PCR primers to study the diversity of expressed fungal genes encoding lignocellulolytic enzymes in soils using high-throughput sequencing. Illumina sequencing of soil fungal expressed genes

Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal Glycoside Hydrolase gene families GH5 and GH11 encoding cellulases and endoxylanases respectively as well as Basidiomycota class II peroxidases active on lignin (AA2). These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 (encoding cellulases), the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the discovery of complex patterns in gene expression soil fungal communities.

土壤中的植物生物质降解是陆地生态系统碳循环的关键步骤之一。腐生真菌群落（fungal saprotrophic communities）通过合成作用于植物有机质主要组分的水解酶，在该过程中发挥核心作用。当前该领域尚存的开放性问题包括：所涉物种的多样性、关联的主要生化通路，以及这些过程如何受凋落物质量、气候变化等外部因素影响。此类问题可通过环境基因组学方法解决：即以土壤提取的RNA为实验材料，对关键酶编码基因家族开展系统性测序。这类方法的实施需要设计并验证基因家族特异性PCR引物，以获得可兼容高通量测序技术的序列片段。

本研究中，我们开发并验证了可特异性扩增真菌糖苷水解酶（Glycoside Hydrolase）GH5、GH11基因家族的PCR引物，这两个家族分别编码纤维素酶与内切木聚糖酶；同时还开发了针对作用于木质素的担子菌门（Basidiomycota）第二类过氧化物酶（AA2）的引物。我们使用提取自多种子囊菌门（Ascomycota）和担子菌门物种的DNA对上述引物进行了实验验证，其中包含27个已完成全基因组测序的物种。结合已发表的糖苷水解酶GH7（编码纤维素酶）引物，新设计的引物被证实可与Illumina MiSeq测序技术兼容。

从山毛榉林或云杉林土壤提取的RNA所获得的测序序列展现出较高的多样性，且在涵盖这些基因家族全球多样性的基因树中呈均匀分布。这种使用多种简并引物的高通量测序方法是一种稳健的技术手段，可同时表征参与植物有机质降解的不同真菌转录本的多样性，或可助力揭示土壤真菌群落基因表达的复杂模式。