The allopolyploid species B. napus originated from interspecific hybridization between B. rapa and B. oleracea about 7500 years ago. Due to this recent polyploidization event, the A and the C subgenomes of oilseed rape share high levels of sequence identity. High homeology among the subgenomes of B. napus results in a plethora of structural variations (SV) in the form of InDels, copy number variations (CNV), translocations or inversions. There have been several studies associating agronomically important traits such as disease resistance, flowering time and seed quality to SV in oilseed rape. These studies revealed the importance of SV in the creation of the de novo genetic variation necessary for adaptation and breeding. In this thesis, I elucidate different approaches for genome-wide detection and analysis of all size ranges of SV in B. napus.For the identification of large-scale SV this dissertation describes an integrated approach combining single nucleotide polymorphism (SNP) arrays, Illumina sequencing and optical mapping using resistance to Verticillium longisporum as an example for a quantitatively inherited trait in B. napus. A significant increase in the resolution of Verticillium resistance quantitative trait loci (QTL) was observed by including the SV in the form of single nucleotide absence polymorphism (SNaP) markers in the genetic map or genome-wide association studies (GWAS) model. Furthermore, presence absence variation (PAV) was observed in 23 to 51% of the genes within the Verticillium resistance QTL. Moreover, every high-priority candidate gene for Verticillium resistance within the QTL was affected by PAV. The widespread PAV in the rapeseed genome suggested that it is an important class of polymorphism and should be exploited more systematically in plant breeding programs.A majority of studies (including the one mentioned above) aimed at the genome wide identification of SV in plants have relied on Illumina sequencing. However, up to 89% false positive rate has been reported for SV calling with Illumina data. Furthermore, it is challenging to unambiguously align short Illumina reads to a reference assembly for a polyploid genome due to the high levels of sequence similarity among the homeologous regions. Therefore, there have been little or no efforts towards cataloguing small to mid-scale SV. This thesis describes the use of long sequencing reads to evaluate the role of small to mid-scale SV in eco-geographical diversification of B. napus into the three predominant ecotypes (winter-type, spring-type and semi-winter type), and survey their extent and impact on genes. Up to 10% of all genes in the rapeseed genome were found to be affected by small to mid-scale SV events. Nearly half of these SV events ranged between 100 bp to 1000 bp, which makes them challenging to detect using short read Illumina sequencing. Furthermore, small SV were also detected in the genes associated with Verticillium resistance in oilseed rape. This thesis also provides first insight and ideas about how new long-read sequencing technologies can help to understand complex SV in large plant genomes by providing additional layers of information, such as methylation signatures, chromatin confirmation, or data from target enrichment strategies implementing long-read sequencing, and describe potential cellular mechanisms that might explain the occurrence of small to mid-scale SV in oilseed rape. Additionally, the dissertation also reviews the challenges and limitations of the third-generation sequencing technologies.The key finding from this dissertation was the surprisingly high level of widespread, small to mid-scale SV in the rapeseed genome. This size range of SV is almost invisible to Illumina sequencing and was therefore completely ignored by the earlier studies aimed at detecting genomic re-arrangements in B. napus. The results from this dissertation suggest that revisiting complex plant genomes using medium-coverage, long-read sequencing might reveal unexpected levels of functional gene variation, with major implications for trait regulation and crop improvement.
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