Gene Mapping And Characterization Of Major Late Blight Resistance Complex In Potato (Solanum Tuberosum)

Potato (Solanum tuberosum ), originated from Andes mountain-forests in South America, is the fourth most worldwide crop in the world after wheat, corn and rice. It plays important role in food security. Late blight caused by the oomycete pathogen Phytophthora infestans, one of the world's most devastating plant diseases, results in tremendous economic losses to global potato production. Due to high evolutionary potential resulted from mixed reproduction and high gene flow of P. infestans, resistance genes (R genes) introgressed into potato cultivars from the wild relative have always been overcome by new isolates that continually emerge in field. At present, R gene polyculture, a mixture of cultivars with different R genes in the same genetic background, is one of promising strategies to control late blight.To creat an R gene polyculture a dozen of R genes need to be isolated.In this study, by inheritance analyses and genetic mapping for R10 gene and R11 gene conferring resistance to late blight, we provide useful insight into resistance gene organization and function in major late blight resistance locus (MLB) in potato on the short arm of chromosome 11. And our work makes the starting point for map-based cloning of the R10 /R11 gene and discovery of resistance mechanism in wild potato population. The main results in this study are as follows:1. The R10 resistance specificity to late blight is conferred by both single dominant R gene and quantitative trait loci. The R10 gene confers broad resistance spectrum and was used widely in breeding programs. Progeny from the cross between the R10 differential and the susceptible cultivar Katahdin were assessed for late blight resistance to three Phytophthora infestans isolates in detached leaf assay. Some progeny displayed differential response to the three isolates. The resistance to the isolates IPO-0 and 99018 are controlled by quantitative trait loci, whereas the resistance to the isolate 89148-9 is inherited as a major dominant R gene, designated as R10 in this study. Statistical analyses revealed that resistance QTLs to isolate IPO-0 and 99018 is linked with the R10 gene that locates on telomeric part of chromosome 11, where the MLB was discovered. The clustering of the qualitative gene R10 with resistance QTLs may explain the field resistance observed on the R10 differential.2. The R10 gene conferring resistance to late blight in potato was mapped by comparative genomics combined with bulked segregant analysis (BSA), and a high-resolution genetic map of the R10 gene was constructed. R10, like R3a conferring resistance to late blight, is a member of MLB and locates on the short arm of chromosome 11. A segregating population of 195 genotypes was inoculated and analyzed. Six markers linked to R10 are developed using a combined strategy of comparative genomics and BSA. R10 resides between the two PCR markers 1001T and C2_At5g59960 that define a genetic interval of 2.6 cM, telomeric to the cloned R3a gene. With flanking markers of the R10 gene, 99 plants as being recombinant in R10 interval were identified from 1942 R10 segregating population, which were then tested for R10 with isolate 89148-9 and analyzed with markers developed. A high-resolution genetic map of the R10 gene was constructed. The R10 gene was delimited into a genetic interval of 0.26 cM between 656T and 1001T, as gives a base for MAS and further R10 cloning.3. The R11 gene conferring resistance to late blight in potato was located by comparative mapping with MLB. A F1 segregating population of 83 genotypes derived from R11 differential MaR11 and the susceptible potato cultivar Katahdin were assessed for late blight resistance in detached leaves assay and genetically analyzed. R11 is inherited as a major dominant R gene and is present in simplex in MaR11. Six markers linked to R11 are developed using a combined strategy of comparative mapping and BSA. R11 is located on the end of the short arm of chromosome 11 and is about 2.4 cM to marker C2_At5g59960. R11 is telomeric to the R3a and R10 gene. The genetic map in this paper gives a base for further construction of high-resolution genetic map of the R11 gene.4. System of magnetic separation for resistance genes and their analogs from potato BAC library was constructed. By alignment for sequences of the R3a gene family, the conserved probes of this gene family were developed. And then, the DNA of BAC SH23G23 containing the R3a gene was partially digested into 7-11kb fragments. By magnetic separation system combined with conserved probes, the 7-11 kb fragments were enriched and cloned into binary vector pBINPLUS. Through identification of positive clones and colony PCR, the ratio of clones including R3a gene in all positive clones reached 82.76%, which was nearly 19 times than without enrichment by magnetic separation system. Positional cloning is the main approach to isolate new R genes. However, it's very difficult to apply positional cloning on cultivated potato that is a auto-tetraploid. In this study, system of magnetic separation for R genes and their analogs provides a new strategy for gene cloning by candidate gene approach from tetraploid potato.