Genome-wide Association Studies And Genome-wide Detection Of Copy Number Variations For Beak Deformity In Chickens

Beak is an important and peculiar structure to birds.Its functions are similar to the lips and teeth in mammals,including eating and drinking.Beak is highly associated with poultry growth and production performance.Beak deformity(normally a crossed beak)has been found in Beijing-You chickens(BJY).Chickens with deformed beaks have reduced feed intake and growth rate,low egg production,and high mortality,causing economic loss in poultry industry.Frequencies of up to 3% of beak deformity were found in various indigenous chickens of China,such as BJY(studied here),Qingyuan Partridge,Huxu,Silkies,and Wenchang chickens.Our previous investigations on the pedigreed individuals with deformed beaks shed light on the heritability of this trait.However,the genetic basis of this trait remains incompletely understood.Therefore,a powerful approach should be used to identify molecular markers,genes and pathways related to beak deformity,which can be used to eliminate beak deformity birds or the birds with teratogenic genes from a chicken population.It will provide theoretical and scientific foundation for the molecular mechanism of beak deformity trait,and also provide guidance for the study of similar genetic diseases on other birds and mammals.In the present study,beak deformity males and females were randomly mated to construct a BJY chicken resource population for revealing the probably heredity law of this trait,providing references for the further study.Forty-eight beak deformity birds and 48 normal ones were selected from the offspring for chicken 600 K high-density(HD)genotyping by Affymetrix.Single SNP-and pathway-based GWAS,and CNV detection were then performed for identifying key SNPs,CNVs,and candidate genes and pathways,combing with the previous transcriptome and proteome studies.The main results of this study were as follows:1.Six beak deformity males and 12 beak deformity females from a BJY chicken population were selected for a randomly mating experiment to produce 921 offspring.The incidence of beak deformity in the progeny(about half males and half females)was 7.82%(Only 0.66% in the normal BJY population of the same batch).A high ratio(88.89%)of beak deformity was observed at early age(before 20 days).The incidence of beak deformity progeny of the same birds mating with mixed semen was 7.87%,which was higher than the progeny of normal birds mating by deformity birds.The backcross test showed that the incidence of beak deformity in the progeny was about 10%,which was higher than the progeny of normal birds mating by deformity birds.The types of crossed beak can be divided into crossed left and crossed right of lower mandible with a ratio of 1:1.The average angle of crossed beaks was larger than 20 degrees.2.ROADTRIPS was employed for the single SNP-based GWAS based on the case-control design.According to the quality control(QC)criteria,95 individuals and 429,539 SNPs were ultimately used in the subsequent analyses with an average marker density of 2.46 Kb per SNP.As a result,one associated SNP(5% genome-wide significance)on GGA 3 and 7 suggestively associated SNPs on GGA 1,3,5,6,6,10,and 23 were detected.Four high-confidence genes,LOC421892,TDRD3,RET,and STMN1,were firstly identified as the most promising candidate genes underlying this complex trait in view of their positions,functions,and overlaps with previous DGE study.In addition,PLINK was employed for the direction of cross and angle analyses.No significant and suggestively SNPs associated with direction of cross were detected.Only 22 suggestively SNPs associated with the angle of all the 96 birds were detected.No significant and suggestively SNPs associated with the angle of 48 beak deformity birds were detected.3.SRT was then employed for the pathway-based GWAS.According to the QC and SNP selection criteria,a total of 149 pathways were finally selected from chicken pathway dataset,covering 128072 SNPs(including 115222 SNPs and 12850 SNPs involved in the unique and multiple pathways,respectively).As a result,we identified six associated pathways,including the ribosome pathway,the oocyte meiosis pathway,the pantothenate and CoA biosynthesis pathway,the pyruvate metabolism pathway,the glycine,serine,and threonine metabolism pathway,and the calcium signaling pathway.Combined with the previous studies of chemical constituents of chicken beaks and iTRAQ,the calcium signaling pathway may be one of the most important pathways associated with beak deformity trait.4.PennCNV was employed to detect genome-wide CNVs among beak deformity and normal birds.Based on the non-strict QC,a total of 33 and 115 CNVRs covering 0.75 Mb and 11.26 Mb on the autosomes were identified in beak deformity and normal birds,respectively.Based on the strict QC,a total of 2 and 8 high confidence CNVRs covering 0.32 Mb and 2.45 Mb on the autosomes were identified in beak deformity and normal birds,respectively.The ratios of 6 CNVRs had significant differences between beak deformity and normal birds(P < 0.01).These 6 CNVRs could be considered as potential specific CNVRs that may distinguish abnormal and normal birds.Further qRT-PCR studies validated 9 of the 10 identified CNVRs.Gene LRIG2 could be firstly selected as a key factor in view of its known functions and the relationship with RET gene identified in GWAS above.In summary,beak deformity could be considered as a complex trait regulated by multiple genes.We performed GWAS and CNV analyses to discuss the genetic mechanism of this trait.Combined with the previous studies,several genes and pathways were identified as the most promising candidate factors underlying this complex trait.