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      Simultaneous monitoring and quantification ofMelampsora allii-populinaandMelampsora larici-populinaon infected poplar leaves using a duplex real-time PCR assay

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          New primers for promising single-copy genes in fungal phylogenetics and systematics

          Developing powerful phylogenetic markers is a key concern in fungal phylogenetics. Here we report degenerate primers that amplify the single-copy genes Mcm7 (MS456) and Tsr1 (MS277) across a wide range of Pezizomycotina (Ascomycota). Phylogenetic analyses of 59 taxa belonging to the Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes and Sordariomycetes, indicate the utility of these loci for fungal phylogenetics at taxonomic levels ranging from genus to class. We also tested the new primers in silico using sequences of Saccharomycotina, Taphrinomycotina and Basidiomycota to predict their potential of amplifying widely across the Fungi. The analyses suggest that the new primers will need no, or only minor sequence modifications to amplify Saccharomycotina, Taphrinomycotina and Basidiomycota.
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            Assessing the performance of single-copy genes for recovering robust phylogenies.

            Phylogenies involving nonmodel species are based on a few genes, mostly chosen following historical or practical criteria. Because gene trees are sometimes incongruent with species trees, the resulting phylogenies may not accurately reflect the evolutionary relationships among species. The increase in availability of genome sequences now provides large numbers of genes that could be used for building phylogenies. However, for practical reasons only a few genes can be sequenced for a wide range of species. Here we asked whether we can identify a few genes, among the single-copy genes common to most fungal genomes, that are sufficient for recovering accurate and well-supported phylogenies. Fungi represent a model group for phylogenomics because many complete fungal genomes are available. An automated procedure was developed to extract single-copy orthologous genes from complete fungal genomes using a Markov Clustering Algorithm (Tribe-MCL). Using 21 complete, publicly available fungal genomes with reliable protein predictions, 246 single-copy orthologous gene clusters were identified. We inferred the maximum likelihood trees using the individual orthologous sequences and constructed a reference tree from concatenated protein alignments. The topologies of the individual gene trees were compared to that of the reference tree using three different methods. The performance of individual genes in recovering the reference tree was highly variable. Gene size and the number of variable sites were highly correlated and significantly affected the performance of the genes, but the average substitution rate did not. Two genes recovered exactly the same topology as the reference tree, and when concatenated provided high bootstrap values. The genes typically used for fungal phylogenies did not perform well, which suggests that current fungal phylogenies based on these genes may not accurately reflect the evolutionary relationships among species. Analyses on subsets of species showed that the phylogenetic performance did not seem to depend strongly on the sample. We expect that the best-performing genes identified here will be very useful for phylogenetic studies of fungi, at least at a large taxonomic scale. Furthermore, we compare the method developed here for finding genes for building robust phylogenies with previous ones and we advocate that our method could be applied to other groups of organisms when more complete genomes are available.
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              Strain-dependent variation in 18S ribosomal DNA Copy numbers in Aspergillus fumigatus.

              Enumerating Aspergillus fumigatus CFU can be challenging since CFU determination by plate count can be difficult. CFU determination by quantitative real-time PCR (qPCR), however, is becoming increasingly common and usually relies on detecting one of the subunits of the multicopy rRNA genes. This study was undertaken to determine if ribosomal DNA (rDNA) copy number was constant or variable among different A. fumigatus isolates. FKS1 was used as a single-copy control gene and was validated against single-copy (pyrG and ARG4) and multicopy (arsC) controls. The copy numbers of the 18S rDNA subunit were then determined for a variety of isolates and were found to vary with the strain, from 38 to 91 copies per genome. Investigation of the stability of the 18S rDNA copy number after exposure to a number of different environmental and growth conditions revealed that the copy number was stable, varying less than one copy across all conditions, including in isolates recovered from an animal model. These results suggest that while the ribosomal genes are excellent targets for enumeration by qPCR, the copy number should be determined prior to using them as targets for quantitative analysis.
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                Author and article information

                Journal
                Plant Pathology
                Plant Pathol
                Wiley
                00320862
                April 2016
                April 2016
                June 26 2015
                : 65
                : 3
                : 380-391
                Affiliations
                [1 ]ANSES Laboratoire de la Santé des Végétaux; Unité de Mycologie; IFR110 EFABA, Domaine de Pixérécourt Malzéville 54220 France
                [2 ]Natural Resources Canada; Canadian Forest Service; Laurentian Forestry Centre; 1055 du PEPS, Stn. Sainte-Foy, PO Box 10380 Québec QC G1V 4C7 Canada
                [3 ]INRA; UMR1136, Interactions Arbres-Microorganismes; IFR110 EFABA; Champenoux 54280 France
                [4 ]Université de Lorraine; UMR1136, Interactions Arbres-Microorganismes; IFR110 EFABA; Vandoeuvre-lès-Nancy 54500 France
                Article
                10.1111/ppa.12426
                09366c32-525e-4868-bb35-71de80636ae4
                © 2015

                http://doi.wiley.com/10.1002/tdm_license_1.1

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