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Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models.pdf

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Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models.pdf

Fungal Genetics and Biology 49 (2012) 95–100 Contents lists available at SciVerse ScienceDirect Fungal Genetics and Biology journal homepage: /locate/yfgbi Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models Maíra Cristina Menezes Freire a, Maria Roméria da Silva a, Xuecheng Zhang b, álvaro Manuel Rodrigues Almeida c, Gary Stacey b, Luiz Orlando de Oliveira a,? a Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Vi?osa, 36570-000 Vi?osa (MG), Brazil b National Center for Soybean Biotechnology, Division of Plant Sciences and Biochemistry, University of Missouri, Columbia, MO, USA c Empresa Brasileira de Pesquisa Agropecuária, Centro Nacional de Pesquisa de Soja, Londrina, PR, Brazil article info Article history: Received 15 July 2011 Accepted 15 December 2011 Available online 3 January 2012 Keywords: Asian soybean rust ITS Phakopsora pachyrhizi Secondary structure abstract The assessment of nucleotide polymorphisms in environmental samples of obligate pathogens requires DNA ampli?cation 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 ?delity 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