MPSeqM, a tool combining multiplex PCR and high-throughput sequencing to study the polymorphism of eightLeptosphaeria maculansavirulence genes and its application to field surveys in France

Abstract

AbstractLeptosphaeria maculansis a fungal pathogen causing stem canker of oilseed rape (Brassica napus). The disease is mainly controlled by the deployment of varieties with major resistance genes (Rlm).Rlmgenes can rapidly become ineffective following the selection of virulent isolates of the fungus, i.e. with deletions or mutations in the corresponding avirulence genes (AvrLm). Reasoned and durable management ofRlmgenes relies on the detection and monitoring of virulent isolates in field populations. Based on previous knowledge ofAvrLmgene polymorphism, we developed a tool combining multiplex PCR and Illumina sequencing to characterise allelic variants for eightAvrLmgenes in fieldL. maculanspopulations.We tested the method on DNA pools of 71 characterisedL. maculansisolates and of leaf spots from 32L. maculansisolates. After multiplex-PCR and sequencing with MiSeq technology, reads were mapped on an in-houseAvrLmsequence database. Data were filtered using thresholds defined from control samples included in each run. Proportions of each allelic variant per gene, including deletions, perfectly correlated with expected ones. The method was then applied to around 1300 symptoms (42 pools of mainly 32 leaf spots) from nineB. napusfields. The proportions of virulent isolates estimated by sequencing leaf spot pools perfectly correlated with those estimated by pathotyping. In addition, the proportions of allelic variants determined at the national scale also correlated with those previously determined following individual sequencing ofAvrLmgenes in a representative collection of isolates. Finally, the method also allowed us to detect still undescribed and rare allelic variants. Despite the diversity of mechanisms generating virulent isolates and the gene-dependant diversity ofAvrLmgene polymorphism, the method proved suitable for large-scale and regular monitoring ofL. maculanspopulations, which will make it possible to choose effectiveRlmgenes and to detect resistance breakdowns at early stages.