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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.

Sex has been revealed to have significant implications for physiology and evolutionary biology by driving beneficial mutations, altering genetic complexity and increasing environmental adaptation 1,2 . Sex determination is the developmental decision of an undifferentiated primordial gonad into a testis or ovary, while sex differentiation is the biological process that differentiates into male or female after sex determination 3 . Sex systems of most gonochoristic vertebrates fall into two categories: genotypic sex determination (GSD) and environmental sex determination (ESD) 4,5 . In GSD animals, such as mammals 6 , birds 7 , amphibians 8 , some reptiles 9 and most fishes 10 , the initial sex is highly determined by genotypic elements carried by sperm and ovum at the time of fertilization. For ESD species, there is no genetic difference between the sexes, and sex development is triggered by external stimuli, such as temperature 11 , humidity 12 , photoperiod 13 and social factors 13 .
Temperature-dependent sex determination (TSD) is the most typical class of ESD, in which the percentage of male or female offspring is determined by the ambient temperature during early embryo or larva development in some reptiles and fishes [14][15][16][17] . The investigation on TSD model was firstly reported in a lizard Agama agama by the French zoologist Madeleine Charnier 18 . However, her research has been questioned for a long time as most biologists believe that TSD is merely a defect in the sexual development of reptiles 19 or a substitute mode of GSD 20 . It was not until 1979 that the research on the TSD model really began, thanks to Bull and Vogt's demonstration of the effect of temperature on the sex ratio of five turtles using laboratory and field data 21 . Recently, great progress in elucidating the TSD mechanism has been made in red-eared slider turtle (Trachemys scripta) 11,22,23 . Moreover, among the published five turtle species including T. scripta, Chrysemys picta, Chelonia mydas, Platysternon megacephalum, and Pelodiscus sinensis [24][25][26] , only the genome of T. scripta has been assembled at the chromosome level.

Material and methods
Sample preparation and genome sequencing. A healthy female M. mutica (estimated age 4 years) was obtained from Guangzhou aquatic thoroughbred base of the Pearl River Fisheries Research Institute. Liver and muscle tissues were flash frozen in liquid nitrogen, and genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. DNA quantity and quality were measured using Qubit 3.0 and 1% agarose gel electrophoresis, respectively. High-quality DNA was used for continuous long-read library construction and sequencing in PacBio platform. Then pair-ended libraries and mate-paired libraries were prepared using the standard Illumina protocol. Library sequencing was performed using the Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA) to further evaluate the PacBio assembly quality (Fig. 1). After discarding low-quality reads, adapter sequences, and contaminant reads, including mitochondrial DNA, plant, bacterial, and viral sequences, clean reads were used for subsequent genome survey, correction, and evaluation. We declare that all animal experiments in this research were performed according to the guidelines established by the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences. Turtles used were treated humanely and ethically, and the experiments were approved by Laboratory Animal Ethics Committee Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

Genome size estimation.
A k-mer depth frequency distribution analysis was performed to estimate the genome size, heterozygosity and repetitive sequences 32 . Under the premise of uniform distribution of sequencing reads, genome size (G) was evaluated based on the following formula: G = k-mer number/mean k-mer depth.

Hi-C library construction and sequencing.
To further obtain a chromosomal-level assembly of the genome, a Hi-C library was created for sequencing of adult female liver tissue. The liver tissue sample was fixed in paraformaldehyde, and DNA molecules were enzymatically digested with MboI, generating sticky ends. After repairing and labeling the 5′ overhang with biotinylated residue, the DNA fragments were ligated to each other to form chimeric circles using DNA ligase. Biotinylated circles, which are chimeras of physically associated DNA molecules from the original cross-linking, were enriched, sheared, and sequenced on the Illumina HiSeq X10 platform (San Diego, CA, United States) in 150 PE mode.
Chromosome assembly using Hi-C data. Illumina reads (119×) and PacBio long reads (10×) should corrected to obtain high-accuracy data. Clean data was corrected using Canu 1.5 33 . After assembly of the corrected subreads using SMARTdenovo software, the draft genome was polished with 50× Illumina short reads using Pilon v1.22 34 . Subsequently, Hi-C data were performed to assembled contigs to chromosome-level scaffolds.
Karyotype analysis. The turtle was intraperitoneally injected with 10 μg/g body weight of phytohemagglutinin (PHA), and 24 h later, injected colchicine with 5ug/g-6ug/g body weight. The spleen was taken after 3.5 h. Then, the tissues were incubated with hypotonic solution (0.0375 M KCl) for 15-20 min, and fixed twice in methanol acetic acid (3:1) for 20 min at 4 °C. The fixed tissue suspension was then dropped onto clean glass slides, air-dried, and stained with 10% Giemsa solution (10 mM potassium phosphate, pH 6.8). The chromosomes were cut out and arranged according to the following standards: Group A consists of macrochromosomes with median (M) or sub-median centromeres (SM); Group B consists of macrochromosomes with terminal centromere (T) or subterminal centromeres (ST); the Group C can be considered microchromosomes (m).

Assessment of the genome assemblies.
To evaluate the quality and completeness of the genome assembly, we first aligned the Illumina reads onto the assembly using BWA v0.7.10-r789 35 to assess the alignment rate. Moreover, CEGMA v2.5 36 was performed to identify conserved core eukaryotic genes (CEGs) with the parameter set as identity > 70%. Finally, BUSCO v2 37 was used to further detect single-copy orthologs to evaluate the completeness, degree of fragmentation and missing genes of the genome assembly.
Functional annotations of the predicted genes were performed by homology alignment to public gene databases, including Kyoto Encyclopedia of Genes and Genomes (KEGG) 53 , KOG (Clusters of orthologous groups for eukaryotic complete genomes) 54 , TrEMBL 55 , NCBI nonredundant protein sequences (NR) using BLAST v2.2.31 with an e-value threshold of 1 e−5 56 , and the GO (Gene Ontology) database using Blast2GO v2.5 57 .

Evolutionary and comparative genomic analyses.
To investigate the phylogenetic relationships between M. mutica and other species, we first used Orthofinder v2.3.7 62 to identify orthologous gene families by comparing protein data of eight other genomes from previously reported reptiles, including C. picta (GCA_000241765.2), C. mydas (GCA_015237465.1), T. scripta (GCA_013100865.1), Platysternon megacephalum (GCA_003942145.1), P. sinensis (GCA_000230535.1), Anolis carolinensis (GCA_000090745.2), Alligator mississippiensis (GCA_000281125.4), Deinagkistrodon acutus, one bird Gallus gallus (GCA_000002315.5) and two mammals Mus musculus (GCA_000001635.9) and Homo sapiens (GCA_000001405.28). The software MUSCLE v3.8.31 63 was applied with default parameters to further extract single-copy orthologous genes shared among these 12 species. Shared gene families were visualized using the upsetr 64 package as implemented in R. Subsequently, the phylogenetic tree was reconstructed based on single-copy orthologous genes using IQ-TREE v1.6.11 65 . Briefly, each single-copy gene family was aligned using MAFFT v7.205 66 , and then the alignment of proteins was converted into codon sequences using PAL2NAL v14 67 . After removing regions with poor sequence alignment or large differences by Gblocks v0.91b 68 , we concatenated well-aligned gene families into a supersequence. Then, a maximum likelihood (ML) phylogenetic tree with the GTR + F + I + G4 best-fit model and 1000 bootstrap replicates were constructed. Moreover, divergence time was estimated using the MCMCTree package of the PAML v4.9 program 69 under the relaxed clock model. Fossil records obtained from the TimeTree database (http:// www. timet ree. org/) including divergence times between C. picta and T. scripta (28.14-29. Positive selection analysis. To identify possible positively selected genes (PSGs) in the Asian yellow pond turtle genome, we first extracted the single-copy orthologous genes shared among the Asian yellow pond turtle and five turtles (T. scripta, C. mydas, C. picta, P. megacephalum and P. sinensis), and then the protein sequences of each gene family were aligned using MAFFT 66 . Subsequently, the ratio (ω) of synonymous (Ks) and nonsynonymous (Ka) substitutions was estimated using a branch-site model of CODEML in PAML v4.4c 69 . The likelihood of the positive selection model M2a was then compared to the null model M1a using the likelihood ratio test (LRT), and the corresponding p-values were calculated. Sites with ω > 1 were then calculated for posterior probability using Bayes Empirical Bayes (BEB) method, and genes with LRT p < 0.01 and at least one codon with a posterior probability > 0.95 were defined as PSGs. Moreover, KEGG annotations of PSGs were conducted based on functional enrichment analysis in KOBAS 3.0 (p < 0.05 by Fisher's exact test).
Quantitative reverse-transcription PCR (qRT-PCR) was performed to further verify the expression level of the positively selected genes. Ovaries and testes were collected from adult M. mutica. Total RNAs were extracted using SV Total RNA Isolation System (Promega) and then reverse-transcribed using a SuperScript™ III First-Strand Synthesis System (Invitrogen) after DNase treatment. Specific primers used were shown in Table S1. Reactions were run with the following program: 95℃ for 2 min, followed by 40 cycles of 95℃ for 15 s, 57℃ for 30 s, and 72℃ for 30 s. The β-actin was amplified as an internal control and the relative expression levels of positively selected genes were calculated using the 2 −ΔCt method. SPSS 20.0 was used to perform statistical analyses and variance at a significance level of 0.05.
Ethics approval and consent to participate. Turtles used were treated humanely and ethically, and the experiments were approved by the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences. We declare that all methods were performed in accordance with ARRIVE guidelines.

Results
Genome sequencing and assembly. The k-mer (k = 21 analyzed here) depth frequency distribution analysis was performed to evaluate the genome size, repeat proportion and rate of heterozygosity ( Fig. S1 and Table S2). The average k-mer depth was 53, sequences with k-mer depths greater than 106 were repeated sequences, and sequences with depths of approximately 26 were heterozygous. Thus, based on 21-mer frequencies, the estimated heterozygosity and repeat sequence content of the M. mutica genome were approximately 0.6% and 52.10%, respectively (Table S2). Moreover, a total of 148,757,912,763 k-mers were obtained from sequencing data. After removing data with abnormal depth, a total of 142,693,993,736 k-mers were used to further estimate the genome size; thus, we first estimated that the genome size of M. mutica was 2.69 Gb (Table S2). A total of 280.42 Gb of high-quality clean data were generated from the PacBio sequencing platform (approximately 118.75×) with a read N50 of 26,874 bp and an average read length of 17,925 bp (Table S3). After Canu 1.5 correction, the data were assembled using SMARTdenovo followed by Pilon polishing, which produced 2.36 Gb total length of the genome assembly with 1530 contigs and contig N50 of 8.61 Mb (Table S4). Subsequently, LACHESIS (http:// shend urelab. github. io/ LACHE SIS/) was used to assemble contigs at the chromosome level based on Hi-C sequencing data. The assembled genome finally resulted in 2.23 Gb, which encompassed 1106 contigs with a contig N50 of 8.53 Mb, scaffold N50 of 141.98 Mb, and anchoring rate of 94.38% (Table S4  and Table 1). This scaffold N50 is the largest compared to other sequenced turtle species (Table 1). In addition, approximately 2,211,083,089 out of 2,330,098,296 (94.89%) of length greater than 100 kb were anchored (Table S4). Chromosome integrity examination revealed that M. mutica were diploids with normal karyotype and chromosome number (2n = 52) ( Fig. 2A,B). It was consistent with the Hi-C analysis of the genome that contained 26 pseudochromosomes (Fig. 2C,D and Table S5). Notably, 12 microchromosome-pairs were identified in M. mutica genome which was in accord with the prevalence of microchromosome in birds and other reptiles 71,72 . The genome-wide Hi-C heatmap of chromosome crosstalk was in accordance with the rule of interaction where the signal strength around the diagonal was obviously stronger than that of other positions, indicating the high quality and completeness of this genome assembly.
Furthermore, Illumina reads were aligned to the reference genome to further evaluate the assembly quality. Approximately 99.69% of the clean reads were mapped to the contigs, and 97.05% of the clean reads were mapped in proper pairs (Table S6). Subsequently, CEGMA v2.5 was performed to assess the completeness of the conserved core eukaryotic genes (CEGs). In total, 449 CEGs, accounting for 98.03% of all 458 CEGs, and 231 CEGs, accounting for 93.15% of 248 highly conserved CEG datasets, were identified (Table S7). Finally, BUSCO v2 was used to examine genome integrity, the degree of fragmentation, and possible loss rates. The results showed that 2,494 (96.44%) and 61 (2.36%) of the 2586 expected conserved core genes in the vertebrate database were identified as complete BUSCOs and fragmented BUSCOs, respectively, suggesting high completeness of the assembled genome and validity for subsequent analysis (Table S8). Genome annotation. The overall genome of M. mutica has a GC content of 45.11%, which is higher than that of the T. scripta (44.21%), P. sinensis (44.4%), C. mydas (43.5%), and C. picta (43%) assembled genomes (Table S4) 24,25 . Approximately 1,448,589,111 bp of repetitive sequences accounting for 61.33% of the genome assembly were identified based on the combined de novo prediction and homology search against the Repbase database (Table S9). RNA transposons (Class I) occupied approximately 45.52% of the genome content, which was higher than that of DNA transposons (Class II) (Table S9). Long interspersed nuclear elements (LINEs) were the most abundant repetitive elements, followed by terminal inverted repeats (TIRs) and penelope-like elements (PLEs) (Table S9). Moreover, several unknown repetitive sequences were also found, which constituted 2.31% of the genome assembly (Table S9).
A total of 24,751 protein-coding genes were obtained in M. mutica, higher than those detected in soft-shell turtles (19,380) and green sea turtles (18,046) 25 (Table S10). Across these genes, 18,126 orthogroups and 176 species-specific orthogroups were identified (Table S11). The average gene length, average coding sequence length, average exon and intron length were 26,645.17, 1521.64, 2491.72 and 24,153.44, respectively ( Fig. 3 and Table S12). Among these predicted genes in M. mutica, 24,066 (~ 97.23%) could be functionally annotated in at least one of the databases, including GO, KEGG, KOG, TrEMBL and NR (Table S13 and Figure S2). Various nonprotein coding genes were also identified, including 219 rRNAs, 8499 tRNAs and 262 microRNA genes (Table S14).

Genome evolution.
To investigate the phylogenetic relationship of M. mutica with other groups, we compared the M. mutica genome with five other turtle species (C. picta, C. mydas, T. scripta, P. megacephalum and P. sinensis) and six other vertebrate species (A. carolinensis, D. acutus, G. gallus, M. musculus, A. mississippiensis and H. sapiens). A total of 16,484 one-to-one orthologous genes were detected in M. mutica, which was similar to C. picta and higher than the remaining 10 organisms (Fig. 4A). Moreover, a total of 10,179 gene www.nature.com/scientificreports/ families were shared by five TSD turtles (Fig. 4B). Among these, 735 M. mutica specific, 312 T. scripta specific, 485 P. megacephalum specific, 381 C. mydas specific and 347 C. picta specific gene families were also identified (Fig. 4B). The ML phylogenetic tree based on an orthologous set of 5134 single-copy coding genes indicated that M. mutica was most closely related to T. scripta, C. picta and P. megacephalum, and turtles were the sister group of crocodilians and birds, consistent with a previous investigation based on the draft genomes of C. mydas and P. sinensis (Fig. 4C) 25   www.nature.com/scientificreports/ selection using PAML software. A total of 805 PSGs (with Ka/Ks > 1) were identified in the M. mutica genome, among which 732 PSGs occurred in M. mutica and the other 4 TSD turtles, and 338 PSGs were shared between M. mutica and GSD P. sinensis (Fig. 5A). Then, the 455 TSD turtle-specific PSGs were used for enrichment analysis in KEGG pathways, mapping to 104 pathways (Table S15). The pathway with most PSGs was calcium signaling pathway, followed by neuroactive ligand-receptor interaction, fatty acid biosynthesis, and so on ( Fig. 5B and Table S15). In the most significantly enriched calcium signaling pathway, nine genes were under positive selection such as Na + /Ca 2+ exchanger (ncx), voltage-dependent P/Q-type calcium channel subunit alpha-1A (cac-na1a), phospholipase C delta (plcδ), neurotensin receptor type 1 (ntsr1), alpha-1A adrenergic receptor (adra1a) and inositol 1,4,5-trisphosphate receptor type 3 (itpr3) (Fig. 5C and Table S15). Based on the transcriptome data of adult gonads 29 , we found out that some of these PSGs displayed sex-biased expression that ncx, cacna1a and plcδ had higher expression in testis than in ovary while adra1a and itpr3 had higher expression in ovary than in testis (Fig. 5D). The qRT-PCR analysis revealed that the trend of these genes expression between ovary and testis were consistent with the transcriptome data (Fig. 5D). Recently, studies on T. scripta have showed that a temperature-sensitive Ca 2+ influx promotes phosphorylation of STAT3 (signal transducer and activator of transcription 3) and then pSTAT3 represses Kdm6b transcription, which blocks the male development 23 . Thus, genes from calcium signaling pathway and neuroactive ligand-receptor interaction under positive selection may be associated with TSD in M. mutica.

Discussion
In this study, we generate the chromosome-level genome assembly of M. mutica by combining the continuous long-read, Illumina, and Hi-C technologies. The assembly yielded a high-quality reference genome with an N50 scaffold length of 141.98 Mb, which is larger than the reported turtle species T. scripta, C. picta, C. mydas, P. megacephalum and P. sinensis (Table S4 and Table 1). Among these genome sequences represented in the NCBI genome database, only the genomes of T. scripta and M. mutica are assembled to the chromosome level, which provide valuable resources for further clarifying and exploring genomic innovations and phylogenetic origin of M. mutica.
Turtles have piqued researchers' interest for a long time, as said by Shaffer et al. 24,73 , 'the chelonians are the most bizarre, and yet in many respects the most conservative, of reptilian groups. Because they are still living, turtles are commonplace objects to us; were they entirely extinct, they would be a cause for wonder' . It was known that turtles contain three hypotheses to their evolutionary origins: (1) they are members of early-diverged reptiles, called anapsids 73 ; (2) they are closely related to the lizard-snake-tuatara (Lepidosauria) lineage 74    www.nature.com/scientificreports/ of multiple biotechnologies and molecular markers, the first two hypotheses have been ruled out. Here, our genome-wide phylogenetic analysis of the list turtle species also robustly confirms a close relationship of turtles to the bird-crocodilian lineage (Fig. 4C). The molecular clock of the time-calibrated phylogeny based on fossil records indicated that the divergence time between turtles and the ancestor of archosaurians was consistent with previous investigations on the origin of shells and the unique body plan of turtles 25,77 . Based on previous known cytogenetic data on chromosome numbers and Hi-C analysis, 12 microchromosome-pairs were identified beside 14 macrochromosome-pairs in M. mutica genome (Fig. 2). Previous work at the cytological level implied that most birds have extremely conserved karyotypes, including 9 pairs of macrochromosomes and 30-32 pairs of microchromosomes 71 . While, turtles and snakes have fewer microchromosomes than birds 78,79 . Recent studies on the origin and fate of microchromosomes in genomes of reptiles, birds, and mammals revealed that microchromosomes retain a high frequency of interchromosome interaction inside thenucleus and regularly locate together at interphase and division 80 . Moreover, we detected several pathways related to temperature sensing/transducing and sex determination/ differentiation, such as the calcium signaling pathway, neuroactive ligand-receptor interaction, oocyte meiosis, progesterone-mediated oocyte maturation and steroid hormone biosynthesis, based on selective pressure analyses (Table S15). Some key functional genes involved in the top 2 significant enrichment pathways, calcium signaling pathway and neuroactive ligand-receptor interaction were positively selected (Fig. 5). Multiple hormone-related genes in neuroactive ligand-receptor interaction pathway play a vital role in mammalian reproduction 81,82 . Moreover, five PSGs, ncx, cacna1a, plcδ, adra1a and itpr3 in the KEGG pathway "calcium signaling" with functions involved in sexual dimorphism have been elucidated in diverse species, such as ascidians 83 , humans 84 and rats 85 . For example, in ascidians, ncx has been revealed to play significant roles in the regulation of sperm-activating and sperm-attracting factor-induced sperm chemotaxis, motility activation and motility maintenance 83 . The prime activation target of Ca 2+ -calmodulin expressed in granulosa-luteal cells of swine can drive the in vitro transcriptional activity of the CYP11A promoter 86 . In closely related species of M. mutica, the influx of intracellular Ca 2+ and increased reactive oxygen species levels could act as a temperature-sensitive factor to activate the pSTAT3-Kdm6b loop to stabilize ovary or testis development 23 in T. scripta. Moreover, intracellular calcium ion www.nature.com/scientificreports/ concentration ([Ca2+]i) also plays significant tool in regulating the dynamics of GnRH neuron burst firing 87 . In our investigation, combined transcriptome analysis indicated that these PSGs also showed differential expression patterns between testes and ovaries in M. mutica (Fig. 5C), suggesting their potential roles in the sexual development or the maintenance or function of the testis versus ovary of M. mutica.

Conclusion
In this study, we present a chromosomal-scale genome assembly of M. mutica using continuous long-read, Illumina, and Hi-C technologies, acquiring a total size of 2.23 Gb, with contig N50 of 8.53 Mb and scaffold N50 of 141.98 Mb. This scaffold N50 is the highest among all currently sequenced turtle genomes. Genome Hi-C scaffolding resulted in 26 pseudochromosomes containing 99.98% of the total assembly. Comparative genomics analysis indicated that the lizard-snake-tuatara clade diverged from the bird-crocodilian-turtle clade at approximately 267.0-312.3 Mya. Moreover, many genes under positive selection are from calcium signaling pathway and neuroactive ligand-receptor interaction that are involved in the process of temperature-dependent sex determination, providing important evolutionary insights into temperature-dependent sex determination system.

Data availability
Chromosome-level data of Mauremys mutica genome are deposited at NCBI Sequence Read Archive database under the BioSample accession number of SRR14883730 (BioProject ID: PRJNA740058). The detailed information of the raw data was shown in the tables below.