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.

Physiological and transcriptome analyses of Chinese cabbage in response to drought stress

Chinese cabbage is an important leafy vegetable crop with high water demand and susceptibility to drought stress.To explore the molecular mechanisms underlying the response to drought,we performed a transcriptome analysis of drought-tolerant and-sensitive Chinese cabbage genotypes under drought stress,and uncovered core drought-responsive genes and key signaling pathways.A co-expression network was constructed by a weighted gene coexpression network analysis(WGCNA)and candidate hub genes involved in drought tolerance were identified.Furthermore,abscisic acid(ABA)biosynthesis and signaling pathways and their drought responses in Chinese cabbage leaves were systemically explored.We also found that drought treatment increased the antioxidant enzyme activities and glucosinolate contents significantly.These results substantially enhance our understanding of the molecular mechanisms underlying drought responses in Chinese cabbage.

Expression changes of mi RNA-regulated genes associated with the formation of the leafy head in cabbage

The vegetative development of cabbage(Brassica oleracea var.capitata)passes through seedling,rosette,folding and heading stages.Leaves that form the rosette are large and mostly flat.In the following developmental stages,the plants produce leaves that curve inward to produce the leafy head.Many microRNAs and their target genes have been described participating in leaf development and leaf curvature.The aim of this study is to investigate the role of miRNA-regulated genes in the transition from the rosette to the heading stage.We compared the mi RNA and gene abundances between emerging rosette and heading leaves.To remove transcripts(miRNAs and genes)whose regulation was most likely associated with plant age rather than the change from rosette to heading stage,we utilized a non-heading collard green(B.oleracea var.acephala)morphotype as control.This resulted in 33 DEMs and 1998 DEGs with likely roles in the transition from rosette to heading stage in cabbage.Among these 1998 DEGs,we found enriched GO terms related to DNA-binding transcription factor activity,transcription regulator activity,iron ion binding,and photosynthesis.We predicted the target genes of these 33 DEMs and focused on the subset that was differentially expressed(1998DEGs)between rosette and heading stage leaves to construct mi RNA-target gene interaction networks.Our main finding is a role for miR396b-5p targeting two Arabidopsis thaliana orthologues of GROWTH REGULATING FACTORs 3(GRF3)and 4(GRF4)in pointed cabbage head formation.

DIA-based proteome profiling with PRM verification reveals the involvement of ER-associated protein processing in pollen abortion in Ogura CMS cabbage

Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

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.

Effect of Foliar Application of Amino Acid on the Quality and Enzyme Activity of Flowering Chinese Cabbage(Brassica parachinensis Bailey)

A pot experiment was conducted to study the influences of foliar application of glycine,alanine,lysine,and glutamic acid in 200 mg/kg or 500 mg/kg upon the quality and enzyme activity of flowering Chinese cabbage（Brassica parachinensis Bailey）.The results showed that all the application of these four amino acids could increase the yield of flowering Chinese cabbage,significantly raise the content of soluble sugar,and reduce the accumulation of nitrate.The applications of three other amino acids except alanine can increase the content of soluble proteins and decrease the accumulation of oxalic acid.However,the application of amino acid has insignificant influences on the SPAD number of chlorophyll,and causes the decrease of Vitamin C content.Meanwhile,the application of amino acid can improve the activity of nitrate reductase（NR） and glutamate dehydrogenase（GDH） as well.It shows that the application of amino acid is beneficial to improve ammonia metabolism,reduce the accumulation of nitrate and oxalic acid,increase the content of soluble sugar and soluble proteins,and improve the quality of flowering Chinese cabbage.

Effects of Combined Application of Biochar-based Organic Fertilizer and Reduced Nitrogen Fertilizer on Soil Enzyme Activity and Yield of Purple Cabbage(Brassica oleracea var.capita rubra)in Yuanmou County

[Objectives]In response to the issue of soil improvement in Yuanmou County,the effects of combined application of biochar-based organic fertilizer and reduced nitrogen fertilizer on soil nutrients,soil enzyme activity,and yield of purple cabbage(Brassica oleracea var.capita rubra)were investigated in the field base of Institute of Thermal Zone Ecological Agriculture,Yunnan Academy of Agricultural Sciences in Yuanmou County.[Methods]A total of 13 treatments were set up by applying biochar-based organic fertilizer at three levels of 15,30 and 45 t/hm^(2)(T_(1),T_(2),T_(3)),combined with top application of nitrogen fertilizer(urea)at four levels:375(N_1),300(N_(2)),225(N_(3))and 0 kg/hm^(2),with non-fertilizing treatment as control check(CK),in order to explore the optimal ratio for the combined application of biochar-based organic fertilizer with nitrogen fertilizer.[Results]The application of biochar-based organic fertilizer could significantly improve soil nutrients,enzyme activity,and purple cabbage yield.The improvement effect of combined application with nitrogen fertilizer was higher than that of single application of biochar-based organic fertilizer,and the improvement effect was enhanced with the application amount of biochar-based organic fertilizer increasing.The contents of organic matter and total nitrogen were the highest in treatment T_(3)N_(3),of which the values increased by 81.39%and 56.09%compared with the CK,respectively.The contents of soil hydrolyzable nitrogen,available phosphorus,and available potassium were all the highest under treatment T_(3)N_(2),with increases of 92.76%,171.01%and 235.50%,respectively.There was a significant positive correlation between the activity of soil catalase,urease,and sucrase and organic matter,total nitrogen,and available nutrients.The overall soil enzyme activity was relatively higher in treatment T_(3)N_(2).The yield of purple cabbage treated with biochar-based organic fertilizer combined with nitrogen fertilizer could reach 85750 kg/hm^(2),which was 94.78%higher than that treated with biochar-based organic fertilizer alone.Based on comprehensive analysis,the optimal combination ratio was 45 t/hm^(2)of biochar-based organic fertilizer and 300 kg/hm^(2)of urea(T_(3)N_(2)).[Conclusions]This study provides data support for the promotion of biochar-based organic fertilizers and reduced fertilizer in agricultural soil in the Dam area of Yuanmou County.

Directional Breeding of An Oval-ecotype Male Sterile Line of Chinese Cabbage(Brassica campestris L. ssp. pekinensis)

[Objective] The study aimed to explore the method for directional breeding of a male-sterile line in oval-ecotype Chinese cabbage. [Method] Based on ＂Multiple Allele Hypothesis of Genic Male Sterile Chinese Cabbage＂, an inbred line ＇06048＇ of oval ecotype was used as the receptor, and male fertile plant of ＇AB12＇ was used as the donor line. Crossing, backcross, selfing, testcross and sibling were ap- plied to transfer the multiple alleles under the directional genetic model. [Result] Segregation ratio of every generation was consistent with theoretical value. A new male sterile line with 100% male sterility and ＇06048＇ horticultural traits was ob- tained successfully, which accomplished the transfer of male sterile multiple allele and horticultural characters of receptor line at the same time. [Conclusion] The re- search verifies that the model of directional transfer is feasible, provides a theoreti- cal basis for the directional transfer of Chinese cabbage with other horticultural traits whose genotype is msms. The model can also be applied to other Brassica crops, to generate genetic male sterile lines with specific botanical traits and high-quality economic traits.

Disease Analysis and Pathogen Identification of Black Spot Disease of Chinese Cabbage [Alternaria brassicae (Berk.) Sace.] in Bijie City

[Objective] Disease analysis and pathogen identification of black spot disease of Chinese cabbage [Alternaria brassicae（Berk.） Sace.] in Bijie City of Guizhou Province was carried out,so as to raise the corresponding prevention measures.[Method] Pathogen was isolated from diseased leaves using tissue surface separation method for cultivation,microscopic examination,spore size determination and pathogen identification.[Result] Microscopic examination showed that the disease was caused by Alternaria sp.,the size of its spores was（15.0-38.5） μm×（7.5-13.5） μm.Combining with the characteristics of host and pathogen,the pathogen caused black spot disease of Chinese cabbage was identified to be Alternaria brassicicola.[Conclusion] A.brassicicola was the pathogenic fungi of black spot disease of Chinese cabbage in Bijie City of Guizhou Province,and damage caused by the disease could be reduced through agricultural measures and chemical control.

Dissection of two QTL clusters underlying yield-related heterosis in the cabbage founder parent 01–20

Cabbage has significant heterosis and most commercial cultivars are hybrids.To explore genetic basis of cabbage heterosis and promote cabbage heterosis utilization,we constructed two populations by crossing 100 DH lines derived from a cabbage hybrid 01–20×96–100 with two female parents.Hybrids exhibited different extents of heterosis,the mean value of economic yield was 2.6 times bigger than parents.We identified 66 and 73 QTLs associated with mid-parent heterosis and transgressive heterosis of twelve yield-related traits,respectively.Some QTLs could be detected under the two-year experiment existed in two populations with different testers,showing relatively high phenotypic contribution rate(15.8%–20.0%).Heterosis QTLs exhibited clustered distribution in several cabbage chromosome regions.Two dominant genetic regions,mk300–316 and mk258–268,originated from the elite parent 01–20,exhibited significant genetic effects for yield-related heterosis,which were first identified.Three elite DH lines(D22,D46,D83)harboring these two dominant regions were selected as having strong heterosis in cabbage production.Candidate gene analysis revealed that some genes participating in biosynthetic processes of carbohydrates and some responses to auxin might affect cabbage yield heterosis.QTL identification and genetic dissection of yield-related traits provide new insights into the genetic effects of cabbage heterosis.

Effects of field application of phosphate fertilizers on the availability and uptake of lead, zinc and cadmium by cabbage (Brassica chinensis L.) in a mining tailing contaminated soil

A field trial was conducted to evaluate the reduction of bioavailability of heavy metals including lead (Pb), zinc (Zn) and cadmium (Cd) in a soil contaminated by mining tailings in Shaoxing, Zhejiang, China. Three commercial phosphate (P) fertilizers including phosphate rock (PR), calcium magnesium phosphate (CMP), and single superphosphate (SSP) were applied to the plot at three P application rates, 50, 300, and 500 g/m2 with 9 treatments and control (CK). Plants, water soluble and exchangeable (WE) extra...

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.

Field demonstration of reduction of lead availability in soil and cabbage (Brassica Chinensis L.) contaminated by mining tailings using phosphorus fertilizers

A field demonstration of reduction of lead availability in a soil and cabbage (Brassica Chinensis L.) contaminated by mining tailings, located in Shaoxing, China was carried out to evaluate the effects of applications of phosphorus fertilizers on Pb fractionation and Pb phytoavailability in the soil. It was found that the addition of all three P fertilizers including single super phosphate (SSP), phosphate rock (PR), and calcium magnesium phosphate (CMP) significantly decreased the percentage of water-soluble and exchangeable (WE) soil Pb and then reduced the uptake of Pb, Cd, and Zn by the cabbage compared to the control (CK). The results showed that the level of 300 g P/m2 soil was the most cost-effective application rate of P fertilizers for reducing Pb availability at the first stage of remediation, and that at this P level, the effect of WE fraction of Pb in the soil de- creased by three phosphorus fertilizers followed the order: CMP (79%)>SSP (41%)>PR (23%); Effectiveness on the reduction of Pb uptake by cabbage was in the order: CMP (53%)>SSP (41%)>PR (30%). Therefore our field trial demonstrated that it was effective and feasible to reduce Pb availability in soil and cabbage contaminated by mining tailings using P fertilizers in China and PR would be a most cost-effective amendment.

Transformation of Cabbage(Brassica oleracea L.var.capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

To obtain transgenic cabbage line with broad insect resistance, a new synthetic Bacillus thuringiensis crylBa3 gene was introduced into white cabbage via Agrobacterium tumefaciens-mediated transformation and 37 transformants were obtained. Polymerase chain reaction （PCR） and Southern blot analyses confirmed that crylBa3 was successfully inserted into the genome of cabbage. Reverse transcription-polymerase chain reaction （RT-PCR） demonstrated that crylBa3 was expressed. Western blot results confirmed the production of insecticidal protein encoded by crylBa3. Insect bioassays showed that transgenic cabbages effectively controlled both susceptible and CrylAc-resistant diamondback moth （DBM） larvae.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Influence of 6-BA on Incidence Rate of Chinese Cabbage Clubroot (Plasmodiophora brassicae Woron)

[Objective] The paper was to study the relationship between cytokinin and Chinese cabbage clubroot, and to explore the incidence mechanism of clubroot. [Method]The spores were extracted from the tumors of diseased plant, and their DNA was extracted for PCR test. The diagnosed spores were inoculated to the culture soils in erlenmeyer flask at seeding stage, at seed germination and 21 d after sowing, and 0.08 μmol/L 6-BA was added to the soil at germination. Spores were extracted from the root tumors developed in inoculated plants and examined by scanning electronic microscope （ SEM）, and the incidence rate of clubroot in 6-BA treatment and control was recorded. [ Result] Plasmodiophora brassicae Woron could cause the clubroot incidence of Chinese cabbage planted in erlenmeyer flasks, the incidence rate of chibroot in the treatment adding with 0.08 μmol/L 6-BA was 100%, while that in the treatment without 6-BA was 57%, and the volume of former tumor was much larger than the latter. SEM showed that the size of resting spore of P. brassicae was 1.5 -4.3μm. [ Conduslon] 6-BA applied at germination could significantly promote the formation of tumor of Chinese cabbage clubroot.

Differential Expression Analysis of Genie 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.

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.