Study Of Embryonic Death Related Genes And Establishment Of Blastodermal Sex Identification Technology In Chicken

In order to study the law of chicken embryonic death,and chicken embryonic death of male or female embryos,and the effects of mutation of Candidate gene that related to embryonic development on embryonic death and the quality of hatching eggs,hatching eggs from Yellow chicken,Aijiaohuang chicken and Zhusi chicken,which were bred by Wenshi Poultry Breeding Company in China,were used.Five candidate genes(Fas, WISP-1,Nmi,RXRαand IGF2R) related to embryonic development in human and mouse were chosen to search for SNPs and to associate gene polymorphisms with the traits of embryonic death.The method of duplex PCR used to detect the sexes of embryo using a small number of cells was established,and then investigated the sex ratio of early death embryos and sex ratio of blastodermal cells using this established method.The main results in this study were listed as follows:1.The method of duplex PCR used to detect the sexes of blastodermal cell was established,which can also been used to identify the sexes of early death embryos.The patent of this method was applied and obtained,the patent ID was 200710052846.Ⅹ.2.The sexes of early embroes of fertile eggs was identified by the method,the initial result showed that the number of females was larger than the males.3.Throughout the incubation period,chicken embryos died in the two deaths peak, in the first death peak,the results show a tread that the number of female embryonic deaths was more than the number of male embryos.4.We have identified 7 SNPs in 5 genes in 200 xingxinghuang chicken embryos,269 Aijiaohuang chicken embryos and 235 Zhusi chicken embryos.Their association was analysed with the death of chicken embryonic death and the quality of hatching egg.5.Three SNPs were discovered in Fas gene,two out of three SNPs that were nonsynonymous mutations were identified in our samples.The XbaⅠ-RFLP in exon-3 of Fas gene significantly associated with the embryonic death in all three chicken breeds(P＜0.05).,while no association with the embryonic death in Aijiaohuang chicken in XbaⅠ-RFLP SNP of exon-2 in Fas gene.6.Five SNPs were detected in Wispl gene,one from five SNPs that was nonsynonymous mutation was identified in our samples.There is no association with the embryonic death in all three chicken breeds in the HaeⅢ-RFLP SNP of exon-5,but the P value in Aijiaohuang chicken and Zhusi chicken were close to significant(P=0.058 and P=0.081).The relative death rate of different genotype in different breeds were different, there existing breeding difference.But there was no significant difference in xingxinghuang chicken(P＞0.05). 7.Five SNPs were discovered in Nmi gene,of which two SNPs that were nonsynonymous mutation were identified in our samples.It was not significant association with the embryonic death in Aijiaohuang chicken in the HindⅢ-RFLP SNP of exon-2 in Nmi gene,while a significant association with the embryonic death in the HinfⅠ-RFLP in exon-5 of Nmi gene in Aijiaohuang chicken(P＜0.05),but the difference was not significant in Zhusi chicken(P＞0.05).8.There was no association with the embryonic death in Aijiaohuang chicken in the HhaⅠ-RFLP genotype in the exon2 of RXRαgene,which had been showed significant difference in yellow chicken.9.It had been showed significant difference with the embryonic death in yellow chicken,but no association in Zhusi chicken in the BlwnⅠ-RFLP genotype in the exon34 of IGF2R gene.10.The results of linkage disequilibrium analysis showed that the two SNPs in Fas gene had higher Linkage disequilibrium(D’=0.9146,r~2=0.3194),and two SNPs in Nmi gene with lower Linkage disequilibrium(D’=0.3303,r~2=0.063).