Resources Protection And Molecular Genetic Basis Of Plumage Color Diversity Of Bashang Long-tail Chickens

Bashang long-tail chicken is a local breed of both egg and meat.It was selected and bred by the people on the dam for a long time.It has the characteristics of strong foraging ability,strong resistance to rough feeding,strong resistance to disease,tender meat and so on.Farmers generally have access to meat and eggs provided by their own,the race consciousness weak.Many exotic breeds make Bashang long-tail chicken of the dam become hybridized.It was in an endangered state at the beginning of this study.Therefore,it is urgent to establish a conservation farm and preserved population.A rich variety of plumage colors including white,black,hemp,reed catkins,silvery grey,and landscape are found in Bashang long-tail chickens.It is a good material for studying the genetic diversity of plumage and preliminarily studying its molecular genetic basis.To test how gene expression influences plumage color,transcriptomes of chicken hair follicles were generated using Illumina sequencing.A research was designed to study the activity and the structure of the EDNRB2,MITF,PMEL,and TYR promoters.Dual-luciferase expression vectors were constructed and transiently transfected to DF1cells with lip2000 liposome.The dual-luciferase detection kit was used to measure the relative luciferase activity.The main results of this study are as follows:1.The core group and base group of Bashang long-tail chicken are established.The appearance features,growth performance and reproductive performance are all in line with the characteristics of the breed.The fitting models of weight growth and development are obtained.The Gompertz model of rooster's weight growth is Y=2015.524e^{-4.218exp(-0.146t)}.The Gompertz model of hen's weight growth is Y=1396.131e^{-3.849exp(-0.155t)}.The egg production rate reaches 50%in 165 days,the weight of the first egg is 39.69 g,the average egg weight is 47 g at 34³8 weeks.The fitting model of egg production curve is obtained,Von Bertalanffy:Y=111.849(1-7.257^e-0.103t)³.The egg weight and egg-shaped index with the highest egg hatching rate are 44.6⁵4.5 g and 1.35¹.44,respectively.2.This study is based on high-throughput transcriptome sequencing technology,six RNA-Seq libraries with over 25 million paired-end clean reads per library with percentage of paired-end clean reads ranging from 96.73%to 96.98%.78%of the reads mapped to the chicken genome,and approximately 70%of the reads are mapped to exons.Transcriptomes of hair follicles producing hemp and land plumage are similar,but these two show moderate differences compared with gray and reed colored plumage.The black and white follicle transcriptomes are most divergent from the other colors.We identify several candidate genes,including GPNMB,PMEL,TYRP1,GPR143,OCA2,SOX10,SLC45A2,KRT75,TYR,EDNRB2,SLC24A5,RAB38,and ASIP.The results suggest that the white chickens due to the homozygous mutation and low expression of the TYR gene.The formation of black area size and color depth may be due to the expression levels of the GPNMB,PMEL,TYRP1,GPR143,OCA2,SOX10,SLC45A2,SLC24A5,KRT75,and TYR.The GO analysis of the differentially expressed genes(DEGs)reveals that DEGs in our transcriptome analysis are enriched in cytoskeleton and cell structure related terms.The KEGG analysis of the DEGs shows significant differences are existed in melanogenesis,tyrosine metabolism,and riboflavin metabolism pathways.3.The 1 616 bp,1 385 bp,1 268 bp,and 1 813 bp of the fragments in 5?flanking region of the EDNRB2,MITF,PMEL,and TYR genes in Bashang long-tail chickens are cloned,respectively.The expression vectors(E1^E8,M1^M6,P1^P9,and T1^T7)with different promoter regions and mutant vectors(mut-E-1^mut-E-3,mut-M-1^mut-M-4,mut-P-1,mut-T-1,and mut-T-2)of the promoter region are constructed.The core promoter region from-814 bp to+59 bp is identified in chicken EDNRB2 gene.The region from-814 bp to-105 bp positively regulates the EDNRB2 gene during the transcription process.The core promoter region from-660 bp to+200 bp is identified in chicken MITF gene.The core promoter region from-840 bp to+68 bp is identified in chicken PMEL gene.The region from-590 to-525 bp negatively regulates the PMEL gene during the transcription process.-840^-590 bp and-525^-266 bp regions are positive regulatory regions.The core promoter region from-810 bp to+65 bp is identified in chicken TYR gene.The region from-810 bp to-213 bp positively regulates the TYR gene during the transcription process.A variety of conservative transcription factors are predicted in the promoter regions of the genes.The 13,10,12,and 6 polymorphic sites are found by sequencing,respectively.The differences of genotype and allele frequency in-631,-615,-600,-577,-513,-397,-392,and-340 sites between black,hemp,and reed catkins feather chicken are significant(P<0.05).The function of-579,-505,-274,-220,-203^-202,-98,-46,-14,+1,and+28 sites in regulating MITF promoter activity is critical.The polymorphic sites(-456,-435,-410,-374,and-341)have a significant effect on the promoter activity of the PMEL gene.Two CpG islands are predicted in the regions of-1152^-483 bp and-150⁺12 bp in the PMEL promoter sequence.The function of-963^-957,-955,-932,-692,-391,and-321 sites in regulating TYR promoter activity is critical.Fifteen haplotypes are predicted in the EDNRB2 gene.The Hap-e6 is not predicted in the black feather chicken.Hap-e2 and Hap-e5 have the highest concentration of black feathers.10 haplotypes are predicted in the TYR gene.The frequency of Hap-t1 in white feather chicken is 100%.The frequency of Hap-t2 in black feather chicken has the highest proportion of haplotype(40%).The frequency of Hap-t1 in hemp feather chicken has the highest proportion of haplotype(40.5%).The frequency of Hap-t5 in hemp feather chicken has the highest proportion of haplotype(32.4%).The decrease of promoter activity may affect the gene transcription,thus affecting the synthesis of melanin.This study proves the activity and the structure of the EDNRB2,MITF,PMEL,and TYR promoters and provides the basis for studying the expression of these genes in hair follicle tissue.