Záveská Drábková Lenka
Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic.
Methods Mol Biol. 2021;2222:69-88. doi: 10.1007/978-1-0716-0997-2_4.
With the expansion of molecular techniques, the historical collections have become widely used. The last boom started with using next- and second-generation sequencing in which massive parallel sequencing replaced targeted sequencing and third-generation technology involves single molecule technology. Studying plant DNA using these modern molecular techniques plays an important role in understanding evolutionary relationships, identification through DNA barcoding, conservation status, and many other aspects of plant biology. Enormous herbarium collections are an important source of material especially for taxonomic long-standing issues, specimens from areas difficult to access or from taxa that are now extinct. The ability to utilize these specimens greatly enhances the research. However, the process of extracting DNA from herbarium specimens is often fraught with difficulty related to such variables as plant chemistry, drying method of the specimen, and chemical treatment of the specimen. The result of these applications is often fragmented DNA. The reason new sequencing approaches have been so successful is that the template DNA needs to be fragmented for proper library building, and herbarium DNA is exactly that. Although many methods have been developed for extraction of DNA from herbarium specimens, the most frequently used are modified CTAB and DNeasy Plant Mini Kit protocols. Nine selected protocols in this chapter have been successfully used for high-quality DNA extraction from different kinds of plant herbarium tissues. These methods differ primarily with respect to their requirements for input material (from algae to vascular plants), type of the plant tissue (leaves with incrustations, sclerenchyma strands, mucilaginous tissues, needles, seeds), and further possible applications (PCR-based methods, microsatellites, AFLP or next-generation sequencing).
随着分子技术的发展,历史标本收藏得到了广泛应用。上一轮热潮始于使用二代和新一代测序技术,其中大规模平行测序取代了靶向测序,而第三代技术涉及单分子技术。利用这些现代分子技术研究植物DNA在理解进化关系、通过DNA条形码进行鉴定、保护状况以及植物生物学的许多其他方面都发挥着重要作用。大量的植物标本馆收藏是重要的材料来源,特别是对于分类学中长期存在的问题、来自难以进入地区的标本或现已灭绝的分类群。利用这些标本的能力极大地促进了研究。然而,从植物标本中提取DNA的过程往往充满困难,这些困难与植物化学、标本干燥方法和标本化学处理等变量有关。这些应用的结果往往是碎片化的DNA。新的测序方法如此成功的原因是,为了构建合适的文库,模板DNA需要被碎片化,而植物标本馆的DNA正是如此。尽管已经开发了许多从植物标本中提取DNA的方法,但最常用的是改良的CTAB法和DNeasy植物微量提取试剂盒方案。本章中选择的九种方案已成功用于从不同种类的植物标本组织中提取高质量的DNA。这些方法的主要区别在于它们对输入材料(从藻类到维管植物)的要求、植物组织的类型(有结壳的叶子、厚壁组织束、粘液组织、针叶、种子)以及进一步可能应用的方面(基于PCR的方法、微卫星、AFLP或新一代测序)。