Establishment Of Evaluation Criteria For Black Character Of Xichou Black Bone Chicken And Study On Transcriptome Of Chicken Comb Black Degree

The Xichu black-boned chicken is a unique breed of black-boned chicken in Xichu County,Yunnan Province,which is popular among consumers for its tender meat and rich nutrition,especially for its melanin and melaningel,which have good health effects.In recent years,our group has investigated the existence of two phenotypes,black and red crowns,in the same breeding condition.In order to establish a model for evaluating the melanin content of the chickens without slaughtering and to investigate the molecular mechanism of the formation of different darkness in the crowns of the chickens,200one-day-old chicks of black and red-crowned males and females were selected from the breeding farm of the chickens.Three measurement methods（colorimetric card,colorimeter,and photograph）were used to determine black degree and different parts of the skin（neck skin,back skin,breast skin,leg skin,under wing skin,and tibia skin）of two different crown color breeds at 30,60,90,120,and 150 days of age,respectively.At the same time,blood was collected for the determination of melanin content.Then,by comparing the black degree determined by different methods at 150 days of age,the best method for determining black degree was screened,and an optimal model for assessing the melanin content in the organism using black degree of comb was initially constructed.In addition,we compared the differences in crowns and development patterns of different crown colors,ages and sexes,and finally selected six chickens（half of each sex）with the highest and lowest crowns at 30 days of age and 150 days of age,respectively.Corresponding comb tissue samples were collected for transcriptome sequencing and analysis to screen out differentially expressed genes associated with comb color,and Q-PCR was used to verify the expression of candidate genes for analysis.The main results were as follows:（1）The optimal selection of three measurement methods:colorimetric card,colorimeter,and photo.The variation patterns of blackness in different tissues of Xichu chicken were the same for colorimetric cards,colorimeter and photographs.The coefficient of variation of colorimeter was the lowest,between 0.05-0.1,and the correlation coefficient with melanin content was the highest,ranging from-0.918 to-0.935,which was the best way to determine the darkness.（2）The correlation analysis between L-value and melanin content in different parts of the chicken comb at 150 days of age showed a positive correlation with other parts.The higher the blackness of the chicken comb,the higher the blackness of other parts.（3）A model was constructed to assess the melanin content in the body using the crowns.The best regression model was a"polynomial regression curve"between L and a values and melanin content,with melanin content as Y,L value as X₁ and a value as X₂.Theregressionmodelforblackcrownswas Y=-2.01X₁²+142.43X₁+3999.50-1.56X₂²+0.001X₂-5397.11,with correlation coefficient R²=0.951.Theredcrownregressionmodelwas Y=1.59X₁²-201.21X₁+12650.90+0.44X₂²-30.81X₂-5314.95,with correlation coefficient R²=0.984.（4）Analysis of the pattern of black and red-crowned Xichuan crowns.The results showed that the L-value of different parts of black-crowned chickens was almost smaller than that of red-crowned chickens under different crown colors of the same species,and the crowns and tibia skins showed significant differences（P<0.05）.The L value of female chickens was smaller than that of male chickens under different sexes of the same crown color,and the crowns showed significant differences（P<0.05）.The overall L value of black crowns,regardless of male and female,showed a pattern of crown<under wing skin<leg skin<breast skin.The a-value of black-crowned chickens was smaller than that of red-crowned chickens under the same sex of different crown colors,and the crowns showed a significant difference（P<0.01）.The a-value of hens was smaller than that of roosters under different sexes of the same crown color.The L and a values of different crown colors and sexes gradually increased with age.（5）Analysis of melanin content in black and red crowned Xichou Black-bone chickens.As the age increases,the content of melanin gradually decreases.Under the same comb color,the melanin content of hens is higher than that of roosters.Under the same gender,the content of melanin in the black comb is higher than that in the red comb.At 150 days of age,the melanin content of the crown was greater than the melanin content of the blood,and the melanin content of the blood was greater than the melanin content of the skin.Under the same comb color,the comb and skin content of black combed hens were higher than that of roosters（P<0.05）,while the comb melanin content of red combed hens was significantly higher than that of roosters（P<0.01）.Under the same gender,the levels of melanin in the comb and skin content of black cockscomb were significantly higher than those of red cockscomb（P<0.01）.（6）Identification of genes related to black quality traits in Xichou Black-bone chickens.TYR,TYRP1,MC1R,DCT and other genes related to melanogenesis and deposition,as well as HBA1,HBBA,HBAD and other genes related to hemoglobin and heme production,may be involved in the regulation of black and red chicken comb color traits.The expression levels of selected genes for black coronavirus TYR,TYRP1,MC1R,and DCT were almost significantly higher than those for red coronavirus（P<0.01）,and the gene expression gradually decreased with age.The expression levels of red coronavirus such as HBA1,HBBA,and HBAD were almost significantly higher than those of black coronavirus（P<0.01）,and the gene expression gradually increased with age.（7）Fluorescence real-time quantitative PCR results.At the age of 30 and 150 days,the expression levels of TYR,TYRP1,MC1R,and DCT genes in the black comb of Xichou Black-bone chickens were upregulated compared to those in the red comb.HBBA,HBAD,and HBA1 were all upregulated compared to those in the red comb.The expression levels of candidate genes in Q-PCR and RNA-Seq showed a consistent upward and downward trend.（8）Analysis of the correlation between the L,a value,melanin content,and gene expression level of chicken comb blackness.At 30 and 150 days of age,the L value was negatively correlated with melanin content,DCT,TYR,MC1R,TYRP1,and other genes related to melanogenesis and deposition,while the a value was positively correlated with genes related to heme and hemoglobin production such as HBBA,HBAD,and HBA1.The above research results have successfully constructed a model for evaluating the melanin content in the body using the crowdsourcing degree of chicken,avoiding the trouble of slaughtering to determine melanin content and identify crowdsourcing degree.And this explains the molecular mechanism behind the formation of different degrees of blackness in chicken combs.Therefore it provides a scientific theoretical basis for further in-depth research on the black quality traits of black bone chickens,and provides important reference value for the determination of melanin related factors in other chicken breeds and chicken breeding.