Detection Of NUDT15 And ADPRHL1 Gene Mutations In Chicken And Development Of Molecular Markers

The growth traits of chickens have important economic significance in the poultry industry.Traditionally,these major economic characteristics have been selected by artificial selective breeding.However,the progress and rapid development of bioinformatics technology and molecular marker-assisted breeding make it more efficient than artificial selection,which not only shortens the breeding process,but also saves resources and improves the breeding efficiency.In this study,local dual-use breeds,fast broiler breeds,high-yield laying hens and "Gushi × Anka" F2 resource population were taken as the main research objects.The conventional experimental techniques of molecular biology were used to analyze the heritage parameters,economic traits association analysis and gene expression level of the mutation sites,in order to screen out the major genes that affect the growth and development of chickens and provide valuable molecular genetic markers for broiler breeding.The main test results are as follows:Research 1: genetic effect of NUDT15 promoter region In Del and its effect on chicken myoblast proliferation1.Two In Del mutations of 23 bp were found and identified in the promoter region of NUDT15,2777 bp and 1673 bp located in the upstream of ATG,respectively,and named NUDT15-In Del-2777 and NUDT15-In Del-1673.NUDT15-In Del-2777 has two genotypes,named N1（113bp）and N2（113bp/90bp）,and NUDT15-In Del-1673 has three genotypes,named N3（150bp）,N4（127-bp）and N5（150bp/127-bp）.2.The expression of NUDT15 gene in different tissues and different developmental stages of AA broilers and Lushi chickens showed that the expression of NUDT15 in leg and chest muscle of 12-embryo AA broilers was significantly higher than that in other tissues,but the expression in skeletal muscle was significantly lower than that in pancreas and liver of chicks.There was no significant difference in the expression of NUDT15 between N1 and N2 genotypes,but the expression level of NUDT15 in N4 was significantly lower than that in N3 and N5.3.The results of population genetic analysis showed that among the 10 breeds except Hubbard broiler and AA broiler,the allele frequency and 1673 Mui I of 2777 Mel I were significantly higher than those of 2777 Mel D and 1673 Mel D.The allele frequencies of 2777-I and 1673 mur D in commercial broilers were significantly higher than those in dual-purpose chickens and commercial laying hens.N1N3 was significantly higher than other genotypes in dual-purpose hens and commercial laying hens,while N2N3 and N2N4 were the least common genotypes,with only three genotypes.N2N4 is the main genotype in commercial broilers.In addition,the frequency of N2N4 and N2N5 genotypes in commercial broilers was significantly higher than that in dual-purpose chickens and commercial laying hens.4.The results of association analysis showed that N2N4 genotype produced the most ideal growth traits,followed by N2N5,N1N3,N1N4 and N1N5,and N2N3 genotypes produced the least ideal growth traits.5.The fluorescence activity of Pro-2777-I and Pro-2777-D showed that the activity of Pro-2777-I and Pro-2777-D was significantly higher than that of p GL4.10,Pro-2777-I and Pro-2777-D,but the difference was not significant.The fluorescence activity of Pro-1673-I and Pro-1673-D showed that the activity of Pro-1673-I and Pro-1673-D was significantly lower than that of p GL4.10.In addition,the promoter activity of Pro-1673-I was significantly higher than that of Pro-1673-D.6.Interference and overexpression experiments showed that NUDT15 could inhibit the proliferation of CPMs.Research 2.detection and association analysis of promoter region 104-bp insertion mutation of ADPRHL1 gene.1.A new 104-bp InDel mutation site（NCBI accession number: NC₀06088.5,Chr1:138752529-138752632）in the 5 ’UTR region of chicken 104-bp In Del gene was identified,and three II,ID,DD genotypes were identified according to the results of electrophoresis.2.Correlation analysis showed that 104-bp In Del locus had significant effects on body weight,shank length and oblique body length at 0,2,4,6 and 10 weeks of age,and had significant effects on slaughter traits such as semi-eviscerated,fully eviscerated,leg weight and leg muscle weight.The above phenotypes of individuals with II genotype were higher than those with DD genotype,that is,II genotype was the dominant genotype.3.The results of population genetic analysis showed that the frequency of DD genotype was low in all breeds and exceedingly rare in commercial broilers.The frequency of I allele in commercial broilers was higher than that in local dual-purpose breeds and laying hens.II genotype is the dominant genotype.4.Tissue expression profile showed that ADPRHL1 gene was expressed in all tissues of 1-week-old Gushi chicken and AA broilers,and the expression level of leg muscle and myocardial tissue was higher.The difference of myocardial expression between 1-week-old Gushi chicken and AA broiler chicken reached an incredibly significant level.The expression of ADPRHL1 gene in leg muscle and chest muscle of Lushi chicken and AA chicken from 10 to 3 weeks old increased at first and then decreased.5.The expression level of ADPRHL1 in leg muscle tissue of 1-week-old AA chickens and 3-week-old Lushi chickens with different genotypes showed that the expression of II genotype was significantly higher than that of ID genotype.6.Double luciferase reporter system showed that the promoter activity of I allele was significantly higher than that of D allele,which was 2.49 times higher than that of D allele.