Abstract
BackgroundZymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing.FindingsWe report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature.ConclusionThis easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.
Highlights
Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely
In order to increase the number of available Single sequence repeat (SSR) from which to choose markers to constitute the panels, microsatellites were mined from the fastaformatted genome of strain IPO-323 using the same pipeline of Perl scripts and C++ program that has been used to obtain microsatellites from EST sequences of higher plants [26]
The proposed multiplex PCR genotyping method, together with the automatic allele assignment procedure, constitutes a reliable tool for population genetics studies on Z. tritici. These ready-to-use methods enable important savings in terms of money, time, reproducibility and accuracy, and produce results in agreement with others previously published about Z. tritici populations
Summary
The proposed multiplex PCR genotyping method, together with the automatic allele assignment procedure, constitutes a reliable tool for population genetics studies on Z. tritici. These ready-to-use methods enable important savings in terms of money, time, reproducibility and accuracy, and produce results in agreement with others previously published about Z. tritici populations. The three markers with null alleles from the multiplexes 1 and 2 (i.e. ST11, ST8, ST1E7) may be as well omitted from certain analyses for the same reason These sets of optimized markers will be of great relevance to the Z. tritici scientific community, as they provide a method that can be applied in any laboratory and will allow facile comparison of studies from various locations, scales, and with different aims.
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