基于性状特征和ISSR分子标记的三棱居群研究

基于药材性状特征和ISSR分子标记技术对7个地区的黑三棱科植物黑三棱进行测量比较及遗传多样性分析,针对湖南三棱建立分子鉴别方法。通过测量各产地三棱药材的质量、长度、宽度和厚度指标,分析三棱药材产地间差异。通过筛选扩增条带清晰、多态性丰富的ISSR引物,构建各产地三棱聚类树状图,分析不同产地三棱遗传多样性,筛选出目标引物并测序特异性片段。从ISSR引物中筛选出9条扩增条带清晰、多态性丰富的引物,扩增出73条清晰的谱带,平均每个引物扩增出8.0条,其中多态性条带38条,多态性比例为52.8%。聚类树状图显示样品间遗传相似系数为0.54~0.94,其中浙赣皖地区的三棱之间的遗传相似性系数较大,差异较小。湖南三棱药材性状特征相比其他产地三棱具有较明显差异,且遗传距离相对较远。筛选获得湖南三棱中扩增出特异性条带(327 bp)的引物ISSR-857,并对扩增得到的特异性片段进行测序,经BLASTn比对后,与该基因片段相似的序列较少,且与植物性状生长过程相关性不大。ISSR分子标记技术为湖南三棱的鉴别方法提供了新思路和方法参考,也佐证了"荆三棱"的产地特殊性。

Study on Sparganium stoloniferum from different regions in China based on appearance characters and ISSR markers

Based on the characteristics and ISSR molecular marker technology, the study is aimed to compare and perform genetic diversity analysis on Sparganium stoloniferum from 7 regions. Molecular identification method was established for S. stoloniferum from Hunan province. Differences among Sparganii Rhizoma samples from seven habitats were analyzed via measuring weight, length, width and thickness of them. Genetic diversity of S. stoloniferum from 7 regions was analyzed by screening out primers amplifying clear band and showing rich polymorphism, then a cultivars dendrogram was built. The target primer was screened out, and the specific band was sequenced. Nine ISSR primers were selected to amplified clear band, rich polymorphism. A total of 73 bands were amplified by nine ISSR primers selected from 27 ISSR primers. On average, each primer produced 8.0 bands. A total of 38 bands were polymorphic, which occupied 52.8% of all bands. The cultivars dendrogram showed the genetic similarity was 0.54-0.94. Genetic similarity coefficient of S. stoloniferum from Jiangsu province, Anhui province and Jiangxi province was big, indicating the differences among them were slight on genetic level. S. stoloniferum from Hunan province is quite different from samples from the other six habitats on appea-rance and genetic level. A specific band(327 bp) in S. stoloniferum from Hunan province was obtained via ISSR-857 primer, and was sequenced. According BLASTn database, there were few sequences similar to the gene fragment and had little correlation with the growth process of plant. ISSR molecular marker technology provides a new idea for the identification of S. stoloniferum. This result confirmed the particularity of S. stoloniferum from ancient Jingzhou.