Comparative Analysis of Transcriptomes Reveals Pathways and Verifies Candidate Genes for Clubroot Resistance in Brassica oleracea.

Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both resistant (R) and susceptible (S) B. oleracea plants infected by P. brassicae. Then the comparative analysis was carried out between the R and S samples at different time points during the infection stages to reveal clubroot resistance related pathways and candidate genes. Compared with 0 days after inoculation, a total of 4991 differential expressed genes were detected from the S pool, while only 2133 were found from the R pool. Gene function enrichment analysis found that the effector-triggered immunity played a major role in the R pool, while the pathogen-associated molecular pattern triggered immune response was stronger in the S pool. Simultaneously, candidate genes were identified through weighted gene co-expression network analysis, with Bol010786 (CNGC13) and Bol017921 (SD2-5) showing potential for conferring resistance to clubroot. The findings of this research provide valuable insights into the molecular mechanisms underlying clubroot resistance and present new avenues for further research aimed at enhancing the clubroot resistance of B. oleracea through breeding.

Genetic analysis and QTL mapping for silique density in rapeseed (Brassica napus L.).

KEY MESSAGE: Genetic models, QTLs and candidate gene for silique density on main inflorescence of rapeseed were identified. Silique density is one of the critical factors to determine seed yield and plant architecture in rapeseed (Brassica napus L.); however, the genetic control of this trait is largely unknown. In this study, the genetic model for silique density on main inflorescence (SDMI) of rapeseed was estimated according to the phenotypic data of P1 (an inbreed line with high SDMI), P2 (an inbreed line with low SDMI), F1, F2, BC1P1 and BC1P2 populations, revealing that SDMI is probably controlled by multi-minor genes with or without major gene. The QTLs for SDMI and its component characters including silique number on main inflorescence (SNMI) and main inflorescence length (MIL) were consequently mapped from a DH population derived from P1 and P2 by using a genetic linkage map constructed by restriction site-associated DNA sequencing (RAD seq) technology. A total of eight, 14 and three QTLs were identified for SDMI, SNMI and MIL under three environments, respectively, with an overlap among SDMI and SNMI in 55.7-75.4 cm on linkage group C06 which corresponding to 11.6-27.3 Mb on chromosome C06. Genomic resequencing was further conducted between a high- and a low-SDMI pool constructed from the DH population, and QTL-seq analysis identified a 0.15 Mb interval (25.98-26.13 Mb) from the C06-QTL region aforementioned. Transcriptome sequencing and qRT-PCR identified one possible candidate gene (BnARGOS) from the 0.15 Mb interval. This study will provide novel insights into the genetic basis of SD in rapeseed.

Sclerotinia sclerotiorum utilizes host-derived copper for ROS detoxification and infection.

Necrotrophic plant pathogen induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Sclerotinia sclerotiorum is a typical necrotrophic pathogen that causes Sclerotinia stem rot in more than 400 species, resulting in serious economic losses. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to S. sclerotiorum and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper flow from healthy area into the necrotic area during infection. A model was proposed that S. sclerotiorum utilizes host copper to detoxify ROS in its cells, whereas the resistant hosts may restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

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.

Introgression and pyramiding of genetic loci from wild Brassica oleracea into B. napus for improving Sclerotinia resistance of rapeseed.

KEY MESSAGE: Resistant rapeseed lines pyramided with multiple resistant QTLs derived from Brassica oleracea were developed via a hexaploidy strategy. Rapeseed (Brassica napus L.) suffers heavily from Sclerotinia stem rot, but the breeding of Sclerotinia-resistant rapeseed cultivar has been unsuccessful. During the study, interspecific hexaploids were generated between rapeseed variety 'Zhongshuang 9' and a wild B. oleracea which was highly resistant to S. sclerotiorum, followed by backcrossing with Zhongshuang 9 and successive selfing. By molecular marker-assisted selection, three major resistant QTLs were transferred and pyramided from B. oleracea into two BC1F8 lines which exhibited ~ 35% higher resistance level than Zhongshuang 9 and produced good seed yield and seed quality. It is the first report on successful development of Sclerotinia-resistant rapeseed lines by introducing multiple resistant loci from wild B. oleracea. This study revealed the effectiveness of pyramiding multiple QTLs in improving Sclerotinia resistance in rapeseed and provided a novel breeding strategy on utilization of B. oleracea in rapeseed improvement.

Simultaneous Transcriptome Analysis of Host and Pathogen Highlights the Interaction Between Brassica oleracea and Sclerotinia sclerotiorum.

White mold disease caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops. Here, we simultaneously assessed the transcriptome changes from lesions produced by S. sclerotiorum on disease-resistant (R) and -susceptible (S) B. oleracea pools bulked from a resistance-segregating F2 population. Virulence genes of S. sclerotiorum, including polygalacturonans, chitin synthase, secretory proteins, and oxalic acid biosynthesis, were significantly repressed in lesions of R B. oleracea at 12 h postinoculation (hpi) but exhibited similar expression patterns in R and S B. oleracea at 24 hpi. Resistant B. oleracea induced expression of receptors potentially to perceive Sclerotinia signals during 0 to 12 hpi and deployed complex strategies to suppress the pathogen establishment, including the quick accumulation of reactive oxygen species via activating Ca2+ signaling and suppressing pathogen oxalic acid generation in S. sclerotiorum. In addition, cell wall degradation was inhibited in the resistant B. oleracea potentially to prevent the expansion of Sclerotinia hyphae. The transcriptome changes in S. sclerotiorum and host revealed that resistant B. oleracea produces strong responses against S. sclerotiorum during early infection.

Understanding the Resistance Mechanism in Brassica napus to Clubroot Caused by Plasmodiophora brassicae.

Exploring the mechanism of plant resistance has become the basis for selection of resistance varieties but reports on revealing resistant mechanism in Brassica napus against Plasmodiophora brassicae are rare. In this study, RNA-seq was conducted in the clubroot-resistant B. napus breeding line ZHE-226 and in the clubroot-susceptible rapeseed cultivar Zhongshuang 11 at 0, 3, 6, 9, and 12 days after inoculation. Strong alteration was detected specifically in ZHE-226 as soon as the root hair infection happened, and significant promotion was found in ZHE-226 on cell division or cell cycle, DNA repair and synthesis, protein synthesis, signaling, antioxidation, and secondary metabolites. Combining results from physiological, biochemical, and histochemical assays, our study highlights an effective signaling in ZHE-226 in response to P. brassicae. This response consists of a fast initiation of receptor kinases by P. brassicae; the possible activation of host intercellular G proteins which might, together with an enhanced Ca2+ signaling, promote the production of reactive oxygen species; and programmed cell death in the host. Meanwhile, a strong ability to maintain homeostasis of auxin and cytokinin in ZHE-226 might effectively limit the formation of clubs on host roots. Our study provides initial insights into resistance mechanism in rapeseed to P. brassicae.

Genome-wide identification of loci affecting seed glucosinolate contents in Brassica napus L.

Glucosinolates are amino acid-derived secondary metabolites that act as chemical defense agents against pests. However, the presence of high levels of glucosinolates severely diminishes the nutritional value of seed meals made from rapeseed (Brassica napus L.). To identify the loci affecting seed glucosinolate content (SGC), we conducted genome-wide resequencing in a population of 307 diverse B. napus accessions from the three B. napus ecotype groups, namely, spring, winter, and semi-winter. These resequencing data were used for a genome-wide association study (GWAS) to identify the loci affecting SGC. In the three ecotype groups, four common and four ecotype-specific haplotype blocks (HBs) were significantly associated with SGC. To identify candidate genes controlling SGC, transcriptome analysis was carried out in 36 accessions showing extreme SGC values. Analyses of haplotypes, genomic variation, and candidate gene expression pointed to five and three candidate genes in the common and spring group-specific HBs, respectively. Our expression analyses demonstrated that additive effects of the three candidate genes in the spring group-specific HB play important roles in the SGC of B. napus.

Genetic characterization and fine mapping for multi-inflorescence in Brassica napus L.

KEY MESSAGE: A major QTL for multi-inflorescence was mapped to a 27.18-kb region on A05 in Brassica napus by integrating QTL mapping, microarray analysis and whole-genome sequencing. Multi-inflorescence is a desirable trait for the genetic improvement of rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait is not well understood. In the present study, a doubled haploid (DH) population derived from a cross between single- and multi-inflorescence lines was investigated for the penetrance of multi-inflorescence across 3 years and genotyped with 257 simple sequence repeat and sequence-related amplified polymorphism loci. A major quantitative trait locus (QTL) for penetrance of multi-inflorescence was mapped to a 9.31-Mb region on chromosome A05, explaining 45.81% of phenotypic variance on average. Subsequently, 13 single-inflorescence and 15 multi-inflorescence DH lines were genotyped with the Brassica microarray, and the QTL interval of multi-inflorescence was narrowed to a 0.74-Mb region with 37 successive single nucleotide polymorphisms between single- and multi-inflorescence groups. A 27.18-kb QTL interval was detected by screening 420 recessive F2 individuals with genome-specific markers. These results will be valuable for gene cloning and molecular breeding of multi-inflorescence in rapeseed.

Mapping of genetic locus for leaf trichome in Brassica oleracea.

KEY MESSAGE: The genetic locus for leaf trichome was identified via marker-based mapping and SNP microarray assay, and a functional marker was developed to facilitate the breeding for hairiness in Brassica oleracea. Plant trichomes are involved in various functions particularly in protecting plants against some biotic and abiotic damages. In the present study, an F2 segregating population was developed from the cross between a glabrous cultivated B. oleracea (CC, 2n = 18) and a hairy wild relative, B. incana (CC, 2n = 18). A 1:3 segregation pattern between glabrous and hairy plants was detected among 1063 F2 genotypes, and the locus for hairiness was mapped in a 4.3-cM genetic region using 267 SSR markers among 149 F2 genotypes, corresponding to a 17.6-Mb genomic region on chromosome C01. To narrow the genetic region for hairiness, the Brassica 60 K SNP Bead Chip Arrays were applied to genotype 64 glabrous and 30 hairy F2 plants, resulting in a 1.04-Mb single peak region located in the 17.6-Mb interval. A candidate gene, BoTRY, was identified by qRT-PCR which revealed significant higher expression in glabrous F2 genotypes as compared with that in hairy plants. A cleaved amplified polymorphic site marker was successfully developed to distinguish the sequence variations of BoTRY between hairy and glabrous plants. Our study will be helpful for molecular breeding for hairiness in B. oleracea.

Development of genic cleavage markers in association with seed glucosinolate content in canola.

KEY MESSAGE: The orthologues of Arabidopsis involved in seed glucosinolates metabolism within QTL confidence intervals were identified, and functional markers were developed to facilitate breeding for ultra-low glucosinolates in canola. Further reducing the content of seed glucosinolates will have a positive impact on the seed quality of canola (Brassica napus). In this study 43 quantitative trait loci (QTL) for seed glucosinolate (GSL) content in a low-GSL genetic background were mapped over seven environments in Germany and China in a doubled haploid population from a cross between two low-GSL oilseed rape parents with transgressive segregation. By anchoring these QTL to the reference genomes of B. rapa and B. oleracea, we identified 23 orthologues of Arabidopsis involved in GSL metabolism within the QTL confidence intervals. Sequence polymorphisms between the corresponding coding regions of the parental lines were used to develop cleaved amplified polymorphic site markers for two QTL-linked genes, ISOPROPYLMALATE DEHYDROGENASE1 and ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE 3. The genic cleavage markers were mapped in the DH population into the corresponding intervals of QTL explaining 3.36-6.88 and 4.55-8.67 % of the phenotypic variation for seed GSL, respectively. The markers will facilitate breeding for ultra-low seed GSL content in canola.

Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step.

KEY MESSAGE: Sclerotinia resistance was transferred into rapeseed from a wild relative of Brassica oleracea (B. incana) using hexaploids derived from crosses between B. incana and rapeseed as a bridge. A high level of resistance against Sclerotinia sclerotiorum has been documented in wild Brassica oleracea, but not in cultivated rapeseed (Brassica napus). To transfer sclerotinia resistance from a wild relative into rapeseed, a strategy was proposed using hexaploids (AACCCC) derived from crosses between the wild B. oleracea-related B. incana genotype 'C01' and the Chinese rapeseed variety 'Zhongshuang 9' as a bridge. Progenies (BC1F1) generated by backcrossing the hexaploid to 'Zhongshuang 9' could be generated with a high crossability (average 18.3 seeds per pod). Seventy-three individuals in BC1F1 were firstly screened for resistance with five molecular markers linked to the major resistance QTL on chromosome C09 in 'C01', and 11 individuals harboring resistance loci were selected to develop vegetative clones. Of these, five exhibited significantly higher resistance than 'Zhongshuang 9' and the most resistant individual was chosen to develop the BC1F2 progeny. Finally, five individual genotypes with nearly twofold higher resistance than 'Zhongshuang 9' were found among 100 BC1F2 individuals by using marker-assisted selection and resistance evaluation. Hereof, one rapeseed-type individual with 38 chromosomes and good self-fertility (15.0 ± 3.56 seeds/pod) was identified. Our results indicate that the proposed strategy is effective for transferring sclerotinia resistance from a wild relative of B. oleracea into rapeseed.

A large-scale introgression of genomic components of Brassica rapa into B. napus by the bridge of hexaploid derived from hybridization between B. napus and B. oleracea.

Brassica rapa (AA) has been used to widen the genetic basis of B. napus (AACC), which is a new but important oilseed crop worldwide. In the present study, we have proposed a strategy to develop new type B. napus carrying genomic components of B. rapa by crossing B. rapa with hexaploid (AACCCC) derived from B. napus and B. oleracea (CC). The hexaploid exhibited large flowers and high frequency of normal chromosome segregation, resulting in good seed set (average of 4.48 and 12.53 seeds per pod by self and open pollination, respectively) and high pollen fertility (average of 87.05 %). It was easy to develop new type B. napus by crossing the hexaploid with 142 lines of B. rapa from three ecotype groups, with the average crossability of 9.24 seeds per pod. The genetic variation of new type B. napus was diverse from that of current B. napus, especially in the A subgenome, revealed by genome-specific simple sequence repeat markers. Our data suggest that the strategy proposed here is a large-scale and highly efficient method to introgress genomic components of B. rapa into B. napus.

Identification of genomic regions involved in resistance against Sclerotinia sclerotiorum from wild Brassica oleracea.

The lack of resistant source has greatly restrained resistance breeding of rapeseed (Brassica napus, AACC) against Sclerotinia sclerotiorum which causes severe yield losses in rapeseed production all over the world. Recently, several wild Brassica oleracea accessions (CC) with high level of resistance have been identified (Mei et al. in Euphytica 177:393-400, 2011), bringing a new hope to improve Sclerotinia resistance of rapeseed. To map quantitative trait loci (QTL) for Sclerotinia resistance from wild B. oleracea, an F2 population consisting of 149 genotypes, with several clones of each genotypes, was developed from one F1 individual derived from the cross between a resistant accession of wild B. oleracea (B. incana) and a susceptible accession of cultivated B. oleracea var. alboglabra. The F2 population was evaluated for Sclerotinia reaction in 2009 and 2010 under controlled condition. Significant differences among genotypes and high heritability for leaf and stem reaction indicated that genetic components accounted for a large portion of the phenotypic variance. A total of 12 QTL for leaf resistance and six QTL for stem resistance were identified in 2 years, each explaining 2.2-28.4 % of the phenotypic variation. The combined effect of alleles from wild B. oleracea reduced the relative susceptibility by 22.5 % in leaves and 15 % in stems on average over 2 years. A 12.8-cM genetic region on chromosome C09 of B. oleracea consisting of two major QTL intervals for both leaf and stem resistance was assigned into a 2.7-Mb genomic region on chromosome A09 of B. rapa, harboring about 30 putative resistance-related genes. Significant negative corrections were found between flowering time and relative susceptibility of leaf and stem. The association of flowering time with Sclerotinia resistance is discussed.

Comparative Transcriptome Analysis Points to the Biological Processes of Hybrid Incompatibility between Brassica napus and B. oleracea.

Improving Brassica napus via introgression of the genome components from its parental species, B. oleracea and B. rapa, is an important breeding strategy. Interspecific hybridization between B. napus and B. rapa is compatible with high rate of survival ovules, while the hybridization between B. napus and B. oleracea is incompatible with the high occurrence of embryo abortion. To understand the diverse embryo fate in the two interspecific hybridizations, here, the siliques of B. napus pollinated with B. oleracea (AE) and B. rapa (NE) were employed for transcriptome sequencing at 8 and 16 days after pollination. Compared to NE and the parental line of B. napus, more specific differentially expressed genes (DEGs) (1274 and 1698) were obtained in AE and the parental line of B. napus at 8 and 16 days after pollination (DAP). These numbers were 51 and 5.8 times higher than the number of specific DEGs in NE and parental line of B. napus at 8 and 16 DAP, respectively, suggesting more complex transcriptional changes in AE. Most of DEGs in the terms of cell growth and cell wall formation exhibited down-regulated expression patterns (96(down)/131(all) in AE8, 174(down)/235(all) in AE16), while most of DEGs in the processes of photosynthesis, photorespiration, peroxisome, oxidative stress, and systemic acquired resistance exhibited up-regulated expression patterns (222(up)/304(all) in AE8, 214(up)/287(all) in AE16). This is in accordance with a high level of reactive oxygen species (ROS) in the siliques of B. napus pollinated with B. oleracea. Our data suggest that the disorder of plant hormone metabolism, retardation of cell morphogenesis, and the accumulation of ROS may be associated with hybrid incompatibility between B. napus and B. oleracea.

Selective modes affect gene feature and function differentiation of tetraploid Brassica species in their evolution and domestication.

The genus Brassica contains a diverse group of important vegetables and oilseed crops. Genome sequencing has been completed for the six species (B. rapa, B. oleracea, B. nigra, B. carinata, B. napus, and B. juncea) in U's triangle model. The purpose of the study is to investigate whether positively and negatively selected genes (PSGs and NSGs) affect gene feature and function differentiation of Brassica tetraploids in their evolution and domestication. A total of 9,701 PSGs were found in the A, B and C subgenomes of the three tetraploids, of which, a higher number of PSGs were identified in the C subgenome as comparing to the A and B subgenomes. The PSGs of the three tetraploids had more tandem duplicated genes, higher single copy, lower multi-copy, shorter exon length and fewer exon number than the NSGs, suggesting that the selective modes affected the gene feature of Brassica tetraploids. The PSGs of all the three tetraploids enriched in a few common KEGG pathways relating to environmental adaption (such as Phenylpropanoid biosynthesis, Riboflavin metabolism, Isoflavonoid biosynthesis, Plant-pathogen interaction and Tropane, piperidine and pyridine alkaloid biosynthesis) and reproduction (Homologous recombination). Whereas, the NSGs of the three tetraploids significantly enriched in dozens of biologic processes and pathways without clear relationships with evolution. Moreover, the PSGs of B. carinata were found specifically enriched in lipid biosynthesis and metabolism which possibly contributed to the domestication of B. carinata as an oil crop. Our data suggest that selective modes affected the gene feature of Brassica tetraploids, and PSGs contributed in not only the evolution but also the domestication of Brassica tetraploids.

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.

Silicon Alleviates the Disease Severity of Sclerotinia Stem Rot in Rapeseed.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a devastating disease in rapeseed. The objective of this study was to investigate the role and the mechanism of silicon (Si) in alleviating the disease severity of S. sclerotiorum in rapeseed. In the absorption assays, the rapeseed that absorbed 10 mM of K2SiO3 exhibited an 86% decrease in lesion size on infected leaves as compared with controls. In the spray assay, the lesion length on rapeseed stems was reduced by 30.5-32.9% with the use of 100 mM of a foliar Si fertilizer as compared with controls. In the pot assay, the lesion length on rapeseed stems was reduced by 34.9-38.3% when using the Si fertilizer as basal fertilizer. In the field assay, both the disease incidence and disease index of sclerotinia stem rot were significantly reduced with the usage of a solid Si fertilizer, Si foliar fertilizer, and the application of both, without negative affection on the main agronomic traits and seed quality of rapeseed. The transcriptome sequencing, quantitative reverse transcription PCR (qRT-PCR), and biochemical assays between K2SO4- and K2SiO3- treated rapeseed leaves revealed that Si promoted the biosynthesis of defense-related substances and enhanced the antioxidation and detoxification abilities of rapeseed after infection. Thus, this study concluded that Si can alleviate the disease severity of S. sclerotiorum in rapeseeds, partially due to the induced defense responses.

Identification of Quantitative Trait Loci for Clubroot Resistance in Brassica oleracea With the Use of Brassica SNP Microarray.

Increasing clubroot resistance (CR) of Brassica oleracea by ascertaining the molecular mechanisms has been the key focus in modern B. oleracea breeding. In order to identify the quantitative trait loci (QTLs) associated with CR in B. oleracea, 94 F2 vegetative lines which were developed by tissue culture of selfed seeds from the F1 generation between a clubroot-resistant B. oleracea inbred line and a susceptible line, were identified for disease incidence and six CR-associated traits under a lab inoculation by Plasmodiophora brassicae and were genotyped with the 60K Brassica SNP array. Significant correlations were detected for numbers of fibrous roots and P. brassicae content in roots with disease incidence. Nine linkage groups were constructed from 565 bins which covered around 3,000 SNPs, spanning 1,028 cM of the B. oleracea genome with an average distance of 1.82 cM between adjacent bins. A total of 23 QTLs were identified for disease incidence and the other two correlated traits, individually explaining 6.1-17.8% of the phenotypic variation. Several overlaps were detected among traits, including one three-traits-overlapped locus on linkage group C08 and two important overlapped regions between the two CR-associated traits on C06. The QTLs were compared with known CR loci/genes and the novelty of our QTLs was discussed.

Ss-Rhs1, a secretory Rhs repeat-containing protein, is required for the virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S. sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.