Distribution of mating‐type alleles and genetic variability in field populations of Leptosphaeria maculans in western Canada

Leptosphaeria maculans is the most important fungal pathogen of canola (Brassica napus, oilseed rape) that causes the devastating stem canker in canola fields of western Canada. The population genetic structure of L. maculans, represented by nine subpopulations from a 6‐year period and three different provinces in western Canada, was determined using ten minisatellite markers. Isolates collected at different locations in six consecutive years had an even distribution of MAT1‐1 and MAT1‐2 across the nine subpopulations. All subpopulations of L. maculans exhibited a moderate gene diversity (H = 0.356–0.585). The majority of the genetic variation occurred within subpopulations. Approximately 8% and 4% of the variations were distributed between sampling year and location, respectively. Genetic distance (F_ST) results, using analysis of molecular variation (AMOVA), indicated that subpopulation pairing within isolates by year ranged from F_ST = 0.010 to 0.109, and the location subpopulation ranged from F_ST = 0.038 to 0.085. Bayesian clustering analyses of multiloci inferred two distinct clusters in all the subpopulations examined. This study indicates a relatively high degree of gene exchange between the different L. maculans isolates. Our results suggest that this can occur in the wide growing areas of canola fields in western Canada. This gene exchange produced different gene allele frequencies and divergence between populations.     

Crystal structure of the effector AvrLm4–7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins

The avirulence gene AvrLm4–7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4–7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4–7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well‐conserved among AvrLm4–7 homologs. Loss of recognition of AvrLm4–7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well‐conserved C–terminal motif or close to the glycine involved in Rlm4‐mediated recognition, resulting in the loss of Rlm7‐mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4–7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4–7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

The Brassica napus receptor‐like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus

Leucine‐rich repeat receptor‐like proteins (LRR‐RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race‐specific R‐genes, including the LRR‐RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line ‘Glacier DH24287’ was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR‐RLP locus, conveying race‐specific resistance to L. maculans isolates harbouring AvrLm2. Several defence‐related LRR‐RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co‐immunoprecipitation of RLM2‐SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co‐expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.     

Characterization of disease resistance genes in the Brassica napus pangenome reveals significant structural variation

Methods based on single nucleotide polymorphism (SNP), copy number variation (CNV) and presence/absence variation (PAV) discovery provide a valuable resource to study gene structure and evolution. However, as a result of these structural variations, a single reference genome is unable to cover the entire gene content of a species. Therefore, pangenomics analysis is needed to ensure that the genomic diversity within a species is fully represented. Brassica napus is one of the most important oilseed crops in the world and exhibits variability in its resistance genes across different cultivars. Here, we characterized resistance gene distribution across 50 B. napus lines. We identified a total of 1749 resistance gene analogs (RGAs), of which 996 are core and 753 are variable, 368 of which are not present in the reference genome (cv. Darmor‐bzh). In addition, a total of 15 318 SNPs were predicted within 1030 of the RGAs. The results showed that core R‐genes harbour more SNPs than variable genes. More nucleotide binding site‐leucine‐rich repeat (NBS‐LRR) genes were located in clusters than as singletons, with variable genes more likely to be found in clusters. We identified 106 RGA candidates linked to blackleg resistance quantitative trait locus (QTL). This study provides a better understanding of resistance genes to target for genomics‐based improvement and improved disease resistance.     

Development and characterisation of Brassica napus-Sinapis arvensis addition lines exhibiting resistance to Leptosphaeria maculans

Blackleg caused by Leptosphaeria maculans is one of the most important diseases affecting oilseed rape worldwide. Sinapis arvensis is valuable for the transfer of blackleg resistance to oilseed rape (Brassica napus) because this species contains high resistance against various aggressive isolates of the blackleg fungus. These include at least one Australian isolate which has been found to overcome resistance originating from species with the Brassica B genome, until now the major source for interspecific transfer of blackleg resistance. Backcross offspring from intergeneric crosses between Brassica napus and S. arvensis were subjected to phytopathological studies and molecular cytogenetic analysis with genomic in situ hybridisation (GISH). The BC₃S progenies included fertile plants exhibiting high seedling (cotyledon) and adult plant resistance associated with the presence of an acrocentric addition chromosome from S. arvensis. In addition, some individuals with adult plant resistance but cotyledon susceptibility were observed to have a normal B. napus karyotype with no visible GISH signals, indicating possible resistant introgression lines. Phytopathological analysis of selfing progenies from 3 different highly resistant BC₃ plants showed that seedling and adult plant resistance are probably conferred by different loci. Received: 20 September 1999 / Accepted: 25 March 2000     

Seedling and adult plant peaetions of Brassica napus-B. juncea recombinant lines towards A- and B-group isolates of Leptosphaeria maculans

The Brassica napus-B. juncea recombinant lines MX and MXS carrying a B. juncea major gene (JLml) in the genetic background of a spring- or a winter type B. napus cultivar, respectively, were tested for their resistance level to Leptosphaeria maculans under controlled conditions. Inoculation with three A-and four B-group individual isolates and with different mixtures of isolates realised within or between these groups was performed on cotyledons, leaves and stems. Cotyledons and leaves of the two recombinant lines were more resistant to A-group isolates than those of B. napus cultivars, except for one isolate recovered from the MX line. The recombinant lines were susceptible at cotyledon stage and resistant on leaves to B-group isolates, as were B. napus cultivars. On stems, severe cortical damage was usually produced on B. napus cultivars by some A-group isolates, whereas B-group isolates induced pith blackening on all genotypes. Stems of the MX line and the resistant donor species (B. juncea cv. Picra) were more resistant than those of the susceptible B. napus (cv. Westar) to the individual A-group isolates. Cultivar Picra was the most susceptible genotype to pith infection caused by the B-group isolates. The consequence of the host pathogen differential interactions on the durability of the monogenic resistance to L. maculans introduced from B. juncea into B. napus is discussed.     

High‐throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus,Brassica rapa and Brassica oleracea

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole‐genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C‐genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.     

A new avirulence gene of Leptosphaeria maculans,AvrLm14, identifies a resistance source in American broccoli (Brassica oleracea) genotypes

In many cultivated crops, sources of resistance to diseases are sparse and rely on introgression from wild relatives. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species, which are sources of diversity for resistance genes. This is the case for Brassica napus (oilseed rape, canola), an interspecific hybrid between Brassica rapa (turnip) and Brassica oleracea (cabbage). B. napus has a narrow genetic basis and few effective resistance genes against stem canker (blackleg) disease, caused by the fungus Leptosphaeria maculans, are currently available. B. rapa diversity has proven to be a valuable source of resistance (Rlm, LepR) genes, while B. oleracea genotypes were mostly considered susceptible. Here we identified a new resistance source in B. oleracea genotypes from America, potentially effective against French L. maculans isolates under both controlled and field conditions. Genetic analysis of fungal avirulence and subsequent cloning and validation identified a new avirulence gene termed AvrLm14 and suggested a typical gene-for-gene interaction between AvrLm14 and the postulated Rlm14 gene. AvrLm14 shares all the usual characteristics of L. maculans avirulence genes: it is hosted in a genomic region enriched in transposable elements and heterochromatin marks H3K9me3, its expression is repressed during vegetative growth but shows a strong overexpression 5–9 days following cotyledon infection, and it encodes a small secreted protein enriched in cysteine residues with few matches in databases. Similar to the previously cloned AvrLm10-A, AvrLm14 contributes to reduce lesion size on susceptible cotyledons, pointing to a complex interplay between effectors promoting or reducing lesion development.     

Variation in abundance of predicted resistance genes in the Brassica oleracea pangenome

Brassica oleracea is an important agricultural species encompassing many vegetable crops including cabbage, cauliflower, broccoli and kale; however, it can be susceptible to a variety of fungal diseases such as clubroot, blackleg, leaf spot and downy mildew. Resistance to these diseases is meditated by specific disease resistance genes analogs (RGAs) which are differently distributed across B. oleracea lines. The sequenced reference cultivar does not contain all B. oleracea genes due to gene presence/absence variation between individuals, which makes it necessary to search for RGA candidates in the B. oleracea pangenome. Here we present a comparative analysis of RGA candidates in the pangenome of B. oleracea. We show that the presence of RGA candidates differs between lines and suggests that in B. oleracea, SNPs and presence/absence variation drive RGA diversity using separate mechanisms. We identified 59 RGA candidates linked to Sclerotinia, clubroot, and Fusarium wilt resistance QTL, and these findings have implications for crop breeding in B. oleracea, which may also be applicable in other crops species.     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Identification of loci contributing to quantitative field resistance to blackleg disease, causal agent Leptosphaeria maculans (Desm.) Ces. et de Not., in Winter rapeseed (Brassica napus L.)

 Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of Brassica napus. Genomic regions controlling blackleg resistance at the adult plant stage were detected using 152 doubled-haploid (DH) lines derived from the F₁‘Darmor-bzh’בYudal’. The rapeseed genetic map used includes 288 DNA markers on 19 linkage groups. Blackleg resistance of each DH line was evaluated in field tests in 1995 and 1996 by measuring the mean disease index (I) and the percentage of lost plants (P). From notations recovered in 1995, ten quantitative trait loci (QTL) were detected: seven QTL for I and six QTL for P, explaining 57% and 41% of the genotypic variation, respectively. Three of them were common to I and P. From data recovered in 1996, seven QTL were identified: five QTL for I and two different QTL for P, accounting for 50% and 23% of the genotypic variation, respectively. One I QTL, located close to a dwarf gene (bzh), was detected with a very strong effect, masking more QTL detection. It was not revealed at the same position and with the same effect in 1995. Four major genomic regions were revealed from 1995 and from 1996 with the same parental contribution. One of them, located on the DY2 group, has a resistance allele from the susceptible parent. Five- and two-year-specific QTL were detected in 1995 and 1996, respectively. Received: 25 April 1997 / Accepted: 5 August 1997