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.

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.

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.

Identification of Micro Ribonucleic Acids and Their Targets in Response to Plasmodiophora brassicae Infection in Brassica napus.

Clubroot disease, which is caused by the soil-borne pathogen Plasmodiophora brassicae War (P. brassicae), is one of the oldest and most destructive diseases of Brassica and cruciferous crops in the world. Plant microRNAs [micro ribonucleic acids (miRNAs)] play important regulatory roles in several developmental processes. Although the role of plant miRNAs in plant-microbe interaction has been extensively studied, there are only few reports on the specific functions of miRNAs in response to P. brassicae. This study investigated the roles of miRNAs and their targets during P. brassicae infection in a pair of Brassica napus near-isogenic lines (NILs), namely clubroot-resistant line 409R and clubroot-susceptible line 409S. Small RNA sequencing (sRNA-seq) and degradome-seq were performed on root samples of 409R and 409S with or without P. brassicae inoculation. sRNA-seq identified a total of 48 conserved and 72 novel miRNAs, among which 18 had a significant differential expression in the root of 409R, while only one miRNA was differentially expressed in the root of 409S after P. brassicae inoculation. The degradome-seq analysis identified 938 miRNA target transcripts, which are transcription factors, enzymes, and proteins involved in multiple biological processes and most significantly enriched in the plant hormone signal transduction pathway. Between 409R and 409S, we found eight different degradation pathways in response to P. brassicae infection, such as those related to fatty acids. By combining published transcriptome data, we identified a total of six antagonistic miRNA-target pairs in 409R that are responsive to P. brassicae infection and involved in pathways associated with root development, hypersensitive cell death, and chloroplast metabolic synthesis. Our results reveal that P. brassicae infection leads to great changes in miRNA pool and target transcripts. More interestingly, these changes are different between 409R and 409S. Clarification of the crosstalk between miRNAs and their targets may shed new light on the possible mechanisms underlying the pathogen resistance against P. brassicae.

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.

The response of transgenic Brassica species to salt stress: a review.

Salt stress is considered one of the main abiotic factors to limit crop growth and productivity by affecting morpho-physiological and biochemical processes. Genetically, a number of salt tolerant Brassica varieties have been developed and introduced, but breeding of such varieties is time consuming. Therefore, current focus is on transgenic technology, which plays an important role in the development of salt tolerant varieties. Various salt tolerant genes have been characterized and incorporated into Brassica. Therefore, such genetic transformation of Brassica species is a significant step for improvement of crops, as well as conferring salt stress resistance qualities to Brassica species. Complete genome sequencing has made the task of genetically transforming Brassica species easier, by identifying desired candidate genes. The present review discusses relevant information about the principles which should be employed to develop transgenic Brassica species, and also will recommend tools for improved tolerance to salinity.