Multiplex SSR analysis of Phytophthora infestans in different countries and the importance for potato breeding

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-19th century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland and other parts of Europe. Late blight remains the most important pathogen to potato and causes a yearly multi-billion US dollar loss globally. In Europe and North America, late blight control heavily relies on the use of chemicals, which is hardly affordable to farmers in developing countries and also raises considerable environmental concerns in the developed countries. The structure of P. infestans populations can change quickly by migration, sexual recombination and sub-clonal variation. Migration and the reconvening of the two mating types considerably raised the level of genetic diversity in the global P. infestans population, leading to a more variable population with a presumed higher level of adaptability as compared to the previously, purely asexually, reproducing population. How can the P. infestans population efficiently be monitored with such diverse genotypes? A high-throughput, high-resolution and easy-handled set of markers would be favorable for this purpose. Few genetic markers, if any, have found such widespread use as SSRs. Sequencing allows the identification of large numbers of microsatellites by bioinformatics. So far, however, only a limited number of informative microsatellite loci had been described for P. infestans and none have been mapped. This thesis first describes the development and mapping of SSR markers in P. infestans and integration with other SSRs to generate a multiplex SSR set and its application in the population analysis of P. infestans from four countries are described with the developed multiplex SSRs. Finally, the use of this knowledge in resistance breeding of potato is shortly indicated and discussed. Chapter 1 describes the historic population changes of P. infestans at the global level and the current population trends. It summarizes microsatellite as favorite molecular markers for studying pathogen population diversity and assesses monitoring of population dynamics in more detail for resistance breeding in potato. The selection and identification of new SSR markers is presented in Chapter 2. From EST and genomic sequences from P. infestans we identified 300 non-redundant SSR loci by a bioinformatic screening pipeline. Based on the robustness, level of polymorphism and map position eight SSR markers were selected, which were assembled in two multiplex PCR sets and labeled with two different fluorescent dyes to allow scoring after single capillary electrophoresis. This successful multiplex SSR approach encouraged the development of fast, accurate and high-throughput genotyping, in an one-step multiplex PCR method to facilitate worldwide screening of P. infestans populations. Published SSRs and the 8 new SSRs were integrated. All these SSR markers were re-evaluated and the 12 most informative SSRs were selected to set up a standard set for global application (Chapter 3). The 12-plex SSRs are distributed over different chromosomes, significantly increasing the resolution of genotyping compared to the previous set of 8 SSRs. The 12-plex SSRs were integrated to one-step fluorescence-based multiplex reaction, which plays a key role to facilitate highly paralleled genotyping and efficient dissection of the more complex P. infestans populations. This multiplex PCR for P. infestans populations is (i) simple, as only one PCR is needed to perform multi-locus typing with twelve markers; (ii) rapid, as the genotyping results can be available in 1 day; and (iii) reproducible and adapted to different laboratories. The genotyping data from different geographic populations were submitted to the Euroblight database. With the same SSR set and the bin set, a comparable global database can easily be achieved. As indicated earlier, more recent analyses of P. infestans populations highlight the appearance of many new genotypes via migration and/or sexual recombination. To practice the newly developed 12-plex SSR set and dissect the current population structure, several P. infestans populations from 4 different continents were selected for analysis. These include Chinese (Chapter 4), Dutch (Chapter 5), Ecuadorian (Chapter 6) and Tunisian (Chapter 7) populations. China has become the largest potato producing country not only for potato cultivation area but also in Megaton potato production. Interprovincial trade of consumption and seed potatoes is very important and frequent in China. Although both, the A1 and A2 mating types are found in China, to this date, no evidence of an active sexual cycle based on changes in allele frequency was found. With the ten SSRs, a large genotypic survey of in nation-wide collection of 228 P. infestans isolates was performed (Chapter 4). One of the three dominant clonal lineages CN-04 (A2) in this Chinese population was genetically similar to a major clonal lineage identified in Europe, called “Blue_13” with A2 mating type. It was not possible to critically assess the origin of this clonal lineage. This study is the first report of “Blue_13” outside Europe. The virulence spectrum of selected Chinese P. infestans isolates showed seven different virulence spectra varying from 3 to 10 differentials. The CN04 genotypes were identified as more aggressive and more virulent genotypes, one of whom had the full virulence pattern after using the potato differential set. Within the Chinese P. infestans population, the genotypes strongly clustered according to their six sampling provinces, which seems not to be influenced by the frequent interprovincial trading activities of seed potatoes. The mating type ratio and the SSR allele frequencies indicate that in China the contribution of the sexual cycle to P. infestans on population dynamics is minimal. It was concluded that the migration through asexual propagules and the generation of sub-clonal variation are the dominant driving factors behind the Chinese P. infestans population structure. The Netherlands has a long history of population studies on local P. infestans isolates and a substantial amount of commercial potato varieties growing in the field. One decade (2000-2009) of isolate sampling in 5 different regions provided the basis for a good understanding of the population dynamics in the Netherlands (Chapter 5). The surveyed population revealed the presence of several clonal lineages and a group of sexual progenies. The major clonal lineage with A2 mating type is known as “Blue_13”, but also two distinct clonal lineages with A1 mating type in this study have been identified. This survey witnesses that the Dutch population was undergoing dramatic changes in the ten years under study. The most notable change was the emergence and spread of A2 mating type strain “Blue_13”. The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation- and dispersal mechanism for successful genotypes. In addition to the neutral SSR markers a molecular marker for the virulence of isolates on potato lines that contain the Rpi-blb1 R-gene has been developed. Using this Avr-blb1 marker and the corresponding virulence assay we report, for the first time, the presence of Rpi-blb1 breaker isolates in the Netherlands even before a Rpi-blb1 containing resistant variety was introduced. The 12 breaker isolates only occurred in sexual progeny. So far the asexual spread of such virulent isolates has been limited because of the absence of Rpi-blb1 containing varieties in the field. Remarkably, on the other end of the world in the Andes, the region of potato origin, the situation is far less complex as far as P. infestans is concerned. There are more than 400 potato landraces in Ecuador and the planting habit by local farmers by traditional cultivation at small scale in the highlands is different from potato cultivation in other potato countries in North America or Europe (Chapter 6). Phytophthora isolates in Ecuador belong to two closely related species, P. infestans (on potato and tomato) and P. andina (on non-tuber bearing host), but SSR analysis of 66 isolates indicated that the two species are separated in two clearly distinguished genetic groups. Two ancient clonal lineages of P. infestans appeared to be dominant in Ecuador one is found only on tomato the other one only on potato. Within the potato isolates, but not in the tomato isolates, there is a large sub-clonal variation caused by (partial) polyploidization and loss of alleles. In Tunisia, potato is cultivated in three to four partly overlapping seasons while tomato is grown either in greenhouses or as aerial crop in most potato producing regions. Chapter 7 revealed, among 165 isolates of five regions, the presence of a major clonal lineage (NA-01, A1 mating type, Ia mtDNA haplotype) that seems to consist of races that are relatively simple. Another highly genetic diverse group of isolates was found containing more complex races and isolates with both mating types. Season clustering indicated that at least some of the new genotypes generated by sexual reproduction overlapped between seasons and such a sexual progeny may play an important role in the next season epidemics. On tomato, mostly asexual progeny was identified with two mtDNA haplotypes but less nuclear genotypes, compared to potato. This study shows that the P. infestans population is currently changing, and the old clonal lineage is being replaced by a more complex, genetically diverse and sexually propagating population in two sub-regions in Tunisia. Despite the massive import of potato seeds from Europe, the P. infestans population in Tunisia is still clearly distinct from the European population. Chapter 8 discusses the application of microsatellites in monitoring genetic diversity of late blight and the potential use in resistance breeding. Monitoring of the local P. infestans population for new virulent genotypes with the differential potato set in combination with screening for effector variation, allows early detection of adaptation of certain genotypes within the P. infestans population to particular resistance genes in a specific region. This provides the possibilities to determine which broad spectrum R-genes are still useful in order to adapt the control strategy by resistance breeding to the new situation. One way of doing that is to replace the existing varieties by other varieties with stacked non-broken R-genes obtained by marker assisted selection or to add additional R-genes to existing (R-gene containing) varieties by transformation. In a transgenic or cisgenic approach, additional broad spectrum R-genes could be added by re-transformation. As we have shown, the right R-gene management strategy in potato breeding, but also in potato production, should include the direct monitoring of local pathogen populations by using the differential set and the 12-plex SSR set.