水稻根系相关微生物群落的分离及其16S rRNA序列分析

Joseph Edwards; Christian Santos-Medellín; Venkatesan  Sundaresan

doi:10.21769/BioProtoc.2884

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Peer-reviewed

Extraction and 16S rRNA Sequence Analysis of Microbiomes Associated with Rice Roots

水稻根系相关微生物群落的分离及其16S rRNA序列分析

JE Joseph Edwards ^*

CS Christian Santos-Medellín ^*

VS Venkatesan Sundaresan email

(*contributed equally to this work) 发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2884 浏览次数: 17289

评审: Joëlle SchlapferYang BaiFrancesco Dal Grande

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Abstract

Plant roots associate with a wide diversity of bacteria and archaea across the root-soil spectrum. The rhizosphere microbiota, the communities of microbes in the soil adjacent to the root, can contain up to 10 billion bacterial cells per gram of soil (Raynaud and Nunan, 2014) and can play important roles for the fitness of the host plant. Subsets of the rhizospheric microbiota can colonize the root surface (rhizoplane) and the root interior (endosphere), forming an intimate relationship with the host plant. Compositional analysis of these communities is important to develop tools in order to manipulate root-associated microbiota for increased crop productivity. Due to the reduced cost and increasing throughput of next-generation sequencing, major advances in deciphering these communities have recently been achieved, mainly through the use of amplicon sequencing of the 16S rRNA gene. Here we first present a protocol for dissecting the microbiota from various root compartments, developed using rice as a model. We next present a method for amplifying fragments of the 16S rRNA gene using a dual index approach. Finally, we present a simple workflow for analyzing the resulting sequencing data to make ecological inferences.

Keywords: Root microbiome (根系微生物群落)

Amplicon sequencing (扩增子测序)

Rhizosphere (根际)

Rhizoplane (根面)

Endosphere (内部层)

Background

Various plant root niches host different microbial communities (microbiota) originating from the soil (Bulgarelli et al., 2012; Lundberg et al., 2012; Edwards et al., 2015; Zarraonaindia et al., 2015; Wagner et al., 2016). Distinct microbiota acquired by each root niche likely have varying metabolic potential and may therefore impact the health of the host plant in different ways (Finkel et al., 2017). Bacterial and archaeal community composition in root-associated microbiota can be inferred through the use of 16S rRNA gene sequencing (Caporaso et al., 2012). The relatively low cost of sequencing now allows for comparative studies across plant species using datasets gathered by different research groups; however, small aberrations in specimen collection, sequencing, and analysis protocols may lead to large differences in the inferred microbial communities (Duvallet et al., 2017). We present this protocol detailing how to collect and analyze root microbiota from rice in an attempt to promote reproducibility across the plant microbiome field.

Materials and Reagents

Falcon 50 ml conical centrifuge tubes (Corning, Falcon^®, catalog number: 352070 )
1.5 ml microfuge tubes (E&K Scientific Products, catalog number: 280150 )
1.5 ml non-stick microfuge tubes (Thermo Fisher Scientific, Ambion^TM, catalog number: AM12450 )
0.2 ml PCR tubes (GeneMate, catalog number: 3235-00-210IS )
Filtered pipette tips (10, 200, 1,000 µl) (VWR, catalog numbers: 89168-750, 89140-936, 89168-754)
Manufacturer: Biotix, catalog number: BT10XLS3 , BT200 , BT1250 .
Gloves (Medline Industries, catalog numbers: large, MDS192086 ; medium, MDS192085 ; small, MDS192084 )
Qubit 0.5 ml assay tubes (Thermo Fisher Scientific, catalog number: Q32856 )
Single edge razor blade (Personna, catalog number: 94-115-71 )
Nuclease-free water (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9939 )
DNeasy PowerSoil kit (QIAGEN, catalog number: 12888-100 )
Primer 515F (GTGCCAGCMGCCGCGGTAA)
Primer 806R (GGACTACHVGGGTWTCTAAT)
HotStar High Fidelity DNA polymerase kit (QIAGEN, catalog number: 202602 )
Agencourt Ampure XP beads (Beckman Coulter, catalog number: A63880 )
Qubit dsDNA HS assay kit (Thermo Fisher Scientific, catalog number: Q32851 )
Ethanol 200 proof (Sigma-Aldrich, catalog number: E7023-500ML )
Agarose (Biotech Sources, catalog number: G01PD-500 )
DNA gel loading dye
NucleoSpin gel and PCR clean-up kit (MACHEREY-NAGEL, catalog number: 740609.250 )
NaCl (Fisher Scientific, catalog number: S271-1 )
KCl (Fisher Scientific, catalog number: P217-500 )
Na₂HPO₄ (Fisher Scientific, catalog number: S374-500 )
KH₂PO₄ (Fisher Scientific, catalog number: P285-500 )
Autoclaved phosphate buffered saline (PBS) solution (~100 ml/plant) (see Recipes)

Equipment

96 well magnetic plate (Alpaqua Engineering, catalog number: A001219R )
1.5 ml tube magnetic rack (Thermo Fisher Scientific, catalog number: MR01 )
Pipettes (2.5, 10, 200, 1,000 µl) (Thermo Fisher Scientific, Finnpipette^TM, catalog numbers: 4641010N , 4641030N , 4641080N , 4641100N )
Ultrasonic cleaning bath, 40 kHz (Branson, model: Branson 1800, catalog number: CPX-952-116R )
Dissection tools (scissors and forceps) (scissors: Bioseal, catalog number: KI011/50 ; forceps: Integra LifeSciences, Miltex, catalog number: 6-184 )
-80 °C freezer
Microcentrifuge (Eppendorf, model: 5417C )
Mini-Beadbeater-96 high-throughput cell disrupter (Bio Spec Products, catalog number: 1001 )
Electrophoresis gel unit (Bio-Rad Laboratories, catalog number: 1704468 )
Qubit fluorometer (Thermo Fisher Scientific, catalog number: Q33226 )
PCR thermal cycler (Bio-Rad Laboratories, model: T100^TM Thermal Cycler , catalog number: 1861096)

Software

Python2 version 2.7.12
R version 3.4.3

Procedure

English

中文翻译

文章信息

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如何引用

Edwards, J., Santos-Medellín, C. and Sundaresan, V. (2018). Extraction and 16S rRNA Sequence Analysis of Microbiomes Associated with Rice Roots. Bio-protocol 8(12): e2884. DOI: 10.21769/BioProtoc.2884.