1. Introduction
Sex determination has received significant research attention in various animals, and numerous genes involved in sex determination have been found in many model species (human, mice, goat, chicken, zebra fish, killifish and cichlid), including sox9, foxl2, wnt4 and dmrt1 [1,2,3]. Facilitated by high conservation, sex-related genes in other species have been identified through homology screening [4,5]. However, the sex-determination mechanisms in fish (especially teleost fishes) are not as well conserved as those in mammals or birds [6,7]. Fishes are extremely diverse in the sex chromosome systems and exhibit a variety of sex-determining systems among vertebrates, resulting in many reproductive strategies [8,9]. Therefore, it is important to screen differentially expressed genes (DEGs) in different sexes and enhance our understanding of sexual differentiation in fishes. Furthermore, growth is often related to gender. Sexual growth dimorphism could be observed in many teleost fish, and these species often show different growth rates between the genders [10,11]. Fish growth is a complex polygenic trait. It is regulated by numerous factors, including the nutrients, energy metabolism, environment and reproductive activity [12]. Thus, elucidating the sex differentiation in fish could help us understand the behavioral, cellular and phenotypic differences between sexes in vertebrates [13].
The lizardfish, Saurida elongata (Temminck & Schlegel, 1846), is a commercially important teleost species that distributes in subtropical and tropical seas of the northwest Pacific, ranging from the coastal waters of Japan to the northern South China Sea (SCS) [14,15]. Lizardfish is primarily used to produce various fish gel products (fish cakes or surimi) due to its high gel-forming ability and meat yield [16]. In addition, it has been reported that lizardfish can be the basic raw material of biologically active peptide production for the treatment of hypertension, heart failure and other cardiovascular disease [17,18]. The lizardfish is abundant, and it has been reported that the production of lizard fish is estimated at more than 120,000 tons in Guangxi of China [17]. As an economic fishery species, there are many studies on the stock resources [19], biology [15], food chemistry [18] and population genetics [20] of S. elongata. However, the biogenetical and molecular studies on S. elongata are insufficient due to limited gene sequence information, as only a few sequences were encountered during searches of public domain nucleotide and protein databases. Moreover, genomic studies on lizardfish are lacking, as these species are difficult to rear. There were only nine records in the SRA (Sequence Read Archive) database of NCBI.
Recently, with rapid advances in next-generation sequencing (NGS) technologies, RNA sequencing has emerged as a useful tool for transcriptome analysis enrichment [9,21]. This technique is ideal for identifying candidate genes and pathways underlying the traits of species whose genome is not yet available [22,23]. Recently, numerous studies have successfully used this approach for genes, single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) discovery in numerous teleost fishes, such as turbot (Scophthalmus maximus) [24], silver sillago (Sillago sihama) [25] and crimson seabream (Parargyrops edita) [26], among others. Despite the economic importance of lizardfish (Synodontidae), no published genome or transcriptome sequence is currently available for these species. In the present study, the first transcriptome of different tissues (muscle, gonad, liver and heart) in male and female S. elongata was reported. We focused on genes related to sex differentiation of S. elongata. In addition, we also screened numerous simple sequence repeats (SSRs) loci in the transcriptome of S. elongata and developed the SSRs markers. The present study provided valuable genomic information of lizardfish and will facilitate further molecular biology research in teleost.
2. Materials and Methods
2.1. Sample Collection
The three female and three male lizardfish used in the present study were collected from the Beibu Gulf of China (N 21.2793°, E 108.9580°). After acclimating in the tank, the lizardfishes were killed immediately after anesthesia. We collected liver, heart, gonads and muscle tissues from each sample.
2.2. Transcriptome Sequencing
Total RNA was extracted from each tissue using TRIzol reagent. Then, we used Agilent 2100 Bioanalyzer to assess integrity of the RNA. Only the RNA sample with the integrity number (RIN) ≥ 7 was used to subsequent analysis [27]. The RNA of tissues of every sample was pooled in equal amounts. Then, cDNA libraries were constructed using 3 μg of RNA from each sample via a conventional protocol. The prepared cDNA libraries were sequenced on a BGISEQ-500 platform (BGI, Shenzhen, China).
2.3. De Novo Assembly and Functional Annotation
In the present study, we used SOAPnuke v 2.10 to control the raw read quality (
2.4. Candidate Sex-Related Genes Analysis
The assembled transcriptome was selected as a reference because of the lack of genome information of S. elongata. All clean reads were aligned to the references with Bowtie 2 [31]. Then, RSEM version v1.2.12 was used to normalize the expression levels of the unigenes [32]. R package DEG-seq was used to identify differentially expressed genes (DEGs) [33]. The thresholds of DEGs were defined as |log2 fold change| ≥ 2 and a p value ≤ 0.001. Enrichment analyses (GO and KEGG) of the DEGs were performed based on the hypergeometric distribution test. A significance test was applied, and a Q-value value ≤ 0.05 indicated significantly enriched terms.
2.5. Potential Simple Sequence Repeat (SSR) Marker Detection
In the present study, we used the MIcroSAtellite identification tool (MISA, version 2.1,
2.6. Quantitative Real-Time PCR (qRT-PCR) Validation
To validate the RNA-seq data, eight DEGs were randomly selected and analyzed via qRT-PCR. The eight selected genes showed significantly different expression between different genders, including four up-regulated genes and four down-regulated genes in the female. According to previous studies, the β-actin gene and 18S gene were proved to be constitutively expressed across different genders in teleost fish species [35,36]. Thus, the two genes were selected as reference genes for internal standardization. We used Primer Premier 6.0 to design the primers (PREMIER Biosoft International, Palo Alto, CA, USA). The amplification reaction system and procedure of qRT-PCR were the same as those described by Lou [23]. We calculated the relative expression levels of eight target unigenes based on the 2−ΔΔCt method (ΔCT = CTtarget unigene − CTreference gene, −ΔΔCT = ΔCTfemale − ΔCTmale), and the log2 fold change was then used for comparison with the log2 fold change of RNA-seq.
3. Results
3.1. Transcriptome Sequencing and De Novo Assembly
In this study, 49.19 million raw read pairs were generated for males and females, respectively. After preprocessing, 44.65 and 45.08 million clean paired-end sequence reads with Q30 percentages of 88.36% and 88.81% were obtained (Table S1). A total of 59,902 unigenes were assembled with an N50 length of 2070 bp and a mean length of 1121 bp (Table S2). The results of BUSCO showed that 94.39% of the genome (73.93% as single genes and 20.46% as duplicated genes) was complete, and 0.01% was missing, indicating the high quality of S. elongata transcriptome (Figure S1).
3.2. Functional Annotation of the S. elongata Transcriptome
In summary, there were only 7627 unigenes (12.73%) annotated to the NT database. Meanwhile, 38,006 were annotated to at least one of the five protein databases (NR, Swiss-Prot, KEGG, KOG, GO) (Table 1). Specifically, 2113 unigenes were annotated with the five protein databases that were searched in the present study (Figure 1).
A total of 30,840 unigenes were annotated to different pathways based on the KEGG pathway analysis; the top two pathways were ‘Human Diseases’ (15,982 unigenes) and ‘Organismal Systems’ (12,845 unigenes). The predominant pathway subcategories were ‘Signal Transduction’ (5188), ‘Immune System’ (3313) and ‘Cancers: Overview’ (3120) (Figure 2). In total, 16,439 unigenes were assigned to three GO categories. As Figure S2 show, the dominant terms were ‘binding’ (8261 unigenes), ‘catalytic activity’ (6036 unigenes) and ‘membrane part’ (4878 unigenes). In addition, 26,122 unigenes were assigned to the KOG. These unigenes were classified into 25 subcategories (Figure 3).
3.3. Candidate Sex-Related Genes and Functional Enrichment Analysis Results
To detect the potential genes and pathways related to sex, comparative analyses were performed to identify differentially expressed unigenes between genders. In the present study, unigenes with |log2 Fold Change| ≥ 2 and p-value ≤ 0.001 were determined to be differentially expressed genes. After filtering, 22,507 DEGs were identified between the sexes, 10,419 of which were female-biased DEGs, while 12,088 were male-biased DEGs (Figure 4 and Figure S3). Many well-known sex-related genes were detected. For instance, zona pellucida sperm-binding protein genes (zps), StAR-related lipid transfer protein genes (start), spermatogenesis-associated protein genes (spatas), doublesex- and mab-3-related transcription factor (dmtr), P43 5S RNA-binding protein-like (42sp43), follicle stimulating hormone receptor (fshr) and forkhead box protein L2 (foxl2) (Table 2).
Furthermore, the results of enrichment analyses of the 22,507 DEGs showed that 7832 unigenes were assigned to 3293 GO terms. The results in GO terms showed that sex-biased genes were predominantly associated with ‘integral component of membrane’ (GO:0016021) (Table S3). In addition, results suggested that the three most-enriched KEGG pathways were ‘Pathways in cancer’ (ko05200), ‘PI3K-Akt signalling pathway’ (ko04151) and ‘Human papillomavirus infection’ (ko05165) (Figure 5). Figure 5 showed the top twenty statistically significant KEGG classifications of the DEGs.
3.4. SSR Markers Detection
We identified 23,941 SSRs, of which 10,592 44.24% were di-nucleotide repeats, followed by mononucleotide (8320 SSRs, 34.75%) and trinucleotide (3458 SSRs, 14.44%), and other types SSRs (6.57%). Among these loci, SSRs with six tandem repeats were the most dominant (Figure 6). Furthermore, we gave the primers and other information of these SSR loci.
3.5. Transcriptome Data Validation
To validate the transcriptomic data, quantitative RT-PCR (qRT-PCR) was performed on eight DEGs. Four were up-regulated and four were down-regulated in the female. Specific primers of each gene were listed in Table S4. As shown in Figure 7, these randomly selected genes displayed similar expression patterns in both RNA-seq and qRT-PCR, confirming the reliability of our RNA-seq data.
4. Discussion
In the present study, 59,902 de novo assembled unigenes were obtained. Moreover, the results of BUSCO showed 94.39% of the genome was complete. These results showed the quality of transcriptome in our study was high, and it was reliable for subsequent analyses. Among the unigenes, 63.45% were annotated to public databases. The match ratio was low because of the lack of genetic data of lizardfish species in the public databases. To date, only three protein sequences have been deposited for species in genus Saurida in the NCBI NR database, explaining the low number of BLAST top hits for lizardfish. The result of main species distribution matched against the NR database showed that 11.99% of the annotated unigenes shared similar sequences with Lates calcarifer, whose draft genome was published in 2015 [37]. Seriola lalandi dorsalis, Larimichthys crocea, Seriola dumerili and Stegastes partitus also shared similar sequences with S. elongata. Thus, unigenes of S. elongata transcriptome matched well to proteins of other teleost fish. It has been reported that the annotation results of the non-model species were highly dependent on the availability of annotated sequence information in the database, and the sizes of their contig sequences [38,39]. Compared to other non-model teleost species, the number of transcripts obtained for S. elongata from this study was at moderate level [24,26,40].
It is evident that genes involved in gonad development and related to sex differentiation play important roles in controlling the sex ratio of teleost fishes [24]. It is crucial to elucidate the mechanisms of sex determination and gonad differentiation. In the present study, we identified numerous DEGs and suspected that these genes may play important roles in the sex differentiation between genders of S. elongata. However, sex determination in teleost fish is an extremely complex process, regulated by numerous genes [9,25]. Due to the limitations of the present study, we could not clarify the exact mechanisms of sex determination or gonad differentiation. The transcriptome data in this study will facilitate further studies of S. elongata. With the annotation results, we identified several well-known genes relate to sex control and gonadal development (zp, dmtr, 42sp43, start, Wee1, foxl2, etc.).
The zona pellucida plays many important biological functions, including postfertilization blockade of polyspermy and relative species-specific binding of sperm to ovulated eggs [41,42]. In fish oocytes, the zona pellucida plays a protective role in sperm binding [43]. It has been reported that zona pellucida protein 3 plays a crucial role in acrosome reaction, and zona pellucida 4 participates in primordial follicle formation [44,45]. In the present study, zp 1-4 showed significantly higher expression in females, showing these genes may play important roles in reproduction and folliculogenesis in S. elongata.
In teleost, dmrt gene families play crucial roles in sexual differentiation and determination [46,47]. It is well known that the dmrt gene family includes many members. These genes encode putative transcription factors with evolutionarily well-conserved Doublesex and Mad-3 (DM) domains [48]. The DM domain is involved in sexual differentiation and development in many species (insects, fish and mammals) [49,50]. Eight dmrt genes (dmrt1 through dmrt 8) have been reported in mammals [51], and some of them have also been detected in teleosts. These include Oreochromis niloticus [52], Gadus morhua [53], Danio rerio [54], Takifugu rubripes [55] and Cynoglossus semilaevis [56]. As an important member, the dmrt2 gene plays a key role in neurogenesis, testicular hypoplasia and even embryonic sex reversal [57,58]. Recently, the dmrt2 gene was found to play a crucial factor in germ cell maturation and gonadal differentiation in male teleost fishes [56]. Consistent with previous studies, the dmrt2 gene showed significantly higher expression in males. Unlike dmrt2, dmrt4 has different expression patterns in different fish. Guan [59] demonstrated that dmrt4 showed female-specific expression pattern in gonads in Oreochromis niloticus, and Cao [60] detected that dmrt4 was expressed in the endbrain, pituitary gland, thalamencephalon and ovary in Oreochromis aureus. However, dmrt4 showed strong expression in the testis but weak expression in the ovary of the adult olive flounder (Paralichthys olivaceus) [61]. In our study, we detected that the dmrt4 gene showed significantly higher expression in female S. elongata. Results showed that the dmrt4 gene may have potentially important roles in ovary development.
Among sex differentiation genes of teleost fish, foxl2 is one of the earliest known markers of ovarian differentiation [62]. The foxl2 is expressed as a transcription regulator of the cytochrome P450 family and maintains cyp19a expression, which could convert androgens into estrogens [63]. Numerous previous studies demonstrated that foxl2 exhibits significant sex-dimorphic expression patterns and plays a key role in fish gonad development and differentiation [64,65]. In our study, foxl2 was highly expressed in females, indicating that foxl2 participates in the development of gonads in S. elongata.
5. Conclusions
In summary, we sequenced the transcriptomes of female and male S. elongata. After assembly, identification and annotation, numerous putative genes related to sex differentiation were detected. Our results will be useful for improving our understanding of sexual differentiation and the molecular mechanism of sex determination in fishes. It will also facilitate future functional analyses of sex-associated genes. Furthermore, SSR loci was detected in the transcriptome of S. elongata, providing valuable markers for future molecular biology on S. elongata. Ours is the first comparative transcriptomic analysis of different genders of S. elongata. The transcriptome in the present study will provide valuable data to enrich the genomic resources of S. elongata.
Conceptualization, B.S. and Y.L.; methodology, B.S. and L.W.; software, B.S. and C.Y.; validation, B.S., Y.L. and C.Y.; formal analysis, B.S. and Y.L.; investigation, B.S. and L.W.; resources, B.S. and M.L.; data curation, Y.L. and C.Y.; writing—original draft preparation, B.S.; writing—review and editing, P.H., C.Y. and D.S.; visualization, B.S.; supervision, M.L. and D.S.; project administration, L.W. and D.S.; funding acquisition, D.S. All authors have read and agreed to the published version of the manuscript.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of Laboratory Animal Welfare and Ethics of South China Sea Fisheries Research Institute (code: nhdf 2021-01 and date of approval: 10 January 2021).
Not applicable.
All raw reads in this study are archived in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) databases under BioProject PRJNA739278, with accession numbers SRR14866270 and SRR14866271. This Transcriptome Shotgun Assembly (TSA) project has been deposited at DDBJ/EMBL/GenBank under the accession GJGB00000000. The version described in this paper is the first version, GJGB00000000.
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 4. Scatter plots of Gene expression profiles in female and male S. elongata. Thresholds for inclusion were defined by p-value ≤ 0.01 and |log2 fold change| ≥ 1.
Enlarge this image.Figure 7. Expression levels of eight unigenes as determined by transcriptome analysis and qRT-PCR.
Summary of unigenes annotations.
| Database | Number of Unigenes | Percentage (%) |
|---|---|---|
| NR | 35,479 | 59.23% |
| Swiss-Prot | 30,201 | 50.42% |
| KEGG | 30,840 | 51.48% |
| KOG | 26,122 | 43.61% |
| GO | 16,439 | 27.44% |
Genes related to the sex differentiation of Saurida elongata.
| Unigene | Annotation | log2 (♂/♀) | p-Value |
|---|---|---|---|
| CL1525.Contig1_All | Zona pellucida sperm-binding protein 1 | −17.663 | 0 |
| Unigene35458_All | Zona pellucida sperm-binding protein 2 | −9.959 | 0 |
| CL1340.Contig1_All | Zona pellucida sperm-binding protein 3 | −8.554 | 8.109 × 10−101 |
| CL4606.Contig2_All | Zona pellucida sperm-binding protein 4 | −8.602 | 3.971 × 10−224 |
| Unigene16088_All | StAR-related lipid transfer protein 4 | 2.207 | 4.498 × 10−28 |
| Unigene42268_All | StAR-related lipid transfer protein 5 | −3.659 | 9.132 × 10−09 |
| Unigene3434_All | StAR-related lipid transfer protein 7 | −2.968 | 2.820 × 10−74 |
| Unigene7294_All | StAR-related lipid transfer protein 13 | 1.237 | 3.248 × 10−08 |
| Unigene10706_All | Spermatogenesis-associated protein 20 | −1.184 | 3.857 × 10−05 |
| CL3615.Contig1_All | Spermatogenesis-associated protein 5-like protein 1 | −8.618 | 1.846 × 10−26 |
| Unigene15846_All | Spermatogenesis-associated protein 7 | −1.838 | 4.243 × 10−26 |
| Unigene3310_All | Spermatogenesis-associated protein 2 | −1.208 | 1.846 × 10−30 |
| Unigene9109_All | Spermatogenesis-associated protein 13 | 1.496 | 6.667 × 10−19 |
| Unigene44347_All | Zteroidogenic acute regulatory protein | −8.039 | 5.755 × 10−05 |
| Unigene12505_All | Meiotic nuclear division protein 1 homolog | −1.587 | 3.245 × 10−26 |
| CL3550.Contig1_All | Wee1-like protein kinase | −8.342 | 0 |
| Unigene21067_All | Doublesex- and mab-3-related transcription factor 2 | 8.150 | 1.289 × 10−04 |
| Unigene40645_All | Doublesex- and mab-3-related transcription factor 4 | −9.348 | 1.183 × 10−204 |
| Unigene39523_All | Forkhead box protein L2 | −8.071 | 2.528 × 10−11 |
| Unigene44534_All | P43 5S RNA-binding protein-like | −10.204 | 0 |
| Unigene8224_All | Sex hormone-binding globulin | 1.450 | 5.326 × 10−34 |
| Unigene43332_All | Follicle stimulating hormone receptor | −8.492 | 1.461 × 10−15 |
Supplementary Materials
The following supporting information can be downloaded at:
References
1. Ottolenghi, C.; Pelosi, E.; Tran, J.; Colombino, M.; Douglass, E.; Nedorezov, T.; Cao, A.; Forabosco, A.; Schlessinger, D. Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells. Hum. Mol. Genet.; 2007; 16, pp. 2795-2804. [DOI: https://dx.doi.org/10.1093/hmg/ddm235]
2. Smith, C.A.; Roeszler, K.N.; Ohnesorg, T.; Cummins, D.M.; Farlie, P.G.; Doran, T.J.; Sinclair, A.H. The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature; 2009; 461, pp. 267-271. [DOI: https://dx.doi.org/10.1038/nature08298] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19710650]
3. Lavery, R.; Chassot, A.A.; Pauper, E.; Gregoire, E.P.; Klopfenstein, M.; de Rooij, D.G.; Mark, M.; Schedl, A.; Ghyselinck, N.B.; Chaboissier, M.C. Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals. PLoS Genet.; 2012; 8, e1003170. [DOI: https://dx.doi.org/10.1371/journal.pgen.1003170]
4. Chandler, J.C.; Elizur, A.; Ventura, T.J.H. The decapod researcher’s guide to the galaxy of sex determination. Hydrobiologia; 2018; 825, pp. 61-80. [DOI: https://dx.doi.org/10.1007/s10750-017-3452-4]
5. Tao, W.J.; Chen, J.L.; Tan, D.J.; Yang, J.; Sun, L.N.; Wei, J.; Conte, M.A.; Kocher, T.D.; Wang, D.S. Transcriptome display during tilapia sex determination and differentiation as revealed by RNA-Seq analysis. BMC Genomics; 2018; 19, 363. [DOI: https://dx.doi.org/10.1186/s12864-018-4756-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29764377]
6. Barske, L.A.; Capel, B. Blurring the edges in vertebrate sex determination. Curr. Opin. Genet. Dev.; 2008; 18, pp. 499-505. [DOI: https://dx.doi.org/10.1016/j.gde.2008.11.004] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19152784]
7. Pan, Q.W.; Anderson, J.; Bertho, S.; Herpin, A.; Wilson, C.; Postlethwait, J.H.; Schartl, M.; Guiguen, Y. Vertebrate sex-determining genes play musical chairs. C. R. Biol.; 2016; 339, pp. 258-262. [DOI: https://dx.doi.org/10.1016/j.crvi.2016.05.010]
8. Kobayashi, Y.; Nagahama, Y.; Nakamura, M.J.S.D. Diversity and plasticity of sex determination and differentiation in fishes. Sex. Dev.; 2013; 7, pp. 115-125. [DOI: https://dx.doi.org/10.1159/000342009]
9. Casas, L.; Saborido-Rey, F.; Ryu, T.; Michell, C.; Ravasi, T.; Irigoien, X. Sex Change in Clownfish: Molecular insights from transcriptome analysis. Sci. Rep.; 2016; 6, 35461. [DOI: https://dx.doi.org/10.1038/srep35461]
10. Saillant, E.; Fostier, A.; Menu, B.; Haffray, P.; Chatain, B. Sexual growth dimorphism in sea bass Dicentrarchus labrax. Aquaculture; 2001; 202, pp. 371-387. [DOI: https://dx.doi.org/10.1016/S0044-8486(01)00786-4]
11. Dutney, L.; Elizur, A.; Lee, P. Analysis of sexually dimorphic growth in captive reared cobia (Rachycentron canadum) and the occurrence of intersex individuals. Aquaculture; 2017; 468, pp. 348-355. [DOI: https://dx.doi.org/10.1016/j.aquaculture.2016.09.044]
12. Wang, N.; Wang, R.K.; Wang, R.Q.; Chen, S.L. Transcriptomics analysis revealing candidate networks and genes for the body size sexual dimorphism of Chinese tongue sole (Cynoglossus semilaevis). Funct. Integr. Genomic.; 2018; 18, pp. 327-339. [DOI: https://dx.doi.org/10.1007/s10142-018-0595-y] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29532338]
13. Cribbin, K.M.; Quackenbush, C.R.; Taylor, K.; Arias-Rodriguez, L.; Kelley, J.L. Sex-specific differences in transcriptome profiles of brain and muscle tissue of the tropical gar. BMC Genom.; 2017; 18, 283. [DOI: https://dx.doi.org/10.1186/s12864-017-3652-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28388875]
14. Yoneda, A.; Sakai, T.; Tokimura, A.; Horikawa, H.; Matsuyama, M. Age and growth of the lizardfish Saurida sp. 1 in the East China Sea using otolith ring marks. Fish. Res.; 2002; 55, pp. 231-238. [DOI: https://dx.doi.org/10.1016/S0165-7836(01)00285-5]
15. Sakai, T.; Yoneda, M.; Shiraishi, T.; Tokimura, M.; Horikawa, H.; Matsuyama, M. Age and growth of the lizardfish Saurida elongata from the Tsushima/Korea Strait. Fish. Sci.; 2009; 75, pp. 895-902. [DOI: https://dx.doi.org/10.1007/s12562-009-0108-y]
16. Shimizu, Y.; Wendakoon, C.N. Agriculture Effects of maturation and spawning on the gel-forming ability of lizardfish (Saurida elongata) muscle tissues. J. Sci. Food Agr.; 1990; 52, pp. 331-338. [DOI: https://dx.doi.org/10.1002/jsfa.2740520306]
17. Wu, S.G.; Sun, J.H.; Tong, Z.F.; Lan, X.D.; Zhao, Z.X.; Liao, D.K. Optimization of hydrolysis conditions for the production of angiotensin-I converting enzyme-inhibitory peptides and isolation of a novel peptide from lizard fish (Saurida elongata) muscle protein hydrolysate. Mar. Drugs; 2012; 10, pp. 1066-1080. [DOI: https://dx.doi.org/10.3390/md10051066]
18. Lan, X.D.; Sun, L.X.; Muhammad, Y.; Wang, Z.F.; Liu, H.B.; Sun, J.H.; Zhou, L.Q.; Feng, X.Z.; Liao, D.K.; Wang, S.F. Studies on the interaction between angiotensin-converting enzyme (ACE) and ACE inhibitory peptide from Saurida elongata. J. Agric. Food Chem.; 2018; 66, pp. 13414-13422. [DOI: https://dx.doi.org/10.1021/acs.jafc.8b04303]
19. Liu, Y.W.; Zhang, C.L.; Zan, X.X.; Sun, M.; Xu, B.D.; Xue, Y.; Ren, Y.P. Distribution of relative abundance of slender lizardfish and its influencing factors in southern coastal waters of Shandong during autumn. Period. Ocean Univ. China; 2020; 50, pp. 45-53.
20. Tu, Z.; Liu, M.; Wang, Y.P.; Xu, S.Y.; Song, N.; Gao, T.X.; Han, Z.Q. The low mitochondrial diversities in lizardfish Saurida elongata: Recent population expansion and selection. Biochem. Syst. Ecol.; 2016; 68, pp. 44-50. [DOI: https://dx.doi.org/10.1016/j.bse.2016.06.011]
21. Lobo, I.K.C.; do Nascimento, A.R.; Yamagishi, M.E.B.; Guiguen, Y.; da Silva, G.F.; Severac, D.; Amaral, A.D.; Reis, V.R.; de Almeida, F.L. Transcriptome of tambaqui Colossoma macropomum during gonad differentiation: Different molecular signals leading to sex identity. Genomics; 2020; 112, pp. 2478-2488. [DOI: https://dx.doi.org/10.1016/j.ygeno.2020.01.022] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32027957]
22. Chatchaiphan, S.; Srisapoome, P.; Kim, J.H.; Devlin, R.H.; Na-Nakorn, U. De novo transcriptome characterization and growth-related gene expression profiling of diploid and triploid bighead catfish (Clarias macrocephalus Gunther, 1864). Mar. Biotechnol.; 2017; 19, pp. 36-48. [DOI: https://dx.doi.org/10.1007/s10126-017-9730-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28181037]
23. Lou, F.R.; Yang, T.Y.; Han, Z.Q.; Gao, T.X. Transcriptome analysis for identification of candidate genes related to sex determination and growth in Charybdis japonica. Gene; 2018; 677, pp. 10-16. [DOI: https://dx.doi.org/10.1016/j.gene.2018.07.044] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30036655]
24. Ma, D.; Ma, A.; Huang, Z.; Wang, G.N.; Wang, T.; Xia, D.; Ma, B. Transcriptome analysis for identification of genes related to gonad differentiation, growth, immune response and marker discovery in the turbot (Scophthalmus maximus). PLoS ONE; 2016; 11, e0149414. [DOI: https://dx.doi.org/10.1371/journal.pone.0149414] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26925843]
25. Tian, C.; Li, Z.; Dong, Z.; Huang, Y.; Du, T.; Chen, H.; Jiang, D.; Deng, S.; Zhang, Y.; Wandia, S. et al. Transcriptome analysis of male and female mature gonads of silver sillago (Sillago sihama). Genes; 2019; 10, 129. [DOI: https://dx.doi.org/10.3390/genes10020129]
26. Shan, B.; Liu, Y.; Yang, C.; Zhao, Y.; Sun, D. Comparative transcriptomic analysis for identification of candidate sex-related genes and pathways in Crimson seabream (Parargyrops edita). Sci. Rep.; 2021; 11, 1077. [DOI: https://dx.doi.org/10.1038/s41598-020-80282-5]
27. Schroeder, A.; Mueller, O.; Stocker, S.; Salowsky, R.; Leiber, M.; Gassmann, M.; Lightfoot, S.; Menzel, W.; Granzow, M.; Ragg, T. The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol.; 2006; 7, 3. [DOI: https://dx.doi.org/10.1186/1471-2199-7-3]
28. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics; 2014; 30, pp. 2114-2120. [DOI: https://dx.doi.org/10.1093/bioinformatics/btu170]
29. Grabherr, M.G.; Haas, B.J.; Yassour, M.; Levin, J.Z.; Thompson, D.A.; Amit, I.; Adiconis, X.; Fan, L.; Raychowdhury, R.; Zeng, Q.J. Trinity: Reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat. Biotechnol.; 2011; 29, 644. [DOI: https://dx.doi.org/10.1038/nbt.1883]
30. Simão, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics; 2015; 31, pp. 3210-3212. [DOI: https://dx.doi.org/10.1093/bioinformatics/btv351]
31. Langmead, B.; Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods; 2012; 9, 357. [DOI: https://dx.doi.org/10.1038/nmeth.1923] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22388286]
32. Li, B.; Dewey, C.N. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform.; 2011; 12, 323. [DOI: https://dx.doi.org/10.1186/1471-2105-12-323] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21816040]
33. Wang, L.K.; Feng, Z.X.; Wang, X.; Wang, X.W.; Zhang, X.G. DEGseq: An R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics; 2010; 26, pp. 136-138. [DOI: https://dx.doi.org/10.1093/bioinformatics/btp612]
34. Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B.C.; Remm, M.; Rozen, S.G. Primer3—New capabilities and interfaces. Nucleic Acids Res.; 2012; 40, e115. [DOI: https://dx.doi.org/10.1093/nar/gks596]
35. Filby, A.L.; Tyler, C.R. Appropriate ’housekeeping’ genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol. Biol.; 2007; 8, 10. [DOI: https://dx.doi.org/10.1186/1471-2199-8-10]
36. Martins, R.S.; Pinto, P.I.; Guerreiro, P.M.; Zanuy, S.; Carrillo, M.; Canário, A.V. Novel galanin receptors in teleost fish: Identification, expression and regulation by sex steroids. Gen. Comp. Endocr.; 2014; 205, pp. 109-120. [DOI: https://dx.doi.org/10.1016/j.ygcen.2014.06.030] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25016048]
37. Domingos, J.A.; Zenger, K.R.; Jerry, D.R. Whole-genome shotgun sequence assembly enables rapid gene characterization in the tropical fish barramundi, Lates calcarifer. Anim. Genet.; 2015; 46, pp. 468-469. [DOI: https://dx.doi.org/10.1111/age.12312]
38. Schmid, R.; Blaxter, M.L. annot8r: GO, EC and KEGG annotation of EST datasets. BMC Bioinform.; 2008; 9, 180. [DOI: https://dx.doi.org/10.1186/1471-2105-9-180]
39. Kanehisa, M.; Sato, Y.; Morishima, K. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J. Mol. Biol.; 2016; 428, pp. 726-731. [DOI: https://dx.doi.org/10.1016/j.jmb.2015.11.006]
40. Rozenfeld, C.; Blanca, J.; Gallego, V.; García-Carpintero, V.; Herranz-Jusdado, J.; Pérez, L.; Asturiano, J.F.; Cañizares, J.; Peñaranda, D.S. De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages. PLoS ONE; 2019; 14, e0218085. [DOI: https://dx.doi.org/10.1371/journal.pone.0218085]
41. Wassarman, P.M.J.S. The biology and chemistry of fertilization. Science; 1987; 235, pp. 553-560. [DOI: https://dx.doi.org/10.1126/science.3027891] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/3027891]
42. Ringuette, M.J.; Chamberlin, M.E.; Baur, A.W.; Sobieski, D.A.; Dean, J.J.D.B. Molecular analysis of cDNA coding for ZP3, a sperm binding protein of the mouse zona pellucida. Dev. Biol.; 1988; 127, pp. 287-295. [DOI: https://dx.doi.org/10.1016/0012-1606(88)90315-6]
43. Dumont, J.N.; Brummett, A.R.J.O. Egg Envelopes in Vertebrates. Oogenesis; Springer: Berlin/Heidelberg, Germany, 1985; pp. 235-288.
44. Litscher, E.S.; Wassarman, P.M. Egg extracellular coat proteins: From fish to mammals. Histol. Histopathol.; 2007; 22, pp. 337-347. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17163408]
45. Meczekalski, B.; Nawrot, R.; Nowak, W.; Czyzyk, A.; Kedzia, H.; Gozdzicka-Jozefiak, A. Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome. J. Endocrinol. Investig.; 2015; 38, pp. 791-797. [DOI: https://dx.doi.org/10.1007/s40618-015-0260-4]
46. Kikuchi, K.; Hamaguchi, S. Novel sex-determining genes in fish and sex chromosome evolution. Dev. Dyn.; 2013; 242, pp. 339-353. [DOI: https://dx.doi.org/10.1002/dvdy.23927]
47. Zhang, X.B.; Wang, H.; Li, M.H.; Cheng, Y.Y.; Jiang, D.N.; Sun, L.N.; Tao, W.J.; Zhou, L.Y.; Wang, Z.J.; Wang, D.S. Isolation of doublesex- and mab-3-related transcription factor 6 and its involvement in spermatogenesis in tilapia. Biol. Reprod.; 2014; 91, 136. [DOI: https://dx.doi.org/10.1095/biolreprod.114.121418]
48. Matson, C.K.; Murphy, M.W.; Griswold, M.D.; Yoshida, S.; Bardwell, V.J.; Zarkower, D. The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev. Cell; 2010; 19, pp. 612-624. [DOI: https://dx.doi.org/10.1016/j.devcel.2010.09.010]
49. Erdman, S.E.; Burtis, K.C. The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. EMBO J.; 1993; 12, pp. 527-535. [DOI: https://dx.doi.org/10.1002/j.1460-2075.1993.tb05684.x]
50. Krentz, A.D.; Murphy, M.W.; Sarver, A.L.; Griswold, M.D.; Bardwell, V.J.; Zarkower, D. DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary. Dev. Biol.; 2011; 356, pp. 63-70. [DOI: https://dx.doi.org/10.1016/j.ydbio.2011.05.658]
51. Veith, A.M.; Klattig, J.; Dettai, A.; Schmidt, C.; Englert, C.; Volff, J.N. Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse. Genomics; 2006; 88, pp. 185-195. [DOI: https://dx.doi.org/10.1016/j.ygeno.2006.01.003]
52. Rather, M.A.; Dhandare, B.C.J.B.R. Genome-Wide identification of doublesex and Mab-3-Related transcription factor (DMRT) genes in nile tilapia (Oreochromis niloticus). Biotechnol. Rep.; 2019; 24, e00398.
53. Johnsen, H.; Andersen, O. Sex dimorphic expression of five dmrt genes identified in the Atlantic cod genome. The fish-specific dmrt2b diverged from dmrt2a before the fish whole-genome duplication. Gene; 2012; 505, pp. 221-232. [DOI: https://dx.doi.org/10.1016/j.gene.2012.06.021]
54. Zhou, X.; Li, Q.; Lu, H.; Chen, H.; Guo, Y.Q.; Cheng, H.H.; Zhou, R.J. Fish specific duplication of Dmrt2: Characterization of zebrafish Dmrt2b. Biochimie; 2008; 90, pp. 878-887. [DOI: https://dx.doi.org/10.1016/j.biochi.2008.02.021]
55. Yamaguchi, A.; Lee, K.H.; Fujimoto, H.; Kadomura, K.; Yasumoto, S.; Matsuyama, M. Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes. Comp. Biochem. Phys. D; 2006; 1, pp. 59-68. [DOI: https://dx.doi.org/10.1016/j.cbd.2005.08.003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20483235]
56. Zhu, Y.; Cui, Z.K.; Yang, Y.M.; Xu, W.T.; Shao, C.W.; Fu, X.Q.; Li, Y.Z.; Chen, S.L. Expression analysis and characterization of dmrt2 in Chinese tongue sole (Cynoglossus semilaevis). Theriogenology; 2019; 138, pp. 1-8. [DOI: https://dx.doi.org/10.1016/j.theriogenology.2019.06.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31279050]
57. Lourenco, R.; Lopes, S.S.; Saude, L. Left-right function of dmrt2 genes is not conserved between zebrafish and mouse. PLoS ONE; 2010; 5, e14438. [DOI: https://dx.doi.org/10.1371/journal.pone.0014438]
58. Yoshizawa, A.; Nakahara, Y.; Izawa, T.; Ishitani, T.; Tsutsumi, M.; Kuroiwa, A.; Itoh, M.; Kikuchi, Y. Zebrafish Dmrta2 regulates neurogenesis in the telencephalon. Genes Cells; 2011; 16, pp. 1097-1109. [DOI: https://dx.doi.org/10.1111/j.1365-2443.2011.01555.x]
59. Guan, G.J.; Kobayashi, T.; Nagahama, Y. Sexually dimorphic expression of two types of DM (Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem. Biophys. Res. Commun.; 2000; 272, pp. 662-666. [DOI: https://dx.doi.org/10.1006/bbrc.2000.2840]
60. Cao, J.L.; Chen, J.J.; Wu, T.T.; Gan, X.; Luo, Y.J. Molecular cloning and sexually dimorphic expression of DMRT4 gene in Oreochromis aureus. Mol. Biol. Rep.; 2010; 37, pp. 2781-2788. [DOI: https://dx.doi.org/10.1007/s11033-009-9820-z]
61. Wen, A.Y.; You, F.; Tan, X.G.; Sun, P.; Ni, J.; Zhang, Y.Q.; Xu, D.D.; Wu, Z.H.; Xu, Y.L.; Zhang, P.J. Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis. Fish Physiol. Biochem.; 2009; 35, pp. 421-433. [DOI: https://dx.doi.org/10.1007/s10695-008-9267-5]
62. Nakamoto, M.; Matsuda, M.; Wang, D.S.; Nagahama, Y.; Shibata, N. Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes. Biochem. Biophys. Res. Commun.; 2006; 344, pp. 353-361. [DOI: https://dx.doi.org/10.1016/j.bbrc.2006.03.137] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16630569]
63. Fan, Z.F.; Zou, Y.X.; Liang, D.D.; Tan, X.G.; Jiao, S.; Wu, Z.H.; Li, J.; Zhang, P.J.; You, F. Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus). Reprod. Fert. Develop.; 2019; 31, pp. 1742-1752. [DOI: https://dx.doi.org/10.1071/RD18233]
64. Li, C.G.; Wang, H.; Chen, H.J.; Zhao, Y.; Fu, P.S.; Ji, X.S. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia. Comp. Biochem. Phys. B; 2014; 177, pp. 36-45. [DOI: https://dx.doi.org/10.1016/j.cbpb.2014.08.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25199961]
65. Shi, H.J.; Gao, T.; Liu, Z.L.; Sun, L.N.; Jiang, X.L.; Chen, L.L.; Wang, D.S. Blockage of androgen and administration of estrogen induce transdifferentiation of testis into ovary. J. Endocrinol.; 2017; 233, pp. 65-80. [DOI: https://dx.doi.org/10.1530/JOE-16-0551] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28148717]
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; Liu, Manting 2 ; Sun, Dianrong 1 ; Huang, Pujiang 3 1 Key Laboratory of Marine Ranching, Ministry of Agriculture Rural Affairs, Guangzhou 510300, China; Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou 510300, China; South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou 510300, China; Scientific Observation and Research Field Station of Pearl River Estuary Ecosystem, Guangzhou 510300, China
2 Key Laboratory of Marine Ranching, Ministry of Agriculture Rural Affairs, Guangzhou 510300, China; Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou 510300, China; South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou 510300, China
3 Shenzhen Fisheries Development Research Center, Shenzhen 518067, China