Transcriptome Analysis Of Chicken Red And White Earlobe And Screening Of Differential Genes

The earlobe of chickens,locating below the earhole,is a typical external trait with a round or oval wrinkled skin structure and no feather covering.The color of earlobes,varying by species,can be found in yellow,blue,purple,and black,but earlobes are more often in red or white.Studies in recent years have shown that the red or white trait of earlobes is controlled by multiple genes in some chicken varieties.However,the molecular and histological mechanisms still remain unclear.Beijing You chicken with red earlobe and Single Comb White Leghorn with white earlobe were selected to insight the red or white earlobe tissue structure by frozen section.In order to screen differentially expressed candidate genes,the earlobe tissue transcriptome data of chickens of these two varieties,at the age of 35 days and 105 days,were analyzed by using high-throughput sequencing technology.Few candidate genes from this study and literatures were tested by fluorescence quantitative method.The preliminary results are as follows:1.The earlobe skin tissue samples were collected from three 105-day-old Beijing You chickens and three 105-day-old Single Comb White Leghorn chickens.Data from the frozen section test revealed that the number of sinusoidal capillaries in the dermis of the red earlobe was more than that in the white earlobe,but there was no significant difference between the epidermis and subcutaneous tissues.2.Based on the analysis of transcriptome data of auricular tissues of three 35-day-old Beijing You chickens（BY35D）,three 35-day-old Single Comb White Leghorn chickens（BLH35D）,three 105-day-old Beijing You chickens（BY105D）and three 105-day-old Single Comb White Leghorn chickens（BLH105D）,there were 2244,914,540and 718 differentially expressed genes found in the four comparisons（BLH105D_BLH35D,BY105D_BY35D,BLH105D_BY105D and BLH35D_BY35D）.GO analysis showed that these differentially expressed genes were involved in various physiological processes,such as metabolic process,developmental process,pigmentation and so on.Hematopoietic cell lineage pathway was significantly enriched in KEGG pathway analysis.These transcriptome data demonstrated that there were five differentially expressed genes detected,and they were related to angiogenesis（IL8L1）,hemoglobin complex（HBBA,HBA1,HBM）and heme binding（CYP3A5）.3.In this study,HBA1 and HBM,the two genes with expression difference of|7)2)₂Fold change|≥2 and the lowest q value（corrected Pvalue）,and CYP3A5,the gene showing expression difference in the four comparisons,were selected to carry out real-time fluorescence quantitative experiments with BNC2,SLC38A4 and SLCO4C1,three candidate genes reported in literatures.The results showed that the up-regulation and down-regulation of these six genes in qPCR and RNA-seq were similar,and there was a significant correlation between the two data,indicating that data from the RNA-seq were reliable.The qPCR data showed that among the six genes,only HBA1 and HBM genes displayed significantly differential expression between red earlobe group and white earlobe group.The tissue expression profile of HBA1 gene showed that HBA1gene was highly expressed in vascular-rich visceral tissues,with the highest expression in lung and the lowest in skin.Data also demonstrated that the expression of HBA1 gene in the skin of red ear was significantly higher than that of white ear,and there was no significant difference in other tissues.To summary,the structural characteristics of the red or white earlobe tissue were analyzed,and candidate genes related to earlobe color formation were selected and detected at the transcriptional level.However further experiments are still needed to verify the function of these genes and to determine the genetic molecular mechanisms of these genes controlling the formation of earlobe color.This finding could provide basic data for revealing the genetic mechanism of the formation of earlobe color traits in poultry.