title: Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models.
abstract: The assessment of nucleotide polymorphisms in environmental samples of obligate pathogens requires DNA amplification through the polymerase chain reaction (PCR) and bacterial cloning of PCR products prior to sequencing. The drawback of this strategy is that it can give rise to false polymorphisms owing to DNA polymerase misincorporation during PCR or bacterial cloning. We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Phakopsora pachyrhizi, an obligate biotrophic fungus that causes the Asian soybean rust. Field-collected samples of P. pachyrhizi were obtained from all major soybean production areas worldwide, including Brazil and the United States. Bacterially-cloned, PCR products were obtained using a high fidelity DNA polymerase. A total of 370 ITS sequences that were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, the pattern of nucleotide polymorphisms, GC content, and the presence of conserved motifs. The sequences exhibited features of functional rRNAs. Overall, polymorphisms took place within less conserved motives, such as loops and bulges; alternatively, they gave rise to non-canonical G-U pairs within conserved regions of double stranded helices. We discuss the usefulness of structural analyses to filter out putative 'suspicious' bacterially cloned ITS sequences, thus keeping artificially-induced sequence variation to a minimum.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, 36570-000 Viçosa (MG), Brazil.
authors.forename: Maíra Cristina Menezes
authors.initials: MC
authors.lastname: Freire
authors.forename: Maria Roméria
authors.initials: MR
authors.lastname: da Silva
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.forename: Álvaro Manuel Rodrigues
authors.initials: ÁM
authors.lastname: Almeida
authors.forename: Gary
authors.initials: G
authors.lastname: Stacey
authors.forename: Luiz Orlando
authors.initials: LO
authors.lastname: de Oliveira
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017433:Protein Structure, Secondary; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013025:Glycine max / Q000235:genetics / Q000469:parasitology
chemical_list: D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S