Using the Microbiome Amplification Preference Tool (MAPT) to Reveal Medicago sativa-Associated Eukaryotic Microbes

Although our understanding of the microbial diversity found within a given system expands as amplicon sequencing improves, technical aspects still drastically affect which members can be detected. Compared with prokaryotic members, the eukaryotic microorganisms associated with a host are understudied due to their underrepresentation in ribosomal databases, lower abundance compared with bacterial sequences, and higher ribosomal gene identity to their eukaryotic host. Peptide nucleic acid (PNA) blockers are often designed to reduce amplification of host DNA. Here we present a tool for PNA design called the Microbiome Amplification Preference Tool (MAPT). We examine the effectiveness of a PNA designed to block genomic Medicago sativa DNA (gPNA) compared with unrelated surrounding plants from the same location. We applied mitochondrial PNA and plastid PNA to block the majority of DNA from plant mitochondria and plastid 16S ribosomal RNA genes, as well as the novel gPNA. Until now, amplifying both eukaryotic and prokaryotic reads using 515F-Y and 926R has not been applied to a host. We investigate the efficacy of this gPNA using three approaches: (i) in silico prediction of blocking potential in MAPT, (ii) amplicon sequencing with and without the addition of PNAs, and (iii) comparison with cultured fungal representatives. When gPNA is added during amplicon library preparation, the diversity of unique eukaryotic amplicon sequence variants present in M. sativa increases. We provide a layered examination of the costs and benefits of using PNAs during sequencing. The application of MAPT enables scientists to design PNAs specifically to enable capturing greater diversity in their system.

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Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

FEMS Microbiology Reviews ◽

10.1111/j.1574-6976.2009.00196.x ◽

2010 ◽

Vol 34 (1) ◽

pp. 59-86 ◽

Cited By ~ 63

Author(s):

Ana Lilia Torres-Machorro ◽

Roberto Hernández ◽

Ana María Cevallos ◽

Imelda López-Villaseñor

Keyword(s):

Ribosomal Rna ◽

Ribosomal Rna Genes ◽

Eukaryotic Microorganisms ◽

Rna Genes

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Structure and variation in ribosomal RNA genes of pea

Plant Molecular Biology ◽

10.1007/bf00016429 ◽

1987 ◽

Vol 8 (1) ◽

pp. 3-12 ◽

Cited By ~ 87

Author(s):

R. A. Jorgensen ◽

R. E. Cuellar ◽

W. F. Thompson ◽

T. A. Kavanagh

Keyword(s):

Ribosomal Rna ◽

Ribosomal Rna Genes ◽

Rna Genes

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Characterization, Comparative Analysis and Phylogenetic Implications of Mitogenomes of Fulgoridae (Hemiptera: Fulgoromorpha)

Genes ◽

10.3390/genes12081185 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1185

Author(s):

Wenqian Wang ◽

Huan Zhang ◽

Jérôme Constant ◽

Charles R. Bartlett ◽

Daozheng Qin

Keyword(s):

Control Region ◽

Ribosomal Rna ◽

Phylogenetic Analyses ◽

Start Codon ◽

Rrna Genes ◽

Rich Region ◽

Protein Coding ◽

Phylogenetic Implications ◽

Rna Genes ◽

Unpaired Base

The complete mitogenomes of nine fulgorid species were sequenced and annotated to explore their mitogenome diversity and the phylogenetics of Fulgoridae. All species are from China and belong to five genera: Dichoptera Spinola, 1839 (Dichoptera sp.); Neoalcathous Wang and Huang, 1989 (Neoalcathous huangshanana Wang and Huang, 1989); Limois Stål, 1863 (Limois sp.); Penthicodes Blanchard, 1840 (Penthicodes atomaria (Weber, 1801), Penthicodes caja (Walker, 1851), Penthicodes variegata (Guérin-Méneville, 1829)); Pyrops Spinola, 1839 (Pyrops clavatus (Westwood, 1839), Pyrops lathburii (Kirby, 1818), Pyrops spinolae (Westwood, 1842)). The nine mitogenomes were 15,803 to 16,510 bp in length with 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and a control region (A + T-rich region). Combined with previously reported fulgorid mitogenomes, all PCGs initiate with either the standard start codon of ATN or the nonstandard GTG. The TAA codon was used for termination more often than the TAG codon and the incomplete T codon. The nad1 and nad4 genes varied in length within the same genus. A high percentage of F residues were found in the nad4 and nad5 genes of all fulgorid mitogenomes. The DHU stem of trnV was absent in the mitogenomes of all fulgorids sequenced except Dichoptera sp. Moreover, in most fulgorid mitogenomes, the trnL2, trnR, and trnT genes had an unpaired base in the aminoacyl stem and trnS1 had an unpaired base in the anticodon stem. The similar tandem repeat regions of the control region were found in the same genus. Phylogenetic analyses were conducted based on 13 PCGs and two rRNA genes from 53 species of Fulgoroidea and seven outgroups. The Bayesian inference and maximum likelihood trees had a similar topological structure. The major results show that Fulgoroidea was divided into two groups: Delphacidae and ((Achilidae + (Lophopidae + (Issidae + (Flatidae + Ricaniidae)))) + Fulgoridae). Furthermore, the monophyly of Fulgoridae was robustly supported, and Aphaeninae was divided into Aphaenini and Pyropsini, which includes Neoalcathous, Pyrops, Datua Schmidt, 1911, and Saiva Distant, 1906. The genus Limois is recovered in the Aphaeninae, and the Limoisini needs further confirmation; Dichoptera sp. was the earliest branch in the Fulgoridae.

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Reciprocal recombination and the evolution of the ribosomal gene family of Drosophila melanogaster.

Genetics ◽

10.1093/genetics/122.3.617 ◽

1989 ◽

Vol 122 (3) ◽

pp. 617-624 ◽

Cited By ~ 1

Author(s):

S M Williams ◽

J A Kennison ◽

L G Robbins ◽

C Strobeck

Keyword(s):

Drosophila Melanogaster ◽

Gene Family ◽

Ribosomal Rna ◽

Natural Populations ◽

Ribosomal Gene ◽

Gene Region ◽

Ribosomal Rna Gene ◽

Reciprocal Recombination ◽

Y Chromosomes ◽

Length Polymorphisms

Abstract The role of reciprocal recombination in the coevolution of the ribosomal RNA gene family on the X and Y chromosomes of Drosophila melanogaster was assessed by determining the frequency and nature of such exchange. In order to detect exchange events within the ribosomal RNA gene family, both flanking markers and restriction fragment length polymorphisms within the tandemly repeated gene family were used. The vast majority of crossovers between flanking markers were within the ribosomal RNA gene region, indicating that this region is a hotspot for heterochromatic recombination. The frequency of crossovers within the ribosomal RNA gene region was approximately 10(-4) in both X/X and X/Y individuals. In conjunction with published X chromosome-specific and Y chromosome-specific sequences and restriction patterns, the data indicate that reciprocal recombination alone cannot be responsible for the observed variation in natural populations.

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