Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

Abstract

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.Key words: multilocus genotyping, polymorphisms, allelic discrimination, primer design. Received: February 16, 2009; Accepted: May 13, 2009. Classical analyses of the distribution of genetic diversity within and among populations have been used to identify patterns of migration and to reveal cryptic recombination in Rhizoctonia solani (Rosewich et al., 1999;Ceresini et al., 2002Ceresini et al., , 2007Ciampi et al., 2005Ciampi et al., , 2008 Linde et al., 2005;Bernardes de Assis et al., 2008), but information on global phylogeography does not exist for any Rhizoctonia pathosystem. Phylogeography is the study of historical processes responsible for the contemporary geographic distributions of individuals. Past events that can be inferred include population expansion, population bottlenecks, vicariance and migration (Karl and Avise, 1993;Avise, 2000).The classical studies on population genetics have used molecular markers, such as RAPD, ISSR, RFLP, and more recently microsatellite loci. However, these molecular markers are not fully suitable for studying the phylogeography of the fungus. A suitable marker would enable the implementation of the genealogical approach and the application of coalescent and phylogenetic tools for population-level questions (Brito and Edwards, 2008). Sequence variation from several stretches of anonymous DNA regions have been suggested as the marker of choice to infer phylogeographical history of species, for containing multiple and linked single nucleotide polymorphisms (SNPs), essential for constructing gene genealogies (Karl and Avise, 1993;Brito and Edwards, 2008). SNPs have simple patterns of variation, the potential for automated de...

Pathotypic and genetic diversity in the population of Rhizoctonia solani AG1‐IA causing rice sheath blight in China

“…Knowledge of the genetic structure of pathogen populations is essential to elucidate their life histories and the evolutionary potential in agricultural ecosystems (McDonald & Linde, ). The genetic structure of R. solani populations has been studied using different molecular markers, such as randomly amplified polymorphic DNA (RAPD) markers (Singh et al ., ; Wang et al ., ), restriction fragment length polymorphism (RFLP; Rosewich et al ., ), analysis of sequence variation in ribosomal DNA (rDNA) and β ‐tubulin genes (Fenille et al ., ; Ceresini et al ., ), inter‐simple sequence repeats (ISSR; Guleria et al ., ); amplified fragment length polymorphism (AFLP; Taheri et al ., ), single nucleotide polymorphisms (SNPs; Ciampi et al ., ) and simple sequence repeats (SSR; González‐Vera et al ., ; Padasht‐Dehkaei et al ., ). Many studies have shown that there is a high level of genetic variability in R. solani populations, and that several AGs possess homothallic and heterothallic mating systems.…”

Biological Activity of Four Trichoderma Species Confers Protection against Rhizoctonia solani, the Causal Agent of Cucumber Damping-Off and Root Rot Diseases

“…However, identification based on these interactions requires experience and may be affected by many factors, which can be time-consuming [14]. Different molecular markers were utilized to differentiate R. solani AGs [15][16][17]. However, the rDNA ITS1-5.8 S-ITS2 region sequence analysis has been verified as an increasingly effective proxy for genetic diversity and phylogenetic studies of R. solani AG and AG-subgroups [18,19].…”

Population Genetics and Anastomosis Group’s Geographical Distribution of Rhizoctonia solani Associated with Soybean

“…To evaluate genetic diversity and to characterize AG of R. solani , numerous molecular markers were used, which included inter-simple sequence repeats (ISSR) [ 23 ], simple sequence repeats (SSR) [ 24 , 25 ], single nucleotide polymorphisms (SNPs) [ 26 ], amplified fragment length polymorphism (AFLP) [ 27 ], restriction fragment length polymorphisms (RFLP) [ 28 ], randomly amplified polymorphic DNA (RAPD) markers [ 29 ], electrophoretic karyotype [ 30 ], DNA-DNA hybridization [ 31 ] and sequence analysis of rDNA ITS1-5.8 S-ITS2region [ 32 , 33 , 34 ]. Among these molecular markers, sequence analysis of rDNA ITS1-5.8 S-ITS2 region, because of their presence of multiple copies of tandem repeats within the genome of all fungi, has proven to be a more powerful tool for genetic diversity and phylogenetic studies of AG and AG subgroups of R. solani [ 15 , 18 , 34 , 35 , 36 , 37 ].…”