Chinese Cabbage Inbred Line Research Articles

Transcriptome Analysis Reveals Association of Photosynthesis and Phytohormone Pathways with Leaf Color in Chinese Cabbage

Chinese cabbage (Brassica rapa L. ssp. Pekinensis) in the genus Brassica of the family Brassicaceae (Cruciferae) originates from China and is one of the most consumed leafy vegetables in East Asian countries. The leaf color is tightly linked with its growth, development, and yield. By screening an EMS mutagenized population of Chinese cabbage inbred line A03, we identified a yellowgreen leaf mutant ygl. This mutant developed abnormal ultrastructure in chloroplasts. Transcriptome and Gene Ontology (GO) analyses unveiled that abiotic stress response and glucan metabolism pathways were enriched in ygl compared to A03. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis reveals that differentially expressed genes were mainly associated with plant hormone signal transduction, photosynthesis, and starch and sucrose metabolism. In ygl, the expression of some photosynthetic pathway genes was significantly up-regulated, while the transcription of key genes related to carbon fixation, starch syntheses, and sugar metabolism pathways, such as RBCS1A, APS1, APL3, and SUS1, was markedly down-regulated. We also found a similar reduction trend in mRNA levels of IAA19, IAA29, and ARR4 associated with auxin and cytokinin pathways. Taken together, we uncovered transcriptional profiles for some important genes that may be responsible for leaf color development, and such gene expression repertoire may be useful for further investigation into the phenotype-to-genotype link in the ygl mutant Chinese cabbage.

Leaf Color Classification and Expression Analysis of Photosynthesis-Related Genes in Inbred Lines of Chinese Cabbage Displaying Minor Variations in Dark-Green Leaves.

The leaves of the Chinese cabbage which is most widely consumed come in a wide variety of colors. Leaves that are dark green can promote photosynthesis, effectively improving crop yield, and therefore hold important application and cultivation value. In this study, we selected nine inbred lines of Chinese cabbage displaying slight differences in leaf color, and graded the leaf color using the reflectance spectra. We clarified the differences in gene sequences and the protein structure of ferrochelatase 2 (BrFC2) among the nine inbred lines, and used qRT-PCR to analyze the expression differences of photosynthesis-related genes in inbred lines with minor variations in dark-green leaves. We found expression differences among the inbred lines of Chinese cabbage in photosynthesis-related genes involved in the porphyrin and chlorophyll metabolism, as well as in photosynthesis and photosynthesis-antenna protein pathway. Chlorophyll b content was significantly positively correlated with the expression of PsbQ, LHCA1_1 and LHCB6_1, while chlorophyll a content was significantly negatively correlated with the expression PsbQ, LHCA1_1 and LHCA1_2. Our results provide an empirical basis for the precise identification of candidate genes and a better understanding of the molecular mechanisms responsible for the production of dark-green leaves in Chinese cabbage.

Integrative analysis of physiology, biochemistry and transcriptome reveals the mechanism of leaf size formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

The leaf, the main product organ, is an essential factor in determining the Chinese cabbage growth, yield and quality. To explore the regulatory mechanism of leaf size development of Chinese cabbage, we investigated the leaf size difference between two high-generation inbred lines of Chinese cabbage, Y2 (large leaf) and Y7 (small leaf). Furtherly, the transcriptome and cis-acting elements analyses were conducted. According to our results, Y2 exhibited a higher growth rate than Y7 during the whole growth stage. In addition, the significant higher leaf number was observed in Y2 than in Y7. There was no significant difference in the number of epidermal cells and guard cells per square millimeter between Y2 and Y7 leaves. It indicated that cell numbers caused the difference in leaf size. The measurement of phytohormone content confirmed that GA1 and GA3 mainly play essential roles in the early stage of leaf growth, and IPA and ABA were in the whole leaf growth period in regulating the cell proliferation difference between Y2 and Y7. Transcriptome analysis revealed that cyclins BraA09g010980.3C (CYCB) and BraA10g027420.3C (CYCD) were mainly responsible for the leaf size difference between Y2 and Y7 Chinese cabbage. Further, we revealed that the transcription factors BraA09gMYB47 and BraA06gMYB88 played critical roles in the difference of leaf size between Y2 and Y7 through the regulation of cell proliferation. This observation not only offers essential insights into understanding the regulation mechanism of leaf development, also provides a promising breeding strategy to improve Chinese cabbage yield.

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.

First Report of Flower Stalk Wilting Caused by Rhizopus oryzae on Chinese cabbage in China.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the main biennial vegetables in China and its flowers can be produced in the second year. In May 2021, approximately 50% of the flower stalks of Chinese cabbage wilted in a field in Laizhou, China. Water-soaked lesions were first observed on the lateral shoots of flower stalks, leading to wilting at a later stage. Small diseased tissues were excised from the margin of lesions, surface disinfected in 75% alcohol, rinsed in distilled water twice, and transferred onto potato dextrose agar (PDA) medium for incubation at 28 ℃. Five fungal isolates were obtained using single spore isolation method. The fungal colonies were initially white and became gray or black within 5 days. The columella was globose to subglobose and 82.86±5.25 μm (n=5) in diameter; sporangiophores were smooth-walled, simple or branched; the globose sporangia were 86.06±15.37 μm (n=5) in diameter and black; the sporangiospores were subglobose and abundant and 5.23±0.98 μm (n=5) in diameter; and the rhizoids were dark brown and 5.69±1.82 μm (n=5) wide. A cetyl tri-methyl ammonium bromide method was used to extract DNA from 3-day-old hyphae (Ausubel et al. 1987). PCR was performed for ITS (White et al. 1990), the RNA polymerase II large subunit (RPB1) gene (Voigt et al. 2000) and the actin (ACT) gene (Stiller et al. 1997). The DNA sequences of the five isolates were identical, therefore, the sequence of Isolate RO21 was submitted to GenBank. According to BLAST search, the ITS (MZ452687), RPB1 (OK431470), and ACT (OK431471) sequences showed 99.66% similarity to Rhizopus oryzae Strain CBS 112.07 (NR103595), 100% to Strain CBS 127.08 (KJ566325) and 100% to Strain CBS 102660 (KJ551423), respectively. A neighbor-joining phylogenetic tree was reconstructed based on the ITS of Isolate RO21 and 14 other Rhizopus species sequences obtained from GenBank. Isolate RO21 was found to be most closely related to R. oryzae and far from other species. Based on morphological and phylogenetic characteristics, Isolate RO21 was identified as R. oryzae (Dolatabadi et al. 2014, Kwon et al. 2015, Palemón-Alberto et al. 2020). Sporangiospores were harvested from 5-day-old PDA cultures, suspended in sterilized distilled water, adjusted to 106 spores/ml and amended with 0.1% Tween-80. Chinese cabbage inbred line "A54-1" was inoculated near the middle of the flower stalk by applying 20 μl of spore suspension (106 spores/ml) to each of three sites wounded using a sterilized knife or to the unwounded site. Sterilized distilled water was used as the control. Forty flower stalks (20 for the inoculation treatment and the rest for the control) selected from ten plants were used for pathogenicity test. All plants were incubated in a growth chamber at 28/22 °C (day/night), with 80 to 90% of relative humidity. Wilting symptoms similar to those in the field were observed in the wounded flower stalks after 5 days and in the non-wounded flower stalks after 15 days. All control flower stalks remained asymptomatic. The fungus was re-isolated from the artificially infected flower stalks and identified as R. oryzae by morphological characteristics and sequencing to fulfill the Koch's postulates. To our knowledge, this is the first report that R. oryzae causes flower stalk wilting on Chinese cabbage in China. The results can provide the basis for future studies on the occurrence, prevention and management of this disease.

Genetic and Transcriptome Analysis of Leaf Trichome Development in Chinese Cabbage (Brassica rapa L. subsp. pekinensis) and Molecular Marker Development

Chinese cabbage (Brassica rapa L. subsp. pekinensis) is one of the vegetables with the largest cultivated area in China and has been a great addition to the daily diet of Chinese people. A genetic map has been constructed in our previous study using the F2 population of two inbred lines of Chinese cabbage, namely "G291" (a hairy line) and "ZHB" (a hairless line), based on which a candidate gene related to trichome traits was identified on chromosome A06 with a phenotypic variance of 47%. A molecular marker was found to co-segregate with the trichome traits of the F2 population, which is in the 5'-flanking region of BrGL1, and a corresponding patent has been granted (NO. CN 108545775 B). Transcriptome analysis was carried out on the cotyledon, the first true leaf and the leaf closest to each inflorescence of F2 individuals of "G291 × ZHB" with or without trichomes, respectively. Ten pathways, including 189 DEGs, were identified to be involved in the development of trichomes in Chinese cabbage, which may be specifically related to the development of leaf trichomes. Most of the pathways were related to the biosynthesis of the secondary metabolites, which may help plants to adapt to the ever-changing external environment. DEGs also enriched the "plant-pathogen interaction" pathway, which is consistent with the conclusion that trichomes are related to the disease resistance of plants. Our study provides a basis for future research on the occurrence and development of trichomes in Chinese cabbage.

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

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.

Screening and Functional Verification of Vernalization-Related Genes in Chinese Cabbage

Flowering is very important for leaf head formation and breeding. Investigating the key genes related to vernalization process can reveal the flowering regulation mechanism and guide production and germplasm breeding. In this study, two Chinese cabbage (Brassica rapa subsp. pekinensis) inbred lines ‘1#’ and ‘2#’ with different winterness were used to identify vernalization-related genes. After low-temperature treatment, the transcriptomes of the shoot apex of the two lines were analyzed. The Arabidopsis thaliana homologues of 2,932 common differentially expressed genes with the same change trend after vernalization of two inbred lines were analysed based on Gene Ontology annotation and description. The results showed that, three genes were identified that may be related to the vernalization: Bra031210, Bra009126 and Bra033615. In which Bra031210 and Bra009126 were cloned, named BrGRP7.1 and BrCPD1, with open reading frames lengths of 507 and 1443 bp, encoding 168 and 480 amino acids, respectively. Through the expression patterns analysis, the expression of BrGRP7.1 showed a trend of initial decreasing (dipping at stage 1) and then increasing (peaking at stage 5) during flower bud differentiation of both inbred lines. Incontrast, BrCPD1 showed a first decreasing, then increasing, and last decreasing trend during flower bud differentiation of both lines. These two genes were transferred into wild-type A. thaliana. The flowering time of T2 generation of BrGRP7.1- and BrCPD1-transgenic plants was 2.31~5.11 days and 2.64~4.35 days earlier than the wild-type, respectively. In conclusion, this study screened three genes that involved in the vernalization process. Through functional verification, BrGRP7.1 and BrCPD1 were proved to have the ability to promote flowering time. This study provided a reference for further study on the flowering mechanism of Chinese cabbage.

Construction of an Intragenic SSR-Based Linkage Map and QTL Mapping for Agronomic Traits in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most widely cultivated and economically important vegetables in China. Constructing an effective genetic linkage map and mapping quantitative trait loci (QTLs) related to yield and leafy head morphology is of great importance for molecular breeding of Chinese cabbage. Using two diverse Chinese cabbage inbred lines, ZHB and G291, as parents, an F2 segregating population consisting of 240 individuals was prepared for genetic map construction and phenotype investigation in this study. The two parents are significantly different in both shape and size. Sixteen important agronomic traits of F2 individuals were investigated. A genetic map of 105 intragenic simple sequence repeat (SSR) markers distributed across 10 linkage groups (LGs) was constructed, which was 2034.1 cM in length and had an average inter-locus distance of 21.75 cM. We identified 48 QTLs for the tested important agronomic traits on the studied LGs, with LOD scores of 2.51–12.49, which explained the phenotypic variance of 3.41–26.66%. The QTLs identified in this study will facilitate further genetic analysis and marker-assisted genetic improvement of Chinese cabbage.

A premature stop codon in BrFLC2 transcript results in early flowering in oilseed-type Brassica rapa plants

Nonsense-mediated mRNA decay (NMD)-mediated degradation of BrFLC2 transcripts is the main cause of rapid flowering of oilseed-type B. rapa 'LP08' plants. Many Brassica species require vernalization (long-term winter-like cooling) for transition to the reproductive stage. In the past several decades, scientific efforts have been made to discern the molecular mechanisms underlying vernalization in many species. Thus, to identify the key regulators required for vernalization in Brassica rapa L., we constructed a linkage map composed of 7833 single nucleotide polymorphism markers using the late-flowering Chinese cabbage (B. rapa L. ssp. pekinensis) inbred line 'Chiifu' and the early-flowering yellow sarson (B. rapa L. ssp. trilocularis) line 'LP08' and identified a single major QTL on the upper-arm of the chromosome A02. In addition, we compared the transcriptomes of the lines 'Chiifu' and 'LP08' at five vernalization time points, including both non-vernalized and post-vernalization conditions. We observed that BrFLC2 was significantly downregulated in the early flowering 'LP08' and had two deletion sites (one at 4th exon and the other at 3' downstream region) around the BrFLC2 genomic region compared with the BrFLC2 genomic region in 'Chiifu'. Large deletion at 3' downstream region did not significantly affect transcription of both sense BrFLC2 transcript and antisense transcript, BrFLC2as along vernalization time course. However, the other deletion at 4th exon of BrFLC2 resulted in the generation of premature stop codon in BrFLC2 transcript in LP08 line. Cycloheximide treatment of LP08 line showed the de-repressed level of BrFLC2 in LP08, suggesting that low transcript level of BrFLC2 in LP08 might be caused by nonsense-mediated mRNA decay removing the nonsense transcript of BrFLC2. Collectively, this study provides a better understanding of the molecular mechanisms underlying floral transition in B. rapa.

Histopathology of the Plasmodiophora brassicae-Chinese Cabbage Interaction in Hosts Carrying Different Sources of Resistance.

Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

ГЕНОТИПИРОВАНИЕ УСТОЙЧИВОСТИ К КИЛЕ И ОЦЕНКА КОМБИНАЦИОННОЙ СПОСОБНОСТИ КАПУСТЫ ПЕКИНСКОЙ

Chinese cabbage is a multivitamin vegetable crop and it is in constant demand due to its taste and dietary qualities, as well as the ability to harvest this crop twice a year due to the short growing season. There are less than 70 cultivars and hybrids in the State Register of Russia, many of them are susceptible to clubroot caused by soil pathogen P.brassicae Wor. In Russia, consumer demand needs to be met with new competitive hybrids that are resistant to major diseases such as clubroot. The aim of the study to evaluate the collection of inbred Chinese cabbage lines with clubroot resistance and to select promising hybrid combinations for further creation of F1 hybrids of the crop. Field trials were carried out in the summer-autumn period of the year 2021. Twenty five Chinese cabbage inbred lines (B.rapa ssp. pekinensis) and 154 hybrid combinations from crossing these lines were used as plant material. To achieve the goal, the following methods were applied: evaluation of combining ability (GCA and SCA) in the system of crossings of two groups of genotypes; clubroot disease test; molecular genotyping using molecular markers of clubroot resistance genes. As a result of a Chinese cabbage hybrids field trials, 14 were identified that exceed the standards in terms of “mass of head” by at least 20%. Three CR inbred lines K7, K19 and P1d4 with high values of GCA were identified, which are recommended for development of a new hybrid combinations. Based on the data of molecular genotyping of the clubroot resistance genes CRa, CRb, and CRA05 and the disease resistance of Chinese cabbage lines, the genetic collection of lines was differentiated by clubroot resistance genes. This will make it possible to use them as resistance donors when pyramiding resistance genes.

Identification of genes related to tipburn resistance in Chinese cabbage and preliminary exploration of its molecular mechanism

BackgroundTipburn, also known as leaf tip necrosis, is a severe issue in Chinese cabbage production. One known cause is that plants are unable to provide adequate Ca2+ to rapidly expanding leaves. Bacterial infection is also a contributing factor. Different cultivars have varying degrees of tolerance to tipburn. Two inbred lines of Chinese cabbage were employed as resources in this research.ResultsWe determined that the inbred line ‘J39290’ was the tipburn resistant material and the inbred line ‘J95822’ was the tipburn sensitive material based on the severity of tipburn, and the integrity of cell membrane structure. Ca2+ concentration measurements revealed no significant difference in Ca2+ concentration between the two materials inner leaves. Transcriptome sequencing technology was also used to find the differentially expressed genes (DEGs) of ‘J95822’ and ‘J39290’, and there was no significant difference in the previously reported Ca2+ uptake and transport related genes in the two materials. However, it is evident through DEG screening and classification that 23 genes are highly linked to plant-pathogen interactions, and they encode three different types of proteins: CaM/CML, Rboh, and CDPK. These 23 genes mainly function through Ca2+-CaM/CML-CDPK signal pathway based on KEGG pathway analysis, protein interaction prediction, and quantitative real-time PCR (qRT-PCR) of key genes.ConclusionsBy analyzing the Ca2+ concentration in the above two materials, the transcription of previously reported genes related to Ca2+ uptake and transport, the functional annotation and KEGG pathway of DEGs, it was found that Ca2+ deficiency was not the main cause of tipburn in ‘J95822’, but was probably caused by bacterial infection. This study lays a theoretical foundation for exploring the molecular mechanism of resistance to tipburn in Chinese cabbage, and has important guiding significance for genetics and breeding.

RETRACTED ARTICLE: BrRNE cleaves RNA in chloroplasts, regulating retrograde signals in Brassica rapa L. ssp. pekinensis

Brassica rapa RNE participates in the processing of polycistronic precursor transcripts into mature monocistronic mRNAs in plastids, thereby sending strong retrograde signals. Leaf color is one of the most important agronomic traits for Chinese cabbage. Not only is it closely linked to photosynthesis, thereby affecting plant growth, but it also influences consumer preference in the marketplace. A pale-green mutant rne was produced by EMS mutagenesis of Chinese cabbage inbred line A03. Chlorophyll content, photosynthetic rate, actual quantum efficiency (φPSII), and maximum quantum efficiency (Fv/Fm) of photosystem II (PSII) were all reduced in rne plants. Genetic analysis indicated that the pale-green trait was controlled by a pair of recessive alleles. Using mixed pool sequencing of F2 individuals derived from an rne × wild-type cross, we identified the essential gene Brassica rapa RNase E (BrRNE), which is responsible for chloroplast development. BrRNE cleaves polycistronic RNA in Chinese cabbage A03 plastids, but rne plants are defective in RNA processing and show reduced translation levels of the seven plastid genes, BrpsaB, BrpsaA, BrpsbA, BrpsbD, BrpsbB, BrpetA, and Brycf4. Abnormal RNA processing in the plastids sends retrograde signals that markedly regulate the expression of nuclear genes, upregulating genes that participate in ribosome and DNA replication pathways and repressing photosynthesis-associated nuclear genes (PhANGs). Our study reveals a new regulatory mechanism by which plastid RNA cleavage influences plastid development and leaf color, sending retrograde signals that affect the expression of nuclear genes in Brassica.

Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Black Rot in Chinese Cabbage.

The nucleotide-binding site–leucine-rich repeat (NBS–LRR) gene family is the largest group of plant disease resistance (R) genes widespread in response to viruses, bacteria, and fungi usually involved in effector triggered immunity (ETI). Forty members of the Chinese cabbage CC type NBS–LRR family were investigated in this study. Gene and protein characteristics, such as distributed locations on chromosomes and gene structures, were explored through comprehensive analysis. CC–NBS–LRR proteins were classified according to their conserved domains, and the phylogenetic relationships of CC–NBS–LRR proteins in Brassica rapa, Arabidopsis thaliana, and Oryza sativa were compared. Moreover, the roles of BrCC–NBS–LRR genes involved in pathogenesis-related defense were studied and analyzed. First, the expression profiles of BrCC–NBS–LRR genes were detected by inoculating with downy mildew and black rot pathogens. Second, sensitive and resistant Chinese cabbage inbred lines were screened by downy mildew and black rot. Finally, the differential expression levels of BrCC–NBS–LRR genes were monitored at 0, 1, 3, 6, 12 and 24 h for short and 0, 3, 5, 7, 10 and 14 days for long inoculation time. Our study provides information on BrCC–NBS–LRR genes for the investigation of the functions and mechanisms of CC-NBS-LRR genes in Chinese cabbage.

Heterosis of plant gross weight and heterotic group classification of inbred lines in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Heterotic groups have been well studied in hybrid breeding of grain crops, such as maize. However, little such information is available for Chinese cabbage. Plant Gross Weight (PGW) is one of the most important yield traits of Chinese cabbage. In this work, 48 F1 hybrids were obtained by incomplete diallel cross (6 × 8) of 14 Chinese cabbage inbred lines, then all parents and F1 hybrids were cultivated to evaluate heterosis performance on the PGW trait in two growing seasons (2017 and 2018) at Yangling, China. A total of five methods were used to determine the heterotic groups of tested inbred lines, which include 1) SCA method based on specific combining ability effect, 2) SCA-Y method based on SCA effect by Yang's method, 3) HSGCA method based on heterotic group specific and general combining ability, 4) HGCAMT method based on general combining ability of multiple traits. Besides, the 14 parental lines also clustered by 5) SSR method based on similarity of a core set of 28 simple sequence repeat (SSR) markers. The result showed that heterosis is obvious on the PGW trait in most F1 hybrids, with the mean value of Mid-parent heterosis (MPH) and Better-parent heterosis (BPH) is 111.17 % and 82.03 % respectively. PGW and MPH of F1 hybrids were used as final indexes to evaluate the efficiency of these five methods on heterotic group classification. The result revealed that HSGCA method and SCA method are most optimal to classify the heterotic groups in Chinese cabbage with identical division results. SSR method based on the core set of 28 SSR markers is also an effective way to predict heterosis in advance which can be used as the basis of field cross design. The 14 inbred lines of Chinese cabbage were classified into five heterotic groups based on common results of HSGCA method and SCA method. Furthermore, five inbred lines were selected as testers for further determining heterotic groups of other new inbred lines. This work is expected to raise the breeding efficiency of Chinese cabbage hybrid breeding and provide reference for classifying heterotic groups in other vegetable crops.

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.

Integrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophora brassicae infection in Pakchoi (Brassica campestris ssp. chinensis Makino).

The biotrophic protist Plasmodiophora brassicae causes serious damage to Brassicaceae crops grown worldwide. However, the molecular mechanism of the Brassica rapa response remains has not been determined. Long noncoding RNA and mRNA expression profiles in response to Plasmodiophora brassicae infection were investigated using RNA-seq on the Chinese cabbage inbred line C22 infected with P. brassicae. Approximately 5,193 mRNAs were significantly differentially expressed, among which 1,345 were upregulated and 3,848 were downregulated. The GO enrichment analysis shows that most of these mRNAs are related to the defense response. Meanwhile, 114 significantly differentially expressed lncRNAs were identified, including 31 upregulated and 83 downregulated. Furthermore, a total of 2,344 interaction relationships were detected between 1,725 mRNAs and 103 lncRNAs with a correlation coefficient greater than 0.8. We also found 15 P. brassicaerelated mRNAs and 16 lncRNA interactions within the correlation network. The functional annotation showed that 15 mRNAs belong to defense response proteins (66.67%), protein phosphorylation (13.33%), root hair cell differentiation (13.33%) and regulation of salicylic acid biosynthetic process (6.67%). KEGG annotation showed that the vast majority of these genes are involved in the biosynthesis of secondary metabolism pathways and plant-pathogen interactions. These results provide a new perspective on lncRNA-mRNA network function and help to elucidate the molecular mechanism of P. brassicae infection.

Transcriptome Arofile of Brassica rapa L. Reveals the Involvement of Jasmonic Acid, Ethylene, and Brassinosteroid Signaling Pathways in Clubroot Resistance

Plasmodiophora brassicae is a protozoan pathogen that causes clubroot disease in cruciferous plants, particularly Chinese cabbage (Brassica rapa). A previous study identified a clubroot resistance gene (CRd) conferring race-specific resistance to P. brassicae. However, the defense mechanisms of B. rapa against virulent vs. avirulent P. brassicae are poorly understood. In this study, we carried out a global transcriptional analysis in the clubroot-resistant Chinese cabbage inbred line “85–74” carrying the CRd gene and inoculated with avirulent (LAB-4) or virulent (SCCD-52) P. brassicae. RNA sequencing showed that “85–74” responded most rapidly to SCCD-52 infection, and the number of differentially expressed genes was much higher in SCCD-52-treated as compared to LAB-4-treated plants (5552 vs. 304). Transcriptome profiling revealed that plant hormone signal transduction and plant–pathogen interaction pathways played key roles in the late stages of P. brassicae infection. Genes relating to the salicyclic acid (SA), jasmonic acid (JA)/ethylene (ET), and brassinosteroid (BR) signaling pathways were up-regulated relative to untreated plants in response to LAB-4 infection at 8, 16, and 32 days post-inoculation (dpi) whereas JA, ET, and BR signaling-related genes were not activated in response to SCCD-52, and SA signaling-related genes were up-regulated in both LAB-4 and SCCD-52, suggesting that SA signaling is not the key factor in host resistance to avirulent P. brassicae. In addition, genes associated with phosphorylation and Ca2+ signaling pathways were down-regulated to a greater degree following LAB-4 as compared to SCCD-52 infection at 8 dpi. These results indicate that effector-triggered immunity in “85–74” is more potently activated in response to infection with avirulent P. brassicae and that JA, ET, and BR signaling are important for the host response at the late stage of infection. These findings provide insight into P. brassicae pathotype-specific defense mechanisms in cruciferous crops.

Construction of pseudomolecule sequences of Brassica rapa ssp. pekinensis inbred line CT001 and analysis of spontaneous mutations derived via sexual propagation.

Chinese cabbage (Brassica rapa ssp. pekinensis) is a major crop that is widely cultivated, especially in Korea, Japan, and China. With the advent of next generation sequencing technology, the cost and time required for sequencing have decreased and the development of genome research accelerated. Genome sequencing of Chinese cabbage was completed in 2011 using the variety Chiifu-401-42, and since then the genome has been continuously updated. In the present study, we conducted whole-genome sequencing of Chinese cabbage inbred line CT001, a line widely used in traditional or molecular breeding, to improve the accuracy of genetic polymorphism analysis. The constructed CT001 pseudomolecule represented 85.4% (219.8 Mb) of the Chiifu reference genome, and a total of 38,567 gene models were annotated using RNA-Seq analysis. In addition, the spontaneous mutation rate of CT001 was estimated by resequencing DNA obtained from individual plants after sexual propagation for six generations to estimate the naturally occurring variations. The CT001 pseudomolecule constructed in this study will provide valuable resources for genomic studies on Chinese cabbage.