Identification Of Candidate Genes Associated With Upright/Pendulous Comb In Chicken

The comb is a secondary ornamental organ and an important packaging trait of chcken which can be used as an indicator of sexual maturity.Moreover,the comb trait has been reported to have effects on egg production and growth.Pendulous combs commonly exist in most single comb chicken breeds.The flops comb can block vision in one eye which has a bad influence on feed intake and mating behaviour in chicken.Furthermore,the risk of localised infection is elevated in the crease that forms when the comb flops over.We had carried out four exprements to research the molecular genetic mechanism of pendulous/upright comb in chicken.First,Phenotypic frequencies of upright(Z),partial pendulous comb(PD)and complete pendulous comb(D)were investigated in five chicken breeds including Huainan chicken(n = 2208),Dongxiang chicken(n = 2829),Wenchang chicken(n = 1905),Jinghong layer(n = 1990),and Nongda-3 layer(n = 2863).Four pure lines including Nongda-3(n = 1893),Huainan(n = 1420),Wenchang(n = 1035)and Dongxiang(n = 1829)were used to measure the 300 d egg number of three comb phenotypes chickens;Huainan breed(n = 600)were used to measure the growth of chickens with different comb phenotypes.Four types of mating were carried out(D♂ × Z♀,D♂ × D♀,Z♂ × Z♀,and Z♂ × D♀)to study the heredity of upright and pendulous comb(Huainan breed).Results show that the phenotypic frequencies of D and PD ranged from 10.1% to 29.0% and 16.8% to 65.3 %,respectively.D males were heavier than PD males at 12 and 16 wk of age and D hens were heavier at 24 wk of age.D hens produced more eggs than PD hens in Nongda-3,Huainan and Wenchang breeds.There was no significant difference in comb phenotypic frequency distribution between the offspring from Z(♂)× D(♀)and D(♂)× Z(♀),however,comb phenotypic frequency distribution differed between offspring from Z(♂)× Z(♀)and D(♂)× D(♀).These results suggesting that the upright or pendulous comb trait may influence chicken growth and egg production.Furthermore,the upright or pendulous comb may be influenced by several major effect loci or some minor effect loci in the autosome or both.Second,three histochemical methods: H&E,Gomori trichrome and safranin-O and fast green,were employed to study the morphology of endulous/upright comb(Huainan roosters at 30 weeks of age,7 chickens with pendulous/upright comb,respectively).We found that there were significant difference in the density of collagenous fibers and mucopolysaccharide content in the dermis between pendulous and upright comb.Thirdly,we investigated the comb transcriptomes architecture of pendulous and upright comb in three Huainan line(4 roosters with pendulous and upright comb,respectively,in each line were used)by using RNA-seq technology.In addition,multilevel bioinformatic analyses(GO and KEGG)of DEGs were used to identify enriched categories that may have been subjected to phenotypes.Many DEGs genes(116)were identified such as CA2,COMP,CEMIP,TNFIP6,GREM1,SPTB,SH2B3 and SIRT1.These candidate genes were related to the category “hemopoiesis”,“immune”,“immune system development”,“cartilage development”,“extracellular matrix”,and “glycosaminoglycan binding”.We focus on the category “extracellular matrix”,and “glycosaminoglycan binding” which may the candidate biological process associate with pendulous comb in chicken.Fourthly,two Huainan breeds were divergent selection for upright/pendulous comb phenotypes.We collected 24 comb samples(12 hens and 12 roosters)from chicken with pendulous(D)and upright comb(Z)phenotypes respectively.We extracted DNA from each sample,made a single genomic DNA library(D and Z)for each population sample,and then sequenced each library within a distinct lane using the Illumina Hi Seq 2000 instrument.A sliding window approach(200 kb windows sliding 50% overlapping steps)was applied to calculate the heterozygosity(HP)and the fixation index(FST)between the two comb phenotypes genomes.Putatively selected regions were located in extracting windows with simultaneously significantly low Z(HP)values(5% right tail)and significantly high Z(FST)values(5% right tail).Morever,multilevel bioinformatic analyses(GO and KEGG)of candidate genes were used to identify enriched categories that may have been subjected to positive selection.A total of 66 candidate genes were identified in Z groups.We searched for significantly overrepresented GO and KEGG pathway among the candidate genes and the most enriched categories were relate to “organic cyclic compound binding” and “Nicotinate and nicotinamide metabolism” pathway.A total of 114 candidate genes were identified in D groups.These candidate genes were relate to 15 GO categories such as “cardioblast proliferation”,“N-acetylglucosamine 6-O-sulfotransferase activity” and “limb development” and “Glycosaminoglycan biosynthesis-keratan sulfate” KEGG pathway.Two genes(CHST4 and CHST6)belonging to the category“N-acetylglucosamine 6-O-sulfotransferase activity” and the KEGG pathway “Glycosaminoglycan biosynthesis-keratan sulfate” which may relate to upright/pendulous comb phenotypes in chicken.