Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica).

BACKGROUND: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. RESULTS: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). CONCLUSIONS: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

Whole-Genome Identification and Characterization of the DKK Gene Family and Its Transcription Profiles: An Analysis of the Chinese Soft-Shell Turtle (Pelodiscus sinensis).

The DKK family is a canonical small family of WNT antagonists. Though recent studies have suggested that the DKK gene family may be involved in sex differentiation in Pelodiscus sinensis, there are still a lot of things about the DKK gene family that we do not know. In this study, we used bioinformatics methods to identify members of the DKK gene family in P. sinensis and analyzed their phylogeny, covariance, gene structure, structural domains, promoter conserved sites, signal peptides, gonadal transcription factors, transcriptional profiles, and tissue expression profiles. Additionally, qRT-PCR results were utilized for the validation and preliminary investigation of the function of the DKK gene family in P. sinensis. The results showed that the DKK gene family is divided into six subfamilies, distributed on six different chromosomal scaffolds containing different gene structures and conserved motifs with the same structural domains, and all of the members were secreted proteins. Our transcriptional profiling and embryonic expression analysis showed that DKKL1 and DKK4 were significantly expressed in the testes, whereas DKK1 and DKK3 were significantly upregulated in the ovaries. This suggests a potential function in sex differentiation in P. sinensis. Our results may provide a basic theoretical basis for the sex differentiation process in P. sinensis.

Assessing and Screening of Female Fertility in Artificially Bred Asian Yellow Pond Turtles (Mauremys mutica) Based on Parentage Assignment.

The Asian yellow pond turtle (Mauremys mutica) is widely traded in China, and its artificial breeding has now become a major industry. However, the insufficient offspring supply and reproductive decline of farmed turtles make the wild turtles more vulnerable. The present study was mainly designed to quantify the fecundity of M. mutica and attempt to screen for good reproductive performance in females. The genetic variability of the population and its genetic structure were also analysed. The parent-offspring relationships of all offspring in four consecutive years were confirmed using sixteen microsatellite loci. The genetic variability between the parents and offspring was low, and offspring of different years also showed little variability. We summarised the reproductive results of all females and counted the annual number of offspring and the variation in the number of offspring. The females were then divided into three types (stable, undulating and levelling off) according to the continuity. We selected seven females with good reproductive ability, which provided 16.94% of the annual contributions, while there were two females that had no offspring in four years. We also analysed the possible reasons for this difference and the importance of carrying out a family survey. This research can provide the basis and materials for the creation of a good reproductive group and the study of the reproductive biology of turtles in M. mutica aquaculture.

Identification of Sex-Specific Markers and Candidate Genes Using WGS Sequencing Reveals a ZW-Type Sex-Determination System in the Chinese Soft-Shell Turtle (Pelodiscus sinensis).

Male and female Chinese soft-shelled turtles (Pelodiscus sinensis) have sex-dimorphic growth patterns, and males have higher commercial value because of their larger size and thicker calipash. Thus, developing sex-specific markers is beneficial to studies on all-male breeding in P. sinensis. Here, we developed an accurate and efficient workflow for the screening of sex-specific sequences with ZW or XY sex determination systems. Based on this workflow, female and male P. sinensis reference genomes of 2.23 Gb and 2.26 Gb were obtained using de novo assembly. After aligning and filtering, 4.01 Mb female-specific sequences were finally identified. Subsequently, the seven developed sex-specific primer pairs were 100% accurate in preliminary, population, and embryonic validation. The presence and absence of bands for the primers of P44, P45, P66, P67, P68, and P69, as well as two and one bands for the PB1 primer, indicate that the embryos are genetically female and male, respectively. NR and functional annotations identified several sex-determining candidate genes and related pathways, including Ran, Eif4et, and Crkl genes, and the insulin signaling pathway and the cAMP signaling pathway, respectively. Collectively, our results reveal that a ZW-type sex-determination system is present in P. sinensis and provide novel insights for the screening of sex-specific markers, sex-control breeding, and the studies of the sex determination mechanism of P. sinensis.

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis).

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17ß-estradiol and 17α-methyltestosterone) and Wnt/ß-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/ß-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17ß-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/ß-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

A chromosome-level genome assembly of the Asian giant softshell turtle Pelochelys cantorii.

The Asian giant softshell turtle Pelochelys cantorii is one of the largest aquatic turtles in China and has been designated a First Grade Protected Animal in China. To advance conservation research, a combination of Illumina short-read, PacBio long-read, and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for P. cantorii. A total of 262.77 Gb of clean data were produced (121.6 × depth) and then the genome was assembled into 2.16 Gb with a contig N50 of 41.44 Mb and scaffold N50 length of 120.17 Mb, respectively. Moreover, about 99.98% assembly genome sequences were clustered and ordered onto 33 pseudochromosomes. Genome annotation revealed that 21,833 protein-coding genes were predicted, and 96.40% of them were annotated. This new chromosome-level assembly will be an enabling resource for genetic and genomic studies to support fundamental insight into P. cantorii biology.

Multi-Effects of Acute Salinity Stress on Osmoregulation, Physiological Metabolism, Antioxidant Capacity, Immunity, and Apoptosis in Macrobrachium rosenbergii.

Salinity stress can trigger a series of physiological changes. However, the mechanism underlying the response to acute salinity stress in Macrobrachium rosenbergii remains poorly understood. In this study, osmoregulation, physiological metabolism, antioxidant capacity, and apoptosis were examined over 96 h of acute salinity stress. Hemolymph osmolality increased with increasing salinity. After 48 h of salinity exposure, the glucose, triglycerides, total protein, and total cholesterol contents in two salinity stress groups (13 and 26‱ salinity) were significantly lower than those in the 0‱ salinity group. The highest levels of these parameters were detected at 6 h; however, superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were the lowest at 96 h in the 13‱ salinity group. The activity of immunity-related enzyme alkaline phosphatase (AKP) showed a decreasing trend with increasing salinity and remained at a low level in the 26‱ salinity group throughout the experiment. No significant differences were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT), or lysozyme (LZM) among the three treatments at 96 h. After 96 h of salinity treatments, the gill filament diameter significantly decreased, and a more pronounced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive signal was detected in the 13‱ and 26‱ groups compared to that in the 0‱ group. Expression levels of apoptosis-related genes, including Cysteine-aspartic acid protease 3 (Caspase 3), Cysteine-aspartic acid protease 8 (Caspase 8), Cytochrome c (Cyt-c), tumor suppressor gene (P53), Nuclear factor kappa-B (NF-κB), and B cell lymphoma 2 ovarian killer (Bok) were significantly higher in the 26‱ salinity group than in the other groups at 24 h, but lower than those in the 0‱ salinity group at 96 h. Cyt-c and P53 levels exhibited a significantly positive relationship with MDA, AST, and LZM activity during salinity stress. In the 13‱ salinity group, Bok expression was significantly correlated with SOD, T-AOC, AKP, acid phosphatase, and LZM activity, whereas in the 26‱ group, the AST content was positively correlated with Caspase 8, Cyt-c, and P53 expression. A significant negative relationship was observed between Caspase 3 expression and catalase (CAT) activity. These findings provide insight into the mechanisms underlying the response to acute salinity stress and will contribute to improving M. rosenbergii aquaculture and management practices.

Exploring the relationship between environmental DNA concentration and biomass in Asian giant softshell turtle (Pelochelys cantorii).

In recent years, environmental DNA (eDNA) technology has become an accepted approach for investigating rare and endangered species because of its economic efficiency, high sensitivity, and non-invasiveness. The Asian giant softshell turtle (Pelochelys cantorii) is a first-class protected aquatic animal in China, and traditional resource survey methods have not identified its natural populations for many years. In this study, primers and a TaqMan probe targeting ND5 were designed, reaction conditions were optimized, a standard curve was constructed using synthetic DNA, and an eDNA quantitative PCR (qPCR) detection method was established. The eDNA detection technology for P. cantorii revealed that the number of species in the experimental pools showed a significant linear relationship with the eDNA concentration (p < 0.05). The eDNA concentration was negatively correlated with the length of time after the removal of P. cantorii and retention in the water body for 9 days. The qPCR detection method for P. cantorii eDNA established in this study can be applied to the qualitative detection of P. cantorii in water bodies, as well as to preliminary evaluation of its relative biomass. This can serve as a baseline for the investigation of natural P. cantorii population and the evaluation of its wild release effects.

Reproductive Output Reveals the Maternal Effects on Offspring Size-Number Trade-Off in Cultured Asian Yellow Pond Turtle (Mauremys mutica).

Offspring size-number trade-off is a critical component of life-history theory and is important for further understanding the reproductive strategies of animals. The relationship between this trade-off and maternal size has been explored in several turtle species, except for the Asian yellow pond turtle, Mauremys mutica. To investigate how the maternal condition affects offspring size and number, we explored the relationships among the maternal body size and the number and size of cultured M. mutica hatchlings using a 4-year dataset. Our results showed that different females not only produced different sizes of offspring but also produced different numbers of offspring. No trade-off in egg size number was detected. According to regression analysis, we did not find that the maternal body size significantly influenced the offspring mass; however, we detected that the offspring size was significantly correlated with the clutch size and maternal age. The mean body mass of offspring increased with maternal age, and the clutch size varied significantly over four years, which was correlated with offspring size, maternal body size and age. However, the number of offspring per female increased with the maternal plastron length rather than age. Our results were inconsistent with the optimal offspring size theory in that females did not increase their offspring size but rather increased the offspring number to increase their fitness, which will also provide a basis for the efficient cultivation management of turtles.

Chromosome-Level Analysis of the Pelochelys cantorii Genome Provides Insights to Its Immunity, Growth and Longevity.

The Asian giant soft-shelled turtle, Pelochelys cantorii (Trionychidae), is one of the largest aquatic turtles in China and was designated as a First-Grade Protected Animal in China in 1989. Previous investigation based on a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies acquired a high-quality chromosome-level genome of Pc. cantorii. In this study, comparative genomic analysis between Pc. cantorii and 16 other vertebrate genomes indicated that turtles separated from the ancestor of archosaurians approximately 256.6 (95% highest posterior density interval, 263.6-251.9) million years ago (Mya) (Upper Permian to Triassic) and that Pc. cantorii separated from the ancestor of Pd. sinensis and R. swinhoei approximately 59.3 (95% highest posterior density interval, 64.3-54.3) Mya. Moreover, several candidate genes, such as VWA5A, ABCG2, A2M and IGSF1, associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. This new chromosome-level assembly has important scientific value in the study of conservation of Pc. cantorii and also enriches the evolutionary investigation of turtle species.

Single-Cell Atlas Reveals the Hemocyte Subpopulations and Stress Responses in Asian Giant Softshell Turtle during Hibernation.

Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation.

Genome-wide identification, evolution and expression analysis of bone morphogenetic protein (BMP) gene family in chinese soft-shell turtle (Pelodiscus sinensis).

Introduction: Bone morphogenetic proteins (BMPs) play a crucial role in bone formation and differentiation. Recent RNA-Seq results suggest that BMPs may be involved in the sex differentiation of P. sinensis, yet more relevant studies about BMPs in P. sinensis are lacking. Methods: Herein, we identified BMP gene family members, analyzed the phylogeny, collinear relationship, scaffold localization, gene structures, protein structures, transcription factors and dimorphic expression by using bioinformatic methods based on genomic and transcriptomic data of P. sinensis. Meanwhile, qRT-PCR was used to verify the RNA-Seq results and initially explore the function of the BMPs in the sex differentiation of P. sinensis. Results: A total of 11 BMP genes were identified, 10 of which were localized to their respective genomic scaffolds. Phylogenetic analysis revealed that BMP genes were divided into eight subfamilies and shared similar motifs ("WII", "FPL", "TNHA", "CCVP", and "CGC") and domain (TGF-ß superfamily). The results of the sexually dimorphic expression profile and qRT-PCR showed that Bmp2, Bmp3, Bmp15l, Bmp5, Bmp6 and Bmp8a were significantly upregulated in ovaries, while Bmp2lb, Bmp7, Bmp2bl and Bmp10 were remarkable upregulated in testes, suggesting that these genes may play a role in sex differentiation of P. sinensis. Discussion: Collectively, our comprehensive results enrich the basic date for studying the evolution and functions of BMP genes in P. sinensis.

Conservation Genetics of the Asian Giant Soft-Shelled Turtle (Pelochelys cantorii) with Novel Microsatellite Multiplexes.

To understand the genetic structure of the protected turtle species Pelochelys cantorii we used transcriptome data to design more than 30,000 tri- and tetranucleotide repeat microsatellite primer pairs, of which 230 pairs were used for laboratory experiments. After two screenings, only 10 microsatellite markers with good specificity, high amplification efficiency, and polymorphisms were obtained. Using the selected primers, two multiplex PCR systems were established to compare and analyze the genetic diversity of artificially assisted breeding generations from four parents (two females and two males) continuously bred over two years. A total of 25 alleles were detected among the 10 microsatellite loci of the offspring. The polymorphism information content (PIC) was 0.313-0.674, with an average of 0.401, among which two loci were highly polymorphic (PIC ≥ 0.5). The number of alleles was 2-5 and the number of effective alleles was 1.635-3.614. The observed heterozygosity was 0.341-0.813, with an average of 0.582, whereas the average expected heterozygosity was 0.389-0.725, with an average of 0.493. Moreover, on the basis of Nei's genetic distance and the Bayesian clustering algorithm, the 182 offspring were divided into two subgroups, and the results corresponded to the two maternal lines. This is the first study to investigate the molecular markers of P. cantorii, and the results obtained can be used to protect genetic resources and provide a genetic basis for the design of population recovery plans.

Characterization of the In Vitro Cultured Ovarian Cells in the Asian Yellow Pond Turtle (Mauremys mutica).

Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes' functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells' culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles.

Status and Analysis of Artificial Breeding and Management of Aquatic Turtles in China.

China is a major country in turtle cultivation and has a long history of artificial breeding of turtles. In this study, a census and statistical analysis of artificially domesticated aquatic turtles in 15 provinces of China were conducted. The results showed that 29 species were aquatic turtles native to China, accounting for approximately 9% of the world's total, and a large number of exotic aquatic turtles are also domesticated in China. The present situation of artificial breeding and protection of aquatic turtles in major provinces of China is shown, and existing problems are also put forward, with suggestions for the protection and management of aquatic turtles.

Temporal variation in DNA methylation during gonadal development in a reptile with temperature-dependent sex determination†.

DNA methylation plays a significant role in transducing external environmental signals to a cellular response in reptiles; however, whether the methylation patterns are conserved across species remains unclear. Here, we examined the genome-wide DNA methylation differentiation between male and female hatchling gonads of the temperature-dependent sex determination (TSD) Mauremys mutica (M. mutica) using methylation-dependent restriction-site associated DNA sequencing (MethylRAD-seq) to test differentially methylated genes underlying sexual development. Several categories, including heat-shock genes (HSP90A, HSP30C), histone- (KDM8) and ubiquitin-related genes (TRIM39), kinases (WNK3), and gonad differentiation or gonadal-development-related genes (HSD17B8, HSD17B12), were identified as candidates for future study. Additionally, we identified several regulatory pathways potentially mediating TSD thermosensitivity such as the GnRH signaling pathway and calcium signaling pathway. These findings provide evidence that sexually dimorphic DNA methylation may be associated with sex determination or sex differentiation in TSD M. mutica.

Expression and Characterization of the Spats1 Gene and Its Response to E2/MT Treatment in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

Spats1 (spermatogenesis-associated, serinerich 1) has been characterized as a male-biased gene which acts an important role in the germ cell differentiation of mammals. Nevertheless, the function of Spats1 in the Chinese soft-shelled turtle (P. sinensis) has not yet been reported. To initially explore the expression of Spats1 in P. sinensis and its response to sex steroid treatment, we cloned the CDS of Spats1 for the first time and analyzed its expression profile in different tissues, including the testes in different seasons. The Spats1 cDNA fragment is 1201 base pairs (bp) in length and contains an open reading frame (ORF) of 849 bp, which codes for 283 amino acids. Spats1 mRNA was highly expressed in the testes (p < 0.01) and barely detectable in other tissues. In P. sinensis, the relative expression of Spats1 also responsive to seasonal changes in testis development. In summer (July) and autumn (October), Spats1 gene expression was significantly higher in the testes than in other seasons (p < 0.05). Spats1 mRNA was found to be specifically expressed in germ cells by chemical in situ hybridization (CISH), and it was mainly located in primary spermatocytes (Sc1), secondary spermatocytes (Sc2) and spermatozoa (St). Spats1 expression in embryos was not significantly changed after 17α-methyltestosterone (MT)and 17ß-estradiol (E2) treatment. In adults, MT significantly induced Spats1 expression in male P. sinensis. However, the expression of Spats1 in testes was not responsive to E2 treatment. In addition, the expression of Spats1 in females was not affected by either MT or E2 treatment. These results imply that Spats1 is a male-specific expressed gene that is mainly regulated by MT and is closely linked to spermatogenesis and release in P. sinensis.

Whole-Transcriptome Analysis Identifies Gender Dimorphic Expressions of Mrnas and Non-Coding Rnas in Chinese Soft-Shell Turtle (Pelodiscus sinensis).

In aquaculture, the Chinese soft-shelled turtle (Pelodiscus sinensis) is an economically important species with remarkable gender dimorphism in its growth patterns. However, the underlying molecular mechanisms of this phenomenon have not been elucidated well. Here, we conducted a whole-transcriptome analysis of the female and male gonads of P. sinensis. Overall, 7833 DE mRNAs, 619 DE lncRNAs, 231 DE circRNAs, and 520 DE miRNAs were identified. Some "star genes" associated with sex differentiation containing dmrt1, sox9, and foxl2 were identified. Additionally, some potential genes linked to sex differentiation, such as bmp2, ran, and sox3, were also isolated in P. sinensis. Functional analysis showed that the DE miRNAs and DE ncRNAs were enriched in the pathways related to sex differentiation, including ovarian steroidogenesis, the hippo signaling pathway, and the calcium signaling pathway. Remarkably, a lncRNA/circRNA-miRNA-mRNA interaction network was constructed, containing the key genes associated with sex differentiation, including fgf9, foxl3, and dmrta2. Collectively, we constructed a gender dimorphism profile of the female and male gonads of P. sinensis, profoundly contributing to the exploration of the major genes and potential ncRNAs involved in the sex differentiation of P. sinensis. More importantly, we highlighted the potential functions of ncRNAs for gene regulation during sex differentiation in P. sinensis as well as in other turtles.

Chromosome-level genome assembly of Asian yellow pond turtle (Mauremys mutica) with temperature-dependent sex determination system.

Knowledge of sex determination has important implications in physiology, ecology and genetics, but the evolutionary mechanisms of sex determination systems in turtles have not been fully elucidated, due to a lack of reference genomes. Here, we generate a high-quality genome assembly of Asian yellow pond turtle (Mauremys mutica) using continuous long-read (PacBio platform), Illumina, and high-throughput chromatin conformation capture (Hi-C) technologies. The M. mutica haplotype has a genome size of 2.23 Gb with a contig N50 of 8.53 Mb and scaffold N50 of 141.98 Mb. 99.98% sequences of the total assembly are anchored to 26 pseudochromosomes. Comparative genomics analysis indicated that the lizard-snake-tuatara clade diverged from the bird-crocodilian-turtle clade at approximately 267.0-312.3 Mya. Intriguingly, positive selected genes are mostly enriched in the calcium signaling pathway and neuroactive ligand-receptor interaction, which are involved in the process of temperature-dependent sex determination. These findings provide important evolutionary insights into temperature-dependent sex determination system.

The Seasonal and Stage-Specific Expression Patterns of HMGB2 Suggest Its Key Role in Spermatogenesis in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

HMGB2, a member of the high-mobility group (HMG) proteins, was identified as a male-biased gene and plays a crucial role in the germ cells differentiation of mammals. However, its role in spermatogenesis of turtle is still poorly understood. Here, we cloned the Pelodiscus sinensis HMGB2 and analyzed its expression profile in different tissues and in testis at different developmental ages. P. sinensis HMGB2 mRNA was highly expressed in the testis of 3-year-old turtles (P < 0.01), but was hardly detected in ovaries and other somatic tissues. The results of chemical in situ hybridization (CISH) showed that HMGB2 mRNA was specifically expressed in germ cells, where it was mainly distributed in round spermatids and sperm, but not detected in somatic cells, spermatogonia, primary spermatocytes, or secondary spermatocyte. The relative expression of HMGB2 also responded to seasonal changes in testis development in P. sinensis. In different seasons of the year, the relative expression of HMGB2 transcripts in the testis of 1 year and 2 year olds showed an overall upward trend, whereas, in the testis of 3 year old, it peaked in July and then declined in October. Moreover, in April and July, with an increase in ages, the expression of HMGB2 transcripts showed an upward trend. However, in January and October, there was a decline in expression in testis in 3-year-old turtles. These results showed that HMGB2 is closely related to spermatogenesis in P. sinensis.