Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi_vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance.
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.
Environmentally sensitive plant gene families like NBS-LRRs, receptor kinases, defensins and others, are known to be highly variable. However, most existing strategies for discovering and describing structural variation in complex gene families provide incomplete and imperfect results. The move to de novo genome assemblies for multiple accessions or individuals within a species is enabling more comprehensive and accurate insights about gene family variation. Earlier array-based genome hybridization and sequence-based read mapping methods were limited by their reliance on a reference genome and by misplacement of paralogous sequences. Variant discovery based on de novo genome assemblies overcome the problems arising from a reference genome and reduce sequence misplacement. As de novo genome sequencing moves to the use of longer reads, artifacts will be minimized, intact tandem gene clusters will be constructed accurately, and insights into rapid evolution will become feasible. Copyright © 2016 Elsevier Ltd. All rights reserved.
Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.
The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.
The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.