The influence of Lactobacillus plantarum fermentation in selenium-enriched Brassica napus L.:changes in the nutritional constituents,bioactivities and bioaccessibility

Selenium(Se)-enriched Brassica napus L.is a valuable organic Se supplement.In this study,the fermentation broth enriched with organic Se(FFS)was prepared using Lactobacillus plantarum to ferment the substrate of Se-enriched Brassica napus L.Significant increases were observed after fermentation in total sugars,reducing sugars,soluble proteins,total phenolic content(TPC),and total flavonoid content(TFC).The organic Se was retained at a concentration of 54.75 mg/g in the freeze-dried sample.Principal component analysis and cluster analysis showed good separation between the FFS and unfermented(FS)groups.Fragrant 2-ethyloxetane had the highest content among all volatiles,while sinapine had the highest content among all phenolic compounds.The fermentation process showed remarkable improvement in the abundance and concentration of volatile compounds and phenolic contents,making FFS exhibit strong antioxidant activity and inhibitory capacity againstα-glucosidase activity.The bioaccessibility of phenolic compounds was significantly greater in FFS compared to FS.ADMET analysis revealed that the majority of phenolic compounds contained in FFS did not exhibit mutagenicity toxicity,hepatotoxicity,skin sensitization,or blood-brain barrier penetration,indicating a favorable level of biosafety.Overall,our study provides a new insight into the further utilization of Se-enriched Brassica napus L.in foods.

Effect of Foliar Fertilizer Spraying at Seedling Stage on the Growth Physiological Characteristics and Yield of Brassica napus L.

In order to select a suitable foliar fertilizer for Brassica napus L.at the seedling stage,using‘Fengyou 958’as the material,different foliar fertilizers including BR,Se,Si,BR+Si,BR+Se,Se+Si and BR+Se+Si were sprayed at the seedling stage to study their effect on the physiological characteristics,growth and yield of Brassica napus L..The results showed that the growth,chlorophyll content,soluble sugar content,soluble protein content,and yield of different treatments at the budding stage improved compared with the control.The effect of the Si+Se treatment was the best,followed by the Se and Si treatments.The chlorophyll content of the flower decreased continuously during the whole flowering period,and the chlorophyll content of the mature silique peel was higher than that of the seed.The content of soluble sugar in flowers was the highest in the early flowering stage,and the content of soluble sugar in leaves at the flowering stage was higher than that at the budding stage.The soluble sugar content in the harvested silique peel decreased gradually with the increase of time,and that in the silique peel and seed was similar at 35 d after pollination.The soluble protein content in the silique peel and seed decreased gradually at the mature stage,and the soluble protein content in the silique peel was higher than that in the seed at the same stage.Si+Se foliar fertilizer spraying at the seedling stage can promote the growth and yield of Brassica napus L.and can be applied in Brassica napus L.production.

Development of a KASP Marker on Chromosome A05 for Seed Oil Content of Brassica napus

In order to identify the molecular markers that can be widely used in the breeding of Brassica napus L.varieties with high seed oil content under different genetic backgrounds,we developed a Kompetitive Allele Specific PCR(KASP)marker for seed oil content on the basis of the results from available studies.The verification in the F_(2) population showed that the marker was closely linked to the quantitative trait locus(QTL)for oil content on chromosome A05.The findings helped to breed the‘Fengyou’varieties with high seed oil content in the middle reaches of the Yangtze River.

Analysis of the Lignin Contents and Related Enzymes Activities in Seed Coat Between Black-Seeded and Yellow-Seeded Rapes (Brassica napus L.)

One pair of near isonegic yellow/black seeded rape （Brassica napus L） were used as experimental materials to study the changes of lignin contents and enzymes activities of 4-coumarate： CoA ligase （4CL）, Cinnamyl alcohol dehydrogenase （CAD） and ferulate 5-hydroxylase （F5H） in seedcoats during the stage of seed development. The variation analysis showed that the changes of lignin contents and enzyme activities of 4CL, CAD and F5H in seed coat had significant differences between black- and yellow-seeded rapes, and also between different development stages. The correlation analysis demonstrated that the lignin contents were positively correlative to the activities of all three enzymes investigated in the study, and the interactions between them in the seed coat of the two lines. For yellow-seeded rape, the correlation coefficient （0.7262018） of lignin content and the interaction between 4CL and F5H was significant, the lignin contents were highly positively correlative to the activities of CAD and F5H, the interaction between 4CL and CAD, and the interaction between CAD and F5H. For the black-seeded rape, only the lignin content was highly positively correlative to the activity of F5H （the correlation coefficient was 0.772949）, the other correlation coefficients, i. e. lignin contents to 4CL, CAD activities, the interactions between the three enzymes were not significant although all the correlation coefficients were above 0.5000. The results suggested that 4CL, CAD and F5H regulated the biosynthesis of lignin in seedcoat of rapes, leading to the lignin contents in the seedcoats of the yellow-seeded rape much lower than that of the black-seeded line, and affecting the thickness of the developing seedcoats in rapes. Therefore, it was likely to change the seedcoat ratio by overexpressing or suppressing the activities of one of the enzymes, both of them or all of them.

Genetic Diversity of Testa Pigments and RAPD Marker of Yellow-Seeded Rapeseed (Brassica napus L.)

14 yellow-seeded rapeseed lines (Brassia napus L.) from different genetic sources were used to analyze diversity of testa pigments content, oil and protein content, and RAPD markers. The results showed that the anthocyanin and melanin were the most important pigments in testa and their content were responsible for the variation in seed color ranging from orange to black yellow, 14 yellow-seeded lines could be classified into 3 groups: high anthocyanin content group with anthocyanin content over 2. 54 mg g-1 DW, the seed color was light yellow or orange; low pigments content group with low content of anthocyanin and melanin, the testa was transparent and the seed color was light yellow, greenish yellow or twany; high melanin content group with melanin content over 178. 4U(A290nm) , the testa was black, the seed color was black yellow. Oil content changed from 36.2% to 45. 5%, protein content from 21.1% to 27.7% , and the correlation analysis revealed that the oil content is highly significantly negatively correlated with the protein content. The cluster analysis showed that the extensive genetic variation existed among 14 yellow-seeded lines by using unweighted paired group method with arithmetic average (UPGMA) based on RAPD markers which were amplified with decamer primers, the genetic similarity among them ranged from 0. 25 to 0.909, and 14 yellow-seeded lines could put into 2 clusters corresponding to genome difference.

Dissecting the genetic architecture of glucosinolate compounds for quality improvement in flowering stalk tissues of Brassica napus

Glucosinolates(GSLs) and their hydrolytic products contribute to the quality traits of rapeseed flowering stalk tissues, such as taste, flavor and anticarcinogenic properties(Glucoraphanin). However, little is known about the genetic mechanisms of GSL accumulation in rapeseed flowering stalks. In this study, the variation and genetic architecture of GSL metabolites in flowering stalk tissues were investigated for the first time among a panel of 107 accessions. All GSL compounds exhibited continuous and wide variations in the present population. Progoitrin,glucobrassicanapin and gluconapin were the most abundant GSL compounds. Five quantitative trait loci(QTL) significantly associated with three GSL compounds were identified by genome-wide association study. GRA_C04 was under selected during modern breeding, in which the ratio of lower GSL haplotype(HAP2) in the accessions bred before 1990(52.56%) was significantly lower than that after 1990(78.95%). Four candidate genes, BnaA01. SOT16, BnaA06. SOT17, Bna A06. MYB51a, and Bna A06. MYB51b, were identified in the GTL_A01 and 4OH_A06 regions.These findings provide new insights into GSL biosynthesis in flowering stalk tissues and facilitate quality improvement in rapeseed flowering stalks.

Physiological Differences Between Yellow-Seeded and Black-Seeded Rapeseeds (Brassica napus L.) with Different Testa Characteristics During Artificial Ageing

Yellow-seeded rapeseed (Brassica napus L.) is a new kind of breeding resources with yellow color, increased oil and protein content and less unwanted crude fiber content due to the thinner and transparent testa compared with traditional black or brown-seeded rapeseed. To analyze the longevity of the yellow-seeded rapeseed during storage, the physiological differences between the yellow and black-seeded near-isogenic lines were studied by artificial ageing method. The testa rate, anthocyanin content and melanin content of yellow-seeded rapeseeds decreased by 20.1, 25.2, and 80.4% respectively than black-seeded rapeseed. During artificial ageing, the yellow-seeded rapeseed showed significantly different effect of ageing compared with the black-seeded, as demonstrated by faster deterioration with lower germination percentage, seed vigour index, reducing sugar and soluble protein contents than the black-seeded, as well as a drastic increase in electrical conductivity, malnodialdehyde (MDA) content and a rapid decrease in activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). The results suggested that the transparent testa of the yellow-seeded rapeseed lost some abilities to protect the embryo against adverse environmental conditions and thus led to a poor storability.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

BnaSD.C3 is a novel major quantitative trait locus affecting semi-dwarf architecture in Brassica napus L.

Plant height is a key plant architectural trait that affects the seed yield,harvest index and lodging resistance in Brassica napus L.,although the genetic mechanisms affecting plant height remain unclear.Here,a semi-dwarf mutant,df34,was obtained by ethyl methanesulphonate-induced mutagenesis.Genetic analysis showed that the semi-dwarf phenotype is controlled by one semi-dominant gene,which was located on chromosome C03 using a bulked segregant analysis coupled with whole-genome sequencing,and this gene was named BnaSD.C3.Then BnaSD.C3 was fine-mapped to a 297.35-kb segment of the“Darmor-bzh”genome,but there was no potential candidate gene for the semi-dwarf trait underlying this interval.Furthermore,the interval was aligned to the Zhongshuang 11 reference genome.Finally,combining structural variation analysis,transcriptome sequencing,phytohormone analyses and gene annotation information,BnaC03G0466900ZS and BnaC03G0478900ZS were determined to be the most likely candidate genes affecting the plant height of df34.This study provides a novel major locus for breeding and new insights into the genetic architecture of plant height in B.napus.

Detection of new candidate genes controlling seed weight by integrating gene coexpression analysis and QTL mapping in Brassica napus L.

Seed weight is a component of seed yield in rapeseed(Brassica napus L.).Although quantitative trait loci(QTL)for seed weight have been reported in rapeseed,only a few causal quantitative trait genes(QTGs)have been identified,resulting in a limitation in understanding of seed weight regulation.We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species.Among the 10 modules in this gene coexpression network,modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein,and photosynthesis.A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19(SCPL19)was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines.Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Effects of Heat Stress during Seed Filling Stage on Brassica napus Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise,the threat of heat stress to rapeseed production is becoming more obvious.Exploring the response characteristics of two important biological pathways,oil accumulation and photosynthesis,to heat stress during B.napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed.The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B.napus germplasms with different oil content and environmental sensitivity,including 6 rapeseed varieties which exhibited environmentsensitive/insensitive and with high,medium or low oil content,were tested by whole plant heat stress or the in vitro silique culture system.Both assay exhibited similar trend on oil content of the rapeseed germplasms.The heat effect on the chlorophyll fluorescence kinetic parameters F_(v)/F_(m),ETR and Y(Ⅱ)were also consistent.Heat stress significantly decreased oil content,although there was abundant genetic variation on heat tolerance among the genotypes.Correlation analysis showed that the decrease rate of F_(v)/F_(m) of silique heat-stressed B.napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed(R=0.9214,P-value<0.01).Overall,the results indicated that heat stress inhibited oil accumulation and photosynthesis in B.napus developing seed.The decrease rate of chlorophyll fluorescence parameter F_(v)/F_(m) of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification.Further,two heat insensitive rapeseed varieties with high oil content were identified.

甘蓝型油菜(Brassica napus L.)千粒重性状遗传体系分析

通过遗传差异较大的 2个甘蓝型油菜 (BrassicanapusL )纯系亲本组合 (HSTC14×宁油 7号 )衍生后代的世代家系群体分析 ,应用主基因 +多基因家系世代联合分离分析方法研究油菜千粒重的遗传体系。结果表明 ,甘蓝型油菜HSTC14×宁油 7号组合千粒重遗传体系系由一对主基因 +多基因构成 ,主基因中只有加性效应 (d =0 10 6 2 ) ,不存在显性效应 ;多基因存在加性 ([d]=0 2 2 2 8)和显性效应 ,但显性效应为负值 ([h]=- 0 2 80 0 )。表明该组合千粒重主要由主基因加性效应和多基因效应决定 ,但无杂种优势。F2∶3家系群体主基因遗传力为 83 39% ,多基因遗传力为 11 71%。HSTC14×宁油 7号组合千粒重的改良可能受主基因无显性效应 ,多基因显性效应为负值的制约。

锰胁迫对甘蓝型油菜Brassica napus L.种子活力和幼苗抗氧化系统的影响

用不同质量分数的锰溶液(0.2%,0.4%,0.8%和1.2%Mn2+)处理甘蓝型胜利油菜Brassica napuscv.Shengli,研究锰对其种子活力、幼苗细胞膜透性、脂质过氧化及抗氧化剂的影响.结果表明:锰胁迫降低了种子活力、幼苗抗坏血酸(Vc)质量分数、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,破坏了细胞膜的完整性,并使丙二醛(MDA)质量分数、脯氨酸(Pro)质量分数和过氧化物酶(POD)活性增加.

激素对甘蓝型油菜(Brassica napus L)矮化突变体幼苗生长及内源GA_3含量的影响

以甘蓝型油菜矮化突变体"DDF-1"为材料,研究了赤霉素(GA3)、生长素(IAA)和油菜素内酯(BR)3种激素对突变体幼苗的影响,并用石蜡切片观察其细胞形态,用酶联免疫吸附技术(ELISA)检测下胚轴内源激素GA3含量.结果表明,外加一定浓度的GA3(7 mg/L)对矮化突变体"DDF-1"的下胚轴伸长作用显著,但不能使之恢复到野生型的长度;萌发早期,BR有明显的促进伸长作用,萌发后期效果不明显;突变体对IAA敏感性较弱.三种激素交互作用对矮秆下胚轴伸长的影响也不显著.施加外源GA3的矮化突变体"DDF-1"的细胞也有明显的伸长.矮化突变体"DDF-1"的内源激素GA3含量高于野生型高秆,但低于施加外源GA3的矮化突变体.矮化突变体"DDF-1"为赤霉素GA3的非应答型矮化突变体.

Correlation Analysis of SPAD Value with Chlorophyll Content and Economic Yield Traits of Brassica napus L.

[Objective] The aim was to compare differences of SPAD value, chloro- phyll content, agronomic characters, economic characters and yield traits to analyze correlation of SPAD value with other indices and establish regression functions. [Method] Based on 34 Brassica napus L. varieties, SPAD value, chlorophyll content, agronomic characters, economic characters and yield traits were measured and re- gression functions were established according to correlations. [Result] SPAD value, chlorophyll content, agronomic and economic characters and yield traits all achieved significant level in differences among 34 varieties. Specifically, SPAD value was of extremely significant correlation with chlorophyll a and b, total chlorophyll and carotenoid, and the correlation from high to low was chl-b〉chl-z〉chl-a〉chl-x. SPAD value was of significantly positive correlation with total pod number per plant, plant height, seed number per pod, yield per plant and harvest yield, and of insignificant correlation with branch point height, effective branch number, pod density of main stem, and pod length. [Conclusion] It is simple and rapid to predict chlorophyll con- tent, economic characters and yields of Brassica napus L. with SPAD value and re- gression functions.

Screening of Drought-tolerant Brassica napus L.Varieties and Analysis on Their Physiologic and Biochemical Variations under Drought Stress

[Objective] The aim of this study was to screen drought-tolerant Brassica napus L. germplasm resources by analyzing their physiological and biochemical changes under drought stress. [Method] Forty varieties of B. napus varieties were cultured under PEG-6000 osmotic stress and extreme drought stress in pots, re- spectively. Then, the contents of chlorophyll, carotenoid, proline, malondialdehyde （MDA）, soluble sugar, soluble protein, and the activities of superoxide dismutase （SOD）, catalase（CAT）, peroxidase（POD） were measured under drought stress. [Result] Sever- al drought-tolerant varieties of B. napus were screened out： YAU200908, Xiangyou No.15, YAU200903, YAU200907, YAU200906 and YAU200904. Physiological and biochemical analysis showed that, the contents of chlorophyll and carotenoid de- creased with drought stress increasing; the contents of proline, soluble sugar, solu- ble protein, MDA and the activities of SOD,CAT, POD raised with drought stress in- creasing. [Conclusion] In the rapeseed varieties with stronger drought tolerance, the decrease in carotenoid content and the increase in proline content, soluble sugar content, MDA content, SOD activity, CAT activity were more obvious, so all these physiological and biochemical indices can be used to evaluate the drought tolerance of rapeseed.

Effects of ABA on Photosynthetic Characteristics of Pods and Yield of Brassica napus

[Objective] The aim was to evaluate the effects of different concentrations of ABA on photosynthetic characteristics of pods and yield of Brassica napus and analyze the relationships between ABA concentration and yield components, to provide theoretical basis for cultivation of high-yield rapeseed. [Method] At the end of flowering stage, the rape plants were separately sprayed with 1, 2, 100 and 200μmol/L of ABA; photosynthetic parameters, water use efficiency （WUE）, yield and yield components of the rape plants were investigated 0, 5, 10 and 15 d later, respectively. [Result] ABA treatment with different concentrations significantly changed pod photosynthetic parameters and yield components of rapeseed. Low concentra- tions of ABA could improve pod＇s net photosynthetic rate and yield components while high concentrations had the contrary effect. [Conclusion] Whether ABA func- tioned in inhibiting or promoting role might depend on the interaction between endogenous hormone levels and exogenous ABA concentration. Improvement of WUE would be the main reason for yield increasing.

Cloning of Brassica napus EIN3 Gene and Its Expression Induced by Sclerotinia sclerotiorum

[Objective] The study was to investigate roles of Brassica napus EINB in （ BnEIN3 ） resistance to Sclerotinia sclerotiorum. [ Methods] Genomic PCR and RT-PCR were carded out to isolate genomic DNA and cDNA sequences of BnEIN3 from oilseed rape, based on the highly conserved region of EIN3 gene from Arabidopsis thaliana and the homologous sequences of oilseed rape ESTs. Expression levels of BnEIN3 were detected in three varieties of oilseed rape inoculated with S. sclerotiorum by real-time quantitative PCR.[Results] A 1 947 bp DNA fragment was obtained from oilseed rape. The fragment shared 82% identity to A. thaliana EIIV3, encoded 614 amino acids containing an EIN3 domain, and was named as BnEIN3. Real-time PCR results showed that expression patterns of BnEIN3 were drastically different in different varieties. In highly resistant oilseed rape variety D083, BnEIN3 expression level was significantly increased 72 h after S. sclerotiorum inoculation whereas in middle resistant and susceptible varieties Zhongshuang 9 and 84039, BnEIN3 expression was suppressed. [ Conclusion ] BnEIIV3 may play an important role in oilseed rape resistance to S. sclerotiorum.

抗倒伏甘蓝型油菜(Brassica napus L.)根和茎解剖学结构分析

对甘蓝型油菜(Brassica napus L.)6个抗倒伏材料和5个易倒伏材料的苗期和苔期的根部和茎部采用石蜡切片的方法切出6μm厚的薄片,用番红-固绿双重染色,借助Olympus显微镜观察不同材料茎的表皮、皮层、厚角组织、维管束和髄的结构,以及根的木质部、导管等,并对茎和根的结构特点进行了比较。结果表明:①在苗期的茎中,与易倒伏材料相比,抗倒伏材料有较多的厚角组织;抗倒伏材料维管束的面积占茎基部横切面面积之比比易倒伏材料平均高13.1个百分点;抗倒伏材料和易倒伏材料间维管束的个数差异不大,但抗倒伏材料的维管束排列相对比较紧密。②在苔期的茎中,抗倒伏材料的厚角组织和厚壁组织比易倒伏材料发达;虽然两者间维管束的个数和面积无明显差异,但抗倒伏材料维管束排列紧密。③在苗期的根部,抗倒伏材料和易倒伏材料木质部的面积占根部横切面面积之比差异不显著;但抗倒伏材料导管数目比易倒伏材料平均多74.7个,易倒伏材料中较大导管的数目多于抗倒伏材料。④在苔期的根部,抗倒伏材料木质部占根横切面面积的之比比易倒伏材料高8.33个百分点,抗倒伏材料单位面积导管的数目多于易倒伏材料。因此,较多的厚角组织和厚壁组织、较多的导管数和排列紧密的维管束是甘蓝型油菜抗倒伏材料苗期和苔期重要的解剖学特点。

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was discussed.