The folding mode of Chinese cabbage leaf ball is the main character that determines the appearance shape,taste and stress resistance of commercial organs.In order to explore the internal molecular mechanism of the formation of the folding mode of Chinese cabbage,we cloned the full length sequence of the transcription factor BrPIF5 gene from overlaping and outward-curling Chinese cabbage as experimental materials,and conducted bioinformatics analysis,constructed the plant overexpression vector,and used Agrobacterium to mediate the transformation into tobacco to obtain positive transformation plants.The expression level of BrPIF5 gene in tobacco was detected by qRT-PCR.The results showed that the protein encoded by BrPIF5 gene was a hydrophilic protein with a continuous and complete open reading frame of 634 bp,containing 210 amino acids.The protein was composed of more α-helical structure and random curl,including an AP2/ERF domain.BrPIF5 protein and the other 9 gene family members contained a conserved motif 1,and the position was different from that of other gene family members,which was located in the front of the protein sequence.Phylogenetic tree showed that BrPIF5 gene had close evolutionary relationship with SoPIF15,BhPIF1,BoPIF4,AtPIF4 and BrPIF4 family members.The tobacco strain with overexpression of BrPIF5 was obtained by Agrobacterum-mediated genetic transformation,and the leaves of the tobacco positive transformation strain showed inward curling.qRT-PCR showed that the expression level of BrPIF5 gene in the overlaping Chinese cabbage was higher than that in the outward-curling Chinese cabbage,and the gene expression level in the positive tobacco plants was higher than that in the control.It was further proved that BrPIF5 gene controlled the inward curling of Chinese cabbage leaves,thus promoting the formation of leaf ball folding type.

In order to quickly obtain homozygous and stable DH lines of Chinese cabbage resistant to clubroot disease,and to provide basic materials for breeding Chinese cabbage resistant varieties to clubroot disease,5 resistant genotypes of Chinese cabbage were used as materials for isolated microspore culture in this study,and phenotype and resistance of the obtained microspore regenerated plants were identified by combining molecular markers,morphological observation,and artificial inoculation of P.brassicae.The results showed that all 5 genotypes were induced to produce embryos,with a variation of 0.02 to 1.72 embryos per bud.Three genotypes,20aCR12,21aCR6,and 21aCR12,obtained regenerated plants,with regeneration rates of 27.09%,1.45%,and 26.07%,respectively.Resistance marker identification showed that molecular markers linked to CRa and CRb^kato all amplified the resistant bands in 50 obtained microspore plants.Phenotypic investigation showed that there were significant differences in the shape and color of basal leaves,bolting timing,and fertility among 50 microspore plants at reproductive growth stage,and 29 DH lines obtained through self-pollination also showed exhibited diversity in plant type,leaf traits,and heading traits at nutritional growth stage.Resistance inoculation showed that the disease index of 11 DH lines to races 4 and 1 of P.brassicae was less than 33.33,indicating resistance or tolerance to clubroot disease.Among them,there were 3 highly resistant DH lines with a disease index less than 5.0 for two races of P.brassicae,namely 20aCR12-23,20aCR12-29,and 21aCR12-39.The research results have shown that the homozygous DH lines resistant to clubroot can be obtained quickly by microspore culture combined with molecular marker assisted identification.

Photoperiod is one of important environmental factors affecting plant bolting and flowering which are regulated by plant phytochrome proteins.The signal network mediated by PHYB has an important inhibitory effect on plant bolting and flowering.Previous studies revealed that there were large segment insertion/deletion differences between the PHYB gene promoter of the Chinese cabbage late-bolting line 06-247 and the easy-bolting line He102.In order to further investigate the impact of promoter mutation on PHYB and the key genes of its downstream pathways,this study was conducted.Based on bioinformatics methods,the redundancy characteristics of phytochrome genes in genome Chinese cabbage were firstly analyzed.It was found that the Chinese cabbage genome contained six phytochrome genes,of which PHYA had two copies,and PHYB,PHYC,PHYD,and PHYE all had only one copy.Then amino acid sequence alignment was used for screening of the specific sequence of PHYB.Antigenic determination clusters were designed based on the specific sequence and the antibodies against PHYB was prepared.The fluorescence quantitative RT-PCR technology and Western Blot technology were used to study the relative content of PHYB in 06-247 and He102.At the same time,the dynamic changes of key regulatory genes such as CCA1,FLC,CO and FT which in the downstream of PHYB pathway were also compared.The results showed that the promoter mutation caused significant differences both in level of mRNA in 06-247 and then significantly increased the protein level of PHYB.At the same time,the downstream regulatory genes such as CCA1,FLC,CO and FT were highly expressed in 06-247,which had an important impact on bolting resistance of 06-247.

CYP79B2 is a related gene that regulates auxin synthesis in Arabidopsis.By cloning the homologous gene BrcCYP79B2-1 of CYP79B2 in B.rapa ssp. cninensis,analyzing its expression in different tissues and periods,and studying its regulatory mechanism on vernal flowering in B.rapa ssp. cninensis,to lay a theoretical foundation for the subsequent functional verification of the auxin-encoding gene in B.rapa ssp. cninensis.The homologous gene of CYP79B2 was cloned from B.rapa ssp. cninensis by qRT-PCR and named as BrcCYP79B2-1.The structure,physicochemical properties and relationship of its protein were analyzed by bioinformatics method,and its protein was analyzed by qRT-PCR method.Expression levels in different tissues and growth stages in B.rapa ssp. cninensis.And its expression in different tissues and growth stages of B.rapa ssp. cninensis was analyzed by qRT-PCR method.The results showed that the full-length coding sequence of BrcCYP79B2-1 gene was 1 623 bp,encoding 540 amino acids.The physicochemical analysis of the protein showed that the molecular mass of the protein was 60.849 73 ku,and the theoretical isoelectric point was 8.71.Compared with the amino acid sequences of other species,it was found that BrcCYP79B2-1 was highly conserved in cruciferous plants,and had the highest homology with turnip,up to 99.52%;the expression levels of different organs of B.rapa ssp. cninensis were analyzed by qRT-PCR, and it was found that the expression level of BrcCYP79B2-1 was the highest in roots, followed by leaves, and the lowest in flower buds.The expression of BrcCYP79B2-1 in seedlings after 0,10,15 and 16 days of low temperature treatment showed that the expresion of BrcCYP79B2-1 reached its peak on the 10th day of low temperature treatment, that is, the vegetative growth period, while the expression of BrcCYP79B2-1 decreased during flower bud differentiation, which indicated that the expression of BrcCYP79B2-1 was related to low temperature vernalization and could affect flower bud differentiation.

To investigate the effects of zeolite and Ca-Mg-based bentonite on the passivation of cadmium in calcareous soil in Northern China and the growth of pakchoi,a pot experiment was conducted to study the effects of two modifiers(mass fraction of zeolite was 0.5%,1.0%,2.0% and mass fraction of calcium-magnesium bentonite was 0.2%,0.3%,0.4%,0.5%,0.6%,0.8%)on pH value,available cadmium content of calcareous cadmium-contaminated soil in Northern China and cadmium content,dry matter accumulation and chlorophyll content of aboveground part of pakchoi.The results showed that compared with the control,adding different doses of zeolite could increase the soil pH value.With the increasing of zeolite application dose,the available cadmium content of soil,the aboveground cadmium content,the dry matter accumulation and chlorophyll content of pakchoi decreased gradually.The germination rate of pakchoi increased gradually,but the effects of zeolite treatments on the indexes determined in the experiment were not significant.The application of Ca-Mg-based bentonite significantly increased the soil pH value(0.70—1.07)and decreased the Cd content in the aboveground part of pakchoi(63.83%—93.62%).The aboveground dry matter accumulation(5.56%—29.22%)and chlorophyll content(5.42%—11.72%)of pakchoi increased,and the soil available cadmium content significantly decreased by a higher dose(≥0.4%).However,the germination rate of pakchoi decreased significantly and inhibited germination.The research showed that Ca-Mg-based bentonite was more suitable for the passivation of cadmium in calcareous soil in Northern China than zeolites.The content of available cadmium in calcareous soil and the content of cadmium in the aboveground part of pakchoi could reduce by applying Ca-Mg-based bentonite.However,the amount of application should be strictly controlled to avoid affecting the germination of pakchoi and reducing yield.The comprehensive analysis of quality and yield factors showed that the addition of 0.3% Ca-Mg-based bentonite could effectively reduce the aboveground cadmium content of pakchoi and increase the aboveground dry matter accumulation and chlorophyll content of pakchoi.

It was conducted to research the effect of Phyto-Cat^TM soil conditioner on the physiological and chemical properties of the soil and the physiological characteristics of non-bearing cabbage, and to explore its effectiveness as an alternative to traditional chemical fertilizers.We take non-heading Chinese cabbage plants as the test object, and adopt a multi-factor single-level method to study the effects of four treatments:study basal fertilizer(CK), basal fertilizer+Phyto-Cat^TM soil conditioner(T₁), basal fertilizer+conventional fertilization(T₂), basal fertilizer+conventional fertilization+The effects of four treatments with Phyto-Cat^TM soil conditioner(T₃)on the physiological characteristics of non-heading Chinese cabbage, soil physical and chemical properties and fertility levels.The results indicated that compared with CK treatment, T₁, T₂ and T₃ treatments could significantly increased the yield, chlorophyll content, leaf soluble sugar and root activity(tetrazole reduction intensity)of non-heading cabbage.Three processing each index had respectively increased by 11.5%-27.9%, 9.8%-22.7%, 7.8%-16.3%, 12.4%-24.8%;compared with CK treatment;T₁ treatment made the test soil pH, water-stable aggregate content, field water holding capacity, soil organic matter, available nitrogen, available phosphorus, and available potassium content increase by 5.9%, 5.3%, 2.1%, 18.42%, 7.32%, 29.89% and 33.97%;Respectively, soil bulk density decreased by 9.5%.The research results indicated that the application of Phyto-Cat^TM soil conditioner had a significant effect on the physiological characteristics and soil properties of non-heading Chinese cabbage. Under the condition of applying no chemical fertilizer, Phyto-cat^TM soil conditioner played an obvious role in improving soil properties and increasing crop yield. It could replace chemical fertilizer application on the premise of ensuring crop yield, and open up a new way for pollution-free agricultural production.

In order to develop closely linked molecular markers for resistance to dry burning heart disease in Qingmaye Chinese cabbage and accelerate the cultivation of high-quality varieties resistant to dry burning heart disease.200 F₂ and 100 BC₁F₁ populations were constructed simultaneously with the excellent Qingmaye high generation inbred line H227 and Baimaye high generation inbred line G83,the resistance and susceptibility phenotypes of the population were identified by in vitro leaf cutting method, and the genetic law of dry burning heart disease in Qingmaye Chinese cabbage was statistically analyzed. At the same time, combined with BSA method, the disease resistance gene of dry burning heart was preliminarily located and labeled by software JoinMap 4.0 and Mapchart.The results showed that the disease grading of F₂ population and BC₁F₁ population constructed by parents with significant difference in resistance and susceptibility to dry burning heart disease showed obvious unimodal distribution, close to normal distribution, and the inheritance law of resistance had the genetic characteristics of quantitative traits.Based on the sequence information of Chinese cabbage genome database and the constructed molecular marker linkage map, the disease resistance gene in the experimental material was located on A07 chromosome, and a molecular marker BrIDCRT07 linked to the dry burning heart disease resistance gene was designed.The genetic distance between the molecular marker and BrID10343 and BrID10349 was 0.13, 0.78 cM respectively.It was verified that the coincidence rate of this marker in F₂ population was 86.8%, and that in BC₁F₁ population was 94.9%. It could be used as an auxiliary screening marker for anti dry burning heart disease resources of Chinese cabbage in the future.

In order to investigate the effect of insitu passivation on the Cd-contaminated soil, the culture experiment was conducted to study the effects of different types of amendment and addition rate on soil Cd availability and the uptake of Cd by Chinese cabbage. The results showed that with the increase of the addition rate of activated carbon, lime and phosphate rock, the available Cd content in soil and the Cd content in the shoot of Chinese cabbage decreased gradually. Compared with the control, all the treatments of activated carbon, lime and phosphate rock significantly reduced the available Cd content in soil and the Cd content in the shoot of Chinese cabbage. Among them, 5% of activated carbon, 4% of lime and 5% of phosphate rock had the best soil passivation effect. The available Cd content in soil decreased by 25.20%, 49.50%,40.09%, respectively. The Cd content in the shoot of Chinese cabbage decreased by 65.13%,54.64%,67.56%, respectively. With the increases of the addition rate of chicken manure organic manure and zeolite, the available Cd content in soil increased gradually, while the Cd content in the shoot of Chinese cabbage decreased. The treatment with 2% of chicken manure organic fertilizer had the best passivation effect, and the Cd content in the shoot of Chinese cabbage decreased 65.64%. The treatment with 5% of zeolite had the best the effect. The Cd content in the shoot of Chinese cabbage decreased 44.46%. To a certain extent, the addition of fly ash, corn straw and mushroom residue had no significant effect on Cd uptake by Chinese cabbage. So the effects of different types of amendment and addition rate on soil Cd passivation were obviously different. Adding amendments, such as activated carbon, lime, phosphate rock and chicken manure organic fertilizer and optimized rate can effectively reduce Cd content in the shoot of Chinese cabbage.

At present, in the identification process of Pol CMS, Ogu CMS and New Brassica napus CMS, two pairs of primers must be used for molecular marker screening from orf138, orf222 and orf224. In order to develop a new type of molecular markers for rapid identification of New Brassica napus CMS, PCR amplification was carried out by orf222 that based on genomic DNA of CMS-AQ29 and CMS Hou36gao which respectively contained Pol CMS and New Brassica napus CMS.The results showed that the main difference genomic DNA between the two varieties was from 180 to 210 bp. A special primer of New Brassica napus CMS was designed and developed by T/G base-based mismatch on the end of the orf222 upstream primer terminal. It was named O-2. It could identify male sterile lines with New Brassica napus CMS immediately, 470 bp nucleotide sequence could be amplified,but nucleotide sequence could't be amplified in other resources without New Brassica napus CMS. The defect was solved that identification of male sterility must be pairs of primer. This method was resulted that is stable and reliable, the CMS-D571 and CMS-D574 are successfully identified by using this method that including New Brassica napus CMS from D571, D574, CMS-D571 and CMS-D574.

In order to identify the characteristics and genotype differences of nitrogen response in different Chinese cabbage cultivars from the main producing areas, screen useful germplasm for Chinese cabbage with high nitrogen use efficiency (NUE), and explore the evaluation indexes for NUE and nitrogen sensitivity of Chinese cabbage, sixty-eight representative Chinese cabbage cultivars from the main producing areas were planted in the field with low(Pure N 54 kg/ha) and normal(Pure N 270 kg/ha) nitrogen inputs, respectively. A total of 15 agronomic traits, including plant width, plant height, total weight, outer leaves number, net weight, leafy head height, leafy head width, head leaves number, maximum leaf length, maximum leaves width, petiole length, petiole width, petiole thickness, central axis length and central axis width, and three nitrogen related traits including nitrogen content, nitrogen agronomic use efficiency(NUE) and nitrogen responsivity were calculated and analyzed at leafy head maturation stage. Finally, the relative values of each trait in two nitrogen levels were calculated, and the classification for NUE and nitrogen responsivity classes of the 68 Chinese cabbage cultivars were conducted according to yield, relative yield, and nitrogen responsivity. The results showed that 13 agronomic traits were significantly different between the two nitrogen levels, except for outer leaves number and central axis length. Nitrogen content and NUE also showed significant differences between the two nitrogen levels. There were significant genotypic variations for all the agronomic traits under both nitrogen input conditions, of which, central column length had the largest coefficient of variation(CV) among cultivars, followed by weight-related traits. Nitrogen content, nitrogen agronomic efficiency and nitrogen responsivity also showed significant genotypic variations, of which, CV of nitrogen responsivity was the largest. The means of relative net weight and relative gross weight were the largest, while those of relative outer leaves number, relative plant width, and relative central column length were much smaller. In addition, the 68 tested Chinese cabbage cultivars were categorized into four NUE classes(Nitrogen efficient under the two nitrogen conditions, NET; Nitrogen efficient under the low nitrogen condition, NEL; Nitrogen efficient under the normal nitrogen condition, NEN; Nitrogen inefficient under the two nitrogen conditions, NIT) according to the yield under both nitrogen supply conditions, and four nitrogen sensitive types(Nitrogen insensitive type, NIS; Nitrogen low-sensitive type, NLS; Nitrogen moderate-sensitive type, NMS; and Nitrogen high-sensitive type, NHS) according to relative yield and nitrogen responsivity. Using both NUE and nitrogen sensitivity classification criteria mentioned in the study, the nitrogen response characteristics of different Chinese cabbage varieties can be more clearly evaluated. The study will provide useful information for further studying the molecular mechanisms regulating NUE in Chinese cabbage and breeding new Chinese cabbage cultivars with high NUE.

In order to identify Chinese cabbage-cabbage translocation line by gene markers, the polymorphism primers were designed according to the nucleotide difference of collinear genes between Chinese cabbage and cabbage, and were screened with Chinese cabbage 85-1 and cabbage 11-1 as test materials. The applicability of the primers was detected using Chinese cabbage-cabbage translocation lines and their self-crossing progenies, Chinese cabbage varieties and cabbage varieties. The results showed that among 171 pairs of primers designed based on 263 collinear-gene sequences of Chinese cabbage and cabbage, 66 pairs of polymorphic primers were confirmed, the ratio being 38.60%. Among the 66 gene markers, two markers from Chinese cabbage gene sequences and 48 markers from cabbage gene sequences could be used as the specific gene markers to distinguish Chinese cabbage and cabbage, which were successfully applied to identify Chinese cabbage-cabbage translocation lines AT1-41 and AT1-47, and the self-crossing progenies of AT1-47. Furthermore, PCR amplification of the 66 gene markers was carried out in four Chinese cabbage varieties and four cabbage varieties, among which 52 markers could distinguished completely the Chinese cabbage varieties and the cabbage varieties, while other 11 markers presented the polymorphism in parts of the varieties. The results established the foundation for further studying the expression, regulation and interaction of exogenous genes in Chinese cabbage-cabbage translocation lines and for marker-assisted breeding.

In order to identify the clubroot resistance gene of Chinese cabbage in G6 and develop linkage markers, high-generation inbred line G4 of Chinese cabbage with high-susceptible to clubroot disease, high-generation inbred line G6 of Chinese cabbage with high-resistant to clubroot disease, F₁ obtained by crossing G4 and G6, and the F₂ generation separation populations constructed by F₁ selfing were used as materials. By artificial inoculation, phenotypic identification and genetic analysis, it was found that clubroot disease resistance in the disease resistance materials were controlled by a dominant single gene. Further expanding the number of F₂ generation populations to map the genes of clubroot disease and screening molecular markers linked to disease resistance genes, the linkage analysis of polymorphic markers was performed using JoinMap 4.0 software, and five InDel markers linked to the resistance gene of Chinese cabbage were obtained. The most recent markers on both sides were BrID10727 and BrID10867, the genetic distance from the disease resistance genes were 4.6,2.5 cM, respectively, and the disease resistance gene was located on the chromosome A08 of Chinese cabbage. In addition, it was found that the newly developed polymorphic marker BrID10381 based on the Crr1 gene sequence was located outside the initial localization range of the newly located clubroot resistance gene, so it could be inferred that the newly located clubroot resistance gene locus was not the same locus as Crr1, and the marker BrID10381 could be used for molecular marker assisted selection of Crr1 gene.

To clarify the resistant relationship between physiological races and Chinese cabbage(Brassica rapa L.ssp. pekinensis) variety, physiological races 4, 7, 10 and 11 were inoculated by injecting. The disease resistance of the 25 Chinese cabbage and CR markers were detected. The results showed that 8 cultivars such as Shandiwang 2 were resistance to only 4 physiological races; 7 cultivars such as Degao CR117 were susceptible to only 4 physiological races; 4 cultivars (including C1) were susceptible to 2 physiological races, and 6 cultivars (including Huakang 301) were only susceptible to 4 physiological race. All materials contained Crr1, CRk was detected in samples except the Degao CR Tiejia 1 and the Dadi CR118, the disease resistance locus CRa and CRb were detected in 21 and 16 samples respectively, 11 cultivars (including Degao CR117) contained Crr2 resistance locus, Crr3 resistance locus was detected only in Jingchun CR3, CRc resistance locus was contained in Xingguan and CR75.In short,different Chinese cabbage materials have different resistance to the same physiological race,and the same material responds differently to different physiological races. The physiological race No.4 was the most serious, followed by the physiological race No.7. The results of molecular marker identification showed that the resistance sites of Chinese cabbage materials were not necessarily resistant to disease. The resistance of Crr1 had little effect on material resistance.

In order to analyze the gene expression more accurately in Chinese cabbage by quantitative Real-time PCR,Chinese cabbage translocation lines added No.4 chromosome fragments from cabbage under Chinese cabbage background were used as materials in this experiment. The leaves of Chinese cabbage-cabbage translocation lines in different growth stages,including seedling stage,rosette stage,folding stage and heading stage,floral buds of different sizes and leaves of folding stage treated with auxin(Indole-3-acetic acid,IAA)and auxin inhibitor(2,3,5-triiodobenzoic acid,TIBA)were selected to evaluate the expressions stability of genes. Expression stability of twenty one candidate reference genes were detected by qRT-PCR,including Apr, BcTIP41, U34559, EF1α, TUB4, CYP, DNAJ, HIS, TUA5, UKN1, SKIP16, CAC, ACTIN, ACTIN-1, ACTIN-2, GAPDH, UBC30, UBQ, PPR, PP2A, MDH. geNorm and NormFinder were used to analyze above these results,which showed that the qRT-PCR most suitable reference genes of Chinese cabbage-cabbage translocation lines had some differences in different development periods,different tissue of the material,and under condition of different hormone treatment. The UKN1 and TUB4 genes were most stable expression in vegetative growth stage(from seedling to heading)of Chinese cabbage-cabbage translocation lines. In folding stage of Chinese cabbage-cabbage translocation lines,the most stable expression reference genes were BcTIP41 and ACTIN after treatment with auxin IAA,and the most stable expression reference genes were UKN1 and BcTIP41 after treatment with auxin inhibitor TIBA. DNAJ, ACTIN and PP2A genes showed the most stable expression in the floral buds with six size levels of Chinese cabbage-cabbage translocation lines. These results will lay the foundation for the accurate analysis of the gene expression of Chinese cabbage-cabbage translocation lines,and further provided a reference for the selection of the reference gene in other plants of Brassica at different developmental stages and hormone treatments.

In order to remedy heavy metal contaminated soil,the culture experiment was conducted to study the effects of organic and inorganic compound amendments on the biomasses of cabbage,the availability of heavy metals Pb and Cd in soils and the absorption of Pb and Cd by pot experiments.The results showed that organic and inorganic compound amendments enhanced the biomasses of cabbage,the single chicken manure treatment showed the best effect.All compound amendments could significantly decrease available Pb and Cd content in soils.The compound amendments treatment of charcoal+phosphate rock powder showed the best effect on soil Pb and Cd immobilizing effects,were 80.60% and 49.21%.The second was chicken manure+phosphate rock powder treatment,the removal efficiencies for soil Pb and Cd were 76.16% and 36.78%.Comparing with control,all compound amendments could significantly decrease Pb and Cd content of cabbage.The compound amendments treatment of chicken manure+phosphate rock powder showed the best effect on Pb of cabbage,was 79.37%.The second treatment was charcoal+phosphate rock powder.The compound amendments treatment of charcoal+phosphate rock powder showed the best effect on Cd of cabbage,was 56.55%.The second treatment was chicken manure+phosphate rock powder.Therefore,the organic and inorganic amendments treatment of charcoal+phosphate rock powder or chicken manure+phosphate rock powder was to be an efficient,cost-effective and environmentally friendly measure to calcareous soil which was slightly contaminated by Pb and Cd.

In order to expand the new purple material resources and breed our own purple Chinese cabbage varieties in China,we focused on a new purple germplasm 15NG28,its F₁ back-crossing F₁ and self-bred progenies to study characteristics,distribution of anthocyanins in leaf,ploidy modes in genome,chromosome patterns and differentially expressed genes of these progenies.Results showed F₁ hybrids crossed by 15NG28 and Chinese cabbage could be obtained,which segregated purple and green colors.At the seedling and adult stages,15NG28 and purple F₁ plants had purple colors in adaxial leaf,abaxial leaf,vein,while the F₁ plants owned purple colors in adaxial,abaxial blade of leaf and 15NG28 had purple color in adaxial blade,green color in abaxial blade.Meanwhile purple F₁ hybrids had different ploidy modes,i.e.,3n,4n,6n,etc.and chromosome patterns,i.e.,24,28,36,etc.Among them,16M-170-21 was similar as B.juncea.16M-170-21 self-bred seeds could be gotten,which showed distinctly different characteristics.While 16M-170-22 was similar as Chinese cabbage.Characteristic of 31 BC₁F₁ progenies of 16M-170-22 were the same as Chinese cabbages,and purple to green ratio was 14:17.Especially self-bred seeds of 14 purple lines were better.RNA-seq results showed twelve genes involved in flavonoid,flavone,flavonol and anthocyanin biosynthesis pathway were apparently up-regulated in purple lines.Interestingly Bra013652 (as LDOX gene) and Bra027457 (as DFR gene) were structural genes,which were up-regulated apparently in purple lines compared to green lines.Especially the two genes were differently from c3563g1i2,a regulator factor R2R3-MYB reported before.Therefore 15NG28 and its progenies could be new germplasm resources and 15NG28 might origin from B.juncea.

Rapid and efficient DNA extraction is the key step in large-scale molecular breeding of crop. To construct a method for rapid extraction of genomic DNA from Chinese cabbage, the Chinese cabbage leaves were used as experimental materials, the quality of DNA extracted by the CTAB method, two-step CTAB method and four alkaline lysis methods was compared, the PCR amplification effects of DNA extracted by different methods were analyzed, and the preservation time and preservation conditions of genomic DNA extracted by different methods were compared, the optimal method was selected for the application of molecular marker-assisted selection against clubroot disease.The results showed that genomic DNA extracted by the CTAB and three kinds of alkaline lysis methods could be used as a template for PCR, and the PCR amplification products could be detected by 8% non-denaturing polyacrylamide gel electrophoresis to clear bands. Among them, the alkaline lysis method Ⅲ, not only the extraction quality was good and the extraction process was simple and fast, but also it could meet the needs of high-throughput DNA extraction of Chinese cabbage, the extracted DNA was stored at 4℃ and -20℃, and the genomic DNA stored for 30 days was used as a template for PCR amplification, the products was still detected by polyacrylamide gel electrophoresis to clear bands, it illustrates that this method has a long preservation time and it has been proved that this method has a good effect on the application of molecular marker-assisted selection against clubroot disease. Alkaline lysis method Ⅲ has significantly improved the efficiency of Chinese cabbage molecular marker-assisted selection, and can be widely used in Chinese cabbage molecular marker-assisted selection breeding.

To explore the root disease resistance genetic regularity,by root disease resistance difference material,G57 and G70,the F₂ segregation population containing 500 per plant was built.Through artificial inoculation of the parents,F₁ and F₂ segregation population phenotypic characterization results were statistically analyzed. The results showed that root disease resistance of materials was controlled by a single dominant gene. In order to further localization of resistance genes in test materials,we used 678 molecular markers of parents,F₁ and F₂ population screening verification,resistance genes was preliminarily located in molecular marker KBRH129J18 and TCR02-F.This test was based on two chain tags,chain development design closer chain of molecular markers,ultimately with resistance genes chain 5 of SSR molecular markers,respectively,Bra0345-1,Bra0235-2,Bra0235-1,Bra19317,Bra019392. Their genetic distance with resistance genes were 2.4,2.4,2.4,2.5,3.3 cM.Validated,development and design of polymorphism markers in Chinese cabbage had good applicability.

Different tipburn resistance materials of Chinese cabbage were used as materials to study the inheritance of resistance gene to tipburn and to conduct QTL mapping so as to provide a theoretical basis for the molecular assistant selection (MAS) breeding and resistance mechanism. This research adopted significantly different resistance tipburn line high inbred Qingmaye type Black 227 (resistant lines) and high inbred Baotou type B120 (susceptible lines) as materials to obtain DH group by microspore culture F₁ generation. We classified degrees of the disease and combined the molecular genetic map of Chinese cabbage. According to disease severity in the field of DH group, QTL analysis on tipnurn resistance genes were conducted by MapQTL 5.0 software. Results showed that the inheritance of the resistance gene to Chinese cabbage tipburn in tested materials fit to the inheritance law of quantitative trait.This study detected two InDel markers BrID10343 and BrID10349 which link closelyed with tipburn resistance gene of Chinese cabbage. Two markers were on the Chr.7,in which the genetic distance of 1.031 cM and genetic contribution rates were above 40%.InDel markers BrID10343 and BrID10349 linked closelyed with tipburn resistance gene of Chinese cabbage. The results would be of great benefit to fine mapping for the major QTL of tipburn resistance gene, also the results will lay a good foundation for Chinese cabbage MAS resistance breeding.

In order to explore the mechanism of super cold resistant varieties of Winter turnip rape,select super cold resistant varieties of Winter turnip rape Longyou No.7 leaves as materials,using two different extraction liquids(Extraction A:0.1 mol/L Tris-HCl (pH=7.6),0.2 mol/L KCl,1 mmol/L PMSF,Extraction B:0.05 mol/L Tris-HCl (pH=7.6),0.01 mol/L EDTA-Na₂,0.02 mol/L Vc,1 mmol/L PMSF) to compare the extracted effect.The experiment showed that the extraction A liquid protein yield reached (0.073±0.004 6) mg/g,and the extraction ratio increased by 42.88% compared to liquid B.Through the Glucose-6-phosphate dehydrogenize (G₆PDH) activity detection and the extraction A was lower than B 0.91 percent;not only the extraction A extract protein was shorter than B 4.5 hours in the first oelectric focusing electrophoresis of two-dimensional electrophoresis,but also was get clear gel map;through the calculation of abundance,there were total 339 differentially expressed proteins discovered in Longyou No.7 under low temperature stress(4℃,48 h)later,changes in expression levels of more than two times the protein spots of 46,which 18 up-regulated protein expression spots,21 down-regulated protein expression spots,7 specific protein expression spots,these differential protein spots were suggested to be related to low temperature stress.This laid the foundation for the research of cold resistant varieties in Winter turnip rape Longyou No.7 proteomics aspect.

The products obtained by long range PCR based on objective sequences were the effective probes for FISH to carry out molecular cytogenetic study.However,it is very difficult to amplify more than 5 kb-long fragments effectively by traditional PCR techniques.Suitable reaction conditions and reaction system are the necessary prerequisite for running effective LR-PCR amplification.In order to obtain LR-PCR products based on the objective sequences as FISH probes,80 pairs of LR-PCR primers were designed based on the sequences of repeat-free regions on the top of Chinese cabbage chromosome A03,and PCR technique system was optimized from the following aspects containing the template quality of Chinese cabbage genomic DNA,dNTPs concentration,annealing temperature and extended time.Results indicated that genomic DNA of seedling leaves and LA Taq polymerase enzyme could improve the amplification quality and efficiency of long-range PCR.And 5-15 kb PCR products could be amplified by the reaction system with 20 μL of total volume,comprising of 50 ng/μL DNA 2 μL,2.5 mmol/L dNTPs 1.6 μL,10 μmol/μL of each primer 1 μL,10×LA PCR Buffer Ⅱ (including Mg²⁺) 2 μL,5 U/μL LA Taq enzyme 0.2 μL.PCR reaction procedure was followed as:98℃ for 15 s;58-64℃ for 10 s,68℃ for 5 min,35 cycles;final extension at 68℃ for 10 min kept at 4℃.Under these conditions,60 fragments with 5-15 kb length were obtained from Chinese cabbage genome.This research would establish the basis for carrying out long range PCR-FISH on meiotic pachytene chromosomes of Chinese cabbage and for clarifying evolutionary by comparing chromosome painting among close related species.

The aim was to study whether xenia effect existed in Chinese cabbage.Taking 14S116 as female parent and 14S443,14S375,14S120,14S502,14S393,14S536 as male parent,and taking 14S193 as female parent and 14S443,14S125,14S116,14S375,14S531 as male parent,and taking 92S24A as female parent and 8407,72M,DaT511,Da164-2-1,14S126,14S375,Shandong number 4,Jiecai,Da qin 3 as male parent,hybrids were prepared,and silique length,silique width,beak length,shape of seed,seed number of per pod,1000-grain weight,from which mid-parent,heterosis and male effect were acquired.Another,JY3 and JY4,JY5 and JY6,JY19 and JY20,Bre and Xiasheng,92S24 and Xiasheng,14S375 and 14S116 were respectively intercrossed,1000-grain weight seed were determined,and shope of seed and color of seed from some hybrids were observed,from which mid-parent,heterosis and male effect were acquired.The results showed that length and width of the silique,beak length,shape of seed,seed number of per pod,1000-grain weight of F₀ affected by pollen from different male parent were detected.Over-parent heterosis and heterosis were presented.

To explore the reasons for difference in mechanism of cold resistance of winter rapeseed of Brassica rape and Brassica napus.The method of combining field and pot culture was adopted,and using 8 different cold resistance of winter rapeseed varieties as materials,before wintering period with 5 to 6 true leaves,field experiments researched botanical morphology and accumulation of dry matter,the physiological characteristics were measured by pot experiment after the treatment of 24℃→10℃→5℃→0℃→-5℃→-10℃ 48 h.The results showed that winter Brassica rape was creeping growth,growth point was under the ground,accumulation of dry matter was mainly concentrated in the underground part,such as fresh matter of underground parts and dry matter of underground parts of winter Brassica rape had increased,which average increased 236.1%,263.0% than winter Brassica napus respectively.It was showed that the photosynthetic organic product of strong cold resistant varieties was preferentially allocated to the underground part during the vegetative growth stage,to establish a large root system to provide metabolic energy for the safe winter.With the change of temperature,the physiological and biochemical indexes of different varieties was vary greatly,SOD activity of Longyou 7 increased 10.7% than CK at low temperature of -5℃,CAT and POD activity of Longyou 7 increased 24.7%,28.6% than CK respectively at 0℃,SP content of winter Brassica rape average increased 32.3% than winter Brassica napus at 0℃,SS content of winter Brassica rape average increased 71.4% than winter Brassica napus,and MDA content of winter Brassica napus average increased 52.8% than winter Brassica rape at low temperature of -10℃,this indicated that the strong cold resistant varieties could protect themselves from damage of low temperature condition,the CAT,POD,SP were protective substance of against chilling injury,but SOD,SS were protective substance of against freezing.Therefore,the winter Brassica rape had obvious advantages in the morphology and physiological level than winter Brassica napus,the morphological advantages was that to withstand cold weather of extreme,to provide metabolic energy of during the wintering period and regreen next year;At physiological levels,the activities of protective enzyme and content of osmotic regulation of winter Brassica rape increased significantly at low temperature stress to protect the structure of cell membrane,and the accumulation of MDA was decreased,to alleviate hurt of winter rapeseed leaves at low temperature,so the over wintering was ensured highly,it was important to provide a theoretical basis for the research on the cold resistance of winter rapeseed of Brassica rape and Brassica napus in the North of China.

For the disease-resistant breeding of Qingmaye Chinese cabbage,five kinds of inoculation methods(Indigenous bacteria,Seal of indigenous bacteria,Dip method,Injection method,Seed soaking method),different soil pH,different inoculation concentration,illumination and root secretion were compared by use Qiulü 60 as material in this article.The results showed that the effect of soil inoculation was more excellent and stable.In contrast,seed-immersion method was the poorest.The acid soil pH was fit to clubroot break out and the disease index reached the maximum when pH value was 6.5.1 g soil inoculated 0.050 g root was the most suitable inoculation concentration.After light treatment,the disease index was increased,it showed that light had a promoting effect on diseases.Root secretion of tomato,resistant and susceptible hosts could promote the happening of disease.Therefore,in the artificial inoculation test of Qingmaye Chinese cabbage can be the above optimum inoculation method and conditions so as to improve the effect of inoculation.