利用SSR标记分析一年生黑麦草遗传多样性的取样策略

一年生黑麦草(Lolium multiflorum)为异花授粉植物，其DNA多态性研究的取样策略与遗传多样性分析的可靠性和效率直接相关。从一年生黑麦草国家审定品种“长江2号”和“特高”中，分别提取50个单株DNA，同时提取5、10、15、20、25和30个单株叶片混合样本的DNA，利用SSR标记进行遗传多态性分析。研究结果表明,取样梯度间多态性信息含量指数(PIC)差异不显著(P>0.05)，但当混合单株样本为20株时，PIC达到最高值；SSR平均等位基因数和单个SSR座位上等位基因种类随样品内单株混合数目增加而增加，当取样量在20株及以上时，等位基因数和等位基因种类趋于稳定，电泳图谱表现基本一致；不考虑稀有等位基因（基因频率≤10%）的漏检，采用20株混合样本能够最大限度保持检出较高频率（＞10%）等位基因，且重演性较好。鉴于此，利用SSR标记分析一年生黑麦草遗传多样性时采用20个单株混合样提取的DNA样本能够有效反映群体间差异。

Bulking strategies for genetic diversity analysis of annual ryegrass based on SSR markers

Since Annual ryegrass (Lolium multiflorum) belongs to the typical cross pollinated species, the sampling strategies are closely related to the authenticity and the efficiency of molecular genetic diversity analysis. Two Annual ryegrass varieties, Changjiang No.2 and Tetragold were selected and their bulked DNA samples obtained from 5, 10, 15, 20, 25 and 30 individual plants respectively. The result of genetic diversity analysis based on simple sequence repeat（SSR）markers showed that there were no differences in the PIC index among seven sampling methods, PIC reached the highest when bulked plant sample were 20. The number of average allele and the alleles, types of per SSR loc increased along with the plant number in the bulked plant samples increasing, which kept relatively steady when bulked plant samples surpassed 20 (including 20) plants, meanwhile the SSR banding patterns were relatively uniform. The bulked samples 20 were able to detect the most high frequency (>10%) alleles with excellent reproducibility, if disregard the undetection of rare alleles (≤10%). It could be concluded that 20 individual plants was the suitable sampling size in genetic diversity analysis of annual ryegrass based on SSR markers.