STRAIN-LEVEL DIAGNOSTICS OF FUNGAL CROP PATHOGENS ENABLED BY FULLY-PHASED ISOLATE GENOMES

Crown rust is the most economically important disease in the domestic oat Avena sativa, and represents a challenge to oat growers worldwide. In the United States, total crop losses average between 2.2-3.1% annually (based on 10 year period 1996-2005) but sporadic outbreaks can have devastating impacts. In 2014, 35% of the oat crop in South Dakota was lost to crown rust while 50% was lost in Minnesota. The principle method for combating crown rust is the breeding of lines of oat resistant to endemic crown rust. However, experimental studies and field observations demonstrate a broadening virulence profile of crown rust races.Proximity ligation sequencing enables phased reference genomes Recent developments in genome sequencing technology have radically reduced the cost and time required to produce high quality reference genomes. One of the key methodologies to have emerged in recent years which has enabled a major step forward in genome assembly is the use of chromosome confirmation capture methods such as Hi-C. Hi-C is a class of proximity ligation sequencing methods that begins with chemically crosslinking DNA in intact nuclei to capture the three-dimensional structure of chromosomes within the nucleus. Crosslinked chromatin is cut by restriction digest and the ends filled-in with biotinylated nucleotides. The digested ends are then ligated together to form chimeric molecules composed of DNA sequences that were crosslinked together in the nucleus. The junctions of the chimeric molecules are enriched by streptavidin pull-down and sequenced using paired-end Illumina sequencing. By sequencing both sides of the chimeric junction, one can infer that these two sequences were in physical proximity in the intact nucleus.Sequences that reside on the same physical chromosome are significantly more likely to be crosslinked to each other simply because of the polymer dynamics of the chromosome molecule. Furthermore, the interaction frequency of any two sequences on the same chromosome follows a power-law decay based on the distance between the two sequences on the linear chromosome. This feature of Hi-C affords the ability to order and orient contigs to create end-to-end chromosome scaffolds by determining the frequency which two sequences are crosslinked together. This is one of the central applications Phase Genomics was founded on and since 2015 this technology has been used to generate hundreds of reference genomes from diverse taxa of plants, animals, and fungi. Furthermore, by combining single nucleotide variant information within the Hi-C read pairs with high contiguity long-read assemblies, we demonstrated that it is possible to fully phase haplotypes in diploid organisms. These features of proximity ligation sequencing enable generation of high-quality reference genome relatively routine.The dikaryotic nature of the Puccinia genomes adds an additional feature that can be uniquely addressed with Hi-C. As the initial crosslinking steps of this method occur prior to cell lysis, Hi-C interactions form exclusively between sequences that occupy the same compartment, be it cell or nucleus. Thus, in a dikaryon, both inter- and intra- chromosomal Hi-C junctions capture the physical location of all chromosomes within their respective nuclei and enable the separation and assembly of complete genomes from both nuclei at the same time. Using the information gathered from interchromosomal crosslinks it is possible to identify which homologs co-segregate within a single dikaryon. This has been recently demonstrated using Hi-C on the stem rust fungus Puccinia graminis f. sp. tritici. The methods used in this study clearly demonstrate the feasibility to generate phased dikaryon reference genomes.From Reference Genomes to Diagnostics Reference genomes for economically destructive pathogens can have important research implications for informing genome-wide association studies, comparative genomics between virulent strains, and understanding gene flow between virulent populations infecting crops across a geographic region. From a more practical standpoint, reference genomes enable the identification of a reduced set of markers which can be used to differentiate pathotypes of fungal species. This approach has recently been applied to Puccinia striiformis f. sp. tritici (Pst, causal agent of wheat stripe rust) where researchers used available reference genomes to identify highly polymorphic regions of Pst genomes to identify a reduced set of markers for lineage-level identification. They identified 242 regions that they amplified by polymerase chain reaction (PCR) and subjected to sequencing using the Oxford Nanopore Minion platform. Authors of this study achieved their goal of a field-deployable diagnostic tool for wheat yellow rust strains using only battery powered equipment and testing the system in the field in Ethiopia. However, the cost associated with the test is outside the range of feasibility other than for very well-funded academic or governmental organizations, with minimum price per sample in $1200-$1500 range based on list prices of reagents used in this publication. The strategy we propose is to put effort into the assembly of high-quality reference Pca genomes up front to reduce the cost of the diagnostic test. Having high confidence polymorphisms that can distinguish between strains can dramatically reduce cost for molecular diagnostics. Instead of the $1200-$1500/sample as above, it is likely that a multi-pool PCR based test could be deployed in $50-$70/sample range making surveillance and diagnosis of crown rusts practical even in a modestly sized agricultural setting.