ORIGINAL PAPER Identifying genetic diversity of avirulence genes in Leptosphaeria maculans using whole genome sequencing Manuel Zander & Dhwani A. Patel & Angela Van de Wouw & Kaitao Lai & Michal T. Lorenc & Emma Campbell & Alice Hayward & David Edwards & Harsh Raman & Jacqueline Batley Received: 8 February 2013 / Revised: 7 May 2013 / Accepted: 12 May 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Next generation sequencing technology allows rapid re-sequencing of individuals, as well as the discovery of single nucleotide polymorphisms (SNPs), for genomic diversity and evolutionary analyses. By sequencing two isolates of the fungal plant pathogen Leptosphaeria maculans, the causal agent of blackleg disease in Brassica crops, we have generated a resource of over 76 million sequence reads aligned to the reference genome. We identi- fied over 21,000 SNPs with an overall SNP frequency of one SNP every 2,065 bp. Sequence validation of a selection of these SNPs in additional isolates collected throughout Australia indicates a high degree of polymorphism in the Australian population. In preliminary phylogenetic analysis, isolates from Western Australia clustered together and those collected from Brassica juncea stubble were identical. These SNPs provide a novel marker resource to study the genetic diversity of this pathogen. We demonstrate that re-sequencing provides a method of validating previous- ly characterised SNPs and analysing differences in im- portant genes, such as the disease related avirulence genes of L. maculans. Understanding the genetic char- acteristics of this devastating pathogen is vital in devel- oping long-term solutions to managing blackleg disease in Brassica crops. Keywords Leptosphaeria maculans . SNPs . Re-sequencing . Molecular markers . Blackleg disease . Brassica Introduction The most economically damaging pathogen of Brassica species, particularly canola (Brassica napus; rapeseed; oil- seed rape), is Leptosphaeria maculans (Howlett et al. 2001). L. maculans, a filamentous ascomycete, is the causal agent of phoma stem canker, commonly referred to as blackleg. In late stages of infection, it spreads through the stem vasculature causing lesions and leading to poor growth, lodging and eventually plant death. This fungus is found in canola-growing regions worldwide such as Australia, Canada and Europe. Increased production of canola in these regions has led to a rise in the severity of the disease. In Australia alone, L. maculans infection is responsible for an estimated Australian $100 million in crop losses each year, with average losses ranging from 15–48 % and significant efforts are underway to improve resistance to this disease (Tollenaere et al. 2012; Kaur et al. 2009). Understanding the characteristics of L. maculans is vital for developing an effective and sustainable approach to the management of blackleg disease on Brassica species. Given the threat imposed by this pathogen on the global rapeseed Electronic supplementary material The online version of this article (doi:10.1007/s10142-013-0324-5) contains supplementary material, which is available to authorized users. M. Zander : D. A. Patel : E. Campbell : A. Hayward : J. Batley (*) School of Agriculture and Food Sciences and Centre for Integrative Legume Research, University of Queensland, Brisbane, Queensland 4072, Australia e-mail: j.batley@uq.edu.au M. Zander : K. Lai : M. T. Lorenc : D. Edwards Australian Centre for Plant Functional Genomics and School of Agriculture and Food Sciences, University of Queensland, Brisbane, Queensland 4072, Australia A. Van de Wouw School of Botany, University of Melbourne, Melbourne, Victoria 3010, Australia H. Raman NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, New South Wales 2650, Australia Funct Integr Genomics DOI 10.1007/s10142-013-0324-5