Scientists have developed an improved method for capturing longer DNA fragments - doubling the size up to 7000 DNA bases that can be analysed for novel genes.
Researchers at the Earlham Institute (EI), The Sainsbury Laboratory (TSL) and the James Hutton Institute, have found a new way to decipher these large stretches of DNA to discover and annotate pathogen resistance in plants.
Using the PacBio, which can read longer stretches of DNA in their entirety, along with the developed NB-LRR gene workflow ‘RenSeq’ (Resistance gene enrichment sequencing), the data not only targets R genes, but also the important regulatory regions of DNA - promoters and terminators that signal when to start making a protein and when to stop.
Each plant typically carries hundreds of potential R gene sequences, encoding NB-LRR proteins, identified by the presence of specific sequence motifs. R genes are often part of families of closely related sequences.
Dr Matt Clark, Head of Technology Development at EI and lead author of the study, said: ‘Wild relatives of crops contain a huge repertoire of novel genes that could be used to breed more resistant varieties that need less pesticide treatments. When it comes to identifying key genes it can be very difficult for researchers to find the exact resistance gene due to the sheer similarity of their DNA sequences. Typical sequencing methods use short reads for example, from the Illumina HiSeq, but these are often too short to prise similar genes apart.’
‘RenSeq diverges from normal DNA sequencing on the PacBio by focusing the sequencing effort on a specific gene family i.e. R-genes stated Clark. 'In this study, by optimising multiple steps in the library construction, we can identify the protein-coding sequences and the neighbouring regulatory regions; indeed in many cases we can reconstruct the entire DNA region even if it contains many similar genes which normally are too hard to tell apart. This means we can identify the exact gene that confers resistance to a certain infection, and used in breeding programmes.’
Professor Jonathan Jones, senior scientist at The Sainsbury laboratory (TSL), said: ‘This improvement to the RenSeq method will greatly facilitate building reliable inventories of R genes in multiple plant species, helping us clone additional genes that could protect our crops.’
Dr Ingo Hein, principal investigator at the James Hutton Institute, added: ‘R genes can control diverse plant diseases including major threats to global crop production. The ability to capture and sequence long genomic DNA fragments that contain full-length R genes enables the rapid identification of novel, functional resistance genes from wild species. These genes, if introgressed into new cultivars via breeding or alternative routes, could significantly reduce the dependency on pesticides for crop production.’
The research has been recently published in a paper, ‘Targeted capture and sequencing of gene-sized DNA molecules’ is published in BioTechniques.