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\begin{document}

\title{De novo sequencing and chromosomal-scale genome assembly of leopard
coral grouper, Plectropomus leopardus}



\author[1]{QIAN ZHOU}%
\author[2]{Xinyu Guo}%
\author[3]{Yang Huang}%
\author[2]{Haoyang Gao}%
\author[1]{Hao Xu}%
\author[4]{Shanshan Liu}%
\author[1]{Weiwei Zheng}%
\author[4]{Tianshi Zhang}%
\author[3]{Changxu Tian}%
\author[3]{Chunhua Zhu}%
\author[5]{Haoran Lin}%
\author[6]{Songlin Chen}%
\affil[1]{Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences}%
\affil[2]{BGI-Qingdao, BGI-Shenzhen}%
\affil[3]{Guangdong Ocean University}%
\affil[4]{Affiliation not available}%
\affil[5]{Sun Yat-Sen University}%
\affil[6]{Yellow Sea Fisheries Research Institute}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
The leopard coral grouper, Plectropomus leopardus, belonging to genus
Plectropomus, family Epinephelinae, is a carnivorous coral reef fish
widely distributing in the tropical and subtropical water of
Indo-Pacific Oceans. Due to its appealing body appearance and delicious
taste, P. leopardus has become a popular commercial fish for aquaculture
in many countries. However, the lack of genomic and molecular resources
for P. leopardus hinders its biological studies and genomic breeding
programs. Here we report the de novo sequencing and assembly of P.
leopardus genome using 10\selectlanguage{ngerman}× Genomics and high-throughput chromosome
conformation capture (Hi-C) technologies. Using 127.36 Gb 10× Genomics
we generated a 902.90 Mb genome assembly with a contig and scaffold N50
of 31.8 Kb and 33.47 Mb, respectively. The scaffolds were clustered and
oriented into 24 pseudo-chromosomes with 13.39 Gb valid Hi-C data. BUSCO
analysis showed that 95.3\% of the conserved single-copy genes were
retrieved, indicating a good entirety of the assembly. We predicted
23,234 protein-coding genes, among which 96.5\% were functional
annotated. The P. leopardus genome provides a valuable genomic resource
for genetics, evolutionary and biological studies of this species.
Particularly, it is expected to benefit the development of genomic
breeding programs in the farming industry.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Abstract}

The leopard coral grouper, \emph{Plectropomus leopardus} , belonging to
genus Plectropomus, family Epinephelinae, is a carnivorous coral reef
fish widely distributing in the tropical and subtropical water of
Indo-Pacific Oceans. Due to its appealing body appearance and delicious
taste, \emph{P. leopardus} has become a popular commercial fish for
aquaculture in many countries. However, the lack of genomic and
molecular resource for \emph{P. leopardus} hinders its biological
studies and genomic breeding programs. Here we report the \emph{de
novo}sequencing and assembly of \emph{P. leopardus} genome using a
combination of 10\selectlanguage{ngerman}× Genomics, high-throughput chromosome conformation
capture (Hi-C) and PacBio long read sequencing technologies. The genome
assembly has a total length of 881.55 Mb with a scaffold N50 of 34.15
Mb, consisting of 24 pseudo-chromosomes scaffolds. BUSCO analysis showed
that 97.2\% of the conserved single-copy genes were retrieved,
indicating a good entirety of the assembly. We predicted 25,248
protein-coding genes, among which 96.5\% were functional annotated.
Comparative genomic analyses revealed that gene family expansions
in\emph{P. leopardus} were associated with immune related pathways. In
addition, we identified 5,178,453 SNPs based on genome resequencing of
54 individuals. The \emph{P. leopardus} genome and variation data
provide valuable genomic resource for genetics, evolutionary and
biological studies of the grouper species. Particularly, it is expected
to benefit the development of genomic breeding programs in the farming
industry.

\textbf{Keywords} : leopard coral grouper, \emph{Plectropomus leopardus}
, genome sequencing, chromosomal assembly, genome annotation

\textbf{Introduction}

Groupers (Perciformes, Epinephelinae), acting as the main carnivorous
predators, are important biological members in the coral reef ecosystem.
The abundance and variety of the groupers have great impact on this
fairly complex ecosystem. The family Epinephelidae is comprised of
approximately 165 species in 16 genus, classified based on 12S, 16S and
histone H3 gene sequences (Craig \& Hastings, 2007). However, the
taxonomy of these species is still in discussion with a previous
classification of family Serranidae (Saad, 2019). Therefore, more
genomic evidence are needed to clarify the taxonomy of groupers.
However, to date, only two genome sequences of grouper species, the
giant grouper (\emph{Epinephelus lanceolatus} ) (Zhou et al., 2019) and
the red-spotted grouper (\emph{Epinephelus akaara} ) (Ge et al., 2019),
are available, which significantly hinders the taxonomical, evolutionary
and biological studies of the groupers.

The leopard coral grouper, \emph{Plectropomus leopardus} , also namely
coral trout or spotted coral grouper, is a representative fish in genus
Plectropomus. It naturally inhabits in tropical or subtropical waters of
the Indo-Pacific Oceans from southern Japan to Australia and eastwards
to the Caroline Islands, Fiji and Tonga (Froese \& Pauly, 2019). Like
most other groupers, \emph{P. leopardus} is protogynous
hermaphroditism,~starting out of females and sex-reverse to male later
in life (Ferreira, 1995). They are also characterized by complex social
structures and a variety of body colour, such as bright red and brown.

Due to low-fat and high-protein flesh, impressive taste and beautiful
skin colour, \emph{P. leopardus} has recently become an important
commercial species worldwide with a high trading price (Fabinyi, 2012).
The increasing demands trigger a rapid development of the aquaculture
of\emph{P. leopardus} in many Asian countries and regions, which
requires advanced aquaculture technology, such as genomic breeding and
metabolic control.

Previous studies in \emph{P. leopardus} mainly focused on species
classification (Harrison et al., 2014; Herwerden et al., 2006),
reproductive biology (Zeller, 1998), physiological stress responses
(Frisch \& Anderson, 2005) and behaviour biology (Leis \& Carsonewart,
1999; Light \& Jones, 1997). Genomic and genetic studies of this species
were reported in the development of microsatellite markers (Zhang et
al., 2010), the transcriptomic comparison in two colour morphs (Wang et
al., 2015), and the muscle metabolic mechanism revealed by gene
expression and metabolome analyses (Mekuchi et al., 2017). The
insufficient exploitation of genomic resource largely limits the genetic
study, conservation and genomic breeding of this species.

Here we report a chromosomal-scale genome assembly and annotation
of\emph{P. leopardus} , which was generated using 10× Genomic and Hi-C
sequencing technology. The well-annotated genome and the massive
sequencing data of leopard coral grouper will provide a crucial resource
for genomic, biological and ecological studies of this species, and will
efficiently promote its genomic breeding program in aquaculture. In
addition, this genome will facilitate future evolutionary, phylogenetic
and comparative studies, as well as resource conservation within family
Epinephelinae.

\section*{Materials and methods}

{\label{materials-and-methods}}

\textbf{2.1 Sampling and sequencing}

Genomic DNA was extracted using a QIAamp DNA purification kit (Qiagen,
Germany) according to the manufacturer's instruction. The integrity and
quality of the extracted DNA was evaluated using 1\% gel
electrophoresis. The DNA concentration was assessed using a Pultton
DNA/Protein Analyzer (Plextech, USA). DNA with a total amount \selectlanguage{english}[?] 20 \selectlanguage{greek}μ\selectlanguage{english}g,
1.8 \textless{} OD\textsubscript{260/280} \textless{} 2.0 and a
concentration\textgreater{} 12.5 ng/\selectlanguage{greek}μ\selectlanguage{english}l were used to construct the
sequencing libraries.

A 10\selectlanguage{ngerman}× Genomics linked-read library was constructed using the standard
protocol (10× Genomics, San Francisco, USA). Raw reads were produced
using BGISEQ-500 platform (BGI, Shenzhen, China), with read lengths of
2×100 bp. The raw reads were then filtered with FastQC
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and QC-Chain
(Zhou et al., 2013). The duplicated reads, the adaptor-contaminated
reads and the reads having a quality value lower than 20 (representing
1\% error rate) were filtered.

To obtain a chromosome-scale genome assembly, we constructed Hi-C
library for sequencing. Genomic DNA in blood samples was fixed with
formaldehyde in a concentration of 1\% and the fixation was terminated
using 0.2 M glycine. A Hi-C library was prepared following the Hi-C
library protocol (Gong et al., 2018) and then sequenced using a
BGISEQ-500 sequencing platform (BGI, Shenzhen, China).

For long-read sequencing, we constructed a SMRTbell library with a
fragment size of 20 Kb using the SMRTBell template preparation kit 1.0
(PacBio, USA) following the manufacturer's protocol. The library was
sequenced with a PacBio Sequel system, and data from one SMAT cell were
generated.

\textbf{2.2 Tissue collection and RNA sequencing}

To facilitate the prediction of protein-coding genes, we extracted total
RNA from six tissues of the leopard coral grouper, including gonad,
liver, skin, spleen, muscle and fin, using TRIzol reagent (Invitrogen,
CA, USA). RNA integrity and quantity was evaluated using the Agilent
2100 Bioanalyzer (Agilent, USA). The six RNA-Seq libraries, which were
prepared using the NEBNext Ultra RNA Library Prep Kit (Illumina, USA)
following the manufacturer's protocol, was sequenced on a BGISEQ-500
sequencing platform (BGI, Shenzhen, China), producing 70.36 Gb raw data.
The quality control of the raw reads were performed with a QC pipeline
for RNA-Seq data, RNA-QC-Chain (Zhou et al., 2018), and 69.34 Gb clean
RNA-Seq data were remained for further analyses (Table 1).

\textbf{2.3 Genome assembly and quality assessment}

We estimated the genome size and heterozygosity of the \emph{P.
leopardus} genome by k-mer analysis, using the quality-filtered reads.
The k-mer counts frequencies was computed with Jellyfish (v2.2.10)
(Marcais \& Kingsford, 2011) using k = 17 and a maximum kmer count of
10,000. The k-mer distribution was measured and plotted using
GenomeScope (Vurture et al., 2017). The genome size was calculated with
the formula G = N\textsubscript{17-mer} /D\textsubscript{17-mer}, where
the N\textsubscript{17-mer} is the total number of 17-mers, and
D\textsubscript{17-mer} denotes the peak frequency of 17-mers.

We \emph{de novo} assembled the 10× Genomics short reads into contigs
and scaffolds using Supernova (v1.2) (Weisenfeld et al., 2017) . Gaps in
the initial assembly were filled with Gapcloser (v1.12) (Luo et al.,
2012) with the parameters of ``avg\_ins=364, max\_ins=500 and
min\_ins=260''. The draft assembly was then anchored and oriented into a
chromosomal-scale assembly using the Hi-C scaffolding approach. Firstly,
the raw Hi-C reads were filtered with HiC-Pro (v2.8.0) (Servant et al.,
2015). Then 3d-dna (v170123) (Dudchenko et al., 2017) with parameters of
``-m haploid -s 0 -c 24'' was used to anchor the primary contigs and
scaffolds into chromosomes. The inter / intra-chromosomal contact maps
were built and visualized with Juicebox (Durand et al., 2016).

To further improve the integrity and accuracy of the genome assembly, we
employed TGS-GapCloser, which uses low depth (\selectlanguage{english}[?] 10x) single molecule
sequencing long reads without any error correction to close gaps in the
draft assembly (Xu et al., 2019). The long sequences were split into
three groups, including total reads (with options --min\_idy 0.2,
--min\_match 200 --r\_round 1), reads with length \selectlanguage{english}[?] 20 kb (with options
--min\_idy 0 --min\_match 0 --r\_round 3) and reads with length in
2-20kb (with options --min\_idy 0 --min\_match 0 --r\_round 3), and each
group were used to fill the corresponding aligned gaps.

The completeness of the genome assembly were assessed by Benchmarking
Universal Single-Copy Orthologs (BUSCO) (Waterhouse et al., 2017) and GC
content analyses. The single copy orthologues of actinopterygii\_obd9
(BUSCO, v2.0) were searched against the assembled genome using BUSCO
tool. The GC content and average sequencing depth across the genome were
also measured with 10 Kb non-overlapping sliding windows and the windows
harboring more than 50\% N's were filtered. No external contamination
was found in the genome.

\textbf{2.4 Repetitive sequence annotation}

We annotated the repetitive sequences in the \emph{P. leopardus} genome
with both homology searching in known repeat database and \emph{de
novo}predictions. Known repeats were identified using RepeatMasker
(v3.3.0) (Taraio-Graovac \& Chen, 2009) with the RepBase TE library
(v14.06) (Bao et al., 2015). RepeatProteinMask (v3.2.2) implemented in
RepeatMasker was used to detect the TE-relevant proteins. Novel repeats
were predicted using RepeatModeler (http://www.repeatmasker.org) based
on the\emph{de novo} repeat library constructed with LTR\_Finder (Xu \&
Wang, 2007) and RepeatScout (Price et al., 2005). In addition, we used
Tandem Repeat Finder (TRF, v4.09) (Benson, 1999) to identify the tandem
repeats in the genome with parameters of ``Match=2, Mismatch=7, Delta=7,
PM=80, PI=10, Minscore=50, and MaxPerid=2000''.

\textbf{2.5 Gene prediction and annotation}

Based on the repeat masked genome, we employed \emph{de novo} ,
homology-based and transcriptome-assisted predictions to detect the
protein-coding genes. \emph{De novo} gene prediction was performed using
Augustus (v2.7) (Stanke et al., 2006) with the \emph{Danio
rerio}training set and default settings. For homology-based prediction,
protein sequences of \emph{Danio rerio, Takifugu rubripes, Gasterosteus
aculeatus, Epinephelus lanceolatus, Epinephelus akaara, Oryzias
latipes}and \emph{Cynoglossus semilaevis} were downloaded from NCBI
database and aligned to the \emph{P. leopardus} genome using tBLASTn
(E-value\selectlanguage{english}[?]1e-5). The homologous genome sequences were then aligned
against the matching proteins using GeneWise (v2.4.0) (Doerks et al.,
2002) for accurate spliced alignments. Transcriptomic data were
generated from six RNA-Seq libraries constructed with six tissues,
including gonad, liver, skin, spleen, muscle and fin, respectively. A
total of 69.34 Gb clean data were aligned to the assembled genome
sequences using HISAT2 (v2.0.10) (Pertea et al., 2016) and the putative
transcript structures were detected using StringTie (v2.1.1) (Pertea et
al., 2016). The candidate protein-coding regions within transcript
sequences were then predicted with TransDecoder (v5.5.0)
(https://github.com/TransDecoder/TransDecoder/). Finally, genes
predicted from the above methods were merged into a consensus gene set
using Glean (Elsik et al., 2007).

\textbf{2.6 Phylogenetic analysis and divergent time estimation}

To define the phylogenetic tree, we identified the orthologous gene
families by comparing the protein and cDNA sequences among the \emph{P.
leopardus} and nine teleosts, including \emph{Takifugu rubripes,
Gasterosteus aculeatus, Oreochromis niloticus, Oryzias latipes, Danio
rerio, Gadus morhua, Lepisosteus oculatus, Epinephelus lancelatus}
and\emph{Epinephelus akaara.} TreeFam (v4.0) (Li et al., 2006) was used
to define the orthologous and paralogous relationships among all the
organisms. Using the single-copy orthologous genes, a phylogenetic tree
was generated with Bayes model using PhyML (v3.0) (Guindon et al., 2010)
with 500 bootstrap replications. The MCMCtree program implemented in the
PAML package (Yang, 2007) was used to predict the divergence times. The
divergent time of \emph{D. rerio} and \emph{T. rubripes} , \emph{G.
aculeatus} and \emph{T. rubripes} , and \emph{O. latipes}
and\emph{G.morhua} were used as calibration time, which was downloaded
from the TimeTree database (http://www.timetree.org/).

\textbf{Comparative genomic analyses}

We compared the genome assembly of \emph{P. leopardus} with the
published genomes of grouper species, including \emph{E. lanceolatus}and
\emph{E. akaara} . Firstly, to reveal their collinearity relationships,
we aligned the chromosomes of the \emph{P. leopardus} to that of the
other Epinephelus species using the LASTZ tool (v1.02.00) with default
options (Harris, 2007). The chromosomal collinearity were constructed
with the mapped regions with lengths \textgreater{}2 Kb for
visualizations using Circos (v0.69) (Krzywinski et al., 2009). Secondly,
we identified the orthologous groups among these three species, using
all-to-all Blast (E-value \selectlanguage{english}[?]1e-5, identity \selectlanguage{english}[?] 80\%) and identified the
expanded and contracted gene families using CAFE (De Bie et al., 2006).
The enrichment analyses based on GO and KEGG annotations were performed
to identify functional implications of the expanded and contracted genes
(Fisher's exact test, adjusted \emph{p} -value \textless{} 0.05).

\textbf{2.8. Genome-resequencing and SNP calling}

For genome resequencing, we sampled a total of 54 individuals of\emph{P.
leopardus} from two farming factories in Hainan Province of China.
Genomic DNA were extracted from fin tissues of each fish. Pair-ended
libraries were constructed according to the standard protocol (Illumina,
USA), with an insert size of 300 bp. The sequencing was conducted on the
Illumina HiSeq 2000 platform. To avoid the potential influence of
low-quality reads in the subsequent analysis, raw reads were checked and
filtered using QC-Chain (Zhou et al., 2013), removing reads in the
following types: (1) reads containing \textgreater{} 10\% unidentified
nucleotides (N's); (2) duplicated reads; (3) reads aligned to adapters,
and (4) reads with 10\% bases having quality score \textless{} 20.

The quality-filtered reads were mapped to the genome assembly using BWA
software with default parameters (Li \& Durbin, 2009). SNP calling were
then performed on a population-scale using GATK (McKenna et al., 2010).
The allele frequencies were calculated using VCFtools. We further
filtered the SNPs, and only SNPs satisfying the criteria of quality of
depth \textgreater{} 2.0, mapping quality \textgreater{} 40, SNP quality
\textgreater{} 30, minor allele frequency (MAF) \selectlanguage{english}[?] 0.05 and missing rate
\selectlanguage{english}[?] 0.1 were kept in the final SNP set.

\textbf{Results and discussion}

\textbf{3.1} \textbf{Sequencing and genome size estimation}

Genomic DNA of a \emph{P. leopardus} individual at around 1 year-old
(0.5 kg) \textbf{(Figure 1)} , which was provided by Mingbo Aquatic
Company (Laizhou, China), was used for genome sequencing. A 10x Genomics
linked-read library was constructed and sequenced on BGISEQ-500 platform
(BGI, Shenzhen, China), producing a total of 152.61 Gb of raw reads.
After quality filtering, we obtained 127.36 Gb clean data (\textbf{Table
1} ). For the Hi-C sequencing, we obtained 631,842,593 raw read pairs,
amounting to 126.37 Gb Hi-C data. Quality control on the Hi-C data
finally resulted to 10.60\% of the total raw reads as valid Hi-C reads,
with two ends mapped to different contigs, which are useful for Hi-C
scaffolding (\textbf{Table 1} ). Single molecule sequencing with PacBio
technology generated 1,255,828 reads for 18.05 Gb (\textbf{Table 1} ).

\textbf{3.2 Chromosomal-level genome assembly}

Based on the clean data, we estimated the genome size to be 871 Mb with
the 17-kmer analysis. A dominant peak of the 17 k-mer distribution
corresponding to the homozygous peak was demonstrated (\textbf{Figure
2a} ) and the heterozygosity was estimated to be 0.635\%.

The 10x Genomics short reads were \emph{de novo} assembled with
Supernova software (v1.2) (Weisenfeld et al., 2017). The contigs and
scaffolds in the draft assembly were then anchored and oriented into a
chromosomal-scale assembly using the Hi-C scaffolding approach
(\textbf{Figure} \textbf{2b} ). As a result, we obtained a draft genome
assembly of 902.46 megabase (Mb) in length, with a contig of 33.60 Kb.
To further improve the completeness and accuracy of the genome assembly,
we used PacBio long sequence reads, with a depth of \textasciitilde{}20
x, to close the gaps in the assembly using TAG-Gapcloser. Finally, the
total length of the \emph{P. leopardus} genome was 881.55 Mb, with a
contig N50 of 855.69 Kb and a scaffold N50 of 34.14 Mb (\textbf{Table 2}
). The genome assembly had 24 pseudo-chromosomes, with chromosome
lengths ranging from 15.72 Mb to 41.71 Mb (\textbf{Supplementary
TableS1} ).

BUSCO analysis showed that the assembly retrieved 97.2\% of the
conserved single copy orthologue genes, including 94.0\% of the complete
and 3.2\% fragmented genes (\textbf{Table 3} ). The distribution of GC
content and sequencing depth were relatively concentrated, with an
average GC content of 39.65\%.

\textbf{3.3 Repetitive sequence annotation}

The consensus and non-redundant repetitive sequences were obtained by a
combination of known, novel and tandem repeats, generating a total of
298.99 Mb of repetitive sequences, occupying 33.38\% of the whole genome
assembly (\textbf{Table 4} ). The repetitive sequences was comprised of
DNA transposons in 146.68 Mb (16.37\% of the assembly), long
interspersed elements in 48.68 Mb (LINEs; 5.43\%), short interspersed
nuclear elements in 3.41 Mb (SINEs; 0.38\%), long terminal repeats in
38.27 Mb (LTRs; 4.27\%), and unknown repeats in 87.71 Mb (9.79\%)
(\textbf{Table 4} ).

\textbf{3.4 Gene prediction and annotation}

Combining the results from \emph{de novo} , homology-based and
transcriptome-assisted predictions, we successfully generated a
non-redundant gene set composing of 25,248 protein-coding genes
(\textbf{Table 5} ). The statistics of the predicted gene models were
compared to other teleost species, including \emph{D. rerio} , \emph{O.
latipes} and \emph{T. rubripes} , showing similar distribution patterns
in mRNA length, CDS length, exon length, intron length and exon number
(\textbf{Figure} \textbf{3)} .

We annotated the predicted genes by comparing the protein sequences in
several public gene databases, including SwissProt, KEGG and TrEMBL,
using BLASTp (E-value\selectlanguage{english}[?]1e-5). As a result, 92.3\%, 84.6\% and 96.4\% of
the predicted genes got positive hits in SwissProt, KEGG and TrEMBL
database, respectively. We also employed InterProScan (v5.0) (Jones et
al., 2014) to identify protein domains in multiple protein domain
databases of InterPro (ProDom, HAMAP, PANTHER, TIGRFAMs, PRINTS, PIRSF,
Gene3D, COILS, PROSITE, Pfam, SMART) (Mitchell et al., 2019) and Gene
Ontology (GO), and 88.9\% and 70.3\% of the predicted genes were
annotated in InterPro and GO database, respectively. Finally, a total of
24,364 genes (96.48\% out of all predicted genes) were successfully
functional annotated in at least one of these databases
(\textbf{Supplementary Table} \textbf{S2} ).

For non-coding genes, 843 tRNAs were identified using tRNAscan-SE (Chan
\& Lowe, 2019). 1,230 rRNA genes and 324 microRNAs were identified by
searching homology against the human rRNA sequence and miRBase (Kozomara
\& Griffiths-Jones, 2014) database, respectively. Small nuclear RNAs
were annotated by the infernal tool (Nawrocki \& Eddy, 2013)
(http://infernal.janelia.org/) using Rfam database (Kalvari et al.,
2018) (\textbf{Supplementary Table S3} ).

\textbf{3.5 Phylogeny and divergent time}

Using the genomes and genes of 10 selected teleosts, we identified a
total of 4,134 were single copy orthologues, based on which a
phylogenetic tree was constructed, revealing the phylogenetic
relationships of the selected teleost (\textbf{Figure} \textbf{4} ). We
found that the \emph{P. leopardus} diverged \textasciitilde{} 57.2
million years ago (mya) from the common ancestor with the linage
of\emph{E. lanceolatus} and \emph{E. akaara} . The most closely related
species to the grouper linage is G. \emph{aculeatus} , which separated
with their common ancestor \textasciitilde{}71.2 mya (\textbf{Figure4}
).

\textbf{3.6 Genomic comparison with other groupers}

Currently, genome sequences are only available for two groupers (genus
Epinephelus), including \emph{E.} \emph{lanceolatus} and \emph{E.akaara}
. To reveal the similarities and differences of the grouper genomes, we
conducted functional comparative genomic analyses. The\emph{P.
leopardus} and the Epinephelus grouper species have the same karyotype
(2\emph{n} =48), and the chromosome syntenic comparisons showed that
they have a high level of genomic collinearity (\textbf{Figure 5a} ).

Furthermore, gene family evolution was analyzed by constructing
orthologous gene families. The numbers of gene families were highly
similar in the three groupers, with 18,336, 18,674 and 18,007 in\emph{P.
leopardus} , \emph{E. lanceolatus} and \emph{E. akaara} , respectively.
A total of 15,497 genes were shared by the three teleost, and 4,427
genes were specific to \emph{P. leopardus} (\textbf{Figure 5b} ). We
also found that \emph{P. leopardus} shared 17,375 genes with\emph{E.
lanceolatus} , and 16,929 genes with \emph{E. akaara} , respectively. In
\emph{P. leopardus} , a total of 799 gene families were expanded
compared to its most recent common ancestor of the Epinephelus linage
(\textbf{Figure 5c} ), among which 126 were significantly expanded
(\emph{p} \textless{} 0.05). The expanded gene families were
significantly enriched in a number of immune systems and immune related
pathways (\textbf{Figure} \textbf{5d} , \textbf{Supplementary Table S4}
), indicating an improved capacity for resistance to diseases in
\emph{P. leopardus} . A total of 12 gene families were contracted,
however, no enriched KEGG pathway was found.

\textbf{3.7 Genome re-sequencing for SNP calling}

The genome re-sequencing of 54 individuals of \emph{P.
leopardus}produced a total of 2,232,057,448 raw sequencing reads. After
quality filtering, we obtained a total of 668.90 Gb clean data, with an
averagely 12.39 Gb for each fish and a mean sequencing depth of 14.0x
(\textbf{Supplementary Table S5} ). The clean reads were aligned to the
assembled genome for each individuals, with 99.63 \% of the total reads
mapped to the genome. Based on these alignments, we identified a total
of 5,178,453 SNPs after quality filtering. The location and effects of
these SNPs were also annotated, showing that 132,709 and 57,082 were
synonymous and nonsynonymous SNPs, respectively, locating in coding
regions (\textbf{Table 6} ). These SNPs will provide an important
genomic resource for the genetic studies, such as population structure
analysis, dissection of agronomical traits, identification of selective
sweeps, and for genomic selective breeding for superior strains. In the
future work, we will use these genomic variations and recorded
phenotypes of the corresponding individuals to dissect the genomic
associations and to identify key genes playing important roles in the
phenotypic differences.

\textbf{Conclusion}

Here we provide a chromosomal-scale genome assembly of the \emph{P.
leopardus} by integration of 10x Genomics, Hi-C and PacBio long read
sequencing technologies. The genome assembly and annotation supplies the
first genome of genus Plectropomus and implement the Epinephelidae
genomes, in addition to \emph{E. lanceolatus} and \emph{E. akaara} ,
thus supplying important genomic data for whole-genome analysis to
elucidate the population genetics, evolution and to dissect the genetic
diversity underlying their phenotypic traits and adaptions. The genomic
variations, together with their functional annotations, will promote
accurate genetic analysis and accelerate the genomic breeding programs
in aquaculture of the \emph{P. leopardus} .

\textbf{Acknowledgements}

This work was supported by the Fund of Southern Marine Science and
Engineering Guangdong Laboratory (Zhanjiang) (ZJW-2019-06), Youth Talent
Program Supported by Laboratory for Marine Fisheries Science and Food
Production Processes, Pilot National Laboratory for Marine Science and
Technology (Qingdao) (2018-MFS-T08), Shandong Superior Variety Project
(2016LZGC009)\selectlanguage{english},the AoShan Talents Cultivation Program Supported by Pilot
National Laboratory for Marine Science and Technology (Qingdao)
{[}2017ASTCP-OS15{]} and Shandong Taishan Scholar Climbing Project.

\textbf{Author contributions}

S.L.C., H.R.L and C.H.Z. conceived the project. S.L.C. and C.H.Z.
managed the project. Q.Z., H.X. and C.X.T. collected the sequencing
samples. X.Y.G. extracted the genomic DNA and performed the genome
sequencing. Q.Z., G.H and Y. H. analyzed the data. Q.Z. wrote the
manuscript. All authors reviewed and approved the final manuscript.

\textbf{Conflicts of interests}

None.

\textbf{Data Accessibility Statement}

The assembled genome has been deposited at DDBJ/ENA/GenBank
(VJNF00000000) and NCBI Assembly database with the GenBank accession
GCA\_008729295.1. Raw sequencing data for \emph{P. leopardus} genome has
been deposited in the Sequence Read Archive (SRA) (SRR9330009,
SRR11482498). Genome re-sequencing data has been deposited at BioProject
PRJNA622646.

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\textbf{Tables}

\textbf{Table 1. Sequencing data for the \emph{P. leopardus} genome
assembly.}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Sequencing technology} & \textbf{Raw data (Gb)} & \textbf{Clean
data (Gb)} & \textbf{Mean} \textbf{read length (bp)}\tabularnewline
10\selectlanguage{ngerman}× Genomics & 152.61 & 127.36 & 100\tabularnewline
Hi-C & 126.37 & 13.39 & 100\tabularnewline
PacBio & 180.46 & / & 14.37k\tabularnewline
RNA-Seq & 70.36 & 69.34 & 100\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2. Statistics of the \emph{P. leopardus} genome assembly.}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Seq type} & \textbf{Total number} & \textbf{Total length (bp)} &
\textbf{N50 (bp)} & \textbf{N90 (bp)} & \textbf{Max length
(bp)}\tabularnewline
\textbf{scaffold} & 50,461 & 881,551,488 & 34,146,761 & 8,092 &
41,705,749\tabularnewline
\textbf{contig} & 54,036 & 865,740,848 & 855,686 & 7,000 &
4,608,261\tabularnewline
\bottomrule
\end{longtable}



\textbf{Table} \textbf{3. BUSCO analysis result of the \emph{P.
leopardus} genome.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{Gene number} & \textbf{Percentage}\tabularnewline
\textbf{Complete BUSCOs (C)} & 4,307 & 94.0\tabularnewline
\textbf{Complete and single-copy BUSCOs (S)} & 4,167 &
90.9\tabularnewline
\textbf{Complete and duplicated BUSCOs (D)} & 140 & 3.1\tabularnewline
\textbf{Fragmented BUSCOs (F)} & 147 & 3.2\tabularnewline
\textbf{Missing BUSCOs (M)} & 130 & 2.8\tabularnewline
\textbf{Total BUSCO groups searched} & 4,584 & 100\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table} \textbf{4. Statistics of repetitive sequences in
the\emph{P. leopardus} genome.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Identification method} & \textbf{Repeat size} & \textbf{\% of
genome}\tabularnewline
TRF & 34,303,179 & 3.83\tabularnewline
RepeatMasker & 49,936,126 & 5.57\tabularnewline
ProteinMask & 11,710,155 & 1.31\tabularnewline
\emph{De novo} & 280,223,461 & 31.29\tabularnewline
Total & 298,989,520 & 33.38\tabularnewline
& \textbf{Combined TEs} & \textbf{Combined TEs}\tabularnewline
\textbf{Biological classification} & \textbf{Length (bp)} & \textbf{\%
in genome}\tabularnewline
DNA & 146,681,333 & 16.37\tabularnewline
LINE & 48,680,003 & 5.43\tabularnewline
SINE & 3,413,395 & 0.38\tabularnewline
LTR & 32,129,629 & 4.27\tabularnewline
Other & 18,438 & 0.002\tabularnewline
Unknown & 87,713,772 & 9.79\tabularnewline
Total & 275,295,833 & 30.74\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table} \textbf{5. Statistics of gene predictions in the \emph{P.
leopardus} genome}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
& \textbf{\#Gene set} & \textbf{Number of genes} & \textbf{CDS+intron
len} & \textbf{CDS len} & \textbf{exon len} & \textbf{intron len} &
\textbf{Exons per gene}\tabularnewline
\textbf{Homolog} & \emph{Danio rerio} & 25,787 & 23,126.00 & 1,524.78 &
181.24 & 2,913.94 & 8.41\tabularnewline
& \emph{Takifugu rubripes} & 21,677 & 15,087.57 & 1,424.34 & 177.75 &
1,948.16 & 8.01\tabularnewline
& \emph{Gasterosteus aculeatus} & 28,700 & 14,201.45 & 1,460.43 & 170.76
& 1,687.02 & 8.55\tabularnewline
& \emph{Epinephelus lanceolatus} & 23,869 & 14,232.13 & 1,400.68 &
169.48 & 1,766.28 & 8.26\tabularnewline
& \emph{Cynoglossus semilaevis} & 33,866 & 19,789.49 & 1,521.26 & 177.79
& 2,417.60 & 8.56\tabularnewline
& \emph{Epinephelus akaara} & 23,142 & 16,439.61 & 1,606.08 & 177.73 &
1,845.79 & 9.04\tabularnewline
& \emph{Oryzias latipes} & 19,672 & 23,455.32 & 1,570.11 & 191.37 &
3,037.64 & 8.20\tabularnewline
De novo & Augustus & 24,499 & 16,070.20 & 1,435.18 & 172.04 & 1,993.26 &
8.34\tabularnewline
\textbf{RNA} & Transdecoder & 31,207 & 17,648.02 & 1,428.34 & 170.77 &
1,501.25 & 8.36\tabularnewline
Glean & Glean & 25,248 & 15,872.47 & 1,632.85 & 181.54 & 1,781.17 &
8.99\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 6.} \textbf{Summary of the single nucleotide polymorphisms
(SNPs) effects in \emph{P. leopardus}}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Type} & \textbf{Number of SNPs}\tabularnewline
Intergenic & 1,740,250\tabularnewline
Intron & 2,453,403\tabularnewline
Upstream & 411,242\tabularnewline
Downstream & 389,553\tabularnewline
Splice\_site & 16,105\tabularnewline
Start\_lost & 82\tabularnewline
Codon\_change & 7\tabularnewline
Synonymous\_coding & 117,662\tabularnewline
Non\_synonymous\_coding & 49,633\tabularnewline
Stop\_gained & 438\tabularnewline
Stop\_lost & 78\tabularnewline
Total & 5,178,453\tabularnewline
\bottomrule
\end{longtable}





\textbf{Figure Legends}

\textbf{Figure 1. A photograph of a red \emph{P. leopardus} (by Qian
Zhou).}

\textbf{Figure 2. Genome size estimation and assembly.} (a) Graph
of\emph{k} -mer frequency distribution (\emph{k} = 17) generated from
127.36 Gb sequencing data of \emph{P. leopardus} . The highest peak at
coverage 66\selectlanguage{ngerman}× corresponds to the homozygous peak. The minor peak at
coverage 35× corresponds to the heterozygous peak. The minor peak at
coverage 138× corresponds to duplications. The illustrations of the
lines are marked in the graph. (b) The Hi-C contact map of the \emph{P.
leopardus} genome. The color bar shows the contact density from white
(low) to red (high).

\textbf{Figure 3. Comparisons of the predicted gene models
between\emph{P. leopardus} genome and other teleost species.} (a) mRNA
length. (b) CDS length. (c) Exon length. (d) Intron length. (e) Exon
number.

\textbf{Figure 4.} \textbf{Phylogenetic tree constructed using 4134
single copy orthologues among the selected teleost, with 500
bootstraps.} The estimated divergent time (Mya, million years ago) and
the 95\% confidential intervals were labeled at each branch. The red
dots indicate the divergent time used for re-calibrations.

\textbf{Figure 5.} \textbf{Comparative genomic analyses of \emph{P.
leopardus}} \textbf{with two grouper species, \emph{E. lanceolatus}and
\emph{E. akaara})} . (a) Chromosomal collinearity between\emph{P.
leopardus} and \emph{E. lanceolatus.} The colorful bars (Chr1-24) and
the green bars (I-IIXIV) indicate each of the 24 chromosomes in \emph{P.
leopardus} and \emph{E. lanceolatus} , respectively. (b) Venn diagram of
the genes from the three grouper species. (c) Gene family expansion and
contraction. (d) The enrichment of KEGG annotations with the expanded
gene families in \emph{P. leopardus} (\emph{p} \textless{} 0.05).\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Fih/Fih}

\end{center}
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