The primer sets of 18S rRNA genes and Internal Transcribed Spacer (ITS) regions are universal primers for amplicon sequencing in soil fungal diversity studies. The selection of proper primer sets has been identified as one of the main drivers affecting effective cross-study comparisons in various types of soils. However, few studies have provided guidance for the selection of fungal primers in soil fungal biodiversity studies. The aim of this study was to evaluate and optimise fungal sequencing methods with three primer sets in desert, forest, grassland and farmland soils. Melting curve analyses were used to evaluate the specificity of each primer set in different soil ecosystems. The quality of fungal sequencing with three primer sets was assessed with the same conditions on the Illumina platform. The melting curve analysis showed that both the FR1/FF390 and the ITS3/ITS4 primer sets have better specificity than the ITS1/ITS primer set. 18S rRNA amplicon sequencing produced less fungal phylotypes compared to the amplicon sequencing data of the ITS1/ITS2 and ITS3/ITS4 primer sets. Our study suggests that the ITS3/ITS4 primer set was more suitable for the analysis of soil fungal biodiversity in desert, forest, grassland and farmland soils on the Illumina sequencing platform.

中文翻译：

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ITS3/ITS4 优于其他 ITS 区域和 18S rRNA 基因引物组用于土壤真菌的扩增子测序

18S rRNA 基因和内部转录间隔 (ITS) 区域的引物组是土壤真菌多样性研究中扩增子测序的通用引物。选择合适的引物组已被确定为影响各种类型土壤中有效交叉研究比较的主要驱动因素之一。然而，很少有研究为土壤真菌生物多样性研究中真菌引物的选择提供指导。本研究的目的是评估和优化沙漠、森林、草原和农田土壤中三种引物组的真菌测序方法。熔解曲线分析用于评估每个引物组在不同土壤生态系统中的特异性。在 Illumina 平台上使用相同的条件评估了三个引物组的真菌测序质量。熔解曲线分析表明，FR1/FF390 和 ITS3/ITS4 引物组均比 ITS1/ITS 引物组具有更好的特异性。与 ITS1/ITS2 和 ITS3/ITS4 引物组的扩增子测序数据相比，18S rRNA 扩增子测序产生的真菌种系型较少。我们的研究表明，ITS3/ITS4 引物组更适合在 Illumina 测序平台上分析沙漠、森林、草地和农田土壤中的土壤真菌生物多样性。