First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed de...

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Bibliographic Details
Published in:PeerJ (San Francisco, CA) Vol. 7; p. e7452
Main Authors: da Silva, Cleiziane Bispo, Dos Santos, Hellen Ribeiro Martins, Marbach, Phellippe Arthur Santos, de Souza, Jorge Teodoro, Cruz-Magalhães, Valter, Argôlo-Filho, Ronaldo Costa, Loguercio, Leandro Lopes
Format: Journal Article
Language:English
Published: United States PeerJ. Ltd 20-11-2019
PeerJ, Inc
PeerJ Inc
Subjects:
16S rDNA
Analysis
Bacteria
Bacterial diversity
Bacterial genetics
Biodiversity
Chimerical sequences
Deoxyribonucleic acid
DNA
DNA sequencing
Ecology
Electrophoresis
Endophytes
Enzymes
Genes
Genetics
Genomes
Genomic databases
Genomics
Identification
Microbiology
PCR limitations
Polyacrylamide
Polymicrobial samples
Population studies
RNA
rRNA 16S
Seeds
Statistical analysis
Taxonomy
Tropical environment
Variation
PCR limitations
Polymicrobial samples
Genomic databases
16S rDNA
Bacterial diversity
Chimerical sequences
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Summary:Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5-V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding I-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the 'number of I bands' and 'type of eletropherogram'. Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the I-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.
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ISSN:2167-8359
2167-8359
DOI:10.7717/peerj.7452