ZymoSoups: A high-throughput forward genetics method for rapid identification of virulence genes in Zymoseptoria tritici

<p>These data were used to generate figures and summarise the number of mutations in 18 re-sequenced genomes of&nbsp;<em>Zymoseptoria tritici.&nbsp;</em>The isolates genotyped in this VCF file are all UV-mutegenized strains of the reference isolate IPO323.</p> <p><strong>Abstract</strong></p> <p><span><span>Septoria tritici blotch is caused by the fungus </span></span><em><span><span>Zymoseptoria</span><span> tritici</span> </span></em><span><span>and</span><span> poses a major threat to wheat </span><span>productivity</span><span>. </span><span>There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance </span><span>against this pathogen</span><span>, h</span><span>owever</span> <span>t</span><span>he corresponding genes in </span></span><em><span><span>Z. tritici</span></span></em><span><span> that confer virulence against distinct </span></span><span><span>R</span></span><span> <span>genes </span><span>remain</span> <span>largely unknown</span><span>. </span><span>In this study, we developed a</span> <span>rapid </span><span>forward genetics </span><span>methodology</span> <span>to </span><span>identify</span><span> genes that enable </span></span><em><span><span>Z. tritici</span></span></em><span><span> to gain virulence </span><span>on </span><span>previously resistant wheat varieties. </span><span>We</span><span> used the known</span><span> gene-for-gene interaction between </span></span><span><span>Stb6</span></span><span><span> and </span></span><span><span>AvrStb6</span></span><span><span> as </span><span>a </span><span>proof of concept that this method could quickly recover single candidat</span><span>e</span><span> virulence genes</span><span>. We subjected the avirulent </span></span><em><span><span>Z. tritici</span></span></em><span><span><em> </em>strain IPO323</span><span>, which carries the recogni</span><span>z</span><span>ed </span></span><span><span>AvrStb6</span></span><span><span> allele,</span><span> to UV mutagenesis</span><span> and</span><span> generat</span><span>ed</span><span> a library of over </span><span>66</span><span>,</span><span>000 </span><span>surviving spores</span><span>. </span><span>We screen</span><span>ed</span><span> these </span><span>survivors</span><span> on leaves of the resistant wheat variety Cadenza</span><span>,</span><span> in</span><span> mixtures</span> <span>(</span><span>soups</span><span>)</span><span> ranging from </span><span>100</span><span>-</span><span>500</span> <span>survivors</span><span> per soup. </span><span>We </span><span>identified</span><span> five soups</span><span> with </span><span>a </span><span>gain</span><span>-</span><span>of</span><span>-</span><span>virulence (</span><span>GoV</span><span>)</span><span> phenotype </span><span>relative</span><span> to the IPO323 parental strain</span><span> and re-sequenced 18 individual isolates, including four control isolates and two </span><span>isolates</span><span> lacking virulence</span><span>,</span><span> when screened </span><span>individually.</span> <span>Of </span><span>the </span><span>12 </span><span>confirmed </span><span>GoV</span> <span>isolates</span><span>, o</span><span>ne had a single nucleotide polymorphism (SNP) in the </span></span><span><span>AvrStb6</span></span><span><span> coding region. </span><span>T</span><span>he other 11 </span><span>GoV</span> <span>isolates</span> <span>exhibited</span> <span>large</span><span> (~70Kb)</span> <span>deletions at the end of chromosome 5, including the </span></span><span><span>AvrStb6</span></span><span><span> locus. </span><span>Our findings </span><span>demonstrate</span><span> the </span><span>efficiency </span><span>of this forward genetic approach in elucidating the genetic basis of qualitative resistance to </span></span><span><span>Z. tritici</span></span><span><span> and the </span><span>potential </span><span>to rapidly </span><span>identify</span><span> other</span><span>,</span><span> currently unknown</span><span>,</span> </span><span><span>Avr</span></span><span><span> genes in this pathogen.&nbsp;</span></span></p> <p><span><strong>Analysis pipeline</strong></span></p> <p><span>The pipeline used to generate this filtered vcf file and tab delimited file is publicly available at https://github.com/megancamilla/GATK4_Zymoseptoria</span></p> <p><span><strong>Isolate Data Accessibility</strong></span></p> <p><span><span>The individual fastq files for each isolate included in this study have been uploaded to NCBI's Short Read Archive (SRA), project number PRJNA1017776. This project </span><span>contains</span><span> 18 re-sequenced samples (SAMN37407989-SAMN37407972),</span> <span>which are UV mutagenized IPO323. Short Read Archive (SRA) accession numbers are SRR26072101-SRR26072118. </span>&nbsp;&nbsp;</span></p>