Biology subjects

Rathjen, J.

Publications and source records attributed to Rathjen, J..

4 recordsLinked to original sources

bioRxiv2018

Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome sequencing from a portable DNA sequencing device as a method for detection of fungal pathogens from wheat (Triticum aestivum). The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), septoria tritici blotch (caused by Zymoseptoria tritici) and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified a bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria infections but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences in the sample. This could be addressed by amplicon-based sequencing approaches in future studies. Our work outlines a new approach for the detection of a broad range of plant pathogens and associated microbes using a portable sequencer, providing the basis for future development of a system for on-site disease monitoring.

plant biology↗

bioRxiv2018

A comprehensive toolkit to enable MinION long-read sequencing in any laboratory

Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realising their full potential relies crucially on extracting high quality, high molecular weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which potentiates all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time consuming and costly. Here we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimising DNA extractions from a challenging plant tissue as a case study. Following the workflow illustrated we were able to reliably and repeatedly obtain > 8.5 Gb of long read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open-source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls, will be useful to others who plan to establish long-read sequencing in their own laboratories.

genomics↗

bioRxiv2017

A near complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high inter-haplome diversity

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here we present a diploidaware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long-reads using the FALCON-Unzip assembler. RNA-seq datasets were used to infer high quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high inter-haplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When investigating genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors, and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.\n\nImportanceCurrent representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organisms evolutionary potential and ability to adapt to stress conditions. Yet it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which leads to allelic variation and the presence of genes lacking allelic counterparts. Non-allelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

microbiology↗

bioRxiv2017

De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust

Oat crown rust, caused by the fungus Puccinia coronata f. sp. avenae (Pca), is a devastating disease that impacts worldwide oat production. For much of its life cycle, Pca is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two Pca isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for a total assembly length of 99.16 Mbp for 12SD80 and 777 primary contigs with a total length of 105.25 Mbp for 12NC29, and approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of co-expressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in Pca.\n\nImportanceDisease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae (Pca), which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding Pca, resources to study the molecular mechanisms underpinning pathogenicity and emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of Pca as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of Pca.

genomics↗