| As one of the important phenotypic characteristics of chickens,the study of the genetic mechanism of feather color is of great significance for the conservation of germplasm resources,product quality control and breeding of new breeds.In the powdered eggs of the Rocky Island Red and White Leghorn lines,the offspring usually have white or nearly white feathers after crossing with colored feathered chickens because of the pure dominant white feather gene I in the maternal line of White Leghorn hens.However,in actual production,it was found that different strains of white laying hens were crossed with red-feathered hens and a different proportion of red-feathered hens appeared in the offspring,suggesting that there may be mutations in white laying hens that disable the dominant white gene,but the molecular genetic mechanism for the formation of such "red feathers" has not yet been explored.In this experiment,we used different strains of White Leghorn(A and B lines,white feathers)hens to cross with Rock Island Red(B line red feathers)roosters,and then used the offspring of different strains of White Leghorn crosses(A X B)to cross with Rock Island Red(1♂:3♀)to clarify the inheritance pattern of the red feather trait in the offspring of White Leghorn and Rock Island Red crosses;then the gold and silver feather gene SLC45A2 and the dominant gene PMEL17 were tested in different populations.The genomic resequencing was then used to analyze the genetic basis of red plumage in the crossed offspring;then the genomic resequencing was used to analyze the differences between the mutant and wild-type white laying hens and to screen for molecular genetic markers closely related to the red plumage trait in the offspring;finally,four full-sibling red and white plumage hens were selected from the crossed offspring of white laying hens and Rock Island red hens,and their back hair follicles were used for The transcriptome sequencing analysis was used to screen the differentially expressed genes,and the results were combined with the genome sequencing results to further screen and validate the genes closely related to the red feather trait and to lay the foundation for elucidating the formation mechanism of this red feather trait.The main results of this study are as follows:(1)The crosses between different strains of White Leghorn hens and Rock Island Red roosters revealed that the crosses between line B and Rock Island Red produced 1,944 red-feathered individuals(1 rooster and 1,943 hens)and 1,819 white-feathered individuals(1,806 roosters and 13 hens),with a red-feathered ratio of 0.516;the crosses between line A and Rock Island Red roosters produced 799 red-feathered individuals(209 roosters and 590 hens)and 6,565 white-feathered individuals(3,557 roosters and 3，008 hens),with a red-feathered ratio of 0.108.The chi-square test revealed that the red feather traits of the offspring of the crosses between the A and B White Leghorn hens and the Rocky Island Red rooster showed a significant correlation with sex(P<0.01);the forward cross and reverse cross between the offspring of the crosses between the A White Leghorn rooster and the B White Leghorn hen(AB)and the Rocky Island Red rooster were tested.It was found that:346 offspring were produced in the forward cross(Rock Island Red ♂ X White Leghorn♀),including 86 white feathered chickens(63 males and 23 females)and 260 red feathered chickens(100 males and 160 females),and the chi-square test showed that the sex of the offspring in the forward cross was significantly correlated with the plumage phenotype(P<0.01);while 427 offspring were produced in the reverse cross(White Leghorn ♂ ×Rock Island Red ♀),with 317 white feathered chickens(130 males)and 427 offspring in the reverse cross(White Leghorn ♂ × Rock Island Red ♀).The chi-square test showed that there was no correlation between sex and plumage phenotype in the offspring of the reverse cross(P>0.05);the chi-square test on the proportion of red-feathered individuals in the offspring of the forward and backcross showed that the difference between the forward and reverse cross was significant(P<0.01).The above results indicated that the red plumage traits represented by the crosses between the white Leghorn hens of the A and B lines and the Rock Island red roosters were companion traits,and the rate of red plumage in the hens of the B line crosses was much higher than that of the A line.(2)By testing the SLC45A2 gene in different White Raihang egg strains,it was found that the fourth exon mutation(C→A)in the SLC45A2 gene was present in all B line White Raihang egg populations,while the fourth exon mutation(C→A)and the third exon mutation(A→G)were both present in A line White Raihang egg populations.The SLC45A2 gene was detected in different plumage color individuals of the crossed offspring of White Leghorn and Rock Island Red,and the third and fourth point mutations of the SLC45A2 gene were A/A haplotypes(only the fourth exon mutation existed),and their plumage color was white(only one red-feathered chicken appeared),indicating that the fourth exon mutation was S.From the orthologous results of Rock Island Red roosters and White Leghorn hens of different genotypes of A X B lines,it was found that The results of the orthologous crosses with different genotypes of A X B lines of White Leghorn hens showed that the red feather rate(0.821)of the third exon heterozygous mutant rooster(G/C-A/C)was similar to that of the hens without the mutation(A/C-W)(0.885),while the red feather rate(0.067)of the fourth exon heterozygous mutant rooster(A/A-A/C)was much lower than that of the hens without the mutation(A/C-W)(0.870),indicating that the third exon mutation was s.The results indicated that the red feather trait in the offspring of crosses between White Leghorn hens and Rock Island red roosters was associated with the SLC45A2 genotype.And the detection of the seventh exon of PMEL17 gene in different strains revealed that the insertion of 60 bp on the seventh exon of PMEL17 gene(genotype Ⅱ)was present in different White Leghorn hens.(3)A total of 9,498,304 SNPS loci were obtained by genome resequencing,including 31,317 pure mutant loci,which were mainly enriched in signaling pathways such as ErbB signaling pathway,ECM-receptor interaction,melanogenesis,and Wnt signaling pathway.After analyzing the known functions and pathways of the genes in which the differential loci are located,18 mutant loci of five genes,FZD10,TUBB3,MC1R,GAS8 and USP3,were selected for validation in A and B lines and in the offspring of different plumage crosses in this experiment.The results showed that mutations at the g.18490748 locus in the TUBB3 gene and the g.18488265 locus on the MC1R gene were fully associated with the red-feathered individuals of the crossed offspring of White Leghorn and Rock Island Red.(4)Dorsal follicle-like transcriptome sequencing of red and white-feathered chicks from crosses between White Leghorn hens and Rock Island Red roosters showed that differential genes were significantly enriched in oxidative phosphorylation,glutathione metabolism,ribosome,and hematopoietic cell lineage pathways.Selected differentially expressed genes,including up-regulated genes KIT,KRAS,SLC45A2,TCF and CALML3,and down-regulated genes DCT,WNT10A,TYRP1 and KITLG,were screened based on differential expression as well as gene function and validated using qPCR.Twenty-eight differential genes,including DEF8,ELF1,TMEM255A and GPR157,were screened by combined genomic and transcriptomic analysis.Further validation revealed that two genes,GPR157 and DEF8,were significantly reduced in expression in the red feather capsule,which was consistent with the transcriptome sequencing results;and the g.18494790 and g.18495822 mutant loci of DEF8 gene were closely associated with the red feather trait in progeny.In summary,the red feather trait present in the crosses between Bai Leghorn and Rock Island Red is a companion trait,which is genetically based on the fourth exon mutation of the companion gene SLC45A2 gene and is affected by autosomal mutations in Bai Leghorn hens.The g.18490748 locus of MC1R gene,the g.18488265 locus of TUBB3 gene and the g.18494790 and g.18495822 loci of DEF8 can be used as genetic markers for this mutation and may act as causal genes causing changes in the expression of genes related to melanin synthesis and thus affecting the formation of plumage color.These findings provide a theoretical basis for the selection and breeding of new strains of white Leghorn egg-laying hens and the breeding of matching lines for pink-shelled egg-laying hens. |
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