Abstract
Selection of optimal primer pairs in 16S rRNA gene sequencing is a pivotal issue in microorganism diversity analysis. However, limited effort has been put into investigation of specific primer sets for analysis of the bacterial diversity of aging flue-cured tobaccos (AFTs), as well as prediction of the function of the bacterial community. In this study, the performance of four primer pairs in determining bacterial community structure based on 16S rRNA gene sequences in AFTs was assessed, and the functions of genes were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results revealed that the primer set 799F–1193R covering the amplification region V5V6V7 gave a more accurate picture of the bacterial community structure of AFTs, with lower co-amplification levels of chloroplast and mitochondrial genes, and more genera covered than when using the other primers. In addition, functional gene prediction suggested that the microbiome of AFTs was involved in kinds of interested pathways. A high abundance of functional genes involved in nitrogen metabolism was detected in AFTs, reflecting a high level of bacteria involved in degrading harmful nitrogen compounds and generating nitrogenous nutrients for others. Additionally, the functional genes involved in biosynthesis of valuable metabolites and degradation of toxic compounds provided information that the AFTs possess a huge library of microorganisms and genes that could be applied to further studies. All of these findings provide a significance reference for researchers working on the bacterial diversity assessment of tobacco-related samples.
Highlights
Microorganisms are dominant drivers of biogeochemical processes (Shinichi et al 2015), yet a large number of microorganisms (≥ 99%) from the environment remain uncultivated in the laboratory, and these regulate ecosystem processes and even affect our lives (Kaeberlein et al.2002; Su et al 2012)
Several researchers have suggested that the primers V3F and V4R, covering the V3V4 amplification region, should be the preferred primer set for future studies to achieve accurate bacterial community diversities, and a large number of studies have focused on this region (Castelino et al 2017; Cai et al 2013; Parulekar et al 2017; Takahashi et al 2014)
Several studies have focused on characterization of bacterial communities in different tobaccos using 16S rRNA gene sequences with the V3V4 and V5V6V7 primers (Huang et al 2010; Tyx et al 2016), few report have showed optimized primers for bacterial microbiome investigation of tobaccos, while there are as many as 10,000 chloroplast DNA copies in tobacco leaf cells (Shaver et al 2006)
Summary
Microorganisms are dominant drivers of biogeochemical processes (Shinichi et al 2015), yet a large number of microorganisms (≥ 99%) from the environment remain uncultivated in the laboratory, and these regulate ecosystem processes and even affect our lives (Kaeberlein et al.2002; Su et al 2012). To exclude the interference of contaminating sequences, a common method is to filter out the chloroplast DNA, mitochondrial DNA, and other unknown sequences during data processing. This might not reveal the real microbiome structures of samples. Several studies have focused on characterization of bacterial communities in different tobaccos using 16S rRNA gene sequences with the V3V4 and V5V6V7 primers (Huang et al 2010; Tyx et al 2016), few report have showed optimized primers for bacterial microbiome investigation of tobaccos, while there are as many as 10,000 chloroplast DNA copies in tobacco leaf cells (Shaver et al 2006). There is, an urgent need to investigate the perfect primers for tobacco bacterial microbiome research
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