Fengqun Yu

Identification of two novel genes for blackleg resistance in Brassica napus

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.

Genetics and breeding for clubroot resistance in Canadian spring canola (Brassica napus L.)

Abstract Clubroot disease caused by Plasmodiophora brassicae Woronin is a concern to the canola (Brassica napus L.) growers in Canada. A crop management strategy that includes deployment of genetic resistance and appropriate cultural practices is needed for long-term management of this disease. Resistance to the P. brassicae pathotypes present in Canada has been found in the primary and secondary gene pools of spring B. napus canola. Some of these sources, such as winter canola ‘Mendel’, rutabaga and Pak Choi (Brassica rapa L.) ‘Flower Nabana’, were used in genetic studies and breeding for the development of clubroot-resistant canola cultivars. A dominant gene in ‘Mendel’ and ‘Flower Nabana’ was found to confer resistance to P. brassicae pathotype 3, while a simple or a complex genetic control of resistance was found in rutabaga. The clubroot resistance (CR) gene in ‘Flower Nabana’ was mapped to chromosome A3, and molecular markers linked to the CR gene were identified for use in marker-assisted breeding (MAB). Using the CR genes from ‘Mendel’ and rutabaga, several clubroot-resistant spring canola lines were developed. Often the CR genes of ‘Mendel’ and rutabaga conferring resistance to pathotype 3 also conferred resistance to other pathotypes of P. brassicae found in Canada, including pathotypes 5, 6 and 8. The CR gene of ‘Flower Nabana’ was introgressed into B. napus and B. rapa canola through MAB. Since single-gene controlled resistance can be eroded, other strategies such as pyramiding different CR genes into B. napus canola should be considered for durable resistance.

Sources of resistance to Plasmodiophora brassicae (clubroot) pathotypes virulent on canola

Abstract A collection of 955 Brassica accessions including B. rapa (718), B. napus (94), B. juncea (93), B. oleracea (30), B. carinata (12) and B. nigra (8) was screened against Plasmodiophora brassicae pathotype 3 (1 × 106 resting spores cc−1 growth medium), the predominant strain of the pathogen on canola in western Canada. A total of 35 accessions (mostly B. rapa) showed at least 50% reduced clubroot severity relative to a susceptible control, with 15 showing complete resistance (clubroot-free). Ten resistant accessions representing Brassica A-, B- and C-genome species were tested further using a 10-fold higher pathogen inoculum dose (1 × 107 resting spores cc−1 growth medium) and by testing them against the five pathotypes (2, 3, 5, 6 and 8) of P. brassicae found in Canada. One B. nigra, two B. oleracea and four B. rapa (oriental vegetable) accessions maintained a high level of resistance under the higher pathogen inoculum pressure, while one B. nigra and two B. rapa (turnip) accessions showed moderate resistance. Most of the selected clubroot-resistant accessions showed consistent resistance to each of the five P. brassicae pathotypes found in Canada, except for one B. nigra and two turnip accessions, which varied slightly against different pathotypes. Several promising sources of clubroot resistance were identified in this study that can be used to develop new canola germplasm with a diverse clubroot resistance background for potentially more durable clubroot resistance.

Identification of Genome-Wide Variants and Discovery of Variants Associated with Brassica rapa Clubroot Resistance Gene Rcr1 through Bulked Segregant RNA Sequencing.

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.

Genotyping-by-sequencing reveals three QTL for clubroot resistance to six pathotypes of Plasmodiophora brassicae in Brassica rapa

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.

Single R Gene Introgression Lines for Accurate Dissection of the Brassica - Leptosphaeria Pathosystem

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.

Shotgun Label-free Proteomic Analysis of Clubroot (Plasmodiophora brassicae) Resistance Conferred by the Gene Rcr1 in Brassica rapa

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 Brassica rapa carrying and not carrying the CR gene Rcr1 in response to P. brassicae infection. A total of 527 differentially accumulated proteins (DAPs) were identified between the resistant (with Rcr1) and susceptible (without Rcr1) samples, and functional annotation of these DAPs indicates that the perception of P. brassicae and activation of defense responses are 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 found to be related to abiotic stresses, especially the cold-stress tolerance in other studies. Both up-regulation of defense-related and down-regulation of pathogenicity-related metabolism was observed in plants carrying Rcr1, and these functions may all contribute to the CR mediated by Rcr1. These results, combined with those of transcriptomic analysis reported earlier, improved our understanding of molecular mechanisms associated with Rcr1 and CR at large, and identified candidate metabolites or pathways related to specific resistance mechanisms. Deploying CR genes with different modes of action may help improve the durability of CR.

Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. “Jazz” using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2.

Evaluating Changes in Cell-Wall Components Associated with Clubroot Resistance Using Fourier Transform Infrared Spectroscopy and RT-PCR

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.