Received: 15 March 2023 | Accepted: 21 April 2023

DOI: 10.1111/age.13329

R E SEA RCH A RTICLE

Integration of GWAS and eGWAS to screen candidate genes

underlying green head traits in male ducks

Jianmei Wang1,2 | Shuaixue Jiang1,2 | Yang Xi1,2 | Jingjing Qi1,2 | Shengchao Ma1,2 |
Liang Li1 | Jiwen Wang1 | Lili Bai1 | Hua He1 | Hengyong Xu1 | Hehe Liu1,2

1
Key Laboratory of Livestock and Poultry
Multi-­omics, Ministry of Agriculture
and Rural Affairs, College of Animal
Science and Technology (Institute of
Animal Genetics and Breeding), Sichuan
Agricultural University, Sichuan, China
2
Farm Animal Genetic Resources
Exploration and Innovation Key
Laboratory of Sichuan Province, Sichuan
Agricultural University, Sichuan, China
Correspondence
Hehe Liu, Key Laboratory of Livestock
and Poultry Multi-­omics, Ministry of
Agriculture and Rural Affairs, College of
Animal Science and Technology (Institute
of Animal Genetics and Breeding), Sichuan
Agricultural University, Sichuan, China.
Email: liuee1985@sicau.edu.cn
Funding information
Key Technology Support Program of
Sichuan Province, Grant/Award Number:
2021JDJQ0008; National Natural Science
Foundation of China, Grant/Award
Number: 31872345 and 32072703
Abstract
Sexually dimorphic plumage coloration is widespread in birds. The male
possesses more brightly colored feathers than the female. Dark green head
feathers comprise one of the most typical appearance characteristics of the male
Ma duck compared with the female. However, there are noticeable individual
differences observed in these characteristics. Herein, genome-­w ide association
studies (GWAS) were employed to investigate the genetic basis of individual
differences in male duck green head-­related traits. Our results showed that
165 significant SNPs were associated with green head traits. Meanwhile, 71
candidate genes were detected near the significant SNPs, including four genes
(CACNA1I, WDR59, GNAO1 and CACNA2D4) related to the individual
differences in the green head traits of male ducks. Additionally, the eGWAS
identified three SNPs located within two candidate genes (LOC101800026 and
SYNPO2) associated with TYRP1 gene expression, and might be important
regulators affecting the expression level of TYRP1 in the head skin of male
ducks. Our data also suggested that transcription factor MXI1 might regulate
the expression of TYRP1, thereby causing differences in the green head traits
among male ducks. This study provided primary data for further analysis of the
genetic regulation of duck feather color.

K EY WOR DS
eGWAS, green head traits, GWAS, male duck, TYRP1

I N T RODUC T ION
Sexually dimorphic plumage coloration is widespread in
birds, and the brighter plumage appears in males rather
than females. The dichromatism of feather colors may
be affected by pigmentary or structural mechanisms in
birds (Roy et al., 2019). However, the most common color-­
producing pigments are eumelanin (brown and black)
and pheomelanin (yellow to reddish brown) (Fuentes-­
López et al., 2022; Wang et al., 2019). Their constitution
ratio is responsible for the difference in feather color be-
tween males and females (Vágási et al., 2010). Melanin
coloration plays a crucial role in social signaling and
mate choice, indicating social dominance and the health
or quality of males (Ernst et al., 2022; Turner et al., 2018).
Therefore, elucidating the genetic basis of melanin-­
based sexual dichromatism makes has significance for
comprehensively understanding the evolution of sexual
dichromatism and the selective forces shaping animal or-
namentation. Remarkably, studies have shown that mel-
anin genes such as MC1R, TYRP1 and ASIP play key
roles in forming the sex dimorphism of bird feather color
(Wang, Liu, et al., 2022a).
Plumage color is considered an essential economic
trait in the poultry industry, which directly affects the
consumer's first impression and purchasing desire. In
Ma duck production, the male duck has a green head
(referred to as green head traits), while the female shows

© 2023 Stichting International Foundation for Animal Genetics.

Animal Genetics. 2023;00:1–10.  wileyonlinelibrary.com/journal/age | 1


2 |   
predominantly dull feathers on the head. Our previous
research found that many pigments remain in the hair
follicles of male ducks during feather growth and the
green head traits of male ducks also have great varia-
tions in formation time and body distribution area, re-
sulting in uneven plumage color of their offspring. Thus,
it is essential to analyze the molecular basis of the forma-
tion speed and color depth of green head traits in male
ducks, which greatly explains the dimorphic plumage
color mechanism in poultry. Additionally, our previous
transcriptome results have found that TYRP1 linked
with Z-­chromosome contributes to duck plumage color
sexual dimorphism in a dose-­dependent manner (Ma
et al., 2021; Wang, Xi, et al., 2022b). The above results
provide a theoretical basis for understanding the differ-
ence in feather color between male and female poultry.
However, the genetic basis of male green head traits dif-
ferences in ducks is still unclear.
With the development of gene chips and genome re-­
sequencing technology, genome-­w ide association studies
(GWASs) have become one of the most effective ways to
detect genetic variations in poultry. Zhou et al. (2018)
found that MITF was closely related to the white feather
traits of the Pekin Duck. Xi et al. (2020) reported that the
14 bp insertion in exon 3 of the EDNRB2 gene is associ-
ated with the white plumage phenotype in Chinese geese.
Nevertheless, limited research is available on regulating
the formation of green head traits of ducks. Meanwhile,
the detection of expression quantitative trait loci has re-
cently been proposed as an excellent strategy to deepen
the study of the genetic architecture of complex traits
(Gilad et al., 2008). This technique identifies genetic
gene transcription-­ related genetic variants (Ballester
et al., 2017; Corominas et al., 2013; Schadt et al., 2008,
2009).
Hence, the main objective of this study was to examine
the green head color depth and formation speed in duck
using a GWAS. We also performed a gene expression-­
based GWAS (eGWAS) of one strong candidate gene
(TYRP1) for green head traits to jointly explore and
discover candidate gene loci that determine the green
head traits in male ducks. This study's results provided
preliminary data for further understanding the genetic
regulatory basis of duck plumage.
M AT E R I A L S A N D M ET HOD S
Animals
In this study, the Waterfowl Breeding Farm of Sichuan
Agricultural University provided 167 male ducks (Tianfu
Nonghua ducks). All ducks for phenotype measure-
ment were raised under the same environmental condi-
tions and had free access to feed and water (Table S1).
At 60 days old, 5 ml of whole blood was collected from
the wing vein using venipuncture and stored at −20°C.
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WANG et al.
At 120 days of age, samples of the head skin were col-
lected, frozen in liquid nitrogen immediately, and then
stored at −80°C. All methods were carried out following
the relevant guidelines and regulations. All animals used
were cared for following the Institutional Animal Care
and Use Committee guidelines of Sichuan Agricultural
University.
Phenotype collection
During the feeding period, the formation process of de-
velopment of male duck green head traits was recorded
for 10 days to obtain phenotypic data. A black or green
area on the head of all ducks was photographed under
the same conditions. Firstly, the digital camera was set
to a manual exposure mode, and each photo was based
on identical exposure conditions, including exposure
time and aperture. Then, the obtained images were im-
ported into IPP 6.0 software (Media Cybernetics), and
afterwards, they were magnified by the identical mul-
tiplier. Next, using an irregular area tool incorporated
into the software, the black or green feather area and
the whole head of each male duck were selected, and
each region's geometric size was measured. The head
area was taken as the area above the white neck ring,
and the black or green feather distribution of the head
of the male duck per unit area was calculated. Finally,
black or green feathers accounting for the entire head
area (BGEH), head area (HA) and black or green
feather area (BGFA), and the black or green feather
ratio to the whole head area reaching 50% (RBGF) were
calculated. Three replicates in each measurement and
the average values were taken as the final phenotype.
The data for each day's age statistics were taken as a
primary phenotype.
Meanwhile, head photographs at 120 days of age were
counted manually using Adobe Photoshop software
22.0.0 (Adobe Systems Software Ireland Limited). The
1 × 1 cm region was selected, and the four corner and di-
agonal junction RGB (red–­g reen–­blue) and HSB (hue–­
saturation–­brightness) values were counted separately
and averaged as the phenotype. All samples were col-
lected at the exact location (Table S2).
SNP genotyping
A total of 167 blood samples were selected for DNA
extraction using the phenol–­ chloroform protocol.
Nanodrop and agarose gel electrophoresis were applied
to examine the quality and quantity of the DNA. The
paired-­end libraries were generated for each eligible sam-
ple using standard procedures. In addition, the average
insert size was 500 bp, and the read length was 150 bp. All
libraries were sequenced on an Illumina®Hiseq X-­Ten
platform in a Bio-­company (Biomarker Technologies,

GWAS AND EGWAS OF GREEN HEAD TRAITS OF MALE DUCKS
Pekin, China) to an average raw read sequence (S) cover-
age of 5× for a paired-­end of 150 bp (PE150).
The raw reads were filtered using the NGS QC (v2.3.3)
Toolkit with default parameters (Kaushik et al., 2017).
The clean reads were mapped to the duck reference ge-
nome ZJU1.0 (https://www.ncbi.nlm.nih.gov/assem​bly/
GCF_01547​6345.1) with Burrows–­ W heeler alignment
(Huang et al., 2018) using the default parameters. Next,
SNP calling was performed using GATK (version 3.5) ex-
clusively (Ariyanayagam-­Baksh et al., 2003), and the out-
put was further filtered using VCFtools (version 0.1.15)
(Olbryt et al., 2011). The SNPs were screened with the pa-
rameters of a minor allele frequency >0.03, a maximum
allele frequency <0.99 and a maximum missing rate <0.1.
Genome-­wide association analysis
The SNPs that were significantly associated with the
phenotypic traits were identified using the mixed linear
model program EMMAX (Eisen et al., 1998):
y = X𝛼 + Z𝛽 + W𝜇 + e,
where y is the vector of observed phenotypes; Xα represents
the fixed effects, including the first three principal compo-
nent values (eigenvectors) derived from the whole-­genome
SNP genotypes, to correct population stratification; Zβ
represents the effect of the tested SNP, with allele substi-
tution effect β; Wμ represents the random animal effect,
with a variance–­covariance structure based on the kinship
matrix estimated using the whole genome SNP genotypes;
and e is the vector of random residual errors. SNPs with a
p-­value that reached a Bonferroni corrected threshold [−
log10 (p) ≥ 8.59] were considered significant.
SNP annotation and functional analysis
The SnpEff software (Cingolani et al., 2012) was used
to annotate SNPs based on the GFF files of the duck
reference genome (ZJU1.0, GCF_015476345.1). Gene
Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment analysis was
performed to annotate candidate genes using KOBAS
3.0 (http://kobas.cbi.pku.edu.cn/).
Real-­time PCR
The expressions of five genes were detected by real-­
time PCR for the head skin of 120-­day-­old male ducks
(n = 167). The primer information of each gene is listed in
Table S3. The total RNA from 167 samples was extracted
using Trizol (Takara). The cDNA was synthesized using
the reverse transcript system (Takara) according to the
manufacturer's instructions.
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   | 3
Next, real-­time PCR was performed using a standard
SYBR Green PCR kit (Takara) and was processed on
Bio-­Rad CFX Manager (Bio-­ Rad Laboratories). The
real-­time PCR conditions were used as follows: 95°C for
5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s,
and 72°C for 30 s. The β-­actin was used as a housekeep-
ing gene, and assays were run in triplicate. The 2−ΔΔCt
method was used to determine the expression level
(Livak & Schmittgen, 2001). All real-­time PCR experi-
ments were repeated in three biological and three tech-
nical replications. An inter-­plate correction is necessary
before calculation.
R E SU LT S
Descriptive statistics of phenotypic traits
The IPP 6.0 software was utilized to evaluate the amount
of black or green in feathers and the area of the duck
head (Figure 1a). During the lateral growth period of the
ducks, the amount of black or green feathers increased
gradually with age (Figure 1b). However, the black or
green feathering on the duck exhibited individual dif-
ferences. The RGB and HSB values were measured to
describe the head color of 120-­day-­old male ducks, and
it was observed that there were large individual differ-
ences in the head color at the same age (Figure 1c). The
phenotype data were collected for 167 male individuals,
including the BGEH, the visual grading of green head
area (VGGHA), the BGFA, the RBGF and the green
head color value of 120-­day-­old ducks (CV120D). These
phenotype values were approximately normal distribu-
tion (Figure S1, Table S4).
Genome-­wide association analysis
The results for all SNPs demonstrated to have genome-­
wide significance are presented in full. The BGEH,
BGFA, VGGHA and RBGF are shown in Figure 2a– ­d,
respectively (Figure 2).
Black or green feathers accounting for the
entire head area
As detailed in Table S5, 17 significant SNPs for the
BGEH40 were detected on chromosome 1, of which 14
significant SNPs contributed to a candidate genomic
region ranging from 186.07 to 204.08 Mb (Table S5).
Regarding the BGEH60, four significant SNPs were de-
tected on two chromosomes (1 and 14). For the BGEH70,
seven significant SNPs were detected on three chromo-
somes (1, 7, 12), whereas for the trait of BGEH50, 80, 90,
100, 110 and 120, non-­significant SNPs were observed
(Figure S2).
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4 |    WANG et al.

F I G U R E 1 Phenotypic observations of black or green feather area in male duck head. (a) IPP 6.0 was used for determining the black or
green feather area (BGFA), head area (HA), and black or green feathers accounting for the entire head area (BGEH). The red dot marked on the
right head showed the black or green feather area distribution by their regular function of IPP 6.0. The red line marked the head in the region
for calculating the head area on the left side. (b) The black or green feather area was accumulated, accompanying the age of ducks. (c) Head
color RGB (red–­g reen–­blue) and HSB (hue–­saturation–­brightness) values of 120-­d ay-­old male ducks. The higher the H value, the darker the
green color is; the larger the G value, the darker the green color is. B1 = B represents brightness.

Black or green feather area
Associations identified with these BGFA traits are
shown in Table S6. For the BGFA40, 24 significant
SNPs were detected on chromosome 1. Thirty significant
SNPs for the BGFA60 were distributed on chromosome
6, of which 24 significant SNPs were clustered within a
5.46 Mb region (24 611 799–­30 075 204 bp). All 22 SNPs
were detected as significantly associated with BGFA70
and contributed a candidate genomic region ranging
from 11.86 to 15.12 Mb on chromosome 12. However,
for the trait of BGFA50, 80, 90, 100, 110 and 120, non-­
significant SNPs were observed (Figure S2).
Visual grading of green head area
chromosome 12. However, for the trait of VGGHA 50,
60, 70, 80, 90, 100 and 110, non-­significant SNPs were
observed (Figure S2).
The black or green feather ratio to the whole
head area reaching 50%
As seen from Table S8, a total of 21 SNPs were detected
as being significantly associated with RBGF40 on chro-
mosome 1. Of these 18 SNPs clustered within an 18 Mb
region (186 085 293–­204 088 514 bp). For the RBGF70
trait, 14 significant SNPs were distributed on two
chromosomes (1 and 12). Nevertheless, for the trait of
BGFA50, 80, 90, 100, 110 and 120, non-­significant SNPs
were observed (Figure S2).

As detailed in Table S7, for VGGHA40, 24 signifi-

cant SNPs were detected on two chromosomes (1 and
3) and most of these significant SNPs were clustered
within a 21.06 Mb region (between 184 256 176 and
205 310 101 bp) on chromosome 1 and a 2.57 Mb region
(114 273 572–­116 844 948 bp) on chromosome 3. One
significant SNP for the VGGHA120 was detected on
The green head color value of 120-­day-­
old ducks
These CV120D traits were observed, including H, S, B,
R, G, B1 and non-­significant SNPs were linked to these
traits (Figure S2).
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GWAS AND EGWAS OF GREEN HEAD TRAITS OF MALE DUCKS    | 5

F I G U R E 2 Manhattan plots of genome-­w ide association analysis results for BGEH (A), BGFA (B), VGGHA (C), and RBGF (D) traits
related to green head. The number represents the age of the duck. The gray line represents the Bonferroni corrected significance threshold (−
log10 p = 8.59). The x-­axis shows the physical positions of each marker along the chromosomes, and the y-­a xis shows the −log10 p values for the
association tests, the same below.

Functional enrichment analysis of candidate TYRP1 expression-­based GWAS

genes located in SNPs
To further study the involved functions and regulation
relationships of the genome-­wide significantly associated
SNP markers and nearby candidate genes, we performed
functional enrichment analysis carried out on the 71 can-
didate genes by GWAS using the KEGG and GO data-
bases. KEGG pathway enrichment analysis showed that
the candidate genes were related to some melanin synthesis
pathways, including the MAPK signaling pathway, melano-
genesis, mTOR signaling pathway, and calcium signaling
pathway. These genes mainly involved CACNA1I, WDR59,
GNAO1 and CACNA2D4 (Figure 3a). Additionally, GO
enrichment analyses suggested that nervous system devel-
opment, cilium assembly and the actin cytoskeleton ac-
counted for a significant proportion (Figure 3b).
identifies male duck green head-­
associated variants
Taking into account that pigmentation is also regulated
by the activation of tyrosinase, a rate-­limiting enzyme
involved in the process of melanin synthesis, previ-
ous studies have shown that TYRP1 is a critical gene
involved in the formation of green head traits in male
ducks. Hence, a GWAS was performed using the gene
expression levels of the TYRP1 of head skin and duck
genotypes among 167 male ducks.
A total of three significant eSNPs were identified, lo-
cated on duck chromosomes 3 and 4 (Figure 4). The SNP
on chromosome 3 is LOC101800026. Two SNPs on chro-
mosome 4 are located within Synaptopodin 2 (SYNPO2)
(Table S9).

6 |   
(a)
F I G U R E 3 KEGG (a) and GO (b) enrichment analysis of candidate genes. The top 20 enriched GO terms and KEGG pathways are listed
in the figure; p < 0.05 was significant.
FIGURE 4 The Manhattan plot of the GWAS analysis of TYRP1 expression in the head skin of 120-­d ay-­old male ducks.
Prediction and correlation analysis of
TYRP1 and its gene transcription factors
Transcription factors (TFs) are important molecules
regulating gene expression, directly controling the
extent of gene expression and participate in an ex-
tensive range of biological processes. Using online
prediction software, we first predicted the transcrip-
tional factor binding sites in the TYRP1 promoter
region (Table S10). Furthermore, we intersected with
73 potential candidate genes screened by GWAS and
eGWAS analysis to narrow down the candidate genes
(Figure S3). Finally, the four transcription factors
(MXI1, PBX1, NCOR2 and ATF6) for RT-­PCR were
selected and analyzed. Subsequently, Pearson correla-
tion analysis was conducted on TYRP1 and the tran-
scription factors, and MXI1 expression was found to
be significantly positively correlated with TYRP1
expression (R = 0.270). Meanwhile, the normalized
MXI1 expression correlates significantly with the
TYRP1 gene expression (R = 0.474). In addition, there
was no correlation between the TYRP1 gene with the
other three transcription factors (PBX1, NCOR2 and
ATF6; Figure 5). We speculate that the difference in
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WANG et al.
(b)
cis-­regulation of the MXI1 transcription factor may be
related to the TYRP1 in male duck head skin.
DI SC US SION
The genetic basis of coloration is the key to understand-
ing the origin and evolution of bird phenotypic varia-
tion in sexual dimorphism feathers (Price-­Waldman &
Stoddard, 2021). In poultry production, feather color,
as a genetic marker, plays an important role in select-
ing high-­quality meat ducks. The green head traits are
the most distinctive appearance characteristic of the Ma
duck, and the feather is crucial to high standards of car-
cass quality and the consumer's choice. However, little
has been known about the development of the male duck
green head for a long time, and the causal molecular and
genetic mechanisms are entirely unclear.
Our study made a preliminary attempt at the pheno-
typic characteristics of the green head of male ducks on
different days during its growth. In terms of the forma-
tion speed of the green head, we found that the area of
black or green feathers on the head of the male duck in-
creased with age and gradually changed from black to

GWAS AND EGWAS OF GREEN HEAD TRAITS OF MALE DUCKS
F I G U R E 5 The correlation coefficient matrix heat map of TYRP1 gene and transcription factors MXI1, PBX1, NCOR2 and ATF6.
Pearson's correlation coefficients and p-­values were calculated using the cor. Test function in the R stats package. *p < 0.05 was considered
statistically significant; **p < 0.01 was considered very significant; and ***p < 0.001 was considered highly significant.
green feathers. Meanwhile, there was also a difference
in the black or green feather ratio to the whole head area
reaching 50%, indicating that the formation time of the
male duck's green head also differed. Secondly, regard-
ing the green head color depth, the measurement of HSB
and RGB values found a large variation in male ducks’
green heads at the same age. These results indicate that
phenotypic diversity is abundant in this study popula-
tion. Moreover, previous studies have shown that the
content of eumelanin plays a dominant role in forming
the green head of male ducks (Eliason & Shawkey, 2012;
Haase et al., 1995; Khudiyev et al., 2014; Ma et al., 2021).
However, melanin synthesis is environmentally and ge-
netically co-­controlled. Galván & Solano (2016) deemed
that the genetic control of avian melanogenesis is exerted
by genes coding for specific enzymes involved in melanin
synthesis and other vital regulatory and structural pro-
teins. Thus, we hypothesized that the difference in the
green head formation of male ducks was related to genes
regulating feather melanin deposition.
Based on GWAS analysis, a total of 165 significant
SNPs and 71 candidate genes were screened. Our find-
ings suggested that genetic factors influence melanin
deposition in duck green feathers. Further functional
enrichment analysis indicated that CACNA1I, WDR59,
GNAO1 and CACNA2D4 genes might be critical for
green heads formation of male ducks. These genes are
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   | 7
mainly involved in the MAPK signaling pathway, mela-
nogenesis, mTOR signaling pathway and calcium signal-
ing pathway, which are closely related to the regulation
of melanin synthesis and metabolism (Li et al., 2018; Ma
et al., 2021; Park et al., 2015; Qi et al., 2022; Wang, Xi,
et al., 2022b). Therefore, we considered that mutations in
genes related to melanin synthesis and metabolism path-
ways would affect melanin synthesis and subsequently
affect individual green head trait differences in male
ducks.
Notably, we identified different SNPs loci at the dif-
ferent growth stages of ducks, and the annotated genes
were completely different, indicating that the genetic
mechanism affecting the green head formation of males
is different in different age stages. Further verification
and study are needed to carry out these results. No
significant loci were identified at some stages with the
male duck's green head traits, which does not provide
evidence to explain the difference between different age
stages. Further search for the loci and genes of this trait
is necessary. In addition, the confirmation of candidate
genes is only based on functional annotation, which is
not convincing to a certain extent. Further verification
of the relationship between all the involved genes and the
green head traits is still necessary.
TYRP1 was the first cloned pigment gene located
on the duck Z chromosome. This gene is an important

8 |   
downstream target gene in the regulation of melanin syn-
thesis and affects melanosome maturation, the melanin
synthesis process and TYR activity (Xu et al., 2022). At
the transcriptome level, we demonstrate that the TYRP1
gene exerts a dose effect on melanin deposition in duck
head feathers (Wang, Xi, et al., 2022b). The eGWAS al-
lows the identification of genetic variants associated
with gene transcript levels (Puig-­Oliveras et al., 2016).
To the best of our knowledge, there was no report about
the sorting performance of eGWAS in the whole blood
of ducks. For the expression of the TYRP1 gene, three
significant SNPs were identified, and two possible can-
didate genes (LOC101800026 and SYNPO2) were ob-
tained after annotation. LOC101800026 is located on
duck chromosome 3, and the biological function in ducks
is currently unknown. Hypermethylation and decreased
expression of SYNPO2 were associated with shortened
melanoma-­ specific survival time (Gao et al., 2015).
However, the transcriptional regulation of the SYNPO2
gene requires further analysis. We suspected that two
genes, LOC101800026 and SYNPO2, might be essential
regulators affecting the expression level of the TYRP1
gene.
Transcription factors are important molecules that
directly regulate gene expression (Hou & Zhong, 2021).
Any interference with the TF-­mediated gene regulatory
mechanism may lead to disturbed and unregulated gene
expressions, which may cause amplified downstream
consequences of genes (Kaushik et al., 2017). Therefore,
it is significant to investigate the transcriptional regu-
lation mechanism of TYRP1 gene expression to reveal
the male duck green head trait color depth and forma-
tion speed. In the present study, the relative expression
of TYRP1 and four TFs (PBX1, NCOR2, MXI1 and
ATF6) in the head skin of male ducks were analyzed,
and a significantly positive correlation was only found
between MXI1 with TYRP1 gene expressions. MXI1,
one of the Max-­associated proteins, is hot spot in cancer
research and has been confirmed to be related to the oc-
currence and development of prostate cancer, glioblas-
toma and lung cancer (Huang et al., 2018). Concurrently,
Ariyanayagam-­Baksh et al. (2003) supported MXI1 as
a putative tumor suppressor gene in conventional mela-
noma progression. This gene is regulated by hypoxia in
human and mouse melanoma cells (Olbryt et al., 2011).
Hence, we speculated that MXI1 might directly or in-
directly control the expression of the pigmentation gene
TYRP1, thereby regulating the formation process of the
greenhead traits among male ducks.
CONC LUSION S
In this study, rich phenotypic variation with green head
traits was observed in the population. Additionally, 165
relevant significant SNP loci and 71 potential candidate
genes of male duck head traits were identified by GWAS,
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WANG et al.
and the DNA variants associated with the phenotypes of
TYRP1 expression were detected using eGWAS analysis.
Meanwhile, it was found that the expression level of the
TYRP1 gene in male duck head skin may be cis-­regulated
by transcription factor MXI1. These results provide new
insights into understanding the molecular mechanisms
underlying greenhead-­related traits, despite further ge-
netic and functional studies to validate our findings.
AC K NO​W L E​D GE​M E N T S
Thank are due to all the authors for their contributions
to the study.
F U N DI NG I N F OR M AT ION
This work was supported by grants from the National
Natural Science Foundation of China (31872345 and
32072703) and the Key Technology Support Program of
Sichuan Province (2021JDJQ0008).
C ON F L IC T OF I N T E R E ST STAT E M E N T
The authors declare that they have no competing
interests.
DATA AVA I L A B I L I T Y STAT E M E N T
The genome sequencing raw data was available in NCBI's
SRA database (https://trace.ncbi.nlm.nih.gov/Trace​s/
sra/sra.cgi?view=studi​es&f=study​&term=&go=Go; ac-
cession number: PRJNA907492 and PRJNA907501).
E T H ICA L A PPROVA L
All methods were carried out following relevant guide-
lines and regulations. The Institutional Animal Care
and Use Committee guidelines of Sichuan Agricultural
University approved all the experiments and protocols.
ORC I D
Hehe Liu https://orcid.org/0000-0002-2537-0425
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10 |    WANG et al.

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Additional supporting information can be found online https://doi.org/10.1111/age.13329
in the Supporting Information section at the end of this
article.