Scientists from the UK published new work detailing important advances in protecting potatoes from the disease that caused the Irish potato famine in the 1800s. It’s not just of historical interest; the team points out that late blight disease is once again endangering the food supply, with global yields of potatoes shrinking in recent years.
“Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing,” published in Nature Biotechnology, comes from lead authors Kamil Witek and Florian Jupe, senior author Jonathan Jones, and collaborators at The Sainsbury Laboratory and The Genome Analysis Centre.
In the paper, the scientists describe an ongoing search among wild potato species for genetic resistance to Phytophthora infestans, the pathogen responsible for late blight disease. For this work, they looked for NLR genes (encoding nucleotide-binding, leucine-rich repeat proteins that are essential for activating plant defense mechanisms) in Solanum americanum, a wild Mexican potato relative. The process of enriching for resistance genes, which they call RenSeq, originally began with short-read sequencing but encountered difficulties in de novo assembly of NLR genes due to highly repetitive sequence and gene copies.
“To accelerate [resistance] gene cloning, and to remove any need for construction of a bacterial artificial chromosome or fosmid libraries, we refined RenSeq to capture and sequence fragments of up to 3.2 kb, which is the average NLR gene length,” the authors report. They incorporated SMRT Sequencing and found that most molecules were covered multiple times in individual reads, leading to highly accurate results.
In a comparison of short-read and long-read NLR assemblies, the scientists found that the short-read versions were as fragmented as they suspected, with just 21% of contigs including a full-length NLR gene. They determined that more than 300 long-read sequences were not represented in short-read data and identified several chimeric sequences in the short-read contigs. “The central positions of matching, aligned CLC or SPAdes NLR assemblies to long-read contigs further confirmed the superiority of SMRT RenSeq by also capturing >1 kb of flanking promoter and terminator sequences,” Witek et al. write. “Capture and sequencing of long fragments can resolve any repetitive gene family or structural genome variation by spanning repeat-rich regions with long reads.”
The team says this approach could be very useful in general for rapid engineering of pathogen-resistant crops. “The SMRT RenSeq method has the potential for use in investigating genetic variation for other important traits likely to involve known multigene families such as metabolic pathways (e.g., cytochromes P450, terpene cyclases) or transcription factors, especially if combined with mutagenesis,” they conclude.
May 26, 2016 | General