A highly multiplexed assay to monitor pathogenicity, 1 fungicide resistance and gene flow in the fungal wheat 2 pathogen Zymoseptoria tritici

and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in 23 the genetic composition of pathogen populations. Here we report the design of a microfluidics-based 24 amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and 25 randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici . The fungus causes 26 one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. 27 We optimized the primer design by integrating polymorphism data from 632 genomes of the same 28 species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of 29 the short-read sequence data generated by the assay showed a fairly stable success rate across samples to 30 amplify a large number of loci. The performance was consistent between samples originating from pure 31 genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed 32 genotypes, we found that the assay recovers variations in allele frequencies. We also explored the 33 potential of the amplicon assay to recover transposable element insertion polymorphism relevant for 34 fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population 35 structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more 36 sustainable crop protection and yields. We developed a microfluidics-based amplicon sequencing assay combining the advantages of high- sequencing and multiplex PCR. We assessed the performance of 798 loci to reliably and sensitively genotype randomly selected genome-wide markers, as well as pathogenicity and fungicide resistance-related genes in a diverse set of Z. tritici samples. We show that a large portion of the designed markers can be amplified consistently across samples, used to monitor the emergence of relevant mutations and provide an assessment of allele frequencies in mixed samples. The set of genome-wide markers provides means to assess the genetic structure of the pathogen directly from field collected wheat leaves.

and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in 23 the genetic composition of pathogen populations. Here we report the design of a microfluidics-based 24 amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and 25 randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes 26 one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance.

27
We optimized the primer design by integrating polymorphism data from 632 genomes of the same 28 species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of 29 the short-read sequence data generated by the assay showed a fairly stable success rate across samples to 30 amplify a large number of loci. The performance was consistent between samples originating from pure 31 genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed 32 genotypes, we found that the assay recovers variations in allele frequencies. We also explored the 33 potential of the amplicon assay to recover transposable element insertion polymorphism relevant for 34 fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population

38
Approximately 30 percent of all crop diseases are caused by fungi [1]. Plant pathogenic fungi affect crops 39 at various life cycle stages and plant tissues, including seeds, root and leaf development, and 40 inflorescence [2][3][4][5]. Yield reductions by pathogenic fungi cause food insecurity and economic losses 41 [6,7]. Crop protection is primarily achieved through the application of a variety of fungicides and 42 resistance breeding [8,9]. However, fungal pathogens have evolved resistance to all major fungicides

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The main advantage is the ability to generate long-reads capturing significant haplotype information of 88 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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For loci associated with pathogenicity on diverse cultivars, we retained a set of 67 amplicons successfully 140 passing primer design ( Table B in File S1). We also randomly selected SNPs at ~50 kb distances to 141 monitor the genetic make-up of populations for a total of 691 designed amplicons across all chromosomes 142 ( Table B in File S1). The random SNP set also included by chance the previously selected fungicide CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. 9 2D). In addition, we retained the amplicon for the mitochondrial resistance locus of CYTB with a read 178 count of 1,372,965. In summary, we retained 521 high-quality loci representing 75% of the randomly 179 selected markers designed for genetic structure analyses, as well as all 67 effector and 24 fungicide 180 resistance loci (Fig. 2D).

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Reproducibility among replicate assays and recovery of allele frequencies 183 To assess the reproducibility of the sequencing assay, we repeated the amplification and sequencing 184 procedure two times. We found that the number of read pairs recovered for each sample were positively  Table E in File S1). Loci without recent strong recent gains 218 more likely retained the IPO323 genotype (i.e. reference allele, Table E in File S1).  Table C in File S1). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. ; https://doi.org/10.1101/2022.07.18.500446 doi: bioRxiv preprint

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We developed a microfluidics-based amplicon sequencing assay combining the advantages of high-251 throughput sequencing and multiplex PCR. We assessed the performance of 798 loci to reliably and 252 sensitively genotype randomly selected genome-wide markers, as well as pathogenicity and fungicide

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made We designed at least one amplicon overlapping the most significantly associated SNP in each of the 359 effector genes. If a significantly associated SNP could not be reproduced in the worldwide isolate 360 collection, a random nearby SNP (within ~200 bp) was selected as the target for the amplicon design. If a 361 different SNP was selected, we filtered for SNPs with a minor allele count of 5 and a minimal genotyping  Table B in File S1 for details on all selected effector and 385 fungicide resistance genes as well as whole genome neutral markers. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. fungicides/sdhi-meeting-minutes/minutes-of-the-2020-sdhi-meeting-21-22th-of-january-521 2020-with-recommendations-for-2020.pdf?sfvrsn=5918499a_2 522 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (Table A)  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. Reference allele proportion sequenced . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 12, 2022. ; https://doi.org/10.1101/2022.07.18.500446 doi: bioRxiv preprint