Metagenomic analysis’ rhizomicrobiome’ Prunus mahaleb based on 16S RNA gene sequencing on the Illumina MiSeq

Year 2025, Volume: 9 Issue: 4, 124 - 131

Serap Çetinkaya , Aysun Baş , Muhammed Safa Çelik , Hüseyin Fatih Çetinkaya , Burak Tüzün

Abstract

The study covered for the first time the investigation of the microbiome of Prunus mahaleb rhizosphere. The methodology included both metagenomic and bioinformatic means, and used the bidirectional sequencing of V3 region of 16S rRNA gene with oligo-primers universal to both Bacteria and Archaea. Results of Illumina MiSeq sequencing indicated the presence of 49 phyla, 104 classes, 242 orders, 353 families and 761 genera. The commonest genus was, not surprisingly, Pseudomonas. This genus was closely followed by uncultured genera and this finding was considered to have very meaningful implications as to the functional aspects of a biome and the importance of it was discussed in the text. A thorough insight into the rhizomicrobiome is therefore envisaged to facilitate the development of microbial fertilizers to improve plant performance and productivity impacting eventually food security.

Keywords

Bioinformatics , metagenomics , Illumina MiSeq sequencing , rhizosphere of Prunus mahaleb , V3 region of 16S rRNA

References

• [1] R.I. Amann, B.J. Binder, R.J. Olson, S.W. Chisholm, R. Devereux, & D. Stahl, Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and environmental microbiology, 56(6) (1990) 1919-1925.
• [2] J. Handelsman, M.R. Rondon, S.F. Brady, J. Clardy, & R.M. Goodman, Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chemistry & biology, 5(10) (1998) R245-R249.
• [3] P. López-García, & D. Moreira, Tracking microbial biodiversity through molecular and genomic ecology. Research in microbiology, 159(1) (2008) 67-73.
• [4] E. Gabor, K. Liebeton, F. Niehaus, J. Eck, & P. Lorenz, Updating the metagenomics toolbox. Biotechnology Journal: Healthcare Nutrition Technology, 2(2) (2007) 201-206.
• [5] E.A. Dinsdale, R.A. Edwards, D. Hall, F. Angly, M. Breitbart, J.M. Brulc, ... & F. Rohwer, Functional metagenomic profiling of nine biomes. Nature, 452(7187) (2008) 629-632.
• [6] K.J. Hoff, M. Tech, T. Lingner, R. Daniel, B. Morgenstern, & P. Meinicke, Gene prediction in metagenomic fragments: a large scale machine learning approach. BMC bioinformatics, 9 (2008) 1-14.
• [7] C.S. Riesenfeld, P.D. Schloss, & J. Handelsman, Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet., 38(1) (2004) 525-552.
• [8] R.D. Sleator, C. Shortall, & C. Hill, Metagenomics. Letters in applied microbiology, 47(5) (2008) 361-366.
• [9] M. Langer, E.M. Gabor, K. Liebeton, G. Meurer, F. Niehaus, R. Schulze, ... & P. Lorenz, Metagenomics: an inexhaustible access to nature's diversity. Biotechnology Journal: Healthcare Nutrition Technology, 1(7‐8) (2006) 815-821.
• [10] D.D. Richter, & D. Markewitz, How deep is soil?. BioScience, 45(9) (1995) 600-609.
• [11] V. Torsvik, F.L. Daae, R.A. Sandaa, & L. Øvreås, L. Novel techniques for analysing microbial diversity in natural and perturbed environments. Journal of biotechnology, 64(1) (1998) 53-62.
• [12] V. Torsvik, & L. Øvreås, Microbial diversity and function in soil: from genes to ecosystems. Current opinion in microbiology, 5(3) (2002) 240-245.
• [13] V. Torsvik, R. Sørheim, & J. Goksøyr, Total bacterial diversity in soil and sediment communities—a review. Journal of Industrial Microbiology and Biotechnology, 17(3-4) (1996) 170-178.
• [14] W.F. Doolittle, Phylogenetic classification and the universal tree. Science, 284(5423) (1999). 2124-2128.
• [15] R. Daniel, The metagenomics of soil. Nature reviews microbiology, 3(6) (2005) 470-478.
• [16] A. Özbey, N. Öncül, Z. Yıldırım, & M. Yıldırım, Mahlep ve mahlep ürünleri. Journal of Agricultural Faculty of Gaziosmanpaşa University (JAFAG), 2011(2) (2011) 153-158.
• [17] Y. Jo, J.K. Cho, H. Choi, H. Chu, S. Lian, & W.K. Cho, Bacterial communities in the phylloplane of Prunus species. Journal of Basic Microbiology, 55(4) (2011) (2015) 504-508.
• [18] C. Oliveira, E. Shakiba, D. North, M. McGraw, E. Ballard, M. Barrett-D’Amico, ... & Y. Rahmatallah, 16S rRNA gene-based metagenomic analysis of rhizosphere soil bacteria in Arkansas rice crop fields. Agronomy, 12(1) (2022) 222.
• [19] G.E. Truett, P. Heeger, R.L. Mynatt, A.A. Truett, J.A. Walker, & M.L. Warman, Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques, 29(1) (2000) 52-54.
• [20] F. Durazzi, C. Sala, G. Castellani, G. Manfreda, D. Remondini, & A. De Cesare, Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota. Scientific reports, 11(1) (2021) 3030.
• [21] E. Fadeev, M.G. Cardozo-Mino, J.Z. Rapp, C. Bienhold, I. Salter, V. Salman-Carvalho, ... & A. Boetius, Comparison of two 16S rRNA primers (V3–V4 and V4–V5) for studies of arctic microbial communities. Frontiers in microbiology, 12 (2021) 637526.
• [22] E.V. Grigorova, N.L. Belkova, U.M. Nemchenko, E.S. Klimenko, A.V. Pogodina, A.I. Romanitsa, ... & L.V. Rychkova, Metasequencing of V3-V4 variable regions of 16S rRNA gene in opportunistic microbiota and gut biocenosis in obese adolescents. Bulletin of Experimental Biology and Medicine, 170(3) (2021) 321-325.
• [23] D. Peterson, K.S. Bonham, S. Rowland, C.W. Pattanayak, R. Consortium, & V. Klepac-Ceraj, Comparative analysis of 16S rRNA gene and metagenome sequencing in pediatric gut microbiomes. Frontiers in microbiology, 12 (2021) 670336.
• [24] Q. Wang, G.M. Garrity, J.M. Tiedje, & J.R. Cole, Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and environmental microbiology, 73(16) (2007) 5261-5267.
• [25] I.F. Spellerberg, & P.J. Fedor, A tribute to Claude Shannon (1916–2001) and a plea for more rigorous use of species richness, species diversity and the ‘Shannon–Wiener’Index. Global ecology and biogeography, 12(3) (2003) 177-179.
• [26] J. Oksanen, F.G. Blanchet, R. Kindt, P. Legendre, P.R. Minchin, R.B. O’hara, ... & M.J. Oksanen, Package ‘vegan’. Community ecology package, version, 2(9) (2013) 1-295.