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A new primer set for Clade I nosZ that recovers genes from a broader range of taxa

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Abstract

Denitrification is an important global N cycle process. The gene encoding NosZ that converts nitrous oxide (N2O) to N2 has been widely used as a biomarker to study denitrifying communities. However, conventional PCR primers target a limited range of the genetically diverse Clade I nosZ, and the amplicons are too long for sequencing on current NGS platforms. To address these issues, we developed a new PCR primer set that amplifies a 355-bp region of Clade I nosZ and captures broader taxonomic coverage than conventional primers in in silico tests. When compared with the widely used nosZF_nosZR_Rich_2003 set using the same soil samples and the same sequencing depth, the new set retrieved genes from four times more unique species, with consistently higher general diversity-based metrics. The new primer set performed well with different sequencing platforms (Ion Torrent and Illumina), and among a wide variety of soils from polar to tropical, desert to agricultural, and surface to a very low biomass subsoil, with significant differences in denitrifying community diversity and composition. This new primer set for Clade I together with the primers recently reported for Clade II by Chee-Sanford et al. (J Microbiol Meth 172:105908, 2020) provides a more comprehensive assessment of denitrifier gene hosts, their ecological patterns, and the degree of novelty in retrieved gene sequences.

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Data availability

Raw sequencing data generated with Ion Torrent PGM and Illumina MiSeq were deposited in the NCBI Sequence Read Archive (SRA) database under the study accession PRJNA640470 and PRJNA640229, respectively.

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Funding

This work was funded in part by the US Department of Energy, Office of Science, Office of Biological and Environmental Research (Awards DE-SC0018409 and DE-FC02-07ER64494), by the National Science Foundation Long-term Ecological Research Program (DEB 1637653) at the Kellogg Biological Station, by Michigan State University AgBioResearch, and by National Natural Science Foundation of China (81800517).

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Authors and Affiliations

  1. Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA

    Bangzhou Zhang, Zhenhua Yu, Chao Xue, Zhangran Chen, Mengxin Zhao, John F. Quensen & James M. Tiedje

  2. Department of Gastroenterology, Zhongshan Hospital, Xiamen University, Xiamen, China

    Bangzhou Zhang

  3. Center for Fundamental and Applied Microbiomics, Biodesign Institute, Arizona State University, Tempe, AZ, USA

    C. Ryan Penton

  4. College of Integrative Sciences and Arts, Faculty of Science and Mathematics, Arizona State University, Mesa, AZ, USA

    C. Ryan Penton

  5. Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin, China

    Zhenhua Yu

  6. Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, China

    Chao Xue

  7. Institute for Microbial Ecology, School of Medicine, Xiamen University, Xiamen, China

    Qiongyun Chen, Zhangran Chen, Changsheng Yan & Qiang Zhang

  8. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

    Mengxin Zhao

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  1. Bangzhou Zhang
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  2. C. Ryan Penton
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Correspondence to James M. Tiedje.

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Zhang, B., Penton, C.R., Yu, Z. et al. A new primer set for Clade I nosZ that recovers genes from a broader range of taxa. Biol Fertil Soils 57, 523–531 (2021). https://doi.org/10.1007/s00374-021-01544-6

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  • DOI: https://doi.org/10.1007/s00374-021-01544-6

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