Screening Of Candidate Genes Of Chicken Feathering

The early and late feathering of chicken can be used for the identification of male and female, and there is a certain association with production traits. It is known that endogenous retrovirus ev21 is close linked to the gene(s) for chicken early and late feathering. In early-feathering chicken (EF), the only ev21 insertion site was located between the prolactin receptor (PRLR) and the sperm flagellar protein 2 (SPEF2) genes. While in late-feathering (LF) chicken, there are two insertion sites of ev27 with one occupied by ev21. Between the sites is a fusion gene (dPRLR/dSPEF2), which consists of partially duplicated PRLR (dPRLR) and SPEF2 (dSPEF2). Due to lack of strong evidence, the three genes (PRLR, SPEF2 and dPRLR/dSPEF2) are all candidate genes for chicken feathering. To further discriminate the three genes for identification of chicken feathering gene(s). We first determined whether the splicing pattern and sequence in the LF chickens used in this study was the same as previously reported by PCR and sequencing. We then determined whether the fusion gene was transcribed using reverse transcription-based quantitative PCR (RT-qPCR), which was followed by strand specific RT-qPCR analysis for determinating whether the fusion gene was bidirectionally transcribed and whether the transcripts formed a double strand RNA (dsRNA). At last, the expression levels of three candidate genes in the skin of the EF and LF chicken were determined by real-time fluorescent quantitative PCR so that we may provide a foundation for identification of genes controlling chicken feathering.1. Using genomic DNA from Suqin GES chicken and Hisex LF chickens, we first confirmed the feathering genotypes of the experimental chickens by PCR with the primers specific for the fusion gene. A 1,429 bp amplicon was only shown in the LF chickens. Sequencing analysis indicated that the sequence of the amplicon was different from previously reported one. In our experimental LF chickens, the junction of the fusion gene was formed by 1,543 bp of SPEF2 intron 4 and 578 bp of PRLR exon 12, suggesting there is a breed-specific variation for the fusion gene.2. RT-qPCR analysis indicated that the dPRLR/dSPEF2 fusion gene could be transcribed and only transcribed in LF chickens. By strand-specific RT-qPCR, we revealed that the fusion gene was bidirectionally transcribed, and the transcripts may form a dsRNA molecule. This dsRNA may regulate the expression of its neighboring genes thus affecting the growth of chicken feather. However, this speculation needs to be validated.3. The expression levels of PRLR, SPEF2 and dPRLR/dSPEF2 genes were determined in the skins of the 1-day-old male/female chickens by real-time fluorescence qPCR analysis. Data showed there was no significant difference in the expression of PRLR between the EF and LF chickens. In other word, the expression of PRLR was not directly related to the formation of EF and LF phenotypes, suggesting that PRLR is not suitable for the candidate gene of chicken feathering. In contrast, the expression of SPEF2 was significantly higher in the skins of the LF chickens than the EF chickens (P<-.01), suggesting SPEF2 is a good candidate gene. Moreover, the fusion gene is also a strong candidate gene for chicken feathering as it is only expressed in the LF chickens. However, the expression level of dPRLR/dSPEF2 was only about 2.9% of PRLR mRNA abundance, suggesting the fusion gene does not regulate the growth of chicken feather in the same way as the PRLR and SPEF2 genes do. In summary, the fusion gene dPRLR/dSPEF2 and SPEF2 gene are strong candidate genes for chicken feathering while PRLR is not. Study on the function, mechanism and regulation of these genes may help address the molecular basis of chicken feathering.