Using the SSR with fluorescent labeling to establish SSR molecular ID code for cultivars of the Chinese herbaceous peony
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摘要: 为了开发芍药品种分子鉴定方法,本研究采用SSR标记技术,在目前芍药SSR引物开发较少的情况下,采用文献相关引物84对,并结合自行设计的27对引物,将6%非变性聚丙烯酰胺凝胶与四重荧光毛细管电泳技术相结合,从111对引物中筛选出29对扩增稳定、多态性高的核心引物;共检测到223个SSR等位位点,平均每对引物为7.69个,有效等位基因数(Ne)为3.658±0.328,Shannon多样性指数(I)为1.371±0.104,观测杂合度(Ho)为0.532±0.045,期望杂合度(He)为0.645±0.036,多态性信息含量PIC值介于0.20~0.84,平均0.60。基于最少引物鉴定最多种质的原则,按照PIC值由高到低顺序串联排序的方式,最终筛选出4对SSR引物(Pmg153、Knu73、Pmg209、J38)。结果表明:筛选出的4对SSR引物组合可以有效鉴定66个芍药品种;采用个位数字和大写英文字母结合对不同带型进行编码,成功绘制了66个中国传统芍药品种的分子身份证。本研究为芍药品种鉴定提供了一种快速、准确与高效的分子检测方法,也有助于中国传统芍药品种的遗传改良与芍药新品种保护。Abstract: Herbaceous peony(Paeonia lactiflora Pall.)is a perennial herbaceous plant which belongs to Sect. Paeonia in family Paeoniaceae. It is a traditional flower with 3000-year history and has hundreds of cultivars in China. However, it is difficult to distinguish cultivars accurately only by the traditional morphological identification method. Establishing the multi-level molecular identification techniques can improve the accuracy of cultivar identification. In order to develop a molecular identification method,we used the SSR technique to establish the molecular ID code of 66 cultivars of Chinese traditional herbaceous peony. In this research, 111 pairs of SSR primers with 84 from literatures and 27 self-designed, were labeled with 6% none-denaturing polyacrylamide gel electrophoresis and quadruple fluorescence capillary electrophoresis. Then 29 pairs of primers with high polymorphism and good repeatability were selected. The results showed that 223 polymorphic alleles were revealed by the 29 primer pairs,with an average of 7.69 alleles per pair. The effective number of alleles (Ne) was 3.658±0.328, Shannon diversity index (I) was 1.371±0.104, observed heterozygosity (Ho) was 0.532±0.045, expected heterozygosity (He) was 0.645±0.036, and the range of PIC at 29 SSR loci of 66 cultivars was 0.20-0.84, 0.60 on average. Based on the principle of distinguishing the most varieties using the minimum pairs of primers, 4 pairs of primers (Pmg153, Knu73, Pmg209, J38) were selected out. This study indicates that the 4 pairs of primers can identify the 66 varieties of Chinese traditional herbaceous peony and can successfully establish 66 molecular ID codes by different single digits and uppercase letters. It not only provides a rapid, accurate and efficient molecular identification method for herbaceous peony cultivars, but also contributes to genetic improvement of cultivars of Chinese traditional herbaceous peony and is significant for protection of the new varieties.
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