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Field surveys indicate blackleg of canola has increased in western Canada since 2010. Earlier studies showed changes in the pathogen (Leptosphaeria maculans (Sowerby) P.Karst.; Lm) population betwe...

Plasmodiophora brassicae, an obligate soilborne pathogen that causes clubroot on Brassica crops, is spreading rapidly in western Canada, threatening canola production in the region. Bioassays and molecular assays have been used to estimate the concentration of P. brassicae resting spores in soil, which can affect clubroot incidence and severity on crops. Droplet digital PCR (ddPCR) is a promising new approach for quantification of pathogen inoculum owing to its low sensitivity to inhibitors and consistency at low target concentrations. The objective of this study was to assess ddPCR against existing quantitative PCR (qPCR) for potential advantage and/or improvement in quantifying P. brassicae resting spores in soil. The new protocol enumerated resting spores accurately in spiked potting mix or soil samples ranging from 102 to 107 spores per gram. At a spore concentration ≥107 spores per gram, however, ddPCR became less accurate, with a tendency of overestimation. The protocol was validated by quantifying the resting spores in spiked brown, dark brown, and black soils using both ddPCR and qPCR simultaneously. These soil types are found commonly on the Canadian Prairies, and they vary in texture, pH, and organic content. ddPCR showed similar results among the different soil types, whereas qPCR often displayed lower counts for the same spore concentration, with the amplification of DNA inhibited completely in black soil samples. The inhibition can be removed by a 10-fold dilution of DNA samples. The results show that ddPCR can be a more versatile tool than qPCR for detection and quantification of P. brassicae resting spores in soil samples.

Blackleg, caused by Leptosphaeria maculans, is a serious disease on canola or rapeseed in Canada, Australia and Europe. Disease levels have increased in western Canada recently and additional tools...

Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in Arabidopsis thaliana (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the Arabidopsis background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of brassica crops worldwide. Vegetable turnip (Brassica rapa L.) have proven to be a source of clubroot resistance genes effective against many pathotypes of P. brassicae. The F

Plasmodiophora brassicae causes clubroot disease in brassica crops worldwide. Brassica rapa, a progenitor of Brassica napus (canola), possesses important sources for resistance to clubroot. A doubled haploid (DH) population consisting of 84 DH lines were developed from a Backcross2 (BC2) plant through an interspecific cross of B. rapa turnip cv. ECD01 (resistant, R) with canola line DH16516 (susceptible, S) and then backcrossed with DH16516 as the recurrent parent. The DH lines and their parental lines were tested for resistance to four major pathotypes (3A, 3D, 3H, and 5X) of P. brassicae identified from canola. The R:S segregation ratio for pathotype 3A was 1:3, and 3:1 for pathotypes 3D, 3H, and 5X. From genotyping by sequencing (GBS), a total of 355.3 M short reads were obtained from the 84 DH lines, ranging from 0.81 to 11.67 M sequences per line. The short reads were aligned into the A-genome of B. napus “Darmor-bzh” version 4.1 with a total of 260 non-redundant single-nucleotide polymorphism (SNP) sites. Two quantitative trait loci (QTLs), Rcr10ECD01 and Rcr9ECD01, were detected for the pathotypes in chromosomes A03 and A08, respectively. Rcr10ECD01 and Rcr9ECD01 were responsible for resistance to 3A, 3D, and 3H, while only one QTL, Rcr9ECD01, was responsible for resistance to pathotype 5X. The logarithm of the odds (LOD) values, phenotypic variation explained (PVE), additive (Add) values, and confidence interval (CI) from the estimated QTL position varied with QTL, with a range of 5.2–12.2 for LOD, 16.2–43.3% for PVE, 14.3–25.4 for Add, and 1.5–12.0 cM for CI. The presence of the QTLs on the chromosomes was confirmed through the identification of the percentage of polymorphic variants using bulked-segregant analysis. There was one gene encoding a disease resistance protein and 24 genes encoding proteins with function related to plant defense response in the Rcr10ECD01 target region. In the Rcr9ECD01 region, two genes encoded disease resistance proteins and 10 genes encoded with defense-related function. The target regions for Rcr10ECD01 and Rcr9ECD01 in B. napus were homologous to the 11.0–16.0 Mb interval of chromosome A03 and the 12.0–14.5 Mb interval of A08 in B. rapa “Chiifu” reference genome, respectively.

The fungal pathogen Leptosphaeria maculans causes blackleg disease on canola and rapeseed (Brassica napus) in many parts of the world. A B. napus cultivar, ‘Quinta’, has been widely used for the classification of L. maculans into pathogenicity groups. In this study, we confirmed the presence of Rlm1 in a DH line (DH24288) derived from B. napus cultivar ‘Quinta’. Rlm1 was located on chromosome A07, between 13.07 to 22.11 Mb, using a BC1 population made from crosses of F1 plants of DH16516 (a susceptible line) x DH24288 with bulked segregant RNA Sequencing (BSR-Seq). Rlm1 was further fine mapped in a 100 kb region from 19.92 to 20.03 Mb in the BC1 population consisting of 1247 plants and a F2 population consisting of 3000 plants using SNP markers identified from BSR-Seq through Kompetitive Allele-Specific PCR (KASP). A potential resistance gene, BnA07G27460D, was identified in this Rlm1 region. BnA07G27460D encodes a serine/threonine dual specificity protein kinase, catalytic domain and is homologous to STN7 in predicted genes of B. rapa and B. oleracea, and A. thaliana. Robust SNP markers associated with Rlm1 were developed, which can assist in introgression of Rlm1 and confirm the presence of Rlm1 gene in canola breeding programs.

Plant cell walls (CW) are composed mainly of cellulose–hemicellulose networks embedded in a pectin matrix. CW apposition, including papilla formation, is often associated with plant defence...

Leptosphaeria maculans is a serious concern for canola production worldwide. For effective disease management, knowledge of the pathogen's genetic variability and population structure is a prerequisite. In this study, whole-genome sequencing was performed for 162 of 1590 L. maculans isolates collected in the years 2007–2008 and 2012–2014 in Western Canada. DNA variants in genome-wide and specific regions including avirulence (Avr) genes were characterized. A total of 31,870 high-quality polymorphic DNA variants were used to study L. maculans genetic diversity and population structure. Cluster analysis showed that 150 isolates were clustered into 2 main groups and 4 subgroups by DNA variants located in either Avr or small secreted protein-encoding genes and into 2 main groups and 6 subgroups by genome-wide variants. The analysis of nucleotide diversity and differentiation also confirmed genetic variation within a population and among populations. Principal component analysis with genome-wide variants showed that the isolates collected in 2012–2014 were more genetically diverse than those collected in 2007–2008. Population structure analysis discovered three distinct sub-populations. Although isolates from Saskatchewan and Alberta were of similar genetic composition, Manitoba isolates were highly diverse. Genome-wide association study detected DNA variants in genes AvrLm4-7, Lema_T86300, and Lema_T86310 associated with the years of collection.

Genetic resistance is a successful strategy for management of clubroot (Plasmodiophora brassicae) of brassica crops, but resistance can break down quickly. Identification of novel sources of resistance is especially important when new pathotypes arise. In the current study, the reaction of 177 accessions of Brassica napus to four new, virulent pathotypes of P. brassicae was assessed. Each accession was genotyped using genotyping by sequencing to identify and map novel sources of clubroot resistance using mixed linear model (MLM) analysis. The majority of accessions were highly susceptible (70–100 DSI), but a few accessions exhibited strong resistance (0–20 DSI) to pathotypes 5X (21 accessions), 3A (8), 2B (7), and 3D (15), based on the Canadian Clubroot Differential system. In total, 301,753 SNPs were mapped to 19 chromosomes. Population structure analysis indicated that the 177 accessions belong to seven major populations. SNPs were associated with resistance to each pathotype using MLM. In total, 13 important SNP loci were identified, with 9 SNPs mapped to the A-genome and 4 to the C-genome. The SNPs were associated with resistance to pathotypes 5X (2 SNPs), 3A (4), 2B (5) and 3D (6). A Blast search of 1.6 Mb upstream and downstream from each SNP identified 13 disease-resistance genes or domains. The distance between a SNP locus and the nearest resistance gene ranged from 0.04 to 0.74 Mb. The resistant lines and SNP markers identified in this study can be used to breed for resistance to the most prevalent new pathotypes of P. brassicae in Canada.

Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools that constructed from 184 F2 cloned-lines derived from cross between clubroot-resistant cabbage ‘GZ87’ (against race 4) and susceptible cabbage ‘263’. QTL-seq analyses identified one CR QTL from group I on chromosome C07 and four QTLs from group II on C04 and C07, among which three QTLs of C07 that found from group II were located within the one detected from group I. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation identified two potential candidate genes (Bol037115 and Bol042270) from the three QTLs interval on C07, which exhibiting up-regulation after inoculation in the resistant pool but down-regulation in the susceptible pool. A functional marker ‘SWU-OA’ was developed from one QTL on C07, exhibiting ~95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on digging resistance genes against P. brassicae and may accelerate breeding process of B. oleracea with CR.Key Message QTLs and potential candidate genes for clubroot resistance were identified in Brassica oleracea

Brassica juncea is an important economic crop of the world; however, the narrow genetic base of this crop has tremendously decreased its crop productivity. As an ancestral species of B. juncea, B. nigra is of great importance in widening the genetic diversity of B. juncea. In the present study, 42 SSR markers were employed to screen the genetic diversity among 83 B. nigra, 16 B. juncea, and other Brassica accessions. The molecular characteristics of 498 virtual B. juncea lines were deduced based on the bands of B. nigra and B. rapa via a virtual allopolyploid strategy, and then compared with natural B. juncea accessions. It was found that B. nigra had rich genetic diversity and could be classified into four subgroups, of which subgroup B-III and subgroup B-IV exhibited the closest and the most distant genetic relationship with B. juncea, respectively. To verify this, a hexaploidy strategy was applied to generated synthetic B. juncea from 20 B. nigra accessions, resulting in 45 new-type B. juncea genotypes. The genetic analyses detected that synthetic B. juncea derived from B. nigra in subgroup B-III was close to natural B. juncea, while B. juncea synthesized with B. nigra from subgroup B-IV exhibited wide genetic diversity and was most distant with current B. juncea. This study revealed a great potential of B. nigra in widening genetic diversity of B. juncea particularly using B. nigra in subgroup B-IV, and is helpful in better understanding of the genetic relationship between B. nigra and B. juncea. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-020-01197-7.

Background: Leptosphaeria maculans is a serious concern for canola production in Canada. For effective management, knowledge of the pathogen’s genetic variability and population structure is a prerequisite. Despite some information on race dynamics of the western Canadian L. maculans population in recent years, genetic diversity based on a large number of genome-wide DNA variants has not been investigated.Results: From 1,590 L. maculans isolates collected from 23 field sites in three provinces: Manitoba, Saskatchewan and Alberta, Canada, in the years 2007-2008 and 2012-2014, 150 representative isolates were selected and whole-genome sequenced, and 31,870 polymorphic DNA variants (SNPs and InDels) were used to study L. maculans genetic diversity and population structure. Cluster analysis showed that the genetic diversity levels and isolate groupings varied with the number and genomic regions of the variants involved; isolates collected in 2012-2014 were more genetically diverse than those collected in 2007-2008 when genome-wide variants were considered. The genome wide association study (GWAS) detected variants in egn4_Lema_T86290 (AvrLm4-7), egn4_Lema_T86300 and egn4_Lema_T86310 associated with the year of collection, but no variants was found to be associated with the province or specific location from which the isolates were collected. Population structure analysis indicated the presence of three distinct sub-populations in western Canada. While isolates from Saskatchewan were mainly of one sub-population (sub-pop1), the Alberta isolates comprised two sub-populations (sub-pop1 and sub-pop2), and all the 3 subpopulations were found in Manitoba.Conclusion: The genetic diversity of the western Canadian L. maculans population varied among provinces. It was highly admixed in Manitoba, followed by that in Alberta. The Saskatchewan population had the lowest genetic diversity. Significant genome variation between 2007-2008 and 2012-2014 occurred in the genes egn4_Lema_T86290 (AvrLm4-7), egn4_Lema_T86300 and egn4_Lema_T86310), with AvrLm4-7 becoming much more common in the L. maculans population in the later period.

Genetic resistance to blackleg (Leptosphaeria maculans, Lm) of canola (Brassica napus, Bn) has been extensively studied, but the mechanisms underlying the host–pathogen interaction are still not well understood. Here, a comparative transcriptome analysis was performed on a resistant doubled haploid Bn line carrying the resistance gene Rlm1 following inoculation with a virulent (avrLm1) or avirulent (AvrLm1) Lm isolate on cotyledons. A total of 6999 and 3015 differentially expressed genes (DEGs) were identified, respectively, in inoculated local tissues with compatible (susceptible) and incompatible (resistant) interactions. Functional enrichment analysis found several biological processes, including protein targeting to membrane, ribosome and negative regulation of programmed cell death, were over-represented exclusively among up-regulated DEGs in the resistant reaction, whereas significant enrichment of salicylic acid (SA) and jasmonic acid (JA) pathways observed for down-regulated DEGs occurred only in the susceptible reaction. A heat-map analysis showed that both biosynthesis and signaling of SA and JA were induced more significantly in the resistant reaction, implying that a threshold level of SA and JA signaling is required for the activation of Rlm1-mediated resistance. Co-expression network analysis revealed close correlation of a gene module with the resistance, involving DEGs regulating pathogen-associated molecular pattern recognition, JA signaling and transcriptional reprogramming. Substantially fewer DEGs were identified in mock-inoculated (control) cotyledons, relative to those in inoculated local tissues, including those involved in SA pathways potentially contributing to systemic acquired resistance (SAR). Pre-inoculation of cotyledon with either an avirulent or virulent Lm isolate, however, failed to induce SAR on remote tissues of same plant despite elevated SA and PR1 protein. This study provides insights into the molecular mechanism of Rlm1-mediated resistance to blackleg.

Genetic resistance is widely used to manage clubroot (Plasmodiophora brassicae) in brassica crops, but new pathotypes have recently been identified on canola (Brassica napus) on the Canadian prairies. Resistance effective against both the most prevalent pathotype (3H, based on the Canadian Clubroot Differential system) and the new pathotypes is needed. BC1 plants of Brassica rapa from a cross of line 96-6990-2 (clubroot resistance originating from turnip cultivar &lsquo, Waaslander&rsquo, ) and a susceptible doubled-haploid line, ACDC, exhibited a 1:1 segregation for resistance against pathotypes 3H and 5X. A resistance gene designated as Rcr3 was mapped initially based on the percentage of polymorphic variants using bulked segregant RNA sequencing (BSR-Seq) and further mapped using Kompetitive Allele Specific PCR. DNA variants were identified by assembling short reads against a reference genome of B. rapa. Rcr3 was mapped into chromosome A08. It was flanked by single nucleotide polymorphisms (SNP) markers (A90_A08_SNP_M12 and M16) between 10.00 and 10.23 Mb, in an interval of 231.6 Kb. There were 32 genes in the Rcr3 interval. Three genes (Bra020951, Bra020974, and Bra020979) were annotated with disease resistance mechanisms, which are potential candidates for Rcr3. Another resistance gene, designated as Rcr9wa, for resistance to pathotype 5X was mapped, with the flanking markers (A90_A08_SNP_M28 and M79) between 10.85 and 11.17 Mb using the SNP sites identified through BSR-Seq for Rcr3. There were 44 genes in the Rcr9wa interval, three of which (Bra020827, Bra020828, Bra020814) were annotated as immune-system-process related genes, which are potential candidates for Rcr9wa.

Certain synthetic herbicides can act synergistically with specific bioherbicides. In this study, a sethoxydim herbicide at 0.1× label rate improved biocontrol of herbicide-sensitive green foxtail (Setaria viridis, GFT) by Pyricularia setariae (a fungal bioherbicide agent), but did not change the efficacy on a herbicide-resistant GFT biotype. Reference transcriptomes were constructed for both GFT biotypes via de novo assembly of RNA-seq data. GFT plants treated with herbicide alone, fungus alone and herbicide + fungus were compared for weed-control efficacy and differences in transcriptomes. On herbicide-sensitive GFT, sethoxydim at the reduced rate induced ABA-activated signaling pathways and a bZIP transcription factor 60 (TF bZIP60), while improved the efficacy of biocontrol. The herbicide treatment did not increase these activities or improve biocontrol efficacy on herbicide-resistant plants. An exogenous application of ABA to herbicide-sensitive plants also enhanced bZIP60 expression and improved biocontrol efficacy, which supported the results of transcriptome analysis that identified the involvement of ABA and bZIP60 in impaired plant defense against P. setariae. It is novel to use transcriptome analysis to decipher the molecular basis for synergy between a synthetic herbicide and a bioherbicide agent. A better understanding of the mechanism underlining the synergy may facilitate the development of weed biocontrol.

BackgroundBlackleg, caused by Leptosphaeria maculans, is a serious disease of canola/ oilseed rape in many parts of the world. An integrated approach is needed to control the disease, with genetic resistance as a key component of the management strategy. Towards this goal, a reliable approach for L. maculans race structure assessment becomes essential to gain understanding of the frequency of avirulence genes and race groups of the pathogen, and provide guidance for deployment of resistant canola cultivars.ResultsA total of 162 representative isolates collected in western Canada were selected for genome re-sequencing with an Illumina platform. Assembly of the short reads against the reference genome of L. maculans 'brassicae' isolate v23.1.3 led to the discovery of 21,016 DNA variants (SNPs and InDels), 93% SNPs and 7% InDels, with a transition/transversion (Ts/Tv) ratio of 3.1 genome wide. InDels occurred mainly in GC-blocks and the Ts/Tv ratio of SNPs in AT-blocks was > 2 times higher than that in GC-blocks. The number of variants were positively correlated with supercontig size, GC-block size and gene numbers. DNA variants in most avirulence genes were SNPs, except a deletion in AvrLm1. The number of SNPs varied from 1–2 in AvrLm2, AvrLmJ1-5-9, AvrLm6, AvrLm10A, AvrLm10B and AvrLm11, 8 in AvrLm3 and 38 in AvrLm4-7. This study is the first report of triallelic SNPs in AvrLm2 and AvrLm4-7, and premature STOP codons in AvrLm4-7. Nine SNP haplotypes were identified in AvrLm4-7, however, only 2 ~ 3 haplotypes occurred in other avirulence genes, and in total 47 haplotype groups were identified from the isolates. The 47 SNP haplotype groups were translated into 44 protein haplotype groups and then isolates of L. macualns collected in western Canada were classified into10 races.ConclusionIn this study, we document the shortcoming of inferring races from SNP genotyping, and propose the use of SNP haplotyping for more reliable and informative analysis of L. maculans race structure.

Field surveys indicate blackleg of canola has increased in western Canada since 2010. Earlier studies showed changes in the pathogen (Leptosphaeria maculans (Sowerby) P.Karst.; Lm) population between 2007 and 2010–2011. In this study, Lm isolates were collected from trap plots (591) and/or commercial fields (372) on the prairies between 2012 and 2014, and tested for the profile of avirulence (Av) genes on a set of host differentials for 10 Lm Av alleles. Up to 35 ‘Westar’ trap plots were set up each year, and commercial fields were surveyed in the same area in 2012 and 2013. A similar trend was observed for Av profile between the two Lm populations; Av1, Av3, Av9 and AvLep2 were at low or very low levels, while Av2, Av4, Av6 and Av7 were present in >60% of the isolates. A total of 82 races were identified in these Lm populations, suggesting a greater diversity than those reported elsewhere. Margalef and Simpson indices also confirmed the greater genetic diversity in Lm. In fact, for each known R gene, there is at least one virulent race already existing in the Lm population. The races carrying Av2, Av4, Av6 or Av7 accounted for >70% of the Lm population, indicating that an R gene corresponding to any of these Av alleles may be effective against blackleg on the prairies. However, Av results alone appeared insufficient for explaining different levels of blackleg observed in canola fields; additional factors, possibly including crop rotation and local weather, may also play a role.

Blackleg [Leptosphaeria maculans (Desm.) Ces. & de Not.] is the most widespread disease of canola (Brassica napus L.) on the Canadian prairies. It has a noticeably increased incidence in recent years possibly due to shifts in the pathogen population and erosion in cultivar resistance. This study was conducted to assess foliar-applied fungicides for mitigating the risk of blackleg. Field trials were conducted at five locations in the Black or Dark-Brown Soil Zones for four years using the susceptible canola ‘Westar’. The fungicides pyraclostrobin (Headline®), azoxystrobin (Quadris®), propiconazole (Tilt®), and azoxystrobin + propiconazole (Quilt®) were applied at the 2–4 leaf stage against early infection. For comparisons, pyraclostrobin was also applied at bolting of ‘Westar’ and at the 2–4 leaf stage of two resistant cultivars (‘43E01’, ‘45H29’). These early treatments, except propiconazole, significantly reduced the mean disease incidence (MDI) and disease severity index (DSI) on ‘Westar’, relative to untreated control, reducing the impact of disease on yield by 16.5–26.9%. Late application of pyraclostrobin at bolting was ineffective. Two-application treatments, with pyraclostrobin at the 2–4 leaf stage and propiconazole at the bolting stage, or vice versa, provided no further efficacy or yield benefit relative to the early application of pyraclostrobin alone. None of the treatments showed substantial disease reduction or yield benefit in two low-disease station years (MDI

Additional file 1.

Background Clubroot of canola ( Brassica napus ), caused by the obligate pathogen Plasmodiophora brassicae Woronin, is a major disease worldwide. Genetic resistance remains the best strategy to manage this disease. The objective of the study was to identify and map new sources of resistance to clubroot in B. napus using genome-wide association mapping. The reaction of a collection of 177 accessions to four highly virulent pathotypes of P. brassicae was assessed. These pathotypes were selected because they were most recently identified and showed different virulence patterns on the Canadian clubroot differential (CCD) lines. The collection was then genotyped using genotyping by sequencing (GBS) method. Multi-locus mixed linear model (MMLM) was used to perform the association analysis. Results The majority of accessions were highly susceptible (70 –100 DSI), while few individual accessions showed strong resistance (0–20 DSI) to 5X (2 accessions), 2B (7 accessions), 3A (8 accessions) and 3D (15 accessions). In total, 301,753 SNPs were mapped to 19 chromosomes. Population structure analysis indicated that the 177 accessions belong to two major populations. SNPs were associated with resistance to each pathotype using MLMM. In total, 23 significant SNP loci were identified, with 14 SNPs mapped to the A-genome and 9 to the C-genome. The SNPs were associated with resistance to pathotypes 5X (4 SNPs), 2B (9), 3A (5) and 3D (5). A blast search of 2 Mb upstream and downstream identified 61 disease resistance genes, of which 24 belonged to TIR-NBS-LRR proteins and 20 belonged to CC-NBS-LRR proteins. The distance between a SNP locus and the nearest resistance genes ranged from 0.11–1.66 Mb. This indicated that NBS-LRR gene family might have an important role in clubroot resistance in B. napus . Conclusion The resistant B. napus lines and the SNP markers identified in this study can be used for breeding for resistance to clubroot and contribute to understanding the genetic mechanism of resistance to clubroot.

Clubroot caused by Plasmodiophora brassicae poses a serious threat to canola production around the world, but sources of clubroot resistance in canola are limited. In this study, turnip cultivar “Purple Top White Globe,” which is highly resistant to pathotype 3 (Williams’ system), was used as the source of resistance. Genetic studies showed that the resistance in this cultivar was controlled by a major gene, Rcr5. Bulked segregant RNA-Seq was used to map the gene. A total of 124.6 M raw reads were generated from the resistant (R) and susceptible (S) pooled samples, and 78 K polymorphic DNA variants between the pooled R and the pooled S samples were identified. The percentage of polymorphic variants (PPV) on A03 was much higher than that on the other chromosomes, which indicated that Rcr5 was located on A03. Rcr5 was further mapped into the 23–31 Mb region of A03 through analysis of PPV in the chromosome. A segregated population consisting of 824 plants was genotyped based on 15 SNP markers in the region using Kompetitive Allele Specific PCR. Rcr5 was finely mapped between SNP_A03_83 and SNP_ A03_100, with 0.2 and 0.3 cM from Rcr5, respectively. The markers were located between 23,339,019 and 23,465,030 bp on A03. Eighteen genes were annotated in the Rcr5 target region, seven genes were expressed, and DNA variants were identified in the genes. The SNP markers developed in this study could be used for marker-assisted selection for resistance to clubroot when the gene is transferred to canola.

Background Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. Results Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8–15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1–6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. Conclusion Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study. Electronic supplementary material The online version of this article (10.1186/s12870-019-1844-5) contains supplementary material, which is available to authorized users.

Additional file 2: Figure S2.Molecular phylogenetic analysis using the maximum likelihood method in RAxML. The percentage of 1000 bootstrap trials (1000 replicates) are shown above the branches. Strain details are summarized in their names: location by province (e.g., AB = Alberta), Williams’ pathotype (e.g., P3) where available, and SS = single-spore isolate, V = vegetable host, and C = canola host.

Two cabbage (Brassica oleracea) cultivars ‘Tekila’ and ‘Kilaherb’ were identified as resistant to several pathotypes of Plasmodiophora brassicae. In this study, we identified a clubroot resistance gene (Rcr7) in ‘Tekila’ for resistance to pathotype 3 of P. brassicae from a segregating population derived from ‘Tekila’ crossed with the susceptible line T010000DH3. Genetic mapping was performed by identifying the percentage of polymorphic variants (PPV), a new method proposed in this study, through bulked segregant RNA sequencing. Chromosome C7 carried the highest PPV (42%) compared to the 30–34% in the remaining chromosomes. A peak with PPV (56–73%) was found within the physical interval 41–44 Mb, which indicated that Rcr7 might be located in this region. Kompetitive Allele-Specific PCR was used to confirm the association of Rcr7 with SNPs in the region. Rcr7 was flanked by two SNP markers and co-segregated with three SNP markers in the segregating population of 465 plants. Seven genes encoding TIR-NBS-LRR disease resistance proteins were identified in the target region, but only two genes, Bo7g108760 and Bo7g109000, were expressed. Resistance to pathotype 5X was also mapped to the same region as Rcr7. B. oleracea lines including ‘Kilaherb’ were tested with five SNP markers for Rcr7 and for resistance to pathotype 3; 11 of 25 lines were resistant, but ‘Kilaherb’ was the only line that carried the SNP alleles associated with Rcr7. The presence of Rcr7 in ‘Kilaherb’ for resistance to both pathotypes 3 and 5X was confirmed through linkage analysis.

Sclerotinia stem rot (SR) is one of the most devastating diseases of canola/rapeseed. Quantitative trait loci (QTL) analyses were carried out to identify loci responsible for resistance to SR in three doubled haploid DH populations (H1, H2 and H3). Petiole inoculation technique PIT was used to evaluate the all populations for resistance to SR. Genetic maps were developed using sequence related amplified polymorphism SRAP and simple sequence repeat SSR markers. Genetic maps of the H1 and H2 populations were developed using 508 and 478 markers, respectively. Previously published genetic map of the H3 population was also used in this study. The QTL analysis was carried out for each replicate separately as well as on the average of all the replicates. The numbers of identified QTL in each analysis varied from four to six in the H1 population, three to six in the H2 population and two to six in the H3 population. A number of common QTL were identified between the replicates of each population. Two common QTL were identified on linkage group A7 and C6 between the H1 and H3 populations and one QTL on A9 between the H2 and H3 populations. We are the first to report, identification of common QTL between different populations of Brassica napus.

Clubroot disease is a serious threat to canola production in western Canada and many parts of the world. Rcr1 is a clubroot resistance (CR) gene identified recently and its molecular mechanisms in mediating CR have been studied using several omics approaches. The current study aimed to characterize the biochemical changes in the cell wall of canola roots connecting to key molecular mechanisms of this CR gene identified in prior studies using Fourier transform infrared (FTIR) spectroscopy. The expression of nine genes involved in phenylpropanoid metabolism was also studied using qPCR. Between susceptible (S) and resistance (R) samples, the most notable biochemical changes were related to an increased biosynthesis of lignin and phenolics. These results were supported by the transcription data on higher expression of BrPAL1. The up-regulation of PAL is indicative of an inducible defence response conferred by Rcr1; the activation of this basal defence gene via the phenylpropanoid pathway may contribute to clubroot resistance conferred by Rcr1. The data indicate that several cell-wall components, including lignin and pectin, may play a role in defence responses against clubroot. Principal components analysis of FTIR data separated non-inoculated samples from inoculated samples, but not so much between inoculated S and inoculated R samples. It is also shown that FTIR spectroscopy can be a useful tool in studying plant-pathogen interaction at cellular levels.

Seven blackleg resistance (R) genes (Rlm1, Rlm2, Rlm3, Rlm4, LepR1, LepR2 & LepR3) were each introgressed into a common susceptible B. napus doubled-haploid (DH) line through reciprocal back-crossing, producing single-R gene introgression lines (ILs) for use in the pathological and molecular study of Brassica—Leptosphaeria interactions. The genomic positions of the R genes were defined through molecular mapping and analysis with transgenic L. maculans isolates was used to confirm the identity of the introgressed genes where possible. Using L. maculans isolates of contrasting avirulence gene (Avr) profiles, we preformed extensive differential pathology for phenotypic comparison of the ILs to other B. napus varieties, demonstrating the ILs can provide for the accurate assessment of Avr-R gene interactions by avoiding non-Avr dependant alterations to resistance responses which can occur in some commonly used B. napus varieties. Whole-genome SNP-based assessment allowed us to define the donor parent introgressions in each IL and provide a strong basis for comparative molecular dissection of the pathosystem.

Background Resistance 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). Results Doubled-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. Conclusion Through 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 material The online version of this article (doi:10.1186/s12870-016-0877-2) contains supplementary material, which is available to authorized users.

Clubroot, caused by the plasmodiophorid pathogen Plasmodiophora brassicae, is one of the most serious diseases on Brassica crops worldwide and a major threat to canola production in western Canada. Host resistance is the key strategy for clubroot management on canola. Several clubroot resistance (CR) genes have been identified, but the mechanisms associated with these CR genes are poorly understood. In the current study, a label-free shotgun proteomic approach was used to profile and compare the proteomes of B. rapa carrying and not carrying the CR gene Rcr1 upon P. brassicae infection. A total of 527 differentially accumulated proteins (DAPs) were identified between the resistant and susceptible samples, and functional annotation of these DAPs indicates that the perception of P. brassicae and activation of defense responses is triggered via an unique signaling pathway distinct from common modes of recognition receptors reported with many other plant-pathogen interactions; this pathway appears to act in a calcium-independent manner through a not-well defined cascade of mitogen-activated protein kinases and may require the ubiquitin-26S proteasome related to abiotic stresses, especially the cold-stress tolerance. Both up-regulation of defense-related and down-regulation of pathogenicity-related metabolism were observed in plants carrying Rcr1, and these functions may all contribute to the clubroot resistance mediated by this CR gene. These results, combined with those of transcriptomic analysis reported earlier, improved our understanding of molecular mechanisms associated with Rcr1 and clubroot resistance at large, and identified candidate metabolites or pathways for further confirmation of specific resistance mechanisms. Deploying CR genes with different modes of action may help improve the durability of clubroot resistance.