Pyramiding of triple Clubroot resistance loci conferred superior resistance without negative effects on agronomic traits in Brassica napus.

Clubroot disease caused by Plasmodiophora brassicae is becoming a serious threat to rapeseed (Brassica napus) production worldwide. Breeding resistant varieties using CR (clubroot resistance) loci is the most promising solution. Using marker-assisted selection and speed-breeding technologies, we generated Brassica napus materials in homozygous or heterozygous states using CRA3.7, CRA08.1, and CRA3.2 loci in the elite parental line of the Zhongshuang11 background. We developed three elite lines with two CR loci in different combinations and one line with three CR loci at the homozygous state. In our study, we used six different clubroot strains (Xinmin, Lincang, Yuxi, Chengdu, Chongqing, and Jixi) which are categorized into three groups based on our screening results. The newly pyramided lines with two or more CR loci displayed better disease resistance than the parental lines carrying single CR loci. There is an obvious gene dosage effect between CR loci and disease resistance levels. For example, pyramided lines with triple CR loci in the homozygous state showed superior resistance for all pathogens tested. Moreover, CR loci in the homozygous state are better on disease resistance than the heterozygous state. More importantly, no negative effect was observed on agronomic traits for the presence of multiple CR loci in the same background. Overall, these data suggest that the pyramiding of triple clubroot resistance loci conferred superior resistance with no negative effects on agronomic traits in Brassica napus.

Functional analysis of the key BrSWEET genes for sugar transport involved in the Brassica rapa-Plasmodiophora brassicae interaction.

Plasmodiophora brassicae, the causative agent of clubroot disease, establishes a long-lasting parasitic relationship with its host by inducing the expression of sugar transporters. Previous studies have indicated that most BrSWEET genes in Chinese cabbage are up-regulated upon infection with P. brassicae. However, the key BrSWEET genes responsive to P. brassicae have not been definitively identified. In this study, we selected five BrSWEET genes and conducted a functional analysis of them. These five BrSWEET genes showed a notable up-regulation in roots after P. brassicae inoculation. Furthermore, these BrSWEET proteins were localized to the plasma membrane. Yeast functional complementation assays confirmed transport activity for glucose, fructose, or sucrose in four BrSWEETs, with the exception of BrSWEET2a. Mutants and silenced plants of BrSWEET1a, -11a, and -12a showed lower clubroot disease severity compared to wild-type plants, while gain-of-function Arabidopsis thaliana plants overexpressing these three BrSWEET genes exhibited significantly higher disease incidence and severity. Our findings suggested that BrSWEET1a, BrSWEET11a, and BrSWEET12a play pivotal roles in P. brassicae-induced gall formation, shedding light on the role of sugar transporters in host-pathogen interactions.

Genome-Wide Identification and Characterization of the Trehalose-6-Phosphate Synthetase Gene Family in Chinese Cabbage (Brassica rapa) and Plasmodiophora brassicae during Their Interaction.

Trehalose is a nonreducing disaccharide that is widely distributed in various organisms. Trehalose-6-phosphate synthase (TPS) is a critical enzyme responsible for the biosynthesis of trehalose, which serves important functions in growth and development, defense, and stress resistance. Although previous studies have found that the clubroot pathogen Plasmodiophora brassicae can lead to the accumulation of trehalose in infected Arabidopsis organs, it has been proposed that much of the accumulated trehalose is derived from the pathogen. At present, there is very little evidence to verify this view. In this study, a comprehensive analysis of the TPS gene family was conducted in Brassica rapa and Plasmodiophora brassicae. A total of 14 Brassica rapa TPS genes (BrTPSs) and 3 P. brassicae TPS genes (PbTPSs) were identified, and the evolutionary characteristics, functional classification, and expression patterns were analyzed. Fourteen BrTPS genes were classified into two distinct classes according to phylogeny and gene structure. Three PbTPSs showed no significant differences in gene structure and protein conserved motifs. However, evolutionary analysis showed that the PbTPS2 gene failed to cluster with PbTPS1 and PbTPS3. Furthermore, cis-acting elements related to growth and development, defense and stress responsiveness, and hormone responsiveness were predicted in the promoter region of the BrTPS genes. Expression analysis of most BrTPS genes at five stages after P. brassicae interaction found no significant induction. Instead, the expression of the PbTPS genes of P. brassicae was upregulated, which was consistent with the period of trehalose accumulation. This study deepens our understanding of the function and evolution of BrTPSs and PbTPSs. Simultaneously, clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae is also of great significance.

Marker-Assisted Pyramiding of CRa and CRd Genes to Improve the Clubroot Resistance of Brassica rapa.

Clubroot, caused by Plasmodiophora brassicae, is an economically important soil-borne disease that threatens Brassicaceae crops worldwide. In recent years, the incidence area of Chinese cabbage (Brassica rapa ssp. pekinensis) clubroot disease has increased, which severely affects the yield and quality of Chinese cabbage. The resistance of varieties harboring the single clubroot-resistance (CR) gene is easily broken through by P. brassicae pathotypes. CRa and CRd, genetically identified in B. rapa, are CR genes known to be highly resistant to different P. brassicaea pathotypes. In our study, we perform the gene pyramiding of CRa and CRd in Chinese cabbages through marker-assisted selection (MAS), and develop homozygous pyramided lines. The newly generated pyramided lines exhibit greater resistance to six different pathotypes than that of two parental lines carrying a single CR gene. This study provides new CR-gene-pyramided lines for the development of clubroot-resistant Brassica varieties for future breeding programs.

Progress on plant orphan genes.

Orphan genes are located in a special evolutionary branch and have no significant sequence similarity with any other identified genes. Orphan genes are prevalent in every species, comparative genomics analyses found that all sequenced species contained a portion of orphan genes, and the number of orphan genes obtained by distinct screening conditions is different. Orphan genes are often associated with various stress responses, species-specific evolution and substance metabolism regulation. However, most of the orphan genes have not been well annotated or even have no recognizable functional domains, which brings some difficulties to the functional characterization of orphan genes. Compared with conserved genes, there is less research on orphan genes, which leads to the possibility that the importance of orphan genes may be "unrewarded". In this review, we summarize the origin and evolution of orphan genes, plant orphan gene screening and functions, and analyse the existing challenges and future research priorities and solutions, which provide theoretical basis for the study of orphan gene function and action mechanisms.

Transferring of clubroot-resistant locus CRd from Chinese cabbage (Brassica rapa) to canola (Brassica napus) through interspecific hybridization.

Clubroot, caused by Plasmodiophora brassicae is one of the most severe threats to brassica species in China and worldwide. Breeding for clubroot resistant varieties is one of the best ways to overcome this disease. In this study, we introduced clubroot resistance (CR) gene CRd from Chinese cabbage (85-74) into elite Brassica napus inbred line Zhongshuang 11 through interspecific hybridization and subsequent backcrossing with whole-genome molecular marker-assisted selection (MAS). The resistant test of CRd to P. brassicae isolates was evaluated in the greenhouse as well as in field conditions. Close linkage markers and the whole-chromosome background marker selection approach improved the recovery rate from 78.3% in BC1 to 100% in BC3F1. The improved clubroot-resistant variety, Zhongshuang11R, was successfully selected in the BC3F2 generation. The greenhouse and field resistant tests revealed that Zhongshuang11R was resistant to P. brassicae pathotypes. The agronomic characteristics of Zhongshuang11R were similar to those of its recurrent parental line, including oil content, composition of fatty acid, plant height, primary effective branches, grain yield per plant and thousand-seed weight. In addition, the oil quality could satisfy the quality requirements for commercial rapeseed oil. Our results will enrich the resistant resources of canola and will certainly accelerate clubroot resistance breeding programs in B. napus.

Fine mapping and candidate gene analysis of CRA3.7 conferring clubroot resistance in Brassica rapa.

KEY MESSAGE: In this study, we fine-mapped a clubroot resistance gene CRA3.7 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding. Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, CRA3.7, was mapped on chromosome A03 of Brassica rapa. A Chinese cabbage line 'CR510', which harbor homozygous resistance locus CRA3.7 was selected from a BC4F3 family. 'CR510' was crossed with a clubroot susceptible Chinese cabbage inbred line '59-1'. Total 51 recombinant plants were identified from an F2 population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F2/3 families was evaluated. Finally, a clubroot resistance gene CRA3.7 was fine-mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine-mapped region. Further, nine candidate's gene expression in parental lines at 7, 14 and 21 days after inoculation of P. brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in 'CR510' than '59-1'. Gene sequence of Bra019410 from 'CR510' was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates for CRA3.7. Our research provides a valuable germplasm resource against P. brassicae Pb3 and CRA3.7 closely linked marker for marker-assisted selection in CR cultivars breeding.

Identification and characterization of putative effectors from Plasmodiophora brassicae that suppress or induce cell death in Nicotiana benthamiana.

Clubroot, caused by Plasmodiophora brassicae, is a major disease of crucifers. Effector proteins are important virulence factors in host recognition of pathogens and the interactions between pathogens and hosts. Secretory proteins, as effector candidates, have been studied in the interaction between Plasmodiophora brassicae and its hosts. In this study, 518 secretary proteins were screened from the Plasmodiophora brassicae genome. A total of 63 candidate effectors that induce or suppress cell death were identified using agroinfiltration-mediated transient expression in Nicothiana benthamiana. The candidate effectors, Pb4_102097 and Pb4_108104 showed high expressing level in the stage of rest spore maturity, could induce cell death and were associated with H2O2 accumulation in N. benthamiana leaves. In addition, 55 candidate effectors that could suppress BAX (Bcl-2-associated X protein) induced cell death, and 21 out of which could suppress the immunity caused by bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 in Arabidopsis. Based on the expression pattern in different stages, 28 candidate effectors showed high expression levels during the primary and secondary infection stage. Five candidate effectors containing the RXLR motif functioned in the cytoplasm and cell membrane.

Research Advances and Prospects of Orphan Genes in Plants.

Orphan genes (OGs) are defined as genes having no sequence similarity with genes present in other lineages. OGs have been regarded to play a key role in the development of lineage-specific adaptations and can also serve as a constant source of evolutionary novelty. These genes have often been found related to various stress responses, species-specific traits, special expression regulation, and also participate in primary substance metabolism. The advancement in sequencing tools and genome analysis methods has made the identification and characterization of OGs comparatively easier. In the study of OG functions in plants, significant progress has been made. We review recent advances in the fast evolving characteristics, expression modulation, and functional analysis of OGs with a focus on their role in plant biology. We also emphasize current challenges, adoptable strategies and discuss possible future directions of functional study of OGs.

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification.

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host's carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.

Identification and Characterization of Circular RNAs in Brassica rapa in Response to Plasmodiophora brassicae.

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of 'BJN 222' at 0, 8, and 23 days post-inoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA-miRNA-mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa.

R gene triplication confers European fodder turnip with improved clubroot resistance.

Clubroot is one of the most important diseases for many important cruciferous vegetables and oilseed crops worldwide. Different clubroot resistance (CR) loci have been identified from only limited species in Brassica, making it difficult to compare and utilize these loci. European fodder turnip ECD04 is considered one of the most valuable resources for CR breeding. To explore the genetic and evolutionary basis of CR in ECD04, we sequenced the genome of ECD04 using de novo assembly and identified 978 candidate R genes. Subsequently, the 28 published CR loci were physically mapped to 15 loci in the ECD04 genome, including 62 candidate CR genes. Among them, two CR genes, CRA3.7.1 and CRA8.2.4, were functionally validated. Phylogenetic analysis revealed that CRA3.7.1 and CRA8.2.4 originated from a common ancestor before the whole-genome triplication (WGT) event. In clubroot susceptible Brassica species, CR-gene homologues were affected by transposable element (TE) insertion, resulting in the loss of CR function. It can be concluded that the current functional CR genes in Brassica rapa and non-functional CR genes in other Brassica species were derived from a common ancestral gene before WGT. Finally, a hypothesis for CR gene evolution is proposed for further discussion.

Brassica rapa orphan genes largely affect soluble sugar metabolism.

Orphan genes (OGs), which are genes unique to a specific taxon, play a vital role in primary metabolism. However, little is known about the functional significance of Brassica rapa OGs (BrOGs) that were identified in our previous study. To study their biological functions, we developed a BrOG overexpression (BrOGOE) mutant library of 43 genes in Arabidopsis thaliana and assessed the phenotypic variation of the plants. We found that 19 of the 43 BrOGOE mutants displayed a mutant phenotype and 42 showed a variable soluble sugar content. One mutant, BrOG1OE, with significantly elevated fructose, glucose, and total sugar contents but a reduced sucrose content, was selected for in-depth analysis. BrOG1OE showed reduced expression and activity of the Arabidopsis sucrose synthase gene (AtSUS); however, the activity of invertase was unchanged. In contrast, silencing of two copies of BrOG1 in B. rapa, BraA08002322 (BrOG1A) and BraSca000221 (BrOG1B), by the use of an efficient CRISPR/Cas9 system of Chinese cabbage (B. rapa ssp. campestris) resulted in decreased fructose, glucose, and total soluble sugar contents because of the upregulation of BrSUS1b, BrSUS3, and, specifically, the BrSUS5 gene in the edited BrOG1 transgenic line. In addition, we observed increased sucrose content and SUS activity in the BrOG1 mutants, with the activity of invertase remaining unchanged. Thus, BrOG1 probably affected soluble sugar metabolism in a SUS-dependent manner. This is the first report investigating the function of BrOGs with respect to soluble sugar metabolism and reinforced the idea that OGs are a valuable resource for nutrient metabolism.

Development of a Sinitic Clubroot Differential Set for the Pathotype Classification of Plasmodiophora brassicae.

Plasmodiophora brassicae, which is known for its broad genetic diversity for virulence, is the causal agent of clubroot disease of Brassica crops worldwide. Studies on pathotype characterization with four differential hosts according to Williams' classification system showed the predominance of pathotype 4 in China. However, the genetic variability within pathotype 4 complicates the breeding of durable clubroot-resistant (CR) cultivars. Herein, a Sinitic clubroot differential (SCD) set was developed using a set of eight differential inbred lines of Chinese cabbage with known or novel CR genes. The presence of immense diversity within pathotype 4 of Williams' system was verified, and 11 pathotypes were characterized using the developed SCD system. The scalability and practicability of the system was further confirmed with a subset of 95 field isolates from different Brassica crops and different regions of China and Korea. Sixteen pathotypes were detected from 132 field isolates, named Pb1 to Pb16, respectively. Among them, Pb1 and Pb4 were prevalent in diverse Brassica crops in the southern and northern regions of China. Pb12, Pb13, Pb14, and Pb16 showed area-specific distribution. The SCD set developed herein will provide important genetic resources for pathogenicity studies of P. brassicae and for CR breeding in Chinese cabbage and other Brassica crops.

Association of Clubroot Resistance Locus PbBa8.1 With a Linkage Drag of High Erucic Acid Content in the Seed of the European Turnip.

Clubroot caused by Plasmodiophora brassicae is a severe threat to the production of Brassica napus, worldwide. The cultivation of resistant varieties is the most efficient and environmentally friendly way to limit disease spread. We developed a highly resistant B. napus line, ZHE226, containing the resistance locus PbBa8.1. However, ZHE226 seeds contain high erucic acid content, which limits its cultivation owing to its low edible oil quality. A segregation population of BC3F2 was developed by crossing ECD04, a resistant European turnip donor, with Huangshuang5, an elite variety with no erucic acid in its seeds, as a recurrent plant. Fine mapping using the bulk segregation analysis sequencing (BSA-Seq) approach detected PbBa8.1 within a 2.9 MB region on chromosome A08. Interestingly, the previously reported resistance gene Crr1a was found in the same region. Genetic analysis revealed that the CAP-134 marker for Crr1a was closely linked with clubroot resistance (CR). Thus, PbBa8.1 and Crr1a might be allelic for CR. Moreover, comparative and genetic analysis showed that high erucic acid in the seeds of ZHE226 was due to linkage drag of fatty acid elongase 1 (FAE1) in the ECD04 line, which was located in the interval of PbBa8.1 with a physical and genetic distance of 729 Kb and 1.86 cm, respectively. Finally, a clubroot-resistant line with a low erucic acid content was successfully developed through gene-specific molecular marker assistant selection from BC4F4. These results will accelerate CR breeding programs in B. napus.

CRb and PbBa8.1 Synergically Increases Resistant Genes Expression upon Infection of Plasmodiophora brassicae in Brassica napus.

PbBa8.1 and CRb are two clubroot-resistant genes that are important for canola breeding in China. Previously, we combined these resistant genes and developed a pyramid-based, homozygous recurrent inbred line (618R), the results of which showed strong resistance to Plasmodiophora brassicae field isolates; however, the genetic mechanisms of resistance were unclear. In the present work, we conducted comparative RNA sequencing (RNA-Seq) analysis between 618R and its parental lines (305R and 409R) in order to uncover the transcriptomic response of the superior defense mechanisms of 618R and to determine how these two different resistant genes coordinate with each other. Here, we elucidated that the number and expression of differentially expressed genes (DEGs) in 618R are significantly higher than in the parental lines, and PbBa8.1 shares more DEGs and plays a dominant role in the pyramided line. The common DEGs among the lines largely exhibit non-additive expression patterns and enrichment in resistance pathways. Among the enriched pathways, plant-pathogen interaction, plant hormone signaling transduction, and secondary metabolites are the key observation. However, the expressions of the salicylic acid (SA) signaling pathway and reactive oxygen species (ROS) appear to be crucial regulatory components in defense response. Our findings provide comprehensive transcriptomic insight into understanding the interactions of resistance gene pyramids in single lines and can facilitate the breeding of improved resistance in Brassica napus.

Transcriptome Sequencing and Metabolism Analysis Reveals the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulbs.

Onion (Allium cepa L.) is an important bulbous vegetable crop that possesses important properties related to health as well as extraordinary colors. Naturally white onion bulbs were used in this study to reveal the complex metabolic mechanisms that underlie phenotypic traits, especially bulb pigmentation. Six libraries (three dark-red and three white) were constructed and analyzed to elucidate differences in cyanidin (Cy) metabolism between dark-red and white onion bulbs. Libraries were screened using RNA-sequencing (RNA-seq) to reveal the differentially expressed genes (DEGs) involved in anthocyanin biosynthesis at the transcriptional level. Comparison with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database shows that a total of 27 unigenes participate in onion anthocyanin biosynthesis and 16 DEGs perform critical roles in flavonoid biosynthesis. Expression patterns of color-related flavonoid compounds associated with the onion anthocyanin biosynthesis pathway (ABP) show that flavonoid 3',5'-hydroxylase (F3'5'H) and dihydroflavonol 4-reductase (DFR) genes play crucial roles in the biosynthesis of dark-red bulbs, the expression levels of flavonol synthase (FLS) and DFR genes may act to block blue pigmentation, and the loss of Cy from white onion bulbs might explain multibranching in the synthesis of this compound. Positive variation in the F3'5'H/F3'H ratio also affects onion bulb color diversity. The transcriptome presented here provides a basis for future onion molecular breeding based on variations in the diversity of ornamental plant pigmentation.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa.

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage (Brassica rapa ssp. pekinensis).

The rapid spread of clubroot disease, which is caused by Plasmodiophora brassicae, threatens Brassicaceae crop production worldwide. Breeding plants that have broad-spectrum disease resistance is one of the best ways to prevent clubroot. In the present study, eight Chinese cabbage germplasms were screened using published clubroot-resistant (CR) loci-/gene-linked markers. A CR gene Crr3 potential carrier "85-74" was detected which linked to marker BRSTS61; however, "85-74" shows different responses to local pathogens "LAB-19," "LNND-2," and "LAB-10" from "CR-73" which harbors Crr3. We used a next-generation sequencing-based bulked segregant analysis approach combined with genetic mapping to detect CR genes in an F2 segregant population generated from a cross between the Chinese cabbage inbred lines "85-74" (CR) and "BJN3-1" (clubroot susceptible). The "85-74" line showed resistance to a local pathogen "LAB-19" which was identified as race 4; a genetic analysis revealed that the resistance was conferred by a single dominant gene. The CR gene which we named CRd was mapped to a 60 kb (1 cM) region between markers yau389 and yau376 on chromosome A03. CRd is located upstream of Crr3 which was confirmed based on the physical positions of Crr3 linked markers. The identification of CRd linked markers can be applied to marker-assisted selection in the breeding of new CR cultivars of Chinese cabbage and other Brassica crops.

Cytological and morphological analysis of hybrids between Brassicoraphanus, and Brassica napus for introgression of clubroot resistant trait into Brassica napus L.

Interspecific hybridization is a powerful tool for improvement of crop species, it has the potential to broaden the genetic base and create new plant forms for breeding programs. Synthetic allopolyploid is a widely-used model for the study of genetic recombination and fixed heterosis in Brassica. In Brassica napus breeding, identification and introgression of new sources of clubroot resistance trait from wild or related species into it by hybridization is a long-term crop management strategy for clubroot disease. Radish (Raphanus sativus L.) is a close relative of the Brassica and most radish accessions are immune to the clubroot disease. A synthesized allotetraploid Brassicoraphanus (RRCC, 2n = 36) between R. sativus cv. HQ-04 (2n = 18, RR) and Brassica oleracea var. alboglabra (L.H Bailey) (2n = 18, CC) proved resistant of multiple clubroot disease pathogen P. brassicae. To predict the possibility to transfer the clubroot resistance trait from the RR subgenome of allotetraploid Brassicoraphanus (RRCC, 2n = 36) into Brassica napus (AACC, 2n = 38), we analyzed the frequency of chromosome pairings in the F1 hybrids produced from a cross between B. napus cv. HS5 and the allotetraploid, characterize the genomic composition of some backcrossed progeny (BC1) using GISH, BAC-FISH and AFLP techniques. The level of intergenomic pairing between A and R genomes in the F1 hybrid was high, allosyndetic bivalents formed in 73.53% PMCs indicative of significant level of homeologous recombination between two genomes and high probability of incorporating chromosomal segments/genes from R-genome into A/C-genomes. The BC1 plants inherited variant extra R chromosomes or fragments from allotetraploid as revealed by GISH and AFLP analysis. 13.51% BC2 individuals were resistant to clubroot disease, and several resistance lines had high pollen fertility, Overall, the genetic material presented in this work represents a potential new genetic resource for practical use in breeding B. napus clubroot resistant cultivars.