Fine mapping and cloning of the sterility gene Bra2Ms in nonheading Chinese cabbage(Brassica rapa ssp.chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage(Brassica rapa ssp.chinensis).However,the molecular mechanisms underlying male sterility in B.rapa are still largely unclear.We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus,Bra2Ms,on the A2 chromosome.Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation.Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B.rapa.The Bra039753 gene encodes a plant homeodomain(PHD)-finger protein and is considered a potential candidate gene for Bra2Ms.Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage.Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants,which led to an amino acid insertion in the Bra039753 protein.In addition,the 369 bp fragment insertion was found to cosegregate with the male sterility trait.This study identified a novel locus related to male sterility in non-heading Chinese cabbage,and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Synergistic effects of carbon cycle metabolism and photosynthesis in Chinese cabbage under salt stress

Chinese cabbage(Brassica rapa ssp. pekinensis) has a long cultivation history and is one of the vegetable crops with the largest cultivation area in China. However, salt stress severely damages photosynthesis and hormone metabolism, nutritional balances, and results in ion toxicity in plants. To better understand the mechanisms of salt-induced growth inhibition in Chinese cabbage, RNA-seq and physiological index determination were conducted to explore the impacts of salt stress on carbon cycle metabolism and photosynthesis in Chinese cabbage. Here, we found that the number of thylakoids and grana lamellae and the content of starch granules and chlorophyll in the leaves of Chinese cabbage under salt stress showed a time-dependent response, first increasing and then decreasing. Chinese cabbage increased the transcript levels of genes related to the photosynthetic apparatus and carbon metabolism under salt stress, probably in an attempt to alleviate damage to the photosynthetic system and enhance CO_(2) fixation and energy metabolism. The transcription of genes related to starch and sucrose synthesis and degradation were also enhanced;this might have been an attempt to maintain intracellular osmotic pressure by increasing soluble sugar concentrations. Soluble sugars could also be used as potential reactive oxygen species(ROS) scavengers, in concert with peroxidase(POD)enzymes, to eliminate ROS that accumulate during metabolic processes. Our study characterizes the synergistic response network of carbon metabolism and photosynthesis under salt stress.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Blue light induces leaf color change by modulating carotenoid metabolites in orange-head Chinese cabbage(Brassica rapa L.ssp. pekinensis)

Carotenoids are involved in the formation of plant leaf color as well as photosystem photoprotection. This study showed that blue light significantly induced up-regulation of the total carotenoid content in the inner leaves of orange-head Chinese cabbage(OHCC). Furthermore, the transcriptomic analysis revealed that blue light treatment induced upregulation of genes in photosynthesis(BrHY5-2, BrCOP1 and BrDET1) and the methylerythritol 4-phosphate pathways(BrGGPS, BrDXS and BrHDR) upstream of the carotenoid metabolic pathway. Carotenoid metabolomic analysis revealed that the accumulation of several orange and red carotenoids(lycopene, zeaxanthin, β-carotene, lutein, and β-cryptoxanthin) after blue light treatment contributed to the deepening of the leaf coloration, suggesting that short-term blue light treatment could be used to boost nutritional quality. The light signal gene BrHY5-2 participated in the blue light-induced transcriptional regulation of carotenoid biosynthesis in OHCC. Overexpression of BrHY5-2 in Arabidopsis significantly increased the total carotenoid content and the sensitivity to blue light. The above findings revealed new insights about blue-light-induced carotenoid synthesis and accumulation in OHCC lines. They suggested a new engineering approach to increase the nutritional value of vegetables.

Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

Fast tracking alien gene discovery by molecular markers in a late flowering Chinese cabbage-cabbage translocation line‘AT7–4'

Flowering time is an important agronomic trait of Chinese cabbage with late flowering being a primary breeding objective.In our previous work,we obtained Chinese cabbage-cabbage translocation lines that contained several beneficial cabbage genes.Cabbage-specific molecular markers show that these genes were coming from chromosome C01 of cabbage.In this study,we investigated the inheritance of flowering time in a couple of translocation lines and analyzed the transmission rate of molecular markers in the offspring.Consequently,we obtained the late flowering Chinese cabbage-cabbage translocation line‘AT7–4’in which the flowering time was later than that of‘85–1’by about 7 days under 4-week vernalization.Based on previous studies of the genomes of Chinese cabbage and cabbage,we located the cabbage-specific molecular markers that were closely linked at the top of the chromosome A01 in the F2mapping population generated by self-crossing F1s derived from a cross between the translocation line‘AT7–4’and Chinese cabbage‘14–36’.Five flowering-related genes in the alien fragment were found by functional annotation and their molecular markers were developed.This study lays the foundation for the future improvement of Chinese cabbage varieties using A-C translocation lines.

Determination and dynamics of difenoconazole residues in Chinese cabbage and soil

An analytical method was developed using a solid phase extraction （SPE） cleanup and gas chromatography for detecting the residues of difenoconazole in Chinese cabbage and soil. The recovery and the relative standard deviation of this method in Chinese cabbage was 87.6-99.0%, 1.71-10.50%, respectively; in soil was 92.4-95.5%, 4.93-10.70%, respectively. Further degradation of difenoconazole residue in Chinese cabbage and soil was studied to evaluate residue behavior and environmental safety of difenoconazole. Degradation rate of difenoconazole in both Chinese cabbage and soil followed the first order kinetics with the half-lives of 6.6-7.8 and 54.2-55.0 days, respectively.

Joint effects of cadmium and lead on seedlings of four Chinese cabbage cultivars in northeastern China

In northeastern China, large area of vegetable land has been simultaneously polluted by cadmium （Cd） and lead （Pb）. Joint effects of Cd and Pb on Chinese cabbage （Brassica pekinensis L.） were investigated using the seed germination and sand culture method. Four Chinese cabbage cultivars including Kangbingjinchun （KB）, Dongyangchunxia （DY）, Qinglvwang （QL） and Qiangshi （QS） from Shenyang in northeastern China were adopted in this study. The results showed that there were positive linear relationships between the inhibitory rate of biomass, root and shoot elongation and the concentrations of Cd and Pb. In particular, root elongation was more sensitive to joint stress of Cd and Pb. The activity of superoxide dismutase and the content of malondialdehyde （MDA）, soluble protein （SP） and proline （PRO） changed significantly with increasing exposure concentration of Cd and Pb. The decrement in the activity of antioxidative enzymes, the content of SP and accumulation of MDA were relatively low in KB and QS. PRO played an important role in resisting Cd and Pb stress.

Effect of Partial Replacement of Nitrate by Amino Acid and Urea on Nitrate Content of Non-heading Chinese Cabbage and Lettuce in Hydroponic Condition

In this paper, the authors studied the effect of different mixtures of glycine (Gly), isoleucine (Iso), proline (Pro), and urea solutions used as a partial (20%) replacment of nitrate in the nitrate content and quality of non-heading Chinese cabbage and lettuce in hydroponice. Five treatments were done 12 d before harvest. Compared to the control group, Gly had the best effect in reducing the nitrate content of both vegetable leaves and petioles; the mixture of Gly, Iso and Pro ranked second and urea the least. Treatments with amino acid could also increase soluble sugar and protein contents and enhance total-N in leaves significantly. In contrast, amino acid enhanced NRA in non-heading Chinese cabbage, while they decreased it slightly in lettuce. The results showed that amino acids and urea could reduce the nitrate content of both vegetables, but they had almost the same effect on non-heading Chinese cabbage. Moreover, amino acids were more effective than urea in lettuce. As a result, it was concluded that partial replacement of nitrate with amino acids not only reduced the nitrate content but also improved the quality of vegetables.

Monosomic Addition Lines of Flowering Chinese Cabbage(B.campestris L.ssp.chinensis var.parachinensis L.H.Bailey)-Chinese Kale(B.oleracea L.var.alboglabra L.H.Bailey)

Interspecific alien addition lines have played significant roles in gene mapping, intergenomic gene transfer and chromosomal homoeological identification between closely related species. Selection of alien addition lines was conducted by karyotype analysis and morphological observation with the reference of parents. Triploid interspecies hybrid （AAC, 2n = 3x = 29） was obtained from Brassica campestris ssp. chinensis var. parachinensis Qinglu 9601 （tetraploid, AAAA, 2n = 4x = 40） x B. oleracea var. alboglabra Baihua 9705 （diploid, CC, 2n = 2x = 18） by immature hybrid embryo culture in vitro. Five different alien monosomic addition lines （AA ＋ C2, AA ＋ C3, AA ＋ C4, AA ＋ C6, AA ＋ C7） were obtained from the backcross progenies of AAC x AA. Each alien monosomic addition line has some specific morphological characters. It is feasible to obtain alien addition lines from the progenies of AAC × AA by karyotype analysis and morphological observation based on the reference of parents

Exogenous Nitric Oxide Alleviated the Inhibition of Photosynthesis and Antioxidant Enzyme Activities in Iron-Deficient Chinese Cabbage (Brassica chinensis L.)

The effects of exogenous nitric oxide （NO） on plant growth, chlorophyll contents, photosynthetic and chlorophyll fluorescence parameters as well as lipid peroxidation and activities of antioxidant enzymes were investigated in Chinese cabbage plants exposed to iron （Fe） deficiency. Iron deficiency led to serious chlorosis in Chinese cabbage leaves, and resulted in significant decrease in plant growth, photosynthetic pigments, net photosynthetic rate, Fv/Fm, Ф ps Ⅱ and activities of antioxidant enzymes, and increase in lipid peroxidation. While treatment with SNP, a NO donor, it could revert the iron deficiency symptoms, increased photosynthetic rate as well as activities of antioxidant enzymes, and protected membrane from lipid peroxidation, as a result, the growth inhibition of Chinese cabbage by Fe deficiency was alleviated.

Differential Expression Analysis of Genic Male Sterility A／B Lines in Chinese Cabbage—Pak—Choi（Brassica campestris ssp.chinensis Makino）

To determine differential expression of genie male sterility A/B lines in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis Makino var. communis Tsen et Lee), we used the RNA fingerprinting technique, cDNA-AFLP analysis, in different developmental stages and different tissues. While no obvious differential expressions were observed in rosette leaves, florescence leaves, and scapes, some differential expressions were found in alabstrums of A/B lines and among leaves, scapes and alabstrums. We analyzed the al-abstrums collected in different developmental stages with 10 primer combinations. We got a unique band between middle size alabstrums and large alabstrums in B line in one of the ten pair primers, and in another one pair, one band reflecting a higher gene-expression level in A line than that in B line was obtained. No unique bands were found with the other primer combinations. The bands reflecting different gene-expression level were confirmed by Northern hybridization. The results indicated that cDNA-AFLP was a suitable tool for studying differential expression of genie male sterility in plants. SDS-polyacrylamide gel electrophoresis patterns of soluble proteins further verified the difference in A/B lines.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Identification of Heat Tolerance Linked Molecular Markers of Chinese Cabbage(Brassica campestris L.ssp.pekinensis)

Genetically stable population of recombination inbred line (RIL) was derived from a cross between a heat tolerant line 177 and a heat sensitive line 276 of Chinese cabbage (Brassica campestris L. ssp. pekinensis) by single seed descent. The RILs were analyzed using isozyme, RAPD and AFLP techniques in order to find molecular markers that are linked to heat tolerance quantitative trait loci (QTL). The results of variance analysis of single factor indicated that there were 9 molecular markers closely linked with heat tolerance QTL, including 5 AFLP markers, 3 RAPD markers and 1 PGM isozyme marker. Total genetic contribution of these makers to heat tolerance was 46.7%. Five of the nine markers distributed in one linkage group, the remaining 4 markers were located in separate groups. Thus the 9 heat tolerance linked markers distributed in 5 independent locations in the genome of Chinese cabbage.

Control Efficiency of Four Biological Pesticides against Pieris rapae Linne and Their Impacts on Yield and Benefit of Chinese Cabbage

[ Objective] The paper was to screen out ideal biological pesticides, in order to provide guidance for pollution-free control against Pieris rapae Linne. [Method] Taking Chinese cabbage variety Taiyuan Erqing as the test material, the field control efficiencies of four pesticides including PrGV · Bt WP 1 000 times dilution, NPV · Bt SC 750 times dilution, 0.5% azadirachtin EC 750 times dilution, and 0.3% matrine AS 500 times dilution against P. rapae were studied, and their impacts on yield and planting benefits of Chinese cabbage were also determined. Using foliar spray method, the pesticides were sprayed for the first time when the second or third instar larvae of P. rapae first occurred in fields, and sprayed for the second time with the interval of 15 d. The fields sprayed with beta-cypermethrin EC （organic chemical pesticide） and water were set as control. [ Result] NPV · Bt SC 750 times dilution had the best effect after spraying for two times： the control efficiency against P. rapae at 15 d after spraying was 90.11% ; the damage rate of Chinese cabbage was only 0.21%, while the commodity rate reached 100% ; compared with chemical pesticide spraying, the commodity yield （177 262.5 kg/hm2 ） and the income after deducting spraying cost （48 858.5 yuan/hm2 ） were increased by 14.7% and 13.75%, respectively. [ Conclusion] Although biological pesticides are more expensive, they have long persistence and good control effect, resulting in green and safe Chinese cabbage with high commodity rate and yield, and higher eventual economic benefit after deducting spraying cost.

Genetic Transformation of Chinese Cabbage (Brassica rapa pekinensis) with Floral-dip Method

[ Objective] This study is aimed to transform gus gene into Chinese cabbage （Brassica rapa pekinensis） by floral-dip method. [ Method] The Chinese cabbage was transformed by floral-dip method for the first time. Then the cabbage seeds were harvested and screened by hygromycin, and the plants with resistance were confirmed by histochemical GUS assay and PCR detection. [ Result] The target gene was successfully integrated into the Chinese cabbage genome, and the transformation rate was 0.1%. [ Conclusion ] This study optimized the genetic transformation system for Chinese cabbage, and laid the foundation for improving the means of genetic transformation of Chinese cabbage.

AFLP Marker Linked to Turnip Mosaic Virus Susceptible Gene in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Turnip mosaic virus (TuMV) which has several strains causes the most important virusdisease in Chinese cabbage in terms of crop damage. In China, Chinese cabbage is infectedby a mixture of strains, breeding of cultivar for the TuMV resistance has become themajor aim. Screening the molecular marker linked to the TuMV-resistance gene formolecular assisted selection is the major method to improve the breeding efficiency. Inthis study, we used AFLP technique and the method of bulked segregant analysis(BSA) tostudy the progeny of Brp0058Brp0108, and identified two DNA molecular marker linked toTurnip mosaic virus-resistance gene with a recombination frequency 7.5 cM and 8.4 cM.

Breeding of a New Clubroot-resistant Chinese Cabbage Variety, CCR11242

In this study, using Japanese radish cytoplasmic sterile line ＂Half Green Cabbage＂ （HGC） and clubfoot-resistant South Korean cabbage variety ＂ CR Cabbage King＂ （CCK） as experimental materials, serf-incompatible DH lines were obtained by mierosporo culture. Through five gnerations of backeross, CCR11239, a clubroot-resistant radish cytoplasmic sterile line, was obtained. Through six generations of serf-crossing, CCR11240, the maintainer line of CCR11239, was bred. After the cross of CCK and Luchunbai 1 （83 - 1 ） and five generations of self-crossing, serf-compatible line CCRl1241 was obtained. Through crossing CCR11240 with CCRl1241, the new cabbage variety CCRl1242 was obtained. After variety comparison test, provincial regional test and production demonstration test, the new variety was registered in 2012. The average yield of CCR11242 reached 60 975 kg/hm2, which was improved by 142% compared with Luchunbai 1 （83 - 1 ）. The disease index of CCR11242 was 5.63, which was 88.13 lower than the control, indicating high resistance （HR） to clubfoot.

Molecular Cloning,Expression Analysis and Localization of Exo70A1 Related to Self Incompatibility in Non-Heading Chinese Cabbage(Brassica campestris ssp.chinensis)

The exocyst is a conserved protein complex,and required for vesicles tethering,fusion and polarized exocytosis.Exo70A1,the exocyst subunit,is essential for assembly of the exocyst complex.To better understand potential roles of Exo70A1 in non-heading Chinese cabbage（Brassica campestris ssp.chinensis）,we obtained the full-length cDNA of Exo70A1 gene,which consisted of 1 917 bp and encoded a protein of 638 amino acids.BlastX showed BcExo70A1 shared 94.9% identity with Brassica oleracea var.acephala（AEI26267.1）,and clustered into a same group with other homologues in B.oleracea var.acephala and Brassica napus.Subcellular localization analysis showed BcExo70A1 was localized to punctate structures in cytosol of onion epithelial cells.Results showed that BcExo70A1 was widely presented in stamens,young stems,petals,unpollinated pistils,roots and leaves of self compatible and incompatible plants.The transcripts of BcExo70A1 in non- heading Chinese cabbage declined during initial 1.5 h after incompatible pollination,while an opposite trend was presented after compatible pollination.Our study reveals that BcExo70A1 could play essential roles in plant growth and development,and is related to the rejection of self pollen in non-heading Chinese cabbage.

Using a Suppression Subtractive Library-Based Approach to Identify Non-Heading Chinese Cabbage Genes Up-Regulated in Early Response to Elicitor PB90

Monitoring expression at the transcriptional level is the first essential step for the functional analysis of plant genes. Genes-encoding proteins directly involved in early response to elicitor constitute only a small fraction of all the genes affected by elicitor. Transcriptional responses to various elicitors have been extensively studied in different plants including Nicotiana and Arabidopsis thaliana; however, corresponding data aren＇t available for non-heading Chinese cabbage. To address this problem, we describe a suppression subtractive library-based approach to isolate the plant＇s ESTs up-regulated in the early induction/execution of the HR induced by elicitor PB90 from Phytophthora boehmeriae. According to their putative identification in BLAST searches against the three genome databases, 70 up-regulated genes were classified into 9 parts： some aspect of primary ＇metabolism＇ or ＇energy＇ production; ＇protein synthesis＇ or ＇protein fate＇; cellular communication/signal transduction mechanism; cell fates including Beclin, SPT1, and SPT2; HLA-B and AGO1 which participate in transcription; cellular transport and hypothetical proteins or proteins for which a function has yet to be determined. Seven selected genes such as Beclin, thioredoxin, HLA-B, MAP3K, SPT1, SPT2, and AGO1 were up-regulated induced by PB90, suggesting that the genes may play an important role in PB90-triggered HR.