BackgroundThe protist Plasmodiophora brassicae is a biotrophic soil-borne pathogen that causes clubroot on Brassica crops worldwide. Clubroot disease is a serious threat to the 8 M ha of canola (Brassica napus) grown annually in western Canada. While host resistance is the key to clubroot management, sources of resistance are limited.ResultsTo identify new sources of clubroot resistance (CR), we fine mapped a CR gene (Rcr1) from B. rapa ssp. chinensis to the region between 24.26 Mb and 24.50 Mb on the linkage group A03, with several closely linked markers identified. Transcriptome analysis was conducted using RNA sequencing on a segregating F1 population inoculated with P. brassicae, with 2,212 differentially expressed genes (DEGs) identified between plants carrying and not carrying Rcr1. Functional annotation of these DEGs showed that several defense-related biological processes, including signaling and metabolism of jasmonate and ethylene, defensive deposition of callose and biosynthesis of indole-containing compounds, were up-regulated significantly in plants carrying Rcr1 while genes involved in salicylic acid metabolic and signaling pathways were generally not elevated. Several DEGs involved in metabolism potentially related to clubroot symptom development, including auxin biosynthesis and cell growth/development, showed significantly lower expression in plants carrying Rcr1.ConclusionThe CR gene Rcr1 and closely linked markers will be highly useful for breeding new resistant canola cultivars. The identification of DEGs between inoculated plants carrying and not carrying Rcr1 is an important step towards understanding of specific metabolic/signaling pathways in clubroot resistance mediated by Rcr1. This information may help judicious use of CR genes with complementary resistance mechanisms for durable clubroot resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1166) contains supplementary material, which is available to authorized users.

Blackleg, caused by Leptosphaeria maculans, is a major disease of Brassica napus. Two populations of B. napus DH lines, DHP95 and DHP96, with resistance introgressed from B. rapa subsp. sylvestris, were genetically mapped for resistance to blackleg disease with restriction fragment length polymorphism markers. Examination of the DHP95 population indicated that a locus on linkage group N2, named LepR1, was associated with blackleg resistance. In the DHP96 population, a second locus on linkage group N10, designated LepR2, was associated with resistance. We developed BC1 and F2 populations, to study the inheritance of resistance controlled by the genes. Genetic analysis indicated that LepR1 was a dominant nuclear allele, while LepR2 was an incompletely dominant nuclear resistance allele. LepR1 and LepR2 cotyledon resistance was further evaluated by testing 30 isolates from Canada, Australia, Europe, and Mexico. The isolates were from B. napus, B. juncea, and B. oleracea and represented different pathogenicity groups of L. maculans. Results indicated that LepR1 generally conferred a higher level of cotyledon resistance than LepR2. Both genes exhibited race-specific interactions with pathogen isolates; virulence on LepR1 was observed with one isolate, pl87-41, and two isolates, Lifolle 5, and Lifolle 6, were virulent on LepR2. LepR1 prevented hyphal penetration, while LepR2 reduced hyphal growth and inhibited sporulation. Callose deposition was associated with resistance for both loci.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of Brassica crops worldwide. F1 progeny from the Brassica rapa lines T19 (resistant) × ACDC (susceptible) were backcrossed with ACDC, then self-pollinated to produce BC1S1 lines, From genotyping-by-sequencing (GBS) of the parental lines and BC1 plants, about 1.32 M sequences from T19 were aligned into the reference genome of B. rapa with 0.4-fold coverage, and 1.77 M sequences with 0.5-fold coverage in ACDC. The number of aligned short reads per plant in the BC1 ranged from 0.07 to 1.41 M sequences with 0.1-fold coverage. A total of 1584 high quality SNP loci were obtained, distributed on 10 chromosomes. A single co-localized QTL, designated as Rcr4 on chromosome A03, conferred resistance to pathotypes 2, 3, 5, 6 and 8. The peak was at SNP locus A03_23710236, where LOD values were 30.3 to 38.8, with phenotypic variation explained (PVE) of 85–95%. Two QTLs for resistance to a novel P. brassicae pathotype 5x, designated Rcr8 on chromosome A02 and Rcr9 on A08, were detected with 15.0 LOD and 15.8 LOD, and PVE of 36% and 39%, respectively. Bulked segregant analysis was performed to examine TIR-NBS-LRR proteins in the regions harboring the QTL.

Clubroot, caused by Plasmodiophora brassicae, is an important disease on Brassica species worldwide. A clubroot resistance gene, Rcr1, with efficacy against pathotype 3 of P. brassicae, was previously mapped to chromosome A03 of B. rapa in pak choy cultivar “Flower Nabana”. In the current study, resistance to pathotypes 2, 5 and 6 was shown to be associated with Rcr1 region on chromosome A03. Bulked segregant RNA sequencing was performed and short read sequences were assembled into 10 chromosomes of the B. rapa reference genome v1.5. For the resistant (R) bulks, a total of 351.8 million (M) sequences, 30,836.5 million bases (Mb) in length, produced 120-fold coverage of the reference genome. For the susceptible (S) bulks, 322.9 M sequences, 28,216.6 Mb in length, produced 109-fold coverage. In total, 776.2 K single nucleotide polymorphisms (SNPs) and 122.2 K insertion / deletion (InDels) in R bulks and 762.8 K SNPs and 118.7 K InDels in S bulks were identified; each chromosome had about 87% SNPs and 13% InDels, with 78% monomorphic and 22% polymorphic variants between the R and S bulks. Polymorphic variants on each chromosome were usually below 23%, but made up 34% of the variants on chromosome A03. There were 35 genes annotated in the Rcr1 target region and variants were identified in 21 genes. The numbers of poly variants differed significantly among the genes. Four out of them encode Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat proteins; Bra019409 and Bra019410 harbored the higher numbers of polymorphic variants, which indicates that they are more likely candidates of Rcr1. Fourteen SNP markers in the target region were genotyped using the Kompetitive Allele Specific PCR method and were confirmed to associate with Rcr1. Selected SNP markers were analyzed with 26 recombinants obtained from a segregating population consisting of 1587 plants, indicating that they were completely linked to Rcr1. Nine SNP markers were used for marker-assisted introgression of Rcr1 into B. napus canola from B. rapa, with 100% accuracy in this study.

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.