Guanidine thiocyanate solution facilitates sample collection for plant rhizosphere microbiome analysis
Guanidine thiocyanate solution facilitates sample collection for plant rhizosphere microbiome analysis
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硫氰酸胍溶液有助于植物根际微生物组分析的样品采集
作者：
Sun Xiaoxiao;Wang meiling;Guo lin;Shu Changlong;Zhang Jie;Geng Lili
根际微生物与植物之间的相互作用对作物的健康和生长发育具有重要意义。分析植物根际细菌的组成，特别是那些与生物/非生物胁迫的抗性，可能会提高其在可持续农业中的应用。通常需要在田间进行大规模的根际取样;然而，这样的样品不能立即冷冻。我们发现，样品在室温下储存2天，导致相当大的减少的操作分类单位（OTU）的数量和根际群落的细菌α多样性指数。为了克服这些问题，本研究建立了一种用硫氰酸胍（GTC）溶液保存根际样品的方法。该方法允许样品在冷冻保存之前在室温下维持至少1天，并且显示与常规DNA分离方案相容。使用Illumina对16 S rRNA基因的V3和V4高变区进行测序，以评估该方法的可行性和可靠性，并且在-70 °C下储存的样品和GTC溶液中储存的样品之间，在OTU的数量以及Chao和Shannon指数方面没有观察到显著差异。此外，假单胞菌属的代表。通过实时定量聚合酶链反应测定，储存在GTC溶液中的样品与储存在−70 °C下的样品无显著差异（p > 0.05）。主坐标分析表明，两种类型的样品聚在一起。此外，与在-70 °C以下储存样品时观察到的情况相比，GTC溶液在不同储存期对细菌分类群分布没有影响。即使在室温下孵育解冻的样品（在-70 °C下冷冻）15分钟，也会引起细菌组成的微小变化。综上所述，我们的研究结果表明，GTC溶液可以提供一个可靠的替代保存根际样品在外地。
The interactions between rhizosphere microorganisms and plants are important for the health and development of crops. Analysis of plant rhizosphere bacterial compositions, particularly of those with resistance to biotic/abiotic stresses, may improve their applications in sustainable agriculture. Large-scale rhizosphere samplings in the field are usually required; however, such samples, cannot be immediately frozen. We found that the storage of samples at room temperature for 2 days leads to a considerable reduction in the operational taxonomic unit (OTU) number and the indices of bacterial alpha-diversity of rhizosphere communities. In this study, in order to overcome these problems, we established a method using guanidine thiocyanate (GTC) solution for the preservation of rhizosphere samples after their collection. This method allowed the maintenance of the samples for at least 1 day at room temperature prior to their cryopreservation and was shown to be compatible with conventional DNA isolation protocols. Illumina sequencing of V3 and V4 hypervariable regions of the 16S rRNA gene was used to assess the feasibility and reliability of this method, and no significant differences were observed in the number of OTUs and in the Chao and Shannon indices between samples stored at −70 °C and those stored in GTC solution. Moreover, the representation of Pseudomonas spp. in samples stored in GTC solution was not significantly different from that in samples stored at −70 °C, as determined by real-time quantitative polymerase chain reaction (p > 0.05). Both types of samples were shown to cluster together according to principal coordinate analysis. Furthermore, GTC solution did not affect the bacterial taxon profiles at different storage periods compared with those observed when storing the samples below −70 °C. Even incubation of thawed samples (frozen at −70 °C) for 15 min at room temperature induced minor changes in the bacterial composition. Taken together, our results demonstrated that GTC solution may provide a reliable alternative for the preservation of rhizosphere samples in the field.
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模板制备影响叶际微生物群落的 16S rRNA 高通量测序分析
影响因子：
5.6
作者：
Tian X;Shi Y;Geng L;Chu H;Zhang J;Song F;Duan J;Shu C
通讯作者：
Shu C
影响因子：
14.9
作者：
Klindworth A;Pruesse E;Schweer T;Peplies J;Quast C;Horn M;Glöckner FO
通讯作者：
Glöckner FO
影响因子：
4.6
作者：
Hosomi K;Ohno H;Murakami H;Natsume-Kitatani Y;Tanisawa K;Hirata S;Suzuki H;Nagatake T;Nishino T;Mizuguchi K;Miyachi M;Kunisawa J
通讯作者：
Kunisawa J
DOI：
10.2174/1874285800903010040
发表时间：
2009
期刊：
The open microbiology journal
影响因子：
--
作者：
Roesch LF;Casella G;Simell O;Krischer J;Wasserfall CH;Schatz D;Atkinson MA;Neu J;Triplett EW
通讯作者：
Triplett EW
影响因子：
56.9
作者：
Jan Dolfing;A. Vos;J. Bloem;P.A.I. Ehlert;Natalia Naumova;P. Kuikman
通讯作者：
Jan Dolfing;A. Vos;J. Bloem;P.A.I. Ehlert;Natalia Naumova;P. Kuikman