Synthesis of low linolenic acid rapeseed (Brassica napus L.) through protoplast fusion

Brassica napus somatic hybrids with low linolenic acid (18:3) content in their seed oil have been produced using fusion partners screened for low 18:3. One somatic hybrid contained only 3.5% 18:3, a level significantly below the mid-parental mean. The low level of 18:3 proved stable in the R₁ generation. Oil content of the lowest 18:3 selection increased from the mid-parental mean (29.3%) in the R₀ generation to 36% in a R₁ field bulk. The R₁ field population also showed some resistance to shattering. This revised version was published online in July 2006 with corrections to the Cover Date.     

原生质体非对称融合获得花椰菜与Ogu CMS甘蓝型油菜种间杂种

甘蓝型油菜Ogu CMS系幼叶原生质体经剂量分别为0.025 0,0.075 0,0.315 0,0.468 0和0.960 0 J/cm2的紫外线辐照后,与花椰菜下胚轴原生质体通过PEG法诱导融合,其中0.025 0~0.468 0 J/cm2获得再生植株68株,0.960 0 J/cm2辐射剂量下没有获得再生株。通过根尖染色体计数、流式细胞仪倍性鉴定、同工酶酶谱分析、RAPD及Ogu CMS线粒体基因orf138 STS(Sequence Tagged Site)标记引物扩增等方法对再生株进行了鉴定。结果表明,其中32株为杂种植株。同时也发现,紫外线辐照剂量的增加对供体染色体的丢失程度影响不大。     

Intertribal somatic hybridization between rapid cycling Brassica oleracea L. and Camelina sativa (L.) Crantz

Black spot, caused by Alternaria brassicae and A. brassicicola, is an important disease in all Brassica oleracea vegetables. Sufficient resistance to the pathogen is not found within the species, nor in species that readily cross to B. oleracea. Camelina sativa (false flax) is highly resistant to Alternaria spp. and has, in addition, other desirable characters for the improvement of B. oleracea. Protoplast fusions were performed between rapid cycling B. oleracea (tribe Brassiceae), which has good regenerability, and C. sativa (tribe Sisymbrieae) by polyethylene glycol (PEG) treatment. The B. oleracea fusion partner was inactivated by treatment with iodoacetate. C. sativa has poor regenerability; hence, no pretreatment was needed for this species. The protoplasts were cultured using a feeder layer system. A total of 2903 calli were isolated from the fusions. Fourteen of these initiated shoots, i.e., 0.5% regeneration frequency. Approximately 110 shoots were excised from 6 of these calli and transferred to rooting medium. Rooted plantlets grew vigorously in vitro and flowering was frequently observed. However, establishment of rooted shoots in soil was unsuccessful. Hybrid identity was confirmed by intermediate shoot morphology, RAPD marker analysis, and flow cytometric estimation of nuclear DNA content. This revised version was published online in August 2006 with corrections to the Cover Date.     

Breeding a genie and cytoplasmic double-MS line of rapeseed (Brassica napus L.)

All rapeseed lines with Polima male sterility (MS) that are applied in hybrid cultivars have the problem that their sterility varies with temperature. To overcome this problem, two double-MS lines with genie (GMS) as well as cytoplasmic male sterility (CMS) genes were synthesized through seven generations of breeding, based on a systematic study of changes in fertility of the genie and cytoplasmic male sterility lines. The fertility of the new sterility lines was determined by observation of the floral organs and by pollen staining. The results showed that, in the double-MS lines, half the plants maintained the features of the Polima CMS line, while the other half behaved like the GMS line. The GMS genes were correctly expressed in the Pol cytoplasm, but there was little interaction between the GMS and CMS genetic systems.     

甘蓝型油菜角果长度性状的全基因组关联分析

角果长度是油菜重要的农艺性状,适度增加角果长度有利于扩大角果库容量,增加光合面积,提高油菜的籽粒产量。本研究利用Illumina60KSNP芯片对496份具有代表性的油菜资源进行基因型分析,考察群体在4个环境中的角果长度表型,利用MLM和GLM模型进行全基因组关联分析。结果表明, MLM模型检测到7个位点,联合解释25.01%的表型变异; GLM模型检测到25个位点,联合解释41.77%的表型变异。合并共同位点后得到27个位点,其中7个与前人报道的QTL重叠,其余20个是新鉴定的位点。效应最大的位点Bn-A09-p29991443位于A09染色体,在MLM和GLM模型中分别解释13.89%和12.86%的表型变异,携带其优异等位基因的材料平均角果长度增加0.89cm。同时,在该位点附近找到已克隆的油菜角果长度基因ARF18和BnaA9.CYP78A9。此外,在5个位点附近发现拟南芥已知角果长度基因GID1b、FUL、EOD3、DOF4.4和GA20ox1的同源拷贝。本研究结果有助于解析角果长度的遗传基础,为研究角果长度的调控机理,指导角果长度的遗传改良打下基础。     

Molecular characterization of novel resynthesized rapeseed (Brassica napus) lines and analysis of their genetic diversity in comparison with spring rapeseed cultivars*

Resynthesized (RS) rapeseed generated from interspecific hybridization between suitable forms of Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and B. oleracea L. (CC, 2n = 18) represents a potentially important resource to expand genetic diversity in the narrow gene pool of oilseed rape (B. napus L., AACC, 2n = 38). In this study 165 RS rapeseed lines originating from crosses between an Indian Yellow Sarson (B. rapa ssp. trilocularis) and five different cauliflower (B. oleracea convar. botrytis) cultivars were studied using amplified fragment length polymorphism (AFLP) markers and their genetic diversity was compared in relationship to an assortment of 40 diverse spring oilseed and fodder rape varieties. Using three AFLP primer combinations, a total of 467 polymorphic bands were scored. Cluster analysis allowed differentiation among the different RS lines, which, as expected, were genetically highly divergent from the cultivars. The genetic diversity of the material is discussed in relation to its morphological variability with a view to the implementation of RS lines in oilseed rape breeding.     

白菜型油菜黄籽沙逊和芥蓝种间杂交合成甘蓝型油菜研究

人工合成甘蓝型油菜是拓宽油菜遗传育种种质资源的有效途径,也是研究芸薹属物种起源与演化的重要手段之一。为了把黄籽沙逊的黄籽性状同芥蓝的优良特性相结合,本研究利用白菜型油菜黄籽沙逊(Brassica. campestris var. Yellow Sarson Pain, 2n=AA=20)与芥蓝(Brassica albograbra, 2n=CC=18)开展了芸薹属种间杂交研究,在MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+7%蔗糖+活性炭1.0 mg/L的培养基上,通过胚珠培养从授粉7 d的140个胚珠中得到6个远缘杂交胚发育而成的植株,其中1个自然加倍成为双二倍体,随后应用形态学、细胞学和分子标记的方法对双二倍体种间杂种植株进行了鉴定,结果表明杂种植株生长势较强,形态属于中间类型;根尖染色体压片观察显示杂种植株具有父母本的染色体数量之和;SSR分子标记分析表明,杂种植株包含了双亲的遗传信息,由此证明了所获杂种的真实性,这为最后自交分离获得黄籽甘蓝型油菜提供了科学依据。     

甘蓝型油菜千粒重全基因组关联分析

千粒重是油菜产量构成的重要因素之一。本研究利用高通量SNP芯片对496份具有代表性的油菜种质资源进行基因型分析,考察群体在3个环境(14NJ、15TZ、16TZ)中的千粒重表型,利用混合线性模型(mixed linear model,MLM)和一般线性模型(general linear model,GLM)进行全基因组关联分析。结果表明,本群体在3个环境中千粒重的广义遗传力为63.12%。MLM模型检测到6个显著位点,解释28.92%的表型变异;GLM模型检测到61个显著位点,解释47.08%的表型变异。合并共同位点后得到62个显著位点,联合解释47.31%的表型变异。这些位点分布在基因组所有染色体上,在A07、A03和C06染色体上分别检测到数目最多的9、8和7个位点。其中效应最大的位点Bn-scaff_17526_1-p1066214位于C09染色体,在MLM和GLM模型中表型贡献值分别为5.55%和15.26%。21个位点与前人报道的QTL重叠,其中8个位点得到至少2个群体的验证。其余41个位点为新鉴定的位点,其中多个位点效应高且在多环境中被检测到,如位点Bn-A03-p560769、Bn-scaff_15743_1-p599416和Bn-scaff_15743_1-p590955等。在11个位点附近找到DGAT、EOD3、AGL61、WRI1、DA2、RAV1等拟南芥已报道千粒重基因的同源基因。本研究结果有助于解析甘蓝型油菜千粒重的遗传基础,为研究千粒重的调控机制、指导千粒重的遗传改良奠定基础。     

Development of self-incompatible Brassica napus: (I) introgression of S-alleles from Brassica oleracea through interspecific hybridization

Cultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.     

油菜不同产量类型品种氮素吸收与利用特性研究

摘要: 【研究目的】探讨甘蓝型油菜不同产量类型品种氮素吸收与利用特性。【方法】在不同土壤肥力条件下以甘蓝型油菜品种（2006-2007年度73个、2007-2008年度98个）为材料,成熟期测定各器官干物重、氮素含量,采用组内最小平方和动态聚类方法对供试品种产量进行聚类。研究不同产量类型品种氮素积累与分配差异。【结果】结果表明：供试品种间产量差异很大,类型间差异显著。随着产量增加,氮素吸收总量、氮素籽粒生产效率增加,籽粒氮素积累量增加,茎枝、果壳氮素分配比例下降,籽粒氮素比例增加。土壤肥力高,植株吸氮总量增加,氮素籽粒生产效率降低。【结论】增加氮素吸收总量,促进氮素向籽粒中输送,使得高产和高氮素利用效率统一。     

Genetic variation in phytosterol content of winter rapeseed (Brassica napus L.) and development of NIRS calibration equations

Rapeseed oil is one of the richest natural sources of phytosterols, known to reduce the LDL-cholesterol levels, one of the major cardiovascular disease risk factors. Increasing the phytosterol content in rapeseed could give an added value to the oil and derived products. Our objective was to analyse the genetic variation of phytosterol content in modern winter rapeseed cultivars and resynthesized lines following field experiments and to develop a near-infrared reflectance spectroscopic (NIRS) calibration for high throughput estimation of phytosterol content. Phytosterol content as analysed by gas–liquid chromatography ranged from 3565 to 4800 mg/kg seed for modern cultivars and from 2079 to 4329 mg/kg seed for resynthesized lines. The NIRS calibration showed a high fraction of explained variance in cross-validation of 0.81 for total phytosterol content and the standard error of cross-validation was 241 mg/kg. The results show that the cultivars contain already high phytosterol contents. The NIRS calibrations developed for total phytosterol content should be useful for germplasm screening and in breeding programmes aimed at increasing the phytosterol content in rapeseed.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

Genetic variation of yellow-seeded rapeseed lines (Brassica napus L.) from different genetic sources

In order to breed yellow-seeded rapeseed, 16 yellow-seeded lines of Brassica napus L. derived from eight genetic sources were used. The genetic variation of the seedcoat ratio, the cellulose content of the seedcoat, the oil content of the seedcoat and of the embryo, and also the correlations between these characters of the yellow- and brown-seeded plants from the same line, were analysed by variance analysis and path analysis. The results show that the seedcoat ratio and cellulose content of brown seeds are 4.2% and 17.74%, respectively, higher than that of yellow seeds and the oil content of the seedcoat of brown seeds is 3% lower than that of the yellow seeds, these differences all being highly significant. However, the differences between yellow and brown seeds in 1000-seed weight and oil content of the embryo were very small. Both characters are determined mainly by the genetic background and not by seed colour or seedcoat thickness. The correlation analysis revealed that the seedcoat thickness has a highly significant positive correlation with the cellulose content of the seedcoat and is highly significantly negatively correlated with the seedcoat oil content and the 1000-seed weight. The oil content of the embryo alone has a highly significant negative correlation with 1000-seed weight. In yellow seeds, the seedcoat thickness has a large and directly positive effect on the oil content of the embryo whereas the 1000-seed weight has a negative one; the opposite was found in brown seeds. Selection objectives in breeding yellow seeds in Brassica napus are also discussed.     

甘蓝型油菜每角粒数的全基因组关联分析

每角粒数是油菜重要的产量构成因子,增加每角粒数有助于提高油菜的籽粒产量。利用Illumina 60K SNP芯片对496份具有代表性的油菜资源进行基因型分析,考察该群体在2个环境中的每角粒数,利用MLM和GLM模型进行全基因组关联分析。结果表明,本群体在2个环境中每角粒数的广义遗传力为57.7%。利用MLM和GLM模型分别检测到9个和20个位点,所有MLM位点均得到GLM结果的验证。6个位点与前人定位的QTL重叠,其中2个位点得到2次验证,其余14个是新位点。在7个位点附近找到了候选基因,其中在C09染色体的位点Bn-scaff155761-p74980附近找到已克隆的油菜每角粒数基因BnaC9.SMG7b,在其余6个位点附近找到GRDP1、SPATULA、HVA22D、DA2等已知的拟南芥每角粒数基因的同源基因。本研究结果有助于解析油菜每角粒数的遗传基础及其调控机制,为每角粒数的遗传改良奠定了基础。     

Resynthesis of Brassica napus L. for self-incompatibility: self-incompatibility reaction, inheritance and breeding potential

Self-incompatibility (SI) in Brassica has been considered as a pollination control mechanism for commercial hybrid seed production, and so far has been extensively used in vegetable types of Brassicas. Oilseed rape Brassica napus (AACC) is naturally self-compatible in contrast to its parental species that are generally self-incompatible. Introduction of S-alleles from its parental species into oilseed rape is therefore needed to use this pollination control mechanism in commercial hybrid seed production. Self-incompatible lines of B. napus , carrying SI alleles in both A and C genomes, were resynthesized from self-incompatible B. oleracea var. italica (CC) cv.'Green Duke' and self-incompatible B. rapa ssp. oleifera (AA) cv. 'Horizon', 'Colt' and 'AC Parkland'. All resynthesized B. napus lines exhibited strong dominant SI phenotype. Reciprocal cross-compatibility was found between some of these self-incompatible lines. The inheritance of S-alleles in these resynthesized B. napus was digenic confirming that each of the parental genomes contributed one S-locus in the resynthesized B. napus lines. However, the presence of two S-loci in the two genomes was found not to be essential for imparting a strong SI phenotype. Possible use of these dominant self-incompatible resynthesized B. napus lines in hybrid breeding is discussed.     

Genetic variation for sinapate ester content in winter rapeseed (Brassica napus L.) and development of NIRS calibration equations

Increasing the meal and protein quality of winter rapeseed (Brassica napus L.) for food and feed purposes is gaining importance in rapeseed breeding programmes. Rapeseed meal has a high content of phenolic acid esters, mainly sinapate esters, which have been shown to cause a dark colour and a bitter taste in rapeseed meal and derived protein products. The aim of the present study was to analyse the genetic variation for individual and total sinapate ester content, to develop Near Infrared Reflectance Spectroscopic (NIRS) calibrations, and to identify genotypes with a low sinapate ester content after testing in the field. The following sinapate esters were analysed by HPLC: sinapoylcholine (sinapine), sinapoylglucose, and a minor group of ‘other sinapate esters’ which includes free sinapate. A genotypically diverse set of seed samples of winter oilseed rape (old and new cultivars, breeding lines, resynthesized rapeseed) from different years and locations was collected, their NIRS spectra recorded and the samples were further analysed by HPLC. The complete NIRS calibration seed sample set (n = 575) showed a large variation in total sinapate ester content, ranging from 3.2 to 12.7 mg sinapate equivalents per g seeds. The NIRS calibration equations showed high fractions of explained variances in cross validation () ranging from 0.75 (other sinapate esters) to 0.85 (sinapoylglucose). The standard errors of cross validation (SECV) ranged from 0.38 (other sinapate esters) to 0.70 mg/g seed (total sinapate esters). In validation and in independent validations the predicted results were not always acceptable, indicating that the NIRS calibrations need to be extended by analysing samples from new populations. Following replicated field experiments, a doubled haploid line obtained from the old Dutch cultivar Mansholts’ Hamburger Raps, and related DH lines from the cross DH Mansholts’ × Express were confirmed to have a 30–40% lower sinapate ester content compared to check cultivars.     

New mutants of winter rapeseed (Brassica napus L.) with changed fatty acid composition

Seeds of the winter oilseed rape (Brassica napus L.) line PN 3756/93 were treated with ethyl methanesulphonate to induce mutations in the fatty acid biosynthetic pathway. The seed mutagenic treatment was repeated in the M₂ generation. After treatments, individual seed and plant selections were made for changes in fatty acid composition during several generations of inbreeding. Self‐pollinated plants with changed fatty acid compositions were inbred to obtain genetically homozygous and stable mutant lines. Two mutants, M‐10453 and M‐10464, with increased levels of oleic acid (approximately 76%) and reduced linoleic and linolenic acid contents (8.5% and 7.5%, respectively) were selected. Gene or genes controlling desaturation of oleic acid were probably mutated in these plants. The third mutant, M‐681 had a very low linolenic acid content (approximately 2.6%) and increased linoleic acid content (approximately 26%). This would suggest the occurrence of mutations in genes controlling linoleic acid desaturation. The results of selection work during several generations showed that the environment had substantial influence on the composition of seed oil. This made the search for mutants with modify fatty acid compositions difficult. The induced mutants are not directly usable as new varieties, but can be used as parents in crosses for the development of high quality rapeseed varieties.     

Effects of Waterlogging Followed by a Salinity Peak on Rapeseed (Brassica napus L.)

Due mainly to alterations in plant metabolism, lack of oxygen and excess salts are disturbances that affect crop yields. In different parts of the world crops are subjected t o those disttirbances, simultaneously or successively. Our objective was to determine the effects of a winter waterlogging followed by a spring salt peak on rapeseed yield, A pot experiment, combining waterlogging and salinization was carried out. The waterlogging duration was: 0 (control), 3, 7 and 14 days and the sahnity treatments were peaks of Electrical Conductivity of 5 and 8 dSm⁻¹ and the control. The yield started decreasingfrotn 3 days during waterlogging, mainly due to the lower number of seeds per plant. The salt peak from 5 dSm⁻¹ affected the yield only in plants which had suffered a waterlogging lower than 7 days, showing interaction between salinity and waterlogging, Only salinity reduced oil content. The saline peak affected the K, Ca and Na concentration in plant tissues, but the effect of salinity on rapeseed could be more related to soil water potential than specific ion toxicities or imbalance.     

人工合成甘蓝型油菜抗旱性及DNA甲基化水平分析

甘蓝型油菜是具有重要经济价值的多倍体物种,是优质食用植物油和饲料蛋白质的重要来源之一。但是其驯化历史较短,遗传背景狭窄,且在整个生命周期中都对干旱胁迫敏感,因此培育高产耐旱品种是甘蓝型油菜的重要育种目标之一。本文用15%PEG-6000模拟干旱胁迫,对人工合成甘蓝型油菜不同世代(S1～S4)及其二倍体亲本进行不同时间的胁迫处理,并结合表型观察,以及叶片中丙二醛(MDA)、可溶性蛋白含量、过氧化物酶(POD)、超氧化物歧化酶(SOD)等生理指标的测定,初步了解上述材料的抗旱性差异。结合表型观察和叶片中相对含水量分析,发现人工合成甘蓝型油菜S1～S4及其亲本的抗旱性表现为甘蓝 Bn-S3 Bn-S4 Bn-S1 Bn-S2白菜型油菜。干旱胁迫后Bn-S3、Bn-S4的POD及SOD活性较高,MDA含量较低,表明Bn-S3和Bn-S4能更加有效地清除活性氧(ROS),对过氧化损伤的防御能力更强。通过HPLC分析发现所有材料的甲基化水平在胁迫12 h时最高,其中亲本白菜型油菜Br的甲基化水平最高, Bn-S1和Bn-S4介于两亲本之间,而Bn-S2和Bn-S3低于两亲本。甲基化敏感多态性分析也显示人工合成甘蓝型油菜在干旱胁迫后,甲基化和去甲基化水平均发生了明显的变化,表明植物的甲基化变化可能有利于提高其抗旱能力。