In-silico identification and differential expression of putative disease resistance-related genes within the collinear region of Brassica napus blackleg resistance locus LepR2’ in Brassica oleracea

Blackleg disease, caused by Leptosphaeria maculans, greatly affects the production of cabbage (Brassica oleracea). However, definitive R-gene(s) are yet to be identified in this crop. In contrast, a number of R-loci have been identified in A- or B-genome crops. Identification of few resistant cabbage genotypes indicates the presence of R-genes in this C-genome crop. High ancestral synteny between Brassica genomes suggests that the collinear regions of known A- or B-genome R-loci may also contain functional R-genes in the C-genome. Strong resistance was observed in the cotyledons of cabbage inbred line SCNU-98 against two L. maculans isolates, 03–02 s and 00–100 s. We investigated the collinear region of the Brassica napus blackleg resistance locus LepR2’ in B. oleracea since both isolates of L. maculans contain corresponding avirulence genes. The locus was collinear to a 5.8 Mbp genomic segment of B. oleracea chromosome C09 containing 13 genes that have putative disease resistance-related domains. High expression of genes Bo9g117290 and Bo9g111510 against isolate 00–100 s, and high expression of genes Bo9g126150 and Bo9g111490 against both isolates in the resistant-line SCNU-98 indicate their putative roles in blackleg resistance, which remained to be functionally verified. This work enhances our understanding of R-gene-mediated resistance to blackleg in cabbage.


Introduction
Blackleg, a disease particularly devastating to canola, is also known to cause substantial economic damage to cabbage (Dilmaghani et al. 2010;Humpherson-Jones 1985;Piliponytė-Dzikienė et al. 2015). The disease is caused by Leptosphaeria maculans (anamorph: Phoma lingam) around the world and by Leptosphaeria biglobosa, a comparatively less damaging species, in Asian countries Liu et al. 2014;Hong et al. 2009;Hao et al. 2015;Mendes-Pereira et al. 2003). Cabbage is an essential ingredient of the daily diet, either in fresh or processed form, in East Asian countries such as Korea, Japan, and China, where 37.8% of the world's total cabbage is produced (FAO Statistics Database 2017).
The possibility of invasion by the more aggressive L. maculans is a growing concern for the East Asian cabbage industry, since L. maculans was previously reported to spread in Canada and Poland, where only L. biglobosa was predominant (Liu 2008;Fitt et al. 2008). Further, both pathogenic species inhabit similar ecological niches and preference for the agro-climatic conditions of this region may be conducive to the establishment of L. maculans (Fitt et al. 2008;West et al. 2001). The complicated life cycle of the pathogen, its ability to reproduce both sexually and asexually Communicated by Inhwa Yeam.
Mohammad Rashed Hossain and Mostari Jahan Ferdous contributed equally to this work.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1358 0-020-00271 -5) contains supplementary material, which is available to authorized users. (Rouxel and Balesdent 2005), multiple disease cycles in a single growing season (Li et al. 2007), longer resting period (several years) in crop residues (West et al. 2001;Li et al. 2007), and substantial global diversity in the pathogenicity of L. maculans strains (Kutcher et al. 2011) make it difficult to control the disease via chemical and agronomic practices alone. Preventing the spread of L. maculans and the development of resistant cultivars are thus prioritized in these countries to safeguard both canola and cabbage industries van den Burg et al. 2008;Zhang and Fernando 2018).
Unlike rapeseed and canola, resistance to blackleg has not been extensively investigated in cabbage. Hence, sources of resistance to the disease are scarce in cabbage. Most of the R-loci against the disease have been identified in the A-or B-genomes of Brassica family crops (Balesdent et al. 2002;Bohman et al. 2002;Rimmer and van den Berg 1992;Chèvre et al. 1997;Christianson et al. 2006;Delourme et al. 2006;Delourme et al. 2004;Leflon et al. 2007;Marcroft et al. 2002;Pang and Halloran 1996;Plieske et al. 1998), while definitive R-loci/genes in C-genome crops such as cabbage are yet to be identified (Larkan et al. 2013;Robin et al. 2017). However, a few cabbage genotypes have been reported to show moderate resistance against the disease (Ananga et al. 2006;Badawy et al. 1991;Ferreira et al. 1992). Very recently, two Korean cabbage lines were found to be resistant at the cotyledon stage against two L. maculans isolates, 03-02 s and 00-100 s, which contain multiple avirulent genes (Robin et al. 2017). These findings suggest the presence of R-gene(s) in cabbage.
Functional analysis indicated that most plant R-genes usually contain several domains such as leucine-rich repeat (LRR), nucleotide-binding site (NBS), coiled-coil (CC), Toll/Interleukin-1 Receptor (TIR), receptor like protein kinase (RLK), F-box domain (FBD), and mitogen-activated protein kinase (MAPK), which have distinct roles in plant defence against phytopathogens (Larkan et al. 2013;Ellis et al. 2000;Liu et al. 2007;Meng and Zhang 2013;Meyers et al. 1999;Sekhwal et al. 2015;van den Burg et al. 2008). Since A-, B-, and C-genomes of Brassica family crops share common evolutionary history and high ancestral synteny, we hypothesized that functional disease resistance-related domain-containing genes may also be present within the collinear regions of known A-or B-genome R-loci in the C-genome Franzke et al. 2011;Chalhoub et al. 2014;Liu et al. 2014). Such approach has been used to identify candidate orthologous genes for Sclerotinia stem rot resistance, seed colour, seed oil content, yield, and efficiency traits in oilseed rape (B. napus) using the corresponding known loci of Arabidopsis thaliana (Ding et al. 2012;Stein et al. 2013;Wu et al. 2013;Zhao et al. 2012). In the case of blackleg resistance, Yu et al. (2013) tracked the collinear region of B. napus blackleg resistance locus LepR4 in B. rapa and identified four putative disease resistancerelated NBS-encoding genes. We have investigated the B. rapa R-loci Rlm1, Rlm2/LepR3, LepR1, and LepR4 from the A-genome against the C-genome (Nou IS, unpublished data). Here, we report disease resistance-related functional domain-containing genes within the collinear region of B. napus blackleg resistance locus LepR2' in the C-genome of B. oleracea along with their putative roles in blackleg resistance in cabbage, determined via differential expression analysis in contrastingly resistant cabbage lines against two L. maculans isolates.

In-silico analysis of the collinear region of B. napus blackleg resistance locus LepR2' in B. oleracea
The collinear region of B. napus blackleg resistance locus LepR2' was identified in the B. oleracea genome (ensembl database) using the homologous segments of B. napus clones that contain the LepR2' flanking markers sN3888Fa and sR6903a (Yu et al. 2012). Sequences of all the genes within this collinear region in B. oleracea were retrieved. The genes which contained putative functional disease resistance-related domains such as NBS, LRR, CC, TIR, MAPK, FBD, RLK, and RLP were identified using the Simple Modular Architecture Research Tool (http://smart .embl-heide lberg .de/) and the Conserved Domain Database Tool (https ://www.ncbi.nlm.nih.gov/cdd/). MEME suite version 5.0.5 (http://meme-suite .org/tools /meme) was used to analyse the conserved motifs in the proteins encoded by these genes. Distribution of exons and introns of these genes were analyzed using the Gene Structure Display Server (GSDS2.0) web tool (http://gsds.cbi.pku.edu.cn/). The microsynteny relationship of these identified putative disease resistance-related genes with B. rapa, B. nigra, and A. thaliana were visualized via Circos v0.69.

L. maculans isolates and preparation of inoculum
L. maculans isolates 00-100 s and 03-02 s, possessing multiple avirulence genes, were cultured on V8 agar media (20%) at 22 °C and 16 h day length under fluorescent light for several weeks. Fungal spores were suspended in 10 mL sterile distilled water by scrapping the spores off culture plates with a sterile glass slide prior to collecting the spores by filtering the suspension with sterile Miracloth (EMD Millipore Corporation, USA). The spore concentration was adjusted to 2.25 × 10 7 spores mL −1 using sterile distilled water prior to inoculation.

Plant materials: inoculation and assessment of disease resistance
Seeds of two Korean cabbage inbred lines, SCNU-72 and SCNU-98, were germinated in multi-pot trays using cocopeat soil in a growth chamber at 24 ± 2 °C, 65% relative humidity, and a 16/8 h (light/dark) photoperiod under 420 µmol photons m −2 s −1 light intensity at bench level. After 12 days of germination, the center of each cotyledon lobe was punctured with a sterile needle prior to inoculation with 10 μL of the spore suspension. The disease responses were evaluated at 10 days post-inoculation (dpi) using a disease rating scale of 0-9 following the procedures described in Robin et al. (2017).

RNA Extraction and cDNA synthesis
Total RNA was extracted from control (0 h), mock-, and pathogen-inoculated cotyledons of resistant and susceptible cabbage lines at 6 h, 24 h, and 48 h using an 'RNeasy mini kit' (Qiagen, CA, USA). RNA purity and concentration were determined using a Nanodrop-2000 (Nanodrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized from the extracted total RNA using 'SuperScript-III First-Strand Synthesis SuperMix' (Invitrogen, CA, USA) as per the manufacturer's guideline.

Expression analysis by qRT-PCR
Expression of the identified putative disease resistancerelated domain (e.g., LRR, CC, FBD, CC, MAPK and RLK) containing genes within the LepR2' collinear region in B. oleracea were analyzed by qRT-PCR in a Roche LightCycler ® 96 System (Roche Applied Science, Penzberg, Germany). Gene-specific primers were designed using 'Primer3Plus'. For each gene, the qRT-PCR reaction was performed with 5 μL of 2x qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems, London, UK), 1 μL of forward and reverse primers each (10 pmol), 2 μL of ultra-pure water, and 1 μL of template cDNA (60 ng μL −1 ) in a final reaction volume of 10 μL. The reaction condition was as follows: initial denaturation at 95 °C for 5 min, 45 cycles of denaturation at 95 °C for 10 s, annealing at specific temperatures for 10 s, and amplification and signal acquisition at 72 °C for 30 s. Each biological replicate was tested with three technical replicates. Relative expression levels were quantified by the 2 −ΔΔCt method using the mean of three actin genes as the internal control.

Statistical analysis
Statistical significance was tested using analysis of variance (ANOVA) and mean separation was performed using Tukey's pairwise comparison in Minitab (v18) (Minitab Inc., State College, PA, USA).

Disease responses of cabbage lines to L. maculans isolates
Significantly different responses to the two L. maculans isolates, 03-02 s and 00-100 s, were observed in the inoculated cotyledons of cabbage inbred lines SCNU-72 and SCNU-98 ( Fig. 1a, b). At 10 dpi, the disease scores against both 00-100 s and 03-02 s isolates ranged from 7 to 8 in the line SCNU-72, having 70% and 80% diseased cotyledon areas, respectively. In contrast, the disease scores in the line SCNU-98 ranged between 2-3 and 2-4 against these isolates, respectively, while having less than 30% diseased cotyledon area. This indicates that line SCNU-98 is resistant against both L. maculans isolates, 00-100 s and 03-02 s. At 30 dpi, overall blackening of the stems of the susceptible line SCNU-72 was observed, whereas the stems of the resistant line SCNU-98 appeared healthy (Fig. 1c).

Collinear region of B. napus blackleg resistance locus LepR2' in B. oleracea
BLAST search of the sequences of the clones containing the flanking markers sN3888Fa and sR6903a of B. napus blackleg resistance locus LepR2' identified a 5.8 Mbp genomic segment of chromosome C09 (C9:36168200-41971165) as the corresponding collinear region in the B. oleracea genome (Fig. 2a, b). This region was flanked by genes Bo9g111470 and Bo9g135890. Mining this collinear region identified a total of 661 genes. The complete list of these 661 genes and their functional annotations are shown in Supplemental Table S1.

Identification of genes containing disease resistance-related functional domains
Functional domain analysis of the 661 genes identified 13 genes as having putative disease resistance-related domains (  distribution along the length of these genes is shown in Fig. 3. Conserved motif analysis of the selected 13 genes identified 15 statistically significant conserved motifs consisting of 18-50 amino acids ( Fig. 4a; Table S3). Among these 15 motifs, 10 of the motifs were associated with LRR domains, while three (namely, motifs 2, 8, and 12) and two (namely, motifs 1 and 4) were associated with MAPK and FBD domains, respectively (Table S3).
Gene structure analysis identified the distribution of exons and introns in the selected 13 genes. Among these genes, a maximum of four exons were found in genes Bo9g111510, Bo9g126150, and Bo9g135700, while four genes (Bo9g113780, Bo9g120720, Bo9g119130, and Bo9g117290) contained only one exon (Fig. 4b).
Along with these 13 genes, another gene, Bo9g169800, encoding a receptor-like protein was also included for differential expression analysis against L. maculans isolates. This gene is orthologous to JX880110 (sequence similarity 92.5%, e-value = 0.0), the first ever cloned B. napus blackleg resistance gene LepR3 on chromosome A10 (Larkan et al. 2013). This gene is included for expression analysis, since it is known to confer resistance in B. napus and is located 8.15 Mbp downstream of the LepR2' collinear region in B. oleracea.

Chromosomal location and microsynteny relationship of the identified LRR, CC, FBD, MAPK, and RLK domain-containing genes
The 13 selected LRR, CC, FBD, MAPK, and RLK domain-containing genes were evenly distributed throughout the LepR2' collinear region in B. oleracea (Fig. 2b). No distinct clustering of the genes was observed, except groups of three genes were found to be closely located: genes Bo9g111490, Bo9g111500 and Bo9g111510 located at 36.2 Mbp (at the flanking end of the collinear region) and genes Bo9g126120, Bo9g126140, and Bo9g126150 located at 39.6 Mbp (at approximately the center of the collinear region).  Table S3 Microsynteny relationship analysis of the identified 13 genes of B. oleracea (a C-genome crop) with B. rapa (an A-genome crop), B. nigra (a B-genome crop), and A. thaliana identified seven orthologous gene pairs with A. thaliana, distributed across all five chromosomes of A. thaliana (Fig. 2c). For the A-genome (B. rapa), fifteen orthologous gene pairs were identified: a maximum of six pairs with chromosome A10, followed by three and two pairs with A03 and A02, respectively, and one pair with each of chromosomes A01, A05, A06, and A07. With B. nigra (a B-genome crop), a total of seven orthologous gene pairs were observed. These results suggest a high microsyntenic relationship for the 13 identified LRR, CC, FBD, MAPK, and RLK genes among A-, B-, and C-genome crops.

Expression profiling of the identified LRR, CC, TIR, FBD, MAPK, and RLK domain-containing genes
Expression of the identified LRR, CC, FBD, MAPK, and RLK domain-containing genes was determined in the resistant and susceptible cabbage genotypes at different time points after inoculation with L. maculans isolates 00-100 s and 03-02 s using gene-specific primers (Table S4). Hierarchical clustering of the expressions of these genes in a heatmap representation identified a cluster of seven genes (Bo9g126150, Bo9g117290, Bo9g111490, Bo9g126120, Bo9g111510, Bo9g111500, and Bo9g126140) that showed significant differential expression in the resistant line SCNU-98 comparted to that in the susceptible line SCNU-72 (Fig. 5). Among these seven genes, only two genes, Bo9g126150 and Bo9g111490, were induced in the resistant line against both isolates, whereas the remaining five genes were only induced against the isolate 00-100 s (Fig. 5, 6). A common feature of these seven genes is that they were significantly induced in the resistant line at 6 h following inoculation with L. maculans isolate 00-100 s. Against this isolate, only the gene Bo9g126150 was consistently highly expressed in the resistant line at later time points as well (i.e., at 24 h and 48 h following inoculation) (Fig. 6). The gene Bo9g117290 also showed higher expression at 24 h, but it did not show significantly higher expression at 48 h following inoculation. The remaining five genes did not show distinctively increased expressions after 6 h of inoculation. Against the isolate 03-02 s, genes Bo9g126150 and Bo9g111490 were only induced at 24 h following inoculation.
Among the significantly expressed genes, the highest expression levels in the resistant line SCNU-98 were observed for the gene Bo9g117290 at 6 h (~ 11-fold) and The gene Bo9g169800 is located 8.15 Mbp downstream of the LepR2' collinear region. This is included for expression analysis since it is orthologous to JX880110, the cloned gene of B. napus blackleg resistance locus LepR3 (Larkan et al. 2013) 1 3 24 h (~ 24-fold) followed by the gene Bo9g111510 (~ sevenfold) at 6 h after inoculation by the isolate 00-100 s (Fig. 6).
Among the genes induced by both isolates, gene Bo9g126150 showed ~ three-fourfold higher expression at 6, 24, and 48 h against isolate 00-100 s and ~ twofold higher expression against isolate 03-02 s at 24 h. On the other hand, the gene Bo9g111490 was highly expressed against isolate 00-100 s only at 6 h (~ fivefold) and against isolate 03-02 s at 24 h (~ fivefold) (Fig. 6).

Discussion
This study reports the identification of putative disease resistance-related LRR, CC, FBD, MAPK, and RLK-domain containing genes within the collinear region of B. napus blackleg resistance locus LepR2' in B. oleracea, while also determining the potential association of those genes with blackleg resistance in cabbage.
R-gene-mediated resistance to L. maculans in Brassica family crops is largely governed by compatible 'gene-forgene' interactions, where a specific avirulence gene (Avr) of the pathogen is recognized by corresponding R-gene of the host (Ansan-Melayah et al. 1998;Flor 1971;Williams and Delwiche 1980). The cabbage inbred line SCNU-98 displayed a high resistance response against both L. maculans isolates, 03-02 s and 00-100 s, at the cotyledon stage. However, the specific R-locus that may confer resistance in this Korean cabbage inbred line could not be determined due to the lack of differential set of L. maculans isolates having different Avr gene profiles (owing to strict import restrictions on L. maculans strains). Mapping the R-genes in segregating populations could be an effective approach, but this is also time consuming, resource demanding, and expensive (Delourme et al. 2018;Miles and Wayne 2008).
A comprehensive meta-analysis of 314 cloned plant R-genes indicated nine different molecular mechanisms of resistance, which were mainly manifested by NBS, LRR, Mbp downstream of the LepR2' collinear region. This is included for expression analysis since it is orthologous to JX880110, the cloned gene of B. napus blackleg resistance locus LepR3 (Larkan et al. 2013) TIR, CC, and RLP/RLKs domains (Kourelis and van der Hoorn 2018). Genome-wide, genes containing such disease resistance-related domains are known for major plant species including rice,potato,soybean,cucumber,melon,peach,grape,apple,. Several studies have identified genome-wide NBS-LRR genes in major Brassicaceae crops (Alamery et al. 2018;Fu et al. 2019;Golicz et al. 2016;Yu et al. 2014), of which Alamery et al. 2018(Alamery et al. 2018 reported the maximum number of NBS-LRR genes in B. rapa (249), B. oleracea (443), and B. napus (641). The recent pan-genome of B. oleracea used an improved pipeline for genome assembly and resistance gene analog (RGA) candidate prediction, identifying a total of 213 RLP, 556 NBS-LRR, and 901 RLK genes (Bayer et al. 2019). A total of 97 NBS-encoding genes were reported to be orthologous between B. oleracea and B. napus (Fu et al. 2019). We reasoned that mining such disease resistance-related, domain-containing genes within the collinear regions of known B. napus R-loci in a C-genome and profiling their differential expressions in the resistant versus susceptible cabbage genotypes might lead to the identification of putative candidate genes for blackleg resistance, which upon validation by functional analysis, could be useful for improving blackleg resistance in elite cabbage cultivars. In this study, we focused on B. napus blackleg resistance locus LepR2', since both isolates (against which the cabbage inbred line SCNU-98 were resistant) contain corresponding avirulence gene (Robin et al. 2017). This suggests the existence of the corresponding R-gene in this line. The locus LepR2', flanked by markers sR8548a and sN2551b on B. napus linkage group A10, was originally introgressed from B. rapa subsp. sylvestris via interspecific hybridization, and its complete expression in B. napus has been speculated to require some chromosomes and genes from the C-genome (Yu et al. 2012). This locus is allelic to the previously identified locus LepR2 and is an incompletely dominant gene that provides strong resistance against a range of L. maculans isolates in B. napus (Yu et al. 2005(Yu et al. , 2012. A total of 13 genes were identified within the collinear region of LepR2' in B. oleracea which contain at least one of the LRR, CC, FBD, MAPK, and RLK domains. Among these genes, the receptor like protein kinase gene Bo9g117290 showed the highest expression level in the resistant line SCNU-98 against the isolate 00-100 s (~ 11and ~ 24-fold induction at 6 h and 24 h following inoculation, respectively). RLKs are major component of membranelocalized pattern-recognition receptors (PRRs) that mediate the first line of plant defence by recognizing the pathogen-associated molecular patterns (PAMPs) and activating PAMP-triggered immunity (PTI) (Jones and Dangl 2006). Sixty out of 314 cloned plant's R-genes have been reported to be RLKs/RLPs (Kourelis and van der Hoorn 2018). RLKs were found to be involved in both broad-spectrum, elicitor-initiated defence responses such as FLS2 (FLAGEL-LIN SENSITIVE 2) in Arabidopsis and pathogen-specific, dominant R-gene-mediated defence responses, such as Xa21 in rice (Sekhwal et al. 2015;Goff and Ramonell 2007;Liu et al. 2017). The gene FLS2 was the first RLK gene found to be involved in perception of the bacterial elicitor Flagellin in Arabidopsis (Gómez-Gómez and Boller 2000), and the gene Xa21 conferred resistance to race 6 of Xanthomonas oryzae pv. oryzae, which causes bacterial blight in rice (Song et al. 1995). Comparison of functional domains revealed that our putative candidate gene Bo9g117290 also contain a cytoplasmic serine/threonine kinase domain along with an LRR domain.
Among known blackleg resistance loci in Brassica genomes, only two R-loci, namely LepR3 and Rlm2, have been cloned so far. These two loci are confirmed to be allelic and to encode LRR-RLP genes (Larkan et al. 2013;Larkan et al. 2015). The corresponding orthologue of this LRR-RLP gene in C-genome is the gene Bo9g169800, which is located approximately 8.15 Mbp downstream of the LepR2' collinear region in B. oleracea (Fig. 2b). We have investigated its expression and found that it is only induced by ~ twofold in the resistant line SCNU-98 against the isolate 00-100 s at 6 h, which is much less than the 11-24-fold higher expression of the RLK domain-containing gene Bo9g117290 against the isolate at 6-24 h after inoculation (Fig. 6).
The two most well-known RLK genes, FLS2 and BAK1 (BRI1-associated receptor kinase 1) are known to initiate the MAP kinase cascade upon recognition of the bacterial PAMP flagellin, flg22 (Deslandes and Rivas 2012;Chinchilla et al. 2007;Kim et al. 2013). Two MAP kinase genes, Bo9g119130 and Bo9g126150, were found within the LepR2' collinear region in B. oleracea. In particular, the gene Bo9g126150 was highly expressed against both isolates: ~ three-fourfold against isolate 00-100 s at all time points following inoculation and ~ twofold against isolate 03-02 s at 6 h, indicating a putative role in blackleg resistance.
Among the 13 selected genes within the collinear region of LepR2' in B. oleracea, six genes had an F-box domain (FBD) along with an LRR domain (Fig. 3). Of these, two genes were highly expressed in the resistant line SCNU-98, Bo9g111510 only against isolate 00-100 s at 6 h following inoculation (~ sevenfold; the second highest expression among the 13 genes) and Bo9g111490 against both isolates (~ fivefold, against isolate 00-100 s at 6 h and against isolate 03-02 s at 24 h following inoculation). Several F-box proteins are known to play roles in plant defence against phytopathogens. For example, MAX2 contributed to resistance against Pectobacterium carotovorum and Pseudomonas syringae in Arabidopsis (Piisilä et al. 2015), CPR1 was involved in controlling the stability of plant NBS-LRR proteins in Arabidopsis (Cheng et al. 2011), and ACRE189/ ACIF1 was involved in activating defence responses and regulating cell death during recognition of Pseudomonas syringae pv. tabaci and Cladosporium fulvum in tobacco and tomato (van den Burg et al. 2008).

Conclusion
In this study, genes containing putative disease resistancerelated domains were identified in the collinear region of B. napus blackleg resistance locus LepR2' in B. oleracea. Very high expression of the LRR-RLK gene Bo9g117290 and the LRR-FBD gene Bo9g111510 against the L. maculans isolate 00-100 s, and high expression of the LRR-MAP kinase gene Bo9g126150 and the LRR-FBD gene Bo9g111510 against both isolates in the resistant cabbage line suggest putative roles of these genes in conferring resistance to blackleg in B. oleracea. A mapping-based approach will be necessary to identify if more than one locus is involved. In addition, functional analyses of these genes will be helpful in identifying the key gene(s) conferring resistance to blackleg in cabbage.