A non-synonymous SNP with the allele frequency correlated with the altitude may contribute to the hypoxia adaptation of Tibetan chicken

<div><p>The hypoxia adaptation to high altitudes is of considerable interest in the biological sciences. As a breed with adaptability to highland environments, the Tibetan chicken (<i>Gallus gallus domestics</i>), provides a biological model to search for genetic differences between high and lowland chickens. To address mechanisms of hypoxia adaptability at high altitudes for the Tibetan chicken, we focused on the Endothelial PAS domain protein 1 (<i>EPAS1</i>), a key regulatory factor in hypoxia responses. Detected were polymorphisms of <i>EPAS1</i> exons in 157 Tibetan chickens from 8 populations and 139 lowland chickens from 7 breeds. We then designed 15 pairs of primers to amplify exon sequences by Sanger sequencing methods. Six single nucleotide polymorphisms (SNPs) were detected, including 2 missense mutations (SNP3 rs316126786 and SNP5 rs740389732) and 4 synonymous mutations (SNP1 rs315040213, SNP4 rs739281102, SNP6 rs739010166, and SNP2 rs14330062). There were negative correlations between altitude and mutant allele frequencies for both SNP6 (rs739010166, r = 0.758, p<0.001) and SNP3 (rs316126786, r = 0.844, <i>P</i><0.001). We also aligned the <i>EPAS1</i> protein with ortholog proteins from diverse vertebrates and focused that SNP3 (Y333C) was a conserved site among species. Also, SNP3 (Y333C) occurred in a well-defined protein domain Per-AhR-Arnt-Sim (PAS domain). These results imply that SNP3 (Y333C) is the most likely casual mutation for the high-altitude adaption in Tibetan chicken. These variations of <i>EPAS1</i> provide new insights into the gene’s function.</p></div

Primer information for detecting SNPs in coding regions.

<p>Primer information for detecting SNPs in coding regions.</p

The sequencing images of 6 SNPs in the coding region of chicken <i>EPAS1</i> gene.

<p>The position and base substitutions in coding regions refer to the sequence of ensemble ENSGALT00000016253.</p

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

<div><p>Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.</p></div

The Hardy-Weinberg equilibrium (HWE) tests of the candidate SNPs in <i>EPAS1</i> in TC and the LC.

<p>The Hardy-Weinberg equilibrium (HWE) tests of the candidate SNPs in <i>EPAS1</i> in TC and the LC.</p

Four synonymous mutations and two non-synonymous mutations in <i>EPAS1</i>.

<p>Four synonymous mutations and two non-synonymous mutations in <i>EPAS1</i>.</p

<i>EPAS1</i> mutation in coding regions.

<p>Structural and evolutionary analysis of the 4 amino acid variants. The protein coordinate is based on Ensembl ID ENSGALP00000016234. The upper layer displayed the Pfam domains of the protein. The orthologous proteins from 17 different vertebrates were aligned with the residues shown as green. Cow, ENSBTAP00000004836; pig, ENSSSCP00000009011; human, ENSP00000406137; dog, ENSCAFP00000003819; elephant, ENSLAFP00000010336; mouse, ENSMUSP00000024954; opossum, ENSMODP00000001136; lizard, ENSACAP00000004025; xenopus, ENSXETP00000031612; zebrafish, ENSDARP00000074832; wallaby, ENSMEUT00000004049; horse, ENSECAT00000015683; cat, ENSFCAT00000013633; rat, ENSRNOT00000034991; panda, ENSAMET00000006660; chimpanzee, ENSPTRT00000022124. The ML tree is shown in the left area. (PAS) Per-Arnt-Sim; (HIF) hypoxia-inducible factor; (ODDD) O₂-dependent degradation domain; (TAD) C-terminal transactivation domain.</p

Altitude, longitude and latitude of the sampling locations for the 8 Tibetan and 7 Lowland chicken populations.

<p>Altitude, longitude and latitude of the sampling locations for the 8 Tibetan and 7 Lowland chicken populations.</p

Genotype of the <i>EPAS1</i> gene SNPs.

<p>Genotype of the <i>EPAS1</i> gene SNPs.</p

Allele frequency, correlation and odds ratio of mutation loci in the <i>EPAS1</i> gene.

<p>Allele frequency, correlation and odds ratio of mutation loci in the <i>EPAS1</i> gene.</p