Quantification of fungal dark matter using qPCR

Fungi are present in a wide variety of natural environments, and in the last years, various studies have shown that they are quite abundant in aquatic ecosystems. In addition, a whole new highly diverse phylum, the Cryptomycota, was discovered. Nevertheless, research on aquatic fungi and a detailed evaluation of their functions and distribution are still sparse. One of the main reasons is a limitation in reliable identification and quantification methods. To bridge part of the research gap, this study aims to implement a quantitative PCR method to detect and quantify the newly discov-ered phylum. We developed and validated a Cryptomycota-specific qPCR primer pair targeting the 5.8S region. The resulting amplicon is 102 bp long. We used different environmental samples to evaluate the primer pair, Chytrodiomycota as negative control and positive control sequences. Ob-tained amplicons were sequenced using Illumina, and the obtained ASV clustered with known Cryptomycota. The qPCR method works reliably and specifically for the quantification of Cryptomy-cota in environmental samples.

真菌广泛分布于各类自然环境中，近年来多项研究证实，水生生态系统中真菌的丰度颇高。此外，学界还发现了一个全新且多样性极高的真菌门——隐真菌门（Cryptomycota）。然而，当前针对水生真菌的相关研究，以及对其功能与分布的详细评估仍较为匮乏。造成这一现状的主要原因之一，是可靠的真菌鉴定与定量方法存在局限。为填补部分研究空白，本研究拟采用定量PCR（qPCR）技术，对该新发现的真菌门开展检测与定量工作。本研究设计并验证了一对靶向5.8S区域的隐真菌门特异性qPCR引物，所获得的扩增子长度为102 bp。我们采用多种环境样本对该引物进行性能评估，并以壶菌门（Chytridiomycota）序列作为阴性对照与阳性对照序列。通过Illumina测序平台对所得扩增子进行测序，结果显示所得到的扩增子序列变体（Amplicon Sequence Variant, ASV）与已知的隐真菌门序列聚为一类。该qPCR方法能够可靠且特异性地对环境样本中的隐真菌门进行定量检测。