Genome-Wide Identification, Sequence Alignment, and Transcription of Five Sex-Related Genes in Largemouth Bass ( Micropterus Salmoides )

Background : Largemouth bass ( Micropterus Salmoides ) is an economically important fish species in China. Most research has focused onits growth, disease resistance, and nutrition improvement. However, the sex-determining genes in largemouthbass are still unclear. The transforming growth factor-beta (TGF-β ) gene family, including amh , amhr2 and gsdf , plays an important role in the sex determination and differentiation of various fishes. These genes are potentially involved in sex determination in largemouth bass. Methods : We performed a systematic analysis of 5 sex-related genes ( amh , amhr2 , gsdf , cyp19a1 , foxl2 ) in largemouth bass using sequence alignment, collinearity analysis, transcriptome, and quantitative real-time polymerase chain reaction (qRT-PCR). This included a detailed assessment of their sequences, gene structures, evolutionary traits, and gene transcription patterns in various tissues including gonads, and at different developmental stages. Results : Comparative genomics revealed that the 5 sex-related genes were highly conserved in various fish genomes. These genes did not replicate, mutate or lose in largemouth bass. However, some were duplicated ( amh , amhr2 and gsdf ), mutated ( gsdf ) or lost ( amhr2 ) in other fishes. Some genes (e.g., gsdf ) showed significant differences in genomic sequence between males and females, which may contribute to sex determination and sex differentiation in these fishes. qRT-PCR was applied to quantify transcription profiling of the 5 genes during gonadal development and in the adult largemouth bass. Interestingly, amh , amhr2 and gsdf were predominantly expressed in the testis, while cyp19a1 and foxl2 were mainly transcribed in the ovary. All 5 sex-related genes were differentially expressed in the testes and ovaries from the 56th day post-fertilization (dpf). We therefore speculate that male/female differentiation in the largemouth bass may begin at this critical time-point. Examination of the transcriptome data also allowed us to screen out several more sex-related candidate genes. Conclusions : Our results provide a valuable genetic resource for investigating the physiological functions of these 5 sex-related genes in sex determination and gonadal differentiation, as well as in the control of gonad stability in adult largemouth bass.


Introduction
The study of sex determination in animals is a challenging but critical task.The sex of diverse animals is determined by a hierarchical gene network, and is considered to be one of the most variable processes in evolution [1,2].Fish are at the intermediate stage of evolution between invertebrates and higher vertebrates.As such they show a much more diverse pattern of sex determination than higher vertebrates, exhibiting both genetic and environmental sex determination systems [3].For example, arapaima (Arapaima gigas) [4] and tiger pufferfish (Takifugu rubripes) [5] have an XX/XY sex determination system, while greater amberjack (Seriola dumerili) [6] and half-smooth tongue sole (Cynoglossus semilaevis) [7] have a ZZ/ZW sex determination system.Barred knifejaw (Oplegnathus fasciatus) [8] has an X1X1X2X2/X1X2Y sex determination pattern.The sex of some fish is also affected by the environment [9][10][11].Almost all types of vertebrate sex determination patterns have been reported in fish [12][13][14][15][16][17].
In vertebrates, sex determination and differentiation are often highly plastic and influenced by a range of different genetic and environmental factors [18][19][20][21].The transforming growth factor-beta (TGF-β) signaling pathway is mainly involved in the formation of tissues and organs, as well as in reproductive development by regulating the growth, proliferation, and differentiation of various cell types [22][23][24].Many recent studies have shown that the TGF-β signaling pathway is inextricably linked to sex determination in a diverse array of fish [25].So far, about 30 sex-determining genes have been reported in fish, of which 20 belong to the TGF-β signaling pathway.These genes are mainly involved in sex determination through gene replication or mutation [2].In particular, anti-Mullerian hormone (amh) induces the degeneration of Mullerian ducts in mammalian males [26].amh was shown to be involved in sex determination in Japanese medaka (Oryzias latipes) [27], European perch (Dicentrarchus labrax) [28], Nile tilapia (Oreochromis niloticus) [29], Northern pike (Esox lucius) [30], and black porgy (Acanthopagrus schlegeli) [31].Anti-Mullerian hormone type-II receptor gene (amhr2) encodes an important receptor for amh and mediates the degeneration of Mullerian ducts [28].Interestingly, some species have lost amhr2 [26,32].However, an extra copy of amhr2, named amhr2y and located on Y chromosome, was recently identified in several fishes and is believed to act as a master male-determining gene in yellow perch (Perca flavescens) [33], tiger puffer [5], Southern catfish (Silurus meridionalis) [34], and ayu (Plecoglossus altivelis) [35].Gonadal soma derived factor (gsdf), another member of the TGF-β superfamily, is ubiquitous in bony fishes but has been lost in many tetrapods [36].Knockout of gsdf in male medaka leads to a sex reversal from male to female [37], suggesting that it plays an important role in male sex determination.
Previous studies have reported that the TGF-beta signaling pathway is also involved in regulating the expression of cytochrome P450 family 19 subfamily A member 1 (cyp19a1) and forkhead box L2 (foxl2).For example, doublesex and mab-3 related transcription factor 1 (dmrt1) can directly inhibit cyp19a1 transcription in Nile tilapia.Overexpression of dmrt1 resulted in decreased expression of cyp19a1 and reduced serum estrogen levels in female tilapia, thereby leading to sex reversal [38].Knockdown of amhy in the embryo of male Patagonian silverside (Odontesthes hatcheri) leads to the upregulation of cyp19a1 and foxl2 mRNAs and to ovarian development [39].In orangespotted grouper (Epinephelus coioides), foxl2 recombinant protein increases the expression of cyp19a1 mRNA, while foxl3 recombinant protein down-regulates the transcription of cyp19a1 but up-regulates the transcription of cyp11b, which is related to androgen synthesis [40].In tilapia, foxl2 up-regulates the expression of cyp19a1 in vivo.In female fish, foxl2 mutation therefore results in sex reversal, decreased expression of cyp19a, and reduced serum 17ßestradiol (E2) level [41,42].However, a systematic analysis of these 5 sex-related genes (amh, amhr2, gsdf, cyp19a1, and foxl2) has not yet been reported in largemouth bass.
Largemouth bass is one of the most economically valuable species in the Chinese aquaculture industry, with a production of over 600,000 tons in 2020.It presents a sexual growth dimorphism during early development, with females often living longer and reaching a larger size than males [43].Our group recently published a draft genome assembly [44] and whole-genome resequencing [45] of largemouth bass.Several previous studies have reported that largemouth bass may have an XX/XY sex determination system [46,47].These basic studies are very helpful for us to study the sex-determining genes of largemouth bass.
Transcriptome is also an effective technique for identifying sex-determining genes.Several studies have in fact identified sex-related genes in various fishes such as in channel catfish [48], platyfish [49] and rainbow trout [50] through the use of transcriptome analysis.Transcriptomic comparisons could also allow prediction of new sex-related genes in largemouth bass.In the present study, we carried out detailed assessments of the sequences, gene structures, evolutionary traits, and gene expression patterns of 5 sex-related genes (amh, amhr2, gsdf, cyp19a1, and foxl2).These findings of this work would improve our understanding of the biological roles of these genes in largemouth bass.They should also contribute substantially to in-depth knowledge of sex-related genes in closely related species, such as smallmouth bass (Micropterus dolomieu).

Phylogenetic and Comparative Genomic Analyses of the Five Sex-Related Genes
A phylogenetic analysis was conducted using the downloaded or extracted amino acid sequences.Multiple protein sequences were aligned using the MUSCLE v.3.7 software (Roque Moraes Drive, Mill Valley, CA, USA) [51] with default parameters.Subsequently, a phylogenetic tree was constructed using IQTREE v.1.6.12 (Center for Integrative Bioinformatics Vienna, Vienna, Austria) [52] based on the maximum likelihood (ML) method and Jones-Taylor-Thornton (JTT+G4) model, with 1000 replicates for evaluation of the branch supports.Detailed locations of upstream and downstream neighboring genes to any sexrelated gene on various chromosomes were compared.

Fish and Sample Collection
One-year-old largemouth bass (five females: body length of 26.6 ± 4.29 cm and body weight of 457.44 ± 45.37 g; five males: body length of 28.8 ± 6.17 cm and body weight of 492.5 ± 40.45 g) were obtained from a local aquaculture base (Huaxuan Aquatic Co. Ltd., Guangzhou, Guangdong, China).Sampled individuals included only mature specimens with a visible ovary or testis.Fish were anesthetized with tricaine methanesulfonate (MS-222, Fuyu Biotechnology Co., Ltd, Shanghai, China, 70-80 mg/L) and then several tissues including brain, muscle, liver and ovary/testis were dissected, immediately frozen in liquid nitrogen, and stored at -80 °C until use.
Sexually mature largemouth bass were harvested for culture in aerated tanks.The general natural spawning, fertilization, and husbandry procedures were performed as previously described [53].Approximately 15-20 fry were collected at each time-point from 13 days post-fertilization (dpf) to 56 dpf (8 time points: 13, 18, 23, 28, 33, 45, 50 and 56 dpf).Fry were euthanized with MS-222 (20-30 mg/L for 2 mins) before sample collection.Each fry was cut into two portions (head and trunk), which were immediately frozen in liquid nitrogen and then stored at -80 °C until use.Male and female samples were distinguished using published molecular markers [43,47] that identify male-allele carrying fish at early, undifferentiated stages (Supplementary Table 1).

RNA Extraction
Total RNA was extracted from each sample (total of 72 samples, 0.5-1 cm 3 of tissue per sample) using the RNeasy Lipid Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions and after RNasefree DNase I (Qiagen) treatment of genomic DNA.The RNA concentration and integrity were examined with a NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively.Only those RNA samples with OD260/280 ≥1.8 and RNA integrity ≥7.0 were selected for transcriptome sequencing.

Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was performed to validate data generated by high-throughput sequencing.Tissue-specific expression patterns for the 5 sex-related genes were compared.qRT-PCR was carried out using gene-specific primers (Supplementary Table 1) designed by routine Primer6 software (PREMIER Biosoft International, San Francisco, CA, USA).
qRT-PCR was performed on an Applied Biosystems 7300 machine (Applied Biosystems, Foster City, CA, USA).The amplification conditions were set as follows: 95 °C for 3 min and 40 cycles of 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s.All qRT-PCRs were conducted with three biological replicates of three individuals.β-actin was used as the internal reference gene, and the 2 −∆∆Ct method [60] was applied to calculate relative transcription values.All data were expressed as the mean ± standard deviation (SD).The Student's t test was applied for statistical analysis, with p < 0.05 for considering as statistical significance.

Ubiquitous Presence of the Five Sex-Related Genes in Eleven Representative Bony Fishes
We studied the 5 sex-related genes in the genomes of 11 representative bony fishes.As in most species, largemouth bass was found to contain all the 5 genes with a single copy, whereas gene loss or duplication was found in some other species (Figs.1,2,3).For example, two gsdf genes were identified in European perch (Fig. 2B), which is consistent with previous reports [61].An earlier study reported a Y-specific duplicate of amh (referred to as amhy) on the Y chromosome of male Nile tilapia [62].However, only one amh was identified in this published male genome assembly.This inconsistency prompted us to reassemble a haplotype-resolved genome using recently released HiFi reads (NCBI SRA: ERR7448120) in order to validate the amh gene (Supplementary Table 2).Interestingly, one amh gene was identified in haplotype X, whereas two amh (amhy and amh∆y) were characterized in haplotype Y (Fig. 1).This suggests that the previously published assembly of Nile tilapia [63] may have certain defects.

Phylogenetic and Synteny Analyses
To elucidate the evolutionary history of each sexrelated gene, we characterized and compared adjacent genomic regions of each gene locus in representative fishes.This synteny analysis indicated that amh, amhr2, cyp19a1, gsdf and foxl2 are conserved throughout the fish tree of life, with only slight modifications of upstream and downstream neighboring genes (Figs. 1,2,3).
The neighbors for amh are peak1 and oaz1 (upstream), and dot1l and ell (downstream) (Fig. 1A).In Nile tilapia, the synteny comparison of X and Y chromosomes confirmed a 10-kb Y-specific insertion within the downstream region of amhy.Annotation of this insertion revealed a single gene that is a duplicate copy of amhy (termed amh∆y; from 2,314,315 bp to 2,317,144 bp in Fig. 1B).amh, amhy, and amh∆y each consists of 7 exons, but amh∆y has an early stop codon in the last exon, resulting in a shorter encoded protein sequence (428 aa) compared to its two counterparts (514 aa; Fig. 1C,D).
Most species have only one gsdf gene in their genomes (Fig. 2), although the European perch genome contains two gsdf genes located on the same chromosome (CAJNNU010000010.1)and sharing 85.71% sequence similarity, but with different neighboring genes (Fig. 2B).Interestingly, in Nile tilapia the protein sequence of gsdf (Fig. 2C) differs between the multiple male and female genomes (three females and two males; Table 1), suggesting its potential participation in sex determination.Compared to other species, the genus Micropterus (including largemouth bass and its relative smallmouth bass) had less conserved upstream and downstream regions around the gsdf gene, with mmr1 located on other chromosomes (Fig. 2A).
As a receptor for amh, amhr2 plays an important role in sex determination and sex differentiation [64].A Yspecific insertion of the amhr2 gene (amhr2by) has been identified in yellow perch and is thought to be a candidate sex-determining gene [33].As in zebrafish, amhr2 is lost in other cyprinid fishes [32], possibly due to chromosome rearrangement during fish evolution (Fig. 3A).Synteny analyses indicate that both cyp19a1 and foxl2 are conserved in the examined species (Fig. 3B,C).However, the foxl2 neighboring genes are less conserved in zebrafish, implying that it is more distantly related to other fish species.

Expression Profiling of the Sex-Related Genes in the Developmental Gonads and Adult Tissues of Largemouth Bass
qRT-PCR was performed to characterize the transcription patterns of amh, amhr2, gsdf, cyp19a1, and foxl2 in the liver, muscle, brain, and gonad (testis or ovary) tissues of mature individuals.All genes showed relatively higher transcription levels in the gonad, while low levels or no expression in the other tissues (Fig. 4).This result suggests that these genes are involved in gonad development and maintenance of gonad stability.
amh, amhr2 and gsdf were predominantly transcribed in the testis and less in the ovary, whereas cyp19a1 and foxl2 were predominantly expressed in the ovary.The transcription levels of cyp19a1 and foxl2 were elevated during gonadal development, with much higher transcription levels observed in female (XX) gonads compared to male (XY) gonads at each of the 8 developmental stages and in the mature gonads.The transcription levels of amh, amhr2 and gsdf were elevated during gonadal development, with much higher levels in XY gonads than XX gonads at the 8 developmental stages and in the mature gonads (Fig. 5).The 5 sex-related genes showed significant differences in expression between males and females, especially at 56 dpf, suggesting this is a critical time-point for the initiation of sex differentiation.In adult gonads, the relative transcription of the 5 sex-related genes (Fig. 4) was consistent with the transcriptome data (Table 2).

Comparative Transcriptomic Analysis of Testes and Ovaries
A differential expression analysis was performed between testes and ovaries to screen for other sex-related candidate genes.A total of 15,962 differentially expressed genes (DEGs) were predicted.The testis showed 9072 up-regulated DEGs and 6890 down-regulated DEGs compared to the ovary (Supplementary Figs.1,2).To identify genes potentially associated with sexual differentiation and gonadal development, we performed sex-related GO annotation and KEGG pathway analysis of these DEGs.This analysis revealed 7656 DEGs, of which 3636 were matched to 264 KEGG subcategories and to 52 GO terms (Supplementary Fig. 3).Many DEGs were predicted to be associated with sex-related GO terms, such as sex differentiation, gamete generation, acrosome assembly, fertilization, and spermatogenesis (Table 3).The transcription of many spermatogenesis-related DEGs, such as sperm sur-face protein Sp17 (sp17) and spermatogenesis-associated protein 22 (spata22), was significantly upregulated in the testis, whereas the transcription of some oogenesis-related genes, such as forkhead box protein N5 (foxn5) and bone morphogenetic protein 15 (bmp15), was markedly upregulated in the ovary.

Discussion
Most of the master sex-determining genes or candidate genes discovered recently in various fishes belong to the TGF-beta signaling pathway.This pathway appears    to play a critical role in the process of sex determination and sex differentiation in diverse fishes.For example, amh is the sex-determining gene for three-spined stickleback (Gasterosteus aculeatus), mandarin fish and Black rockfish (Sebastes schlegelii) [65][66][67].gsdf for sablefish [68], and bmpr1bby for Atlantic herring (Clupea harengus) [69].TGF-beta signaling pathway may also be able to influence sex determination by inhibiting aromatase activity [70].Knockdown of the sex determination gene amhy in male Patagonian silverside can activate the expression of downstream foxl2 and cyp19a1a.These are important genes in the female signaling pathway and can lead to increased estrogen synthesis, thereby causing sexual reversal from male to female [39].However, the detailed mechanisms involved in sex determination, sex differentiation, and gonadal development in largemouth bass have thus far remained unclear.
In our current study, evolutionary and collinear analyses revealed that the 5 sex-related genes (amh, anhr2, cyp19a1, gsdf and foxl2) were generally conserved in 11 representative teleost fishes.However, some genes were found to be duplicated (amh, amhr2 and gsdf ), mutated (gsdf ) or lost (amhr2) in several fish genomes (Figs.1,2,3).In certain Perciformes species, some of these genes (amh, amhr2 and gsdf ) were reported to be replicated or altered to become the master sex-determining genes [67].Therefore, most sex-determining genes may be derived from their orthologous genes (usually sex-related genes) by altering the structures of encoded proteins.
In male Nile tilapia, for example, an extra amh∆y gene is inserted into the Y-specific sequence (Fig. 1B).Knockout of this gene results in male-to-female sex reversal [62], suggesting that it is a master sex-determining gene.Sablefish have different X and Y chromosome copies of gsdf, with gsdfY possessing a spatio-temporal expression profile that is characteristic of a male master sex determination gene [68].The present study also found differences in the gsdf gene coding sequence between male and female Nile tilapia.However, their roles and regulatory mechanisms in sex determination need further investigation.amhr2 is an amh receptor involved in the sex determination and sex differentiation of various fishes [34].In yellow perch, a male-specific duplicate of amhr2, considered to be a candidate sex determining gene, was inserted at the proximal end of the Y chromosome (NC_041339.1)[33].amhr2 SNPs in tiger pufferfish are sex-linked, with heterozygous males and homozygous females [5].These sex-specific SNPs are also identified in Panther puffer (Takifugu pardalis) and spotted puffer (Takifugu poecilonotus).However, cyprinid species such as zebrafish, common carp, grass carp and golden-line barbels appear to have lost amhr2 during evolution.The amh receptor role might be replaced by another type II receptor, most likely bone morphogenetic protein receptor type 2 (bmpr2) [26].Of note, zebrafish have a different sex-determination mechanism compared to other teleost fishes, partially due to the absence of amhr2 [32].In contrast to cyprinid fishes, results from the present study show the amhr2 gene was present in all Perciformes, and even duplicated in some other species (Fig. 3A).It appears therefore that the amhr2 gene may have gradually evolved during the fish evolutionary process.These results also confirm the diversity and complexity of sex-determining genes in fish.This complexity poses a great challenge to the practical identification of sex determination genes.
Compared with other fishes, largemouth bass and smallmouth bass are conservative in that they contain all five sex-related genes studied here, without duplication, loss, or differences in gene features between males and females.Nevertheless, having no differences at the gene level does not rule out that they are sex-related, since many studies have reported that differences at the expression level can also determine a fish's sex.For example, the sex-determining gene in channel catfish, breast cancer anti-resistance 1 (bcar1), shows similar male and female genome sequences.However, a comparative transcriptome analysis revealed that sex-specific isoform expression through alternative splicing may underlie the sex determination processes [71].Based on our comparative transcriptome data of gonads from adult largemouth bass, we found no new alternative splicing transcripts for the five sex-related genes, suggesting that their transcript sequences were also conserved in the mature gonads.Interestingly, the sex-determining region (~1.7 Mb) in largemouth bass was previously reported on Chr7.However, this large nonrecombinant region of the Chr7 does not contain any obvi-ously potential master genes for sex determination [43].In our current study, the 5 sex-related genes were distributed on different chromosomes of the largemouth bass genome (i.e., not just on the Chr7).This finding suggests that the identification of sex-determining genes in largemouth bass remains difficult.
To further investigate the 5 sex-related genes, we conducted expression analyses of adult tissues and gonads at different developmental stages using transcriptome sequencing and qRT-PCR.The 5 sex-related genes displayed significant differences in transcription between the ovary and testis at different developmental stages and in adult gonads (Figs.4,5).The gsdf gene in particular exhibited the highest transcription level in males (XY) (Table 2 and Fig. 4), consistent with those findings reported in Nile tilapia [32].Overexpression of gsdf in the developing gonads of medaka and Nile tilapia converted XX individuals into functional males [72,73].Furthermore, knockout of gsdf in XY fish resulted in male-to-female sex reversal [37,74], suggesting that gsdf could be involved in male sex differentiation, sex maintenance, and testis development in medaka and Nile tilapia.Both amh and amhr2 exhibited higher transcription levels in the testis than the ovary, implying that these genes may play a role in testis development in largemouth bass.
During the natural sex reversal process of ricefield eel (Monopterus albus), increased expression of amh and decreased expression of dosage-sensitive sex reversal (dax1) in the ovaries are important for the activation of testis development.High expression of amh and low expression of dax1 are necessary for the maintenance of testis functions [75].In contrast, cyp19a1 and foxl2 are expressed at higher levels in the ovary than in the testis.In olive flounder (Paralichthys olivaceus), foxl2 may play an important role in ovarian differentiation by maintaining cyp19a1 expression and antagonizing the expression of dmrt1 [76].These results indicate that foxl2 and cyp19a1 promote ovarian development and male-related gene expression.In largemouth bass, the expression of the 5 sex-related genes was significantly different between adult male and female gonads, and during gonadal development.amh, amhr2 and gsdf were predominantly expressed in the testis, whereas cyp19a1 and foxl2 were mainly transcribed in the ovary.These differences indicate the genes may have important roles in the process of sex differentiation and the sex stability of largemouth bass.At 86 dpf in Senegalese sole (Solea senegalensis), the follicle stimulating hormone receptor (fshr) can activate other gonadal marker genes (such as amh and cyp19a1) to promote gonad differentiation [77].
In the present study, amh, amhr2, gsdf, cyp19a1 and foxl2 were significantly expressed in the testes and ovaries on 56 dpf.We therefore speculate that differentiation between males and females in largemouth bass starts at about the 50th day after fertilization.
In the present study, we also performed comprehensive transcriptomic analyses of the testis and ovary in largemouth bass.Differential expression analysis allowed us to identify multiple sex-biased genes that may be associated with steroidogenic hormones, ovarian steroidogenesis, and sex differentiation.In particular, several additional members of the TGF-beta signaling pathway were identified that showed significantly different expression between the testis and ovary.These include bmp7, bmpr1a, bmp8a, fsrp, fsrp3, tgfb2, ir, gdf9, SMAD family member 1 (smad1), smad2, smad6b, smad7, inhibitor of DNA binding 3 (id3), repulsive guidance molecule BMP co-receptor a (rgma), and activin A receptor type 1 (acvr1).A comparative transcriptome analysis of yellowfin bream (Acanthopagrus latus) identified several genes in the TGF-beta signaling pathway that were differentially expressed, including bmp1-8, smad1-8, follistatin and transcription factor e2f4 [78].id2 and id3 are members of the dominant-negative, basic helixloop-helix transcription factor family.The id2bbY gene is a duplicated copy of the autosome id2b gene on the Y chromosome, which has been reported as a sex-determining gene in arapaima [4].Gonadal transcriptome analyses of Channa argus and C. maculate showed that the transcription level of id2 in the testis was significantly lower than that in the ovary [79].The id3 expression level was also significantly different between the male and female gonads of largemouth bass, suggesting that it may be critical for gonad development in this fish.
Dmrt and the transcription factor Sox (sox) are wellknown sex-related gene families in various animals.Dmrt1 plays an important role in sex determination, male germ cell differentiation, and sex maintenance in diverse mammals, birds, and fishes [42,[80][81][82].Moreover, dmrt2-5 are important for testis development in many fish species [83][84][85].Our results showed that dmrt1, dmrtb1, dmrta1, dmrt2 and dmrt3 were highly expressed in largemouth bass testis (Table 4).The sox family has multiple roles in several biological processes, including gonad development and sex determination.sox3 is a sex-determining gene in Indian ricefish (Oryzias dancena) [72], and sox9 cooperates with sox8 to protect the adult testis from male-to-female genetic reprogramming and complete degeneration [86,87].High expression levels of sox2, sox5, sox7, sox9a and sox10 in the testis, and of sox3, sox11 and sox19b in the ovary suggests that sox genes may have complex roles in the sex differentiation of largemouth bass.Interestingly, similar results were reported in yellowfin bream [88].

Conclusions
This study characterized 5 sex-related genes (including amh, amhr2, cyp19a1, foxl2 and gsdf ) in largemouth bass and in 11 other teleost representatives, including their transcription profiles.Phylogenetic and comparative genomics analyses provided additional evidence for validating the orthologs of these genes.Transcriptome sequencing and qRT-PCR revealed distinct transcription levels for these sex-related genes in various tissues of largemouth bass and at different stages of development.In summary, this study provides a comprehensive overview of these sex-related genes in largemouth bass.These interesting data establishes a basic foundation for in-depth functional analyses of these genes in relation to sex determination and sex differentiation.

Fig. 1 .
Fig. 1.Characterization of a Y-specific duplication or insertion of the amhy gene in the male Nile tilapia genome.(A) The phylogenetic tree and synteny comparison of amh in various fishes.The maximum likelihood (ML) tree (left panel) was constructed by IQTREE.Bootstrap values are shown on branches.The right panel presents the synteny of the amh gene in each fish species.(B) A synteny comparison between X and Y contigs.Note the Y-specific insertion at the 10-kb region.(C) ClustalW alignments of amh, amhy and amh∆y proteins.(D) Comparison of gene structures among amh, amhy and amh∆y.Rectangles represent exons and lines represent introns.Abbreviations: dot1l, DOT1-like histone-lysine N-methyltransferase; ell, RNA polymerase II elongation factor ELL; oaz1, Ornithine decarboxylase antizyme 1; peak1, pseudopodium-enriched atypical kinase 1.

Fig. 5 .
Fig. 5. Relative transcription levels of the five sex-related genes at eight different developmental stages in the gonads of largemouth bass.Data were normalized to ß-actin.Values are expressed as relative gene expression (mean ± SD).

Table 2 . FPKM values of the five sex-related genes in largemouth bass gonads.
FPKM, fragments per kilobase of transcript per million mapped reads; FC, fold change.