Analysis of cytosine methylation in early generations of resynthesized Brassica napus

DNA methylation, an important epigenetic modification, serves as a key function in the polyploidization of numerous crops. In this study, early generations of resynthesized Brassica napus （F1,S1-S3）, ancestral parents B. rapa and B. oleracea were analyzed to characterize their DNA methylation status during polyploidization, applying DNA methylation-sensitive amplifica- tion polymorphism （MSAP） and high-performance liquid chromatography methods. In F, 53.4% fragments were inherited from both A- and C-genomes. Besides, 5.04 and 8.87% fragments in F were inherited from A- and C- genome, respectively. 5.85 and 0.8% fragments were newly appeared and disappeared in resynthesized B. napus, respectively. 13.1% of these gene sites were identified with methylation changes in F, namely, hypermethylation （7.86%） and hypomethylation （5.24%）. The lowest methylation status was detected in F （38.7%） compared with in S1-S3. In S3, 40.32% genes were methylated according to MSAP analysis. Sequencing of methylated fragments indicated that genes involved in multiple biological processes were modified, including transcription factors, protein modification, and transporters. Expression ananlysis of DNA methyltransferase I and DNA methyltransferase chromomethylase 3 in different materials was consistent to the DNA methylation status. These results can generally facilitate dissection of how DNA methylation contributes to genetic stability and improvement of B. napus during polypLoidization.

Factors limiting seed yield potential in F1 hybrids between natural and resynthesized Brassica napus

Resynthesized Brassica napus lines are of great potential in hybrid breeding for their higher genetic basis. An 8×8 complete diallel experiment was designed using 4 B. napus cultivars （ BN） and 4 resynthesized rapeseed lines （ RS） as parents, and all 56 were divided into BN × BN, NR （ RS × BN/ BN × RS ） and RS × RS types. Their heterosis for agronomic characteristics were investigated, especially on NR hybrids. Results showed that NR hybrids had higher plants, longer branches, more siliques and fewer seeds per silique at highly significant level when compared with conventional rapeseed cultivars. For yield per plant, all 32 NR cross combinations showed positive mid - parent heterosis and 25% of the combinations （8 from 32 NRs） showed positive high - parental heterosis. On average of the 32 NRs, the heterosis was 30. 56% over mid - parents and - 8. 57% over high - parents. Compared with BN x BN hybrids, NRs had more siliques per plant （437. 0 vs 370. 9） and fewer seeds per silique （11.6 vs 17.3）. There was no significant difference on thousand seed weight between NR and BN × BN hybrids. NR hybrids showed abnormal flower organs, including A： 59% of NR combinations had more or less ab-normal perianth or anthers; B：pollen fertility reduced, ranging from 56. 2% to 95. 5% and 80.1% on average; C ：poor silique - set （33. 4% on average） and seed set （7. 8 ±3.6 seeds per silique） after self -pollination ; D： frequent univalents and multivalents in metaphase I of meiosis; E ： they showed highly significant self - incompatibility.

双低油菜湘油15(B.napus)对菌核病抗性的研究

经苗期菌丝块接种鉴定和花期带菌牙签鉴定表明 ,双低油菜湘油 15对菌核病有高抗性。当病原危害后 ,湘油15体内酚类物质含量和几丁质酶活性迅速升高 ,且维持时间长 ,湘油 15与感病品种 98C4 0和中抗品种中油 82 1杂交后代抗性表现为双亲平均值 。

Reproduction of the Three-line Genic Male Sterile Line Parent Mian 7MB-1 （Brasscia Napus L.） and Seed Production of F1 Based on Somatic Tissue Culture

[Objective] The aim was to study the reproduction of the three-line genic male sterile （GMS） lineparent Mian7MB-1 （B. NapusL.） and the seed production of F1 through somatic tissue culture. [Methed] Through hybridization, a new breeding material Mian 7MB-1 in three-line genic temporary maintainer line propagated by tissue culture was used to improve the sterile plant rate of rapeseed in dual-purpose recessive GMS line, such as Mian 7AB type, S45AB type, and etc. And then the variety comparative test was performed. [Result] In order to avoid some fertility restoration phenomena occurring during the process of self-reproduction, Mian 7AB was propagated in bulk with somatic tissue culture of temporary maintainer line plant stem. The propagated temporary maintainer line seedlings were applied to the breeding and seed production of net room male sterile line parent, promoting the sterile plant rate of the male sterile line parent to 91.7% -93.5%. The male sterile line parents per hectare were enough for the seed production of hybrid F1 in 7 500 -15 000 hm^2. [ Conclusion ] Compared with the original dual-purpose GMS line, the seed production ultilizing male sterile line with high sterile plant rate greatly reduced the labor, significantly improved the seed yield, ensuring the seed quality and forming a perfect breeding and seed production system.

Production of Partial New-typed Brassica Napus by Introgression of Genomic Components from B. rapa and B. carinata

A breeding strategy for widening the germplasm of Brassica napus was proposed by introgression of the A^r subgenome of B. rapa （A^rA^r） and C^c of B. carinata （B^cB^cC^cC^c） into natural B. napus （AnAncncn）. The progenies with 38 chromosomes that were derived from the self-pollinated seeds of pentaploid hybrids （A^rA^nB^cC^cC^n） were used for further research. Some of the partial new-typed B. napus showed normal meiotic behavior, high portion of germinated pollen and normal embryological development. This indicates that the selected new-typed B. napus had a balanced genetic base. Molecular analysis showed that about 50% of the genome in the new-typed B. napus was replaced by A^r and C^c subgenome from B. rapa and B. carinata. Considering the genetic diversity among different lines of new-typed B. napus, it was deduced that the introgression of the genomic components from B, rapa and B. carinata could widen the genetic diversity of rapeseed.

Effects of Nitrogen Fertilization on the Growth and N Uptake of Late-sowing Winter Oilseed Rape (Brassica napus L.)

[Objective] The aim of this work was to analyze the N fertilization on the vegetative growth and N uptake of different winter rapeseed varieties at wintering stage. [Method] In two consecutive years （2009-2011）, two winter rapeseed varieties （B. napus L.）, an early maturity variety Zhongyou 116 （ZY116） and a middle-late application maturity variety Zhongyouza 12 （ZYZ12） were employed. Field experiments with different N levels （0, 90, 180, 270, 360 kg N/hm 2 ） were designed. At the wintering stage, the dry matter weight, the nitrogen content and concentration of plants, leaf nitrate reductase activity （NRA） and seed yields were investigated. [Result] The shoot dry matter of ZY116 increased rapidly when N rate ranged from 0 to 180 kg/hm 2 , and it raised slightly when N rate ranged from 180 to 360 kg/hm 2 . The shoot dry matter of ZYZ12 were changed in a single peak curve; the peak of shoot dry matter appeared at 270 kg N/hm 2 . The N concentration and N content in shoot and root increased rapidly when the N rate changed from 90 to 180 kg/hm 2 . Moreover, the N concentration and N content root of in ZYZ12 were much higher than that of ZY116. Present study revealed that the changed trend of leaf nitrate reductase activities （NRA） were significantly increased at the N rate of 180 kg/hm 2 in ZY116 and ZYZ12 compared with the N rate of 90 kg/hm 2 in two years. [Conclusion] Optimal nitrogen application significantly increased the dry weights and N uptake at wintering stage as well as increasing the yield of winter oilseed rape.

Geometric Modeling of Rape (Brassica napus L.) during Seedling Stage

[Objective] The paper was to study the geometric modeling of rape（Brassica napus L.） during seedling stage.[Method] Based on the analysis and observation of morphological structure and growth process of rape during seedling stage,a characteristic parameters-based three-dimensional mathematical model of rape and its visible method was proposed.The individual control parameters were extracted according to the morphological structures of various organs of rape.Different sizes of leaf and petiole model were constructed by using cubic Bézier surface.The cylinder with different upper and lower bottom area was adopted as the main stem model.Finally,three-dimensional reconstruction of whole Rape plant during seedling stage was achieved through the operations of rotation,scaling and splicing.[Result] This method had certain controllability,which was also easy and convenient,and could quickly use to build the geometric model of rape during seedling stage.[Conclusion] The results provided reference for study on structural model of rape.

Preliminary investigation and detection based on loop-mediated isothermal amplification(LAMP)of phytoplasmas associated with diseases in B.napus L.

In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detection results pointed to the possible pathogen as phytoplasma.The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B.napus L.,including witches’broom,multi-stems,aggregate main inflorescences,and flat stems.The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61%in 2010 to 6.00%in 2021.Some phytoplasma infected plants died without any growing points.These studies would be helpful for detecting phytoplasmas diseases,selecting disease resistant germplasm and improving varieties with disease resistances in B.napus L.

Nitrogen Metabolising Enzymes and Photosynthetic Pigments as Influenced by Nitrogen Application in Oilseed Rape(Brassica napus L.)

Nitrogen(N)assimilation is a wide pathway in plants because of its fundamental importance for growth and development.The transport,assimilation and recycling of nitrogen is a highly complex and regulated process,as it is the mineral nutrient that is required in great abundance by the plants.Basic approach to enhance agriculture sustainability is dependent on exploration of the elite germplasm where new cultivars could perform better even under low N.To test the effect of nitrogen levels at 100 and 125 kg ha-1 on photosynthetic pigments and N assimilation in oilseed rape(B.napus L.)canola variety GSC-7 and hybrid Hyola PAC 401 were selected for comparison with new hybrid PGSH-52 in pipeline.N assimilating enzymes were assayed at vegetative,flowering and siliquing stages of crop growth to visualise the impact of N on the productivity.Nitrogen assimilating enzymes were highest at flowering stage and enhanced with increased N level.Nitrate reductase(NR)activity improved by 11.5%,nitrite reductase(NiR)by 24.2%,glutamine synthetase(GS)by 12.2%and glutamate synthase(GOGAT)by 35.2%over recommended N dose(N100)at flowering stage.Hyola PAC 401 registered maximum enzymatic activities trailed by GSC-7 at all the three stages of crop growth.Differences existed within the genotypes for photosynthetic pigments which varied with N levels.Chlorophyll a,chlorophyll b,total chlorophyll and carotenoids increased with nitrogen at 125 kg N ha^(-1)whereas chlorophyll a/b declined at three stages of crop growth.At flowering total chlorophyll and carotenoids were maximum and enhanced by 12.8%and 5%respectively with higher nitrogen level.

Brassinosteroids promote seed development and physiological maturity of oilseed rape (Brassica napus L.)

Long developmental stage and late harvest time of winter rapeseed (Brassica napus L.) have great negative effects on rice planting of rice-rapeseed farming system in China. Early maturity improvement of rapeseed is necessary. ‘Zhongshuang 11’, an elite winter rapeseed cultivar, was used in consecutive field experiments during 2010-2012. At initial flowering stage, plants were consecutively sprayed with 0.1 mg/L 2-4-Epibrassinolide(BR) for 3 d. Two hundred sampling pods from different plants were randomly collected to measure seed related indexes with a 4 d interval from 7 to 47 d after peak anthesis (DAPA).Seed color turned light brown at 31 or 35 DAPA after BR treatment, seed dry weight (DWT)was increased while seed moisture content (SMC) was decreased during seed development.DWT almost reached the maximum value when SMC was 33.20% at 31 DAPA in 2010-2011 and 35.29% at 35 DAPA in 2011-2012 growing season after BR treatment. Similarly,the maximum values of standard germination test (SGT), accelerated aging test (AAT)and cold test (CT) were observed at 31 or 35 DAPA after BR treatment respectively. The high yield and seed oil content appeared at 31 or 35 DAPA accompanied with rapid decrease in total non-structural carbohydrate (TNC) in stems and leaves. Our study indicated that BR application advanced maturity of winter rapeseed by 4 to 8 days.

不同甘蓝型油菜幼苗期对Ag^＋和Pb^2＋的耐性研究

采用室内培养方法,对9个甘蓝型油菜进行了重金属Ag+和Pb2+耐受梯度实验。结果表明,秦油7号、湘油15及2N43表现出较强的耐性。进一步实验证明甘蓝型油菜对重金属的耐受程度和MDA的含量呈现显著的负相关。此外,根中重金属的含量在耐受程度较强的秦油7号中的含量超过耐受程度较差的Jan-95。本研究可为今后筛选耐受重金属的油菜品种作出有益探索。

油菜优质新不育系黔油2AB选育及遗传研究

利用双低油菜品种油研2号的天然不育可遗传突变株,采取田间选择和品质筛选相结合、不育株后代连续兄妹交保持纯化的定向培育方法,育成甘蓝型油菜双低不育系——黔油2AB。经田间性状和遗传鉴定表明:其恢保关系有别于其它不育源,育性受两对细胞核显性基因(杂合型两型系)控制,属细胞核显性不育系;该不育系芥酸含量低于1%、硫甙含量低于30μmol/g,含油量达40%以上;其育性分明、不育性稳定而彻底;具有田间长势强、经济性状好,较抗(耐)油菜菌核病和配合力较强等优点,是开展优质杂交油菜品种选育的重要亲本材料。

应用^(10)B研究不同油菜品种花器官的硼素基因型差异

应用^(10)B固体径迹蚀刻技术,测定4个油菜品种花器官中的含硼量。结果表明:(1)缺硼时浠水白的花萼和花瓣的含硼量分别为5.8和5.9ppm,均极显著高于三个甘蓝型品种。3个甘蓝型品种的含硼量依次为:甘油4号>821>84004,差异均达极显著水平。(2)缺硼时浠水白的雄蕊和雌蕊含硼量最高,分别为10.4和10.3ppm。甘蓝型品种依次为甘油4号、中油821、84004。(3)不同品种缺硼时蕾的含硼量依次为浠水白、甘油4号、中油821、84004,除甘油4号与中油821之间的差异不显著外,其余品种间差异极显著。

体细胞组织培养用于甘蓝型油菜核三系保持系绵7MB-1亲本繁殖与F_1制种的研究

利用自育的核三系艋保系新材料绵7MB-1，可以将油菜绵7AB类型、S45AB类型等臆性核不育两用系的不育株率从50％提高到90％以上。针对该材料在自交繁殖过程中，发生部分育性回复的现象，采用临保系植株茎段体细胞组织培养技术，实现了绵7MB-1的批量繁殖，将繁殖的临保系苗用于网室亲本不育系繁制，可使不育系亲本不育株率达到91．7％-93．5％。每繁制公顷不育系亲本，可供7500—15000hm^2杂交F1的制种生产使用。用高不育率不育系代替原核不育两系进行制种，大大减少了用工，同时使制种产量显著提高，制种质量也更有保证，形成了完善的繁殖与制种体系。

双低油菜纯合两型系48AB的转育

首例以油菜杂合型不育株２０４Ａ（Ｍｓｍｓｒｆｒｆ）与恢复系８３２４８（ｍｓｍｓＲｆＲｆ）为材料，通过杂交、自交、兄妹交，成功地转育成纯合两型系４８ＡＢ（１ＭｓＭｓｒｆｒｆ：１ＭｓＭｓＲｆｒｆ）．该方法具有技术简便、见效快的特点，是目前想获得纯合两型系而只有杂合两型系（Ｍｓｍｓｒｆｒｆ×ｍｓｍｓｒｆｒｆ）的育种者可借鉴的一种快捷方法。

黔油12号双低杂交油菜制种技术研究 Ⅷ.母本隐性核不育两型系SAB-3的保优扩繁技术规程

通过隐性核不育两型系双低杂交油菜品种黔油12号多年的亲本繁育实践,根据其母本隐性核不育两型系SAB-3的特点,总结出其高产保优扩繁技术规程,以确保品种的制种质量。

油菜B-box型锌指蛋白基因BnSTO的克隆和表达

B-box型锌指蛋白参与植物多种生长发育和非生物逆境胁迫响应过程。笔者通过RT-PCR克隆得到甘蓝型油菜2个B-box型锌指蛋白基因Bn STO-1和Bn STO-2。Bn STO-1的ORF长度为723 bp,推测编码240个氨基酸,定位于A8染色体。Bn STO-2的ORF长度为726 bp,推测编码241个氨基酸,定位于C8染色体。2个基因均由3个外显子和2个内含子构成,在氨基酸水平上的相似性达到了96.68%。系统进化分析表明Bn STO与拟南芥At STO的亲缘关系较近,同源性较高。荧光实时定量PCR结果表明Bn STO在检测的各个组织中均有表达,其中叶和蕾中的表达量最高;高盐、干旱和高温能显著诱导Bn STO-1和Bn STO-2的表达,但出现峰值的时间不同,推测2个基因均可能参与了油菜的非生物逆境胁迫响应。

甘蓝型油菜三隐性核不育材料L7AB及临保系的选育

用从贵州省油料研究所选育的高不育株率材料中发现的异型不育株作母本,自育另一黄籽双低高不育株率材料2822B作父本杂交,其后代经多年测交、自交选育、鉴定和恢复系的筛选,已成功选育了稳定的双低三基因隐性核不育系、保持系和优良恢复系,从而实现了三系配套。

人工合成甘蓝型油菜游离小孢子培养及其植株再生研究初报

利用游离小孢子培养技术对5个人工合成甘蓝型油菜品系进行了离体小孢子培养。从大田非控温控光条件下生长的供体植株第一朵花开后3～12d，取长度2．5～3．5mm的花蕾分离小鬼子，在NLN13液体培养基中进行薄层培养诱导成胚状体，然后用无激素的MS培养基诱导植株再生。对不同的供试材料、取蕾时期和培养基蔗糖浓度对小孢子胚胎发生能力的影响作了初步研究。供试的5个材料中有4个对培养有反应，但不同材料的小孢子胚胎发生能力有较大的差异。小孢子取样的时期以主花序第一朵花开后6d左右为宜，培养基蔗糖浓度以13％的效果最好。

用SRAP标记分析中国甘蓝型油菜品种的遗传多样性和遗传基础

【目的】探讨中国甘蓝型油菜遗传多样性和遗传基础。【方法】采用SRAP（sequence-related amplifled polymorphiSill）标记对建国以来不同时期选育的130个品种进行分析。【结果】25个SRAP引物组合共扩增到509个谱带、123个多态性带；多态性带的比例为24％。每对引物组合的谱带数和多态性带数分别为20．4个和4．9个。在遗传距离为0．12处，将130个甘蓝型油菜品种（系）分为A、B、C、D4个类群，其中78．5％的品种归入C类。C类又可在遗传距离0．10处分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ5个亚群，又有58．5％的品种归入Ⅲ亚群。说明我国近60%的甘蓝型油菜品种遗传多样性较匮乏。遗传基础分析结果表明，20世纪80年代前育成的甘蓝型油菜品种的遗传基础最窄，80年代最宽，90年代略有下降。进入21世纪，品种间的遗传基础进一步下降。差异显著性测验结果表明，1991～2000年间与2000年以后育成的品种间的平均遗传距离差异不显著，80年代前育成品种与80年代育成的品种平均遗传距离间差异达到0．01显著水平，80年代与90年代育成的品种间遗传距离差异达到0．05的显著水平。我国育成的品种间的遗传距离与引进品种间的遗传距离差异达到0．01的极显著水平。【结论】SRAP标记是一种经济、有效和可靠的分子标记手段。