Subtracted Diversity Array identifies novel molecular markers including retrotransposons for fingerprinting Echinacea species

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationships within the species of this genus are unclear, which makes difficult the authentication of the species used for the medicinal industry. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes, including four that were not used in the array construction, could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic sequences. Five out of the eleven most discriminatory features matched to known retrotransposons.

紫锥菊属（Echinacea）原产于加拿大草原及美国草原各州，北美原住民将其作为民间药物已有悠久历史。目前，紫锥菊属植物因治疗普通感冒的应用日益广泛，且具备刺激免疫系统的功效，跻身美国与欧洲销量前十的草药之列。然而，该属物种间的遗传关系尚不明确，这给药用产业所用物种的鉴定带来了困难。本研究报道了一种针对紫锥菊属物种的新型消减多样性阵列（Subtracted Diversity Array, SDA）的构建方法，并证实了该阵列用于分离高度多态性序列的潜力。为选择性分离紫锥菊特异性序列，研究以24份紫锥菊基因型混合池为试验组，以其他被子植物与非被子植物的混合池为对照组，开展了抑制性消减杂交（Suppression Subtractive Hybridization, SSH）实验。共扩增得到283个消减基因组DNA（genomic DNA, gDNA）片段并进行阵列点样。研究成功区分了27份紫锥菊基因型，其中包含4份未参与阵列构建的材料。值得注意的是，通过聚类分析可对未知的包舌紫锥菊（E. paradoxa）与紫锥菊（E. purpurea）样本进行明确鉴定。此外，该紫锥菊特异性SDA同样可用于分离高度多态性序列。在11个最具区分能力的特征中，有5个与已知的逆转录转座子（retrotransposons）相匹配。