Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China.
PLoS One. 2012;7(4):e33851. doi: 10.1371/journal.pone.0033851. Epub 2012 Apr 9.
Abundant evidence indicates that chicken reproduction is strictly regulated by the hypothalamic-pituitary-gonad (HPG) axis, and the genes included in the HPG axis have been studied extensively. However, the question remains as to whether any other genes outside of the HPG system are involved in regulating chicken reproduction. The present study was aimed to identify, on a genome-wide level, novel genes associated with chicken reproductive traits.
METHODOLOGY/PRINCIPAL FINDING: Suppressive subtractive hybridization (SSH), genome-wide association study (GWAS), and gene-centric GWAS were used to identify novel genes underlying chicken reproduction. Single marker-trait association analysis with a large population and allelic frequency spectrum analysis were used to confirm the effects of candidate genes. Using two full-sib Ningdu Sanhuang (NDH) chickens, GARNL1 was identified as a candidate gene involved in chicken broodiness by SSH analysis. Its expression levels in the hypothalamus and pituitary were significantly higher in brooding chickens than in non-brooding chickens. GWAS analysis with a NDH two tail sample showed that 2802 SNPs were significantly associated with egg number at 300 d of age (EN300). Among the 2802 SNPs, 2 SNPs composed a block overlapping the GARNL1 gene. The gene-centric GWAS analysis with another two tail sample of NDH showed that GARNL1 was strongly associated with EN300 and age at first egg (AFE). Single marker-trait association analysis in 1301 female NDH chickens confirmed that variation in this gene was related to EN300 and AFE. The allelic frequency spectrum of the SNP rs15700989 among 5 different populations supported the above associations. Western blotting, RT-PCR, and qPCR were used to analyze alternative splicing of the GARNL1 gene. RT-PCR detected 5 transcripts and revealed that the transcript, which has a 141 bp insertion, was expressed in a tissue-specific manner.
CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that the GARNL1 gene contributes to chicken reproductive traits.
大量证据表明，鸡的繁殖受到下丘脑-垂体-性腺（HPG）轴的严格调控，HPG 轴包含的基因已得到广泛研究。然而，HPG 系统之外的其他基因是否参与调控鸡的繁殖仍存在疑问。本研究旨在在全基因组水平上鉴定与鸡繁殖性状相关的新基因。
方法/主要发现：利用抑制性消减杂交（SSH）、全基因组关联研究（GWAS）和基因中心 GWAS 鉴定鸡繁殖的新基因。利用大群体进行单标记-性状关联分析和等位基因频率谱分析，以验证候选基因的效应。利用两个全同胞宁都黄鸡（NDH）鸡，通过 SSH 分析鉴定出 GARNL1 是一个与鸡就巢性相关的候选基因。在就巢鸡的下丘脑和垂体中，其表达水平显著高于非就巢鸡。用 NDH 两尾样本进行 GWAS 分析显示，2802 个 SNP 与 300 日龄产蛋数（EN300）显著相关。在 2802 个 SNP 中，2 个 SNP 组成了一个重叠 GARNL1 基因的块。用另一个 NDH 两尾样本进行基因中心 GWAS 分析表明，GARNL1 与 EN300 和首次产蛋日龄（AFE）密切相关。在 1301 只雌性 NDH 鸡中进行的单标记-性状关联分析证实，该基因的变异与 EN300 和 AFE 有关。在 5 个不同群体中，SNP rs15700989 的等位基因频率谱支持上述关联。Western blot、RT-PCR 和 qPCR 用于分析 GARNL1 基因的可变剪接。RT-PCR 检测到 5 种转录本，并显示具有 141bp 插入的转录本以组织特异性方式表达。
结论/意义：我们的研究结果表明，GARNL1 基因与鸡的繁殖性状有关。
