Identification of Genome-Wide DNA Variants and SNP Haplotypes Associated with Avirulence Genes of Leptosphaeria Maculans in Western Canada

Abstract

BackgroundBlackleg, caused by Leptosphaeria maculans, is a serious disease of canola/ oilseed rape in many parts of the world. An integrated approach is needed to control the disease, with genetic resistance as a key component of the management strategy. Towards this goal, a reliable approach for L. maculans race structure assessment becomes essential to gain understanding of the frequency of avirulence genes and race groups of the pathogen, and provide guidance for deployment of resistant canola cultivars.ResultsA total of 162 representative isolates collected in western Canada were selected for genome re-sequencing with an Illumina platform. Assembly of the short reads against the reference genome of L. maculans 'brassicae' isolate v23.1.3 led to the discovery of 21,016 DNA variants (SNPs and InDels), 93% SNPs and 7% InDels, with a transition/transversion (Ts/Tv) ratio of 3.1 genome wide. InDels occurred mainly in GC-blocks and the Ts/Tv ratio of SNPs in AT-blocks was > 2 times higher than that in GC-blocks. The number of variants were positively correlated with supercontig size, GC-block size and gene numbers. DNA variants in most avirulence genes were SNPs, except a deletion in AvrLm1. The number of SNPs varied from 1–2 in AvrLm2, AvrLmJ1-5-9, AvrLm6, AvrLm10A, AvrLm10B and AvrLm11, 8 in AvrLm3 and 38 in AvrLm4-7. This study is the first report of triallelic SNPs in AvrLm2 and AvrLm4-7, and premature STOP codons in AvrLm4-7. Nine SNP haplotypes were identified in AvrLm4-7, however, only 2 ~ 3 haplotypes occurred in other avirulence genes, and in total 47 haplotype groups were identified from the isolates. The 47 SNP haplotype groups were translated into 44 protein haplotype groups and then isolates of L. macualns collected in western Canada were classified into10 races.ConclusionIn this study, we document the shortcoming of inferring races from SNP genotyping, and propose the use of SNP haplotyping for more reliable and informative analysis of L. maculans race structure.

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

“…However, they represented only 0.7% of the detected alleles in average, but could reach up to 7.8% in pools displaying numerous new alleles. Altogether, alleles encoding the AvrLm11_0 isoform were largely prevalent in the population (94.6% in average), while isoforms AvrLm11_1 or AvrLm11_2, previously described in Canada, USA or New Zealand (Chen et al, 2020; Van de Wouw et al, 2023) were never detected. AvrLm11 deletion was estimated at 3.1% in average, ranging from 0% to 15.4% depending on the sample.…”

“…Finally, whole genome sequencing (WGS) of individual isolates gives direct access to the sequences of the full set of AvrLm genes in an isolate. For instance, Chen et al (2020) studied the polymorphism in nine avirulence genes following the WGS of 162 Canadian isolates. Similarly, sequences of avirulence genes in an international collection of 205 L. maculans isolates were recently published (Van de Wouw et al, 2023).…”