Abstract
Tilletia species cause diseases on grass hosts with some causing bunt diseases on wheat (Triticum). Two of the four species infecting wheat have restricted distributions globally and are subject to quarantine regulations to prevent their spread to new areas. Tilletia indica causes Karnal bunt and is regulated by many countries while the non-regulated T. walkeri is morphologically similar and very closely related phylogenetically, but infects ryegrass (Lolium) and not wheat. Tilletia controversa causes dwarf bunt of wheat (DB) and is also regulated by some countries, while the closely related but non-regulated species, T. caries and T. laevis, both cause common bunt of wheat (CB). Historically, diagnostic methods have relied on cryptic morphology to differentiate these species in subsamples from grain shipments. Of the DNA-based methods published so far, most have focused on sequence variation among tested strains at a single gene locus. To facilitate the development of additional molecular assays for diagnostics, we generated whole genome data for multiple strains of the two regulated wheat pathogens and their closest relatives. Depending on the species, the genomes were assembled into 907 to 4633 scaffolds ranging from 24 Mb to 30 Mb with 7842 to 9952 gene models predicted. Phylogenomic analyses confirmed the placement of Tilletia in the Exobasidiomycetes and showed that T. indica and T. walkeri were in one clade whereas T. controversa, T. caries and T. laevis grouped in a separate clade. Single copy and species-specific genes were identified by orthologous group analysis. Unique species-specific genes were identified and evaluated as suitable markers to differentiate the quarantine and non-quarantine species. After further analyses and manual inspection, primers and probes for the optimum candidate genes were designed and tested in silico, for validation in future wet-lab studies.
Highlights
There are approximately 140 known species in the fungal genus Tilletia (Tilletiales, Ustilaginomycotina), all causing diseases on grass hosts in the Poaceae family
DNA/RNA extraction and sequencing Ten Tilletia strains representing five Tilletia species were selected for this study: T. caries DAOMC 238032, T. controversa DAOMC 236426 & DAOMC 238052, T. indica DAOMC 236414, DAOMC 236408 & DAOMC 236416, T. laevis DAOMC 238040 & American Type Culture Collection (ATCC) 42080, and T. walkeri DAOMC 236422 & DAOMC 238049
Paired end sequencing (2 × 300 bp) of genomic libraries of T. controversa DAOMC 236426 and DAOMC 238052, T. laevis DAOMC 238040 and ATCC 42080, T. indica DAOMC 236408 and DAOMC 236414, and T. walkeri DAOMC 238049 was performed on an Illumina MiSeq at the Molecular Technologies Laboratory at Agriculture and Agri-Food Canada
Summary
There are approximately 140 known species in the fungal genus Tilletia (Tilletiales, Ustilaginomycotina), all causing diseases on grass hosts in the Poaceae family. Tilletia species vary in their infection process but sporulation for most occurs in the plant ovary with host tissues in the kernel gradually replaced by masses of darkly pigmented teliospores, so-called “bunt balls”. Phylogenetic analyses of nuclear ribosomal (rDNA) large subunit sequences (LSU) demonstrated that the four pathogens on wheat and other grasses, and other species occurring on hosts in the subfamily Pooideae, form a large and well-supported clade within a monophyletic genus (Castlebury et al 2005). This large clade was subdivided, with T. indica and its closest relative T. walkeri on ryegrass (Lolium) in their own well-supported sub-clade with identical sequences. The other three wheat-infecting species were grouped with a dozen other species occurring on other hosts, in a well-supported subclade but with few nucleotide differences amongst species
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