Dissection of the major late blight resistance cluster on potato linkage group IV

Potato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context.