Molecular Cloning,Polymorphisms Of Melaninrelated Genes And Their Association With Plumage Color In Duck (Anas Platyrhynchos)

Plumage color is an important breed characteristic and individual characteristic,and it is also considered to be an important economic trait and genetic marker.In our previous research,the interaction of alleles at two different loci were reported to determine black(grey)plumage color in duck.Moreover,we found that plumage color of black(grey)ducks displayed great variation.To date,the underlying mechanisms of duck plumage color are poorly understood.In order to identify the genes that control duck plumage color,the second generation of Liancheng × white Kaiya were used as experimental animals.EDNRB2,EDN3,RAB27 A,MLPH,MYO5 A and MITF were selected as candidate genes.We characterized the transcript and genomic structure of these genes in duck,performed mutation screening,and investigated their association with plumage color and plumage pattern.The main results are indicated as follows:1.The entire coding region of EDNRB2 gene was cloned by the reverse transcription polymerase chain reaction(RT-PCR).Sequence analysis showed that duck EDNRB2 c DNA contained a 1311 bp open reading frame and encoded a putative protein of 436 amino acids residues,which is a typical membrane protein and contains seven transmembrane domains.The entire coding region of EDNRB2 were detected by PCRSSCP method and 13 SNPs were identified.Two of them were non-synonymous substitutions(c.940 G>A→p.Val314 Met,c.995G>A→p.Arg332His),and were genotyped by PCR-RFLP method.The results of association analyses indicated that c.995G>A mutation was completely associated with spot phenotype(P<0.01),c.995G>A and c.940G>A were significantly associated with plumage color(P<0.05).Mating tests were designed based on the genotype of c.995G>A site,segregation ratios and genotypes of offspring of 39 testing combinations were in agreement with our hypothesis,thus the cosegregation of the spot locus with the c.995G>A mutation was confirmed.In addition,MITF gene was used as cell type marker of melanocyte progenitor cells while TYR and TYRP1 gene were used as cell type markers of mature melanocytes.Transcripts of MITF,TYR and TYRP1 gene with expected size were identified in all pigmented skin tissues while PCR products were not obtained from non-pigmented skin tissues.These results indicated that melanocytes are absent in non-pigmented skin tissues of spot ducks.It was inferred that the distribution of melanocytes were affected by the c.995G>A mutation of EDNRB2 gene,leading to the change of plumage color in duck.2.Partial transcript and genomic structure of duck EDN3 gene were cloned.5 SNPs were identified using the method of DNA poolings.The g.3578A>T site and g.3601C>T site can be detected by Tru1 I and Ssp I,respectively.The results of association analyses indicated that g.3578A>T was significantly associated with plumage pattern of colored ducks(P<0.05),while it is not associated with plumage color(P>0.05);g.3601C>T was not significantly associated with plumage color and plumage pattern of colored ducks(P>0.05).3.The entire coding region of duck RAB27 A gene was cloned by RT-PCR based on the conserved domain of RAB27 A gene in other species.Sequence analysis showed that duck RAB27 A c DNA contained a 666 bp open reading frame and encoded a putative protein of 221 amino acids,which contains the conserved domains of Ras proteins.RAB27 A gene were detected by PCR-SSCP method and 4 SNPs were identified in the introns,namely g.2426T>C,g.3885A>T,g.3893A>G and g.3931A>G;g.2426T>C and g.3885A>T were genotyped by PCR-RFLP method.The results of association analyses indicated that these two sites were not associated with plumage color and pattern in ducks(P>0.05).4.Ten exons of duck MYO5 A gene was cloned and sequenced using the method of DNA poolings.As a result,24 SNPs were identified including 8 SNPs in coding region and 16 SNPs in introns.The g.7014C>A,c.1139C>A and g.48935C>T site can be detected by Alw26 I,Bc1I and Taq I,respectively.The results of association analyses indicated that g.7014C>A site was close to be significantly associated with plumage color and pattern in ducks(P>0.05),while c.1139C>A and g.48935C>T were not associated with plumage color and pattern in ducks(P>0.05).5.The c.711A>G and c.1334T>G mutation sites of duck MLPH gene reported by Yu(2012)both can be detected by restriction enzyme Mbo I.These two sites were genotyped and the results of association analyses indicated that c.711A>G and c.1334T>G were not associated with plumage color and pattern in ducks(P>0.05).6.Part of the genomic sequence of duck MITF gene were cloned,8 SNPs were identified using the method of DNA poolings.The g.18361G>T,g.20591A>G and g.20708 A>G sites were genotyped by PCR-RFLP method.The results of association analyses indicated that these three sites were significantly associated with plumage color(P<0.01),while they were not associated with plumage pattern of colored ducks(P>0.05).