Long-read sequencing reveals widespread intragenic structural variants in a recent allopolyploid crop plant.

Genome structural variation (SV) contributes strongly to trait variation in eukaryotic species and may have an even higher functional significance than single-nucleotide polymorphism (SNP). In recent years, there have been a number of studies associating large chromosomal scale SV ranging from hundreds of kilobases all the way up to a few megabases to key agronomic traits in plant genomes. However, there have been little or no efforts towards cataloguing small- (30-10 000 bp) to mid-scale (10 000-30 000 bp) SV and their impact on evolution and adaptation-related traits in plants. This might be attributed to complex and highly duplicated nature of plant genomes, which makes them difficult to assess using high-throughput genome screening methods. Here, we describe how long-read sequencing technologies can overcome this problem, revealing a surprisingly high level of widespread, small- to mid-scale SV in a major allopolyploid crop species, Brassica napus. We found that up to 10% of all genes were affected by small- to mid-scale SV events. Nearly half of these SV events ranged between 100 bp and 1000 bp, which makes them challenging to detect using short-read Illumina sequencing. Examples demonstrating the contribution of such SV towards eco-geographical adaptation and disease resistance in oilseed rape 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.

A novel deletion in FLOWERING LOCUS T modulates flowering time in winter oilseed rape.

KEY MESSAGE: A novel structural variant was discovered in the FLOWERING LOCUS T orthologue BnaFT.A02 by long-read sequencing. Nested association mapping in an elite winter oilseed rape population revealed that this 288 bp deletion associates with early flowering, putatively by modification of binding-sites for important flowering regulation genes. Perfect timing of flowering is crucial for optimal pollination and high seed yield. Extensive previous studies of flowering behavior in Brassica napus (canola, rapeseed) identified mutations in key flowering regulators which differentiate winter, semi-winter and spring ecotypes. However, because these are generally fixed in locally adapted genotypes, they have only limited relevance for fine adjustment of flowering time in elite cultivar gene pools. In crosses between ecotypes, the ecotype-specific major-effect mutations mask minor-effect loci of interest for breeding. Here, we investigated flowering time in a multiparental mapping population derived from seven elite winter oilseed rape cultivars which are fixed for major-effect mutations separating winter-type rapeseed from other ecotypes. Association mapping revealed eight genomic regions on chromosomes A02, C02 and C03 associating with fine modulation of flowering time. Long-read genomic resequencing of the seven parental lines identified seven structural variants coinciding with candidate genes for flowering time within chromosome regions associated with flowering time. Segregation patterns for these variants in the elite multiparental population and a diversity set of winter types using locus-specific assays revealed significant associations with flowering time for three deletions on chromosome A02. One of these was a previously undescribed 288 bp deletion within the second intron of FLOWERING LOCUS T on chromosome A02, emphasizing the advantage of long-read sequencing for detection of structural variants in this size range. Detailed analysis revealed the impact of this specific deletion on flowering-time modulation under extreme environments and varying day lengths in elite, winter-type oilseed rape.

Effect of breeding on nitrogen use efficiency-associated traits in oilseed rape.

Oilseed rape is one of the most important dicotyledonous field crops in the world, where it plays a key role in productive cereal crop rotations. However, its production requires high nitrogen fertilization and its nitrogen footprint exceeds that of most other globally important crops. Hence, increased nitrogen use efficiency (NUE) in this crop is of high priority for sustainable agriculture. We report a comprehensive study of macrophysiological characteristics associated with breeding progress, conducted under contrasting nitrogen fertilization levels in a large panel of elite oilseed rape varieties representing breeding progress over the past 20 years. The results indicate that increased plant biomass at flowering, along with increases in primary yield components, have increased NUE in modern varieties. Nitrogen uptake efficiency has improved through breeding, particularly at high nitrogen. Despite low heritability, the number of seeds per silique is associated positively with increased yield in modern varieties. Seed weight remains unaffected by breeding progress; however, recent selection for high seed oil content and for high seed yields appears to have promoted a negative correlation (r= -0.39 at high and r= -0.49 at low nitrogen) between seed weight and seed oil concentration. Overall, our results reveal valuable breeding targets to improve NUE in oilseed rape.

Frequent spontaneous structural rearrangements promote rapid genome diversification in a Brassica napus F1 generation.

In a cross between two homozygous Brassica napus plants of synthetic and natural origin, we demonstrate that novel structural genome variants from the synthetic parent cause immediate genome diversification among F1 offspring. Long read sequencing in twelve F1 sister plants revealed five large-scale structural rearrangements where both parents carried different homozygous alleles but the heterozygous F1 genomes were not identical heterozygotes as expected. Such spontaneous rearrangements were part of homoeologous exchanges or segmental deletions and were identified in different, individual F1 plants. The variants caused deletions, gene copy-number variations, diverging methylation patterns and other structural changes in large numbers of genes and may have been causal for unexpected phenotypic variation between individual F1 sister plants, for example strong divergence of plant height and leaf area. This example supports the hypothesis that spontaneous de novo structural rearrangements after de novo polyploidization can rapidly overcome intense allopolyploidization bottlenecks to re-expand crops genetic diversity for ecogeographical expansion and human selection. The findings imply that natural genome restructuring in allopolyploid plants from interspecific hybridization, a common approach in plant breeding, can have a considerably more drastic impact on genetic diversity in agricultural ecosystems than extremely precise, biotechnological genome modifications.

Dissection of Quantitative Blackleg Resistance Reveals Novel Variants of Resistance Gene Rlm9 in Elite Brassica napus.

Blackleg is one of the major fungal diseases in oilseed rape/canola worldwide. Most commercial cultivars carry R gene-mediated qualitative resistances that confer a high level of race-specific protection against Leptosphaeria maculans, the causal fungus of blackleg disease. However, monogenic resistances of this kind can potentially be rapidly overcome by mutations in the pathogen's avirulence genes. To counteract pathogen adaptation in this evolutionary arms race, there is a tremendous demand for quantitative background resistance to enhance durability and efficacy of blackleg resistance in oilseed rape. In this study, we characterized genomic regions contributing to quantitative L. maculans resistance by genome-wide association studies in a multiparental mapping population derived from six parental elite varieties exhibiting quantitative resistance, which were all crossed to one common susceptible parental elite variety. Resistance was screened using a fungal isolate with no corresponding avirulence (AvrLm) to major R genes present in the parents of the mapping population. Genome-wide association studies revealed eight significantly associated quantitative trait loci (QTL) on chromosomes A07 and A09, with small effects explaining 3-6% of the phenotypic variance. Unexpectedly, the qualitative blackleg resistance gene Rlm9 was found to be located within a resistance-associated haploblock on chromosome A07. Furthermore, long-range sequence data spanning this haploblock revealed high levels of single-nucleotide and structural variants within the Rlm9 coding sequence among the parents of the mapping population. The results suggest that novel variants of Rlm9 could play a previously unknown role in expression of quantitative disease resistance in oilseed rape.