Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages
Anaerobic fungi from the herbivore digestive tract (<i>Neocallimastigomycetes</i>) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs...
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MDPI AG
2022-08-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/9/1749 |
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| author | Diana Young Akshay Joshi Liren Huang Bernhard Munk Christian Wurzbacher Noha H. Youssef Mostafa S. Elshahed Christina D. Moon Katrin Ochsenreither Gareth W. Griffith Tony M. Callaghan Alexander Sczyrba Michael Lebuhn Veronika Flad |
| author_facet | Diana Young Akshay Joshi Liren Huang Bernhard Munk Christian Wurzbacher Noha H. Youssef Mostafa S. Elshahed Christina D. Moon Katrin Ochsenreither Gareth W. Griffith Tony M. Callaghan Alexander Sczyrba Michael Lebuhn Veronika Flad |
| author_sort | Diana Young |
| collection | DOAJ |
| description | Anaerobic fungi from the herbivore digestive tract (<i>Neocallimastigomycetes</i>) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a <i>Neocallimastigomycetes</i>-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new <i>Neocallimastigomycetes</i> clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent <i>Dolichotis patagonum</i> (mara). |
| first_indexed | 2024-03-09T23:06:33Z |
| format | Article |
| id | doaj.art-12821430819445b6b4c8436082556803 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T23:06:33Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-12821430819445b6b4c84360825568032023-11-23T17:52:37ZengMDPI AGMicroorganisms2076-26072022-08-01109174910.3390/microorganisms10091749Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel LineagesDiana Young0Akshay Joshi1Liren Huang2Bernhard Munk3Christian Wurzbacher4Noha H. Youssef5Mostafa S. Elshahed6Christina D. Moon7Katrin Ochsenreither8Gareth W. Griffith9Tony M. Callaghan10Alexander Sczyrba11Michael Lebuhn12Veronika Flad13Micro and Molecular Biology, Central Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, 85354 Freising, GermanyBiocatalysis, Environment and Process Technology Unit, Life Science and Facility Management, ZHAW, 8820 Wadenswil, SwitzerlandCenter for Biotechnology (CeBiTec), University of Bielefeld, 33615 Bielefeld, GermanyChair of Urban Water Systems Engineering, Technical University of Munich (TUM), 85748 Garching, GermanyChair of Urban Water Systems Engineering, Technical University of Munich (TUM), 85748 Garching, GermanyDepartment of Microbiology and Molecular Genetics (OSU), Oklahoma State University, Stillwater, OK 74074, USADepartment of Microbiology and Molecular Genetics (OSU), Oklahoma State University, Stillwater, OK 74074, USAAgResearch, Grasslands Research Centre, Palmerston North 4442, New ZealandProcess Engineering in Life Sciences 2: Technical Biology (KIT), Karlsruhe Institute of Technology, 76131 Karlsruhe, GermanyDepartment of Life Sciences (DoLS), Aberystwyth University, Aberystwyth SY23 3DD, Wales, UKNiskus Biotec Limited Co., F92 K314 Donegal, IrelandCenter for Biotechnology (CeBiTec), University of Bielefeld, 33615 Bielefeld, GermanyMicro and Molecular Biology, Central Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, 85354 Freising, GermanyMicro and Molecular Biology, Central Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, 85354 Freising, GermanyAnaerobic fungi from the herbivore digestive tract (<i>Neocallimastigomycetes</i>) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a <i>Neocallimastigomycetes</i>-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new <i>Neocallimastigomycetes</i> clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent <i>Dolichotis patagonum</i> (mara).https://www.mdpi.com/2076-2607/10/9/1749anaerobic gut fungi<i>Neocallimastigomycetes</i>environmental screeningQuantitative Real-Time PCRlarge ribosomal subunitbarcoding |
| spellingShingle | Diana Young Akshay Joshi Liren Huang Bernhard Munk Christian Wurzbacher Noha H. Youssef Mostafa S. Elshahed Christina D. Moon Katrin Ochsenreither Gareth W. Griffith Tony M. Callaghan Alexander Sczyrba Michael Lebuhn Veronika Flad Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages Microorganisms anaerobic gut fungi <i>Neocallimastigomycetes</i> environmental screening Quantitative Real-Time PCR large ribosomal subunit barcoding |
| title | Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages |
| title_full | Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages |
| title_fullStr | Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages |
| title_full_unstemmed | Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages |
| title_short | Simultaneous Metabarcoding and Quantification of <i>Neocallimastigomycetes</i> from Environmental Samples: Insights into Community Composition and Novel Lineages |
| title_sort | simultaneous metabarcoding and quantification of i neocallimastigomycetes i from environmental samples insights into community composition and novel lineages |
| topic | anaerobic gut fungi <i>Neocallimastigomycetes</i> environmental screening Quantitative Real-Time PCR large ribosomal subunit barcoding |
| url | https://www.mdpi.com/2076-2607/10/9/1749 |
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