卷丹及主要食用百合EST-SSR鉴别体系的构建

目的开发卷丹Lilium lancifolium、兰州百合L. davidii var. willmottia、岷江百合L. regale、香水百合L. casa和龙牙百合L. brownie var. viridulum的EST-SSR分子鉴别体系,分析EST-SSR分子标记技术对百合近缘种的开发效率和鉴别能力。方法利用MISA.pl程序对NCBI公布的百合属EST基因序列进行SSR位点的识别,通过Primer3程序模块批量生成EST-SSR引物,经PCR扩增和琼脂糖凝胶电泳对引物进行初筛,用聚丙烯酰胺凝胶电泳验证初筛引物,标记并分析不同种质的特征泳带,构建鉴别体系。结果共设计了199对SSR引物,经过26对引物的筛选,获得了2对高效鉴别引物,其中引物JZ391构建的分子鉴别体系,可有效鉴别混掺的商品百合,具有一定的实用价值。结论基于研究材料具有极端的遗传特征,本实验鉴定标记开发非常高效,研究结果证实了物种的遗传基础是影响其EST-SSR分子标记开发效率的重要因素,同时,该案例可为其他类似百合遗传基础的中药材进行EST-SSR标记开发,提供参考尺度。

Construction of EST-SSR identification system for Lilium lancifolium and main edible Lilium

Objective To develop the EST-SSR molecular identification system of Lilium lancifolium, Lilium davidii var. willmottiae, Lilium regale, Lilium casa blanca and Lilium brownie var. viridulum, and analyze the development efficiency and identification ability of EST-SSR molecular marker technology for Lilium genus. Methods The MISA.pl program was used to identify the SSR locus of the EST gene sequence published by NCBI. The EST-SSR primers were generated by Primer3 program module, and the primers were screened by PCR amplification and agarose gel electrophoresis. The primary screening primers were verified by polyacrylamide gel electrophoresis, and the characteristic bands of different germplasms were labeled and analyzed to construct an identification system. Results A total of 199 pairs of SSR primers were designed. After screening 26 pairs of primers, two pairs of highly efficient primers were obtained. The molecular identification system constructed by primer JZ391 can effectively identify the mixed commercial materials, which had certain practical value. Conclusion Based on the extreme genetic characteristics of the research materials, this identification marker development is very efficient. The results confirm that the genetic basis of the species is an important factor affecting the development efficiency of its EST-SSR molecular marker. At the same time, this case can be used as a reference for the development of EST-SSR markers for other Chinese medicinal herbs similar to Lilium genus.