A Ca2+ sensor BraCBL1.2 involves in BraCRa-mediated clubroot resistance in Chinese cabbage.

Clubroot disease caused by Plasmodiophora brassicae (P. brassicae) severely threatens the cultivation of Cruciferous plants, especially Chinese cabbage. Recently, resistance genes in plants have been reported to encode for a Ca2+-permeable channel in the plasma membrane, which can mediate the cytosolic Ca2+ increase in plant cells upon pathogen attack. However, the downstream Ca2+ sensor and decoder are still unknown. In this study, we identified the virulent and avirulent P. brassicae isolates (Pbs) of two near isogenic lines, CR 3-2 and CS 3-2, with CR 3-2 harboring clubroot resistant gene BraCRa. The transcriptomic analysis was then conducted with CR 3-2 after inoculating with virulent isolate PbE and avirulent isolate Pb4. From the differentially expressed genes of transcriptomic data, we identified a Ca2+-sensor encoding gene, BraCBL1.2, that was highly induced in CR 3-2 during infection by Pb4 but not by PbE. Moreover, GUS histochemical staining and subcellular localization analysis revealed that BraCBL1.2 was specifically expressed in the root hair cells of Arabidopsis and encoded a putative Ca2+ sensor localized in the plasma membrane. We also developed an assay to investigate the BraCRa-mediated hypersensitive response (HR) in tobacco leaves. The results suggest that BraCBL1.2 is involved in the BraCRa-mediated plant ETI immune response against P. brassicae. In addition, we verified that overexpression of BraCBL1.2 enhanced clubroot resistance in Arabidopsis. Collectively, our data identified the involvement of a Ca2+ sensor in BraCRa-mediated clubroot resistance in Chinese cabbage, providing a theoretical basis for further research on the resistance of Chinese cabbage to P. brassicae.

Effects of Exogenous Ergothioneine on Brassica rapa Clubroot Development Revealed by Transcriptomic Analysis.

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae that leads to a serious yield reduction in cruciferous plants. In this study, ergothioneine (EGT) was used to culture P. brassicae resting spores, the germination of which was significantly inhibited. Further exogenous application of EGT and P. brassicae inoculation in Chinese cabbage showed that EGT promoted root growth and significantly reduced the incidence rate and disease index. To further explore the mechanism by which EGT improves the resistance of Chinese cabbage to clubroot, a Chinese cabbage inbred line BJN3-2 susceptible to clubroot treated with EGT was inoculated, and a transcriptome analysis was conducted. The transcriptome sequencing analysis showed that the differentially expressed genes induced by EGT were significantly enriched in the phenylpropanoid biosynthetic pathway, and the genes encoding related enzymes involved in lignin synthesis were upregulated. qRT-PCR, peroxidase activity, lignin and flavonoid content determination showed that EGT promoted the lignin and flavonoid synthesis of Chinese cabbage and improved its resistance to clubroot. This study provides a new insight for the comprehensive prevention and control of cruciferous clubroot and for further study of the effects of EGT on clubroot disease.

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.

Fungus Pichia kudriavzevii XTY1 and heterotrophic nitrifying bacterium Enterobacter asburiae GS2 cannot efficiently transform organic nitrogen via hydroxylamine and nitrite.

Heterotrophic nitrification is a process of organic nitrogen degradation completed by the participation of heterotrophic nitrifying microorganisms, which can accelerate the nitrogen transformation process. However, the current research mainly focuses on heterotrophic nitrifying bacteria and their ammonium degradation capacities. And there is little accumulation of research on fungi, the main force of heterotrophic nitrification, and their capacities to transform organic nitrogen. In this study, novel heterotrophic nitrifying fungus (XTY1) and bacterium (GS2) were screened and isolated from upland soil, and the strains were identified and registered through GenBank comparison. After 24 h single nitrogen source tests and 15N labeling tests, we compared and preliminarily determined the heterotrophic nitrification capacities and pathways of the two strains. The results showed that XTY1 and GS2 had different transformation capacities to different nitrogen substrates and could efficiently transform organic nitrogen. However, the transformation capacity of XTY1 to ammonium was much lower than that of GS2. The two strains did not pass through NH2OH and NO2 - during the heterotrophic nitrification of organic nitrogen, and mainly generated intracellular nitrogen and low N2O. Other novel organic nitrogen metabolism pathways may be existed, but they remain to be further validated.

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.

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.

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.

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.

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.

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.

Identification of Novel Locus RsCr6 Related to Clubroot Resistance in Radish (Raphanus sativus L.).

Clubroot is a devastating disease that causes substantial yield loss worldwide. However, the inheritance and molecular mechanisms of clubroot resistance during pathogen infection in radish remain largely unclear. In this study, we investigated the inheritance of clubroot resistance in the F2 population derived from crossing clubroot-resistant (CR) and clubroot-susceptible inbred lines "GLX" and "XNQ," respectively. Genetic analysis revealed that a single dominant gene controlled the clubroot resistance of "GLX" with a Mendelian ratio of resistance and susceptibility of nearly 3:1. Bulked segregant analysis combined with whole-genome resequencing (BSA-seq) was performed to detect the target region of RsCr6 on chromosome Rs8. Linkage analysis revealed that the RsCr6 locus was located between two markers, HB321 and HB331, with an interval of approximately 92 kb. Based on the outcomes of transcriptome analysis, in the RsCr6 locus, the R120263140 and R120263070 genes with a possible relation to clubroot resistance were considered candidate genes. In addition, three core breeding materials containing the two reported quantitative trait loci (QTLs) and our novel locus RsCr6 targeting clubroot resistance were obtained using marker-assisted selection (MAS) technology. This study reveals a novel locus responsible for clubroot resistance in radishes. Further analysis of new genes may reveal the molecular mechanisms underlying the clubroot resistance of plants and provide a theoretical basis for radish resistance breeding.

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.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

Spatiotemporal Quantification of Plasmodiophora brassicae Inoculum in Relation to Clubroot Development Under Inoculated and Naturally Infested Field Conditions.

Clubroot caused by Plasmodiophora brassicae is a destructive disease of cruciferous plants worldwide. A quantitative PCR (qPCR) system specific to P. brassicae was developed. Analysis of the qPCR sensitivity indicated that the lower limit of detection was 1 × 101 resting spores/ml, 1 × 102 spores/g of soil, and 1 × 103 spores/g of roots and seeds. The regression curves generated from the qPCR data of different samples had a parallel relationship. The difference between the theoretical and actual concentrations was lowest at 1 × 105 spores/g of sample, compared with other concentrations. The P. brassicae biomass in soil and plant root tissues after inoculated with different spore concentrations was correlated. A correlation analysis confirmed that the clubroot incidence and disease index at 6 weeks after inoculation increased as the spore concentration increased. Under field conditions, the natural inoculum density of the P. brassicae population decreased at the early stage and then increased, with P. brassicae mainly being detected at a soil depth of 0 to 50 cm. The horizontal distribution of P. brassicae varied in the field with occurrences of hot spots. This study established a qPCR-based method for quantitative detection of clubroot. The developed assay is useful for monitoring the spatiotemporal dynamics of P. brassicae in the field. It may also be applicable for clubroot forecasting as a part of proactive disease management.

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.

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.