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\begin{document}

\title{Chromosome-level genome assembly of burbot (Lota lota) provides insights
into the evolutionary adaptations in freshwater}



\author[1]{Zhiqiang Han}%
\author[2]{Manhong Liu}%
\author[3]{Qi Liu}%
\author[2]{Hao Zhai}%
\author[3]{shijun xiao}%
\author[4]{Tianxiang Gao}%
\affil[1]{Affiliation not available}%
\affil[2]{Northeast Forestry University}%
\affil[3]{Wuhan Gooalgene Technology Company}%
\affil[4]{Zhejiang Ocean University}%


\vspace{-1em}



  
  \date{June 09, 2024}


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\selectlanguage{english}
\begin{abstract}
The burbot (Lota lota) is the only member of the cod family (Gadidae)
that is adapted solely to freshwater. This species shows the widest
longitudinal range of freshwater fish in the world. The burbot is a good
model for studies on adaptive genome evolution from marine to freshwater
environment. However, no high-quality reference genome has been
released. Here, the first chromosome-level genome of the burbot was
constructed using PacBio long sequencing and Hi-C technology. A total of
95.24 Gb polished PacBio sequences were generated, and the preliminary
genome assembly was 575.83 Mb in size with a contig N50 size of 2.15 Mb.
The assembled sequences were anchored to 22 pseudo-chromosomes by using
the Hi-C data. The final assembled genome after Hi-C correction was
575.92 Mb, with a contig N50 of 2.01 Mb and a scaffold N50 of 22.10 Mb.
A total of 22,067 protein-coding genes were predicted, 94.82\% of which
were functionally annotated. Phylogenetic analyses indicated that burbot
diverged with the Atlantic cod about 44.4 million years ago. In
addition, 377 putative genes that appear to be under positive selection
in burbot were identified. These positively selected genes might adapt
to the freshwater environment. These genome data provide an invaluable
resource for the ecological and evolutionary study of the order
Gadiformes.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The order Gadiformes includes some of the most important commercial fish
(e.g., cod, hake, and haddock) in the world and accounts for
approximately 18\% of the world's total marine fish catch (FAO, 2004).
Gadiform fish inhabit cold waters in every high-latitude ocean from
deep-sea benthic habitats to coastal waters. Only two species in this
order are known in freshwater habitats (Nelson, 2006). However, to date,
only one high-quality genome sequence of the Gadiformes species, i.e.,
the Atlantic cod (\emph{Gadus morhua~} ) (Star et al., 2011), is
available, and this limitation significantly hinders the taxonomical,
evolutionary, and biological studies of the order Gadiformes.

The burbot \emph{Lota lota~} is the only member of the cod family
(Gadidae) that is adapted solely to freshwater (Schaefer et al., 2016).
This fish has a wide holarctic distribution, showing the widest
longitudinal range of freshwater fish in the world. The burbot is
distributed in nearly all suitable freshwater basins of North America,
Europe, and north Asia (Lehtonen, 1998). Although this fish thrives in
freshwater, \emph{L. lota} has retained many characteristics of its
marine ancestors (Blabolil et al., 2018), such as preference for cold
water, spawning at low temperatures, high fecundity, and a pelagic
larval stage. This species spawns during winter or early spring,
typically when the water is still ice-covered, and the water
temperatures are between 1 \selectlanguage{ngerman}°C and 4 °C (Bergersen et~al., 1993).
Spawning occurs on fine to gravel substrate in shallow bays or groyne
fields in water depths of 0.3~m to 3.0~m (Fredrich \& Arzbach,~2002;
Eick et al., 2013).

The burbot is apparently an excellent ``indicator'' species. This
species is vulnerable to many environmental changes, in particular,
warming water temperatures and pollution (Stapanian et al., 2010). The
burbot in marginal habitats may serve as an early indicator of the
impacts of climate change on cold-water fish species (Stapanian et al.,
2010). However, stocks of the burbot have severely declined in number
and distribution during the past century. Many populations are
threatened, have been extirpated, or are otherwise in need of
conservation measures (Maitland \& Lyle, 1990). For example, in Finland,
burbot populations have declined or have been destroyed completely in
16\% of the lakes (Tammi et~al., 1999). A series of threats, including
pollution, habitat fragmentation, exploitation, and invasive species,
have caused the decline or extirpation of many burbot populations
(Stapanian et al., 2010). Genomics resources will support the
conservation studies of burbot. Given the widest holarctic distribution
of this species, the burbot may undergo some degree of local adaptation,
which can be resolved with genome-wide high-quality SNPs. However, the
available genetic information for this fish remains scarce. At present,
only limited genetic studies have been conducted on the microsatellite
loci isolation and population structure of the burbot (Houdt et al.,
2005; Sanetra, 2005). Thus, sequencing the genome of the burbot is
essential. This process may help to reveal insights into the
evolutionary history of the burbot and the role of environmental changes
in shaping the genome evolution from marine to freshwater.

The burbot\selectlanguage{english},the only freshwater species in the cod family
(Gadidae)\selectlanguage{english},represents a classical transition from marine to freshwater.
Fossil evidence suggests that the \emph{Lota} genus has already
inhabited European rivers in the early Pliocene (Houdt et al., 2005).
This phenomenon indicates that the burbot left the ocean and migrated to
the freshwater. The transition from an oceanic to a freshwater habitat
provides an opportunity for drastic environmental changes in the
ecology, morphology, and behavior of fish. This transition should select
numerous functional genes. Marine to freshwater transition events rarely
occurs (Finnegan, 2017), which is likely due to physiological and
ecological barriers associated with changing environmental conditions.
These factors include lower salinity, relatively high levels of UV
radiation, dramatic fluctuations in freshwater temperature, and
competition from primary freshwater fish lineages. Despite these
challenges, several freshwater fish from marine-derived lineages have
completed this transition from ocean to freshwater and invaded
successfully freshwater habitats. Concerted effort, such as the
convergence of their morphological and physiological characters, has
been reported to freshwater adaptations (Jara, 1988). The genomic
changes underlying a convergent evolution may be reproducible to some
extent, and convergent phenotypic traits may commonly arise from the
same genetic changes. Physiological convergence is strong in freshwater
fish of marine-derived lineages and provides a practical way to identify
freshwater adaptations.

In this study, a chromosome-level genome assembly of burbot was
constructed by combining short reads, PacBio long reads, and Hi-C
sequencing data. The assembly was used to identify the genetic
signatures of evolution related to freshwater adaptation in burbot and
Perciformes by comparative genomics of 13 distantly related species,
including three freshwater Percomorpha species. This study will provide
a genomic resource to further address the key evolutionary process of
freshwater adaptation for marine-originated species.

\textbf{Materials and methods}

\textbf{Sample and DNA extraction}

A single female fish (\textasciitilde{}800 g) was collected in November
2019 from Heilong River, the northeastern part of China. Muscle, eye,
gonad, gill, liver, and spleen tissues were collected and stored in
liquid nitrogen until DNA and RNA extraction. Muscle tissues were used
for DNA sequencing for genome assembly, while all tissues were used for
transcriptome sequencing. Genomic DNA from the white muscle tissue was
extracted using the standard phenol/chloroform extraction method to
construct the DNA sequencing library. The integrity and concentration of
the genomic DNA molecules were checked using 1\% agarose gel
electrophoresis and Pultton DNA/Protein Analyzer (Plextech, USA).

\textbf{Library construction and genome sequencing}

The Illumina NovaSeq-6000 and PacBio Sequel II platforms were applied
for genomic sequencing to generate short and long genomic reads,
respectively. Illumina sequencing libraries were prepared to estimate
the genome size, correct the genome assembly, and evaluate assemblies. A
paired-end library was constructed with an insert size of 300 bp
according to the Illumina standard protocol. After discarding reads with
low-quality bases (reads with more than 10\% N bases or low-quality
bases\selectlanguage{english}[?]5), adapter sequences, and duplicated sequences, the clean reads
were used for subsequent analysis.

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

\textbf{Genome size estimation and genome assembly}

The Kmer-based method of the Illumina short read data was used to
analyze the genome survey to estimate the genome size, heterozygosity,
and repeat content of the burbot genome (Liu et al., 2013). The
NextDenovo package (https://github.com/Nextomics/NextDenovo) was
performed to assemble the burbot genome with PacBio long reads by using
the following parameters: parallel\_jobs, 300; seed\_cutoff, 15,000;
pa\_correction, 320; and random\_round, 100. To correct the random
sequencing errors in the NextDenovo output, two steps of genome sequence
polishing were applied. The arrow was used to polish the genome by using
the long sequencing data (Chin et al., 2013), and two rounds of
polishing using Illumina short reads were then applied with Pilon
(Walker et al., 2014).

\textbf{Hi-C analysis and chromosome assembly}

To obtain a chromosome-scale genome assembly, the Hi-C library for
sequencing was constructed. The muscle tissue of the burbot was used to
prepare the library, according to Rao et al. (2014). High-quality Hi-C
fragment libraries were sequenced for the Illumina NovaSeq-6000
platform. The sequencing reads were mapped to the polished burbot genome
with Bowtie 1.2.22. The two read ends were independently aligned to the
genome, and only the read pairs with both ends uniquely aligned to the
genome were selected. Lachesis (Burton et al., 2013) with default
parameters was then applied to perform the chromosomal-level genome
assembly by using the corrected contigs and valid Hi-C reads. The
ggplot2 in the R package was applied to generate a genome-wide Hi-C
heatmap to evaluate the quality of the chromosomal-level genome
assembly.

Two methods were performed to assess the completeness and accuracy of
the genome assembly. First, the Illumina and PacBio reads were aligned
to the burbot assembly genome by using BWA-MEM (version 0.7.10-r789) (Li
\& Durbin, 2009) and BLASR (Mark et al., 2012), respectively. Second,
the completeness of the genome assembly was evaluated by using BUSCO
(version 2.0) (Simao et al., 2015) to search the genome against the
Actinopterygii database, which consisted of 4,584 single copy orthologs.

\textbf{Repeat annotation, gene prediction and functional annotation}

Repeat elements were annotated in the burbot genome before the gene
prediction. Tandem Repeat Finder (Benson, 1999) and LTR\_FINDER (Zhao et
al., 2007)~were applied for the~\emph{ab initio} ~prediction of repeat
elements in this genome. RepeatMasker and RepeatProteinMask
(\emph{http://www.repeatmasker.org}) were executed to search the genome
sequences for known repeat elements, with the genome sequences used as
queries against the Repbase database (Jurka et al., 2005). The ribosomal
RNA (rRNA) and microRNA genes were predicted using the Infernal v.1.1
software based on the Rfam and miRBase databases, respectively
(Griffiths-Jones et al., 2005). Transfer RNA (tRNA) genes were
identified using the tRNAscan-SE v. 1.3.1 software (Lowe \& Eddy, 1997).

Based on the repeat-masked genome, three strategies based on
\emph{abinitio} , homologs, and RNA-sequencing were applied to predict
the protein-coding genes in the burbot genome assembly. \emph{Ab
initio}gene prediction was performed using Augustus (v2.7) (Stanke et
al., 2006) and GenScan (Burge \& Karlin, 1997) with default settings.
For the homology-based prediction, protein sequences of \emph{Gadus
morhua} ,\emph{Acanthochromis polyacanthus} , \emph{Oryzias latipes}
,\emph{Amphiprion ocellaris} , \emph{Anabas testudineus}
,\emph{Astatotilapia calliptera} , \emph{Astyanax mexicanus}
,\emph{Austrofundulus limnaeus} , \emph{Lepisosteus oculatus} ,
and\emph{Notothenia coriiceps} were downloaded from the NCBI database
and aligned to the burbot genome by using tBLASTn (E-value \selectlanguage{english}[?] 1e\selectlanguage{english}-5). The
homologous genome sequences were then aligned against the matching
proteins by using GeneWise (v2.4.0) (Doerks et al., 2002) for accurately
spliced alignments. Transcriptomic data (2x150 bp, NovaSeq-6000
platform) generated from a mixture of six tissues, including the muscle,
eye, gonad, gill, liver, and spleen, were aligned to the assembled
genome sequences by using HISAT2 (v2.0.10) (Pertea et al., 2016), and
the putative transcript structures were detected using gene structure,
which was predicted by Cufflinks (Ghosh et al., 2016). All gene models
were merged, and redundancy was removed by MAKER (Cantarel et al., 2008)
and HiCESAP.

The NCBI nonredundant protein (NR), InterPro, the SwissProt, TrEMBL
(Boeckmann et al., 2003), eukaryotic orthologous groups of proteins
(KOG) (Tatusov et al., 2003), and Kyoto Encyclopedia of Genes and
Genomes (KEGG) (Kanehisa \& Goto, 2000) databases with an E-value
threshold of 1e--5 were used for the functional annotation of the
protein-coding genes by using BLASTX and the BLASTN utility (Lobo,
2008). Functional ontology and pathway information from the Gene
Ontology (GO) database was assigned to the genes by using Blast2GO
(Conesa et al., 2005).

Comparative genomic analyses and testing for genomic selection

The protein sequences of 12 species of teleost fish (Supporting
Information Table S1) were downloaded from Ensembl (release version
100). Only the longest transcript was selected for each gene locus with
alternative splicing variants. Orthologous groups were constructed by
ORTHOMCL (14) v2.0.9 by using the default settings based on the filtered
BLASTP results. The single\selectlanguage{english}-copy orthologous genes shared by all 13
species were further aligned using~MUSCLE (version 3.8.31) (Edgar,~2004)
and concatenated to construct a phylogenetic tree
with~RaxML~(Stamatakis, 2014). The divergence time among species was
estimated by the r8s (Sanderson, 2003). Divergence times
of\emph{Larimichthys crocea-Notothenia coriiceps} (88--114),\emph{~L.
crocea-Gambusia affinis} (96.9--150.9), \emph{Clupea harengus-G.
affinis} (149.85--165.2) from the TimeTree database (Kumar et al., 2017)
were used as the calibration times. Gene family expansion and
contraction analyses were performed using CAFE 3.1 (De Bie et al., 2006)
with the estimated phylogenetic tree information. \emph{P} value
\textless{} 0.05 was used to indicate significantly changed gene
families. The expanded and contraction gene families in burbot, the
shared contraction gene families of three freshwater species
(burbot,\emph{Monopterus albus,} and \emph{G. affinis} ) in GO terms,
and KEGG pathways were enriched, and the Benjamini and Hochberg FDR
correction was applied. Significantly overrepresented GO terms and KEGG
pathways were identified with corrected \emph{P} values \selectlanguage{english}[?]0.05.

To construct multiple sequence alignments among the ortholog genes, the
CODEML program in PAML 4.5 was used to estimate the dN/dS ratio (\selectlanguage{greek}ω\selectlanguage{english})
(Yang, 2007). Two different branch-site likelihood ratio tests were
applied to find genes under positive selection. First, the
species-specific positively selected genes (PSGs) in burbot were
identified with burbot as foreground species and other species,
excluding zebrafish \emph{Danio rerio} , \emph{Clupea harengus}
and\emph{Esox lucius} as background species. Second, three freshwater
species (i.e., burbot, \emph{M. albus,} and \emph{G. affinis} ) were
selected as foreground species and other species, excluding
zebrafish\emph{Danio rerio} , \emph{Clupea harengus} and \emph{Esox
lucius} , as background species. GO and KEGG categories were assigned to
orthologous groups according to the zebrafish genome reference for the
functional enrichment analyses.

\textbf{3 Results and discussion}

\textbf{3.1Genome size estimation and initial characterization of the
genome}

A total of 79 Gb of Illumina data werer generated from the Illumina 150
insert-size library, representing 143.64-fold coverage of the burbot
genome (Table 1, Supplementary Table S2). The total number of f\emph{k}
-mers was 62,863,455,719, with a \emph{k} -mers peak at a depth of 112
(Supplementary Figure S1 and Table S3). The genome size was estimated to
be \textasciitilde{}550 Mb with heterozygosity of 0.57\% and repeat
content of 36.60\% (Supplementary Table S3).

\textbf{3.2 Genome assembly and completeness of the assembled genome}

The PacBio Sequel II platform generated 95.24 Gb high-quality data from
the long-read library, covering 173.16-fold of the genome assembly
(Table 1, Supplementary Table S4). These data were assembled using
NextDenovo followed by racon and pilon polishing, which produced a
575.83 Mb genome assembly with a contig N50 of 2.15 Mb (Table 2). The
length of this assembly was consistent with the genome size estimated by
k-mer analysis.

The Illumina reads and PacBio long reads were aligned to the burbot
assembly to evaluate the quality of the initial genome assembly. The
results showed that 99.23 \% of the Illumina reads and 97.55\% of the
PacBio long reads were successfully mapped to the assembled genome
(Supplementary Table S5 and S6). The BUSCO analysis showed that 94.67\%
(4344/4584) of the complete BUSCO were found in the genome assembly
(Table 3), including 91.93\% of the complete and single copy and 2.84\%
duplicated genes.

The contigs in the draft assembly were then anchored and oriented into a
chromosomal-scale assembly by using the Hi-C scaffolding approach. The
Hi-C library generated 69.51 Gb (126.38\selectlanguage{ngerman}×) clean data (Table 1,
Supplementary Table S7). With the use of LACHESIS, 88.66\% of the
assembled sequences were anchored to 22 pseudo-chromosomes, with
chromosome lengths ranging from 15.18 Mb to 51.8 Mb (Table 4). Based on
the heatmap, the 22 pseudochromosomes could be distinguished easily and
the interaction signal strength around the diagonal was considerably
strong, which indicated a high quality of this genome assembly (Figure
2). The final assembled genome after Hi-C correction was 575.92 Mb, with
a contig N50 of 2.01 Mb and a scaffold N50 of 22.10 Mb (Table 5).

\textbf{3.3 Repeat annotation, gene prediction and gene annotation}

A total of 384.29 Mb of repeat sequences were detected, accounting for
66.74\% of the assembly genome (Table 6). This repeat content was
obviously larger than the value (36.60\%) obtained from the k-mer
analysis. The repetitive sequences mainly consisted of the DNA
transposable element (289.32 Mb; 50.24\% of the assembly), long terminal
repeats (66.95 Mb; 11.63\%), and long interspersed elements in 30.96Mb
(5.38\%) (Table 7).

A total of 21,664 protein-coding genes were predicted by the combination
of strategies based on \emph{ab} \emph{initio} , homologs, and RNAseq.
The average values of the gene length, exon length, and average intron
length were 14,606, 292.38, and 1,223 bp, respectively (Table 8). The
statistics of the predicted gene models were compared to other ten
teleost species,including :\selectlanguage{english}\emph{Acanthochromis polyacanthus,Oryzias
latipes,Amphiprion ocellaris,Anabas testudineus,Astatotilapia
calliptera,Astyanax mexicanus,Austrofundulus limnaeus,Gadus
morhua,Lepisosteus oculatus,Notothenia coriiceps} , showing similar
distribution patterns in mRNA length, CDS length, exon length, intron
length and exon number (Supplementary Figure S2). The summary of genome
characteristics of burbot was shown in Figure 3. A total of 20658
predicted genes (95.36\%) were successfully annotated by alignment to
the nucleotide, protein, and annotation databases InterPro, NR,
Swissprot, TrEMBL, KOG, GO, and KEGG (Table 9). A total of 6390 tRNAs,
300 rRNAs, and 519 microRNAs were identified by noncoding RNA prediction
(Supplementary Table S8).

3.4 \textbf{Comparative genomics and the mechanism of adaption to
freshwater}

A total of 19,998 gene families and 2,650 single-copy orthologous genes
were identified using the genomes and genes of 13 selected teleosts. In
addition, 21,664 genes of burbot could be clustered into 14,504 gene
families, including 132 unique gene families (Supplementary Table S9).
Based on the single-copy orthologous genes, the ML phylogenetic tree was
constructed and showed that burbot and Atlantic cod were clustered
together, and the divergence time between two cod species was
\textasciitilde{}44.4 Mya (Figure 4). The divergence time was consistent
with the estimated time by Hughes et al. (2018). The burbot genome
displayed 639 expanded and 1564 contracted gene families compared with
the common ancestor of burbot and Atlantic cod (Figure 4). The expanded
gene families of burbot were significantly enriched in 73 GO terms and
34 KEGG pathways, mainly including DNA integration (GO:0015074,
corrected \emph{P} value =0.00E+00), DNA metabolism process (GO:0006259,
corrected \emph{P} value =2.05E\selectlanguage{english}-06), apoptosis process (GO:0006915,
corrected \emph{P} value =5.22E\selectlanguage{english}-05), zinc ion binding (GO:0008270,
corrected \emph{P} value =2.02E\selectlanguage{english}-96), transition metal ion binding
(GO:0046914, corrected \emph{P} value =1.19E\selectlanguage{english}-91), natural killer
cell-mediated cytotoxicity (ko04650, corrected \emph{P} value
=4.200299E\selectlanguage{english}-20), and hematopoietic cell lineage (ko04640,
corrected\emph{P} value=3.04E\selectlanguage{english}-18) that were associated with cell damage
repair, ion binding, and immune system (Supplementary Tables S10 and
S11). Conversely, the burbot clearly showed contracted gene families in
homophilic cell adhesion via plasma membrane adhesion molecules
(GO:0007156, corrected \emph{P} value =2.69E\selectlanguage{english}-29), cell-cell adhesion via
plasma-membrane adhesion molecules (GO:0098742, corrected \emph{P} value
=2.69E\selectlanguage{english}-29), membrane (GO:0016020, corrected \emph{P} value=1.18E\selectlanguage{english}-10) GO
terms, amino sugar and nucleotide sugar metabolism (ko00520,
corrected\emph{P} value=1.52E\selectlanguage{english}-04), and NOD-like receptor signaling
(ko04621, corrected \emph{P} value=2.41E\selectlanguage{english}-02) pathways (Supplementary
Tables S12 and S13).

Notably, three freshwater species shared no expanded gene families and
two contracted gene families associated with cell adhesion (GO:0007155:
corrected \emph{P} value=0.00E+00) and membrane (GO:0016020,
corrected\emph{P} value =0.00E+00) (Supplementary Table S14). These
functions are critical for adjusting the ion concentrations inside and
outside the cell. However, no enriched KEGG pathway was found for the
contracted gene families. Such gene families may reflect the reduced
functional requirements of a stable ionic environment in freshwater for
cell membrane permeability. These findings are consistent with the
different components of omega-3 fatty acids between marine and
freshwater fish (Ta\selectlanguage{polish}ş\selectlanguage{english}bozan \& G\selectlanguage{ngerman}ökçe, 2017). Marine fish have higher
levels of omega-3 fatty acids than freshwater species. Compared with the
omega-6 fatty acids, omega-3 fatty acids help improve cell membrane
fluidity and provide osmoregulatory capabilities.

To identify the genes evolving under positive selection for freshwater
adaptation, two different likelihood ratio tests (branch-site model)
were performed. A total of 377 genes were identified as PSGs in the
burbot genome (Supplementary Table S15). The burbot PSGs were
functionally enriched in the organic cyclic compound metabolic process
(GO:1901360, corrected \emph{P} value =1.83E\selectlanguage{english}-02), cellular nitrogen
compound metabolic process (GO:0034641, corrected \emph{P} value
=4.11E\selectlanguage{english}-03), RNA metabolic process (GO:0016070, corrected \emph{P} value
=4.13E\selectlanguage{english}-03), and nucleic acid metabolic process (GO:0090304,
corrected\emph{P} value =6.16E\selectlanguage{english}-03 ) (Supplementary Table S16).
Additionally, 38 PSGs were detected with three freshwater lineages
(burbot, \emph{M. albus} and \emph{G. affinis} ) as foreground branch
(Supplementary Table S17). Four PSGs (\emph{stk33} , \emph{ino80e} ,
\emph{nabp1a} and\emph{znf385a} ) were related to DNA damage repair.
Genes \emph{stk33 and nabp1}participate~in~the~mitotic DNA damage
checkpoint\emph{.} \emph{znf385a} is located upstream in the p53
activating pathway. \emph{znf385a} ~interacts with p53/TP53 and promotes
DNA damage-induced cell cycle arrest (Das et al., 2007). Protein ino80e
is a component of the chromatin remodeling INO80 complex and contributes
to the DNA double-strand break repair (Yao et al., 2008).

The exposure of freshwater fish to UV radiation may cause DNA damage.
The presence of a group of genes involved in DNA repair under positive
selection was consistent with the high levels of exposure to UV
radiation in freshwater environment compared with that in the ocean
environment. This finding suggests that these genes had functionally
convergent in three freshwater lineages.

The PSGs of freshwater lineages were enriched in folic acid transport
(GO: 0015884\selectlanguage{english}, \emph{slc19a1} , corrected \emph{P} value =8.10E\selectlanguage{english}-05) GO
terms, amino acid metabolism, replication, and repair pathways
(Supplementary Tables S18 and S19). \emph{slc19a1} has an important role
in folate transmembrane transport. Low osmotic~pressure has been
previously shown to affect the efficiency of folic acid absorption in
the intestine (Zhao et al.,2011). The positive selection
on\emph{slc19a1} may improve folic acid absorption for freshwater
species. These data will serve as valuable resources for future
evolution studies of burbot.

4. Conclusion

A chromosomal-scale genome assembly of the burbot was provided by
integrating the Hi-C and PacBio long read sequencing data. The burbot is
the only freshwater member of the cod family and represents the widest
longitudinal range of freshwater fish in the world. The genome assembly
and annotation supplied the second high-quality genome of the order
Gadiformes and important genomic data for whole genome analysis to
further investigate the evolution of burbot with other cod species. A
series of candidate genes involved in freshwater adaptation were
identified in these comparative genomics analyses. The results were
beneficial in elucidating the evolution process in order Gadiformes
under environment change. These data are also useful for diverse
conservation applications, including identifying conservation units,
assessing gene flow, detecting local adaptation of the populations and
elucidating the evolutionary history of burbot.

\textbf{Acknowledgements}

This work was supported by the National Key Research and Development
Program of China (2017YFA0604904) and Natural Science Foundation of
Heilongjiang Province of China (LH2019C070).

\textbf{Author contributions}

T.X. G. and Z.Q.H. conceived and managed the project. M.H.L. and H.Z.
collected the sequencing samples. Q.L. and X.S.J. extracted the DNA/RNA
and performed the genome sequencing. Z.Q.H. analyzed the data. Z.Q.H.
and T.X. G. wrote the manuscript. All authors reviewed and approved the
final manuscript.

\textbf{Conflicts of interests}

No

\textbf{Data Accessibility Statement}

Raw sequencing data (PacBio, Illumina, Hi-C and RNA-seq data) for burbot
genome has been deposited at the Sequence Read Archive (SRA)
(SRR12549297, SRR12549430, SRR12550374 and SRR12577979). The assembled
genome and annotations have been deposited at NCBI Assembly database
with the GenBank accession PRJNA663985. In addition, the genome data is
also openly available in figshare
at\emph{https://doi.org/10.6084/m9.figshare.12927203}(Liu, 2020).

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\textbf{Figure legend}

Figure 1 The burbot (\emph{Lota lota} )

Figure 2 Genome-wide Hi-C heatmap of the burbot.

Figure 3 Genome characteristics of burbot. From outer circle to inner
circle: gene distribution, GC content of the genome, short read depth,
long read depth, DNA TE, and long tandem repeats (LTR).

Figure 4 Phylogenetic analysis and divergence time tree of the burbot
with other teleost species.\selectlanguage{english}
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