Genome Sequence and Heterokaryosis in the Oomycete Pathogen, Bremia lactucae

Bremia lactucae is a highly specialized obligate biotrophic oomycete pathogen that causes downy mildew of lettuce. Genome assembly has been challenging due to high levels of heterozygosity. Several technologies have been used to sequence and de novo assemble multiple isolates. High quality assemblies have been generated of isolates SF5 (Illumina, Moleculo & Pac Bio) and C82P24 (Illumina only). The draft assembly of SF5 currently contains 667 scaffolds spanning 116 Mb with an N50 of 529 Kb. To ensure that these scaffolds were not over-assembled, we re-sequenced and de novo assembled both strains using Pac Bio (RSII) long read technology and both assemblies were aligned to check for collinearity. Mapping Illumina reads of multiple modern and historical field isolates to a reference assembly of SF5 revealed multiple distributions of allele frequencies. Haploid genome size estimates measured by flow cytometry were consistently ~150 Mb, providing no evidence for polyploidy in B. lactucae. Additionally, allele frequencies in eight F₁ progeny derived from SF5 (1:1 allele frequencies) and C82P24 (1:3 allele frequencies) were 1:1. Kinship analysis identified two sets of progeny from this cross, indicating that they were derived from three different parental nuclei. Sequencing of single spore isolations of C82P24 is under way to investigate the possibility of heterokaryosis.