Exploring Potential Relationship Underlying Blue-green Eggshell Color Of Duck Eggs Associated With Candidate Geges And Major SLCO2B1 Gene

The eggshell color of poultry is one of the most important traits in modern poultry breeding and has attracted great attention.Using blue-green duck eggs,this study expects to provide insight into the mechanisms by comparative transcriptome analysis and candidate gene approach.Exp.1: Functional analysis of candidate genes underlying blue-green eggshell color of duck eggsTwelve libraries(n=3 per group)from shell glands of two 35-weeks-old blue-shelled(BSD)and two 35-weeks-old white-shelled ducks(WSD)groups were sequenced using Illumina RNA-Seq platform.The results as follows:(1)RNA-Seq generates mean 41 million clean reads per library,of which 55.9 % are mapped to duck reference genome and assembled into 31542 transcripts.Mean length of transcripts is 1839 ± 86 bp.(2)A total of 464 candidate genes(CG)associated with blue-green eggshell color are differentially expressed uniquely between BSD and WSD.Of these CGs,230 genes are upregulated in the BSD,and 234 genes are downregulated.(3)Gene Ontology(GO)annotation shows that 390 CGs were annotated by GO terms,of which 359 CGs were assigned into 1515 biological process(BP)categories,363 CGs were annotated into the 250 cellular component(CC)categories,and 339 CGs were classified into the 469 molecular function(MF)categories.Overrepresentation test shows that a total of 9 BP,6 CC,and 1 MF categories are significantly(P < 0.05)overrepresented.(4)Here,a total of 13 Ca2+,10 Na+,8 for K+,5 H+,1 Cl-transporter and two HCO3-synthesis genes are found among these CGs,mainly participate in the expression of ion transporters related to eggshell mineralization process.Exp.2: Study on the relationship between blue-green eggshell color of duck eggs and SLCO2B1 geneEighteen samples(n=3 per group)from livers of three BSD and three WSD in three duck groups from different areas were analyzed using reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of c DNA ends(RACE)to clone the total transcript sequence of SLCO2B1.Then,this experiment analyzed the structure of SLCO2B1 gene and its protein so as to provide insight into the mechanisms.The results as follows:(1)This experiment obtains the interspecific difference of SLCO2B1 gene structure.The sequence alignment results shows that the sequence homology of SLCO2B1 among three different duck breeds is 98.71 % and there are great differences in the region of 5’UTR of the gene among BSD and WSD.(2)The total transcript sequence of SLCO2B1 is cloned.The mean 2372 bp transcript extends towards 5’ end by 188 bp compared with the published data and has 94.11% similarity to reference transcript.(3)This experiment obtains the complete protein sequence of SLCO2B1.The sequence homology of SLCO2B1 protein among three different duck breeds is 99.45 % and the protein length is 663 aa,which begins with methionine(Met)and extends towards the N-terminal by 21 aa compared with the published data.Prediction of protein structure finds that SLCO2B1 protein of three duck breeds mainly consisted of Alpha helix and Random coil.This protein has 12 transmembrane domains and 1 Kazal Serine protease inhibitor domain.Conclusions:(1)RNA-Seq finds 464 CGs associated with blue-green duck eggshell color whereas none of them are directly implicated into biliverdin biosynthesis or transport.These CGs are involved in the integral components of membrane,metal ion transport,cholesterol biosynthesis,signal transduction,skeletal system development,and chemotaxis,and then regulates the blue-green eggshell color.(2)This study obtains the total transcript sequence of SLCO2B1 by RT-PCR and RACE and there are great differences in the region of 5’UTR of the gene among BSD and WSD.Structural analysis of SLCO2B1 protein provides insight into further study on the genetic mechanisms underlying blue-green duck eggshell color.