Genome structural variation associates with fungal quantitative disease resistance in oilseed rape (Brassica napus L.)

Abstract

Brassica napus L. (oilseed rape/canola) originated through spontaneous interspecific hybridisation between turnip rape (Brassica rapa L., syn. campestris; genome AA, 2n = 20) and cabbage (Brassica oleracea L.; genome CC, 2n = 18), resulting in an amphidiploid genome comprising the full chromosome complements of its two progenitors. Because no wild B. napus forms are known, it is assumed that the species arose relatively recently, when the parental species began being cultivated in geographical proximity due to anthropogenic influences. The occurrence of spontaneous chromosome doubling in crosses among closely related Brassica diploid species and the high homoeology between the diploid progenitor genomes (A and C subgenomes) have led to extensive structural genome variation within the crop genome. New genotyping methods that can generate large datasets from individual genotypes are available nowadays also for oilseed rape. Technologies like the 60k Illumina SNP array, short-read next-generation sequencing and long-read 3rd generation sequencing can provide valuable, extremely detailed insight into genome structural rearrangements generated during interspecific hybridisation between related diploid species. The impact of genome wide structural variations on plant phenotypes is of crucial importance to scientists and plant breeders in order to optimize parental crosses with maximal genetic gain. To address this issue, the thesis describes effects of small and long range structural variation on three major fungal pathogens of oilseed rape. A diverse nested association mapping (NAM) population of 200 lines, representing crosses among five resynthesized B. napus accessions with a common elite B. napus donor, was genotyped using the 60K single-nucleotide-polymorphism (SNP) Infinium array. Phenotypic evaluations for disease resistance to Phoma stem canker/blackleg (Leptosphaeria maculans), Sclerotinia stem rot (Sclerotinia sclerotiorum) and Verticillium stem striping (Verticillium longisporum) were performed in greenhouse experiments and field trails across France and Germany in order to identify qualitative and quantitative resistances. After analysis on population structure, detailed measurements of genetic diversity and linkage disequilibrium, a genome-wide association study (GWAS) was performed for each disease. Major QTL regions were identified on various chromosomes that were highly associated to disease resistance. Additionally, the presence of co-localising QTL regions among the three studied pathogens was observed, indicating putative multiple-disease resistance mechanisms. The key finding of the dissertation is a novel strategy to recover valuable information from single nucleotide absence polymorphisms (SNaP) by population-based quality filtering of SNP hybridization data to distinguish patterns associated with genuine deletions from those caused by technical failures. Standard data quality filtering approaches can remove large numbers of potentially useful marker information that can mask QTL caused by structural variations. The thesis results indicate that presence-absence variation has a strong influence on quantitative disease resistance in B. napus and that SNaP analysis using cost-effective SNP array data can provide extensive added value from missing data . For blackleg disease, 50 QTL were mapped in this study using SNP and SNaP markers. Partial resistance to Sclerotinia stem rot was found, and 37 QTL were mapped using SNP and SNaP markers. From these, only seven were previously reported in the literature. After adding SNaP markers, a 1.6 -to 3.5-fold increase in detected QTL regions associated with blackleg and Sclerotinia stem rot resistance was observed, corresponding to a total of 57 new QTL detected. Most of the additional QTL for V. longisporum resistance detected in the NAM panel contained only SNaP markers (18 out of 28 QTL), suggesting that regions affected by long-range presence/absence variation strongly affect quantitative disease resistance. Re-analysis and integration of SNP array data, short-range next generation sequencing data and long-range BioNano optical mapping data with QTL data provided new insights into the importance of structural variations for disease resistance expression against major fungal pathogens of oilseed rape. The frequent localization of new QTL in regions affected by structural genome variations confirms the hypothesis that copy-number and presence-absence variations have particular relevance for disease resistance. Offspring of resynthesized B. napus with high rates of presence-absence and other structural variations may therefore have an increased potential for use in resistance breeding of B. napus. This strategy might also be applicable for improving the precision of genetic mapping in many important crop species.