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Abstract：
Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method\'s potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.
摘要：
分子序列数据已经改变了密码的研究(例如，地衣,微藻,真菌,及其共生体），但方法仍然受到目标生物的小尺寸和混合生长的强烈阻碍，其次级代谢产物的可培养性差和有害作用。这里,我们的目标是展示一些例子，在这些例子上，改进了直接PCR方法，用于环境样品中涉及生物锈病的分子工作的各个方面，生物膜，和密码为研究界提供了新的选择。与传统方法不同，这种方法只需要相当于直径0.2毫米的菌落和碎片的生物量，可以直接从环境样本中提取，并且包括快速DNA裂解和随后的标准化PCR循环,其允许在同一运行中各种生物体/靶区域的共循环。我们证明了这种改进的方法可以（i）从自由生活的蓝细菌的单个菌落和细丝中扩增最广泛使用的分类学基因区域以及用于应用和环境科学的基因区域，苔藓植物,真菌,还有地衣,包括他们的真菌，绿藻,以及在共循环过程中来自分离物和原位材料的蓝藻；（ii）充当工具，以确认从原始样品中分离出主要的地衣光生物；（iii）任选地去除抑制性次级地衣物质。我们的结果代表了一些例子，这些例子突出了该方法对涵盖真菌学的未来应用的潜力，植物学，生物锈蚀，和地衣学，特别是。IMPORTANCECyanobacteria,绿藻,地衣,和其他隐藻在复杂的微生物系统中起着至关重要的作用，例如干旱生物群落的生物土壤结皮或洞穴中的生物膜。对这些微生物的环境样品或分离物的分子研究通常因它们的密集聚集而受到阻碍。小尺寸,或使DNA提取和随后的PCR复杂化的代谢产物。我们的工作提供了依赖低生物量插入物的直接DNA提取和PCR方法如何克服这些常见问题的各种示例，并讨论了工作流程的其他应用，包括改编。
