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.

New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida)

“…When using ITS for phylogenetic analysis, verification and annotation of amplicons are critical. Nonfunctional pseudogenes or chimeric sequences are readily recognizable by irregularities in the 5.8S rDNA and/or by the absence of some or all of the conserved regions of the ITS spacers (Freire et al., ; Harpke & Peterson, ; Hřibová et al., ; Rampersad, ). Only the GenBank clitellate sequences with recognizable 5.8S region were selected for primer design.…”

Asian Soybean Rust Resistance: An Overview

“…The high capacity of dispersion over long distances enables virulent genotypes of P. pachyrhizi move quickly between different populations [36]. In Nigeria, P. pachyrhizi has approximately 90% of their genetic diversity within the soybean fields, with little diversity distributed among the fields [35,[37][38][39].…”

The origin and genetic diversity of the causal agent of Asian soybean rust, Phakopsora pachyrhizi, in South America

“…This strategy imposed requirements for specialized laboratory resources and greenhouse facilities to handle the collection free of cross‐contamination. In other circumstances, environmental samples were taken directly from field‐collected leaves bearing uredinia and contained a pool of individuals of P. pachyrhizi with an unknown level of genetic relatedness (see Freire et al ., , ; Zhang et al ., ). When used as template for PCR, the pool of mixed DNA genotypes gave rise to DNA amplicons of distinct sizes.…”

“…pachyrhizi . In general, molecular data from distinct markers – microsatellites (Twizeyimana et al ., ) and DNA sequencing analyses (Freire et al ., , ; Zhang et al ., ) – were congruent in showing that genetic diversity in P. pachyrhizi displayed little structure over large geographic areas; most of the variation resided within locations. These findings suggested the existence of efficient mechanisms for long‐distance dispersal and large gene flow of P. pachyrhizi amongst soybean fields.…”

“…Under these circumstances, direct sequencing of PCR products yielded electropherograms bearing overlapping peaks and unreadable results. The strategy required bacterial cloning to obtain high‐quality electropherograms free of double peaks; each clone recovered a unique sequence from the pool of distinct PCR amplicons (Freire et al ., , ). Thus far, genetic diversity studies in P. pachyrhizi have been carried out using small sampling sizes per location.…”