利用微卫星多态性鉴别不同马品种试验

Test on Identifying Different Horse Breeds with Microsatellite Polymorphism

【目的】选取3个马匹品种(柯尔克孜马、俄罗斯速步马、纯血马),对其5个微卫星DNA座位AHT4、ASB2、HMS3、HMS7、LEX3进行多态性分析,利用SPASS分析软件,对3个马品种进行初步鉴别。【方法】以柯尔克孜马、俄罗斯速步马、纯血马为实验对象,分别采集血样10 mL,枸橼酸钠抗凝,实验室中对所选取的5个微卫星DNA座位进行非变性丙烯酰胺电泳(PAGE)检测,用Popgene32分析数据。比较品种间有效等位基因数(Ne)、等位基因频数分布间的差异,辅以各品种特有的基因型条带对三个马品种进行初步鉴别。【结果】对5个微卫星标记的有效等位基因数(Ne)数值进行分析,发现纯血马与俄罗斯速步马、柯尔克孜马与俄罗斯速步马呈显著差异关系(P<0.05),成为初步鉴别三个马品种的重要依据;对5个微卫星标记进行非变性丙烯酰胺电泳(PAGE)检测,发现ASB2微卫星标记的基因型中,柯尔克孜马有区别于其他两个品种马的基因型:CC、CD、EG、EH基因型,纯血马所特有的基因型为BI、JJ、KK基因型;AHT4微卫星标记的等位基因型中,柯尔克孜马所特有的基因型为DF、EF、GG基因型,纯血马所特有的基因型为BC基因型;HMS3微卫星标记的等位基因型中,纯血马所特有的基因型为CC基因型;HMS7微卫星标记的等位基因型中,纯血马所特有的基因型为AA基因型。【结论】3个马品种的鉴别可以通过筛选出的微卫星位点的有效等位基因数(Ne),以及所选微卫星标记的等位基因,辅以各品种特异的基因型电泳条带,从而达到初步鉴别三个马匹品种的目的。

[ Objective]The purpose of this project is to conduct preliminary identification of the 3 horse breeds by choosing the three horses breeds （Kirghiz horse, Russian trotter, thoroughbred analysis of the polymorphism of 5 microsatellite DNA markers （ AHT4, ASB2, HMS3, horse） through the HMS7 and LEX3 ） with the SPASS analysis software. [ Method] Kirghiz horse, Russian trotter, thoroughbred horse were taken as experimental subjects, from which blood samples （10 ml） were collected to anticoagulant with sodium citrate. The polymorphism of 5 microsatellite DNA makers went through PAGE. Popgene 32 computer software was used to analyze the data. The difference of Ne and allele frequency distribution among three breeds was com- pared, and preliminary identification of the three horse breeds was conducted supplemented with the unique zenotvoe bar for each breed. [ Result ]Through the analysis the Ne of 5 microsatellite makers, it was found that： there were significant difference between Thoroughbred, Russian trotter and Kirghiz horse （ P 〈 0.05）, which was a important basis for the preliminary identification of three horse breeds; 5 microsatellite markers were tested with PAGE, which showed that if the genotype was marked with ASB2 microsatellite markers, there were specific genotype CC, CD, EG, EH in the Kirgiz horse, specific genotype BI, JJ, KK in the thor- oughbred horses; If the genotype was marked with AHT4 microsatellite markers, there were specific genotype DF, EF, GG in the Kirgiz horses, there were specific genotype BC in the thoroughbred horses. If the genotype was marked with HMS3 microsatellite markers, there was specific genotype CC in the thoroughbred horses. If the genotype was marked with HMS7 microsatellite markers, there were specific genotype AA in the thorough- bred horses. [ Conclusion ] The identification of 3 horse breeds can be screened by the effective number of al- leles （Ne）, the distribution of microsatellite marker allele frequency, supplemented with unique genotype bar for each breed to conduct preliminary identification of the three horse breeds.