基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

Identification of Atractylodes chinensis and A.japonica Based on SCAR Molecular Markers and DNA Barcoding Technology

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

Objective To develop a molecular marker method that can identify Atractylodes chinensis(Bunge) Koidz and A. japonica Koidz.ex Kitam. and to investigate the genetic evolution of Atractylodes from different germplasm resources.Methods The genomic DNA fragments of Atractylodes chinensis(Bunge) Koidz and A. japonica Koidz.ex Kitam from different regions were sequenced, combined with SRAP, ISSR and DAMD molecular markers to optimize the PCR reaction system, screened and converted into specific markers. At the same time, interspecific sequence differences were analyzed by barcode method. Results By PCR amplification with SRAP, ISSR and DAMD, a total of 198 pairs of primers with stable amplification and good reproducibility were screened out, and 7 pairs of SCAR primers were converted to identify Atractylodes chinensis and A. japonica. The ITS2 sequences were 454 bp in length for Atractylodes chinensis and 453 bp in length for A. japonica, indicating that they were genetically distant from other Atractylodes spp.The NJ tree results showed that Atractylodes chinensis, Atractylodes japonica and other Atractylodes spp. were all clustered into a single species, showing good monophyly. Based on the ITS2 secondary structure, the number, size and position of stem loops in the helical region of the four Atractylodes spp. differed significantly and could be distinguished visually.Conclusion The developed specific SCAR marker provides a new method for screening excellent varieties of Atractylodes.The ITS2 sequence can be used as a DNA barcode primer to identify Atractylodes chinensis stably and accurately.