| Hybridization between chicken and quail are more typical distant hybridization, the high mortality of early female hybrid embryos are concentrated in the3-5days of incubation.It is accordingly with the time for gender differentiation. Studies have shown that multiple gene regulation mechanism of sexual differentiation are related to their death, While researches on ncRNA can find out much more genes that involved in the regulation from a number of ways, and therefore it lays foundation for better studies on the mechanism between death of early female embryonic and sex differentiation.So in this study, embryonic at early stage of chickens, quail, and their hybrids were used as experimental material, after accurately sex identification were carried on for combining the morphological observation with molecular biology method, Illumina Solexa sequencing technology to detecting differential expression miRNAs at incubation for three days’male and female embryos, and selected part of differentially expressed miRNAs to analyze the expression trends with sequencing results, and analyzed the alternative splicing form of genes that possiblely regulated sex differentiation, the aim of this study is to compare the relationship between early embryonic death with the chicken-quail hybrids. The experimental results are as follows:(1)Chicken, quail and its hybrid embryos that incubated for3-5days (sampling every12h) were collected to morphology observation for using the stereo microscope. The morphologic changing of hybrid embryos were between chicken and quail embryos, and the morphological characteristics at third day of incubation of the three species are almost the same; wing vein blood sampling for quails were used for extraction DNA, unknown gender hybrid embryos were used as experimental subjects, with the reference to quail W chromosome conserved region of CHD gene primers2550F/2718R, at the same time Wpkci gene was furtherly verify the reliability of the sex identification at mRNA level. The results showed that accuracy of CHD2550F/2718R in early embryo sexing was100%, so it can be applied to the poultry early embryonic sex identification and to ensure the accuracy sample in this study.(2)Based on the morphological characteristics and CHD gene for sex identification, Illumina sequencing method for Small RNA deep sequencing on male and female hybrid embryos incubation of third day, combining with bioinformatic means to analysis type, length of abundance, as well as participating GO analysis and KEGG pathways of known and predicted miRNAs in the male and female embryos. The result of Solexa sequencing provided a total of16,058,009and17,943,294reads of3nt-30nt from the female and male embryo tissue libraries and their length is22nt primarily.In both two libraries,there are significant differences for117sequences(P<0.01). Randomly selected differentially expressed four known miRNAs (miR-202*, miR-202, miR-1579and miR-31) and three candidate miRNAs (novel-miR-227, novel-miR-348, and the novel-miR-439)for Real-time PCR showed that the expression trend consistent with the sequencing results,it proved the authenticity of the sequencing results.After target gene prediction, GO analysis and KEGG pathway analysis for part of miRNAs, showed that these target genes of differential miRNAs involved in the Apoptosis signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway and other pathways that associated with embryonic development. There are also some sex differnetiation target genes that have been reported, such as DMRT1, FET1and SOX9, and so on. It is suggested that these differentially expressed miRNA target genes may be involved in the chicken and quail hybrid early embryonic sex differentiation occurred.(3)According with the reported DMRTlb transcription designed specific primers, quail, chicken, and their hybrids of early embryonic tissue were used to extracted RNA,than reverse transcription to cDNA as a template, PCR amplified gained two target bands, found after their recovery sequencingthe fragment size of the two amplification products of547bp and105bp.Compared with the original sequence of915bp are deficiencies,its functions in gender differentiation need further analysis. In summary, our study accomplished the constructed small RNA libraries for the hatching of the first three days of chicken and quail hybrids female and male embryos for using Illumina sequencing technology.Random screening differentially expressed miRNAs through quantitative PCR validation, consistent with the Solexa sequencing results.Combined with GO and KEGG pathway analysis of differentially expressed miRNAs and their target genes (DMRT1, SOX9, et al.) involved in regulatory pathways, may impact on sexual differentiation between the male and female embryos of the hybrid; the DMRT1gene of PCR amplification foundthere are two different spliceosome, the result provide a theoretical basis and foundation for in-depth experimental chicken(♂)×quail (♀) hybrids early embryonic death and sex differentiation. |
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