Utility of large subunit for environmental sequencing of arbuscular mycorrhizal fungi: a new reference database and pipeline
Utility of large subunit for environmental sequencing of arbuscular mycorrhizal fungi: a new reference database and pipeline
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DOI：
10.1111/nph.17080
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发表时间：
2020-12-04
期刊：
影响因子：
9.4
通讯作者：
Bever, James D.
中科院分区：
文献类型：
--
作者：
Delavaux, Camille S.;Sturmer, Sidney L.;Bever, James D.
背景丛枝菌根真菌（AMF-phylum Glomeromycota）与世界范围内的大多数植物物种形成共生，并且在植物营养和水吸收、病原体抗性和土壤聚集中起关键作用（Smith & Read，2008; Delavaux et al.，2017; Brundrett & Tedersoo，2018）。由于AMF群落组成影响生态功能（货车der Heijden等人，1998; Vogelsang等人，2006; Koziol等人，2018），了解AMF组成的模式是研究的优先事项。AMF物种的菌丝在形态上不可区分，因此AMF物种多样性和群落组成的定量越来越依赖于来自田间样品的核糖体RNA（rRNA）基因序列的元条形码（Opik et al.，2014年）。然而，迄今为止，没有一个rRNA基因的单一区域被普遍接受为AMF环境测序的最佳选择。rRNA基因的内部转录间隔区（ITS）区域已被认为是通用真菌标记（Schoch et al.，2012; Lindahl等人，2013）并已用于AMF生物地理学研究（Tedersoo et al.，2014）和环境测序（Opik等人，2014年）。然而，该区域作为AMF的标记基因是次优的（Stockinger等人，2010; Schoch等人，2012年）。用于ITS序列与其他真菌的序列匹配方法对于AMF的效用有限，因为ITS序列数据库中AMF序列的代表性差和管理差（Bidartondo，2008; Stockinger等人，2010年）。这个数据库的问题不能很容易地纠正，因为环境样品中遇到的AMF的比例很高，是未描述的。虽然系统发育方法可用于鉴定新序列作为AMF，但该方法不能用于ITS扩增子，因为其快速序列进化（Nilsson et al.，#20008;，并不可靠。用于AMF的环境测序的rRNA基因的最常用区域是小亚基或SSU（Opik等人，2014年）。该区域的效用通过针对AMF的良好开发和策划的数据库而增强（Opik等人，2010年; Davison等人，2015年）。然而，SSU区域的缺点是进化缓慢，因此没有足够的可变性来充分解析AMF种类（Krüger等人，2009; Bruns & Taylor，2016; Schlaeppi等人，2016年）。相比之下，大亚基（LSU）区域始终显示出对AMF的分类学解析的更大效用（Krüger等人，2012;哈特等人，2015; House等人，2016），使其在环境AMF测序中可能更有用。到目前为止，LSU区域很少用于AMF的环境测序（Golvet al.，2004; Lekberg等人，2013; House &贝弗，2018;维埃拉等人，2018; Schütte等人，2019年），也许是因为实施中的生物信息学挑战。在这里，我们的目标是通过提供一个精心策划的LSU参考数据库，一个参考骨干树的系统发生位置和一个计算管道容易实现使用当前的生物信息学工具，以扩大实用性和简化采用的LSU扩增子测序的AMF。
BackgroundArbuscular mycorrhizal fungi (AMF–phylum Glomeromycota) form symbioses with most plant species worldwide and play critical roles in plant nutrient and water uptake, pathogen resistance and soil aggregation (Smith & Read, 2008; Delavaux et al., 2017; Brundrett & Tedersoo, 2018). Because AMF community composition influences ecological function (van der Heijden et al., 1998; Vogelsang et al., 2006; Koziol et al., 2018), understanding patterns of AMF composition is a research priority. Hyphae of AMF species are not morphologically distinguishable, and therefore quantification of AMF species diversity and community composition has increasingly relied on metabarcoding of ribosomal RNA (rRNA) gene sequences from field samples (Opik et al., 2014). However, to date, no single region of the rRNA gene has been universally accepted as optimal for AMF environmental sequencing. The internal transcribed spacer (ITS) region of the rRNA gene has been suggested as the universal fungal marker (Schoch et al., 2012; Lindahl et al., 2013) and has been used for AMF biogeographical studies (Tedersoo et al., 2014) and environmental sequencing (Opik et al., 2014). However, this region is suboptimal as a marker gene for AMF (Stockinger et al., 2010; Schoch et al., 2012). The sequence matching approach used for ITS sequences with other fungi is of limited utility for AMF because of the poor representation and poor curation of AMF sequences in ITS sequence databases (Bidartondo, 2008; Stockinger et al., 2010). This database problem cannot be easily rectified because a high proportion of AMF encountered in environmental samples are undescribed. While phylogenetic approaches can be used to identify new sequences as AMF, this approach cannot be used for ITS amplicons because its rapid sequence evolution (Nilsson et al., 2008) does not generate reliable trees. The most commonly used region of the rRNA gene for environmental sequencing of AMF is the small subunit, or SSU (Opik et al., 2014). The utility of this region is enhanced by a well-developed and curated database for AMF (Opik et al., 2010; Davison et al., 2015). However, the SSU region has the disadvantage of being slow-evolving and therefore not sufficiently variable to adequately resolve AMF species (Krüger et al., 2009; Bruns & Taylor, 2016; Schlaeppi et al., 2016). By contrast, the large subunit (LSU) region consistently shows greater utility for taxonomic resolution for AMF (Krüger et al., 2012; Hart et al., 2015; House et al., 2016), making it potentially more useful in environmental AMF sequencing. Thus far, the LSU region has rarely been used in environmental sequencing of AMF (Gollotte et al., 2004; Lekberg et al., 2013; House & Bever, 2018; Vieira et al., 2018; Schütte et al., 2019), perhaps because of bioinformatical challenges in implementation. Here, we aim to expand the utility and ease the adoption of the LSU for amplicon sequencing of AMF by providing a well-curated LSU reference database, a reference backbone tree for phylogenetic placement and a computational pipeline easily implemented using current bioinformatical tools.