Using The DGE Technology Screening Chicken×quail Hybrid Embryos Early Death Related Genes

Hybridization between chicken and quail are very typical case of distanthybridization between their female offspring appears a lot of early embryonic death,and are concentrated in the first3-5days of incubation, the time period exactlycoincide with their sex differentiation, Studies have shown that gender differentiationassociated gene there is a certain correlation between their deaths, the regulatorymechanism is more complex and gender differentiation genes involved in very many,through the establishment of digital expression profiling, screening associated withsex differentiation genes specifically expressed in the period for better researchchicken and quail hybrids mechanism to lay the foundation of early embryonicdevelopment and female sex differentiation of embryonic death in.Therefore, this study chickens, quail and their offspring early embryodevelopment as test material, its accurate sex identification by morphological andmolecular biological methods, using Illumina sequencing technology to the first threedays of hatching chickens, quail, hybrid embryos for high-throughput sequencing,male and female were constructed ontogeny first three days of six DGE libraries,screening differentially expressed genes between them, analyze their expressionsequencing results are consistent with trends in, and be associated with sexualdifferentiation gene analysis, which is designed to contrast with the chicken and quailamong offspring of early embryonic death-existing relationship, the specific contentsand results are as follows:(1) Collecting chickens, quail and hybrid hatch3-5days (every12h sampling)embryos, developmental morphology was observed by optical microscopy of eachperiod. The results showed that hybrid embryos morphological changes during theintervening parent of each of the chicken and female quail, morphologicaldevelopment of each period are consistent with previous studies, and the chickens,quail and hybrid embryos on day3of the form features are basically the same. At thesame time, an unknown gender chicken and quail hybrid embryos for the study, takingadult quail blood DNA as a control, the amplified with specific primers quail helixprotein gene on chromosome W chromosome (CHD) conserved regions identifiedgender as a control the results showed that incubation of early CHD gene method foraccurate sexing embryos was100%, can be applied to the early embryo sexing poultry,while ensuring the accuracy of the study sample.(2) in morphology and the level of DNA amplification accurate genderidentification of CHD gene as the foundation, through the Illumina sequencing platform to hatch chickens, quails,3days hybrid embryos for high-throughputsequencing, build the female and male respectively ontogenesis3days6DGE library,screening of differentially expressed genes between them, and on the GO forstatistical analysis and KEGG pathways, etc.Results show that the male and female,male and female quail, male and female chicken embryos than in hybrid birds toreference genome sequences respectively2840108,2817776,1343632,1279590,1844727and1824082;Male and female chickens, male and female quail, sexualhybridization birds, hen and female female hybrid birds, quail and female hybrid birdsthere were66differentially expressed genes,1340,72,130and1340, differentiallyexpressed genes related to gender differentiation respectively4,2,3,9and8, containsHINTW, HINT1, cFOXL2gene, etc.Randomly selected six differentially expressedgenes in real-time fluorescent quantitative PCR (Real-time PCR), the expressiontrend consistent with the results of sequencing, prove the authenticity of thesequencing results.And then GO on them and KEGG pathway analysis showed thatthe main differentially expressed genes involved in Wnt, p53, Osteoclast, PPAR, TGF-β and MAPK, growth and development, reproduction, cell proliferationdifferentiation related signaling pathway.With differentially expressed genes related togender differentiation analysis found that the genes involved in sex differentiationprocess not only, and are involved in cell cycle and differentiation process, combinedwith previous studies, suggest that chicken and quail hybrid offspring early embryodeath and the female gender differentiation related genes expressed in cell cycle anddifferentiation process related to the disorder.To sum up, using Illumina sequencing platform to hatch chickens, quails,3dayshybrid embryos for high-throughput sequencing, build the female and malerespectively ontogenesis3days6DGE library, screening of differentially expressedgenes between them, and the random screening differentially expressed genes of Real-time PCR test, consistent with the results of sequencing, combined with the GO andKEGG pathway analysis, find the differentially expressed genes related to genderdifferentiation (HINTW, HINT1, etc.), for further research on chicken（♂） quail (♀)hybrid offspring early embryo death and gender differentiation to provide theoreticalfoundation and basis.