Inverted duplication including <i>Endothelin 3</i> closely related to dermal hyperpigmentation in Silkie chickens

doi:10.15302/J-FASE-2014026

Front. Agr. Sci. Eng.

2014, Vol. 1

Issue (2) : 121-129 https://doi.org/10.15302/J-FASE-2014026

RESEARCH ARTICLE

Inverted duplication including Endothelin 3 closely related to dermal hyperpigmentation in Silkie chickens

Ming TIAN,Suyun FANG,Yanqiang WANG,Xiaorong GU,Chungang FENG,Rui HAO,Xiaoxiang HU(

),Ning LI(

)

State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100193, China

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Abstract

The dermal hyperpigmentation phenotype in chickens is controlled by the dominant fibromelanosis allele. One of the ten unique characteristics of Silkie chickens is the fibromelanosis phenotype, which is pigmentation in the dermal layer of the skin and connective tissue. In this study, we found a mutation of fibromelanosis, a genomic rearrangement that included an inverted duplication of endothelin3 (EDN3), is responsible. We show that, as a stimulator of melanoblast proliferation, EDN3 expression was increased in silkie embryos and in both skin and muscle throughout adulthood. EDN3 expression led to an increase in expression of the downstream genes EDNRB2 and TYRP2, and was closely relate with the hyperpigmentation phenotype. We examined eight different Chinese chicken breeds showing hyperpigmentation and conclude that this structural genetic variant exists in all fibromelanosis chicken breeds.

Keywords dermal hyperpigmentation duplication endothelin 3 Silkie chicken

Corresponding Author(s): Xiaoxiang HU

Online First Date: 14 July 2014 Issue Date: 10 October 2014

Cite this article:

Ming TIAN,Suyun FANG,Yanqiang WANG, et al. Inverted duplication including Endothelin 3 closely related to dermal hyperpigmentation in Silkie chickens[J]. Front. Agr. Sci. Eng. , 2014, 1(2): 121-129.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2014026
https://academic.hep.com.cn/fase/EN/Y2014/V1/I2/121

Fig.1 Founding breeds of the mapping populations and chick pigmentation phenotypes. Individuals of the chicken breeds used to develop the population for pigmentation: (a) Silkie, (b) Gushi and (c) Youxima; after hatching, four-week-old chickens from the mapping populations displaying different pigmentation phenotypes in the comb, which represents the color of the skin: (d) a putative Fm/fm⁺ (black comb) individual and (e) a putative fm⁺/fm⁺ (white comb) individual.

Tab.1 Primer sequences for locating the boundary with PCR

Tab.2 Chinese chicken breeds used for confirmation of copy number variation regions

Fig.2 Results of qPCR analysis of genes and fragments for Dup-CNV-1 detection. Dup-CNV-1: (a) PCCA (control); (b) EDN3; (c) C20orf174-1; (d) ATP5e; (e) TUBB1. Genomic copy number was detected with qPCR in 11 breeds of chicken. The heterozygotes (FM G) showed an estimated copy number of approximately 1.5 × compared to wild-type individuals (YX, QY, FM W). Homozygotes (FM B, WJ, JH, TC, YJ, WD, CX) showed an estimated copy number of approximately 2 × compared to wild-type individuals. The numbers after the hyphen mean the label when feeding; “N”means the individual has no Fm loci; the data are the mean ± SD.

Fig.3 Genome Walker PCR. PCR products were digested with four restriction enzymes, namely Dra II, EcoR V, Pvu II, Stu I, producing fragments of different lengths from the same lines and indicating differential amplification of segments with the same primers (i.e., polymorphic segments).

Fig.4 Results of qPCR analysis of the expression of duplicated genes in Dup-CNV-1. (a): END3 mRNA; (b) SLMO2 mRNA; (c) ATP5e mRNA; (d) TUBBa mRNA. Gene expression analysis in Silkie (FM, WJ) and White Leghorn (fm⁺, BLH) chickens with SYBR Green qPCR normalized to expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Genes in the first duplicated region (EDN3, SLMO2, ATP5e, TUBB1) showed significantly increased expression from embryonic tissue (E3d) through adult skin and muscle tissue (4D) of the Silkie chicken. HL: hind leg; S: skin; M: muscle; L: liver.

Fig.5 Expression of genes downstream of EDN3. Gene expression analysis of the EDN3 receptor genes EDNRB and EDNRB2 and the melanin biosynthesis pathway gene TYRP2. EDNRB expression in skin or muscle showed no significant difference between Silkie and White Leghorn chickens at either embryonic day 13 (a) or adult day 4 (b). However, the expression of EDNRB2 and TYRP2 was strongly increased in Silkie skin and muscle tissue in both the embryo and adult. In contrast, the expression of EDNRB2 and TYRP2 was very low in White Leghorn chickens. HL: hind leg; S: skin; M: muscle; L: liver.

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