2061498,"identity":"536338cb-8294-4341-b430-7b80cc0d1c16","order_by":2,"name":"Likang Lyu","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Likang","middleName":"","lastName":"Lyu","suffix":""},{"id":2061499,"identity":"ebe64431-ac88-4508-9489-c574effe81c2","order_by":3,"name":"Yun Li","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Li","suffix":""},{"id":2061500,"identity":"e8526ea3-d0a2-42e8-a2fa-f6b102103acc","order_by":4,"name":"Xiaojie Wang","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaojie","middleName":"","lastName":"Wang","suffix":""},{"id":2061501,"identity":"c4b474d1-b83c-4650-9c86-f3d66556f67b","order_by":5,"name":"Ying Zhang","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":2061502,"identity":"27a8aaf4-e692-4cd1-8767-55d3ad1832ee","order_by":6,"name":"Yijia Yao","email":"","orcid":"","institution":"Ocean University of China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yijia","middleName":"","lastName":"Yao","suffix":""},{"id":2061503,"identity":"67e1c9d2-4af2-412d-812e-cc25d4e47a2a","order_by":7,"name":"Xin Qi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYFAC5gMQ+gDxWtgSSNbCY0CiFr4bOR8f89QwyPHdSGD8XECMFskzZzcb8xxjMJa8kcAsPYMYLQbHe7dJ8zYwJG64kcDGzEOUlsM8z0Ba6knQcryHDaQlwYBoLZJnjhkbzjkmYTjzzMNmaaK08N1IfvjgTY2NPN/x5IOfidICjQ4JIGZsIEoDSZE+CkbBKBgFIxUAAHF2Lp+YY++NAAAAAElFTkSuQmCC","orcid":"","institution":"Ocean University of China","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Qi","suffix":""}],"badges":[],"createdAt":"2020-09-01 11:19:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-70073/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-70073/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":2297974,"identity":"3d18536a-b420-4896-aa19-17a8e718418c","added_by":"auto","created_at":"2020-09-08 15:49:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284120,"visible":true,"origin":"","legend":"H\u0026E stain of gonad development stage of black rockfish. III♂, IV♂ and V♂represent stage III, IV and V testis, respectively. And III♀, IV♀ and V♀represent stage III, IV and V ovary, respectively.","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.1.jpg"},{"id":2297975,"identity":"4f2f45b9-6ae0-4c9d-bdfc-499a96777616","added_by":"auto","created_at":"2020-09-08 15:49:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91035,"visible":true,"origin":"","legend":"Annotation and functional classification of transcripts in gonad of black rockfish. (a) Top-hit species distribution of BLASTX matches of assembled transcripts. (b) Function annotation of assembled transcripts based on gene ontology (GO) analysis. GO analysis was performed at level 2 for the three main categories (biological process, cellular component and molecular function). The x-axis shows the specific terms. The y-axis shows the number of transcripts in each term. (c) Pathway assignment based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Transcripts were classified into five main categories (A: cellular process, B: environmental information processing, C: genetic information processing, D: metabolism and E: organismal systems). ","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.2.jpg"},{"id":2297976,"identity":"da94b5e0-5ac3-4a96-89f7-f06430c7d8cf","added_by":"auto","created_at":"2020-09-08 15:49:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":463379,"visible":true,"origin":"","legend":"Venn diagram of the DEGs of 4 comparisons in the gonad of black rockfish.","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.3.jpg"},{"id":2297977,"identity":"a8a3a38a-c30b-4525-ad36-75e401bc3306","added_by":"auto","created_at":"2020-09-08 15:49:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401285,"visible":true,"origin":"","legend":"Identification and annotation of DEGs in different development stage in testis of black rockfish. (a) Venn diagram of the DEGs of M_III vs M_IV and M_IV vs M_V. (b) Expression values of 32,772 DEGs in 3 libraries (M_III, M_IV, M_V) are presented in heat map. Red and blue colors indicate up- and down- regulated transcripts, respectively. (c) Gene ontology (GO) analysis for the 32,772 DEGs. The x-axis shows the number of genes in each term. The y-axis shows the specific terms. The asterisk represents the corrected p-value \u003c0.05 in each GO term.","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.4.jpg"},{"id":2297978,"identity":"be7ff07a-fc7e-4b11-97c2-1153735f67b7","added_by":"auto","created_at":"2020-09-08 15:49:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107848,"visible":true,"origin":"","legend":"Regulation of pathways in M_III vs M_IV group of black rockfish. Red and green orthogon represent up and down regulated KEGG pathways and DEGs, respectively(45, 49, 82).","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.5.jpg"},{"id":2297979,"identity":"8b08860b-65b9-4944-99ab-5aa0193cf33d","added_by":"auto","created_at":"2020-09-08 15:49:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":86879,"visible":true,"origin":"","legend":"Regulation of pathways in M_IV vs M_V group of black rockfish. Red and green orthogon represent up and down regulated KEGG pathways and DEGs, respectively(45, 49, 82).","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.6.jpg"},{"id":2297980,"identity":"c64d6b11-649d-4ea6-8677-d0e213c27658","added_by":"auto","created_at":"2020-09-08 15:49:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82618,"visible":true,"origin":"","legend":"Identification and annotation of DEGs in different development stage in ovary of black rockfish. (a) Venn diagram of the DEGs of F_III vs F_IV and F_IV vs F_V. (b) Expression values of 765 DEGs in 3 libraries (F_III, F_IV, F_V) are presented in heat map. Red and blue colors indicate up- and down- regulated transcripts, respectively. (c) Gene ontology (GO) analysis for the 765 DEGs. The x-axis shows the number of genes in each term. The y-axis shows the specific terms. The asterisk represents the corrected p-value \u003c0.05 in each GO term.","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.7.jpg"},{"id":2297981,"identity":"39c8b54a-70ea-4124-8544-14f0d2892ee7","added_by":"auto","created_at":"2020-09-08 15:49:39","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":90740,"visible":true,"origin":"","legend":"qRT-PCR validation of 10 differentially expressed genes generated from RNA-Seq results from gonad. The expression levels of the selected genes were normalized to the 18s gene. A: F_III vs F_IV; B: F_IV vs F_V; C: M_III vs M_IV; D: M_IV vs M_V. Gene abbreviations are: nuclear receptor corepressor 1 (ncor1); collagen alpha-1(XXVII) chain B (col27a1); cathepsin Z (ctsz); steroidogenic acute regulatory protein (star); sodium/myo-inositol cotransporter (slc5a11); cyclooxygenase 2 (cox2); transcriptional regulator ATRX (atrx); syndecan-2 (sdc2); importin-4 (ipo4); pericentriolar material 1 protein (pcm1).","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/Fig.8.jpg"},{"id":13589720,"identity":"e6003258-c216-4675-bb18-b961ea5dcb95","added_by":"auto","created_at":"2021-09-17 05:01:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1529695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/f3f50ebf-0951-4c9e-860a-0a6c289acb9b.pdf"},{"id":2297983,"identity":"119e6aee-2ea7-42ae-8026-df54075a3f12","added_by":"auto","created_at":"2020-09-08 15:49:39","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66579,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.zip","url":"https://assets-eu.researchsquare.com/files/rs-70073/v1/SupplementaryFile.zip"}],"financialInterests":"","formattedTitle":"\u003cp\u003eComparative Transcriptomic Analysis of Gonad Development and Renewal of Ovoviviparous Black Rockfish (Sebastes Schlegelii)\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eSpermatozoa and oocyte in fish species are highly specialized cells fusing into an embryo which will develop into a mature organism producing gametes again\u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e. Germ cell development and renewal are required by gonad development during the entire reproductive lifespan of teleost. Spermatogenesis and oogenesis are both highly organized processes and crucial for transmitting the genetic information to next generation\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e. Many aspects of gametogenesis unfold their difference between female and male despite the development of spermatozoa and oocyte follow common principles\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. Teleost represent the most diverse and numerous group in vertebrates and the researches on spermatogenesis in teleost are mainly focused on a few species such as Atlantic cod (\u003cem\u003eGadus morhua\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e, tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e, rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e, zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e and so on. The molecular mechanism of fish spermatogenesis has been reviewed as well\u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e. Besides, the studies of oogenesis in teleost fish have been reported in recent decades, including the related gene in tilapia\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e, rainbow trout\u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e, Least killifish (\u003cem\u003eHeterandria formosa\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e and pejerrey (\u003cem\u003eOdontesthes bonariensis\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, some molecular techniques and tools are rising their importance in identification of key pathways and genes in some biological process. RNA-seq platform based on the next-generation sequencing technology (NGS) is considered to be a revolutionary and efficient tool for investigating the key pathways and genes in gonad development. It has been already applied in study of reproduction system in many species including pig (\u003cem\u003eSusscrofa domestica\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e, American alligator (\u003cem\u003eAlligator mississippiensis\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e, swimming crab (\u003cem\u003ePortunus trituberculatus\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e. A lot of researches also focused on gonad development and germ cell renewal in teleost revealed the potential mechanism in germ cell and gonad development\u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e, sex determination and differentiation\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e, sex-related genes in both sexes\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFishes take different reproductive strategies including oviparity which is common in most teleost for laying eggs before fertilization, viviparity in some Chondrichthyes species whose embryos develop inside ovary with direct nutrition connect with maternal\u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e, and the recent well-studied ovoviviparity which eggs fertilized in ovary with development of lecithotrophic larva in some of teleost including the black rockfish (\u003cem\u003eSebastes schlegelii\u003c/em\u003e)\u003csup\u003e(\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/sup\u003e in the present study. Previous studies on black rockfish were mainly focused on environmental toxicology and immune\u003csup\u003e(\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/sup\u003e, response to stress \u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/sup\u003e, whole genomic data analysis\u003csup\u003e(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/sup\u003e and reproductive physiology\u003csup\u003e(\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/sup\u003e. While the reproductive studies of black rockfish were most on single gene identification analysis, \u003csup\u003e(\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e and function\u003csup\u003e(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u003c/sup\u003e. While little is known about the comprehensive understanding of gonad development of black rockfish. In this study, RNA-seq was performed on both testis and ovary to characterize key pathways and genes during development and gamete maturation in black rockfish. The transcripts were de novo assembled and annotated greatly enriched the black rockfish gene database. The identified pathways and genes in this study also provided a novel insight into the reproductive biology of ovoviviparious teleost.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Basic physiological data and histology analysis identified different stages of ovary and testis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E stain was processed to identified the different stages of gonad development from October to March of the next year. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, testis was characteristic as stage III which spermatogenesis was not observed yet in October until early November when spermatogenesis was completed as a mark for stage IV. After mating in January of the next year, sperm was totally emptied from testis, showed the testis was in stage V. Stage III oocyte was observed in early November with lipid drop and yolk accumulation initiate. Lipid drop and yolk were showed to fuse together in oocyte at stage IV. Completion of the first meiotic division and first polar body discharge were observed at stage V oocyte.\u003c/p\u003e\n\u003cp\u003eAccording to the identification by H \u0026amp; E stain, 3 different stages of both gonads were distinguished and collected and named as F_III, F_IV and F_V for stage III, IV and stage V ovary, while M_III, M_IV and M_V for stage III, IV and stage V testis. Average bodyweight and gonad weight were showed in \u003cstrong\u003eAdditional file 1.\u003c/strong\u003e In testis, both high body weight (795.09g\u0026thinsp;\u0026plusmn;\u0026thinsp;58.63\u0026nbsp;g) and gonadosomatic index (GSI) (1.26) were conventionally observed in winter which mating behavior occurs in ovoviviparous black rockfish. While high body weight (880.68g\u0026thinsp;\u0026plusmn;\u0026thinsp;99.48\u0026nbsp;g) and GSI (7.57) of ovary were showed in middle March of the next year when the oocytes mature and fertilization was coming.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2. de novo\u003c/em\u003e assembly and annotation of black rockfish gonad transcripts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-Seq was performed on both ovary and testis samples from 3 different stages. A total of 1,029,619,820 raw reads (150\u0026nbsp;bp) were obtained from 18 gonad samples on the Illumina HiSeq X ten platform. After preprocessing and filtration of low-quality sequences, clean read count was 998,950,272 (\u003cstrong\u003eAdditional file 2\u003c/strong\u003e). The \u003cem\u003ede novo\u003c/em\u003e assembled transcriptome included 517,848 genes with the N50 of 1,660 indicated a high-quality assembly. The genes of black rockfish gonads were annotated in 7 public databases including Nr, Nt, KO, SwissProt, PFAM, gene ontology (GO)and KOG with 61.63% of genes annotated in at least 1 database. The Nonredundant (NR) annotation showed that 72.6% of genes were annotated in 5 fish species with the highest sequence similarity compared to large yellow croaker (\u003cem\u003eLarimichthys crocea\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eThe functional classification of transcriptome data is the basic requirement for the application of functional genomic approaches in fishery research. Analysis based on GO and KEGG database are the common methods in functional classification of transcriptomic sequence. Result showed Blast2Go assigned 123,181 genes into 56 functional GO terms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Regarding the 3 primary ontology categories, Biological Process (BP) represents the majority (26 terms) of annotations, followed by Cell Component (CC) (20 terms) and Molecular Function (MF) (10 terms). Based on the analysis of level 2 GO terms, cellular process (GO:0009987) showed the most annotations genes in BP. And for CC, cell (GO:0005623) and cell part (GO:0044464) contained the highest numbers of annotations. The GO terms related to MF with the highest number of annotations were binding (GO:0005488), catalytic activity (GO:0003824). KEGG analysis was performed to understand the higher order functional information of biological system\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/sup\u003e. Based on the analysis, totally 70,174 genes were annotated into 5 categories on 32 significantly enriched KEGG pathways (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Analysis different expression genes in gonad development stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 33,393 DEGs were obtained from 4 different gonad development libraries (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and absolute log\u003csub\u003e2\u003c/sub\u003efoldchange\u0026thinsp;\u0026gt;\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Among them, 464 DEGs (151 up-regulated and 313 down-regulated) were significantly expressed in F_III vs F_IV, and 329 DEGs (109 up-regulated and 220 down-regulated) were significantly expressed in F_IV vs F_V. While male showed less time in reproductive period revealed much more DEGs than female, which 3,858 DEGs (1,611 up-regulated and 2,247 down-regulated) were significantly expressed in M_III vs M_IV and 30,160 DEGs (24,446 up-regulated and 5,714 down-regulated) were significantly expressed in M_IV vs M_V.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1 Identification of DEGs in testis development of black rockfish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on these DEGs mentioned above, 3,858 and 30,160 annotated DEGs were obtained from M_III vs M_IV group and M_IV vs M_V group, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Heatmap analysis of these 32,772 DEGs expressed in all 3 stages of testis revealed that most of these genes in both stage III and IV testis showed similar expression pattern, which up-regulated genes in stage III (M_III) and stage IV testis (M_IV) showed down-stream in stage V (M_V) testis, suggested that the expression profile of these DEGs and male reproductive process including gamete mature were directly proportional (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). GO analysis of these DEGs showed most of them were mapped onto MF term (\u003cem\u003ep v\u003c/em\u003ealue\u0026thinsp;\u0026lt;\u0026thinsp;0.01), especially molecule binding GO terms (anion binding, GO:0043168, small molecule binding, GO:0036094 and so on) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003e11 and 14 significantly enriched KEGG pathways were mapped between M_III vs M_IV group and M_IV vs M_V group (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), respectively (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn M_III vs M_IV group, 11 KEGG pathways were classified into 3 categories, including \u003cstrong\u003eintercellular interaction and cytoskeleton\u003c/strong\u003e, \u003cstrong\u003emolecule amplification and repairment in cell cycle\u003c/strong\u003e, and \u003cstrong\u003eother\u003c/strong\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The DEGs in category \u003cstrong\u003eintercellular interaction and cytoskeleton\u003c/strong\u003e, including extracellular matrix (ECM)-receptor interaction, focal adhesion and regulation of actin cytoskeleton, were basically up-regulated in stage IV compared with stage III. DEGs in category \u003cstrong\u003emolecule amplification and repairment in cell cycle\u003c/strong\u003e, including cell cycle, ubiquitin mediated proteolysis, DNA replication, Fanconi anemia pathway, RNA transport and mRNA surveillance pathway, were significantly enhanced in stage III, when the spermatogenesis start, cell division and protein biosynthesis are processing, compared with stage IV. In addition, some other pathways such as steroid hormone biosynthesis was up-regulated in stage IV testis.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, 14 KEGG pathways were classified into 5 categories, including \u003cstrong\u003eprogesterone induced gamete mature\u003c/strong\u003e, \u003cstrong\u003emolecule amplification and repairment in cell cycle\u003c/strong\u003e, \u003cstrong\u003eendoplasmic reticulum-related protein processing and exocytosis in nervous system\u003c/strong\u003e, \u003cstrong\u003einfection and immune related\u003c/strong\u003e and other in M_IV vs M_V group. In category \u003cstrong\u003eprogesterone induced gamete mature\u003c/strong\u003e, most DEGs showed up-regulated in stage IV testis due to the importance of steroid hormone to germ cell division. Category \u003cstrong\u003emolecule amplification and repairment in cell cycle\u003c/strong\u003e, which also showed up-regulated in both stage III and stage IV, implied the drastic changes in cell metabolism continue the whole reproductive process. The protein processing in endoplasmic reticulum, synaptic vesicle cycle and retrograde endocannabinoid signaling in categories \u003cstrong\u003eendoplasmic reticulum-related protein processing and exocytosis in nervous system\u003c/strong\u003e showed the most interest and abundant DEGs. DEGs, such as \u003cem\u003esar1b\u003c/em\u003e, \u003cem\u003esec13\u003c/em\u003e, \u003cem\u003esec24c\u003c/em\u003e and \u003cem\u003eslc18a2\u003c/em\u003e are involved in transport vesicles, \u003cem\u003estx2\u003c/em\u003e is related to epithelial morphogenesis due to exocytosis, \u003cem\u003ecacna1b\u003c/em\u003e regulates hormone or neurotransmitter release, while \u003cem\u003egria1\u003c/em\u003e and \u003cem\u003egrm1\u003c/em\u003e for receipting glutamate neurotransmitter message. These results suggested these genes expressing variated significantly between stage III and IV testis were inseparable with the intense reproductive stages.\u003c/p\u003e\n\u003cp\u003eAs shown in \u003cstrong\u003eAdditional file 3\u003c/strong\u003e, 3 KEGG pathways cell cycle, mRNA surveillance pathway and RNA transport, which were both up-regulated in M_III vs M_IV and M_IV vs M_V, indicated that the activity process in the whole reproductive cycle. Interestingly, Ubiquitin mediated proteolysis, which was highly expressed in M_III vs M_IV and M_IV vs M_V, presented totally different DEGs, suggested that the pathway may play different roles in different development stages. In Oocyte meiosis pathway, \u003cem\u003esgo1\u003c/em\u003e, \u003cem\u003estag3\u003c/em\u003e, \u003cem\u003esmc1b\u003c/em\u003e and \u003cem\u003esmc3\u003c/em\u003e were up-regulated in stage IV, suggested the exit of meiosis of gamete in final reproductive stage. Besides, DEGs in p53 signaling pathway, \u003cem\u003eccne1\u003c/em\u003e, \u003cem\u003eccnd2\u003c/em\u003e, \u003cem\u003ecdk2\u003c/em\u003e with up-regulated and \u003cem\u003esrsf10\u003c/em\u003e with down-regulated expressed profile, which \u003cem\u003esrsf10\u003c/em\u003e repressed cell cycle G1 and G2 phase arrested by inhibition of \u003cem\u003eccne1\u003c/em\u003e, \u003cem\u003eccnd2\u003c/em\u003e, \u003cem\u003ecdk2\u003c/em\u003e, resulting cell cycle was active in stage III and IV testis.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003esignificant enriched KEGG pathways in M_IIIvsM_IV group and M_IVvsM_V group.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStage\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG pathway\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eInput gene\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRegulation\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM_IIIvsM_IV\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eIntercellular interaction and cytoskeleton\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eECM-receptor interaction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04512\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFocal adhesion\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04510\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRegulation of actin cytoskeleton\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04810\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" align=\"left\"\u003e\n\u003cp\u003eMolecule amplification and repairment in cell cycle\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04110\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eUbiquitin mediated proteolysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA replication\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03030\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFanconi anemia pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03460\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003emRNA surveillance pathway\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSteroid hormone biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko00140\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycosylphosphatidylinositol (GPI)-anchor biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko00563\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM_IVvsM_V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eProgesterone induced gamete mature\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProgesterone-mediated oocyte maturation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04914\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e117\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOocyte meiosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04114\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e129\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"6\" align=\"left\"\u003e\n\u003cp\u003eMolecule amplification and repairment in cell cycle\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003emRNA surveillance pathway\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04110\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e140\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eUbiquitin mediated proteolysis\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e143\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA transport\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e139\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ep53 signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04115\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eApoptosis - multiple species\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04215\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eendoplasmic reticulum-related protein processing and exocytosis in nervous system\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProtein processing in endoplasmic reticulum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04141\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e159\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSynaptic vesicle cycle\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04721\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e102\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRetrograde endocannabinoid signaling\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko04723\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e179\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eInfection and immune related\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStaphylococcus aureus infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko05150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTranscriptional misregulation in cancer\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko05202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e174\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePertussis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko05133\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eRNA transport\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko03013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDOWN\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuntington's disease\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eko05016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e205\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Identification of DEGs in ovary development of black rockfish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the special reproductive strategy in female black rockfish, the transcriptomic level changes in ovary seemed not as much drastic as testis. Annotation and enrichment analysis only achieved 464 and 329 DEGs (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and absolute log\u003csub\u003e2\u003c/sub\u003efoldchange\u0026thinsp;\u0026gt;\u0026thinsp;2) in F_III vs F_IV group and F_IV vs F_V group, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). The expression pattern of all these 765 DEGs were different from testis, which up-regulated in stage IV (F_IV) and V (F_V) ovary, suggested the delayed development in ovary compared with testis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eInterestingly, GO analysis of these 765 DEGs with a total of 205 DEGs were annotated in membrane (GO:0016020) in CC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). \u003cstrong\u003eAdditional file 4\u003c/strong\u003e showed the detail of these 205 DEGs, most of which participated in membrane-anchored enzymatic reaction or molecular transport. 10 accurately biological function classifications and several subclassifications were accomplished with \u003cstrong\u003ecell cycle\u003c/strong\u003e, \u003cstrong\u003ecell junction\u003c/strong\u003e, \u003cstrong\u003ecell structure\u003c/strong\u003e, \u003cstrong\u003emetabolism\u003c/strong\u003e, \u003cstrong\u003eDNA binding and transcription regulated\u003c/strong\u003e, \u003cstrong\u003eimmune system\u003c/strong\u003e, \u003cstrong\u003enervous system\u003c/strong\u003e, \u003cstrong\u003emolecular transport\u003c/strong\u003e, \u003cstrong\u003eprotein modification\u003c/strong\u003e, \u003cstrong\u003eRNA binding\u003c/strong\u003e, and \u003cstrong\u003esignal transduction\u003c/strong\u003e including. Among these classifications, \u003cstrong\u003eMolecular transport\u003c/strong\u003e showed the most abundant (40 DEGs) of all, indicated the importance of matter transport process during ovary development stage of black rockfish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Validation of RNA-Seq results by qRT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10 DEGs were randomly selected for q-PCR analysis to validate the present RNA-Seq data. The results showed that the q-PCR expression pattern of the selected genes was significantly correlated with the RNA-Seq results (R\u003csup\u003e2\u003c/sup\u003e: 0.933\u0026ndash;0.9559). Totally, the RNA-Seq data were confirmed by the q-PCR results, implying the reliability and accuracy of the RNA-Seq analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThe black rockfish, as an economically crucial fish species, has been processed a lot of works, especially in reproductive and toxic physiology and molecular biology due to the unusual ovoviviparous strategy\u003csup\u003e(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/sup\u003e. While the transcriptomics profile of the black rockfish gonad development was less clear. In order to investigate underlying mechanisms, a series of sex-related genes and biological pathways should be determined for further exploration. The present study mainly focused on identification of the key genes and pathways in both ovary and testis development and germ cell renewal by histology and RNA-seq.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and terminology of gonad development of the black rockfish\u003c/h2\u003e \u003cp\u003eReproduction of most teleost is an annual and cyclic event, while gonads present different development stages for germ cells renewal. Critical phases of fish reproductive cycle were determined by a conceptual model based on specific histological and physiological markers\u003csup\u003e(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u003c/sup\u003e. Black rockfish testis go through 4 phases during development, including the developing phase when no spermatozoa present in lumen of lobules or sperm ducts (described as stage III in the present study), spawning phase when spermatogenesis active (described as stage IV), and regressing phase with few or no sperm (described as stage V) \u003csup\u003e(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e)\u003c/sup\u003e. Different from the oviparity strategy, black rockfish ovulated eggs fertilized and retained in ovary during the gestation phase\u003csup\u003e(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u003c/sup\u003e. In the present study, the description of the developmental stage of ovary as stage III, stage IV and stage V ovary indicated the terminology of developing phase, early developing subphase and actively spawning subphase, respectively\u003csup\u003e(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eKey pathways in testis development and gametogenesis of male black rockfish\u003c/h2\u003e \u003cp\u003eTestis of male black rockfish showed drastic variation from stage III to stage V which reflected in both histology results and transcriptomic profile. During the development from stage III to stage IV, one category of pathways \u003cb\u003eintercellular interaction and cytoskeleton\u003c/b\u003e showed upregulation significantly. Similar to mammals, cellular interactions among sertoli cells and germ cells of fish in the seminiferous epithelium play important structural and functional roles as well. Testis structure undergo dramatic morphofunctional changes during reproductive season to activate spermatogenic arrangement\u003csup\u003e(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003c/sup\u003e. It suggested that actin-related adhesion among sertoli cells and sertoli cells to germ cells are associated with multiple events crucial for spermatogenesis and normal fertility, especially for spermatid elongation in stage IV testis\u003csup\u003e(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e)\u003c/sup\u003e. In the transcriptomics analysis of spotted knifejaw (\u003cem\u003eOplegnathus punctatus\u003c/em\u003e), similar intercellular interaction pathway (cell adhesion molecules) was significantly enriched in testis as well\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn fish, cytoplasmic extensions of sertoli cells form a envelop of a single synchronously developing group of germ cells derived from a single spermatogonium. The form of the group of germ cells, namely spermatogenesis is a highly organized and coordinated process in which diploid spermatogonia proliferate and differentiate to mature spermatozoa \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. Both processes required a large amount of molecule, including DNA, RNA, protein, lipid and steroid, for cell amplification. In the present study, category \u003cb\u003eMolecule amplification and repairment in cell cycle\u003c/b\u003e, including cell cycle, ubiquitin mediated proteolysis, DNA replication, fanconi anemia pathway, mRNA surveillance pathway, RNA transport, p53 signaling pathway and apoptosis, showed significant up-regulated in both stage III and IV testis. Different from mammals, fish show a cystic type of spermatogenesis and go through different stages by an accompanying group of sertoli cells. The shape shifting of a cystic which is enveloped by sertoli cells is accompanied by strong proliferation of sertoli cells\u003csup\u003e(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e)\u003c/sup\u003e. In African catfish and Nile tilapia, sertoli cell proliferation occurred primarily during spermatogonial proliferation\u003csup\u003e(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e)\u003c/sup\u003e. In addition, in \u003cem\u003eLeporinus taeniatus\u003c/em\u003e spermatogenesis is also processed as which diploid spermatogonia proliferate and differentiate to haploid spermatozoa\u003csup\u003e(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e)\u003c/sup\u003e. On the other hand, fanconi anemia pathway was confirmed as an efficient DNA repairment pathway\u003csup\u003e(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e)\u003c/sup\u003e. In fanconi anemia disease causing genes mutant zebrafish, the DNA damage repairment failed and caused a series of abnormal development.\u003c/p\u003e \u003cp\u003eTestis development and spermatogenesis required a mass of metabolites, especially for androgens. In fish, transduction of the gonadotropin signal stimulates the production of 11-ketotestosterone (11-KT), a major androgen for fish \u003csup\u003e(\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e)\u003c/sup\u003e, functionally activity the androgen receptors\u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e)\u003c/sup\u003e. To complete the process, enzymes and other proteins are required for biosynthesis\u003csup\u003e(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e)\u003c/sup\u003e. In the present study, the steroid hormone biosynthesis pathway and the key genes \u003cem\u003e3b-hsd\u003c/em\u003e and \u003cem\u003ecyp11a1\u003c/em\u003e were significant up-regulated in stage IV testis, indicating the importance of steroidogenesis for spermatogenesis \u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e)\u003c/sup\u003e. A few pathways related to RNA transcription and translation transportation, namely RNA transport, mRNA surveillance pathway and ubiquitin mediated proteolysis, were also significantly enriched in stage IV testis, suggested the metabolism and synthesis under gonadotropin and steroid hormone have been crucial for maintenance of spermatogenesis\u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e)\u003c/sup\u003e. Interestingly, these pathways (progesterone-mediated oocyte maturation and oocyte meiosis) were both enriched in stage IV testis, implying the importance of the role that progestogen plays in spermatogenesis. Serum 17α, 20β-dihydroxy-4-pregnen-3-one (DHP), as fish specific progestin, exhibited higher in stage IV concomitant with testicular development in male turbot (\u003cem\u003eScophthalmus maximus\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e)\u003c/sup\u003e. Knock out of the nuclear receptor of DHP resulted a smaller testis and a lower GSI compared with normal testis in Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eKey genes in ovary development and gametogenesis of female black rockfish\u003c/h2\u003e \u003cp\u003eDue to the special reproductive strategy in female black rockfish, the transcriptomic level changes in ovary seemed not as much drastic as testis. A total of 765 DEGs were enriched significantly which classified into 10 classifications including cell cycle, cell junction, cell structure, metabolism, DNA binding and transcription regulated, immune system, nervous system, molecular transport, protein modification, RNA binding, signal transduction and unclassified genes.\u003c/p\u003e \u003cp\u003eSimilar to the testis of black rockfish, ovary development presented significantly enriched DEGs in cell cycle and junction and cytoskeleton as well. The matrix metalloproteinases (MMPs) are a family of extracellular proteinases which plays a role in the ECM remodeling associated with many physiological and pathological processes\u003csup\u003e(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e)\u003c/sup\u003e. Previous studies in mammal have shown increased MMP19 expression in preovulatory follicles\u003csup\u003e(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e)\u003c/sup\u003e, and estrogen receptor knockout lead to down-regulation of \u003cem\u003eMMP19\u003c/em\u003e and failure in release of mature oocyte\u003csup\u003e(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e)\u003c/sup\u003e. In the present study, \u003cem\u003emmp19\u003c/em\u003e was also up-regulated in stage IV ovary, which may be due to follicles mature and rupture afterward in black rockfish.\u003c/p\u003e \u003cp\u003eBlack rockfish \u003cem\u003evipr\u003c/em\u003e gene showed a significant increase in stage IV ovary implied its critical role in folliculogenesis. Both neuropeptides vasoactive intestinal polypeptide (vip) and pituitary adenylate cyclase-activating polypeptide (pacap) are considered to stimulate the ovarian functions such as steroidogenesis, cAMP accumulation in rat granulosa cells\u003csup\u003e(\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e)\u003c/sup\u003e through the PACAP/VIP receptors. VIPR has been found equal affinity with both PACAP and VIP\u003csup\u003e(\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e)\u003c/sup\u003e. In zebrafish, the \u003cem\u003evipr\u003c/em\u003e expression maintained high during follicle until full-grown stage\u003csup\u003e(\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe classification \u003cb\u003emolecular transport\u003c/b\u003e showed the most DEGs in ovary development of black rockfish with 20 genes of solute carrier (SLC) gene superfamily enriched. The SLC gene superfamily encodes series of membrane transporters\u003csup\u003e(\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e)\u003c/sup\u003e including passive transporters, cotransporters and exchangers in various cellular membranes\u003csup\u003e(\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e)\u003c/sup\u003e. The members of \u003cem\u003eslc6\u003c/em\u003e gene family (\u003cem\u003eslc6a11\u003c/em\u003e, \u003cem\u003eslc6a15\u003c/em\u003e, \u003cem\u003eslc6a6\u003c/em\u003e, \u003cem\u003eslc6a8\u003c/em\u003e) which performs Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e dependent neurotransmitter transport for γ-aminobutyric acid (GABA), creatine and taurine were differentially expressed along ovary development. The result was coincident with the transcriptomic analysis of hapuku (\u003cem\u003ePolyprion oxygeneio\u003c/em\u003es)\u003csup\u003e(\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e)\u003c/sup\u003e. However, these transport proteins were more commonly mentioned in central nervous system of vertebrates\u003csup\u003e(\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e)\u003c/sup\u003e, which imply the SLCs may also functional in ovary. Zinc is necessary for meiotic in zebrafish and mammals\u003csup\u003e(\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e)\u003c/sup\u003e, the zinc transport proteins \u003cem\u003eslc30\u003c/em\u003e gene family was found expressed in oocyte and cumulus cells during maturation in mouse (\u003cem\u003eMus musculus\u003c/em\u003e)\u003csup\u003e(\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e)\u003c/sup\u003e, which may be a proper explanation that \u003cem\u003eslc30a9\u003c/em\u003e showed up-regulated in early oocyte mature for zinc transport.\u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusions","content":" \u003cp\u003eThe present study is the first report of the transcriptomic information of the ovoviviparous black rockfish gonad development. Several important candidate pathways and genes in both testis and ovary development have been identified. Among these pathways and genes, the categories \u003cb\u003eintercellular interaction and cytoskeleton\u003c/b\u003e, \u003cb\u003emolecule amplification and repairment in cell cycle\u003c/b\u003e were revealed to be crucial in testis development and spermatogenesis along with a series of metabolite biosynthesis. Some key genes emerged in ovary development such as \u003cem\u003emmp19\u003c/em\u003e and neuropeptide receptor \u003cem\u003evipr\u003c/em\u003e in follicles mature and rupture and the membrane transporter family \u003cem\u003eslc6\u003c/em\u003e and \u003cem\u003eslc30\u003c/em\u003e in various ways. These data provided a comprehensive insight into the black rockfish gonad development for further study of reproductive physiology and molecular biology in ovoviviparity.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Animal and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTogether, 27 adult male and 27 adult female black rockfish cultured in northern Yellow Sea were obtained from October to March of the next year. Nine individuals were sampled for each development stage in all 3 development stages in both sexes. Fish were acclimatized at a density of 10 individuals per tank (diameter 1\u0026nbsp;m, height 1.5\u0026nbsp;m) under laboratory conditions for 2\u0026nbsp;days without feeding. After acclimation, individuals were anesthetized with MS222 (200\u0026nbsp;mg /L). Body weight and gonad weight were measured and GSI was calculated. Gonad were also collected immediately for both histology and RNA isolation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Histology analysis and RNA isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTestis and ovary of different development stages were fixed in Bouin\u0026rsquo;s solution, dehydrated and embedded in paraffin. Tissue sections were cut into 6\u0026nbsp;\u0026micro;m by a microtome (Leica, Wetzler, Germany) and stained with hematoxylin-eosin. All section photos were taken by Olympus bright field light microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eGonads were collected and frozen in liquid nitrogen for further total RNA isolation with TRIzol reagent (Invitrogen, USA). The quality and concentration of the total RNA were assessed by agarose gel electrophoresis and Agilent 2100 Bioanalyzer system (Agilent Technologies, USA), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Library construct and transcriptome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to mask the difference among sample repetitions, equal amount of total RNA from 3 individual ovaries or testis in same development were pooled together. 18 sequencing libraries were generated NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (NEB, USA) following manufacturer\u0026rsquo;s instructions and index codes were added to attribute sequences to each sample. Samples were sequenced on an Illumina Hiseq X ten platform and 150\u0026nbsp;bp paired-end reads were generated. Raw sequences were deposited in the Short Read Archive of the National Center for Biotechnology Information (NCBI) with accession numbers of PRJNA573572.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4. De novo\u003c/em\u003e assembly and annotation of sequencing reads\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e assembly was performed on gonad clean reads using the Trinity assembly software suite\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/sup\u003e without a reference genome. Transcripts (both contigs and singletons) were annotated by BLASTx searches\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e)\u003c/sup\u003e using NCBI non-redundant (Nr), NCBI nucleotide sequences (Nt) and Swiss-Prot databases with a cutoff \u0026ldquo;e-value\u0026rdquo; of \u0026lt;\u0026thinsp;1e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. Domain-based comparisons with Protein family (Pfam) and KOG (a eukaryote-specific version of the Clusters of eukaryotic Ortholog Groups) databases were performed by RPS-BLAST tool from locally installed NCBI BLAST\u0026thinsp;+\u0026thinsp;v2.2.28 and HMMER 3.0 program, respectively. Annotated transcripts were analyzed to GO classification with the aid of Blast2Go program\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e)\u003c/sup\u003e. These gene terms were then enriched on the three GO categories (Biological Process, Cellular Component and Molecular Function at level 2) using the GOseq R package\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/sup\u003e. KEGG, which is a database of biological systems, maps were retrieved by online KEGG Automatic Annotation Server for the overview of metabolic pathway analysis\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Differential gene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reads of each library were mapped to the \u003cem\u003ede novo\u003c/em\u003e assembled transcripts with the bowtie 2 program for mismatch check\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/sup\u003e. Count numbers of mapped reads and FPKM (expected number of Fragment Per Kilobase of transcript sequence per Millions base pairs sequenced) were achieved and normalized by RSEM V1.2.15\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e)\u003c/sup\u003e. Differential expression statistical analysis of different development stage of gonad was conducted by the DEGSeq R package\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e)\u003c/sup\u003e with a cutoff \u0026ldquo;q-value\u0026rdquo; of 0.01 and |log\u003csub\u003e2\u003c/sub\u003e(fold change)|\u0026gt;2. Transcripts with absolute fold change values over 2.0 were marked as significantly differential expressed genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Experimental validation by quantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression analysis of 10 selected DEGs were performed by quantitative real-time PCR (qPCR) with specific primers validate our Illumina sequencing data. Primers were listed in \u003cstrong\u003eAdditional file 5\u003c/strong\u003e. Samples were generated from F_III, F_IV, F_V ovary and M_III, M_IV, M_V testis in the preceding experiment. After RNA extraction and reverse transcription, all the cDNA products were diluted to 500\u0026nbsp;ng/\u0026micro;L. The 20\u0026micro;L qPCR reaction mixture consisted of 2\u0026micro;L cDNA template, 0.4\u0026micro;L of both primers, 10\u0026micro;L of KAPA SYBR\u0026reg;FAST qPCR Master Mix (2X), 0.4\u0026micro;L of ROX and 6.8\u0026micro;L of RNAase-free water. PCR amplification was performed as that incubated in a 96-well optical plate at 95\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s, followed by 40 cycles of 95\u0026nbsp;\u0026deg;C for 5\u0026nbsp;s, 58\u0026nbsp;\u0026deg;C for 30\u0026nbsp;s, and a final extension at 72\u0026nbsp;\u0026deg;C for 2\u0026nbsp;min. qPCR was performed using the StepOne Plus Real-Time PCR system (Applied Biosystems) and 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method was used to analysis the expression level of genes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDEGs: Different expression genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; NGS: The next-generation sequencing technology; GSI: Gonadosomatic index; GO: Gene ontology; NR: Nonredundant; BP: Biological Process; CC: Cell Component; MF: Molecular Function; ECM: Extracellular matrix; 11-KT: 11-ketotestosterone; DHP: 17\u0026alpha;, 20\u0026beta;-dihydroxy-4-pregnen-3-one; MMPs: Matrix metalloproteinases; vip: vasoactive intestinal polypeptide; pacap: pituitary adenylate cyclase-activating polypeptide; SLC: Solute carrier; GABA: \u0026gamma;-aminobutyric acid; NCBI: the National Center for Biotechnology Information; Nr: non-redundant; Nt: nucleotide sequences; Pfam: Protein family; KOG: a eukaryote-specific version of the Clusters of eukaryotic Ortholog Groups; qPCR: quantitative real-time PCR;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involved in dealing of fish in this study were approved by Animal Research and Ethics Committees of Ocean University of China (Permit Number: 20141201) prior to the initiate of the study. The studies did not involve endangered or protected species. And all experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during this study were deposited in the Short Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra) of the National Center for Biotechnology Information (NCBI) with accession numbers of PRJNA573572.\u003c/p\u003e\n\u003cp\u003eAnd other data supporting the conclusion of this article is included within the article, and can be found in the additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by The National Natural Science Funds (41676126). Our funding agencies did not play a role in the study design, data collection, analysis, interpretation of the data, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWHS, LJF and QX designed the study; LJS performed the transcriptome and qRT-PCR experiment; LLK, WXJ, YYJ and LJS performed in samples collection; LJS wrote the manuscript and QX provided manuscript editing and feedback; All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLabb\u0026eacute; C, Robles V, Herraez MP. 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Biol Reprod. 2019; 100: 468-478.\u003c/li\u003e\n\u003cli\u003eKim AM, Vogt S, O'halloran TV, Woodruff TK. Zinc availability regulates exit from meiosis in maturing mammalian oocytes. Nat Chem Biol. 2010; 6: 674.\u003c/li\u003e\n\u003cli\u003eLisle RS, Anthony K, Randall M, Diaz FJ. Oocyte-cumulus cell interactions regulate free intracellular zinc in mouse oocytes. 2013; 145: 381-390.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016; 44: D457-D462.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Black rockfish, Transcriptomic, Gonad development, RNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-70073/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-70073/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Black rockfish (\u003cem\u003eSebastes schlegelii\u003c/em\u003e) has an ovoviviparous reproductive pattern and long-term sperm storage, which resulting in asynchronous gonadal development between the sexes. While the comprehensive understanding of gonad development of black rockfish has not been well studied. Here, we study the gonad development and germ cell renewal by histology and RNA-seq.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, RNA-seq was performed on both testis and ovary to characterize key pathways and genes during development and gamete maturation in black rockfish. Different expression genes (DEGs) were identified and annotated in 4 comparisons (F_III vs F_IV, F_IV vs F_V, M_III vs M_IV and M_IV vs M_V). Based on enriched analysis of DEGs in testis, 11 and 14 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mapped in M_III vs M_IV group and M_IV vs M_V group, respectively. And DEGs in ovary development periods were also classified into 10 biological function group. The results of the q-PCR expression pattern of the selected genes was significantly correlated with the RNA-Seq results, implying the reliability and accuracy of the RNA-Seq analysis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The categories intercellular interaction and cytoskeleton, molecule amplification and repairment in cell cycle were revealed to be crucial in testis development and spermatogenesis along with a series of metabolite biosynthesis. Our results provided a comprehensive insight into the black rockfish gonad development for further study of reproductive physiology and molecular biology in ovoviviparity.\u003c/p\u003e","manuscriptTitle":"Comparative Transcriptomic Analysis of Gonad Development and Renewal of Ovoviviparous Black Rockfish (Sebastes Schlegelii)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-08 15:49:35","doi":"10.21203/rs.3.rs-70073/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-04-05T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2021-04-05T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-17T00:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-10-25T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-03T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-22T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-09-01T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-08-31T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-08-31T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-08-30T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e73d81b-4861-4c9b-b527-bda243bbdd1a","owner":[],"postedDate":"September 8th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":455386,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2020-09-08T15:49:35+00:00","versionOfRecord":[],"versionCreatedAt":"2020-09-08 15:49:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-70073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-70073","identity":"rs-70073","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}