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\begin{document}

\title{Chromosome-level Genome Assembly for Takin (Budorcas taxicolor) Provides
Insights into Its Taxonomic Status and Genetic Diversity}



\author[1]{Anning Li}%
\author[1]{Qimeng Yang}%
\author[1]{Ran Li}%
\author[1]{Xuelei Dai}%
\author[1]{Keli Cai}%
\author[2]{Yinghu Lei}%
\author[2]{Kangsheng Jia}%
\author[1]{Yu Jiang}%
\author[3]{Linsen Zan}%
\affil[1]{Northwest A\&F University}%
\affil[2]{Shaanxi Academy of Forestry Sciences}%
\affil[3]{Northwest Agriculture and Forestry University}%


\vspace{-1em}



  
  \date{May 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The takin (Budorcas taxicolor) is one of the largest bovid herbivores
across caprinae subfamily. The takin is at high risk of extinction,
however, its taxonomic status is still unclear. In this study, we
constructed the first reference genome of B. taxicolor using PacBio long
High-Fidelity reads and Hi-C technology. The assembled genome is
\textasciitilde{}2.95 Gb with a contig N50 of 68.05 Mb and a scaffold
N50 of 101.27 Mb, which were anchored onto 25+XY chromosomes. Compared
to the common ancestral karyotype of bovidae (2n=60), we found the takin
(2n=52) experienced four chromosome fusions and one large translocation.
We also found that the takin was most closely related to muskox, not
other caprinae species. Further, we re-sequenced nine golden takins from
the main distribution area, Qinling Mountains, and identified 3.3
million SNPs. The genetic diversity of takin was very low (\selectlanguage{greek}θπ\selectlanguage{english}=0.00028
and heterozygosity=0.00038), which was among the lowest detected in the
domestic and wild mammals. We also found takin genomes showed high
inbreeding coefficient (FROH=0.217) suggesting severe inbreeding
depression. The genome analysis show that the effective population size
of takins declined significantly from \textasciitilde{}100,000 years
ago. Our results provide valuable information for protection of takins
and insights into its evolution.%
\end{abstract}%



\sloppy


\textbf{Chromosome-level Genome Assembly for Takin (\emph{Budorcas
taxicolor}) Provides Insights intoIts Taxonomic Status and Genetic
Diversity}

\textbf{Running title: \emph{De novo} assembly of Takin (\emph{B.
taxicolor})}

Anning Li\textsuperscript{1\#}, Qimeng Yang\textsuperscript{1,2\#}, Ran
Li\textsuperscript{1,2}, Xuelei Dai\textsuperscript{1,2}, Keli
Cai\textsuperscript{1}, Yinghu Lei\textsuperscript{3}, Kangsheng
Jia\textsuperscript{3}, Yu Jiang\textsuperscript{1,2*}, Linsen
Zan\textsuperscript{1,3*}

\textsuperscript{1} College of Animal Science and Technology, Northwest
A\&F University, Yangling 712100, Shaanxi, P. R. China

\textsuperscript{2} Center for Ruminant Genetic and Evolution, Northwest
A\&F University, Yangling 712100, Shaanxi, P. R. China

\textsuperscript{3} Research Center for the Qinling Giant Panda (Shaanxi
Rare Wildlife Rescue Base), Shaanxi Academy of Forestry Sciences,
Zhouzhi 710402, Shaanxi, P. R. China

\textsuperscript{\#} These authors contributed equally to this study.

* Corresponding E-mail: \emph{yu.jiang@nwafu.edu.cn}
;\emph{zanlinsen@163.com} .

\textbf{Abstract:} The takin (\emph{Budorcas taxicolor} ) is one of the
largest bovid herbivores across caprinae subfamily. The takin is at high
risk of extinction, however, its taxonomic status is still unclear. In
this study, we constructed the first reference genome of \emph{B.
taxicolor} using PacBio long High-Fidelity reads and Hi-C technology.
The assembled genome is \textasciitilde{}2.95 Gb with a contig N50 of
68.05 Mb and a scaffold N50 of 101.27 Mb, which were anchored onto 25+XY
chromosomes. Compared to the common ancestral karyotype of bovidae
(2n=60), we found the takin (2n=52) experienced four chromosome fusions
and one large translocation. We also found that the takin was most
closely related to muskox, not other caprinae species. Further, we
re-sequenced nine golden takins from the main distribution area, Qinling
Mountains, and identified 3.3 million SNPs. The genetic diversity of
takin was very low (\selectlanguage{greek}θπ\selectlanguage{english}=0.00028 and heterozygosity=0.00038), which was
among the lowest detected in the domestic and wild mammals. We also
found takin genomes showed high inbreeding coefficient (FROH=0.217)
suggesting severe inbreeding depression. The genome analysis show that
the effective population size of takins declined significantly from
\textasciitilde{}100,000 years ago. Our results provide valuable
information for protection of takins and insights into its evolution.

\textbf{Keywords} : Takin; PacBio HiFi; Hi-C; Chromosomal evolution;
Inbreeding depression

\textbf{Introduction}

The takin (\emph{Budorcas taxicolor} ) is a large bovid herbivore,
belonging to the Caprinae subfamily. The number of takins was estimated
only about 7,000 \textasciitilde{} 12,000 in the world (Cheng et al.
2020). Takin has been listed as vulnerable by the International Union
for Conservation of Nature (IUCN) (Zeng and Song 2002). It was divided
into four subspecies according to the physiological characteristics and
geographical location (Wu 1986). Qinling takin (\emph{B. t. bedfordi} )
and Sichuan takin (\emph{B. t. tibetana} ) are only confined in China,
while the Mishmi takin (\emph{B. t. taxicolor} ) has a distributed area
from Gaoligong Mountains in northwestern Yunnan province, China to India
and Myanmar (Wu 1986). In addition, Bhutan takin (\emph{B. t. whieti} )
is found in Bhutan and the Yarlung Zangbo River in Tibet, China (Li et
al. 2003). Qinling takin (\emph{B. t. bedfordi} ), also known as golden
takin, is mainly distributed in the Qinling Mountains of China
(\textbf{Figure 1a} ) (Li et al. 2021).

Compared to other Caprinae animals, the morphology, ecological traits
and G-banded karyotype of takin were found to be similar to muskox
(\emph{Ovibos moschatus} ) (Pasitschniak-Arts et al. 1994). However, the
analysis of mitochondrial cytochrome b genes (\emph{Cytb} ) sequences
showed that muskox and takin were not close with each other but under
convergent evolution (Groves and Shields 1997, Ren et al. 2012).
Recently, based on the complete mitochondrial genome, takin was found to
be closely related to goat (\emph{Capra hircus} ) rather than sheep
(\emph{Ovis aries} ) (Feng et al. 2016, Kumar et al. 2019, Zhou et al.
2019). In our previous study, takin was found to be closely related to
sheep rather than goat according to the transcriptome analysis (Qiu et
al. 2021). Thus, to clarify on its taxonomic status, further analysis is
needed at the whole genomic level.

Recently, highly accurate long high-fidelity (HiFi) reads was generated
by PacBio single-molecule real-time (SMRT) sequencing with circular
consensus sequencing (CCS) (Wenger et al. 2019). HiFi reads combined
with Hi-C technology have been used to construct chromosome-level
reference genome of various animals (Wu et al. 2021, Zhou et al. 2021)
and plants (Chen et al. 2021, Huang et al. 2021, Ma et al. 2021, Sharma
et al. 2021, Wang et al. 2021). In this study, we performed the PacBio
long HiFi reads and Hi-C technology to construct a high-quality
chromosome-level reference genome of takin. The phylogenetic
relationship, chromosomal evolution, genetic diversity, demographic
history and inbreeding depression of takin were analyzed. These
information will be helpful for understanding the evolution of the takin
and working towards its conservation.

\textbf{Materials and Methods}

\emph{2.1} \emph{Sample collection and ethics statement}

The liver sample from a dead adult male golden takin was used
for\emph{de novo} genome sequencing. Seven Blood samples (alive) and two
muscle samples (dead) from nine golden takins were used for genome
resequencing. All of the samples were collected from the Rare Wildlife
Rescue and Breeding Research Center in Qinling Mountains. All the
experimental procedures were carried out according to the guidelines of
the China Council on Animal Care and approved by the Experimental Animal
Management Committee (EAMC) of Northwest A\&F University.

\emph{Genome sequencing and assembly}

A 15~Kb DNA SMRTbell library was constructed for SMRT sequencing
according to a standard protocol (Ardui et al. 2018) using PacBio Sequel
II platform with circular consensus sequencing (CCS). HiFi reads were
assembled in \emph{de novo} using Hifiasm (v0.13) (Cheng et al. 2021).
Then the restriction enzyme \emph{Mbo} I was used to digest cross-linked
chromatin to construct the Hi-C library, which was sequenced on an
Illumina NovaSeq6000 platform. The Hi-C contigs were anchored onto the
chromosomes using Juicer (v1.6) (Durand et al. 2016) and 3D-DNA
(v201008) (Dudchenko et al. 2017) combined with Juicebox
(\emph{https://github.com/theaidenlab/juicebox} ). BUSCO (v3.0.2) was
used to assess the completeness of assembled genome (Simao et al. 2015).
Genome resequencing was performed by the Illumina NovaSeq6000 platform.
All of the sequencing services were provided by the Berry Genomics
Biotechnologies Co., Ltd. (Beijing, China). A resequencing genome of
golden takin (TX-11) was selected to estimate the genome size and
heterozygosity rate by gce (v1.0.2)
(\emph{https://arxiv.org/abs/1308.2012v2} ).

\emph{Repeats and gene annotation}

The RepeatMasker software (v4.1.2) (http://www.repeatmasker.org) and the
RepBase library (2018.10.26) were used to identify the repeats in takin
genome. Homology-based method was used to predict the protein-coding
genes with RNA-seq data. Firstly, we annotated the protein-coding genes
based on the protein sequences from \emph{Bos taurus} , \emph{C. hircus}
,\emph{O. aries} and \emph{Homo sapiens} using the BRAKER (v2.1.5)
pipeline (Hoff et al. 2016, Hoff et al. 2019). Secondly, we aligned
three RNA-seq datasets (PRJNA720167) to the takin's genome using STAR
software (v2.7.1a) (Dobin et al. 2013), which were predicted by the
BRAKER pipeline. Next, the BRAKER with TSEBRA module (Gabriel et al.
2021) was used to integrate the prediction results. Finally, the
prediction results were used to align against the non-redundant (Nr)
database using the DIAMOND (v2.0.11.149) software (Buchfink et al. 2015,
Buchfink et al. 2021). GC content distribution of genome was performed
by bedtools (v2.26.0) with a window of 500 kb (Quinlan 2014).

\emph{Mitochondrial genome assembly}

All the HiFi reads (Average length 14 kb) were blasted to the
mitochondrial genome of Sichuan takin (GenBank accession No.
NC\_039686.1) and assembly reference genome of golden takin using the
Minimap2 (v2.22-r1101) (Li 2018). The HiFi reads aligned to NC\_039686.1
were selected to assemble the mitochondrial genome of golden takin. The
sequence similarity was analyzed using the BLAST search program. The
annotation of mitochondrial genome was completed by the AGORA online
platform (Jung et al. 2018).

\emph{Phylogenetic analysis and divergence time estimation}

We downloaded nine mitochondrial genome from GenBank database to
construct a phylogenetic tree, including \emph{B. taurus} (GenBank
accession No. NC\_006853.1), \emph{C. hircus} (GenBank accession No.
NC\_005044.2), \emph{O. aries} (GenBank accession No.
NC\_001941.1),\emph{Pantholops hodgsonii} (GenBank accession No.
NC\_007441.1),\emph{O. moschatus} (GenBank accession No. NC\_020631.1),
\emph{Oreamnos americanus} (GenBank accession No. NC\_020630.1),
\emph{Ammotragus lervia} (GenBank accession No. NC\_009510.1),
\emph{Pseudois nayaur}(GenBank accession No. NC\_020632.1), and \emph{B.
taxicolor} (GenBank accession No. NC\_039686.1, NC\_013069.1,
NC\_043930.1, KY399869.1, KU361169.1 and OM237313 ). Firstly, the
mitochondrial genomes were aligned by MUSCLE (v3.8.31) (Edgar 2004) to
exclude the ambiguous regions. Then, the neighbor-joining (NJ) method
was selected using MEGA (v11) (Tamura et al. 2021) with 1000 bootstrap
replicates.

The genome of~\emph{B. taurus} ~(ARS-UCD1.2) (Rosen et al. 2020) was
selected as reference, and that of \emph{O. americanus} (ASM975805v1)
(Martchenko et al. 2020),\emph{O.
aries}(Oar\_rambouillet\_v1.0),\emph{C. hircus} (ARS1) (Worley
2017),\emph{P. hodgsonii} (PHO1.0) (Ge et al. 2013),\emph{A.
lervia}(ALER1.0) (Chen et al. 2019),\emph{Pseudois nayaur}
(ASM318257v1) (Chen et al. 2019), \emph{O. moschatus} (ASM2146233v1) and
\emph{B. taxicolor}(Takin1.0) were separately aligned to the reference
genome using LAST software (v942) (Kielbasa et al. 2011). The pair-wise
alignments were merged into multiple genome alignments using the MULTIZ
software (v11.2) (Blanchette et al. 2004). The consensus coding
sequences of nine species were extracted using the Perl scripts from RGD
(v2.0). 5, 862 single-copy homologous genes were identified using the
DIAMOND (v2.0.11.149) and RAxML (v8.2.12) (Stamatakis 2014)~with Maximum
Likelihood (ML) method to construct the phylogenetic tree. The
estimations of divergence time were calculated using PAML MCMCtree
(v4.9j) (Yang 2007) with calibrated time from the previous study (Chen
et al. 2019).

\emph{Chromosomal evolution}

The ancestral chromosome karyotype was reconstructed using the genomes
of \emph{B. taurus} (Rosen et al. 2020), \emph{C. hircus} (Li et al.
2021), \emph{O. aries} (Davenport et al. 2021), \emph{B. taxicolor}
and\emph{Physeter catodon} (Fan et al. 2019) (as outgroup). The genome
of\emph{B. taurus} was selected as the reference, and other genomes were
aligned to the reference genome using LAST (v942) (Kielbasa et al.
2011). Next, ``chain'' and ``net'' files were generated from axtChain
and ChainNet, which were used as input for DESCHRAMBLER at a 350 kb
resolution (Kim et al. 2017). Lastly, we analyzed the collinearity of
the genomes of \emph{B. taurus} , \emph{O. aries} and \emph{B.
taxicolor}using mummer (v4.0beta2) (Marcais et al. 2018). We visualized
the collinearity region and detected the chromosome fusion events using
the RectChr (\emph{https://github.com/BGI-shenzhen/RectChr} ).

\emph{Heterozygosity estimation}

Genome resequencing datasets from nine golden takins were aligned with
the assembly reference genome using BWA-MEM (v0.7.17)
(\emph{http://arxiv.org/abs/1303.3997} ). The SAMtools (v1.7) (Li et al.
2009) was used to convert sam to bam files. The single nucleotide
polymorphisms (SNPs) were called and filted using the GATK (v4.1.7.0)
(McKenna et al. 2010) with parameters ``QD \textless{} 2.0, QUAL
\textless{} 30.0, FS \textgreater{} 60.0, MQ \textless{} 40.0, MQRankSum
\textless{} -12.5, ReadPosRankSum \textless{} -8.0''. The nucleotide
diversity (\selectlanguage{greek}θπ\selectlanguage{english}) was calculated using the VCFtools (v0.1.16) with a 50 kb
window (Danecek et al. 2011). Genome resequencing datasets 8 giant
pandas (Zhao et al. 2013) were used to calculate \selectlanguage{greek}θπ. \selectlanguage{english}The genome-wide
heterozygosity rate was calculated as previously described (Liu et al.
2021). Genome resequencing datasets from 26 snub-nosed monkeys (Yu et
al. 2016) were used to calculate heterozygosity. The heterozygosity
rates of other species were obtained from previous reports (Li et al.
2010, Cho et al. 2013, Dobrynin et al. 2015, Liu et al. 2021).

\emph{ROH estimation and inbreeding coefficient}

The runs of homozygosity (ROH) were estimated across autosomes for nine
golden takins using PLINK (v1.90b6.21) (Chang et al. 2015). The
following PLINK parameters (Purcell et al. 2007) were applied to define
a ROH: (i) a 50 SNPs sliding window across the genome; (ii) the
proportion of homozygous overlapping windows was 0.05; (iii) minimum
number of consecutive SNPs included in a ROH was 50; (iv) required
minimum density was set at one SNP per 50 kb; (v) maximum gap between
consecutive homozygous SNPs was 1000 kb; and (vi) maximum of five SNPs
with missing genotypes and up to three heterozygous genotype were
allowed in a ROH. Inbreeding coefficient based on ROH (FROH) for each
individual was calculated according to previous study (McQuillan et al.
2008). The number of generations was estimated to the common ancestor of
these homologous sequences as previously described (Thompson 2013).

\emph{2.9 Demographic history analysis}

The golden takin (TX-11, \textasciitilde{}42X), Milu (\emph{Elaphurus
davidianus} , SRR5762659- SRR5762666, \textasciitilde{}50X) (Chen et al.
2019) and Qinling snub-nosed monkey (SRR2017686, \textasciitilde{}15X)
(Yu et al. 2016) were selected for inferring the effective population
size trajectory using the Pairwise Sequentially Markovian Coalescence
(PSMC) model (v0.6.5-r67) (Li and Durbin 2011). The neutral mutation
rate (\selectlanguage{greek}μ\selectlanguage{english}) was estimated using r8s (v1.81) (Sanderson 2003). The remaining
steps were performed as previously described (Yin et al. 2021).

\textbf{Results}

\emph{Chromosome-level de novo genome assembly of golden takin}

To estimate the genome size of \emph{B. taxicolor} , 124.37 Gb clean
reads were used for k-mer analysis. The genome size was estimated at
2.79 Gb (k=17, \textbf{Figure 1b} ). We constructed a chromosome-level
reference genome for \emph{B. taxicolor} by PacBio HiFi combined with
Hi-C sequencing technology (\textbf{Figure 1c} ). A total of 68.56 Gb
HiFi reads (\textasciitilde{}23\selectlanguage{ngerman}×) were generated by PacBio Sequel II
platform (\textbf{Table S1} ). We assembled 4671 contigs using hifasm
software. The \emph{de novo}assembled reference genome was about 2.95 Gb
with a contig N50 of 68.05 Mb (\textbf{Table 1} ), which had a longer
contig N50 as compared to goat, sheep and cattle reference genomes
(\textbf{Table S2} ). Further, 344.7 Gb Hi-C reads
(\textasciitilde{}117×) spanned to 4,512 scaffolds with a N50 of 101.27
Mb (\textbf{Table 1} ), which anchored onto 25+XY chromosomes
(\textbf{Figure 1d} ). The genome of takin had a good collinearity
relationship with that of the goat (Saanen\_v1) (\textbf{Figure S1} )
and sheep (ARS-UI\_Ramb\_v2.0) (\textbf{Figure S2} ). Based on the
assembled genome, we evaluated its completeness with mammalia\_odb9
database using BUSCO program. The completeness of assembled genome
reached 94.2\%, of which a single-copy reached 93.1\% (\textbf{Table S3}
).

\emph{Genome annotation}

A total of 49.48\% of the assembled genome was repeat sequences. Long
interspersed nuclear elements (LINEs) accounted for 25.08\%, which was
the highest among all types of repeats, followed by short interspersed
nuclear elements (SINEs, 9.92\%), satellites (6.97\%), long terminal
repeats (LTRs, 4.33\%), and DNA elements (1.97\%) (\textbf{Table S4} ).
In addition, 554,037 simple sequence repeats (SSRs, 0.82\%) were
identified (\textbf{Table S4} ). The dimers were the largest proportion
(29.70\%) of SSRs, followed by quadmers (14.72\%), monomers (13.75\%),
pentamers (9.73\%), hexamers (9.20\%) and trimers (8.90\%)
(\textbf{Table S5} ). 21,301 protein-coding genes were predicted by the
homology-based method combined with RNA-seq data. The average gene
length was 26,238 bp with 8.82 exons on average. 99.15\% (20,282) and
87.69\% (18,679) were functionally annotated in the Nr and InterPro
databases, respectively.

\emph{Phylogenetic analysis at the mitochondrial and genomic level}

The assembled mitochondrial genome was about 16,584 bp (GenBank
accession No. OM237313), which shared 98.10\%, 99.05\% and 99.69\%
sequence similarity with Mishmi (GenBank accession No. NC\_043930.1),
Sichuan (GenBank accession No. NC\_039686.1) and golden takin (GenBank
accession No. KY399869.1 and KU361169.1), respectively. At the
mitochondrial genome level, takin was closely related to goat branch as
compared to muskox (\textbf{Figure 2a} ), which is consistent with
previous studies (Feng et al. 2016, Kumar et al. 2019, Zhou et al.
2019).

To reveal the evolution of takin genome, we constructed the phylogenetic
tree with representative subfamily Caprinae species. Interestingly,
takin was most closely related to muskox (\textbf{Figure 2b and Figure
S3} ). Molecular dating using PAML MCMCtree showed that takin had split
from the ancestor of sheep and goat at \textasciitilde{}10.06 million
years ago (Mya), while takin showed a split from the ancestor of muskox
at \textasciitilde{}8.26 Mya (\textbf{Figure 2b} ).

\emph{Chromosome evolution analysis}

Using the sperm whale as outgroup, we reconstructed the ancestral
karyotype (2n=60, \textbf{Table S6} ) of the cattle, goat, sheep and
takin. 559 conserved segments were identified and 30 ancestral
chromosomes with a total length of \textasciitilde{}2.60 Gb were
predicted. Compared to the ancestral karyotype of bovid (2n=60), the
karyotype of cattle (2n=60) was most conserved. The karyotype of goat
(2n=60) occured only one translocation, however the karyotype of sheep
(2n=54) occured three fusions and one translocation. The karyotype of
takin decreased to 2n=52 after occurring four fusions and one
translocation (\textbf{Figure 3a} ). Further, we constructed the
collinear relationship among takin, sheep and cattle. There were three
chromosome (Chr) fusions from cattle to sheep including Chr 1 and 3 to
Chr 1, Chr 2 and 8 to Chr 2, and Chr 5 and 11 to Chr 3. In contrast,
there were four fusions from cattle to takin including Chr 1 and 22 to
Chr 1, Chr 2 and 25 to Chr 2, Chr 5 and 28 to Chr 5, and Chr 11 and 23
to Chr 11. Interestingly, the translocation occurred from bovine Chr 9
to takin's Chr 14 and sheep's Chr 9 (\textbf{Figure 3b} ).

\emph{Genetic diversity and population history of golden takins}

Nine additional golden takins were carried out by genome resequencing,
442.36 Gb clean reads were generated (\textbf{Table S7} ). In total of
3,328,260 SNPs were identified by GATK software. To examine the extent
of inbreeding of golden takins, we estimated the \selectlanguage{greek}θπ, \selectlanguage{english}heterozygosity and
identified the runs of homozygosity (ROH). Golden takins displayed much
lower single nucleotide variants (SNVs) (\selectlanguage{greek}θπ\selectlanguage{english}=0.00028) as compared to
Qinling giant pandas (\selectlanguage{greek}θπ\selectlanguage{english}=0.00133). The average heterozygosity of nine
golden takin genomes was about 0.00038, which was lower than domestic
and most protected wild animals including Qinling giant pandas and
snub-nosed monkeys, while was a little higher than that of snow leopards
and cheetahs (\textbf{Figure 4a} ).

The heterozygosity of golden takin genomes and proportion of the genome
in ROH were shown as \textbf{Figure 4b.} Based on the length of
ROH \textgreater{} 1 Mb (eliminating the effects of linkage
disequilibrium (LD)), the average of genomic inbreeding coefficient
(FROH) was found to be 0.217 (\textbf{Table S8} ). The number of short
ROH (2.5-4.17 Mb) was more than 20, while the number of long ROH
(6.25-10 Mb) was at least one in all of the nine golden takins
(\textbf{Figure 4c} ). The result indicated that inbreeding occurred
from 5\textsuperscript{th} to 20\textsuperscript{th} generations.

To investigate the demographic history of golden takins, the PMSC was
performed. Effective population size dropped significantly from
\textasciitilde{}1 million years ago (the Xixiabangma Glaciation) for
golden takins, Milu and snub-nosed monkeys. The golden takins declined
more significantly than Milu and snub-nosed monkeys from
\textasciitilde{}100,000 years ago (\textbf{Figure 4d} ).

\textbf{Discussion}

\emph{4.1 High-quality chromosome-level assembly reference genome of
takin}

The genome size of takin was about 2.95 Gb, which was slightly larger
than that of the cattle (\textasciitilde{}2.72 Gb) (Rosen et al. 2020),
goat (\textasciitilde{}2.70 Gb) (Li et al. 2021) or sheep
(\textasciitilde{}2.63 Gb) (Davenport et al. 2021). The contig N50 of
takin assembly genome reached 68.05 Mb, which was also larger compared
to cattle (\textasciitilde{}25.90 Mb), goat (\textasciitilde{}46.21 Mb)
and sheep (\textasciitilde{}43.18 Mb). Moreover, the takin assembly
genome, which only has 151 gaps, had a good collinearity relationship
with goat and sheep. Further, Hi-C reads with a N50 of 101.27 Mb were
anchored onto 25+XY chromosomes. These results showed a high-quality
chromosome-level reference genome of takin was assembled. 49.48\% of
reference genome was repeat sequences. Among these repeat sequences,
554,037 SSRs were identified which were more than that of the takin
transcriptome (Qiu et al. 2021). These SSRs could be used as molecular
markers for population genetic analysis and conservation of takins.

\emph{4.2 Taxonomic status and chromosome evolutionof takin}

The mitochondrial genome was assembled from HiFi reads, which showed
high similarity with that of the GenBank database. The HiFi reads can
reach to an average length of 14 kb, thus the assembled mitochondrial
genome was highly credible. Phylogenetic results of subfamily Caprinae
showed that takin was closely related to goat and Bharal at the
mitochondrial genome level. However, it was more closely related to
muskox at the genomic level. It indicated that mitochondrial evolution
is different from genome evolution. Previous studies showed that takin
was most closely related to goat (Feng et al. 2016, Kumar et al. 2019,
Zhou et al. 2019) at the mitochondrial genome level, which was
consistent with the present study. However, based on the morphology,
ecological traits and G-banded karyotype, takin showed similarity to
muskox (Pasitschniak-Arts et al. 1994). Combined with the phylogenetic
analysis at the genomic level, we speculated that it is more reasonable
to classify takin and muskox into subfamily Ovibovini.

Chromosome evolution is linked to phenotypic evolution, gene family
evolution and speciation (Eichler and Sankoff 2003, Damas et al. 2021).
In the Bovidae family, the number of chromosomes dramatically varied in
case of cattle (2n=60), goat (2n=60), sheep (2n=54), takin (2n=52) and
muskox (2n=48). Based on the chromosome-level reference genome in
muskox, the ancestral karyotype (2n=60) was reconstructed with cattle,
goat, sheep and takin. The karyotype of takin (2n=52) occurred when
there were four chromosome fusions and one translocation from the
ancestor (2n=60). As a result, the number of chromosomes in takin has
been reduced to 2n=52. Previous studies showed chromosome evolution was
related to chromosome fission and fusion events (Chen et al. 2019, Liu
et al. 2021, Yin et al. 2021). Interestingly, a functional
single-chromosome yeast was successfully created by end-to-end
chromosome fusions and centromere deletions (Shao et al. 2018).
Therefore, further research on chromosome arrangements is required in
the Bovidae family to reveal the relation of chromosome evolution as
well as rapid evolution of genomic sequences.

\emph{4.3 Genetic diversity and conservation status of takin}

Being a vulnerable species (Zeng and Song 2002), the genetic diversity
of golden takin was very low (\selectlanguage{greek}θπ\selectlanguage{english}=0.00028 and heterozygosity=0.00038),
which was even lower than that of the Qinling giant pandas and
snub-nosed monkeys who live in the same habitat. The high inbreeding
coefficient (FROH=0.217) indicated that golden takins presented
inbreeding depression. The long ROH represents recent inbreeding events,
while the shorter ROH stems from an ancient process or mating between
distant relatives (van der Valk et al. 2019). We speculated that the
golden takins in our study had always been inbreeding from
5\textsuperscript{th} to 20\textsuperscript{th} generations. These
results indicated that the golden takin was in a highly inbreeding state
(Kardos et al. 2021). It has been reported that the number of golden
takin is only about 3, 500 in Qinling mountains (Zeng et al. 2003). It
is probably that the golden takin is currently endangered. Therefore, it
is urgent to establish the protection scheme of golden takin.

\textbf{5. Conclusion}

The high-quality chromosome-level reference genome of the takin
was\emph{de novo} assembled for the first time, with its phylogenic
relationship resolved. The karyotype of takin (2n=52) was formed via
four chromosome fusions and one translocation from the common ancestral
karyotype of bovidae family (2n=60). The very low genetic diversity
(\selectlanguage{greek}θπ\selectlanguage{english}=0.00028 and heterozygosity=0.00038) and high inbreeding coefficient
(FROH=0.217) indicated that takins were in severe inbreeding depression
and endangered. These results provide valuable information to protect
the takin and insights into its evolution.

\textbf{Acknowledgments:} We are very grateful to the Rare Wildlife
Rescue and Breeding Research Center, Shaanxi Academy of Forestry
Sciences for providing the samples. This research was funded by Shaanxi
Province Forestry Science and Technology Innovation Special Project (No.
SXLK2021-0102 and SXLK2020-0301).

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\textbf{Data Availability Statement:} The data presented in this study
are available in the Sequence Read Archive (SRA) database, BioProject:
PRJNA776710 and PRJNA778655. The Takin1.0 genome has been submitted to
the GenBank database.

\textbf{Author Contributions:} Conceptualization, Y. J. and A.~L.;
Methodology and Validation, Q. Y., K. C., R. L. and~X. D.; Formal
Analysis, A.~L. and Q. Y.; Resources, Y. L., K. J. and L.~Z.; Writing --
Original Draft Preparation, A.~L.; Writing -- Review \& Editing, Y. J.,
R. L., Q. Y.~and X. D.; Supervision, Y. J. and L. Z.; Project
Administration, Y. J. and A.~L.; Funding Acquisition,~L. Z. All authors
have read and agreed to the published version of the manuscript.

\textbf{Conflicts of Interest:} The authors declare no competing
financial interest.

Supplemental Figures and Tables (Captions)

\textbf{Figure S1.} The collinearity relationship between takin and goat
genome.

\textbf{Figure S2.} The collinearity relationship between takin and
sheep genome.

\textbf{Figure S3.} Phylogeny of the subfamily Caprinae in the genomic
level with bootstrap.

\textbf{Table S1} Summary of HiFi reads.

\textbf{Table S2} Comparison of well-assembled genomes.

\textbf{Table S3} Assessment of genome completeness.

\textbf{Table S4} Repeat content of Takin.

\textbf{Table S5} Simple repeats content of Takin.

\textbf{Table S6} Lengths of reconstructed ancestral chromosome (chr)
karyotypes of Bovidae

\textbf{Table S7} Genome resequencing of 9 golden takins.

\textbf{Table S8} The statistics of ROH.

\textbf{Table 1.} Assembly statistics of takin genome\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Assembly} & \textbf{Number/length}\tabularnewline
Total assembly length (bp) & 2,947,084,417\tabularnewline
Gap number & 151\tabularnewline
Number of contigs & 4,671\tabularnewline
N50 contig length (bp) & 68,053,581\tabularnewline
Contig L50 & 16\tabularnewline
Number of scaffolds & 4,512\tabularnewline
N50 scaffold length (bp) & 101,265,939\tabularnewline
Scaffold L50 & 11\tabularnewline
Chromosome number & 25+XY\tabularnewline
GC content (\%) & 43.22\tabularnewline
Protein-coding genes & 21,301\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. High-quality chromosome-level reference genome
assembly.}

\textbf{(a)} Golden takin; \textbf{(b)} K-mer analysis of the genome
size by gce (v1.0.2) (\emph{https://arxiv.org/abs/1308.2012v2}
);\textbf{(c)} Circos plot showing the visualization of genomic details
with a window of 500 kb. a. gene density; b. repeat content; c. GC
content; \textbf{(d)} The Hi-C interaction heatmap of takin.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Phylogeny of the subfamily Caprinae in the
mitochondrial and genomic level.}

\textbf{(a)} Takin was closely related to goat clade, but far to muskox
in the mitochondrial genomic level. The neighbor-joining (NJ) method
with 1000 bootstrap replicates using MEGA (v11) (Tamura et al.
2021);\textbf{(b)} Takin was most closely related to muskox in the
genomic level. 5862 single-copy homologous genes were used to construct
the phylogenetic tree by RAxML (v8.2.12) with Maximum Likelihood (ML)
method. The divergence time were calculated using PAML (v4.9j) with
calibrated from divergence time of goat and sheep (3.9-8.1Mya) (Chen et
al. 2019). The estimated divergence time (Mya) was marked on each node
(95\% confidence intervals).

\textbf{Figure 3. Reconstruction of ancestral chromosomes of cattle,}
\textbf{goat, sheep and takin.}

\textbf{(a)} The distribution of ancestral chromosome segments in the
genomes of cattle, goat, sheep and takin, including chromosome fission
and fusion events. The asterisks represent chromosome fission
events;\textbf{(b)} The collinear relationship of takin, sheep and
cattle. Sperm whale as outgroup.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Genetic diversity and demographic history of nine
golden takins.}

\textbf{(a)} The average heterozygosity of different domestic and wild
animals. Genome resequencing datasets from nine golden takins were used
to calculate heterozygosity as previously described (Liu et al. 2021).
The heterozygosity rates of other species were reported in previous
reports (Li et al. 2010, Cho et al. 2013, Dobrynin et al. 2015, Liu et
al. 2021). \textbf{(b)} The heterozygosity and proportion of the genome
in ROH from nine golden takins. \textbf{(c)} The distributions of ROH
statistics. With the length of ROH increasing, the number of ROH
gradually decreased. \textbf{(d)} Demographic history analysis of golden
takins, Milu and snub-nosed monkeys estimated by PSMC. The gray box
marks the time range of the Xixiabangma Glaciation (0.8-1.17 million
years ago). ``g'' represents generation, ``\selectlanguage{greek}μ\selectlanguage{english}'' represents neutral
mutation rate.

\selectlanguage{english}
\FloatBarrier
\end{document}

