Miller Andrew N, Karakehian Jason, Raudabaugh Daniel B
Illinois Natural History Survey, University of Illinois at Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820, USA.
Department of Plant Biology, University of Illinois at Urbana-Champaign, 505 South Avenue, Urbana, IL 61801, USA.
J Fungi (Basel). 2022 Sep 2;8(9):932. doi: 10.3390/jof8090932.
Fungaria are an unmatched resource for providing genetic data from authoritative, taxonomically-correct fungal species, especially type specimens. These specimens serve to anchor species hypotheses by enabling the correct taxonomic placement of taxa in systematic studies. The DNA from ancient specimens older than 30 years is commonly fragmented, and sometimes highly contaminated by exogenous, non-target fungal DNA, making conventional PCR amplification and Sanger sequencing difficult or impossible. Here, we present the results of DNA extraction, PCR amplification of the ITS2 region, and Illumina MiSeq Nano sequencing of nine recent and 11 ancient specimens, including seven type specimens. The taxa sampled included a range of large and fleshy, to small and tough, or small, melanized specimens of , , , , and , with a culture of serving as a positive control. DNA was highly fragmented and in very low quantity for most samples, resulting in inconclusive or incorrect results for all but five samples. Taxonomically-correct sequences were generated from the holotype specimens of , , and , from the neotype of , and from the positive control. Taxonomic assignments were confirmed through morphology, top BLASTn hits, and maximum likelihood phylogenetic analyses. Though this study was not cost-effective due to the small number of samples submitted and few generating correct sequences, it did produce short DNA barcode fragments for four type specimens that are essential for their correct taxonomic placement in our ongoing systematic studies.
真菌标本是获取来自权威的、分类正确的真菌物种，特别是模式标本的遗传数据的无与伦比的资源。这些标本通过在系统研究中实现分类单元的正确分类定位，为物种假说提供支撑。超过30年的古老标本的DNA通常会碎片化，有时还会受到外源非目标真菌DNA的高度污染，使得传统的PCR扩增和桑格测序变得困难甚至无法进行。在这里，我们展示了对9个近期标本和11个古老标本（包括7个模式标本）进行DNA提取、ITS2区域的PCR扩增以及Illumina MiSeq Nano测序的结果。所采样的分类单元包括一系列大型肉质、小型坚韧或小型黑化的标本，分别属于、、、、和，其中一种的培养物用作阳性对照。大多数样本的DNA高度碎片化且数量极少，除了5个样本外，其余样本均得到不确定或错误的结果。从、和的模式标本、的新模式标本以及阳性对照中生成了分类正确的序列。通过形态学、顶级BLASTn比对结果和最大似然系统发育分析确认了分类归属。尽管由于提交的样本数量少且产生正确序列的样本少，本研究不具有成本效益，但它确实为4个模式标本生成了短DNA条形码片段，这些片段对于在我们正在进行的系统研究中正确分类定位它们至关重要。
