Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant 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 enables 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 optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 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 and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long-read sequencing in their own laboratories.

Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f.sp. tritici, is a major threat to wheat production worldwide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole-genome comparative genomics of two P. striiformis f.sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural, and allele levels. At the whole-genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates, whereas 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall, this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f.sp. tritici is the first step in understanding the evolution of dikaryotic rust fungi at a whole-genome level.

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Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 h after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.