title: Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).
abstract: Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), Biotechnology Department, University of the Western Cape, Bellville 7535, Cape Town, South Africa.
authors.forename: Mapula Kgomotso Annah
authors.initials: MK
authors.lastname: Maropola
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), Biotechnology Department, University of the Western Cape, Bellville 7535, Cape Town, South Africa; Center for Microbial Ecology and Genomics (CMEG), Genomics Research Institute, University of Pretoria, South Africa.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Ramond
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), Biotechnology Department, University of the Western Cape, Bellville 7535, Cape Town, South Africa. Electronic address: ituffin@uwc.ac.za.
authors.forename: Marla
authors.initials: M
authors.lastname: Trindade
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D060026:Endophytes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D020459:Genes, rRNA; D056186:Metagenomics / Q000379:methods*; D018517:Plant Roots / Q000382:microbiology; D018547:Plant Stems / Q000382:microbiology; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D045868:Sorghum / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004247:DNA
keywords: Endophytic bacteria; Metagenomic DNA extraction; Pyrosequencing; Sorghum root and stem; t-RFLP