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\begin{document}

\title{High-quality genome assembly of Chinese shrimp (Fenneropenaeus
chinensis) suggests genome contraction and adaptation to the environment}



\author[1]{Qiong Wang}%
\author[1]{Xianyun Ren}%
\author[2]{Ping Liu}%
\author[2]{Jitao Li}%
\author[1]{Jianjian Lv}%
\author[1]{Jiajia Wang}%
\author[1]{Haien Zhang}%
\author[1]{Wei Wei}%
\author[1]{Yuxin Zhou}%
\author[2]{Yuying He}%
\author[2]{Jian Li}%
\affil[1]{Chinese Academy of Fishery Science Yellow Sea Fisheries Research Institute}%
\affil[2]{Chinese Academy of Fishery Sciences}%


\vspace{-1em}



  
  \date{May 01, 2024}


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\selectlanguage{english}
\begin{abstract}
A high-quality reference genome is necessary to determine the molecular
mechanisms underlying important biological phenomena; therefore, in the
present study, a chromosome-level genome assembly of the Chinese shrimp
Fenneropenaeus chinensis was performed. Muscle of a male shrimp was
sequenced using PacBio platform, and assembled by Hi-C technology. The
assembled F. chinensis genome was 1,465.32 Mb with contig N50 of 472.84
Kb, including 57.73\% repetitive sequences, and was anchored to 43
pseudochromosomes, with scaffold N50 of 36.87 Mb. In total, 25,026
protein-coding genes were predicted. The genome size of F. chinensis
showed significant contraction in comparison with that of other penaeid
species, which is likely related to migration observed in this species.
However, the F. chinensis genome included several expanded gene families
related to cellular processes and metabolic processes, and the
contracted gene families were associated with virus infection process.
The findings signify the adaptation of F. chinensis to the selection
pressure of migration and cold environment. Furthermore, the selection
signature analysis identified genes associated with metabolism,
phototransduction, and nervous system in cultured shrimps when compared
with wild population, indicating targeted, artificial selection of
growth, vision, and behavior during domestication. The construction of
the genome of F. chinensis provided valuable information for the further
genetic mechanism analysis of important biological processes, and will
facilitate the research of genetic changes during evolution.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

The Chinese shrimp \emph{Fenneropenaeus chinensis} is one of the most
commercially important cultured shrimp species in China (Figure 1a). It
is mainly distributed in the Yellow Sea and Bohai Sea of China, and west
and south coast of the Korean Peninsula (Wang et al.,
2017).\emph{Fenneropenaeus chinensis} is an annual species; they migrate
to warmer sea areas to overwinter after mating and swim back to the
original coast for oviposition. With the development of aquaculture
techniques, \emph{F. chinensis} became the most important cultured
shrimp species in China in the 1990s. However, in 1993, the production
of \emph{F. chinensis} decreased sharply owing to an outbreak of the
white spot syndrome virus (WSSV) disease. Since 1997, breeding efforts
have been made to increase the production and disease resistance of
\emph{F. chinensis} . After continuous artificial selection, several
cultured varieties possessing excellent characteristics, such as high
yield, disease resistance, and stress resistance, have been developed
and cultured during the past two decades in China.

Because of their structural complexity and high heterozygosity (Yu et
al., 2015), only a few crustacean genomes have been completely
characterized. Recently, third-generation sequencing, characterized as
long reads, has helped to ameliorate the difficulties engendered by
heterozygosity and repetitive sequences in genome assembly (van Dijk,
Jaszczyszyn, Naquin, \& Thermes, 2018). An assembled genome of \emph{F.
chinensis} was published recently using Illumina short reads, PacBio
long reads and Hi-C technology; the assembled genome covered 1.58 Gb in
8768 scaffolds, with N50 length of 28.92 Mb (Yuan et al., 2021).
Furthermore, the reference genome of \emph{Litopenaeus vannamei} ,
which---similar to \emph{F. chinensis---} previously belonged to the
genus \emph{Penaeus} , has been reported also using both Illumina short
reads and PacBio long reads (X. Zhang et al., 2019), covering 1.66 Gb in
4,683 scaffolds, with N50 length of 605.56 Kb, and was improved to 31.30
Mb by using Hi-C technology (Yuan et al., 2021). Two genomic resources
for another \emph{Penaeus} species, \emph{Penaeus monodon} , was
released lately (Uengwetwanit et al., 2021; Van Quyen et al., 2020); the
improved genome was generated using long-read PacBio and long-range
Chicago, producing a final genome assembly of 2.39 Gb, with contig N50
length of 79 Kb (Uengwetwanit et al., 2021).

A high-quality reference genome is essential for resolving the molecular
mechanism of important biological processes (You, Shan, \& Shi, 2020).
In this research, an improved chromosome-level genome of \emph{F.
chinensis} was assembled using the PacBio sequencing platform and Hi-C
technology, in an attempt to explain the genetic changes during
evolution and domestication. Quality of the genome assembly could affect
the accuracy of following studies (You et al., 2020). Therefore, the new
version assembled genome provides a high-quality reference, and will be
a valuable resource for the further investigations of biological process
and mechanism in \emph{F. chinensis} .

\textbf{2 Materials and Methods}

\textbf{2.1 Sample collection and genome sequencing}

\emph{Fenneropenaeus chinensis} shrimp were obtained from the
conservation base of Haifeng Aquaculture Co., Ltd. (Weifang, Shandong
Province, China). Muscle of a 7-months-old male shrimp was collected and
frozen in liquid nitrogen immediately. Total genomic DNA was extracted
and sequenced for genome survey and genome construction. Genome
construction contains PacBio sequencing (Eid et al., 2009) and Hi-C
assembly (Lieberman-Aiden et al., 2009). DNA sample used for genome
survey and Hi-C assembly was sequenced by Illumina HiSeq platform
(Illumina, San Diego, USA), and used for PacBio sequencing was sheared
to 20 Kb and sequenced by PacBio Sequel platform (Pacific Biosciences,
Menlo Park, USA).

\textbf{2.2 Genome survey and assembly}

The genome size, repetitive sequence proportion and heterozygosity was
estimated by K-mer frequency distribution method with K-mer=17. The
genome size was revised by error rate: Revised size = Genome size
(1-error rate), the error rate refers to the proportion of K-mer with
depth of 1.

After adapter removal and filtered by minimum length of 50 bp, the
subreads from PacBio platform were assembled using wtdbg2 (Ruan \& Li,
2020), which uses the Fuzzy Bruijn Graph (FBG) approach. The FBG is not
as sensitive to small duplications as the De Bruijn Graph. To solve the
problem of high error rate, the Gapped Sequence Alignment method was
used. After quality control, the high-quality Hi-C sequencing data were
mapped to the draft genome by BWA software (H. Li \& Durbin, 2010), and
Samtools (H. Li et al., 2009) was used to remove duplicate and unmapped
data to obtain high-quality data. Next, the reads near the restriction
sites were extracted for assisted assembly (Burton et al., 2013).

\textbf{2.3 Genome annotation}

Structural annotation of the genome incorporates ab initio prediction,
homology-based prediction, and RNA-Seq assisted prediction. For gene
predication based on Ab initio, Augustus (v3.2.3) (Hoff \& Stanke,
2019), GeneID (v1.4) (Parra, Blanco, \& Guigo, 2000), Genescan (v1.0)
(Aggarwal \& Ramaswamy, 2002), GlimmerHMM (v3.04) (Majoros, Pertea, \&
Salzberg, 2004), and SNAP (2013-11-29) (Korf, 2004) were used in our
automated gene prediction pipeline. Six species, \emph{Litopenaeus
vannamei} , \emph{Hyalella azteca} , \emph{Eurytemora affinis}
,\emph{Daphnia pulex} , \emph{Drosophila hydei,} and \emph{Bombyx mori}
, were used for homology-based prediction. Sequences of homologous
proteins were downloaded from Ensembl and NCBI. Protein sequences were
aligned to the genome using tBLASTn (v2.2.26; E-value [?]
1e\textsuperscript{-5}), and then the matching proteins were aligned to
the homologous genome sequences for accurately spliced alignments using
GeneWise (v2.4.1) software (Birney, Clamp, \& Durbin, 2004). To optimize
the genome annotation, the RNA-Seq reads from different tissues (NCBI
BioProject: PRJNA558194) were aligned to the genome. Hierarchical
indexing for spliced alignment of transcripts (HISAT; v2.0.4) (Kim,
Langmead, \& Salzberg, 2015) and TopHat (v2.0.11) (Cole Trapnell,
Pachter, \& Salzberg, 2009) were used with default parameters to
identify exons and splice positions. The alignment results were then
used as input for Stringtie (v1.3.3) (Pertea et al., 2015) and Cufflinks
(v2.2.1) (C. Trapnell et al., 2010) with default parameters for
genome-based transcriptome assembly.

Gene functions were assigned according to the best match by aligning the
protein sequences to the SwissProt database using BLASTp (Altschul et
al., 1997) (E-value [?] 1e\textsuperscript{-5}). The motifs and domains
were annotated using InterProScan70 (v5.31) (Mulder \& Apweiler, 2007)
by searching against publicly available databases, including ProDom,
PRINTS, Pfam, simple modular architecture research tool (SMART), PANTHER
and PROSITE. The GO IDs for each gene were assigned according to the
corresponding InterPro entry. We also mapped the gene set to the KEGG
pathway database and identified the best match for each gene.

\textbf{2.4 Comparative genomics analysis}

The following 17 species were used for comparative genomic
analysis:\emph{F. chinensis} , \emph{P. monodon} , \emph{L. vannamei}
,\emph{Portunus trituberculatus} , \emph{Trinorchestia longiramus}
,\emph{H. Azteca} , \emph{Eurytemora affinis} , \emph{Drosophila
melanogaster} , \emph{Acyrthosiphon pisum} , \emph{Apis mellifera}
,\emph{Bombyx mori} , \emph{Zootermopsis nevadensis} ,
\emph{Cryptotermes secundus} , \emph{Pediculus humanus} ,
\emph{Tribolium castaneum} ,\emph{Anopheles gambiae} , and \emph{Limulus
polyphemus} . Genomic sequences were downloaded from NCBI. The gene set
of each species was filtered. In brief, when a gene possessed multiply
spliced transcripts, only the longest protein-coding transcripts were
retained for further analysis. Furthermore, genes were excluded if the
proteins encoded by them consisted of less than 30 amino acids or
contained degenerate bases or termination codons. The similarity between
protein sequences of all species was assessed using BLASTp (E-value [?]
1e\textsuperscript{-7}). The results were clustered using OrthoMCL (L.
Li, Stoeckert, \& Roos, 2003), with an expansion coefficient of 1.5.
Single-copy and multiple-copy homologous genes were filtered by these
analyses.

A phylogenetic tree was constructed using single-copy homologous genes
in the 17 species. MUSCLE (Edgar, 2004) was used for sequence alignment.
The final dataset was used to construct the phylogenetic tree with RAxML
(Rokas, 2011) using the maximum likelihood method. The best tree was
used as an input tree for divergence time estimation using MCMCTREE in
the PAML package (Yang, 2007), with the following parameters: burn in =
700, sample number = 1000,000, sample frequency = 2. Fossil calibrations
were used as priors for the divergence time estimation, as
below:\emph{P. monodon} and \emph{L. vannamei} {[}58--108 million years
ago (Mya) {]}, \emph{Acyrthosiphon pisum} and \emph{Eurytemora
affinis}(452--557 Mya), \emph{Zootermopsis nevadensis} and
\emph{Cryptotermes secundus} (103--156 Mya), \emph{Zootermopsis
nevadensis} and\emph{Pediculus humanus} (330--398 Mya), \emph{Anopheles
gambiae} and\emph{Drosophila melanogaster} (217--301 Mya),
\emph{Drosophila melanogaster} and \emph{Bombyx mori} (243--317 Mya),
\emph{Tribolium castaneum} and \emph{Apis mellifera} (308--366 Mya). In
the gene family expansion and contraction analysis, we filtered the gene
families with the results of the clustering analysis of gene families
using CAFE software (De Bie, Cristianini, Demuth, \& Hahn, 2006).
Protein sequences of single-copy homologous genes in \emph{F. chinensis}
, \emph{L. vannamei} , \emph{P. trituberculatus} and \emph{H. azteca}
were subjected to multiple alignment using MUSCLE to detect positive
selection. The ratios of nonsynonymous substitution per nonsynonymous
site (dN) to synonymous substitution per synonymous site (dS) were
calculated using the branch-site model of the Codeml tool included in
the PAML package. Likelihood ratio tests were applied to test for
positive selection.

\textbf{2.5 Selection signature analysis}

Cultured \emph{F. chinensis} shrimps were obtained from Haifeng
Aquaculture Co., Ltd, which has undergone continuous, high-intensity
artificial selection for growth traits for more than ten generations.
Wild \emph{F. chinensis} were captured from the northeast region of the
Huanghai Sea. We used 22 cultured and 22 wild shrimps to collect muscle
tissue and extract DNA. The DNA samples were sequenced using the BGISEQ
platform (BGI, Wuhan, China). Sequencing data were mapped to the
reference genome, and VCFtools (Danecek et al., 2011) was used to
calculate the fixation index (\emph{F} \textsubscript{ST}) and
nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}) in 40 kb sliding windows with a step size of 20
kb. Windows with the top 5\% values of both \emph{F} \textsubscript{ST}
and \selectlanguage{greek}π \selectlanguage{english}were considered as outliers. Genes in these windows were picked
for the subsequent Gene ontology (GO) and KEGG pathway enrichment
analysis.

\textbf{3 Results}

\textbf{3.1 Genome survey analysis}

A male shrimp (\emph{F. chinensis} ) was sequenced using the Illumina
HiSeq platform, and 125.99 Gb raw data were obtained. The K-mer analysis
(Figure S1) revealed that the genome size was 1,384.88 Mb, with 54.79\%
repetitive sequences, and the genome heterozygosity was 1.04\% (Table
1). The first six comparison species using 10,000 high quality reads
randomly were all homologous comparisons (Table S1), which indicates an
absence of exogenous contamination in our sample.

\textbf{3.2 Genome assembly and assessment}

A total of 350.81 Gb polymerase reads were obtained from the PacBio
platform. After adapter removal and quality control, we obtained 220.53
Gb subreads (coverage depth approximately 159 X), with average read
length of 16,922 bp (Table S2). The genome assembled using these
subreads was 1,465.32 Mb in size and contained 9,015 contigs with an N50
length of 472,841 bp (Table 1). According to the Benchmarking Universal
Single-Copy Orthologs (BUSCO) notation analysis using 1,066 lineal
homologous single-copy genes, 95.3\% of the genes were assembled
(complete: 94\%, fragmented: 1.3\%, missing: 4.7\%) (Table S3).
Furthermore, Core Eukaryotic Genes Mapping Approach (CEGMA) analysis
revealed that 234 genes were assembled from 248 Core Eukaryotic Genes
(CEGs), which accounted for 94.35\% (Table S4). Both analyses indicated
that the genome assembly was relatively complete. The mapping rate of
short reads from the Illumina platform was approximately 89.62\%, and
the coverage rate reached 96.15\%, indicating that the short reads and
the assembled genome had a good consistency (Table S5).

We obtained 203.8 Gb clean, non-duplicate data from the Illumina HiSeq
platform by Hi-C technology. The contigs were anchored to 1,063
scaffolds with N50 of 36.87 Mb (Table 1, Table S6), including 43
pseudochromosomes (Figure 1b, c) and 1,020 unplaced scaffolds. The total
length of the 43 pseudochromosomes was 1,451.52 Mb (Table S7), covered
99.00\% of the assembly, whereas the length of unplaced scaffolds was
14.60 Mb (Table S8).

\textbf{3.3 Genome annotation}

There were 57.73\% repetitive sequences in the \emph{F. chinensis}genome
(Table S9). A total of 25,026 genes, with average length of 11,290 bp
(including untranslated regions {[}UTRs{]}), average coding sequence
(CDS) length of 1,230 bp, and 5.94 exons per gene were predicted with
three methods---de novo prediction, homolog prediction, and RNA-seq
prediction (Table S10). Gene function of 76.7\% of the predicted genes
was annotated using multiple databases (Table S11). By comparing with
the non-coding RNA (ncRNA) database, 72,517 ncRNA genes were annotated,
including 59,026 miRNA genes, 2,592 tRNA genes, 24 rRNA genes, and
10,875 snRNA genes (Table S12).

\textbf{3.4 Comparative genomics}

According to the gene family clustering analysis of 17 Arthropoda
species, a total of 27,512 gene families were clustered, and 443 of them
were single-copy genes in all species (Figure 2a). Compared with\emph{P.
monodon} , \emph{L. vannamei,} and \emph{P. trituberculatus} ,\emph{F.
chinensis} possessed 593 unique gene families (Figure 2b). In general,
gene families unique to a species are responsible for the biological
characteristics of the species. GO enrichment analysis of the 593 unique
gene families indicated that they were enriched in 47 GO terms,
including structural constituent of cuticle, sodium and potassium ion
transport, and chitin metabolic process (Table S13). These genes were
enriched in nine KEGG pathways, including RNA polymerase, Huntington's
disease, and endocrine and other factor-regulated calcium reabsorption
pathways (Table S14).

Phylogenetic analysis based on the 443 single-copy homologous genes
revealed that \emph{F. chinensis} and \emph{P. monodon} diverged
approximately 44 Mya, after they diverged from \emph{L. vannamei} 70 Mya
(Figure 2c). The three penaeid shrimp species diverged from \emph{P.
trituberculatus} , which belongs to Family \emph{Portunidae} ,
approximately 271.5 Mya. The \emph{F. chinensis} genome showed 49
expanded and 51 contracted gene families in comparison with the \emph{P.
monodon} genome. The GO and KEGG enrichment analysis indicated that the
expanded gene families were mostly related to cellular process and
metabolic process, including chitin metabolism (Figure S2, Table
S15-16), whereas the contracted gene families were mostly associated
with infection with certain pathogens and phototransduction (Figure S3,
Table S17-18). Compared with \emph{L. vannamei} , \emph{P.
trituberculatus} and \emph{H. azteca} , a total of 63 genes were subject
to positive selection. These genes were mostly related to basic cellular
process (Table S19). One KEGG pathway named mRNA surveillance pathway
was enriched.

\textbf{3.5 Selection signature analysis}

A total of 447.23 Gb clean resequencing data of 42 shrimps (21 wild and
21 cultured) was obtained, which were mapped to the assembled genome.
Fixation index (\emph{F} \textsubscript{ST}) between wild shrimp and
cultured shrimp and nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}) in both populations were
calculated in 40 kb windows across the genome (Figure 3a). A total of
534 outlier genes were identified according to the\emph{F}
\textsubscript{ST}-\selectlanguage{greek}π \selectlanguage{english}conjoint analysis. Most of these genes were
involved in metabolic process (Figure S4). In addition, the
phototransduction-fly pathway and neuroactive ligand-receptor
interaction pathway were enriched by the target genes (Figure 3b).

\textbf{4 Discussion}

\textbf{4.1 Quality of this assembled genome improved}

Compared to the published genome of \emph{F. chinensis} (Yuan et al.,
2021), the quality of our assembled genome improved significantly (Table
S20). Primarily, the contig N50 increased from 59.00 Kb to 472.84 Kb,
approximately eight times, and scaffold N50 increased from 28.92 Mb to
36.87 Mb. Secondly, the assembled complete ortholog proportion enhanced
from 83.58\% to 94.00\% according to the BUSCO assessment, and the
genome coverage enhanced from 82.46\% to 96.15\% according to the
Illumina short read mapping test. In short, our assembled genome
improved in both fragment length and completeness.

Initially, we assembled the contigs to 44 pseudochromosomes, but the
heatmap revealed a low correlation between the contigs on the last
pseudochromosome (Figure S5). Therefore, only 43 pseudochromosomes were
assembled in the final version of the \emph{F. chinensis} genome. We
speculate that there are only 43 pairs of chromosomes in the somatic
cell of \emph{F. chinensis} , same as that in \emph{Marsupenaeus
japonicus} (Yuan et al., 2017).

\textbf{4.2 The Genome of \emph{F. chinensis} contracted compared to
other penaeid shrimps}

The K-mer analysis revealed that the \emph{F. chinensis} genome is 1.38
Gb in size. Compared to the genomes of other three penaeid
shrimps,\emph{L. vannam} ei (2.60 Gb) (X. Zhang et al., 2019), \emph{P.
monodon}(2.59 Gb) (Yuan et al., 2017), and\emph{M. japonicus} (2.28 Gb)
(Yuan et al., 2017), the genome size of\emph{F. chinensis} contracted
sharply (Table 2). Although \emph{F. chinensis} and \emph{L. vannam} ei
genomes contain a similar number of genes (25,026 vs. 25,596,
respectively), the proportion of repetitive sequences in \emph{F.
chinensis} is markedly lower (57.73\% vs. 78.00\%, respectively) (X.
Zhang et al., 2019). The lower repetitive sequences proportion could
contribute to the genome contraction, but not be the dominant factor,
because the \emph{P. monodon} , which closer to \emph{F. chinensis} in
evolution, has a similar repetitive sequences proportion with \emph{F.
chinensis} (62.5\%), but the genome size is also bigger (2.59 Gb)
(Uengwetwanit et al., 2021).

Research suggests that genome size is under selective pressure to
contract owing to constraints placed by an elevated metabolism on cell
size (Hughes \& Hughes, 1995; Olmo, 1983; Szarski, 1983). A similar
phenomenon is observed in vertebrates. The two living groups of flying
vertebrates, birds and bats, have constricted genome sizes compared with
their close relatives (Hughes \& Hughes, 1995). Research shows that
genome size contraction preceded flight during evolution (Organ \&
Shedlock, 2009). Likewise,\emph{F. chinensis} shrimp has a special
characteristic relative to \emph{L. vannamei} , \emph{P. monodon} , and
\emph{M. japonicus} , which is migration. Same as flight, migration
requires a high metabolic intensity, which closely linked with genome
size (Andrews, Mackenzie, \& Gregory, 2009; Vinogradov \& Anatskaya,
2006). The nucleotypic theory suggests that genome size affects nucleus
size and cell size (Bennett, 1971; Gregory, 2001). The cell size may
further influence housekeeping dynamics, cellular metabolism, and the
rate of cell division, leading to small cells with higher metabolic
rates (Kozlowski, Konarzewski, \& Gawelczyk, 2003). The results of gene
family expansion and contraction analysis and positive selection
analysis support this conjecture. The \emph{F. chinensis} shrimp genome
showed expanded several gene families related to cellular process and
metabolic process, and genes involved in cellular process had undergone
positive selection during evolution.

\textbf{4.3 Pathogen infection related gene families contracted
in\emph{F. chinensis}}

Some of the contracted gene families in \emph{F. chinensis} are
associated with pathogen infection. Most of the contracted gene families
involving in these pathways encoded actin-like protein. The actin
protein is a fundamental component of the host cellular cytoskeleton,
playing an important role in viral replication (Roberts \& Baines, 2011;
Spear \& Wu, 2014). Previous research suggests that the actin gene is
upregulated in \emph{M. japonicus} after infection with \emph{Vibrio
parahaemolyticus} and WSSV (Ren, Zhang, Liu, \& Li, 2019), the pathogens
that cause the two most serious diseases affecting shrimp cultivation
(Escobedo-Bonilla et al., 2008). The \selectlanguage{greek}β-\selectlanguage{english}actin gene is also involved in
WSSV infection in \emph{L. vannamei} (J. Feng, Li, Liu, Tang, \& Du,
2019). We speculate that the contraction of the actin gene family
made\emph{F. chinensis} more sensitive to viral infections because the
viruses could destroy the force-generating and macromolecular
scaffolding properties of the actin cytoskeleton to drive the infection
process (Spear \& Wu, 2014). The lower number of actin genes may make it
difficult to repair damaged cell in \emph{F. chinensis} . This finding
is consistent with those of previous studies, which indicate that
\emph{F. chinensis} exhibit lower resistance for WSSV than other shrimp
species (Y. Feng et al., 2017; Jiang, Yu, \& Zhou, 2006). This is one of
the reasons why \emph{L. vannamei} has replaced \emph{F. chinensis} to
become the dominant cultured shrimp species in recent years.

Higher temperatures often increase the severity of disease
susceptibility (Cohen \& Leach, 2020). Pathogen infection needs a
suitable temperature, for example, the WSSV needs an optimum temperature
to successfully enter the host hematopoietic stem cells, and the virus
entry is blocked at 6 \selectlanguage{ngerman}°C (Korkut, Noonin, \& Soderhall,
2018).\emph{Fenneropenaeus chinensis} is mostly distributed in the
northern Pacific, a colder environment relative to that of other
\emph{Penaeus}species, where pathogen infectivity is weaker. We
hypothesize that, during evolution, \emph{F. chinensis} sacrificed an
aspect of immunity to preserve more energy for subsistence in the cold
environment, because immunoreaction is an energy-consuming activity
(Alwarawrah, Kiernan, \& MacIver, 2018; Hosomi \& Kunisawa, 2020; Loftus
\& Finlay, 2016).

\textbf{4.4 Targeted artificial selection during domestication
of\emph{F. chinensis}}

Domestication of animals was driven by both natural and artificial
selection. Under these directed selection pressures, change in the
allele frequency in specific regions may be accumulated in the animal
genome after domesticated for several generations (Nielsen, Hellmann,
Hubisz, Bustamante, \& Clark, 2007). The cultured shrimps were derived
from the wild population by continuous, high-intensity artificial
selection for growth traits and is characterized by faster growth and
higher body weight than wild individuals. Genes responsible for these
characteristics may be detected by identifying unique selection
signatures in the genome. Selection signature is characterized by the
decrease in polymorphism and increase in linkage disequilibrium in
certain loci. Identification of genetic variations by selection
signature reveals the genetic mechanism of the formation of phenotypic
trait diversity during selection (Lopez, Neira, \& Yanez, 2014). In the
present study, most genes with selection signatures were identified to
participate in metabolic processes. Metabolism is the basis of growth
and is related to growth rate (Krieger, 1978; Vahl, 1984). The changes
in the allele frequency of genes related to metabolic processes suggest
artificial selection on growth. However, owing to the short history of
shrimp domestication, the genetic divergence between the wild and
cultured shrimp populations is not significant.

A pathway named the phototransduction-fly pathway was enriched by genes
with selection signatures; of the 27 background genes of this pathway,
six genes were located in the selection signature regions. This enriched
pathway signifies a visual change in domesticated
shrimp.\emph{Fenneropenaeus chinensis} juveniles exhibit an intense
attack behavior (P. Zhang, Zhang, Li, \& Meng, 2008). We propose that
the high-density culture environment required them to develop better
vision to survive cannibalism.

Among the pathways enriched by the candidate genes, there was a pathway
related to the nervous system, neuroactive ligand-receptor interaction
pathway, which suggests that genes affecting neuronal development were
also targeted during domestication. Animal behavior is regulated by the
nervous system. Compared with aggressive animals, docile animals survive
more easily in a culture environment (Carneiro et al., 2014; Darwin,
1860). We speculate that tame shrimps were selected for during
domestication, and therefore the related genes were targeted.

\textbf{4.5 Conclusion}

In summary, an improved chromosome-level genome of \emph{F.
chinensis}was reported in this article. The assembled genome was 1.47 Gb
in size, and was anchored to 43 pseudochromosomes, with N50 length of
36.87 Mb. The contraction of the genome size was speculated relating to
the migration. Gene families related to cellular processes and metabolic
processes expanded, while gene families associated with virus infection
contracted to adapt to the cold environment. Furthermore, genetic
variations in genes associated with metabolism, phototransduction, and
nervous system were detected by selection signature, indicating targeted
artificial selection on growth, vision, and behavior during
domestication.

\textbf{Acknowledgements}

We would like to appreciate our colleagues of the Key Laboratory for
Sustainable Utilization of Marine Fisheries Resources of Yellow Sea
Fisheries Research Institute, for their assistance on sample collection
and helpful comments on the manuscript. This work was supported by the
National Key R \& D Program of China (No. 2019YFD0900403), Modern
Agro-industry Technology Research System (No. CARS-48), National Natural
Science Foundation of China (No. 31902367) and Central Public-interest
Scientific Institution Basal Research Fund of CAFS (No. 2020TD46).

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\textbf{Data accessibility}

Genomic sequences, PacBio data used for genome assembly and Illumina
data used for genomic survey and Hi-C were deposited in NCBI under
accession number: PRJNA691453. The RNA-seq data used for genome
annotation in this study were deposited in NCBI under accession number:
PRJNA558194 and PRJNA694186. The resequencing data used for selection
signature analysis were deposited in NCBI under accession number:
PRJNA628752.

\textbf{Author contributions}

QW, JW and HZ collected samples. WW and YZ extracted DNA and RNA for
sequencing. QW, XR and YH carried out genome assembly and assessment,
and performed gene prediction and annotation. PL, JTL and JJL carried
out comparative genome analysis. QW and JL performed selection signature
analysis. JL and YH conceived this project. QW and XR wrote the
manuscript. All authors contributed to the final manuscript editing.



\textbf{Tables and Figures}

\textbf{Tables}

\textbf{Table 1 General information regarding \emph{Fenneropenaeus
chinensis} genome assembly}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Genome survey} &\tabularnewline
\midrule
\endhead
Genome size (Mb) & 1384.88\tabularnewline
Heterozygosity & 1.04\%\tabularnewline
Repetive sequence proportion & 54.79\%\tabularnewline
\textbf{PacBio assembly} &\tabularnewline
Total length (Mb) & 1,465.32\tabularnewline
GC content & 37.53\%\tabularnewline
Contig number & 9,015\tabularnewline
Max length (bp) & 3,793,399\tabularnewline
N50 (bp) & 472,841\tabularnewline
N90 (bp) & 77,864\tabularnewline
\textbf{Hi-C assembly} &\tabularnewline
Total length (Mb) & 1,466.12\tabularnewline
Scaffold number & 1,063\tabularnewline
Max length (bp) & 48,777,264\tabularnewline
N50 (bp) & 36,870,704\tabularnewline
N90 (bp) & 24,342,607\tabularnewline
Pseudochromosome number (2n) & 86\tabularnewline
unplaced scaffold (Mb) & 14.60\tabularnewline
Place rate & 99.00\%\tabularnewline
\textbf{Annotation} &\tabularnewline
Repetitive sequence proportion & 57.73\%\tabularnewline
Total gene number & 25,026\tabularnewline
Average length (bp) & 11,290\tabularnewline
Annotated gene number & 19,192\tabularnewline
\bottomrule
\end{longtable}



\textbf{Table 2 Genome comparison among four penaeid shrimp species}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Species & Genome size (K-mer analysis) & Chromosome number (2n) &
Protein-coding gene number & Repetitive sequence proportion &
Citation\tabularnewline
\midrule
\endhead
\emph{L. vannam}ei & 2.60 Gb & 88 & 25,596 & 78\% (K-mer); 49.38\%
(assembly) & Zhang, 2019\tabularnewline
\emph{P. monodon} & 2.59 Gb & 88 & 30,038 & 50.92\% (K-mer); 62.5\%
(assembly) & Yuan, 2017; Uengwetwanit, 2021\tabularnewline
\emph{M. japonicas} & 2.28 Gb & 86 & \textbackslash{} & 47.73\% & Yuan,
2017\tabularnewline
\emph{F. chinensis} & 1.38 Gb & 86 & 25,026 & 54.79\% (K-mer); 57.73\%
(assmbly) & This article\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure Legends}

\textbf{Figure 1. Basic information of \emph{Fenneropenaeus
chinensis}.}(a) \emph{F. chinensis.} Male above and female below, both
are sexual maturity\emph{.} (b) Heatmap of anchored chromosomes (c)
Genomic characteristics of \emph{F. chinensis} . Track 1 (from the
outer-ring): 43 pseudochromosomes; Track 2: Distribution of gene density
with sliding windows of 1 Mb. Higher density is indicated by darker
color (the same below). Track 3: Distribution of genes on the forward
strand. Track 4: Distribution of genes on the reverse strand. Track 5:
Distribution of single nucleotide polymorphism (SNP) density (based on
resequencing of 42 shrimps). Track 6: Distribution of simple sequence
repeats (SSRs). Track 7: Distribution of GC content (only values between
20\% and 50\% are displayed). Track 8: Distribution of proportion of
repetitive sequences. Track 9: Schematic representation of
interchromosomal relationships in the genome.

\textbf{Figure 2. Comparative genomic analysis of \emph{Fenneropenaeus
chinensis} and homologous species.} (a) The distribution of genes in
different species. The horizontal axis represents 17 species, and the
vertical axis represents the number of genes. (b) Common and unique gene
families among four homologous species. (c) Phylogenetic tree of 17
species. Green numbers indicate the number of expanded gene families,
and red numbers indicate the number of contracted gene families.

\textbf{Figure 3. Selective sweeps on the genome of
domestic\emph{Fenneropenaeus chinensis}.} (a) Conjoint analysis
of\emph{F} \textsubscript{ST} and \selectlanguage{greek}θπ; \selectlanguage{english}the red dots indicate the top 5\%
of rank level values. (b) Results of the KEGG enrichment analysis of the
genes under artificial selection pressure in \emph{F. chinensis} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-1/figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-2/figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-3/figure-3}

\end{center}
\end{figure}

\selectlanguage{english}
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