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Genome-wide association study of chicken plumage pigmentation.


Poultry industry, in the process of rapid industrialization, developed commercial chicken strains from a small number of breeds. To increase the productivity of native chickens, they were bred for economic traits. Although this process resulted in higher productivity, at the same time it decreased genetic diversity (Tadano et al., 2007). In recent years, it has become increasingly important to protect national endemic genetic resources and use local breeds to create commercial strains that can adapt to the changing environment.

In Korea, the National Institute of Animal Science (NIAS) has been studying the process of indigenization of foreign breeds in to Korea and methods to restore Korean native chicken breeds. Korean native chickens (KNCs) as defined by NIAS in 2008 are chickens that have been bred true for at least seven generations. The commercial KNC called Woorimatdag (WR CC) was developed by crossing three native chicken breeds (Heo et al., 2011). Woorimatdag has contributed to the industrialization of KNCs because of its rapid growth and the texture of the meat in comparison to the native chickens (Park, 2010). However, the use KNC H strain in the paternal line to create Woorimatdag has led to the decrease in plumage uniformity. Unlike typical white broilers, KNCs usually have colored feathers and various pigmentation patterns. Plumage color is an important factor that is used by consumers to distinguish between KNC strains. Although plumage color is easily observed, the genetics behind the feather coloration is governed by both qualitative and quantitative features (Klungland and Vage, 2000). In chickens, mutations in MC1R and TYR genes have been shown to be associated with feather pigmentation (Kerje et al., 2003; Liu et al., 2010). However, there is a lack of research on the genetics of plumage coloration in Korean chicken at the genomewide scale. The purpose of this study is to characterize the genetic polymorphism underlying different plumage color using the chicken 60K SNP chip through GWAS (genomewide association study) and to increase plumage color uniformity of Woorimatdag. The results will also be used for selecting and breeding KNC H strain.


Sample and phenotype collection, and genotyping

A total of 274 samples from four KNC strains were collected from NIAS. It comprised of 245 KNC H strains (KNCH), 9 KNC S stains (KNCS), 9 KNC R stains (KNCR), and 11 KNC L strains (KNCL). The plumage colors of these strains range from black, black with brown, brown, red-brown, and black. KNC H strain chickens can have black to black and brown plumage and the individuals were classified into seven categories according to the number of body parts it exhibited brown plumage. Plumage color was scored for six specific body parts: head, neck, breast, back, wings, and tail. If the individual only had black feather, it was given a score of zero, however, if an individual showed brown plumage, for every body part it had brown it received 1 point. This classified the individuals into seven categories, ranging from all black to brown in all scored body parts. Blood samples were collected in EDTA tubes and DNA was extracted using Wizard genomic DNA purification kit (Promega, USA) according to the manufacturer's instruction. The genomic DNA samples were genotyped using the 60K SNP Illumina iSelect chicken array (Illumina Inc., USA).

Genome-wide association test

The 60K SNP Illumina iSelect chicken array contains 57,637 SNPs that are distributed across the chicken genome. SNPs were excluded if it had a missing rate of >5%, a minor allele frequency (MAF) of <0.01, or a Hardy-Weinberg equilibrium (HWE) test p-value of <[10.sup.-6] using PLINK 1.07 (Purcell et al., 2007). After the quality control, 53,257 SNPs were retained for further analysis. GWAS analyses on plumage coloration of whole body and the body parts traits were performed using mixed model of GEMMA (v0.93) (Zhou and Stephens, 2012), which accounts for population stratification and sample structure.

y = W[alpha]+x[beta]+u+[micro],

u~MV[N.sub.n] (0,[lambda][[tau].sup.-1]K), [epsilon]~MVNn (0,[[tau].sup.-1][I.sub.n])

Where y is an n-vector of traits (plumage coloration) for n individuals; W = ([w.sub.1], [w.sub.2], ... ,[w.sub.c]) is an nxc matrix of covariates (fixed effects) including a column of 1s; [alpha] is a c-vector of the corresponding coefficients including the intercept; x is an n-vector of marker genotypes; [beta] is the effect size of the marker; u is an n-vector of random effects; [epsilon] is an n-vector of errors; [[tau].sup.-1] is the variance of the residual errors; [lambda] is the ratio between the two variance components; K is a known nxn relatedness matrix and [I.sub.n] is an nxn identity matrix. MV[N.sub.n] denotes the n-dimensional multivariate normal distribution. Relatedness matrix K was calculated as following:

K = 1/p [p.summation over (t=1)] ([x.sub.i] - [1.sub.n] [[bar.x].sub.i]) [([x.sub.i] - [1.sub.n][[bar.x].sub.i]).sup.T]

[x.sub.i] as its ith column representing genotypes of ith SNP, [[bar.x].sub.i] as the sample mean, and [1.sub.n] as a nx1 vector of 1's. GEMMA tests the alternative hypothesis [H.sub.1]:[beta][not equal to]0 against the null hypothesis [H.sub.0]:[beta] = 0 for each SNP. To correct for multiple hypothesis testing, we obtained adjusted p values by using the Benjamini and Hochberg false discovery rate procedure (Benjamini and Hochberg, 1995), adjusted p-value 0.2 significance level is used. An overview of the test results was shown as a Manhattan plot constructed by the statistical package R. Base pair position of SNP markers were given based on the chicken genome assembly build WASHUC2. Inflation factor was calculated by the R package GenABEL with "median" option (Aulchenko et al., 2007).

Estimating genetic variance

We estimated the genetic variance of plumage by using GCTA (Yang et al., 2011). After calculating the genetic relationship matrix (GRM) between all pairs of samples using all the autosomal SNPs, we estimated the genetic component, or heritability, for each trait by REML analysis of an Mixed Linear Model y = X[beta]+[g.sub.G]+[epsilon], where y is a vector of phenotypes, [beta] is a vector of fixed effect such as sex, age with its incidence matrix X, [g.sub.G] is a vector of aggregate SNP effects as random effect with var ([g.sub.G]) = [A.sub.G][[sigma].sup.2.sub.G], and [A.sub.G] is the GRM estimated from all autosomal SNPs. We defined heritability or the proportion of variance explained by all autosomal SNPs as [h.sup.2.sub.G] = [[sigma].sup.2.sub.G] / [[sigma].sup.2.sub.P].


Plumage color of KNC H strain

Each of the 245 KNC (H strain) was investigated individually for plumage coloration (Figure 1). The predominant plumage color of KNC H strain chickens was black, but 88 out of 245 had brown feathers in addition to the black. This mixing of brown plumage causes the uniformity of Woorimatdag to decrease. Plumage color was investigated in six body-parts: head, neck, breast, back, wings and tail. One point was given for each body part that showed brown plumage (Table 1). Out of the 207 KNC H strain hens, 157 hens only had black plumage color, while 41 hens had brown plumage on the neck and 9 hens had brown plumage on both the head and neck. None of the 38 KNC H strain roosters were pure black.

Roosters and hens, respectively, have ZZ and ZW sex chromosome, which may be the cause of the differential plumage color between sexes. Sex-linked silver locus have been shown to control silver and wild type/gold color and interfere with the coloration of red (Gunnarsson et al., 2007). This result is estimated to be associated with the difference in the color of the hen and rooster. It is possible that the sex-linked plumage coloration is related to the fact that rooster with colorful plumage has an advantage when it comes to mating success (Brawner III et al., 2000).

The SNPs associated with feather pigmentation

The genome-wide association study revealed 12 significantly associated SNPs that surpassed the significance level (Figure 2, Table 2). As genomic inflation factor is 0.987, it can be concluded that the GWAS result is not inflated by considering relatedness using GEMMA (Figure 3). Among the significant SNPs, we identified 4 susceptibility SNPs: rs14339964 (Gga3:36327458, p = 4.07 x[10.sup.-9]), GGaluGA344987 (Gga3:705798, p = 1.12x[10.sup.-6]), rs14641648 (Gga8:12987908, p = 2.06x[10.sup.-6]), and GGaluGA193591 (Gga24:5696828, p = 2.38x[10.sup.-6]) in the population (Table 2). SNP rs14339964 at Gga3:36327458 is located in an intron region of AKT3 which is known to be regulators of cell signaling in response to insulin and growth factors and involved in a wide variety of biological processes. AKT3 is one of the key genes in the formation of melanoma cells (Tsao et al., 2012). Previous studies reported that through gene-environment interactions pigmentation pathways can contribute to the formation for melanoma and tumours (Gudbjartsson et al., 2008; Ibarrola-Villava et al., 2012). Thus, we indirectly infer that AKT3 mutations may be related to plumage pigmentation. Both SNPs, GGaluGA344987 at Gga3:705798 and rs14641648 at Gga8:12987908, are located in an intergenic region around KRT7 and PAP2 which are associated with pigmentation. PAP2 is another name of LPPR5 which has been found to increase pigmentation (Shan et al., 2009). KRT7 is a member of the keratin gene family and is related with melanocytic tumors (Blum et al., 2010). DDX6 encodes a member of the DEAD box protein family, which has multiple functions including translation suppression and mRNA degradation (Weston and Sommerville, 2006). DDX6 is a previously confirmed gene for vitiligo which is a disease related with pigmentation of skin (Tang et al., 2012). Interestingly, although rs15175679 (Gga20: 8397089, p = 3.91 x[10.sup.-6]) is not significant, the variant exits in gga-mir-668 which is a region that harbors a small RNA. Previous studies of chicken embryogenesis has shown that this small RNA regulates developmental signaling pathways (Shao et al., 2012). The results of GWAS of head plumage, wing plumage, breast plumage, back plumage, neck plumage and tail plumage traits, separately identified the same SNPs: rs14339964 and rs15616451 (near gene: AKT3, ENSGALG00000020136) as the result of GWAS with the whole body trait. Through the concordant result, we infer that quantitative analysis of whole body plumage is not a simple trait (Table 3).

The feather pigmentation related genes including MC1R, TYR, PMEL, MLPH, ASIP, SOX10, and SLC34A2 are well known. However, the related loci of these genes were not found in this study. The chicken 60K SNP chip does not contain SNPs of the MC1R region, and so we could not identify the effects of MC1R in this study. The results of this study are nevertheless meaningful in that novel loci affecting pigmentation at genome-wide level were found. Estimated genetic heritability was 18.2%, but estimated genetic heritability of significant SNPs was 3.1%. The results support a polygenic effect in feather pigmentation. This means previously reported genes MC1R, TYR, PMEL, MLPH, ASIP, SOX10, and SLC34A2 as well as the reported loci in this study are important in plumage coloration. The results may contribute to selecting and breeding of KNC H for plumage color uniformity.


The work was supported by a grant from the AGENDA project (No. PJ907057) in the National Institute of Animal Science, Rural Development Administration (RDA), Republic of Korea.


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Mi Na Park, Jin Ae Choi, Kyung-Tai Lee, Hyun-Jeong Lee, Bong-Hwan Choi, Heebal Kim (1), Tae-Hun Kim, Seoae Cho (2), and Taeheon Lee (1) * Division of Animal Genomics and Bioinformatics, National Institute of Animal Science, Rural Development Administration, Suwon, Korea

* Corresponding Author: Taeheon Lee. Tel: +82-2-880-4822, Fax: +82-2-883-8812, E-mail:

(1) Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea.

(2) C&K genomics, Seoul National University Research Park, Seoul 151-919, Korea.

Submitted Jul. 10, 2013; Accepted Sept. 4, 2013; Revised Oct. 10, 2013

Table 1. Plumage color pattern of KNC H strain

         Feather color                          Point
                                                           Number of
Head   Neck   Back   Breast   Wings   Tail                 chickens


0       1      0       0        0      0          1            3
0       1      1       0        0      0          2            2
1       1      0       0        1      0          3            9
1       1      1       0        0      0          3            1
1       1      1       0        1      0          4           14
1       1      1       0        1      0          4            4
1       1      1       1        1      0          5            5

                  Female                     Shank color

0       0      0       0        0      0          0           157
0       1      0       0        0      0          0           41
1       1      0       0        0      0          0            9

Feather color: 0 = black, 1 = black+brown.

Table 2. Top SNPs associated with plumage coloration

rs               CHR     Position    /Maj (1)    Freq

rs15304667        1      70248953      G/A      0.0623
rs15408789        1      125252900     G/A      0.115
GGaluGA172731     2      149522175     G/A      0.421

rs14339964        3      36327458      A/C      0.210
GGaluGA239670     3      110847381     G/A      0.206
rs15616451        4      75015502      A/G      0.053

rs16445392        4      85858579      A/C      0.025
rs15790835        6      19031740      A/C      0.132

rs14641648        8      12987908      A/G      0.115
rs15047928        19      5092926      A/G      0.329
GGaluGA193591     24      5696828      G/A      0.121
GGaluGA344987   E22C19    705798       G/A      0.287

rs              Beta     SE     p value    Q value *

rs15304667      1.221   0.230   2.66E-07     0.014
rs15408789      0.548   0.116   3.58E-06     0.186
GGaluGA172731   1.180   0.244   2.43E-06     0.126

rs14339964      1.186   0.194   4.70E-09     0.000
GGaluGA239670   1.366   0.241   4.50E-08     0.002
rs15616451      0.597   0.125   3.30E-06     0.171

rs16445392      0.908   0.186   1.85E-06     0.096
rs15790835      1.087   0.227   2.89E-06     0.150

rs14641648      1.165   0.239   2.06E-06     0.107
rs15047928      1.325   0.251   2.87E-07     0.015
GGaluGA193591   1.162   0.240   2.38E-06     0.123
GGaluGA344987   1.110   0.222   1.12E-06     0.058

rs              gG SNP **0   Gene       Location

rs15304667        0.143     STK38L       Intron
rs15408789        0.112     AP1S2      Intergenic
GGaluGA172731     0.575   ENSGALG000   Intergenic
rs14339964        0.394      AKT3        Intron
GGaluGA239670     0.447     TFAP2B     Intergenic
rs15616451        0.060   ENSGALG000   Intergenic
rs16445392        0.044      MXD4        Intron
rs15790835        0.249   ENSGALG000   Intergenic
rs14641648        0.237      PAP2        Intron
rs15047928        0.585    FAM211A       Intron
GGaluGA193591     0.247      DDX6        Intron
GGaluGA344987     0.454      KRT7      Intergenic

(1) Minor allele/Major allele. * Adjusted
p value. ** Estimated variance.

Table 3. Top SNPs associated with plumage coloration
by each part

Plumage                                     Min
part      rs ID           CHR   Position    /Maj (1)   Freq

Head      rs14398623      3     97933258    A/G        0.115
          rs318020030     7     19055437    A/G        0.350
          rs16669242      9     14516013    A/C        0.296
          GGaluGA105119   14    14228307    G/A        0.321

          rs14112979      18    7210201     G/A        0.341
Wing      rs13982792      1     1.83E+08    A/G        0.463
          rs14131527      2     4895894     A/G        0.346
          rs14188826      2     59639362    A/G        0.058
          rs14339964      3     36327458    A/C        0.210
          rs14404313      3     103537929   C/A        0.146
          GGaluGA110134   15    9605683     G/A        0.333
          rs15027075      17    10503821    A/G        0.357
Breast    rs14316836      3     7925459     G/A        0.293
Back      rs14401050      3     100155987   A/G        0.204
          rs15616451      4     75015502    A/G        0.053

          GGaluGA287070   5     49185847    G/A        0.393
          GGaluGA095084   13    11601056    G/A        0.082
          rs15022353      15    7826821     A/G        0.216

part      rs ID           Beta     SE      p value    Q value *

Head      rs14398623      -0.149   0.030   1.56E-06   0.081
          rs318020030     0.220    0.045   1.79E-06   0.093
          rs16669242      0.235    0.041   2.74E-08   0.001
          GGaluGA105119   0.154    0.032   2.76E-06   0.144

          rs14112979      0.219    0.042   3.26E-07   0.017
Wing      rs13982792      0.153    0.033   3.48E-06   0.181
          rs14131527      0.114    0.022   4.14E-07   0.022
          rs14188826      0.163    0.034   2.70E-06   0.140
          rs14339964      0.146    0.029   1.19E-06   0.062
          rs14404313      0.203    0.036   3.75E-08   0.002
          GGaluGA110134   0.175    0.037   3.52E-06   0.184
          rs15027075      0.111    0.023   2.70E-06   0.141
Breast    rs14316836      0.062    0.013   2.49E-06   0.129
Back      rs14401050      0.174    0.036   2.23E-06   0.116
          rs15616451      0.118    0.024   1.71E-06   0.089

          GGaluGA287070   0.198    0.038   4.53E-07   0.024
          GGaluGA095084   0.175    0.033   1.81E-07   0.009
          rs15022353      0.178    0.029   1.84E-09   9.56E-05

part      rs ID           Gene         Location

Head      rs14398623      RNF144A      Intron
          rs318020030     PPP1R9B      Intron
          rs16669242      FGF12        Intergenic
          GGaluGA105119   ENSGALG000   Intergenic
          rs14112979      HELZ         Intron
Wing      rs13982792      XPO4         Intron
          rs14131527      XIRP1        Intron
          rs14188826      PRL          Intergenic
          rs14339964      AKT3         Intron
          rs14404313      NT5C1B       Intergenic
          GGaluGA110134   MSI1         Intron
          rs15027075      PBX3         Intron
Breast    rs14316836      LCLAT1       Intron
Back      rs14401050      E2F6         Intergenic
          rs15616451      ENSGALG000   Intergenic
          GGaluGA287070   VRK1         Intergenic
          GGaluGA095084   SGCD         Intron
          rs15022353      TTC28        Intron

(1) Minor allele/Major allele. * Adjusted p value.
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Author:Park, Mi Na; Choi, Jin Ae; Lee, Kyung-Tai; Lee, Hyun-Jeong; Choi, Bong-Hwan; Kim, Heebal; Kim, Tae-H
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9SOUT
Date:Nov 1, 2013
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