Janik Paulina, Ronikier Michał, Ronikier Anna
W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków, Poland.
PeerJ. 2020 Jan 24;8:e8406. doi: 10.7717/peerj.8406. eCollection 2020.
Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.
植物标本馆的馆藏为广泛的生物学研究提供了重要基础，随着基于DNA方法的发展，它们已成为遗传分析的宝贵材料。然而，此类材料的使用受到与DNA降解和生物材料数量相关的技术限制。对于某些生物类群来说，生物材料数量有限是其固有特点，黏菌就是最好的例证，它们形成微小的子实体。据估计，约三分之二的黏菌分类单元所代表的材料极其稀少。由于目前在黏菌研究中应用的DNA分离方法需要对多个子实体进行破坏性采样，该类群中很大一部分已描述的多样性仍无法用于系统发育研究或条形码分析。在此，我们测试了几种DNA分离和扩增程序，以寻找一种高效且可能非破坏性的采样方法。测试基于代表不同分类目19个物种的植物标本馆标本。我们检测了基于硅胶膜柱的几种分离变体，以及一种新设计的程序，该程序使用极少量的生物材料（一小部分孢子），基于孢子的精细破碎和直接PCR。虽然最常用的基于柱的方法在使用大量材料时，89.5%的样本PCR成功，但基于单个子实体时，其成功率降至52%。使用专门用于少量DNA样本的试剂盒时，单个子实体在89.5%的样本中提供了扩增子。我们的新程序似乎最有效（94.7%），同时它仅使用了一小部分孢子，几乎是非破坏性的；它也是最具成本效益的。因此，我们证明，对孢子进行适当的微破碎处理并结合直接PCR的应用，可以成为克服黏菌遗传研究技术限制的有效方法，从而可以显著改善系统发育和条形码分析的分类单元采样。此外，这种方法提供了一种独特的可能性，即对同一结构（子实体）同时应用分子和形态学分析，然后该结构可进一步作为文献保存。
