Key message “We identified both quantitative and quantitative resistance loci to Leptosphaeria maculans, a fungal pathogen, causing blackleg disease in canola. Several genome-wide significant associations were detected at known and new loci for blackleg resistance. We further validated statistically significant associations in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance in canola.” Blackleg, caused by Leptosphaeria maculans, is a significant disease which affects the sustainable production of canola (Brassica napus). This study reports a genome-wide association study based on 18,804 polymorphic SNPs to identify loci associated with qualitative and quantitative resistance to L. maculans. Genomic regions delimited with 694 significant SNP markers, that are associated with resistance evaluated using 12 single spore isolates and pathotypes from four canola stubble were identified. Several significant associations were detected at known disease resistance loci including in the vicinity of recently cloned Rlm2/LepR3 genes, and at new loci on chromosomes A01/C01, A02/C02, A03/C03, A05/C05, A06, A08, and A09. In addition, we validated statistically significant associations on A01, A07, and A10 in four genetic mapping populations, demonstrating that GWAS marker loci are indeed associated with resistance to L. maculans. One of the novel loci identified for the first time, Rlm12, conveys adult plant resistance and mapped within 13.2 kb from Arabidopsis R gene of TIR-NBS class. We showed that resistance loci are located in the vicinity of R genes of Arabidopsis thaliana and Brassica napus on the sequenced genome of B. napus cv. Darmor-bzh. Significantly associated SNP markers provide a valuable tool to enrich germplasm for favorable alleles in order to improve the level of resistance to L. maculans in canola.

BackgroundResistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS).ResultsDoubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response.ConclusionThrough multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0877-2) contains supplementary material, which is available to authorized users.

The hemibiotrophic fungus, Leptosphaeria maculans is the most devastating pathogen, causing blackleg disease in canola (Brassica napus L). To study the genomic regions involved in quantitative resistance (QR), 259–276 DH lines from Darmor-bzh/Yudal (DYDH) population were assessed for resistance to blackleg under shade house and field conditions across 3 years. In different experiments, the broad sense heritability varied from 43 to 95%. A total of 27 significant quantitative trait loci (QTL) for QR were detected on 12 chromosomes and explained between 2.14 and 10.13% of the genotypic variance. Of the significant QTL, at least seven were repeatedly detected across different experiments on chromosomes A02, A07, A09, A10, C01, and C09. Resistance alleles were mainly contributed by ‘Darmor-bzh’ but ‘Yudal’ also contributed few of them. Our results suggest that plant maturity and plant height may have a pleiotropic effect on QR in our conditions. We confirmed that Rlm9 which is present in ‘Darmor-bzh’ is not effective to confer resistance in our Australian field conditions. Comparative mapping showed that several R genes coding for nucleotide-binding leucine-rich repeat (LRR) receptors map in close proximity (within 200 Kb) of the significant trait-marker associations on the reference ‘Darmor-bzh’ genome assembly. More importantly, eight significant QTL regions were detected across diverse growing environments: Australia, France, and United Kingdom. These stable QTL identified herein can be utilized for enhancing QR in elite canola germplasm via marker- assisted or genomic selection strategies.

Summary In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one‐stage assay are more than double than those by two‐stage assay. This may be due to the longer incubation times (10–12 min) in the two‐stage assay. This study aimed to determine the time course of the activation phase of the two‐stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one‐stage and two‐stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short‐ or long‐incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23–56%, mean 41%) than after 10 min (19–41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21–64%, mean 37%) than with the longer incubation times usually used (13–29%, mean 23%). These time‐course experiments have verified that the longer incubation time used in the two‐stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

European winter canola (Brassica napus L.) cultivars harbour genes for durable resistance to the fungus Leptosphaeria maculans, which causes blackleg disease under Australian environmental conditions. Previous studies have shown that resistance in winter-type cultivars Maxol and Columbus is controlled by two genes, Rlm1 and Rlm3, which have been mapped using randomly amplified polymorphic DNA markers onto chromosome A7. We mapped a doubled-haploid population that consisted of 101 lines from a cross between Maxol*1 and Westar-10 using diversity arrays technology and simple sequence repeat (SSR)-based markers. Two SSR marker loci, Xol12-e03 and Xra2-a05b, flanked the Rlm1 locus at an interval of 6.7 cM, which corresponds to ~3.2 Mb of the Brassica rapa genomic sequence; this region contains several genes encoding putative kinase and leucine-rich repeat-type disease-resistance proteins. SSR markers were further tested for their linkage with the Rlm1 locus in an independent population derived from Columbus*3/Westar-10. Our results showed that SSR markers linked to Rlm1 can be useful for monitoring Rlm1 gene introgression in breeding populations derived from Maxol and Columbus.

Genomic prediction is becoming a popular plant breeding method to predict the genetic merit of lines. While some genomic prediction results have been reported in canola, none have been evaluated for blackleg disease. Here, we report genomic prediction for seedling emergence, survival rate, and internal infection), using 532 Spring and Winter canola lines. These lines were phenotyped in two replicated blackleg disease nurseries grown at Wickliffe and Green Lake, Victoria, Australia. A transcriptome genotyping-by-sequencing approach revealed 98,054 single nucleotide polymorphisms (SNPs) after quality control. We assessed various genomic prediction scenarios based on Genomic Best Linear Unbiased Prediction (GBLUP), BayesR and BayesRC, which can make use of prior quantitative trait loci information, via cross-validation. Clustering based on genomic relationships showed that Winter and Spring lines were genetically distinct, indicating limited gene flow between sets. Genetic correlations within traits between Spring and Winter lines ranged from 0.68 and 0.90 (mean = 0.76). Based on GBLUP in the whole population, moderate to high genomic prediction accuracies were achieved within environments (0.35-0.74) and were reduced across environments (0.28-0.58). Prediction accuracy within the Spring set ranged from 0.30-0.69, and from 0.19-0.71 within the Winter set. The BayesR model resulted in slightly lower accuracy to GBLUP. The proportion of genetic variance explained by known blackleg quantitative trait loci (QTL) was < 30%, indicating that there is a large reservoir of genetic variation in blackleg traits that remains to be discovered, but can be captured with genomic prediction. However, providing prior information of known QTL in the BayesRC method resulted in an increased prediction accuracy for survival and internal infection, particularly with Spring lines. Overall, these promising results indicate that genomic prediction will be a valuable tool to make use of all genetic variation to improve blackleg resistance in canola.

Blackleg disease causes yield losses in canola (Brassica napus L.). to identify resistance genes and genomic regions, genome-wide association studies (GWAS) of 585 diverse winter and spring canola accessions were performed using imputed whole-genome sequence (WGS) and transcriptome genotype-by-sequencing (GBSt). Blackleg disease phenotypes were collected across three years in six trials. GWAS were performed in several ways and their respective power was judged by the number of significant single nucleotide polymorphisms (SNP), the false discovery rate (FDR), and the percentage of SNP that validated in additional field trials in two subsequent years. WGS GWAS with 1,234,708 million SNP detected a larger number of significant SNP, achieved a lower FDR and a higher validation rate than GBSt with 64,072 SNP. A meta-analysis combining survival and average internal infection resulted in lower FDR but also lower validation rates. The meta-analysis GWAS identified 79 genomic regions (674 SNP) conferring potential resistance to L. maculans. While several GWAS signals localised in regions of known Rlm genes, fifty-three new potential resistance regions were detected. Seventeen regions had underlying genes with putative functions related to disease defence or stress response in Arabidopsis thaliana. This study provides insight into the genetic architecture and potential molecular mechanisms underlying canola L. maculans resistance. Abbreviations AFLP Amplified fragment length polymorphism BLAST Basic local alignment score tool BLUEs Best linear unbiased estimates DH Doubled haploid EMMAX Effective mixed model association expedited FDR False discovery rate GWAS Genome-wide association study LD Linkage disequilibrium MAF Minor allele frequency QC Quality control SNP Single nucleotide polymorphism GBSt Transcriptome genotype-by-sequencing VS Validation strategy WGS Whole-genome sequence

Sustainable canola production is essential to meet growing human demands for vegetable oil, biodiesel, and meal for stock feed markets. Blackleg, caused by the fungal pathogen, Leptosphaeria maculans is a devastating disease that can lead to significant yield loss in many canola production regions worldwide. Breakdown of race-specific resistance to L. maculans in commercial cultivars poses a constant threat to the canola industry. to identify new alleles, especially for quantitative resistance (QR), we analyzed 177 doubled haploid (DH) lines derived from an RP04/Ag-Outback cross. DH lines were evaluated for QR under field conditions in three experiments conducted at Wagga Wagga (2013, 2014) and Lake Green (2015), and under shade house conditions using the 'ascospore shower' test. DH lines were also characterized for qualitative R gene-mediated resistance via cotyledon tests with two differential single spore isolates, IBCN17 and IBCN76, under glasshouse conditions. Based on 18,851 DArTseq markers, a linkage map representing 2,019 unique marker bins was constructed and then utilized for QTL detection. Marker regression analysis identified 22 significant marker associations for resistance, allowing identification of two race-specific resistance R genes, Rlm3 and Rlm4, and 21 marker associations for QR loci. At least three SNP associations for QR were repeatedly detected on chromosomes A03, A07 and C04 across phenotyping environments. Physical mapping of markers linked with these consistent QR loci on the B. napus genome assembly revealed their localization in close proximity of the candidate genes of B. napus BnaA03g26760D (A03), BnaA07g20240D (A07) and BnaC04g02040D (C04). Annotation of these candidate genes revealed their association with protein kinase and jumonji proteins implicated in defense resistance. Both Rlm3 and Rlm4 genes identified in this DH population did not show any association with resistance loci detected under either field and/ or shade house conditions (ascospore shower) suggesting that both genes are ineffective in conferring resistance to L. maculans in Australian field conditions. Taken together, our study identified sequencebased molecular markers for dissecting R and QR loci to L. maculans in a canola DH population from the RP04/Ag-Outback cross. Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a major disease of the canola (Brassica napus L., A n A n C n C n , 2n = 4× = 38) industry especially in Australia, Canada, and France. Developing new cultivars resistant to blackleg is recognized as the most economically and environmentally safe strategy to control yield losses. Traditional breeding for blackleg resistance largely relies on the selection of elite 'good looking' lines in disease nurseries where there is high disease pressure from a diverse pathogen population. Utilizing this strategy, several canola varieties have been developed and released for commercial cultivation with ratings of moderate-to highly-resistant (to blackleg) without any prior knowledge of either r...