Genomics 114 (2022) 110518

Contents lists available at ScienceDirect

Genomics
journal homepage: www.elsevier.com/locate/ygeno

Chromosome-level genome assembly of the Muscovy duck provides insight

into fatty liver susceptibility
Ming-Min Xu a, b, 1, Li-Hong Gu c, 1, Wan-Yue Lv a, d, 1, Sheng-Chang Duan e, 1, Lian-Wei Li b, f,
Yuan Du e, Li-Zhi Lu g, Tao Zeng g, Zhuo-Cheng Hou h, Zhanshan Sam Ma b, f, Wei Chen i, j,
Adeniyi C. Adeola a, Jian-Lin Han k, l, Tie-Shan Xu m, *, Yang Dong i, j, **, Ya-Ping Zhang a, b, d, n, ***,
Min-Sheng Peng a, b, n, ***
a
State Key Laboratory of Genetic Resources and Evolution & Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming 650223, China
b
Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, China
c
Institute of Animal Science & Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, China
d
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming 650091, China
e
Nowbio Biotechnology Company, Kunming 650201, China
f
Computational Biology and Medical Ecology Lab, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, China
g
Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
h
National Engineering Laboratory for Animal Breeding and Key Laboratory of Animal Genetics, Breeding and Reproduction, MARA; College of Animal Science and
Technology, China Agricultural University, Beijing 100193, China
i
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China
j
Key Laboratory for Agro-Biodiversity and Pest Control of Ministry of Education, Yunnan Agricultural University, Kunming 650201, China
k
CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing
100193, China
l
Livestock Genetics Program, International Livestock Research Institute (ILRI), Nairobi 00100, Kenya
m
Tropical Crops Genetic Resources Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
n
KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming
650223, China

A R T I C L E I N F O A B S T R A C T

Keywords: The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty
Fatty liver liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver
Muscovy duck disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In
Genome assembly
this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb
Evolutionary by-product
and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak
Accelerated CNEs
lipid catabolism capabilities (fatty acid β-oxidation and lipolysis). Furthermore, conserved noncoding elements
(CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of
genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an
evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the suscepti­
bility of Muscovy duck to fatty liver.

* Corresponding author.
** Correspondence to: Y. Dong, State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming
650201, China.
*** Correspondence to: Y.-P. Zhang and M.-S. Peng, State Key Laboratory of Genetic Resources and Evolution & Yunnan Laboratory of Molecular Biology of Do­
mestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.
E-mail addresses: xutieshan760412@163.com (T.-S. Xu), loyalyang@163.com (Y. Dong), zhangyp@mail.kiz.ac.cn (Y.-P. Zhang), pengminsheng@mail.kiz.ac.cn
(M.-S. Peng).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ygeno.2022.110518
Received 14 August 2022; Received in revised form 1 November 2022; Accepted 4 November 2022
Available online 5 November 2022
0888-7543/© 2022 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.-M. Xu et al.
1. Introduction
The Muscovy duck (Cairina moschata) is a tropical bird native to
Central and South America [1]. The drake is characterized by a nuchal
crest and fleshy caruncles distributed around the orbital region to the
base of the bill [2]. Wild Muscovy ducks prefer to live in marshy forests,
roosting in trees and nesting in tree hollows [3], are highly opportunistic
omnivores, feeding primarily on native vegetation and corn as well as
small fish, reptiles, invertebrates, crustaceans, and insects [4,5]. The
Muscovy duck is one of only two large birds (including the turkey) that
were originally domesticated in the New World [6]. During the second
voyage of Columbus, Muscovy ducks were brought back to Europe, with
subsequent dispersal to Africa, Asia, and Australia [2,6]. As an
economically important poultry breed, Muscovy ducks are now farmed
worldwide for meat consumption due to their leanness, tenderness, and
taste [7,8], and are an essential source of income in many rural com­
munities, especially in developing countries in Africa [9,10].
The liver is a primary site of lipid metabolism in birds [11]. In
waterfowl, non-pathological hepatic steatosis occurs spontaneously for
energy storage prior to migration [12]. Muscovy and Peking ducks (Anas
platyrhynchos) are two common domesticated waterfowls, which
diverged from their common ancestor ~13.8 million years ago [13] and
Fig. 1. Overview of the Muscovy duck genome assembly. (a) Pipeline used for assembly of the Muscovy duck genome. (b) Hi-C heat map of Muscovy duck showing
chromosome interactions. (c, d) Dot plots of whole-genome alignment between Muscovy and Peking duck genomes show a high degree of synteny, including
macrochromosomes (c) and microchromosomes (d).
2
Genomics 114 (2022) 110518
show marked differences in fatty liver susceptibility. Notably, Muscovy
ducks are prone to develop severe fatty liver when overfed high carbo­
hydrate diets, while Peking ducks tend to develop mild fatty liver [14].
These differences in fatty liver susceptibility are mainly attributed to
genetic differences between the two species [15,16]. However, the ge­
netic mechanism underlying the susceptibility of Muscovy ducks to fatty
liver remains unclear, hindered by the lack of a high-quality genome.
However, this unique susceptibility to fatty liver is also utilized. Mule
ducks produced from crossing male Muscovy ducks with female Peking
ducks have replaced geese in foie gras production [17]. Moreover, due to
the shared fatty liver phenotype, the Muscovy duck is proposed as a
potential animal model for studying non-alcoholic fatty liver disease
(NAFLD) in humans. With an estimated global prevalence of 25%,
NAFLD is regarded as a hepatic manifestation of metabolic syndrome
[18,19], and is the fastest growing cause of hepatocellular carcinoma,
liver transplantation, and liver-related mortality [20,21]. Fatty liver is
characterized by lipid accumulation and deposition in hepatocytes and
can evolve into NAFLD [22]. Hence, understanding the susceptibility of
Muscovy ducks to fatty liver may provide insight into the study of
NAFLD in humans.
Herein, we de novo assembled a chromosome-level genome of the
Muscovy duck. We performed comparative genomic and transcriptomic
M.-M. Xu et al. Genomics 114 (2022) 110518

analyses to investigate the genetic mechanism underlying susceptibility
to fatty liver in Muscovy duck, emphasizing the contribution of accel­
erated evolution of conserved noncoding elements (CNEs). This study
provides novel insights into the genetic mechanism underlying fatty
liver from an evolutionary medicine perspective and highlights the po­
tential of the Muscovy duck as a model animal for studying NAFLD.
2. Results
2.1. Genome assembly and annotation
Genome assemblies with a high degree of completeness and conti­
guity are desirable for comparative genomic analyses. The recently
published draft Muscovy duck genome was assembled exclusively by
linked-read sequencing, a cost-effective approach at the expense of
genome contiguity [13]. Therefore, we de novo assembled a Muscovy
duck genome combining Oxford Nanopore long reads, Hi-C reads,
linked-reads (10× Genomics), and Illumina short reads (Table S1). The
assembled contigs were scaffolded into 40 pseudochromosomes
(Figs. 1a, b and 2) according to previous study [23], resulting in a
chromosome-level genome assembly (KizCaiMos1.0) with a contig N50
of 11.81 Mb and scaffold N50 of 83.16 Mb. The contig N50 was ~37
times higher than the recently published assembly (ASM1810499v1)
[13]. The KizCaiMos1.0 assembly showed excellent collinearity with
both the chicken and duck genome references [24] (Fig. 1c, d, and S3).
Syntenic analyses revealed misplacements and inversions in the recently
published draft Muscovy duck genome [13], which we aligned to the
KizCaiMos1.0 assembly (Figs. S4). Results showed that 94.3% of the
Aves Benchmarking Universal Single-Copy Orthologs (BUSCO) could be
aligned to the Muscovy duck genome (Table 1). Compared with the
previous assemblies [13], the KizCaiMos1.0 genome exhibited higher
continuity and completeness (Table 1) and was therefore used for sub­
sequent analyses.
We performed de novo and homology-based gene prediction of the
Muscovy duck genome (Fig. S5; Table S2). In total, 15,829 protein-
coding genes were annotated in the KizCaiMos1.0 assembly. We

Fig. 2. Landscape of the Muscovy duck genome. Tracks (from outer to inner circles) indicate: (a) contigs and gaps as blank; (b) GC content (window size of 100 kb);
(c) mobile elements content (window size of 100 kb with a step size of 20 kb); (d, e) DNA methylation level (window size of 100 kb; darker colour indicates higher
methylation), including 5mC (d) and 6 mA (e); (f) gene density (gene numbers per 100 Kb); (g) gene expression level (FPKM; highest expression of 15 sequenced
tissues are shown; darker colour indicates higher expression level); and (h, i) variants density (window size of 100 kb), including single nucleotide polymorphisms
(SNPs) (h) and indels (i). Outer gray circle represents Muscovy duck chromosome length.

3
M.-M. Xu et al.
Table 1
Quality comparison for different versions of Muscovy duck assemblies.
Assembly Largest contig (Mb)a N50 contigs (Mb)a
Cairina moschata (Muscovy duck)
0.87 0.09
[CaiMos1.0]
Cairina moschata (Muscovy duck)
2.05 0.32
[ASM1810499v1]
Cairina moschata (Muscovy duck)
53.95 11.81
[KizCaiMos1.0]
Anas platyrhynchos (mallard)
28.50 5.68
[ZJU1.0]
Gallus gallus (chicken)
65.77 17.49
[GRCg6a]
a
Statistics were calculated using stats.sh script in BBMap(v.38.45).
b
Sum of all “N” nucleotides present in the genome assembly.
c
BUSCO assessment of genome assembly. C: Complete BUSCOs; S: Complete and single-copy BUSCOs; D: Complete and duplicated BUSCOs; F: Fragmented BUSCOs;
M: Missing BUSCOs; n: Total BUSCO groups searched.
further annotated non-coding RNA genes, including 176 microRNAs
(miRNAs), 270 ribosomal RNAs (rRNAs), 227 small-nucleolar RNAs
(snRNAs), and 360 transfer RNAs (tRNAs) (Table S3). Repeat sequences
were also identified (Table S4). Transposable elements comprised 10.3%
of the genome, 5% of which were long interspersed nuclear elements. In
addition, C5-methylcytosine (5mC) and N6-methyldeoxyadenosine
(6mA) sites, two important types of DNA methylation, were first
detected in the genome by decoding the raw electric signal during
Nanopore sequencing. The average levels of genome methylation for
5mC and 6mA were 10.73% and 0.076%, respectively (Figs. 2 and S6).
2.2. Genomic selection and expansion of gene families in Muscovy ducks
To systematically investigate the genomic variations underlying fatty
liver susceptibility in Muscovy ducks, we conducted comparative
genomic analysis of 10 species (Fig. S7). No positively selected genes
(PSGs) and only five rapidly evolving genes (REGs) were retained ac­
cording to the false discovery rate (FDR)-adjusted P-value <0.05. Thus,
we used the threshold of P-value <0.05 (chi-square test without FDR
correction) and identified 38 putative PSGs and 190 putative rapidly
REGs (Tables S5 and S6). However, only a few PSG and REG hits were
found in lipid metabolism items catalogued in the Reactome database
(Fig. S8). To some extent, this implies weak selective signals against
lipid metabolism on the coding regions in the Muscovy ducks. Analysis
of gene family evolution detected 170 expanded and 1396 contracted
gene families in the Muscovy duck genome (Fig. S9a). Functional
enrichment analysis revealed that the expanded gene families were
enriched in carbohydrate digestion and absorption (Fig. 3a; Table S7),
suggesting improved carbohydrate digestion and absorption capacity for
adaptation to a plant-based diet. However, functional enrichment
analysis of the rapidly expanded gene families specific to Muscovy ducks
(Fig. S9b; Table S8) failed to detect any enriched signals directly related
to lipid metabolism. Taken together, these findings suggest that selec­
tion on coding regions and expanded gene families is unlikely to be the
major evolutionary force driving susceptibility to fatty liver in Muscovy
ducks.
2.3. Evolution of CNEs in Muscovy ducks
CNEs are conserved DNA regions that evolved under purifying se­
lection and do not encode proteins [25–27]. CNEs act as cis-regulatory
elements, including enhancers, insulators, and repressors, to regulate
the expression of target genes [28,29]. Divergence and loss of CNEs
during evolution account for a larger portion of phenotypic diversity
among species [27,30–32]. Here, we conducted a genome-wide
screening of CNEs that were specifically lost or underwent rapid evo­
lution in the Muscovy duck. We identified 272,251 CNEs from whole-
genome alignment of 10 species. Among these CNEs, 669 were
Genomics 114 (2022) 110518
Largest scaffold (Mb)a N50 scaffolds (Mb)a “N” gaps (Mb)b BUSCO assessmentc
C:94.5% [S:93.1%, D:1.4%],
177.54 58.54 28.77
F:3.3%, M:2.2%, n:4915
C:93.7% [S:92.0%, D:1.7%],
194.81 77.35 15.03
F:3.1%, M:3.2%, n:4915
C:94.3% [S:93.2%, D:1.1%],
200.48 83.16 0.34
F:2.8%, M:2.9%, n:4915
C:94.3% [S:92.6%, D:1.7%],
207.24 76.27 4.23
F:3.4%, M:2.3%, n:4915
C:91.1% [S:90.0%, D:1.1%],
197.60 91.31 9.78
F:5.4%,M:3.5%,n:4915
considered lost during the Muscovy duck evolution. Interestingly, a CNE
located in an intronic region of CDH13 was specifically lost in the
Muscovy duck (Fig. 3b). The CDH13 gene encodes for cadherin 13, a
vascular adiponectin receptor [33]. Furthermore, in humans, CDH13 is
reported to influence adiponectin levels in different ethnic populations
[34–36]. Adiponectin is an adipokine secreted by adipocytes and can
promote fatty acid β-oxidation [37,38]. Thus, we speculated that the loss
of the CNE in CDH13 may alter fatty acid β-oxidation in the Muscovy
duck by regulating adiponectin levels. We also identified 1379 CNEs
showing accelerated evolution specific to the Muscovy duck lineage
(hereafter referred to as Muscovy-accelerated CNEs; Table S9). The
typical cases of these Muscovy-accelerated CNEs will be elucidated later
through combination of other analyses.
2.4. Distinctive lipid metabolism in Muscovy ducks
Given their different fatty liver susceptibility, we considered that the
Muscovy duck may be characterized by a unique lipid metabolism
profile compared to the Peking duck. To investigate differences in lipid
metabolism between these species, we retrieved publicly available
transcriptomic data of liver tissues from both ducks, fed under two
conditions (ad libitum vs. overfeeding) [14]. The downloaded RNA
sequencing (RNA-seq) data were used in the following analyses. Inter-
specific transcriptomic analysis was performed to characterize the he­
patic gene expression profiles of the Muscovy duck, using Peking duck as
a reference, under the two feeding conditions (ad libitum vs. over­
feeding), based on 13,417 orthologous genes (Table S10).
Overall, we identified 3780 differentially expressed genes (DEGs)
between the two species independent of feeding condition (Fig. S10;
Table S11). The significant differences in gene expression were mainly
ascribed to genetic divergences between the two duck species. Among
the DEGs, 2183 (58%) were lowly expressed (hereafter referred to as
Muscovy-low DEGs; Table S12) and 1597 were highly expressed (Mus­
covy-high DEGs; Table S13) in the Muscovy duck liver compared to the
Peking duck liver. However, no functional enrichments related to lipid
or fatty acid metabolism were detected in the Muscovy-high DEGs
(Table S14). In contrast, functional enrichment analysis revealed that
the Muscovy-low DEGs were significantly enriched in fatty acid
biosynthesis (Fig. S11; Tables S15 and S16). In addition, several
Muscovy-low DEGs were functionally associated with lipid catabolism
(fatty acid β-oxidation and lipolysis) (Figs. 4a and S12).
Gene set enrichment analysis (GSEA) revealed that fatty acid
biosynthesis was up-regulated in Peking ducks, while fatty acid
β-oxidation was down-regulated in Muscovy ducks in response to
overfeeding (Fig. 4b and c; Tables S17 and S18). Weighted gene coex­
pression network analysis (WGCNA) was applied to characterize the
transcriptomes of the two species. We identified seven modules with
gene sizes ranging from 117 to 3405, among which the brown module
4
M.-M. Xu et al. Genomics 114 (2022) 110518

(a)
Biosynthesis of secondary metabolites
Neutrophil extracellular trap formation
Shigellosis
Human papillomavirus infection
Systemic lupus erythematosus
Focal adhesion
cAMP signaling pathway
Biosynthesis of amino acids
Lysine degradation
Amoebiasis p.adjust

Arginine and proline metabolism 0.01

0.02
Alcoholism
0.03
Adrenergic signaling in cardiomyocytes 0.04
Transcriptional misregulation in cancer
Toxoplasmosis
Phospholipase D signaling pathway Count
10
Small cell lung cancer 20
Herpes simplex virus 1 infection 30
ECM−receptor interaction
Necroptosis
Dopaminergic synapse
Glucagon signaling pathway
Aldosterone synthesis and secretion
Glioma
Insulin secretion
Phototransduction−fly
Carbohydrate digestion and absorption
0.025 0.050 0.075 0.100 0.125
GeneRatio

(b) CDH13
100
Muscovy duck
50
Peking duck
Nucleotide sequence similarity
to the chicken counterpart (%)

Goose
Turkey

Guineafowl
Collared flycatcher
Zebra finch
Emu
Alligator
(chr11:16,261,486-16,271,486)

Fig. 3. Comparative genomic analysis. (a) Functional enrichment analysis of expanded gene families in Muscovy duck genome. (b) VISTA sequence conservation plot
of specific CNE loss in Muscovy duck. Using the chicken genome as a reference, sequence alignment for nine species was performed to predict conserved regions with
≥80% identity and ≥ 50 bp window size. Exonic region is indicated by blue shadowed column. Lost CNE was located in the 7th intronic region of the CDH13 gene
indicated by gray shadowed column. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

5
M.-M. Xu et al. Genomics 114 (2022) 110518

(a)
condition
2
species
1

}
HSD17B12
ABCD3 0
CROT -1
ACSL1 fatty acid oxidation -2
CPT1A
CDH13
ADIPOR2 condition

}
ATGL fed ad libitum
ABHD5 lipolysis overfed
MGLL
species

}
FOXO1
FOXO3 Peking duck
FOXOs
FOXO4 Muscovy duck
FOXO6

(b) (c)
Peking duck (fed ad libitum vs. overfed) Muscovy duck (fed ad libitum vs. overfed)

fatty−acyl−CoA biosynthetic process fatty acid oxidation

Running Enrichment Score

Running Enrichment Score

0.6 unsaturated fatty acid biosynthetic process fatty acid beta−oxidation

0.0

0.4 −0.2

0.2 −0.4

0.0 −0.6
Ranked List Metric
Ranked List Metric

7.5 8

5.0 4
2.5
0
0.0
−2.5 −4

3000 6000 9000 3000 6000 9000

Rank in Ordered Dataset Rank in Ordered Dataset
(d) (e)
Module-trait relationships
1
−0.22 0.86 −0.7 0.055
MEgreen (0.2) (6e−12) (1e−06) (0.7)

−0.62 0.52 −0.47 0.57

MEyellow (4e−05) (9e−04) (0.003) (2e−04)

0.5
0.55 0.6 −0.6 −0.54
MEturquoise (4e−04) (7e−05) (6e−05) (5e−04)

0.41 0.14 0.26 −0.83

MEblack (0.01) (0.4) (0.1) (1e−10)

0
0.83 −0.27 0.11 −0.66
MEred (2e−10) (0.1) (0.5) (7e−06)

−0.53 −0.61 0.6 0.54

MEblue (6e−04) (5e−05) (6e−05) (5e−04)

−0.5
0.33 −0.79 0.64 −0.18
MEbrown (0.04) (4e−09) (1e−05) (0.3)

−0.57 0.52 −0.51 0.57

MEgrey (2e−04) (9e−04) (0.001) (2e−04)

−1
Muscovy duck Muscovy duck Peking duck Peking duck
fed ad libitum overfed fed ad libitum overfed

(caption on next page)

6
M.-M. Xu et al.
Fig. 4. Comparative transcriptomic analysis. (a) Heat map of gene expression for Muscovy-low DEGs. (b, c) Gene set enrichment analysis (GSEA) of lipid metabolism
pathway in livers of Peking (b) and Muscovy ducks (c). (d) Relationships between module and traits. Every cell contains the corresponding correlation coefficient and
P-value shown in brackets. Cell is colored by correlation value according to colour legend. (e) Gene expression network for brown module. Edges with weight > 0.1
are plotted. Circles in network represent genes. Hub genes are represented by solid circles and labelled by gene names. Red solid circles indicate hub genes also
characterized as Muscovy-low DEGs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
was the most negatively associated with overfed Muscovy ducks with
severe fatty liver (Fig. 4d). Genes in the brown module were enriched in
fatty acid β-oxidation (Table S19). We detected 37 hub genes in the
brown module, several of which functionally promote fatty acid
β-oxidation (PPARGC1A [39], CROT [40], ACSL1 [41], ACOT13 [42],
CPT1A [43], and ACADM [44]), with CPT1A, CROT, and ACSL1 also
characterized as Muscovy-low DEGs (Figs. 4e and 6b; Table S20). These
results suggest marked differences in fatty acid metabolism, especially in
fatty acid synthesis and β-oxidation, between the two species. Compared
to the Peking duck, the Muscovy duck showed insufficient fatty acid
biosynthesis and β-oxidation abilities.
2.5. Rapidly evolving CNEs shape lipid metabolism in Muscovy ducks
To explore and compare the potential functions of Muscovy-
accelerated CNEs of DEGs in Muscovy and Peking ducks, we collected
a Muscovy duck liver sample to generate Hi-C sequencing, assay for
transposase-accessible chromatin sequencing (ATAC-seq), and RNA-seq
data (Table S1). Comparative genomic and transcriptomic analyses were
performed using Hi-C, ATAC-seq, and RNA-seq data. We firstly gener­
ated a three-dimensional (3D) genome map of the Muscovy duck with a
resolution of 3.35 kb. We identified 835 topologically associating do­
mains (TADs) and 11,740 loops. Additionally, 107,367 ATAC-seq peaks
and 10,254 commonly expressed genes (TPM ≥ 1) were detected in the
Muscovy duck liver tissue.
In total, 90.14% (1243 in 1379) of the Muscovy-accelerated CNEs
were associated with 7803 potential target genes by colocalization
within the same TADs, of which 71.15% (5552 in 7803) were commonly
expressed in the Muscovy duck liver (Table S21). A total of 1538 po­
tential target genes were characterized as DEGs and commonly
expressed in the Muscovy duck liver (Fig. 5a). These results indicate that
the accelerated CNEs were more likely to account for the distinctive
hepatic gene expression profiles in the Muscovy duck, treating CNEs as a
proxy for cis-regulatory elements. Furthermore, 59.82% (920 in 1538) of
potential target genes were Muscovy-low DEGs and were enriched in
biological processes relevant to fatty acid synthesis and oxidation
(Fig. 5b; Table S22). However, limited enrichment in fatty acid or lipid
metabolism was detected for potential target genes characterized as
Muscovy-high DEGs (Table S23).
For Muscovy-low DEGs, we identified a Muscovy-accelerated CNE
located in the intronic region of MGLL (Fig. 5c, d, and e), a key gene that
catalyzes the final step of lipolysis [45]. Mus.CNE.2447 was proximal to
significant ATAC-seq peaks and interacted with the initial region of
MGLL in the loop within the same TAD (Fig. 5f). The reporter assay
results confirmed that Mus.CNE.2447 may exhibit enhancer activity, but
the enhancer activity was weaker in the Muscovy duck than in the
Peking duck (Fig. 5g), implying weaker lipolysis ability. Collectively,
our findings suggest that the accelerated evolution of CNEs may have
shaped the distinctive lipid metabolism of Muscovy ducks by down-
regulating genes involved in fatty acid synthesis, fatty acid oxidation,
and lipolysis. The rapidly evolving CNEs may account for the suscepti­
bility to fatty liver in Muscovy ducks.
3. Discussion
3.1. Weak lipid catabolism contributes to fatty liver susceptibility in
Muscovy ducks
Reduced fatty acid oxidation (especially β-oxidation) leads to excess
7
Genomics 114 (2022) 110518
lipid storage in the liver and hepatic steatosis [46–48]. Here, several hub
genes in the brown module known to promote fatty acid oxidation (e.g.,
CROT, ACSL1, and CPT1A) were also characterized as Muscovy-low
DEGs (Fig. 4a, e, and S12). For instance, the protein encoded by
CPT1A transfers fatty acids into the mitochondria, which is a rate-
limiting step in subsequent fatty acid β-oxidation [43]. Importantly,
we also found that fatty acid β-oxidation was exclusively down-
regulated in overfed Muscovy ducks (Fig. 4c), consistent with previous
research showing low expression of fatty acid β-oxidation-related genes
in the livers of overfed Muscovy ducks [14]. Hepatic steatosis develops
when there is an imbalance between lipid storage and lipolysis in lipid
droplets, resulting in triglyceride accumulation in hepatocytes [49]. The
ATGL gene initiates the first step in lipolysis, with enzymatic activity
activated by the protein product of CGI-58 (also known as ABHD5)
[50,51]. The enzyme encoded by the MGLL gene catalyzes the last step
in triglyceride hydrolysis [45] (Fig. 6b). In our study, ATGL, ABHD5, and
MGLL were identified as Muscovy-low DEGs (Figs. 4a, 5e, and S12). In
conclusion, reduced fatty acid β-oxidation increased intrahepatic fatty
acid accumulation and impaired lipolysis increased accumulated lipids
storage in lipid droplets, resulting in greater lipid retention in the liver of
Muscovy ducks (Fig. 6a).
3.2. Accelerated evolution of CNEs drives lipid metabolism in Muscovy
ducks
Previous studies in birds have shown that CNEs are associated with
developmental genes and that convergent evolution of CNEs likely
contributed to loss of powered flight [32,52]. Our work provides addi­
tional evidence that accelerated CNEs also played a dominant role in
shaping avian lipid metabolism. Notably, accelerated CNEs promoted
fatty liver in Muscovy ducks by down-regulating a series of genes
involved in fatty acid β-oxidation and lipolysis (Figs. 6b and S13).
Interestingly, the CPT1A gene, a potential target gene of Muscovy-
accelerated CNEs and characterized as a Muscovy-low DEG in the liver
(Figs. 4a and S12), showed no expression differences between the two
duck species in the muscle [53]. This suggests that accelerated CNEs
shape avian lipid metabolism in a tissue-specific manner. In contrast,
our results show weak selective signals against lipid metabolism on
coding regions (Tables S5 and S6; Fig. S8). This further supports the
hypothesis that cis-regulatory mutations causing morphological muta­
tions are dominant in long-term evolution, as compared to coding re­
gions [54].
3.3. Fatty liver susceptibility is an evolutionary by-product in Muscovy
ducks
Fatty acids, as a high-energy fuel, are generally preferred by
migratory birds that require excessive energy consumption for extended
flight [11,55]. The Muscovy duck is non-migratory throughout its wild
range [5,56]. In this species, reduced lipolysis leads to lower free fatty
acid production, while attenuated fatty acid β-oxidation results in lower
acyl-CoA for energy generation. Therefore, we hypothesize that Mus­
covy ducks require less fatty acid for energy consumption due to their
non-migratory habits, in contrast to migratory mallards (Anas pla­
tyrhynchos, wild ancestor of Peking ducks) [57,58]. The digestion and
absorption capabilities of animals typically reflect the main nutrients
and energy sources (such as carbohydrates, proteins, and fats) in their
diets [59–61]. In the wild, Muscovy ducks mainly feed on plant food,
which is generally rich in carbohydrates [4,61]. In the current study,
M.-M. Xu et al. Genomics 114 (2022) 110518

(caption on next page)

8
M.-M. Xu et al. Genomics 114 (2022) 110518

Fig. 5. Integrations of comparative genomic and transcriptomic analyses. (a) Venn diagram showing overlap in DEGs between Peking and Muscovy ducks,
commonly expressed genes (TPM ≥ 1) in Muscovy duck liver, and potential target genes of Muscovy-accelerated CNEs. (b) Selected enriched GO terms relevant to
lipid metabolism for potential target genes of Muscovy-accelerated CNEs, which were also Muscovy-low DEGs and commonly expressed in Muscovy duck liver. (c)
VISTA visualization of Muscovy-accelerated CNE (Mus.CNE.2447). Using the chicken genome as a reference, sequence alignment for nine species was performed to
predict conserved regions with ≥80% identity and ≥ 50 bp window size. Mus.CNE.2447 was located in the 2nd intronic region of the MGLL gene, indicated by gray
shadowed column. (d) Sequence alignment for Mus.CNE.2447 among nine species. Sequences in each row represent CNEs for species in the same order as those in
VISTA visualization. (e) Expression of the MGLL gene between Peking and Muscovy ducks under two feeding conditions. Gene expression was measured by
normalized gene counts. P-values for comparisons were determined by Wilcoxon test. (f) Integrative map of Mus.CNE.2447 and MGLL locus. TAD and loop are shown
with triangles and red arcs, respectively. (g) Results of dual-luciferase reporter assays for Mus.CNE.2447. Significance was tested by Student's t-test and data are
represented as mean ± standard error of the mean (SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)

gene families enriched in carbohydrate digestion and absorption were
significantly expanded (Fig. 3a), suggesting that Muscovy ducks prefer
carbohydrates to fatty acids as their major energy source, in part due to
dietary adaptation. This appears to be a common strategy adopted by
birds. For example, the ATGL protein, which catalyzes the first step in
lipolysis, shows reduced hydrolytic activity in flight-degenerate bird
species, which tend to utilize carbohydrates as their main energy source
[62]. In our study, both the ATGL and MGLL genes were characterized as
Muscovy-low DEGs (Figs. 4a and S12). Thus, we propose that the
adaptation of Muscovy ducks to carbohydrate-dominated diets and non-
migratory habits led to weak lipid catabolism, and that fatty liver sus­
ceptibility is an evolutionary by-product.
3.4. Muscovy ducks as a potential animal model of NAFLD
Muscovy ducks share several molecular characteristics of hepatic
steatosis with human patients and genetically manipulated model ani­
mals. Notably, several genes characterized as Muscovy-low DEGs are
associated with hepatic steatosis (Figs. 4a and S12). For example, CPT1A
expression is significantly decreased (by 50%) in NAFLD patients [63]
and liver-specific CPT1A knockout mice display severe hepatic steatosis
[64]. Liver-specific ATGL or ABHD5 knockout mice show triglyceride
accumulation and steatosis in the liver [51,65] and liver specific
FOXO1/3/4 triple knockout mice develop severe hepatic steatosis [66].
Muscovy ducks can also develop hepatic steatosis after only for two
weeks of overfeeding [17], which is helpful for experimental research.
Thus, our study highlights the potential of Muscovy ducks as a model
animal for NAFLD.
4. Conclusions
We presented a high-quality chromosome-level genome and high-
resolution 3D genome map for the Muscovy duck, providing an impor­
tant genetic resource for evolutionary and functional genomic studies in
waterfowl. By integrating RNA-seq, Hi-C, and ATAC-seq data with the
newly assembled genome, we revealed that the weak fatty acid
β-oxidation and lipolysis ability of Muscovy ducks likely contributes to
their fatty liver susceptibility. The Muscovy duck-specific accelerated
CNEs down-regulated the expressions levels of many hepatic genes
related to fatty acid β-oxidation and lipolysis. Importantly, the suscep­
tibility of Muscovy ducks to fatty liver may be an evolutionary by-
product of their adaptation to a carbohydrate-dominated diet and non-
migratory habit.
5. Materials and methods
5.1. Sample collection and sequencing
All animal experiments were approved by the Ethics and Experi­
mental Animal Committee of the Kunming Institute of Zoology, Chinese
Academy of Sciences, China (Approval ID: IACUC-OE-2021-06-001).
Genomic DNA was extracted from whole blood and muscle of a male
Muscovy duck collected in Yunnan in southwestern China (Fig. S1a). For
10× Genomics data, sample-indexed and barcoded libraries were
prepared using a Chromium Genome Reagent Kit. Paired-end libraries
with insert sizes ranging from 500 to 700 bp were sequenced on the
Illumina HiSeq X Ten platform. For genome sequencing, short-read data
were obtained on the Illumina HiSeq X platform (400-bp insert size).
Long-read data were sequenced on the Oxford Nanopore GridION plat­
form. For Hi-C data, the Hi-C library was prepared following standard
procedures and sequenced using the Illumina HiSeq X Ten platform
(400-bp insert size).
For RNA-seq used in assembly annotation, we extracted RNA from a
total of 15 tissue or organ samples from the same Muscovy duck,
including the cerebellar vermis, cerebral cortex, mesencephalon, pons,
striatum, eye, tongue, heart, kidney, liver, lung, spleen, stomach, rectum
and testis using TRIzol Reagent. Total RNA-seq libraries were prepared
using a TruSeq RNA Library Prep Kit v2 according to the manufacturer's
instructions and subsequently sequenced on the HiSeq 4000 platform.
We sampled liver tissues from another male Muscovy duck collected
in Jilin in northeastern China to investigate regulation of gene expres­
sion profiles (Fig. S1b). For ATAC-seq, the libraries of three biological
replicates were prepared according to standard protocols [67] and
sequenced on the Illumina HiSeq 4000 platform. The Hi-C library was
constructed, and sequencing data were obtained using the MGI
DNBSEQ-T platform. The prepared RNA-seq library was sequenced on
the MGI DNBSEQ-T platform according to the recommended protocols.
5.2. Genome de novo assembly
The Muscovy duck genome size was estimated based on k-mer
analysis (Fig. S2). We followed the recommended protocols and used the
Supernova software package (10× Genomics, CA) for de novo assembly
of the chromium-prepared sequencing linked-reads. Scaffolding of the
produced contigs was then completed by using fragScaff software.
Integrity of the primary assembly was further improved using a
hybrid strategy, as reported in previous study ([68] Fig. 1). In brief,
DBG2OLC software [69] was used to perform hybrid assembly as fol­
lows: a) The DBG contigs were mapped to the long reads. Each long read
was converted into a compressed read, an order list of contigs. b) The
compressed reads were chained together based on the best calculated
overlaps. c) The assembly backbone was constructed from the best
overlaps. Finally, we aligned all longs reads and 10× contigs to the
backbone and calculated the most likely sequence as output using Sparc
[70]. DBG2OLC was executed by using the following commands:
DBG2OLC k 17 KmerCovTh 10 MinOverlap 150 AdaptiveTh 0.005 LD1
0 Contigs Contigs.fa RemoveChimera 1 f Nanopore.fasta; Consensus step
was run with following parameters: split_and_run_sparc.sh backbone.
fasta DBG2OLC_Consensus_info.txt tg_na.fasta consensus_batch_dir 2 10.
The contigs generated by hybrid assembly were polished using
NextPolish (v1.0.2) [71], with three rounds of alignment with long reads
and three rounds of alignment with short reads. To generate an assembly
with chromosome-length scaffolds, clean Hi-C reads were mapped to the
draft assembly with Juicer (v1.5.6) [72], and a candidate chromosome-
length assembly was then generated automatically using the 3D-DNA
(v180114) [73] pipeline. Manual review and refinement of the candi­
date assembly was performed in Juicebox Assembly Tools [72] for
quality control and interactive correction. The genome was then re-

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M.-M. Xu et al. Genomics 114 (2022) 110518

Fig. 6. Formation of fatty liver in Muscovy duck. (a) Schema of metabolic causes of fatty liver of Muscovy duck. (b) Metabolic pathway of weak lipid catabolism in
Muscovy duck liver. Genes in red indicate Muscovy-low DEGs. FA: fatty acid; LFA: long-chain fatty acid; VLFA: very long-chain fatty acid; FA acyl-CoA: fatty acyl-
CoA; M/L FA acyl-CoA: medium/long-chain fatty acyl-CoA; MAG: monoacylglycerol; DAG: diacylglycerol; TAG: triacylglycerol; HSD17B12: hydroxysteroid 17-beta
dehydrogenase 12; ACSL1: acyl-CoA synthetase long chain family member 1; ACOT13: acyl-CoA thioesterase 13; ABCD3: ATP binding cassette subfamily D member
3; CROT: carnitine O-octanoyltransferase; CPT1A: carnitine palmitoyltransferase 1A; ACADM: acyl-CoA dehydrogenase medium chain; LDAH: lipid droplet asso­
ciated hydrolase; ATGL: adipose triacylglycerol lipase; MGLL: monoglyceride lipase; AKT3: AKT serine/threonine kinase 3; PGC-1α: peroxisome proliferator-activated
receptor-gamma coactivator 1 alpha; FOXO1: forkhead box O proteins 1; ADIPOR2: adiponectin receptor 2. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

assembled using 3D-DNA [73] according to manual adjustment. The
chromosomes were anchored using 3D-DNA and Juicebox workflow.
5.3. Genome annotation and evaluation
Transposable elements (TEs) in the genome were identified by a
combination of de novo and homology-based approaches. For homology-
based prediction, known repeats were identified using RepeatMasker
(v4.0.9) [74] and RepeatProteinMask [74] against Repbase [75]
(Repbase Release 20,181,026). RepeatMasker was applied for DNA-level
identification using a custom library. At the protein level, RepeatPro­
teinMask was used to perform an RMBLAST search against the TE pro­
tein database. For de novo prediction, RepeatModeler (http://repeatma
sker.org/) and LTR FINDER (v1.07) [76] were used to identify de novo

10
M.-M. Xu et al.
evolved repeats inferred from the assembled genome.
For gene prediction and functional annotation, we employed EVi­
dence Modeler (EVM, v1.1.0) [77] to consolidate RNA-seq data, protein
alignments with ab initio gene predictions and homology-based anno­
tations into a final gene set. For RNA-seq data, reads were cleaned with
Trimmomatic (v0.32) [78] and aligned to the genome with Tophat2
(v2.1.1) [79]. Alignments were then assembled independently with
Cufflinks (v2.0.0) [80] and de novo assembled with Trinity
(vr20140413p1) [81]. RNA-seq assemblies were combined and further
refined using PASA (v2.2.0) [77]. The ab initio predictions were per­
formed using AUGUSTUS (v3.3.1) [82] with a chicken training set.
Protein sequences of Anas platyrhynchos, Gallus gallus, Meleagris gallo­
pavo, and Taeniopygia guttata were used in the homology-based gene
annotation. In brief, tBLASTn was first performed to map protein se­
quences of the aforementioned species to the genome with the param­
eter “E-value = 1e-5”. For a single protein, all matching DNA sequences
in the reference genome were concatenated by Solar after low-quality
records were filtered. A protein-coding region was obtained by extend­
ing 2000 bp upstream and downstream of the concatenated sequence.
GeneWise (v2.2.0) [83] was then used to predict gene structure within
each protein-coding region. All lines of evidence were then fed into EVM
using intuitive weighting (RNA-seq > protein > ab initio gene pre­
dictions). Finally, the EVM models were updated into PASA. Gene
functions were assigned according to the best match alignment using
BLASTP against the Swiss-Prot [84], TrEMBL and Kyoto Encyclopedia of
Genes and Genomes (KEGG) [85] databases. InterProScan functional
analysis and Gene Ontology (GO) IDs were obtained using InterProScan
[86]. The pathway to which the gene may belong was derived from the
matching genes in KEGG.
For non-coding gene annotation, tRNAscan-SE [87] was specified for
Eukaryotic tRNA and tRNA annotation. We used the homology-based
method to identify rRNA. The rRNA sequence data downloaded from
the Rfam [88] database were used as a reference. INFERNAL [89] was
used to identify snRNA and miRNA.
To evaluate genome quality, completeness of Muscovy duck assem­
bly was assessed against the avian lineage BUSCO (v3.0.2) [90]. The
syntenic relationship between Muscovy duck, chicken, and Peking duck
genomes was conducted using Mummer (v3.23) [91] with default pa­
rameters and visualized using gnuplot (v5.2.8) (http://www.gnuplot.
info). The genome assembly statistics were calculated using stats.sh
script in BBMap (v38.45) (https://sourceforge.net/projects/bbmap/).
5.4. Phylogenetic tree construction
The Muscovy duck phylogenetic tree was constructed based on nine
additional species (Anas platyrhynchos, Anser cygnoides, Gallus gallus,
Meleagris gallopavo, Numida meleagris, Ficedula albicollis, Taeniopygia
guttata, Dromaius novaehollandiae, and Alligator mississippiensis). Except
for Anser cygnoides [92], Numida meleagris [93], and Gallus gallus
(downloaded from ENSEMBL v104), the genomes of the other species
were downloaded from the NCBI database. The single-copy orthologous
genes of 10 species were identified by the reciprocal best bit (RBH)
method. The coding sequences of each orthologous pair were aligned
using prank (v170427) [94] and trimmed using Gblock (v 0.91b) [95].
The filtered sequences were joined into supergenes and used as input in
RAxML (v8.2.12) [96] to infer a maximum likelihood (ML) tree
(Fig. S6), with parameters: -f a -m GTRGAMMA -p 2021 -x 20210808 -N
100 -T 10 -n ex.
5.5. Gene family evolution analysis
The protein sequences of the 10 species were clustered with Ortho­
Finder (v2.3.11) [97]. The r8s (v1.81) [98] program was used to obtain
an ultrametric tree of the 10 species with fossil records from the
TIMETREE website (http://www.timetree.org/). Expanded and con­
tracted gene families were identified by CAFÉ (v4.2) [99]. Rapidly
11
Genomics 114 (2022) 110518
evolving gene families for each species were determined based on P-
value ≤0.01. GO and KEGG enrichment analyses of expanded and con­
tracted gene families were conducted using clusterProfiler (v 4.0.5)
[100]. GO and KEGG items with P-value <0.05 and q-value <0.05 were
regarded as significantly enriched.
5.6. Detection of gene evolution
We applied the same method described in previous study [101] to
detect gene evolution signals. Single copy orthologous genes of the 10
species (same as used for phylogenetic tree construction) were extrac­
ted, aligned, and trimmed as described above. PSGs and REGs were
analyzed using the Codeml program in the PAML package (v 4.9e)
[102]. A positive selection signal on a gene within the Muscovy duck
lineage was detected using the branch-site model. The branch model was
used to detect fast evolution signal of genes. P-values were computed
based on Chi-square statistics, and genes with P-values <0.05 were
treated as candidates undergoing positive selection or rapid evolution.
The PSGs and REGs were cross-referenced against the Reactome data­
base [103] (Release 78, Pathway browser 3.7) based on seven
metabolism-related categories, including lipid metabolism, carbohy­
drates metabolism, metabolism of amino acids and derivatives, pyruvate
metabolism and citric acid cycle, metabolism of vitamins and cofactors,
and integration of energy metabolism.
5.7. Identification of conserved noncoding elements (CNEs)
Pairwise whole genome alignments (WGAs) were constructed for the
10 species above using LAST (v983) [104] (parameter: -E0.05), with the
chicken as a reference genome. The pairwise WGAs were merged and
used to build multiple alignments with the roast program in Multiz
(v11.2) [105].
To identify CNEs, fourfold degenerative sites were extracted from the
whole-genome alignments and used to estimate a neutral model with the
phylogenetic tree described above as input using phyloFit (v1.4) [106].
Conserved elements were detected using the phastCons (v1.4) package
[107]. Conserved elements within 5 bp were merged into single ele­
ments and subsequently removed when their lengths were < 50 bp. After
filtering misassembled CNEs that split into two or more discrete regions
on the Muscovy duck genome, a final set of CNEs was obtained that did
not intersect the coding regions of the annotated genes in the chicken
genome.
Muscovy-accelerated CNEs were screened using two methods. First,
phyloP (v1.4) was used to test each CNE for lineage-specific accelerated
evolution in the Muscovy duck branch. CNEs showing false discovery
rate (FDR)-adjusted P-values <0.05 were considered to show signifi­
cantly accelerated evolution. Second, CNEs for each species were
concatenated into super-sequences as input in PhyloAcc (v1.0) [108], a
new Bayesian method to detect genomic region with rate shifts and
identify genomic elements accelerated in target species from a set of
conserved elements. The accelerated CNEs in the Muscovy duck were
obtained with practical thresholds [32]: BF ≥ 10 and BF2 ≥ 1. After
removing misassembled CNEs, the Muscovy-accelerated CNEs were
identified by the intersection of results of the two methods.
To identify CNEs specifically lost in the Muscovy duck, we searched
the chicken CNEs against the genomes of the remaining species using
BLASTN (v.2.9.0) (E-value <1e-10; ≥ 80% identity; ≥ 30% coverage).
Those CNEs that only had no significant match in the Muscovy duck
genome were considered as lost. In addition, we used AVID (v 2.1) [109]
to perform alignments between each reference and query sequence with
the parameter “-nm = both”. The input sequences were prepared by
extending 50 kb upstream and downstream of the genes. VISTA
(v1.4.26) [110] was used to visualize and plot the Muscovy-accelerated
CNEs and lost CNEs after alignment.
M.-M. Xu et al.
5.8. ATAC analysis
The data were trimmed and adapters were removed using Cutadapt
(v3.5) [111] (parameters: -a CTGTCTCTTATA -A CTGTCTCTTATA -q 10
–trim-n –minimum-length 30). The clean data were aligned to the
Muscovy duck genome, with no mitogenome sequences, using bowtie2
(v2.3.5.1) [112] (parameters: –very-sensitive -X 2000 -p 5). Polymerase
chain reaction (PCR) duplicated reads were filtered by Picard (htt
ps://broadinstitute.github.io/picard/, v1.131) and low-quality map­
ped reads were removed. SAMtools (v1.9) [113] was used to merge the
aligned bam files of the three replicates. The merged bam files were fed
into MACS2 (v2.2.7.1) [114] (parameters: -f BAMPE –nomodel –shift
-100 –extsize 200 -B –SPMR –keep-dup all -g 1091823599) for narrow
peak calling.
5.9. HiC analysis
The Hi-C data were processed using the HiC-Pro (v2.11.1) [115]
pipeline to generate a contact matrix. TADs were called at 40-kb reso­
lution using hicFindTADs wrapped in HiCExplorer (v3.6) [116] (pa­
rameters: –minDepth 300,000 –maxDepth 3,000,000 –step 300,000
–minBoundaryDistance 400,000 –correctForMultipleTesting fdr
–thresholdComparisons 0.01 –numberOfProcessors 5). Loop calling was
conducted by FitHiC2 (v 2.0.8) [117] with default parameters at 5-kb
resolution.
5.10. Transcriptional analysis
One-to-one orthologous genes between Muscovy and Peking ducks
were identified by RBH using BLASTN. The quantification of gene
expression was limited to orthologous genes between the two species.
Transcriptional sequencing data of the liver were downloaded from
previous study [14]. RNA-seq reads were aligned to the reference
genome with HISAT2 (v2.1.0) [50]. The read counts were calculated
using featureCounts (v1.6.2) [51]. The expression matrix was loaded
into R and DEGs were selected using the DESeq2 [52] package (v1.32.0)
with thresholds: padj <0.05 and |log2FoldChange| ≥ 1. GSEA was
performed using the gseGO function in the clusterProfiler package. The
GSEA results were filtered with parameters: q-values <0.25 and P-values
<0.05.
The WGCNA (v1.68) package [118] was used to conduct weighted
gene coexpression network analyses. The significant module was iden­
tified and imported into Cytoscape (v3.8.2) [119] for network con­
struction and visualization. Hub genes were selected with the highest
degree of connectivity within a module. GO enrichment analysis was
completed using the clusterProfiler package [100].
5.11. Dual-luciferase assay
The CNE sequences of Peking duck-MGLL (NCBI: NC_051784.1:
12,092,780-12,092,874) and Muscovy duck-MGLL (HiC_scaffold_11:
10,559,921-10,560,015) were synthesized chemically and inserted into
the pGL4.23 vector (Promega, USA) and sequenced (Tsingke Biotech,
China). Duck embryo hepatocytes (DEHCs) were seeded in 24-well
plates with six replicates for every plasmid. The constructed pGL4.23
(500 ng) vectors were co-transfected with 25 ng of pRL-TK (internal
control) into the DEHCs using Turbofect reagent (ThermoFisher Scien­
tific, USA). Cellular lysates were collected 24–48 h after transfection
using passive lysis buffer. Luciferase activity was measured using the
Dual-Luciferase Reporter Assay System (Promega, USA) with a multi­
mode microplate reader (Spark Tecan, Switzerland). Light output from
transcriptional activity was divided by the output from Renilla luciferase
activity to normalize the samples.
12
Genomics 114 (2022) 110518
Data availability
The assembled genome and annotation were deposited in the
Genome Warehouse of the National Genomics Data Center (https://
ngdc.cncb.ac.cn/gwh/) under BioProject accession code: PRJCA009398
(accession code: GWHBJBF0000000). All raw data were submitted to
the Genome Sequences Archive (GSA) of the National Genomics Data
Center under BioProject accession code: PRJCA007991. In addition,
transcriptomic data of the livers from Peking and Muscovy ducks under
two feeding conditions were downloaded from the Sequence Read
Archive (SRA) database of the NCBI under accession number
SRP144764 (https://www.ncbi.nlm.nih.gov/sra/SRP144764).
Acknowledgments
This study was supported by the Spring City Plan: the High-level
Talent Promotion and Training Project of Kunming (2022SCP001),
Chinese Modern Technology System of Agricultural Industry (CARS-42-
50), Yunnan Revitalization Talent Support Program, and the Animal
Branch of the Germplasm Bank of Wild Species, Chinese Academy of
Sciences (the Large Research Infrastructure Funding). We thank Prof.
Qiang Qiu and Cheng-Long Zhu (School for Ecological and Environ­
mental Sciences, Northwestern Polytechnical University, Xi'an, China)
for their helpful advice on comparative genomic analysis. We are also
grateful to all volunteers who assisted in sampling.
Author contributions
M.S.P. and Y.P.Z. led the project, and designed and conceived the
study. M.M.X. and M.S.P. prepared the manuscript. Y.P.Z., Y.D., T.S.X.,
and A.C.A. revised the paper. L.W.L., S.C.D., and Y.D. assembled and
annotated the genome assembly. M.M.X. and L.H.G. performed subse­
quent data analysis. W.Y.L. conducted experiments. L.Z.L., T.Z., Z.C.H.,
Z.S.M., W.C., and J.L.H. provided technical assistance. All authors read
and approved the final manuscript. The authors declare that they have
no competing interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ygeno.2022.110518.
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