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

Leptosphaeria maculansis a pathogen of oilseed rape (Brassica napus), mainly controlled by major resistance genes (Rlm) in varieties.Rlmgenes can rapidly be overcome following the selection of virulent isolates with deletions or mutations in the corresponding avirulence genes (AvrLm). Reasoned management ofRlmgenes relies on the detection and monitoring of virulent isolates in populations. Based on previous knowledge ofAvrLmgene polymorphism, we developed a tool combining multiplex PCR and Illumina sequencing to characterize allelic variants for eight avirulence genes in fieldL. maculanspopulations.We tested the method on DNA pools of 71 characterizedL. 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 applied to around 1300 symptoms (42 pools of mainly 32 leaf spots) from nine fields. The proportion 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 of eightAvrLmgenes in a representative collection of isolates. Finally, the method also allowed us to detect still undescribed and rare allelic variants. Altogether, the method appeared suitable for large-scale and regular monitoring ofL. maculanspopulations.