Cloning Sequence Of MC1R Gene In Duck And AFLP Markers Of Plumage Color In Mule Duck

Plumage color is an important quality trait of mule duck.The water fowl breeding researchers applied oneself to the improvement of the plumage color trait.However, conventional methods were greatly restricted on the breeding precede.The application of molecular marker-assisted selection had become an inevitable trend on duck breeding.Based on the original selection,molecular technology as a assistant means was introduced to further improve the purity of white feather on mule duck and rapidly breed female parent line of big white mule duck,which possessed local characteristics.Melanocortin 1 receptor (MC1R)gene of mule duck and Beijing duck were cloned and sequenced respectively,and the AFLP marker was used to screen plumage color of mule duck.This study would provide foundational genetic information for the research of molecular marker-assisted selection.Experiment 1,Sequence of MC1R gene:Genomic DNA was extracted from the Beijing duck and mule duck blood,and primers for amplification of MC1R gene was designed according to the conserved region of MC1R gene sequence of different bird,such as gallus, coturnix japonica,anser caerulescens,and pavo cristatus.PCR products were recovered and sequenced.Sequence analysis shows that PCR product was 855 bp in length.This is the basic information for further study on plumage color MC1R gene SNP markers of mule duck in the screening and selection of duck lines.The results of similarity search through BLAST show that the amplification products share high identity with other MC1R genes submitted in GenBank.Alignment analysis shows that the mule duck MC1R gene shares the highest identity with pekin's,reaching in 98.9%,and with anser caerulescens in 97.6%,while shares a identity with gallus,meleagris gallopal,cotumix japonica,pavo cristatus between 91.0%～95.0%.All of above demonstrate that the MC1R genes of different birds are relatively conserved.The MC1R gene of Beijing duck and mule duck exist mutations at nine positions,four of them were non-synonymous mutations(229(A/G),385(A/G),634(A/ G))which led to the amino acid replacement on the peptide of MC1R at 77(K/E),129(I/ V),212(T/A),respectively.Experiment 2,AFLP molecular marker associated with the mule duck plumage color trait was screened repeatedly with 64 sets of primers selected from the EcoRⅠ/ TaqⅠseries. The results show that:(1)After the first round,10 sets of primers were selected which amplified 13 bands exist especially in the white feather duck.Finally,3 sets of primers (E-AAG/T-AAC,E-AAG/T-AGT,E-AGC/T-AAT)were picked out as the credible markers after several round screening.All of the 3 sets primers could amplify 3 bands associated specifically with white feather.(2)The AFLP PCR products of the 3 markers were sequenced and analyses respectively.The results showed that the differential fragment EST of product of the primer set E-AGC/T-AAT share the similarity of 73%with gene WAG-9005 of gallus, while the products of primer set of E-AAG/T-AAC and E-AAG/T-AGT were new genes fragments which had low identity with the ESTs sequences submitted in GenBank.According to the results,these two differential fragments might be newly genome diversity fragments detected in mule duck.