Beom-Soon Choia,#, Jeonghoon Hanb,#, Min-Sub Kimb,#, Yoshitaka Sakakurac,d, Bo-Young Leeb,* and Jae-Seong Leeb,*
< BMC Genomics>
Beom-Soon Choia,#, Jeonghoon Hanb,#, Min-Sub Kimb,#, Yoshitaka Sakakurac,d, Bo-Young Leeb,* and Jae-Seong Leeb,*
a Phyzen Genomics Institute, Seongnam 13558, South Korea
b Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea
c Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852–8521, Japan
d Organization for Marine Science and Technology, Nagasaki University, Nagasaki 852–8521, Japan
_______________________________________
# These authors contributed equally to this work.
*Corresponding authors: [email protected] (B.-Y. Lee) or [email protected] (J.-S. Lee)
Abstract
Background: The selfing fish Kryptolebias hermaphroditus has unique reproductive system for self-fertilization, making genetically homozygous offsprings. Here, we report on high density genetic map-based genome assembly for the K. hermaphroditus Panama line (PanRS). Results: The numbers of scaffolds were 5,212 and the genome was 683,992,224 bp (N50 = 27.45 Mb). The length of anchored scaffold onto 24 linkage groups was 652,231,070 bp (95.3% of genome) with the gap 0.01% and GC content 39.33% and complete Benchmarking Universal Single-Copy Orthologs value was 96.6%. The numbers of annotated genes were 36,756 (average gene length 1,368 bp) with the GC content of 54.1%. To examine the difference of two sister species in the genus Kryptolebias, we compared the genomes of K. hermaphroditus PanRS and Kryptolebias marmoratus PAN line on the composition of transposable elements. Also we identified 63 cytochrome P450 (CYP) genes from K. hermaphroditus PanRS with their basal expression levels and compared the syntenies containing loci of CYP genes on five linkage groups.
Conclusions: In this paper, we discussed the potential use of the genome of K. hermaphroditus PanRS with the potential role of duplicated CYP genes, studying in defense mechanism in the view of molecular evolutionary ecotoxicology. This K. hermaphroditus genome information will be helpful for a better understanding on genome-wide mechanistic view of CYP genes over evolution in the view of fish environmental ecotoxicology.
Keywords: Genome annotation, Kryptolebias, synteny, linkage group, cytochrome P450
Background
The selfing fish Kryptolebias hermaphroditus (formerly, known as Kryptolebias marmoratus PanRS) reproduces by self-fertilization [1–4], as reported in the sister species Kryptolebias marmoratus [5]. Thus, two sister species show a high degree of genome homogenization and have at least two genetically and geographically distinct lineages such as the Panama and others [6]. Although many questions arise how these selfing fishes can be evolved over time under selection pressure, extensive comparison of two genomes will provide more clear answer on the way how they are coping with the weakness of genetic homozygosity in defending in response to environmental stressors.
As shown in the mangrove killifish K. marmoratus [5, 7], the kin K. hermaphroditus has similar beneficial characters as a model organism for environmental studies such as small size (~5–6 cm), short generation time (~4 months), and ease of maintaining in the laboratory condition. These characters make K. hermaphroditus potentially important model organism too for environmental toxicology, reproductive biology, and genetics [2–4]. To use this species more useful, the availability of a high-quality reference genome is required, whereas approximately 900 Mb and 680 Mb of the genome sequence were available from K. marmoratus RHL (27,328 genes) and K. marmoratus PAN (36,767 genes) lines, respectively [8, 9].
To examine the robustness of two selfing fishes in response to environmental stressors, we compared the representative defence genes such as cytochrome P450 (CYP) genes in their genomes. The CYPs are major enzymes in phase I detoxification metabolism in all living organisms and play an essential role in biotransformation of endogenous compounds (e.g., steroids, fatty acid, and prostaglandins) and diverse xenobiotics (e.g., drugs, toxin, and polycyclic aromatic hydrocarbons [PAHs]) [10–13], as shown in aquatic organisms [14–16]. In particular, a full complement and some members of CYPs have been identified and characterized in fish species [17–19]. For example, 94 CYPs were identified in zebrafish Danio rerio and their expression patterns were analyzed during early development [20]. In the channel catfish Ictalurus punctatus, 61 CYPs were identified and their expression profiles were investigated after bacterial infection [21]. In the mangrove killifish K. marmoratus PAN, 74 CYPs were identified with their expression in response to benzo[α]pyrene (B[α]P) and endocrine-disrupting chemicals (EDCs) [22, 23]. Thus, the identification of the full CYP complements can provide a better understanding of CYPs-mediated defense mechanism in fishes, whereas the identification of CYPs in diverse fish species will be of great asset for examining the first line of defense in response to environmental stressors.
In this paper, we report the genome assembly and gene annotation (36,756 genes) of the PanRS line of the mangrove killifish K. hermaphroditus and identified the full complements of 63 CYP genes in this species. This paper will provide the better understanding on comparative genomics focusing on defence mechanism in two closely related fish species in the view of environmental evolutionary ecotoxicology.
Results
Genome assembly and the integration of genetic map
After the trimming and quality control a total of 101 Gb of genomic data were used for the de novo assembly (Table 1). Do novo genome assembly of K. hermaphrodictus consisted of 5,911 scaffolds (>1kb) with N50 value of 2,341,580 bp, which made a total genome length 684 Mb (Table 2).. Using the marker sequences in the high-density genetic linkage map of Kryptolebias spp. (K. hermaphroditus PanRS x K. marmoratus Dan) [1], we anchored the assembled genome scaffolds onto the genetic map. Of 9,904 SNP markers, 9,123 markers (92%) were anchored to 723 scaffolds (Table 2). Among 723 scaffolds, 71 were split into 1 to 3 positions and re-assembled into a total of 808 integrated scaffolds (Table 2). After anchoring, the length of the final genome assembly was 683,992,224 bp with 78,400 bp gaps (0.01%) (Table 2). Among the 808 integrated scaffolds, the orientation was determined in 292 scaffolds spanning 498,445,335 bp, which accounted for 76.4% of the total physical length in the linkage map (Table 3). Overall, the final genome assembly including unanchored scaffolds consisted of 5,212 scaffolds; 24 linkage map-based scaffolds (95.4%) and 5,188 unanchored scaffolds (4.6%) (Table 4). The total genome length was 684 Mb with an N50 value of 27.46 Mb (GenBank accession no. VDMH00000000) (Table 4). The genome JBrowses are available at http://rotifer.skku.edu:8080/Kh for K. hermaphroditus PanRS and http://rotifer.skku.edu:8080/Km for K. marmoratus PAN, respectively.
BUSCO analysis indicated that the final genome assembly of K. hermaphroditus represented 94.7% of the complete copy in the vertebrate gene model (Table 5). The genome annotation pipeline in the final assembly identified 36,756 genes (http://rotifer.skku.edu:8080/Kh) (Table 6 and Suppl. Table 1).
Comparison of orthologous gene families in the small teleost fishes
Based on gene annotation data of K. hermaphroditus, analysis of orthologous gene clusters has been carried out among closely related teleost species including K. marmoratus, O. melastigma, and X. maculatus. Numbers of clusters are 23,477 in K. hermaphroditus, 22, 897 in K. marmoratus, 19,524 in X. maculatus, and 18,868 in O. melastigma (Fig. 2).. In total, 15,001 clusters were shared among the compared teleost species, while 413 genes were specific to K. hermaphroditus (Fig. 2).. Among 15,001 clusters shared in the four genomes, genes related to nucleosome assembly (GO:0006334) of biological process was significantly enriched (p = 2.15e–72) (Suppl. Table 2). The most enriched GO terms of K. hermaphroditus specific gene clusters were nucleosome assembly (GO:0006334, p = 1.15e–08), protein heterodimerization activity (GO:0046982, p = 3.60e–06), transposition, DNA-mediated (GO:0006313, p = 6.29e–06), inflammatory response (GO:0042110, p = 0.000643), T cell activation (GO:0042110, p = 0.001009), and RNA-directed DNA polymerase activity (GO:0003964, p = 0.001834) (Suppl. Table 2).
Comparison of transposable elements in the genus Kryptolebias spp.
DNA transposons were the most abundant TEs in the genome of K. hermaphroditus PanRS, and followed by long terminal repeats (LTRs), long interspersed nuclear elements (LINEs), and unknown, in parallel to RHL and PAN strains (Fig. 3; Suppl. Table 3).
Identification of cytochrome P450 genes and syntenic analyses
In this study, we found 63 putative CYPs, consisting of 48 full sequences and 15 partial sequences. Of 63 CYPs, full length of 58 K. hermaphroditus CYPs were separated into nine different clans: 2, 3, 4, 7, 19, 20, 26, 46, 51, and mitochondrial (Fig. 4).. In detail, 63 K. hermaphroditus CYPs were divided into 16 CYP families (families CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP46, and CYP51) (Table 7).. In particular, K. hermaphroditus CYPs belonging to clan 2 showed highest in the numbers compared to other clans and phylogenetic analysis, indicating further diversification of clan 2 CYPs into seven smaller branches. In addition, we found the steroidogenic CYPs genes (e.g., CYP17A2, CYP17A2, CYP11A1, CYP19A1, and CYP19A2 [cytochrome P450 aromatase]), suggesting a possible role of these CYPs in the production of steroid hormones. All gene information was registered to the GenBank database and the accession numbers of each gene are appended in Table 8.
When comparative syntenic analysis was made for CYP genes in K. hermaphroditus PanRS with those of K. marmoratus, the genome of K. marmoratus contained higher number of CYP genes for which of those additional genes mostly seemed to be generated by tandem duplication (Figs. 5A and 5B). In particular, genes belong to CYP5, CYP2K, CYP4T, CYP2K, CYP45A, CYP2P, and CYP2Z subfamilies were expanded in K. marmoratus by tandem duplication, compared to K. hermaphroditus, while, in turn, CYP11C1V genes were specifically tandemly duplicated in K. hermaphroditus. Thus, it is plausible that those lineage-specifically duplicated CYP genes would play an important role to adapt their environmental pressures as the adjacent genes for those duplicated CYP genes were mostly conserved between species.
Basal expression of cytochrome P450 genes
Of 63 CYP genes in K. hermaphroditus, 38 CYPs were mapped to show the basal levels of expression. Of them, CYP2N, CYP2X24, CYP3A30, CYP2AF12, and CYP2X27 were highly expressed, compared to others (Fig. 6)..
Discussion
We made the 24 chromosome-based genome assembly of K. hermaphroditus, while a couple of reference genome have been developed in different lineages of the sister species K. marmoratus [8, 9]. High density genetic linkage map of a hybrid between two species can be facilitated to make a chromonome (a chromosome-level genome assembly) [24]. In this study, the length of final genome assembly in K. hermaphroditus was 684 Mb, which was a little bigger than those published in two lineages of K. marmoratus (654 Mb for RHL and 680 Mb for PAN) [8, 9]. Of 9,904 genetic markers, 98% markers were mapped to K. marmoratus genome [9], while 92% markers (9,123) were mapped to K. hermaphroditus genome.
TEs are known to be involved in making the genome structure by mediating duplication, shuffling, and recruitment of host genes [25], leading to lineage-specific genetic diversity. Comparison of TEs in the genus Kryptolebias showed that K. hermarphroditus contained ~2.1% more repetitive sequences in the genomes (Suppl. Table 3),, affecting on the genome size of K. hermaphroditus, although overall compositions of the TEs were similar in three Kryptolebias genomes. Although TEs are known to be the major contributor of genome expansion, there were no significant correlation between the TEs composition and genome sizes; the proportion of TEs in the genome of K. hermaphroditus PanRS (29.95%) was rather higher than K. marmoratus RHL (26.85%) and PAN (27.84%) strains despite its largest genome size among three strains [8, 9] (Fig. 3), indicating that there would be another factors responsible for the variance of genome sizes in Kryptolebias spp. Interestingly, although all the Kimura substitution profiles of the genus Kryptolebias spp. strains analyzed in the present study followed the pattern of “one or two general bursts of transposition” in teleost fish [26], K. hermaphroditus PanRS strain had intraspecific distribution of TEs, as it had more stronger recent transposition bursts, compared to other strains, indicating that K. hermaphroditus PanRS strain had rather recent amplification of TEs than other two K. marmoratus strains (Fig. 3).
In this study, total 63 CYPs were identified from the genome of K. hermaphroditus. To date, CYPs have been studied extensively for over 40 years as one of the major enzymes involved in biotransformation of endogenous and exogenous chemical compounds [27–30]. In general, CYPs consist of 11 clans (clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74, and mitochondrial) and 19 families (families 1, 2, 3, 4, 5, 7, 8, 11, 16, 17, 19, 20, 21, 24, 26, 27, 39, 46, and 51) in vertebrates [28, 31, 32]. Of CYPs, CYPs in families 1 to 4 are well-known as major enzymes involved in detoxification metabolisms of xenobiotics such as drugs and environmental pollutants, whereas CYP families belonging to 5–51 have important role in endogenous metabolisms such as steroid, fatty acids, and hormones [18, 33, 34]. In this study, five steroidogenic CYPs (e.g., CYP17A2, CYP17A2, CYP11A1, CYP19A1, and CYP19A2) were found from 63 identified K. hermaphroditus CYPs. In general, steroid hormones are synthesized via steroidogenesis and play a number of important physiological roles in the regulation of reproduction, sex differentiation, pheromone signaling, and osmoregulation [35]. In particular, three CYPs (CYP11A; cytochrome P450 side chain cleavage, CYP17; cytochrome P450 17a-hydoroxylase, and CYP19; cytochrome P450 aromatase) and steroidogenesis-related genes (e.g., two types of hydroxylated dehydrogenases [HSDs]; 3ß-HSD and 17ß-HSD, steroidogenic acute regulatory protein [StAR]) are responsible for estradiol/testosterone biosynthesis metabolism that paly important role in reproduction and sex differentiation in fish species [36, 37].
Of syntenies containing 63 CYPs genes of K. hermaphrodititus, we found the vestiges of transposon-related genes (e.g. Tcl, Tnp, and Tx1) in LGs 11, 12, and 19 particularly in the regions with tandem duplicated CYP genes in K. hermaphrodititus (Fig. 5B), although the number of CYP genes were less in K. hermaphrodititus than in K. marmoratus. Also TEs are enriched within or in close proximity to CYP genes in insects [38, 39]. Thus, TE insertions around CYPs can alter their regulation and cause the over-expression of CYP enzymes, resulting in insecticide resistance [40–43]. The transpositional activity of TEs in normal conditions is relatively low but is increased in response to environmental stress [44, 45]. However, further studies are required to understand the relationship between TE diversity and ecological pressure on the molecular mechanisms in Kryptolebias spp.
CYP genes were sporadically expressed at basal level in K. hermaphroditus PanRS. CYPs are known to play important role in maintaining cellular homeostasis, and also involved in phase II detoxification process as a major defense system in response to xenobiotics [13]. Previously, in the marine medaka Oryzias melastigma, 65 CYPs were identified and their expression profile in response to water accommodated fractions (WAFs) of crude oil and benzo[α]pyrene (B[α]P) was reported [46, 47]. In particular, significant increase of CYPs belonging to clans 2 and 3 mRNA expression were observed in response to WAFs and B[α]P. In addition, a full complement of CYPs was identified in Kryptolebias marmoratus (n = 74), whereas significant modulation of some member of K. marmoratus-CYPs (clan 2) were observed in response to benzo[α]pyrene (B[α]P), bisphenol A (BPA), 4-octylphenol, and 4-nonylphenol (NP) [22, 23]. Thus, those CYP genes highly expressed at basal level in K. hermaphroditus PanRS would play important role in cellular homeostasis, although it is difficult to draw a conclusion in terms of functional aspects of CYP genes identified in the present study.
Taken together, we identified total 63 CYPs from the genome of K. hermaphroditus. These findings will contribute to the better understanding of expansion on CYPs’ involvement in detoxification mechanism, steroidogenesis, and sex differentiation in response to environmental pollutants in K. hermaphroditus.
MATERIALS AND METHODS
Fish culture
Kryptolebias hermaphroditus (order Cyprinodontiformes; family Rivulidae; formerly known as Kryptolebias marmoratus PanRS) were kindly provided by Dr William P. Davis (U.S. Environmental Protection Agency, Gulf Breeze, FL). All PanRS individuals were the descendants of a single hermaphrodite collected near Bocas del Toro in the Republic of Panama in 1994. The K. hermaphroditus were maintained in 20-L glass tanks containing artificial seawater (ASW) (TetramMarine, Cincinnati, OH, USA) with confined environmental conditions of 25±1°C, 14 h/10 h light/darkness, 15 ppt salinity, and pH 8.0, controlled through an automated flow-through system. Each tank in the system housed 40 fish larvae that were maintained on a diet of the brine shrimp Artemia sp. provided twice a day.
All animal handling and experimental procedures were approved by the Animal Welfare Ethical Committee and the Animal Experimental Ethics Committee of the Sungkyunkwan University (Suwon, South Korea). Experiments were carried out in accordance with the approved guidelines of the Animal Experimental Ethics Committee of the Sungkyunkwan University.
Genomic DNA isolation
Liver (approximately 10 mg) from a single individual was homogenized in a sterile container with genomic DNA isolation buffer (10 mM TrisCl, pH 8.0; 100 mM NaCl; 25 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0; 100 μg/ml proteinase K; 0.5% sodium dodecyl sulfate; 1 μg/ml RNase). The sample was incubated in a water bath at 55°C overnight. The genomic DNA was isolated with phenol/chloroform (Sigma, St. Louis, MO, USA) and chloroform (Sigma) and precipitated with 10 M ammonium acetate (0.2 volumes, Sigma) and isopropanol (0.5 volumes, Sigma). After washing with 70% ethanol, the genomic DNA was dissolved in TE (10 mM TrisCl, pH 8.0; 1 mM EDTA) buffer and stored at 4°C. Finally, genomic DNA was qualified and quantified using a spectrophotometer (Qiaxpert®, Qiagen, Hilden, Germany) and electrophoresed with 0.8% agarose gels.
Genome sequencing
We sequenced genomic DNA using the Illumina HiSeq 2500 platform (GenomeAnalyzer, Illumina, San Diego, CA, USA) with the recommended protocols from the manufacturer. We randomly sheared five μg of K. hermaphroditus genomic DNA using the nebulizer (GenomeAnalyzer) following the manufacturer’s instructions. The fragmented DNA was end-repaired using T4 DNA polymerase and Klenow polymerase with T4 polynucleotide kinase for phosphorylation of 5′ ends of the DNA. To ligate Illumina paired-end adaptor oligonucleotides with the sticky ends of DNA, a 3′-overhang was created using a 3′->5′ exonuclease-deficient Klenow fragment. Products were electrophoresed on an agarose gel and the fragments of each size were stabbed with a scalpel blade. Subsequently, the GenomeAnalyzer paired-end flow-cell was prepared and the clusters of PCR colonies were then sequenced on the GenomeAnalyzer platform according to the manufacturer’s instructions. FASTQ sequence files were reproduced from raw images.
Genome assembly
For paired-end reads, sequencing adapters and low quality (< Q20) were removed using Trimmoatic v.0.33 [48]. Before assembly of the clean reads, we excluded highly repetitive, non-informative reads, which consisted of short tandem repeats. Whole genome assembly was conducted using Platanus v1.2.4 [49] with error-corrected sequences from paired end (PE) libraries (PE 400 and PE 600). All cleaned reads from mate pair (MP) libraries (MP 3kb and MP 8kb) were mapped onto contigs to build scaffolds and were used to fill gaps that were represented by “N” in scaffolds. Scaffolds were built using SSPACE v3.0 [50] and the remaining gaps in the resulting scaffolds were closed using GapCloser (a module of SOAPdenovo2) [51].
Integration of genetic map with the reference genome
To make a chromonome (a chromosome-level genome assembly) [24], we anchored 9,904 restriction site-associated DNA (RAD)-tag (SNP markers) [1] to the reference genome of the selfing fish K. hermaphroditus. Using Chromonomer v. 1.08 (http://catchenlab.life.illinois.edu/chromonomer/), we corrected the misassembly of scaffolds by aligning scaffolds on the linkage groups. The genetic map integrated genome assembly was assessed with benchmarking universal single-copy orthologs (BUSCO) v.3.0 (http://busco.ezlab.org) [52] using the vertebrate database (OrthoDB v.9.0; https://www.orthodb.org/?page = filelist).
Repeat analysis
We used the RepeatMasker fish library together with a de novo generated repeat library to perform repetitive sequence analysis. To identify transposable elements (TEs) at the DNA and protein levels, homologous repeat family annotation was conducted by employing the programs RepeatMasker (ver. 4.0.7) and RepeatProteinMask (http://www.RepeatMasker.org) with default parameters against the TE database Repbase (version 20160829) [53]. Thede novo repeat family was analyzed with RepeatModeler (ver. 1.0.10; http://www.RepeatMasker.org) using default parameters. To obtain consensus sequences from the alignments, the entire identified TEs sequences were aligned with Muscle software [54]. All TE sequences were classified with RepeatClassifier in the RepeatModeler package against Repbase [55]. Tandem repeats were also analyzed using TRFfinder (ver. 4.04) [56]. The above procedure was applied to all fish genomes in this study.
Genome annotation
For gene annotation, evidence file was constructed with RNA-seq transcripts using Trinity V2.1.1 [57] and protein sequences of closely relative species (Fig. 1). The initial gene model was constructed using SNAP 2006–07–28 [58] and AUGUSTUS v.3.3.2 [59]. The resultant gene prediction was used to annotate the assembled genome using the Maker2 v.2.31.8 pipeline [60].
RNA-Seq
Whole tissues of five K. hermaphroditus larvae (approximately 1 cm) were homogenized in TRIZOL reagent (3 volumes, Invitrogen, Paisley, Scotland). Total RNA was isolated according to the manufacturers’ protocols. DNA digestion was performed using DNase I (Sigma). Total RNA was quantified by UV absorption at 260 nm and quality checked by analyzing the ratios A230/260 and A260/280 using a spectrophotometer (QIAxpert®, Qiagen, Hilden, Germany). A paired-end library was synthesized and sequenced using the Genomic Sample Preparation Kit (Illumina, San Diego, CA, USA) and Illumina HiSeqTM 2500 (Illumina) according to the manufacturer’s instructions at the National Instrumentation Center for Environmental Management (NICEM, Seoul National University, Seoul, South Korea). Briefly, short fragments were isolated with the MinElute PCR Purification Kit (Qiagen, Chatsworth, CA, USA). Adaptor-ligated fragments were separated by size on an agarose gel, and the desired range of cDNA fragments (200 ± 25 bp) was excised from the gel. Suitable fragments were purified as templates for PCR amplification and subsequently, PCR amplified to create the final cDNA library template. The image data output was transformed by base calling into sequence data. Image deconvolution and quality value calculations were conducted using Illumina HCS 1.1 software based on the Illumina GA pipeline (ver. 1.6) following the protocol of the manufacturer (Illumina).
Transcriptome assembly
Low-quality sequences (reads containing more than 50% bases with Q-value ≤ 20), adapter-only reads, empty nucleotides (‘N’ in the end of reads), and adaptor sequences were totally removed from raw reads in the clean process. All the clean reads were subsequently assembled to generate contigs, unigenes, and non-redundant unigenes using the de novo assembler Trinity (ver. 2.0.6) [57]. Candidate coding regions from the assembled transcripts and/or contigs were analyzed with TransDecoder (http://transdecoder. sourceforge.net). The regions were used for BLAST analysis against the NCBI non-redundant (nr) protein database. The presence of conserved domains in the assembled transcripts was identified and annotated using InterProScan5 [61]. Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of the contigs were performed using Blast2GO [62]. Three main categories of GO such as cellular component, biological process, and molecular function were analyzed after comparing for similarities using default parameters at the NICEM, Seoul National University (Seoul, South Korea). Transcriptome assembly with RNA-seq was registered at the GenBank accession no. GHNN00000000.
Comparison of orthologous gene families in the small teleost fishes
In order to visualize the overlapping orthologous genes of K. hermaphroditus with the small teleost fishes, we used the web-based program OrthoVenn [63]. All protein information of closely related species was extracted from the annotated information of the genome assemblies (K. hermaphroditus PanRS: PRJNA547668, K. marmoratus PAN: GCF_001649575.1, Xiphophorus maculatus: GCA_002775205.2, and Japanese medaka Oryzias latipes HdrR: GCA_002234675.1)..
Identification of cytochrome P450 genes and interspecific comparison
To obtain CYPs of K. hermaphroditus RNA-Seq information was performed. Genes were subjected to BLAST analysis in non-redundant (NR; including all GenBank, EMBL, DDBJ, and PDB sequences except EST, STS, GSS, and HTGS) amino acid sequence database (http://blast.ncbi.nlm.nih.gov/). All acquired contigs were mapped to the genome for obtaining the complete DNA sequence using Geneious (v.10.0.7; Biomatters Ltd., Auckland, New Zealand). Annotation and nomenclature of all CYPswere completed based on amino acid sequence similarities and phylogenetic analysis.
To analyze the evolutionary relationships of CYPs in K. hermaphroditus, the translated amino acid sequences of CYPs were first subjected to multiple alignments with ClustalW algorithm. To establish the best-fit substitution model for phylogenetic analysis, the model with the lowest score according to the Bayesian Information Criterion (BIC) [64] and Akaike Information Criterion (AIC) [65, 66] was analyzed by maximum likelihood (ML) analysis. The phylogenetic tree was constructed using MEGA software (ver.7.0) under the best-fit model (LG+G+I) (Center for evolutionary Medicine and Informatics, Tempe, AZ, USA) [67]. Full lengths protein sequences were aligned and a phylogenetic tree was obtained as described above and the reliability of tree topology was evaluated by bootstrapping test (1000 replicates).
CYP genes in the 24 LGs-level were compared between the genomes of K. hermaphroditus PanRS and K. marmoratus PAN after we compare all the CYP genes into 24 LGs.
Basal expression of cytochrome P450 genes
The preprocessed RNA-Seq reads were aligned against the scaffold assembly by using the STAR program (ver. 2.5.1b) with gene annotation data and default parameters [68]. The numbers of mapped reads in exons were counted by using the HTSeq program (ver. 0.6.1) [69]. The expression level of exons overlapping with CYP genes was calculated by the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped)measure [70]. Three FPKM scores (1, 0.1, and 0.001) were used as a threshold to count the number of expressed exons.
Additional files
Suppl. Table 1. Blast results to NR.
Suppl. Table 2. List of GO Enrichment
Suppl. Table 3. Repeat analysis of three Kryptolebias spp.
Funding
This work was supported by the Collaborative Genome Program of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) (No. 20180430) to Jae-Seong Lee and also supported by a grant from the National Research Foundation (2017R1D1A1B03036026) to Bo-Young Lee.
Availability of data and materials
The datasets supporting the results in this article are available in the NCBI. Raw sequencing reads are available under the following SRA numbers: SRX5913042-SRX5913045. Genome assembly data have been deposited in GenBank under Accession number VDMH00000000.
Authors’ contributions
B.-S. C., J. H., M.-S. K., B.-Y. L., and J.-S. L. designed the experiments, analyzed the data, and wrote the manuscript. B.-S. C., J. H., and M.-S. K. performed the experiments. B.-S. C., Y. S., and B.-Y. L. discussed the experiments and worked through potential problems during their execution.
Ethics approval
All animal handling and experimental procedures were approved by the Animal Welfare Ethical Committee and the Animal Experimental Ethics Committee of the Sungkyunkwan University (Suwon, South Korea). Experiments were carried out in accordance with the approved guidelines of the Animal Experimental Ethics Committee of the Sungkyunkwan University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Table 1. Statistics of whole genome sequencing raw data for Kryptolebias hermaphroditus PanRS
Library Name
Insert size
Read length (bp)
Raw data
Cleaned reads
Coverage
(×)
Accession numbers
No. reads
Total length (bp)
No. reads
%
Total length (bp)
%
PE 400
400 bp
251
278,588,212
69,918,111,212
225,680,212
81.01%
45,128,796,583
64.55%
66
SRX5913044
PE 600
600 bp
251
232,611,706
58,385,538,206
178,496,242
76.74%
35,693,695,178
61.13%
52.2
SRX5913045
MP 3kb
3 kb
151
155,247,206
23,442,328,106
84,259,104
54.27%
6,892,185,668
29.40%
10.1
SRX5913042
MP 8kb
8 kb
151
389,939,598
58,880,879,298
164,429,478
42.17%
13,354,978,366
22.68%
19.5
SRX5913043
Total
1,056,386,722
210,626,856,822
652,865,036
61.80%
101,069,655,795
47.90%
147.7
Table 2. Summary of the genome assembly and anchoring genetic markers in Kryptolebias hermaphroditus PanRS
Categories
values
No. of de novo scaffolds
5,911
Length of scaffolds (bp)
684,109,229
N50 (bp)
2,341,580
Largest scaffold (bp)
14,448,439
Gap (%)
2.33
GC content (%)
39.33
Anchored genetic markers
9,123
No. of original scaffolds mapping to the genetic map
723
No. of scaffolds after anchoring
808
Gap (bp)
78,400
Total length of scaffolds in the genetic map (bp)
652,152,670
Total length of integrated genome sequence (bp) + gap
652,231,070
Total length of scaffolds unplaced (bp)
31,761,154
Total length (bp) of integrated and unplaced sequences
683,992,224
Table 3. Physical length of linkage map integrated with the reference genome assembly in Kryptolebias hermaphroditus RanRS
LG
Physical length (bp)
No. of anchors
No. of scaffolds
No. of oriented scaffolds
Length of oriented scaffolds (bp)
Kh1
34,104,283
488
40
19
27,238,493
Kh2
37,058,898
482
56
22
24,738,365
Kh3
29,655,158
448
30
11
21,116,543
Kh4
31,024,993
443
39
12
23,441,365
Kh5
25,266,975
421
27
4
18,329,414
Kh6
31,987,661
396
42
12
20,336,443
Kh7
24,780,988
384
41
13
14,877,042
Kh8
29,411,091
371
39
9
23,728,346
Kh9
29,158,035
409
27
7
21,396,788
Kh10
23,713,822
370
27
11
18,013,240
Kh11
27,456,164
400
34
11
19,418,923
Kh12
23,899,941
389
35
14
18,324,496
Kh13
26,746,140
379
35
8
20,357,121
Kh14
25,579,387
352
27
15
21,561,108
Kh15
33,609,228
361
39
14
26,815,765
Kh16
32,124,555
356
31
11
26,610,076
Kh17
24,994,607
365
29
13
21,077,549
Kh18
20,417,116
369
35
13
17,057,629
Kh19
22,807,870
355
29
12
17,028,349
Kh20
27,156,570
362
22
7
21,699,507
Kh21
23,020,402
331
49
21
18,190,731
Kh22
29,098,290
360
22
10
25,555,585
Kh23
16,351,084
278
37
13
11,158,228
Kh24
22,729,412
254
16
10
20,374,229
Total length
652,152,670
9,123
808
292
498,445,335
Table 4. Statistics of the final genome assembly in K. hermaphroditus PanRS including 24 LGs and unanchored scaffolds.
Scaffold information
K. hermaphroditus
Number of scaffolds
5,212
Length of scaffolds (bp)
683,992,224
N50 (bp)
27,459,464
Largest scaffold (bp)
37,064,398
Gap (%)
2.31
GC content (%)
39.33
No. of unanchored scaffolds
5,188
Length of unanchored scaffolds (bp)
31,761,154
Table 5. Assessment of de novo assembly in Kryptolebias spp.
K. hermaphroditus
K. marmoratus PAN
K. marmoratus RHL
Nos.
%
Nos.
%
Nos.
%
Complete BUSCOs
287
94.7
286
94.4
270
89.1
Complete and single-copy BUSCOs
278
91.7
277
91.4
264
87.1
Complete and duplicated BUSCOs
9
3.0
9
3.0
6
2.0
Fragmented BUSCOs
6
2.0
4
1.3
20
6.6
Missing BUSCOs
10
3.3
13
4.3
13
4.3
Table 6. Genome annotation statistics for the assembled Kryptolebias hermaphroditus genome
Statistics
K. hermaphroditus
AED: 1
Number of genes
36,756
Total coding sequence length (bp)
50,296,725
Average CDC length (bp)
171
Largest gene length (bp)
67,113
GC content (%)
54.1
Average gene length (bp)
1,368
Average intron length (bp)
9,970
Table 7. CYP clan membership of the 63 Kryptolebias hermaphroditus CYPs assigned to CYP families.
Clan
Family
Genes
Total
Clan 2
1, 2, 21 (3 families)
33
Clan 3
3, 5 (2 families)
3
Clan 4
Clan 7
Clan 19
Clan 20
Clan 26
Clan 46
Clan 51
Mitochondrial
4 (1 family)
7, 8 (2 families)
19 (1 family)
20 (1 family)
26 (1 family)
46 (1 family)
51 (1 family)
11, 24, 27 (3 families)
3
5
2
1
3
2
1
10
Table 8. Classification of 63 identified cytochrome P450 genes and steroidogenesis-related genes in Kryptolebias hermaphroditus. Matched species and E-values of NCBI non-redundant blast analysis are listed below.
Gene (Accession no.)
Clan
Matched species
Matched gene (Accession no.)
E-value
CYP1A1 (MK947934)
2
Kryptolebias marmoratus
CYP1A1 isoform X1 (XM_017428724.2)
0.0
CYP1A1-like (MK947935)
2
Kryptolebias marmoratus
CYP1A1-like (XM_017417374.2)
0.0
CYP1B1 (MK947936)
2
Kryptolebias marmoratus
CYP1B1 (XM_017435073.2)
0.0
CYP1C1 (MK947937)
2
Kryptolebias marmoratus
CYP1C1 (MF326084.1)
0.0
CYP1C2 (MK947938)
2
Kryptolebias marmoratus
CYP1C2 (MF326085.1)
0.0
CYP2G1 (MK947939)
2
Kryptolebias marmoratus
CYP2G1 (XM_017431083.2)
0.0
CYP2K1 (MK947946)
2
Kryptolebias marmoratus
CYP2K1 (XM_017425333.2)
0.0
CYP2K1–1 (MK947947)
2
Kryptolebias marmoratus
CYP2K1 (XM_017427375.2)
0.0
CYP2K1-like (MK947948)
2
Kryptolebias marmoratus
CYP2K1-like (XP_024863631.1)
0.0
CYP2K1-like2 (MK947949)
2
Kryptolebias marmoratus
CYP2K1-like (XM_017425325.2)
1e–92
CYP2K1-like3 (MK947950)
2
Kryptolebias marmoratus
CYP2K1-like (XP_017280814.1)
6e–174
CYP2K1-like4 (MK947951)
2
Kryptolebias marmoratus
CYP2K1-like (XP_017280819.1)
7e–70
CYP2K1-like5 (MK947952)
2
Kryptolebias marmoratus
CYP2K1-like isoform X1 (XM_017427407.2)
0.0
CYP2K4 (MK947953)
2
Kryptolebias marmoratus
CYP2K4 isoform X1 (XP_017280823.1)
0.0
CYP2K4-like (MK947954)
2
Kryptolebias marmoratus
CYP2K4 isoform X1 (XM_017425334.2)
0.0
CYP2K48 (MK947955)
CYP2N (MK947943)
2
2
Kryptolebias marmoratus
Chaetodon punctatofasciatus
CYP2K48 (MF326099.1)
CYP2N (XP_027146356.1)
0.0
0.0
CYP2N22 (MK947956)
2
Kryptolebias marmoratus
CYP2N22 (MF326104.1)
0.0
CYP2N22-like (MK947957)
CYP2P18 (MK947941)
CYP2P19 (MK947940)
CYP2P20 (MK947944)
2
2
2
2
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
CYP2N22 (AUX14899.1)
CYP2P18 (AUX14903.1)
CYP2P20 (AUX14905.1)
CYP2P20 (AUX14905.1)
4e–31
0.0
0.0
0.0
CYP2R1 (MK947958)
2
Kryptolebias marmoratus
CYP2R1 (MF326111.1)
0.0
CYP2U1 (MK947959)
2
Kryptolebias marmoratus
CYP2U1 (XM_017412525.2)
0.0
CYP2X24 (MK947960)
2
Kryptolebias marmoratus
CYP2X24 (MF326113.1)
0.0
CYP2X25 (MK947961)
2
Kryptolebias marmoratus
CYP2X25 (MF326114.1)
2e–119
CYP2X26 (MK947962)
2
Kryptolebias marmoratus
CYP2X26 (MF326115.1)
0.0
CYP2X27 (MK947963)
CYP2Z6 (MK947945)
CYP2AD12 (MK947942)
2
2
2
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
CYP2X27 (MF326116.1)
CYP2Z6 (XP_017262783.1)
CYP2AD12 (AUX14882.1)
0.0
0.0
0.0
CYP17A1 (MK947964)
2
Kryptolebias marmoratus
CYP17A1 (MF326142.1)
0.0
CYP17A2 (MK947965)
CYP21A1 (MK947966)
2
2
Kryptolebias marmoratus
Kryptolebias marmoratus
CYP17A2 (MF326143.1)
CYP21A1 ( MF326147.1)
0.0
0.0
CYP3A30 (MK947967)
3
Kryptolebias marmoratus
CYP3A30 isoform X2 (XM_017430134.2)
0.0
CYP3A40-like (MK947968)
3
Kryptolebias marmoratus
CYP3A40-like (XM_017431035.2)
0.0
CYP5A2 (MK947969)
3
Kryptolebias marmoratus
CYP5A2 (MF326128.1)
0.0
CYP4B1-like (MK947970)
4
Kryptolebias marmoratus
CYP4B1-like (XM_017415248.2)
0.0
CYP4F128 (MK947971)
4
Kryptolebias marmoratus
CYP4F128 (MF326123.1)
0.0
CYP4V2 (MK947972)
CYP7A1 (MK947973)
CYP7C1 (MK947974)
CYP8A1 (MK947975)
CYP8A2 (MK947976)
CYP8B15 (MK947977)
CYP19A1 (MK947978)
CYP19A2 (MK947979)
CYP20A1 (MK947980)
CYP26A1(MK947981)
CYP26B1 (MK947982)
CYP26B1-like (MK947983)
CYP46A1 (MK947984)
CYP46A2 (MK947985)
CYP51A1 (MK947986)
CYP11A1 (MK947987)
CYP11C1v1 (MK947988)
CYP11C1v2 (MK947989)
CYP11C1v3 (MK947990)
CYP24A1 (MK947991)
CYP27A1 (MK947992)
CYP27A3 (MK947993)
CYP27B1 (MK947994)
CYP27C1 (MK947995)
4
7
7
7
7
7
19
19
20
26
26
26
46
46
51
MT
MT
MT
MT
MT
MT
MT
MT
MT
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
Kryptolebias marmoratus
CYP4V2 (XM_017408443.2)
CYP7A1 (MF326132.1)
CYP7C1 (MF326133.1)
CYP8A1 (MF326134.1)
CYP8A2 (MF326135.1)
CYP8B15 (MF326139.1)
CYP19A1 (MF326144.1)
CYP19A2 (MF326145.1)
CYP20A1 (XM_017438094.2)
CYP26A1 isoform X1 (XM_017431830.2)
CYP26B1 (XM_017439209.2)
CYP26B1-like (XM_017434276.2)
CYP46A1 (MF326156.1)
CYP46A2 (MF326157.1)
CYP51A1 (MF326160.1)
CYP11A1 (MF326140.1)
CYP11A1 (MF326141.1)
CYP11A1 (MF326141.1)
CYP11A1 (MF326141.1)
CYP24A1 (MF326148.1)
CYP27A1 (MF326152.1)
CYP27A3 (MF326153.1)
CYP27B1 (MF326154.1)
CYP27C1 isoform X2 (XM_017440589.2)
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1e–40
7e–139
3e–22
0.0
0.0
0.0
0.0
0.0
CYP27C1-like (MK947996)
MT
Oreochromis niloticus
CYP27C1 isoform X1 (XP_005452352.1)
0.0