Asset Tag: RenSeq

April 21, 2020  |  

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease of wheat throughout the world. One of the most important environmental-friendly and economical methods to reduce wheat loss caused by Bgt is to develop highly resistant varieties (Kuraparthy et al., 2007). Pm21 from the wild species Haynaldia villosa (also known as Dasypyrum villosum) confers high resistance to Bgt in wheat throughout all growth stages. It has now become one of the most highly effective genetic loci introgressed into wheat from wild species, and the commercial varieties harboring Pm21 have been widely used in wheat production with more than 4 million hectares in China.

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July 19, 2019  |  

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing.

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

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July 19, 2019  |  

SMRT RenSeq protocol

R gene enrichment and Sequencing (RenSeq, Jupe et al. 2013) is a genome complexity reduction method which allows to enrich for nucleotide-binding, leucine reach repeat (NLR) type plant disease resistance genes prior to sequencing. RenSeq was established and successfully used with Illumina platforms (Jupe et al. 2013, Andolfo et al. 2014), however the repetitive nature of NLR genes hampered de novo assembly of this family. Here we describe a protocol which enables to prepare long enriched libraries that are suitable for Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. Reads Of Inserts (ROI) generated with this protocol are around 3-4 kb in length (longer than the average NLR sequence). These long reads are especially well suited for de novo assembly of whole NLR genes including their regulatory elements

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July 19, 2019  |  

Targeted capture and sequencing of gene-sized DNA molecules.

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.

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July 7, 2019  |  

Resistance from relatives.

Crops are made resistant to pathogens such as wheat stem rust, Asian soybean rust and potato late blight by methods to access the pool of resistance genes present in related plants.

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July 7, 2019  |  

Sustaining global agriculture through rapid detection and deployment of genetic resistance to deadly crop diseases.

Contents Summary 45 I. Introduction 45 II. Targeted chromosome-based cloning via long-range assembly (TACCA) 46 III. Resistance gene cloning through mutational mapping (MutMap) 47 IV. Cloning through mutant chromosome sequencing (MutChromSeq) 47 V. Rapid cloning through resistance gene enrichment and sequencing (RenSeq) 49 VI. Cloning resistance genes through transcriptome profiling (RNAseq) 49 VII. Resistance gene deployment strategies 49 VIII. Conclusions 50 Acknowledgements 50 References 50 SUMMARY: Genetically encoded resistance is a major component of crop disease management. Historically, gene loci conferring resistance to pathogens have been identified through classical genetic methods. In recent years, accelerated gene cloning strategies have become available through advances in sequencing, gene capture and strategies for reducing genome complexity. Here, I describe these approaches with key emphasis on the isolation of resistance genes to the cereal crop diseases that are an ongoing threat to global food security. Rapid gene isolation enables their efficient deployment through marker-assisted selection and transgenic technology. Together with innovations in genome editing and progress in pathogen virulence studies, this creates further opportunities to engineer long-lasting resistance. These approaches will speed progress towards a future of farming using fewer pesticides.© 2017 Commonwealth of Australia. New Phytologist © 2017 New Phytologist Trust.

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