According to our previous research, there was an obvious relationship between the early death of chicken(Gallus gallus)-quail(Cotumix cotunux) hybrids and sex differentiation. Meanwhile, current widespread adapted methods to indentify sex differentiation stayed at RNA level, experimental steps complicated and easy to make mistakes, and RNA samples, which are needed to be measured, was quite difficult to preserved longer, but could not be simply employed. So it is necessary to find a more simple and accurate way to identify the early embryo's sexing. In this study, there were two sections for CHD (chromobox-helicase-DNA binding) gene DNA level sex determination, at first,a total of 116 chicken-quail hybrid embryos at different incubate stages (2.5~5 d) was treated as experiment group and 10 mg embryonic organs were used to DNA extraction; then the DNA extraction from blood of 60 sex-known quails (male and female were half-and-half)rnwas regarded as control. Wpkci (W-linked protein kinase C inhibitor) for mRNA level was known as a maturernmethod to identify sexing, and then it is used to check the result of embryos' sex determination in our research.rnThe result showed that CHD 2550F/2718R could identify the sex of chicken-quail hybrid embryos accurately.rnThe amplication size of male embryo tissues was 613 bp and two bands in female were 613 and 446 bp,rnrespectively. The experimental results provide basic data for the chicken-quail hybrids sex determinationrnmechanism.%鸡(Gallus gallus)与鹌鹑(Coturnix coturnix)属间杂交胚胎早期死亡与性别分化存在着一定的关系,寻找简单、准确的早期胚胎性别鉴定方法是深入研究其死亡分子机制的前提.本实验室前期使用Wpkci引物从mRNA水平对早期胚胎进行准确的性别鉴定,而RNA提取对样品质量要求较高,鉴定程序较复杂.因此需要建立更加简单快捷的方法,对鸡与鹌鹑属间杂交早期胚胎进行准确性别鉴定.本研究采集孵化不同时期(2.5～5 d)杂交活胚116枚,以10 mg胚胎组织DNA为模板(3 d时,约占整个胚胎的1/20),依据CHD(chromobox-helicase-DNA binding)基因保守区设计引物2550F/2718R进行PCR反应；用相同胚胎组织提取的RNA为模板,用Wpkci(W-linked protein kinase C inhibitor)基因设计为特异性引物,对杂交早期胚胎进行RNA水平的验证.结果表明,2550F/2718R引物可以准确鉴定杂交胚胎的性别,雄性胚胎组织中扩增出1条约613 bp片段,雌性胚胎组织中扩增出2条分别为613和446 bp的片段,与预期结果一致.本研究为鸡与鹌鹑属间杂交种性别决定机制的研究提供了基础研究资料.
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