毛红椿AFLP体系优化及引物筛选

Optimization verification and primer selection of AFLP reaction system of Toona ciliata var.pubescens

以江西九连山国家级自然保护区毛红椿天然林为研究材料,对毛红椿AFLP反应体系中的DNA提取方法、酶切与连接步骤、连接产物稀释倍数、预扩增产物稀释倍数等影响因子进行优化,并筛选AFLP多态性高的引物。结果表明:不同提取方法对DNA质量有很大影响,试剂盒法提取的DNA杂质少,质量高;酶切与连接采用一步法完成;连接产物最适稀释倍数为3倍,预扩增产物最适稀释为10倍;利用4个天然居群的毛红椿基因组DNA从64对引物组合中筛选出4对具有多态性扩增条带的引物,总扩增出147个位点,其中多态性位点122个,平均多态位点百分率84.36%,多态性较高,适用于毛红椿AFLP分析的4对引物组合分别是:E1/M6、E4/M7、E5/M3、E5/M8,为毛红椿遗传多样性和种质资源研究奠定了基础。

The leaves of the natural forest of Toona ciliata var.pubescens in Jiulianshan National Nature Reserve in Jiangxi Province were used as experimental materials.Study on influencing factors of AFLP reaction system of T.ciliata var.pubescens,from DNA extraction methods,enzyme digestion and ligation steps,ligation product dilution multiples,and pre-amplification product dilution multiples to study,establish T.ciliata var.pubescens AFLP system,and screen out polymorphic primer combinations.The results showed that different genome extraction methods have a great impact on DNA quality,and the DNA extracted by the DP305 kit method has less impurities and high quality;And the results of one-step and two-step methods were no different for enzyme digestion and linkage,so one-step method was chosen as the follow-up step;The optimal dilution of the ligation product was 3 times,and the optimal dilution of the pre-amplification product was 10 times;Four pairs of primers with polymorphic amplification bands were screened from 64 pairs of primer combinations by using the genomic DNA of 4 natural populations of T.ciliata var.pubescens,and a total of 147 amplified bands were obtained,the polymorphism sites was 122,the percentage of polymorphic loci was 84.36%.According to the established AFLP reaction system of T.ciliata var.pubescens,four pairs of polymorphic primers(E1/M6,E4/M7,E5/M3,E5/M8)were screened to provide support for the subsequent studies on the genetic diversity and breeding system of T.ciliata var.pubescens.