Study On The SNPs And Expression Difference Of Chicken Polydactyly Candidate Genes

Polydactyly is a character in Several chicken breeds, which is a good material to study the interaction among genes in the process of limb growth for it is easy to operate and observate .Considering the interaction among polydactyly relevant genes and the polydactyly homologous region in human and mouse, we selected En2, Shh, Lmbr1 , Hlxb9 and Gli3 as polydactyly candidate genes in chicken. The SNPs related to toes and the difference in the genes expression were found and detected, in order to uncover the functions of candidate genes and explain the mechanism of chicken polydactyly..The materials used for finding SNPs included six groups:Silkies(5-toed), A strain of broilers (4-toed), Juhua chickens (4-teod and 5-teod) and Beijing-youji chickens (4-teod and 5-teod) .With 5 chickens from each group, exons and intron-conserved-domains (ICDs ) of five candidate genes, and 5' upstream of Lmbr1 were amplified separately. Then each sequence were purified and compared by 6 groups to search for the SNPs which could strictly distinguish 4-toed from 5-toed. The exons of the 5 genes were conservative and only two SNPs were found , one SNP (C/T) in exonl6 of Lmbr1 , the other SNP (G/A) in exon15 of Gli3. More sequences comparison and SSCP results exclude their relation to toes. But in ICDs, only one SNP(C/A) in Lmbrl intron5 was found and mapped to chr2:7901586bp, C variation correlating with 4-toed and A variation correlating with 5-toed, which was consistent with SSCP typing results from 517 DNAs of Juhua chickens, Beijing-youji chickens, CAU resource reference population(established by Silkies and A strain of broilers) .From the 5'-upstream of Lmbrl , 6 out of 26 SNPs were found related to toes, mapped to Chr2:7922655~ Chr2:7928742bp.Accordong to internet MatInspector prediction, 3 of the 6 SNPs could change homeodomain factor or protein binding sites that relevant to embryogenesis; the SNP in the intron5 of Lmbrl could change three homeodomain transcription factors binding sites. Their coding genes. Hox gene family, play important roles in the anterior-posterior axis in the development of polydactyly in human and mouse. The prediction results supported the influence of Lmbrl in the development of toes.To detect the differences in candidate genes' expression, RNAs wereused, which were extracted from heads, bodies and limb buds of White Leghon embryos (4-toed) and Silkie embryos (5-toed) at the same stage.The expression of GHS, Lmbrl and Shh was initially detected in Id (24h incubation) by RT-PCR, ahead of the first detectable time 2.5d~3d in Whole-mount in situ hybridization. The three genes were all expressed in 1 d~7d, distinct in some stages and weak in other stages.The expression of GH3^ Lmbrl and Shh varied with time. In Silkies, GH3 expression was higher in the heads and bodies (4d up to 7.18 times) than limb buds (4d up to 2.15 times) . In White Leghorns, GH3 expression in the limb buds after 4.5d rose rapidly up to 7.55 times, forming a sharp contrast to those of their heads and bodies (little variation , 5d up to 2.54 times) as well as three parts of the Silkies intensively. Repeated experiments got the same results. There has been no such similiar report until now. Lmbrl and Shh in limb buds of two breeds varied little with time, although Lmbrl reached higher (3.5d up to 6.27 times) in the heads and bodies of Silkies before 4.5d.The expression of three genes was also different between the two breeds. The expression of GH3 in the heads and limb buds was higher in 3d~4d and lower in 4.5d~6d in Silkies than that in White Leghorns. The expression of Shh in the limb buds was higher in Silkies than that in White Leghorns (except 6d), which is in reverse to that of the bodies. When GU3 rose rapidly, Shh descended quickly all the time, presenting antagonistic relationship. 4d~6d is the critical stage of toe development. At the very time, higher GH3 and lower Shh expression in the limb buds of White Leghorns might avoid forming 5-toed, while higher Shh and lower GH3 in the limb buds of Silkies might help to form 5-toed.The little expression difference of Lmbrl in two breeds and the SNPs in the introns of Lmbrl related to toes, Linking up other studies on Lmbrl in orientation, expression and variation in vertebrate, indicated that Lmbrl was a key gene in chicken toes development, likely to regulate toes growth by changing the binding sites of transcription factors.Chicken polydactyly is formed by extra Shh expression, which can be arose by the expression quantity of GH3 or intron variation of Lmbrl.