De novo transcriptome assembly of four organs of Collichthys lucidus and identification of genes involved in sex determination and reproduction

DOI: https://doi.org/10.21203/rs.2.9879/v1

Abstract

Background Spinyhead croaker (Collichthys lucidus) is an important kind of fish for commercial fishery because of its delicious meat and abundant resource. However, the resource has been declining with the overfishing and environmental disruption. Unfortunately, the mechanism of sex determination and reproduction in Collichthys lucidus is still confusing, which has largely hindered molecular regulation and reproduction studies. Results Based on transcripts sequencing in brain, liver, ovary and testis, a de novo transcriptome assembly of Collichthys lucidus was performed. Totally, 131,168 non-redundant unigenes with an average length of 644 bp and an N50 value of 1033 bp, among which 2,143, 304, 333 and 556 were found specifically expressing in brain, liver, ovary and testis, respectively. Importantly, 1288 genes were differently expressed between ovary and testis, including 442 up- and 846 down-regulated in ovary. The differently expressed genes were involved in function of sexual reproduction, sex differentiation, development of primary male sexual characteristics, female gamete generation, and male sex differentiation. HYAL and SYCP3, two genes which were reported to play dominant roles in reproduction, may regulated the key of fertilization and meiosis in Collichthys lucidus. Meanwhile, 35,476 simple sequence repeats (SSRs) were identified, which would contribute to further genetic and mechanism researches. Conclusions The studies of genetic information, molecular regulation and reproduction of Collichthys lucidus would be expand due to the sequencing and de novo assembling on the transcripts of major organs. In addition, the identification of candidate genes involved in reproduction will be useful for protecting the resource of Collichthys lucidus.

Background

Collichthys lucidus, which belongs to perciformes, is a kind of economically important fish with small somatotype usually inhabiting in the benthic zone of coastal waters[ 1]. It is mainly captured in northwestern Pacific that ranges from Kyushu of Japan to the south China sea and makes a major contribution to demersal fish catches, supporting an important commercial fishery[ 2]. For its good quality of meat, this species is appreciated by customers. However, because of the overfishing and the damage of environment, the resource of Collichthys lucidus has been declining in recent years[ 3]. Protecting Collichthys lucidus resource is quite necessary.

Hypothalamic–pituitary–gonadal axis (HPG) play a dominant role in the reproductive manipulation[ 4, 5]. As previous article reported, a variety of natural or synthetic compounds, such as monocrotophos pesticide (MCP), semicarbazide (SMC) and thyroid hormones (THs) [ 6-8], could lead to significant influence on fish reproduction through HPG. With the environment pollution pricking up, zebrafish, Japanese flounder and goldfish were all injured by contaminants[ 7-9]. However, the regulation of reproduction through this axis in Collichthys lucidus is still unknown.

With the rapid development of next-generation high-throughput sequencing technology and ubiquitous application of RNA-seq, gene profiles of many species such as mice, human, and rice[ 10, 11] were constructed. Moreover, as the paired-read tag sequencing strategy has been widely used, the efficiency of sequencing has been much higher. The transcriptomic information can be used in a really wide field of biological researches, including SSR mining and SNP discovery[ 12], as well as provides an in-depth understanding of biological processes.

In this study, 4 organizations of Collichthys lucidus were sequenced and de novo the transcriptome using Illumina Hiseq 2500 platform. Differentially expressed genes were detected and screened using RSEM and Deseq. The analysis of transcriptome sequences expanded the comprehension of functional genomic studies. Additionally, the differentially enrichment in GO terms contributed to understand the mechanism of the reproductive development and be helpful for the subsequent research on sex universality of Collichthys luciduss.

Methods

Ethical statement

The collection of samples and the handling of animals in this article were approved by the Chinese Academy of Fishery Sciences Welfare Committee, Shanghai, China. All procedures conducted with the fishes were performed in accordance with relevant guidelines and regulations. All efforts were made to minimize animal suffering.

Sample collection and RNA extraction

Collichthys lucidus were obtained from Ningde sea area, fujian province, and once the capture from the sea, fishes were euthanized by intraperitoneal injection with excessive pentobarbital (150mg/kg). Brain, liver, ovary and testis tissues from five healthy fishes were collected randomly and saved in RNAlater (TransGen Biotech, Beijing, China). Moreover, 0.3-0.3 grams of each tissue was taken from each individual and mix into a mix pool for each organ. Animal Total RNA Isolation Kit (Foregene, Chengdu, China) was used to extract the total RNA of each sample following the introductions by manufacture and samples of same organization were pooled.

cDNA library preparation and sequencing

RNA quality and quantity were evaluated by Agilent 2100 Bioanalyzer (Agilent, Shanghai, China). Qualified RNA was processed using Genomic Sample Preparation kit (Illumina, Beijing, China) for library construction and sequenced on Hiseq TM 2500 (Illumina) with a paired-end 150 bp read run.

De novo assembly

To remain high-quality clean reads, reads which had adaptors, poly-N or Q-values <20 were removed. De novo assembly with clean reads was performed using Trinity (version r20140717, K-mer 25 bp) software. Transcriptome assembly was accomplished using a part of Trinity called Butterfly and the longest transcript of each gene was designed as unigenes.

Functional annotation

All unigenes were distributed into public databases, containing NCBI nonredundant protein sequences (NR), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genome (KEGG), evolutionary genealogy of genes: Nonsupervised Orthologous Groups (eggNOG) and Swiss-Prot, for functional annotation to find the relationship between Collichthys lucidus and other fishes and detect the functions of unigenes.

Simple sequence repeat (SSR) identification

MISA(Version 1.0, http://pgrc.ipkgatersleben.de/misa/misa.html)was used to identify SSR motifs. For di-, tri-, tetra-, penta- and hexa-nucleotides, the lowest number of base repeats was 10, 6, 5, 5, 5 and 5, respectively. In addition, if the distance between two SSRs was less than 100 bp, they would be merged into a composite SSR.

Quantification of gene expression levels

Clean reads were mapped to assembled transcriptome using RSEM to obtain the read count for each gene and the expression level of each gene was estimated using fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM). Differentially expressed genes were identified with a standard of fold change and p-vale (| log2(fold change) | > 1 & p-value< 0.05)

Quantitative real-time PCR (qRT-PCR)

Gene-specific primers were designed by Primer Premier5.0. First strand cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit (ThermoFisher). FastStart Universal SYBR Green Master mix was used to amplify cDNA on QuantStudio 6 Flex Real-Time PCR System (ThermoFisher). The primers were displayed in (Additional file 1). The 2-ΔΔCT method was used to determine the relative gene expression with internal control of B2M.

Investigation of SSR polymorphism

A total of 10 primer pairs (Additional file 1) were synthesized and 210 Collichthys luciduss from 7 different locations were selected for polymorphism investigation with the SSRs. The 7 locations including Lianyungang (LYG), Zhoushan (ZS), Chongming (CM), Wenzhou (WZ), Xiamen (XM), Ningde (ND), Dafeng (DF). Total DNA was extracted from dorsal muscles by the CTAB method. The total volume of PCR reactions were co 20 μL containing 1 μL template DNA,1 μL each primer (10 μM), 1 μL 2.5 mM dNTPs, 1 μL EasyTaq DNA polymerase (Beijing Trans Gen Biotech Co., Ltd. China), 2 μL 10×EasyTaq buffer and 13 μL ddH2O. Amplification program of PCR was as follows: denaturation for 5 min at 94℃, 35 cycles of 94℃ for 30 s, 59℃ for 30 s, and 72℃ for 1min, and a final step at 72℃ for 10 min. Alleles were detected on a 12% polyacrylamide gel. UPMGA evolution tree was constructed using MEGA 5.1 based on Nei genetic distance.

Statistical analysis

Statistical analysis was performed bia the SPSS 13.0 statistical software (Chicago, IL, USA). The data were expressed as mean ± standard deviation (SD). Comparison between two difference independent groups were performed by two-sided Student’s test. A p-value < 0.05 was considered to be a statistically significant difference.

Results

Sequencing and de novo assembly

Totally, 60,954,060, 57,468,802, 61,486,012, 57,611,124 paired-end reads were generated by samples from brain, liver, ovary and testis of Collichthys luciduss, respectively. After removing low quantity reads and trimming adaptor reads, 60,322,004 (98.96%), 57,044,284 (99.26%), 60,867,978 (98.99%) and 57,087,688 (99.09%) clean reads were obtained (8.0 G). De novo assembly of clean reads generated 189,181 transcripts with an average length of 818 bp and a N50 length of 1463 bp. The longest transcript of each gene was selected and yielded 131,168 unigenes. The average length of unigenes was 644 bp and the N50 length was 1033 bp (Table 1). Most transcripts and unigenes were 200 to 299 bp in length (Fig 1A, B). These unigenes were retained for subsequent functional annotation.

Functional annotation

Unigenes were annotated using NR, GO, KEGG and Swissport databases, and a 10-5 e-value cut-off value was used. 51,590 (39.33%), 27,708 (21.12%), 12,883 (9.82%) and 44,051 (33.58%) significant hits were produced respectively. Among these unigenes, 53,200 (40.56%) were annotated at least in one database and 8851 (6.75%) unigenes were annotated in all five databases (Table 1). The similarity distribution of the top hits showed that 61% of the mapped sequences had similarities higher than 80%, while 37%of the hits had similarities ranging from 40% to 80%. (Fig 2A). The E-value distribution had a comparable pattern with 51% of the mapped sequences with high homologies (<1e-60), whereas 48% of the homological sequences ranged between 1e-5 to 1e-60 (Fig 2B). The species distribution of NR BLAST matches showed that the top two species was Maylandia zebra (26%) and Oreochromis niloticus (21%) (Fig 2C).

GO and KEGG analysis of Collichthys lucidus transcriptomes

The roles of unigenes were investigated by GO and eggNOG analysis. In GO database, 27,708 unigenes were clustered into 67 terms of three different domains (biological process (BP), cellular component (CC) and molecular function (MF). The number of unigenes in each term are exhibited in Fig 3A. The top 3 terms ranked by number of unigenes in BP were cellular process (17,477), single-organismal process (16,219) and biological regulation (11,558). In CC, unigenes were mainly converged on cell (16,229), cell part (16,151) and membrane (11,815). In MF, the most prominent terms were binding (16,123), nucleic acid binding catalytic activity (10,504) and signal transducer activity (1,974). On the other hand, eggNOG analysis annotated 48,042 (36.63%) unigenes, and the result revealed that unigenes mainly contributed to signal transduction mechanisms. The top 3 terms were signal transduction mechanisms, general function prediction only and function unknow (Fig 3B).

To further explore the biological functions of unigenes, KEGG database was mapped and 12,883 unigenes were distributed into 32 pathways, among which signal transduction (1,877), endocrine system (849), immune system (820), signaling molecules and interaction (814) and transport and catabolism (668) were the largest five groups (Fig 4).

Differentially expressed unigenes

The expression pattern of each unigene in brain, liver, ovary and testis was assessed by expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM). The result of examination of unigenes in each organ revealed that brain expressed the most unigenes (74.83%), while liver expressed the least (46.18%) (Fig 5 A). Additionally, a gene was identified as organ-specific when the FPKM was at least 10 in one organ whereas less than 1 in other organs. Under this standard, 2,143, 304, 333, 556 unigenes were found specifically expressing in brain, liver, ovary and testis, respectively. The most genes were specifically expressed in brain, followed by ovary and testis, according with our hypothesis that HPG play a dominant role in Collichthys lucidus.

The expression profiles were compared between each two organs. Between brain and liver, 992 genes were upregulated in brain and 264 genes were up regulated in liver. Between brain and ovary, 749 genes were upregulated in brain and 317 genes were upregulated in ovary. Between brain and testis, 685 genes were upregulated in brain and 388 genes were upregulated in testis. Between liver and ovary, 211 genes were upregulated in liver and 114 genes were upregulated in ovary. Between liver and testis, 138 genes were upregulated in liver and 315 genes were upregulated in testis. Between ovary and testis, 442 genes were upregulated in ovary and 846 genes were upregulated in testis (Table 2). The differential genes of group ovary vs testis are shown in Fig 5B and the differential genes of other groups are shown in Additional file 2 (Fig S1).

Potential roles of differentially expressed unigenes

GO enrichment analysis were established for differentially expressed unigenes between groups and we focused on terms related to reproduction, sex determination and sexual development. Differentially expressed unigenes between ovary and testis were primarily involved in sexual reproduction (GO: 0019953), development of primary sexual characteristics (GO: 0045137), sex differentiation (GO: 0007548), male gonad development (GO:0008584), female gamete generation (GO: 0007292), male gamete generation (GO: 0048232), development of primary male sexual characteristics (GO: 0046546), male sex differentiation (GO: 0046661) (Additional file 3). Interestingly, these GO terms were also enriched in other organs, implying their high correlation with reproduction and sex in Collichthys lucidus. For instance, sexual reproduction (GO: 0019953), multicellular organism reproduction (GO: 0032504), reproduction (GO: 0000003) were enriched in differentially expressed unigenes between brain and liver. Developmental process involved in reproduction (GO: 0003006), cellular process involved in reproduction in multicellular organism (BP, GO: 0022412) and sexual reproduction (GO: 0019953) were enriched in differentially expressed unigenes between brain and ovary. Developmental process involved in reproduction (GO:0003006), sexual reproduction (GO: 0019953) and cellular process involved in reproduction in multicellular organism (GO: 0022412) were enriched in differentially expressed unigenes between brain and testis. Regulation of growth (GO: 0040008), Sexual reproduction (GO: 0019953) and multicellular organism (GO: 0022412) were enriched in differentially expressed unigenes between liver and ovary. Multicellular organism reproduction (GO: 0032504), sexual reproduction (GO: 0019953) and cellular process involved in reproduction in multicellular organism (GO: 0022412) were enriched in differentially expressed unigenes between liver and testis.

Furthermore, through KEGG pathway enrichment analysis, several pathways were enriched in each group. To investigate pathways connected to reproduction and gonad development, we particularly focused on differentially expressed unigenes between ovary and testis. 165 pathways, including PPAR signaling pathway, ovarian steroidogenesis, oocyte meiosis, steroid hormone biosynthesis, apoptosis – multiple species and proximal tubule bicarbonate reclamation were significantly enriched in differentially expressed unigenes between ovary and testis, which might participate in the regulation of reproduction. The profile of group ovary vs testis is exhibited in Fig 5C and others are shown in Additional file 4 (Fig S2).

Identification and characterization of genes involved in reproduction and sexual development

Based on the de novo assembly and annotation, 652 and 650 candidate unigenes were identified for reproduction (GO: 0000003) and reproductive process (GO: 0022414) respectively. Furthermore, 8 unigenes were enriched in both terms of reproduction, sex differentiation and development, implying that they dominated the regulation of reproduction and sex development. Among the 8 unigenes, HYAL (hyaluronidase), KLHL10 (Kelch-like protein 10), ROPN1L (rhophilin associated tail protein 1 like), ODF3L2 (outer dense fiber of sperm tails 3 like 2), SYCP3 (synaptonemal complex protein 3) and SRPK3-like (serine/threonine-protein kinase 3-like ) were upregulated in testis, whereas, BMP15 (bone morphogenetic protein 15) and RGS14 (regulator of G protein signaling 14) were upregulated in ovary (Table 3).

Identification of SSRs

Using MISA 1.0 (http://pgrc.ipkgatersleben.de/misa/misa.html), 35,476 SSRs were predicted and 2,455 compound formations were composed. Of the 35,476 detected SSRs, the most affluent repeat motif was mono- and di-nucleotide repeats, constituting 41.18% (14,608) and 38.23% (13,563), respectively, followed by tri- (18.74%, 6,648), tetra- (1.73%, 614), penta- (0.09%, 31) and hexa-nucleotide (0.03%, 12). In general, the most abundant repeat motif was poly-T (18.66%, 6617). Among di-nucleotide repeat motifs, (GT/TG) was the dominant type with frequency of (12.24%, 4342) and (CG/GC) was the least type which only occupied (0.09%, 31). These SSRs were valuable for further genetic and mechanism researches (Table 4).

SSR marker polymorphism

In this study, 10 primer pairs were randomly designed to validate the amplification and polymorphism in Collichthys lucidus collected from 7 different regions. Seven of the primer pairs successfully amplified fragments. Among the successful primer pairs, one pair exhibited polymorphisms among the seven colonial Collichthys lucidus (Figure 6 A and B). The polymorphic SSR marker was then used to perform genetic correlation analysis among the Collichthys lucidus groups. The UPGMA clustering produced a dendrogram (Figure 6 C) that separated theCollichthys lucidus into two main groups, from which ND completely separate from the others. Moreover, LYG and ZS formed a sub-cluster, while CM and DF formed a sub-cluster.

Validation of gene expression using qRT-PCR

To further verify the expression of genes identified by transcriptome sequencing experimentally, 8 genes enriched in reproduction terms were validated by qRT-PCR analysis. In accordant with the RNA-seq, qRT-PCR showed ROPN1L, KLHL10, ODF3L2, SYCP3 and SRPK3-like were specific expressed in testis whereas BMP15 and RGS14 were specific expressed in ovary (Fig 7).

Discussion

De novo has been constructed in various of fishes according to published articles. In salmonid fish species including Atlantic salmon, Brown trout, Arctic charr and European whitefish, 191,977,874, 190,239,319, 180,232,708 and 209,578,198 paired-end reads were obtained and assembling 235,515, 242,899, 200,760 and 209,920 transcripts, respectively[ 13]. However, little is known about the transcriptome of Collichthys lucidus, especially the genes involved in the reproduction and sex differentiation remain unclear. In this study, we gathered 60,322,004 (98.96%), 57,044,284 (99.26%), 60,867,978 (98.99%) and 57,087,688 (99.09%) paired-end clean reads from samples of brain, liver, ovary and testis of Collichthys lucidus., assembled 189,181 transcripts and identified 131,168 unigenes. Moreover, HPG axis is the sex endocrine system of fish and plays an important role in fish reproduction via a cascade of hormones [ 14]. Transcriptome analysis of HPG axis from Lateolabrax maculatus showed 748, 349 and 319 unigenes were uniquely expressed in the brain, ovary and testis tissues, respectively, while 26 623 unigenes were commonly expressed in all HPG tissues [ 15]. Previous study has defined the overall gene expression of HPG axis in the transgenic fish with blocked gonad development, which displayed 9, 28, and 212 differentially expressed genes in hypothalamus, pituitary, and ovary, respectively [ 16]. In the present study, the vast majority of the organ-specific expression is found in brains, containing 2,143 unigenes, whereas, only 304, 333 and 556 in liver, ovary and testis of Collichthys lucidus, respectively. Moreover, 1288 genes were differently expressed between ovary and testis. These data may provide a basis for understanding the molecular regulation of gonadal development, sex determination, and reproduction.

In view of that no obvious secondary sexual characteristic has been found in Collichthys. lucidus and the mechanism of gender development is still not clear, profile of ovary and testis were compared to do help in subsequent researches. We noticed that hyaluronidase was an important component of fertilization in mammals for acrosomal reaction [ 17], and, HYAL, the gene encoding hyaluronidase, was exactly upregulated in testis. Furthermore, this gene was involved in glycosaminoglycan degradation pathway (ko00531), indicating that hyaluronidase may also contribute to fertilization in Collichthys lucidus through giving a hand in dissolving zona pellicuda though there are great differences in fertilization between mammal and fish. On the other hand, SYCP3, the gene encoding synaptonemal complex protein 3, which closely connect to synapsis in meiosis[ 18] was also upregulated in testis. According to published articles, this gene was examined to reflect the level of meiosis and spermatogenesis[ 19-21] in zebrafish, tilapia and catfish. Considering the Collichthys lucidus resource decline, this gene can be a biomarker in researches about the influence of environmental damage on Collichthys lucidus reproduction. Additionally, SYPC3 was involved in homologous recombination pathway (ko03440), meaning that it might a potential target in genetic engineering for reproduction and breeding of Collichthys lucidus.

Various genes that master sex determinants have been identified in vertebrates, most of which are known to perform basic functions during sexual development. However, the mechanism of sex determination in teleost fish is largely different from that in mammals and birds. Since their sex chromosomes are less differentiated and often recombine along the length of X and Y chromosomes. In fishes without Y sex chromosomes, gonad development of female is triggered via Wnt4, and female differentiation is induced by Foxl2, alpha, Rspo1 and Ctnnb1. While in fishes with Y chromosomes, the gene that determines sex is located on the Y chromosome, such as dmy, gdf6y, sdy, amhy, gsdfy, sox3y, and amhr2, are over-expressed during sex determination[ 22, 23]. In the presence of Foxl2, SdY is transported to the nucleus, and interacts synergistically with Nr5a1 to block the activation of the cyp19a1a promoter in rainbow trout [ 24]. Therefore, SdY blocked the pathway of female differentiation, thus enabling testicular differentiation. Recently, a novel sex determination mechanism was revealed in teleost fish by comparing RNA-Seq between females and males of the channel catfish [ 25], which exclusive male sex-specifically expressed BCAR1 gene during the early sex differentiation. Moreover, knockout of BCAR1 significantly converted genetic male (XY) into phenotypic female. At present, little is known about gender determination in perciformes, especially in the genus Collichthys. Vasa RNA is only detected in germ cells from the gonads, and its expression reflects crucial stages of oogenesis in a key stage of oocytogenesis in Collichthys lucidus [ 26]. The present study displayed that a class of differently expressed genes between ovary and testis were involved in functions of development of primary sexual characteristics, sex differentiation, male gonad development, female gamete generation, male gamete generation, development of primary male sexual characteristics, and male sex differentiation, indicating theses differently expressed genes may contribute to sex-determination of Collichthys lucidus.

Conclusion

The studies of genetic information, molecular regulation and reproduction of Collichthys lucidus would be expand due to the sequencing and de novo assembling on the transcripts of major organs. The identification of candidate genes involved in reproduction will be useful for protecting the resource of Collichthys lucidus. In addition, HYAL and SYCP3 may play dominant roles in the regulation of reproduction and are valuable in further research.

Abbreviations

SSRs: simple sequence repeats HPG: Hypothalamic–pituitary–gonadal axis

MCP: monocrotophos pesticide SMC: semicarbazide

THs: thyroid hormones NR: NCBI nonredundant protein sequences

GO: Gene Ontology KEGG: Kyoto Encyclopedia of Genes and Genome

eggNOG: evolutionary genealogy of genes: Nonsupervised Orthologous Groups

FPKM: fragments per kilobase of transcript sequence per millions base pairs sequenced qRT-PCR: Quantitative real-time PCR

LYG: Lianyungang ZS: Zhoushan CM: Chongming WZ: Wenzhou

XM: Xiamen ND: Ningde DF: Dafeng

BP: biological process CC: cellular component MF: molecular function

Declarations

Ethics approval and consent to participate

All procedures conducted with the fishes were performed following the relevant guidelines and regulations. This study was approved by the Ethics Committee of Chinese Academy of Sciences. No associated permit number was required since commercial fishes sampling was approved.

Consent for publication

All the authors listed have read and approved the manuscript for publication.

Availability of data and material

All data can be obtained by email to the corresponding author.

Competing interests

The authors have declared that no competing interests exist.

Funding

This work was supported by the Basic Research Fund for State-Level Nonprofit Research Institutes of ESCFRI, CAFS(#Dong2018Z01). This funding body has no role in the design of the study, collection, analysis, interpretation of data or in writing the manuscript.

Authors' contributions

Conceptualization and funding: JF G; Data curation: W S, YB Z, and XJ Z; Investigation: W S, YB Z, and XJ Z; Preparation of original draft: W S. All authors have read and approved the manuscript.

Acknowledgments

We show appreciation to Shanghai YingBiotech Company for the dynamic help provided during the analysis of the sequencing data.

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Tables

Table 1 Summary of the assembly and analysis of Collichthys lucidus transcriptome

 

Assembly

Number of sequences

Total number of unigenes

131,168

Average unigene length (bp)

644

N50 (bp)

1033

Annotation

Number of matched unigenes (percentages)

Transcript BLASTx against NR

51,590 (39.33%)

Transcript BLASTx against GO

27,708 (21.12%)

Transcript BLASTx against KEGG

12,883 (9.82%)

Transcript BLASTx against Swissport

44,051 (33.58%)

All annotated transcripts

53,200 (40.56%)

Transcripts identified in all four databases

8851 (6.75%)

 

Table 2 unigenes differentially expressed in each two organs

 

Case

control

Up-regulateed unigenes

Down-regulated unigenes

Total DE unigenes

B

L

992

264

1256

B

O

749

317

1066

B

T

685

388

1073

L

O

211

114

325

L

T

138

315

453

O

T

442

846

1288

 

Table 3 different genes in GO terms related to reproduction

 

id

O

T

foldChange(T/O)

log2FoldChange

pval

c35502_g1

(HYAL)

0

1950

Lnf

Inf

0.003

c55252_g1

KLHL10

2

1765

882.5

9.79

0.008

c55445_g1

BMP15

9245

20

0.002

-8.85

0.015

c28789_g1

ROPN1L

15

4292

286.13

8.16

0.019

c47852_g1

ODF3L2

0

217

Inf

Inf

0.023

c65782_g1

SYCP3

53

20287

382.77

8.58

0.024

c66108_g1

RGS14

19878

48

0.003

-8.18

0.026

c54644_g1

SRPK3-like

0

157

inf

Inf

0.028

 

 

 

 

 

 

 

Tbale 4 SSRs identified in Collichthys. Lucidus

item

number

Total number of SSRs

34,476

Number of mono-nucleotide repeats

14,608

Number of di-nucleotide repeats

13,563

Number of tri-nucleotide repeats

6,648

Number of tetra-nucleotide repeats

614

Number of penta-nucleotide repeats

31

Number of hexa-nucleotide repeats

12

 

 

Additional File Legend

Additional file 1: Information of primers used in this study.

Additional file 2: Fig S1. Volcano plot shows differential genes between Brain (B), liver (L), ovary (O) and testis (T) tissues

Additional file 3: GO terms of differently expressed genes that were related to reproduction.

Additional file 4: Fig S2. KEGG pathway enrichment analysis of differently expressed genes between Brain (B), liver (L), ovary (O) and testis (T) tissues.