Identification, characterization and high-resolution mapping of resistance genes to Phytophthora infestans in potato

Potato ( Solanum tuberosum L.) is one of the most important crops in the world. The oomycete Phytophthora infestans (Mont. de Bary) is the causal agent of late blight which is the most devastating disease of the cultivated potato. It causes economic losses of several billion US dollars in crop production and protection annually worldwide. To control this disease, the application of enormous amounts of chemicals is required, which is expensive and environmentally unsafe. Also, late blight is increasingly more difficult to control because the pathogen is developing tolerance to the chemicals and new strains of the pathogen are evolving. Therefore, introduction of resistance genes from wild Solanum species into potato cultivars is considered as the most promising and environmental-safe approach to achieve durable late blight resistance.<o:p></o:p></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman"'>Late blight resistance exists in a wide range of Solanum wild species of which 75 % have a diploid constitution (2n=2x=24) although the majority of the cultivated potato is tetraploid (2n=4x=48). Therefore, several different approaches such as the use of 2n gametes, colchicine treatment, intraspecific hybridization, somatic hybridization, embryo rescue and gene transformation are used to overcome the difficulties caused by different ploidy levels. Of these approaches, gene transformation is the most controlled and time-efficient method to introduce resistance into the cultivated potato. However, gene identification and gene isolation are the first prerequisites for this approach.<o:p></o:p></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman"'>During the last century, most effort for late blight resistance was focused on the wild species S. demissum which contains at least 11 race-specific resistance genes ( R1 - R11 ). However, these resistances were rapidly overcome by new virulent pathogens which evolved. Therefore, new sources of late blight resistance are required and several resistances from different wild species such as S. microdontum , S. bulbocastanum , S. berthaultii<span style='mso-bidi-font-style:italic'>and</span>S . pinnatisectum<span style='mso-bidi-font-style:italic'>were characterized. In the present research, we investigated both foliage and tuber blight resistances in different mapping populations derived from different sources of Solanum species.<o:p></o:p></span></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>The pathogen P. infestans can infect both foliage and tuber of potato. To study tuber blight resistance, a wounded tuber assay was developed and the correlation between tuber and foliage blight resistance in four different mapping populations CE, SHRH, RH94-076 and RH4X-103, in which foliage blight resistances were identified, was examined (Chapter 2). Tuber blight resistance was found to be correlated with foliage blight resistance in three populations CE, SHRH and RH94-076, while it was independent in the RH4X-103 population. Further investigation to explore late blight resistances using molecular markers and resistance assays on both foliage and tuber with four different isolates was focused on the RH4X-103 population. Three specific resistance genes in foliage, tuber or both were segregating. The tuber blight resistance locus was closely linked to marker GP179 and co-segregating with the R1 -derived marker 76_2s on chromosome 5. The R1 gene functioned both as a foliage and tuber blight R gene, whereas t</span><span lang=EN-US style='font-family: "Times New Roman"'>he Rpi-abpt and R3a genes acted only on foliage.<o:p></o:p></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman"'>S. bulbocastanum</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>is one of the most promising wild</span><span lang=EN-US style='font-family:"Times New Roman"'>Solanum</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>species possessing late blight resistance. ABPT material, which is a quadruple hybrid created by bridge crosses using the four</span><span lang=EN-US style='font-family:"Times New Roman"'>Solanum</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>species;</span><span lang=EN-US style='font-family:"Times New Roman"'>S. <u>a</u> caule</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>,</span><span lang=EN-US style='font-family:"Times New Roman"'>S. <u>b</u> ulbocastanum</span><span lang=EN-US style='font-family:"Times New Roman"; mso-bidi-font-style:italic'>,</span><span lang=EN-US style='font-family:"Times New Roman"'>S. <u>p</u> hureja</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>and</span><span lang=EN-US style='font-family:"Times New Roman"'>S. <u>t</u> uberosum</span><span lang=EN-US style='font-family:"Times New Roman"'>,<span style='mso-bidi-font-style: italic'>to overcome incompatibility between</span>S. bulbocastanum<span style='mso-bidi-font-style:italic'>and</span>S. tuberosum<span style='mso-bidi-font-style: italic'>were used to introduce late blight resistance. A tetraploid mapping population (RH4X-103) containing the late blight resistance gene</span>Rpi-abpt<span style='mso-bidi-font-style: italic'>was created by a 4x-2x cross and was originally derived from ABPT material (Chapter 3). Resistance assay and AFLP marker analysis allowed localization of the</span>Rpi-abpt<span style='mso-bidi-font-style:italic'>locus on chromosome 4. The origin of the resistance was confirmed by the analysis of</span>Rpi-abpt<span style='mso-bidi-font-style:italic'>locus-linked AFLP markers and the resistance assay of ABPT-related wild species accessions. An extended population of 1383 offspring was screened to construct a high-resolution map where the Rpi-abpt locus was consequently localized in a 0.5 cM interval between flanking AFLP markers and was co-segregating with one AFLP marker. The translated protein sequence of the co-segregating AFLP marker appeared to be highly homologues to several disease resistance proteins.</span><o:p></o:p></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>The investigation of the race-specific R2-like gene was performed in a diploid mapping population (Chapter 4) primarily used for mapping nematode resistance. R2-like was phenotypically indistinguishable from the S. demissum -derived R2 gene although S. demissum is not directly involved in the pedigree of the population and</span><span lang=EN-US style='font-family:"Times New Roman"'>R2-like</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>is expected to originate from another species. The R2-like locus was placed in a 0.4 cM interval, flanked by two AFLP markers on chromosome 4 using the extended population of 1586 offspring. Four AFLP markers were also identified to be co-segregating with the R2-like locus.<o:p></o:p></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>A further late blight R gene ( Rpi-blb3</span><span lang=EN-US style='font-family: "Times New Roman"'>)<span style='mso-bidi-font-style:italic'>on chromosome 4 was identified in an intraspecific hybrid population derived from S. bulbocastanum (Chapter 5). The Rpi-blb3 locus was localized in a 0.9 cM interval between flanking AFLP markers and co-segregated with one AFLP marker in a population of 1396 offspring. According to the results of AFLP marker allele similarity tested by all co-segregating markers with four different R genes Rpi-abpt , R2-like , Rpi-blb3 and R2 , the four R genes appeared to be genetically very close and could be members of the same R gene cluster although the four R genes showed different race-specificities and origins.<o:p></o:p></span></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>Additional interesting scientific results came from the 4x-2x population (RH4X-103) used for late blight resistance. In this population, the male parent produced unreduced 2n pollen by FDR (First Division Restitution), allowing the localization of centromeres by using half-tetrad analysis (Chapter 6). 130 male parent-derived AFLP markers were analyzed and the genetic linkage group and location of 95 AFLP markers was determined by comparing them with the reference markers of the ultra high density (UHD) map of potato. Depending on the heterozygosity (simplex) or homozygosity (nulliplex or duplex) of the AFLP markers of the offspring, the position of centromeres and the genetic distance of the AFLP markers from the centromere were determined.</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-weight:bold'>In FDR 2n pollen, all the parental heterozygous loci from centromere to first crossover are expected to be heterozygous (Aa) resulting in simplex genotype (Aaaa) of their offspring, when they are combined with homozygous recessive gametes (aa) from the female parent. In contrast, a single crossover between the locus and centromere releases 50 % homozygous and 50 % heterozygous gametes in FDR.</span><span lang=EN-US style='font-family:"Times New Roman"; mso-bidi-font-style:italic'>The centromeres of 10 chromosomes were accurately localized.</span><span lang=EN-US style='font-family:"Times New Roman"; mso-bidi-font-weight:bold'>The centromere positions were compared with those, which were putatively identified in the UHD map by the fact that markers are dense due to suppression of recombination, and proved to be identical. The centromere position of two chromosomes could not precisely be determined by the half-tetrad analysis approach due to the absence of the 100 % heterozygous markers. However, their positions could be inferred</span><span lang=EN-US style='font-family:"Times New Roman"'>based on the combination of the results between the increasing or decreasing rate of heterozygosity in the half-tetrad analysis and marker density in the UHD map.<span style='mso-bidi-font-weight: bold'>Additional investigation of three chromosomes with sufficient numbers of markers proved that only one crossover occurs per chromosome arm, as a result of interference of recombination via centromere and telomere.<o:p></o:p></span></span><p class=MsoNormal style='line-height:16.0pt'><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>In conclusion, three different</span><span lang=EN-US style='font-family:"Times New Roman"'>R</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>genes against</span><span lang=EN-US style='font-family: "Times New Roman"'>P. infestans</span><span lang=EN-US style='font-family: "Times New Roman";mso-bidi-font-style:italic'>in foliage were identified and the loci were accurately determined. These R loci are genetically very close and could be allelic. Our research for tuber blight resistance showed that</span><span lang=EN-US style='font-family:"Times New Roman"'>R</span><span lang=EN-US style='font-family:"Times New Roman";mso-bidi-font-style:italic'>genes are not always active in both foliage and tuber. The results of the research described in this thesis demonstrate a great potential for further research. This will be directed towards the isolation of the R genes which can be used to introduce resistance into cultivated potato and to explore tuber blight resistance using a genetic transformation approach