Van Caenegem Warre, Haelewaters Danny
Research Group Mycology, Department of Biology, Ghent University, Ghent, 9000, Belgium.
Meise Botanic Garden, Meise, 1860, Belgium.
IMA Fungus. 2024 Jun 11;15(1):14. doi: 10.1186/s43008-024-00146-9.
Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.
多年来，对虫囊菌目（子囊菌门，粪壳菌纲）真菌的分子研究一直受到阻碍，原因在于它们体积微小、无法在无菌培养条件下生长，且缺乏可靠且经济高效的DNA提取方案。特别是，Laboulbenia属在DNA提取和聚合酶链反应（PCR）扩增方面成功率极低，声名狼藉。这归因于细胞中存在黑色素，已知该分子会抑制PCR。我们评估了一种基于单细胞的标准DNA提取方案的效果，通过将推荐的试剂用量减半以降低每次提取的成本，并在多重置换扩增步骤中添加牛血清白蛋白（BSA）来抵消黑色素的影响。总共进行了196次提取，其中111次成功。我们发现，将基于单细胞的提取试剂盒中使用的试剂用量减半，对成功提取DNA的概率没有显著影响。使用减半后的方案可降低成本和资源消耗。此外，添加BSA与否对成功提取DNA的概率没有显著差异，这表明菌体细胞中存在的黑色素量对PCR没有重大抑制作用。我们从五个基因座生成了277个序列，但内转录间隔区、线粒体小亚基rDNA和蛋白质编码基因的扩增仍然具有挑战性。从虫囊菌目成功提取DNA的概率还受到标本保存方法的影响，与保存在70%乙醇和干燥材料中的材料相比，保存在>95%乙醇中的材料成功率更高。我们强调妥善保存材料的重要性，并建议设计虫囊菌目特异性引物，以克服引物错配和污染问题。我们的新见解不仅适用于Laboulbenia属；一般来说，虫囊菌目研究不足，绝大多数物种仍未测序。新的、易于操作的分子技术发展将有利于虫囊菌目的研究，有助于阐明这些奇特微真菌的真正多样性和进化关系。
