Resynthesizing Brassica napus with race specific resistance genes and race non-specific QTLs to multiple races of Plasmodiophora brassicae.

Clubroot disease in canola (Brassica napus) continues to spread across the Canadian prairies. Growing resistant cultivars is considered the most economical means of controlling the disease. However, sources of resistance to clubroot in B. napus are very limited. In this study, we conducted interspecific crosses using a B. rapa line (T19) carrying race-specific resistance genes and two B. oleracea lines, ECD11 and JL04, carrying race non-specific QTLs. Employing embryo rescue and conventional breeding methods, we successfully resynthesized a total of eight B. napus lines, with four derived from T19 × ECD11 and four from T19 × JL04. Additionally, four semi-resynthesized lines were developed through crosses with a canola line (DH16516). Testing for resistance to eight significant races of Plasmodiophora brassicae was conducted on seven resynthesized lines and four semi-resynthesized lines. All lines exhibited high resistance to the strains. Confirmation of the presence of clubroot resistance genes/QTLs was performed in the resynthesized lines using SNP markers linked to race-specific genes in T19 and race non-specific QTLs in ECD11. The developed B. napus germplasms containing clubroot resistance are highly valuable for the development of canola cultivars resistant to clubroot.

A CRISPR/Cas9-based vector system enables the fast breeding of selection-marker-free canola with Rcr1-rendered clubroot resistance.

Breeding for disease resistance in major crops is of crucial importance for global food security and sustainability. However, common biotechnologies such as traditional transgenesis or genome editing do not provide an ideal solution, whereas transgenic crops free of selection markers such as cisgenic/intragenic crops might be suitable. In this study, after cloning and functional verification of the Rcr1 gene for resistance to clubroot (Plasmodiophora brassicae), we confirmed that the genes Rcr1, Rcr2, Rcr4, and CRa from Brassica rapa crops and the resistance gene from B. napus oilseed rape cv. 'Mendel' on chromosome A03 were identical in their coding regions. We also determined that Rcr1 has a wide distribution in Brassica breeding materials and renders potent resistance against multiple representative clubroot strains in Canada. We then modified a CRISPR/Cas9-based cisgenic vector system and found that it enabled the fast breeding of selection-marker-free transgenic crops with add-on traits, with selection-marker-free canola (B. napus) germplasms with Rcr1-rendered stable resistance to clubroot disease being successfully developed within 2 years. In the B. napus background, the intragenic vector system was able to remove unwanted residue sequences from the final product with high editing efficiency, and off-target mutations were not detected. Our study demonstrates the potential of applying this breeding strategy to other crops that can be transformed by Agrobacterium. Following the streamlined working procedure, intragenic germplasms can be developed within two generations, which could significantly reduce the breeding time and labor compared to traditional introgression whilst still achieving comparable or even better breeding results.

Identification of QTLs for resistance to 10 pathotypes of Plasmodiophora brassicae in Brassica oleracea cultivar ECD11 through genotyping-by-sequencing.

KEY MESSAGE: Two major quantitative trait loci (QTLs) and five minor QTLs for 10 pathotypes were identified on chromosomes C01, C03, C04 and C08 through genotyping-by-sequencing from Brassica oleracea. Clubroot caused by Plasmodiophora brassicae is an important disease in brassica crops. Managing clubroot disease of canola on the Canadian prairie is challenging due to the continuous emergence of new pathotypes. Brassica oleracea is considered a major source of quantitative resistance to clubroot. Genotyping-by-sequencing (GBS) was performed in the parental lines; T010000DH3 (susceptible), ECD11 (resistant) and 124 BC1 plants. A total of 4769 high-quality polymorphic SNP loci were obtained and distributed on 9 chromosomes of B. oleracea. Evaluation of 124 BC1S1 lines for resistance to 10 pathotypes: 3A, 2B, 5C, 3D, 5G, 3H, 8J, 5K, 5L and 3O of P. brassicae, was carried out. Seven QTLs, 5 originating from ECD11 and 2 from T010000DH3, were detected. One major QTL designated as Rcr_C03-1 on C03 contributed 16.0-65.6% of phenotypic variation explained (PVE) for 8 pathotypes: 2B, 5C, 5G, 3H, 8J, 5K, 5L and 3O. Another major QTL designated as Rcr_C08-1 on C08 contributed 8.3 and 23.5% PVE for resistance to 8J and 5K, respectively. Five minor QTLs designated as Rcr_C01-1, Rcr_C03-2, Rcr_C03-3, Rcr_C04-1 and Rcr_C08-2 were detected on chromosomes C01, C03, C04 and C08 that contributed 8.3-23.5% PVE for 5 pathotypes each of 3A, 2B, 3D, 8J and 5K. There were 1, 10 and 4 genes encoding TIR-NBS-LRR/CC-NBS-LRR class disease resistance proteins in the Rcr_C01-1, Rcr_C03-1 and Rcr_C08-1 flanking regions. The syntenic regions of the two major QTLs Rcr_C03-1 and Rcr_C08-1 in the B. rapa genome 'Chiifu' were searched.

Canola with Stacked Genes Shows Moderate Resistance and Resilience against a Field Population of Plasmodiophora brassicae (Clubroot) Pathotype X.

Genetic resistance is a cornerstone for managing clubroot (Plasmodiophora brassicae). However, when used repeatedly, a clubroot resistance (CR) gene can be broken rapidly. In this study, canola inbred/hybrid lines carrying one or two CR genes (Rcr1/CRaM and Crr1rutb) were assessed against P. brassicae pathotype X by repeated exposure to the same inoculum source under a controlled environment. Lines carrying two CR genes, either Rcr1 + Crr1rutb or CRaM + Crr1rutb, showed partial resistance. Selected lines were inoculated with a field pathotype X population (L-G3) at 5 × 106 resting spores/g soil, and all clubs were returned to the soil they came from six weeks after inoculation. The planting was repeated for five cycles, with diseased roots being returned to the soil after each cycle. The soil inoculum was quantified using qPCR before each planting cycle. All lines with a single CR gene were consistently susceptible, maintaining high soil inoculum levels over time. The lines carrying two CR genes showed much lower clubroot severity, resulting in a 10-fold decline in soil inoculum. These results showed that the CR-gene stacking provided moderate resistance against P. brassicae pathotype X, which may also help reduce the pathogen inoculum buildup in soil.

Identification of a genomic region containing genes involved in resistance to four pathotypes of Plasmodiophora brassicae in Brassica rapa turnip ECD02.

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 F1 progeny from the cross B. rapa canola ACDC (susceptible, S) × B. rapa turnip ECD02 (resistant, R) were backcrossed with ACDC, then self-pollinated to produce BC1 S1 lines. All the F1 plants were resistant to four pathotypes (3A, 3D, 3H, and 5X) of P. brassicae. Segregation for R and S in BC1 to each pathotype was 1:1 and resistance reactions were highly correlated. From whole genome sequencing, 192.1 M sequences with 96% template coverage from ECD02, and 478.9 M sequences with 92% coverage from ACDC, were aligned with the reference genome of B. rapa. Genotyping-by-sequencing was performed on the BC1 population. The number of aligned short reads per plant in the BC1 ranged from 1.4 to 8.5 M sequences with 4-8% template coverage. We obtained 1,344 high-quality single-nucleotide polymorphism (SNP) loci with a mean missing rate at 0.27% and distributed them on 10 chromosomes. A single co-localized quantitative trait loci (QTL), designated as Rcr9ECD02 on chromosome A08, conferred resistance to the four pathotypes. The QTL explained 68.9-74.4% of phenotypic variation with the logarithm of the odds values of 24.3 to 31.1. Bulked segregant analysis was performed, and 14 SNP markers linked to the gene were developed using the Kompetitive Allele Specific PCR. Rcr9ECD02 was mapped into an interval of 2.2 cM, flanked by CF_A08_10664692 and CF_A08_12230973, which spanned 1.51 Mb on the chromosome and included 219 B. rapa genes. Four of these genes (BraA08g012910.3C, BraA08g012920.3C, BraA08g013130.3C, and BraA08g013630.3C) encoded disease resistance proteins.

Ethylene Plays a Dual Role during Infection by Plasmodiophora brassicae of Arabidopsis thaliana.

Plasmodiophora brassicae infection leads to hypertrophy of host roots and subsequent formation of galls, causing huge economic losses to agricultural producers of Cruciferae plants. Ethylene (ET) has been reported to play a vital role against necrotrophic pathogens in the classic immunity system. More clues suggested that the defense to pathogens in roots may be different from the acrial. The ET pathway may play a positive role in the infection of P. brassicae, as shown by recent transcriptome profiling. However, the molecular basis of ET remains poorly understood. In this study, we investigated the potential role of ethylene against P. brassicae infection in an ein3/eil1 double-mutant of Arabidopsis thaliana (A. thaliana). After infection, ein3/eil1 (Disease Index/DI: 93) showed more susceptibility compared with wild type (DI: 75). Then, we inoculated A. thaliana Columbia-0 (Col-0) with P. brassicae by 1-aminocyclopropane-1-carboxylic acid (ACC) and pyrazinamide (PZA), respectively. It was found that the symptoms of infected roots with ACC were more serious than those with PZA at 20 dpi (day post infection). However, the DI were almost the same in different treatments at 30 dpi. WRKY75 can be directly regulated by ET and was upregulated at 7 dpi with ACC, as shown by qRT-PCR. The wrky75-c mutant of A. thaliana (DI: 93.75) was more susceptible than the wild type in Arabidopsis. Thus, our work reveals the dual roles of ET in infection of P. brassicae and provides evidence of ET in root defense against pathogens.

A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond.

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.

Identification of Candidate Genes for Clubroot-Resistance in Brassica oleracea Using Quantitative Trait Loci-Sequencing.

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, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage "GZ87" (against race 4) and susceptible (S) cabbage "263." Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker "SWU-OA" was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.

Genetic diversity and population structure of Leptosphaeria maculans isolates in Western Canada.

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.

Identification of resistance loci against new pathotypes of Plasmodiophora brassicae in Brassica napus based on genome-wide association mapping.

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.

Genome-wide transcriptome reveals mechanisms underlying Rlm1-mediated blackleg resistance on canola.

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.

Identification of Two Major QTLs in Brassica napus Lines With Introgressed Clubroot Resistance From Turnip Cultivar ECD01.

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), Rcr10 ECD01 and Rcr9 ECD01 , were detected for the pathotypes in chromosomes A03 and A08, respectively. Rcr10 ECD01 and Rcr9 ECD01 were responsible for resistance to 3A, 3D, and 3H, while only one QTL, Rcr9 ECD01 , 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 Rcr10 ECD01 target region. In the Rcr9 ECD01 region, two genes encoded disease resistance proteins and 10 genes encoded with defense-related function. The target regions for Rcr10 ECD01 and Rcr9 ECD01 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.

Investigating genetic relationship of Brassica juncea with B. nigra via virtual allopolyploidy and hexaploidy strategy.

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.

Transcriptome analysis identified the mechanism of synergy between sethoxydim herbicide and a mycoherbicide on green foxtail.

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.

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies.

BACKGROUND: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). RESULTS: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10- 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. CONCLUSION: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

Two Clubroot-Resistance Genes, Rcr3 and Rcr9wa, Mapped in Brassica rapa Using Bulk Segregant RNA Sequencing.

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 'Waaslander') 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.

Quantification of Plasmodiophora brassicae Resting Spores in Soils Using Droplet Digital PCR (ddPCR).

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.

Whole-genome DNA similarity and population structure of Plasmodiophora brassicae strains from Canada.

BACKGROUND: Clubroot is an important disease of brassica crops world-wide. The causal agent, Plasmodiophora brassicae, has been present in Canada for over a century but was first identified on canola (Brassica napus) in Alberta, Canada in 2003. Genetic resistance to clubroot in an adapted canola cultivar has been available since 2009, but resistance breakdown was detected in 2013 and new pathotypes are increasing rapidly. Information on genetic similarity among pathogen populations across Canada could be useful in estimating the genetic variation in pathogen populations, predicting the effect of subsequent selection pressure on changes in the pathogen population over time, and even in identifying the origin of the initial pathogen introduction to canola in Alberta. RESULTS: The genomic sequences of 43 strains (34 field collections, 9 single-spore isolates) of P. brassicae from Canada, the United States, and China clustered into five clades based on SNP similarity. The strains from Canada separated into four clades, with two containing mostly strains from the Prairies (provinces of Alberta, Saskatchewan, and Manitoba) and two that were mostly from the rest of Canada or the USA. Several strains from China formed a separate clade. More than one pathotype and host were present in all four Canadian clades. The initial pathotypes from canola on the Prairies clustered separately from the pathotypes on canola that could overcome resistance to the initial pathotypes. Similarly, at one site in central Canada where resistance had broken down, about half of the genes differed (based on SNPs) between strains before and after the breakdown. CONCLUSION: Clustering based on genome-wide DNA sequencing demonstrated that the initial pathotypes on canola on the Prairies clustered separately from the new virulent pathotypes on the Prairies. Analysis indicated that these 'new' pathotypes were likely present in the pathogen population at very low frequency, maintained through balancing selection, and increased rapidly in response to selection from repeated exposure to host resistance.

Fine mapping of Brassica napus blackleg resistance gene Rlm1 through bulked segregant RNA sequencing.

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.

Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa.

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.