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.     

利用DH和IF_2群体检测油菜籽粒油酸、亚油酸、亚麻酸含量QTL

以德国冬性甘蓝型油菜Express和中国半冬性甘蓝型油菜SWU07为亲本构建包含261个株系的DH群体和包含234个株系的IF2群体,检测不同年份条件下油菜籽粒油酸、亚油酸、亚麻酸含量相关的QTL。在DH群体4年环境下共检测出71个QTL,在IF2群体2年环境下共检测出4个QTL。去掉在不同年份和群体中置信区间相互重叠的QTL之后,共得到3个品质性状的51个QTL,其中有15个在2年以上环境中被检测到。这些QTL分别分布在13个连锁群上,其中与油酸含量相关的18个,分布于A01、A02、A04、A05、A07、A09、C01连锁群,揭示3.44%～13.97%的表型变异;与亚油酸相关的12个,分布于A02、A06、A09、C01、C02连锁群,揭示3.84%～19.51%的表型变异;与亚麻酸相关的21个,分布于A01、A02、A03、A04、A05、A08、A09、C01、C02、C03、C06连锁群,揭示2.86%～11.91%的表型变异。这些结果将为油菜脂肪酸品质改良提供更多遗传信息。     

Estimating the fatty acid composition of the oil in intact-seed rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The objective of this work was to evaluate the potential of near-infrared reflectance spectroscopy (NIRS) as a rapid method to estimate the fatty acid composition of the oil in intact-seed samples of rapeseed. A total of 549 samples (3 g intact seed) from selected mutant and breeding lines were scanned by NIRS, and 220 of them were selected and scanned again by using two different adapters, which reduced the sample size to 300 and 60 mg, respectively. Selected samples were analysed by gas liquid chromatography and calibration equations for individual fatty acids were developed. Calibrations for oleic, linoleic, linolenic, and erucic acid were highly accurate, with values of r² in cross validation from 0.95 to 0.98 (samples of 3 g), from 0.93 to 0.97 (300 mg), and from 0.84 to 0.96 (60 mg). Calibrations for palmitic and stearic acid were less accurate, with values of r² in cross validation always lower than 0.8, probably because of the narrow range available for these fatty acids. The accuracy of the calibration equations for eicosenoic acid was very low (r² = 0.69 in 3 g samples), although improved equations were developed (r² from 0.78 to 0.91) when the relationship between erucic and eicosenoic acid was taken into account. We conclude that NIRS is a powerful technique to estimate the fatty acid composition of the oil in rapeseed, provided that samples covering a wide range of fatty acid levels are available, with the advantage that such estimation is possible with few additional costs when NIRS is used for the determination of other seed quality traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estimation of seed weight, oil content and fatty acid composition in intact single seeds of rapeseed ( Brassica napus} L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) for the simultaneous analysis of seed weight, total oil content and its fatty acid composition in intact single seeds of rapeseed was studied. A calibration set of 530 single seeds was analysed by both NIRS and gas-liquid chromatography (GLC) and calibration equations for the major fatty acids were developed. External validation with a set of 75 seeds demonstrated a close relationship between NIRS and GLC data for oleic (r = 0.92) and erucic acid (r = 0.94), but not for linoleic (r = 0.75) and linolenic acid (r = 0.73). Calibration equations for seed weight and oil content were developed from a calibration set of 125 seeds. A gravimetric determination was used as reference method for oil content. External validation revealed a coefficient of correlation between NIRS and reference methods of 0.92 for both traits. The performance of the calibration equations for oleic and erucic acid was further studied by analysing two segregating F2 seed populations not represented in the calibration set. The results demonstrated that a reliable selection for both fatty acids in segregating populations can be made by using NIRS. We concluded that a reliable estimation of seed weight, oil content, oleic acid and erucic acid content in intact, single seeds of rapeseed is possible by using NIRS technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Impact of low linolenic acid content on seed yield of winter oilseed rape (Brassica napus L.)

An essential quality improvement of rapeseed oil can be obtained by reduction of its linolenic acid (C18:3) content from about 10% to less than 3% of the total fatty acids. Genotypes low in C18:3 have been developed by mutagenesis. The initial summer rapeseed mutant had been low yielding and highly susceptible to various diseases. It has been debated whether the low C18:3 character can be successfully combined with high seed yield for physiological reasons. Therefore, the low linolenic character of mutant M48 was transferred into high-yielding genotypes by repeated backcrossing to well-adapted low erucic acid, low glucosinolate (00-) winter rapeseed cultivars. Lines with low C18:3 content were selected from BC3 and BC4 generations and examined in 1990–95. Positive selection response for seed yield was shown to continue over the years. Presently, the best lines are yielding as well as the control cultivars being equivalent also in oil and glucosinolate contents. In order to test the effect of a low C18:3 content on seed yield, plants with low and with high C18:3 content, respectively, were selected from 16 segregating BC5-F₂ populations and bulked to form 32 F₃ populations. These ‘isogenic’ bulk populations were tested for field performance at four locations in 1995. The results show that C18:3 content of the seed oil is not associated with seed yield, oil content, beginning of flowering, plant height and disease resistance. Means of relative seed yield for the high and the low linolenic F₃ bulk populations were not significantly different with 88.0% and 86.9% of the control cultivars, respectively. There was a significant interaction between genotypes with high or low C18:3 content and location. This shows that under specific environmental conditions a low C18:3 content may be either favourable or unfavourable. The results indicate that the low C18:3 character of the original mutants per se does not cause a decrease in seed yield, oil content or general field performance.     

Selection on pollen for linolenic acid content in rapeseed, Brassica napus L.

In order to enhance the economic value of edible rapeseed oil, an improvement of quality is necessary. Mutagenesis of rapeseed resulted in a low linolenic acid content and a low ‘linolenic acid (CIS: 3) level to linoleic acid (CIS: 2) level’ ratio, that is, the linoleic desaturation ratio (LDR), in the seeds of the Canadian variety ‘Stellar’. As an early breeding marker for low linolenic acid content, the pollen fatty acid composition was determined on 80 doubled haploid plants derived from a single F₁ hybrid obtained from a cross between ‘Stellar’ and a high CIS: 3 variety ‘Drakkar’. Fatty acid analysis on seed and pollen showed that the low CIS: 3 and the low LDR traits from the ‘Stellar’ variety were expressed in pollen and in seeds, and that a very close correlation (r = 0.88) existed between seed and pollen for these two traits. The inheritance of these traits is controlled by two major genes with additive effects, both in seed and pollen. However, minor genes also appeared to be expressed in pollen and seed. These genes may allow the production of plants with lower CIS: 3 levels than that of the low linolenic acid content parent. The efficiency of this new tool for early screening in breeding programmes is discussed.     

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.     

Prospects for the Development of Rapeseed (B. napus L.) with Improved Linoleic and Linolenic Acid Content

The special aspects of the Western Australian rape-seed breeding programme for the improvement of C₁₈ fatty acids (FA) have been highlighted. Progress made through the use of ‘Oro’-mutant and IXLIN (interspecific X derived) as sources of genes fur improved C₁₈ FA is discussed. These two donor lines were crossed or intercrossed with high yielding, disease resistant B. napus lines (summer and winter type) or their early generation progenies from interspecific crosses with B. juncea or B. carinata. Tins provided suitable genetic diversity and favourable agronomic background for the introgression of target genes or gene system for improved C₁₈ fatty acids. Many of the polyenoic lines selected from these crosses have indicated scope for combining high linoleic and low linolenic acid levels, with maintenance of good growths and seed development in plants well adapted to the environment.     

SCAR and RAPD markers associated with 18-carbon fatty acids in rapeseed, Brassica napus

Breeding rapeseed for enhanced oil quality includes the development of varieties with low linolenic acid content. The breeder also aims to develop varieties with a high linoleic acid content because of its nutritional value. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers have been developed for linolenic acid content, but they are not best suited for a direct application in marker-assisted selection. The RFLP technique is too complex and time-consuming and RAPD markers lack codominance, precluding the distinction of homozygous from heterozygous individuals. In this report the conversion of a RAPD marker to a codominant sequence characterized amplified region (SCAR) marker named L1L9 is described. One of the alleles consisting of an 899 bp fragment (allele A), is associated with low linolenic acid content. The other allele consists of an 641 bp fragment (allele B) and is associated with high linolenic acid content. This marker explains approximately 25% of the genetic variation for this trait. Linkage analysis in the mapping population indicates that the SCAR marker probably tags an ω-3 desaturase gene in B. napus. Two RAPD markers were found to be associated with oleic/linoleic acid content. Markers M14-350 and I06-650 explained approximately 10% and 7% of the genetic variation for linoleic acid content, respectively. These two markers were found linked at 12.3cM in the segregating B. napus F₂ progeny used for mapping. All the markers reported in this paper should be useful in breeding programmes for developing high linoleic and low linolenic acid rapeseed varieties.     

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。     

Expression of a cDNA encoding palmitoyl-acyl carrier protein desaturase from cat's claw (Doxantha unguis-cati L.) in Arabidopsis thaliana and Brassica napus leads to accumulation of unusual unsaturated fatty acids and increased stearic acid content in the seed oil

A cDNA encoding palmitoyl‐acyl carrier protein (ACP)‐desaturase from cat's claw (Doxantha unguis‐cati L.) was expressed in Arabidopsis thaliana and Brassica napus L. with the goal of decreasing the saturated fatty acid (FA) content of the seed oil. In general, transformation of Arabidopsis resulted in a greater change in the FA composition of the seed oil than for B. napus. An increase in palmitoleic acid (16:1cisΔ⁹) was obtained in transgenic lines, suggesting that the 16:0‐ACP‐desaturase cDNA was expressed in the manner originally intended. Other effects on lipid metabolism, however, were observed in the seed of transgenic plants. In Arabidopsis, there was a large increase in the proportions of cis‐vaccenic acid (18:1cisΔ¹¹) and cis‐13‐eicosenoic acid (20:1cisΔ¹³), possibly generated through elongation of 16:1cisΔ⁹. Elongation of 18:1cisΔ¹¹ to 20:1cisΔ¹³, however, was not observed in B. napus indicating that certain aspects of lipid metabolism in the model plant, Arabidopsis, may not apply to B. napus. As well, the appearance of 18:1cisΔ¹¹ was accompanied by a decrease in the proportion of oleic acid (18:1cisΔ⁹). Although the introduced ACP‐desaturase resulted in synthesis of some unsaturated FAs, the overall saturated FA content was maintained at similar levels to the control or was enhanced. Increased levels of saturation were mainly associated with an increase in stearic acid, which unlike 16:0, is considered non‐atherogenic. The results suggest that a mechanism exists further downstream in oil biosynthesis to counteract the decrease in saturation brought about by the 16:0‐ACP‐desaturase action.     

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.     

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。     

Identification of molecular markers associated with oleic and linolenic acid in spring oilseed rape (Brassica napus)

The quality of the oil derived from oilseed rape is determined by its fatty acid composition. Breeding oilseed rape for enhanced oil quality includes the development of cultivars with high oleic and low linolenic acid. Random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) techniques were investigated for the development of molecular markers for genes controlling oleic and/or linolenic acid. Markers that were identified were converted to sequence characterized amplified region (SCAR) markers for use in breeding. Molecular markers associated with these two fatty acids were identified in a doubled haploid population derived from a cross between the oilseed rape lines TO99‐5318‐20, very high oleic (>79%) and very low linolenic acid (<2%) × DH12075, high oleic (68%) and higher linolenic acid (>7%). Eight RAPD markers were associated with oleic and linolenic acid contents. The RAPD marker UBC 2₈₃₀ accounted for 43% and 13% of the genetic variation for oleic and linolenic acid levels, respectively. The RAPD marker UBC 153₅₅₀ accounted for 19% of the genetic variation for linolenic acid. The UBC 2₈₃₀ fragment was converted to a SCAR marker. The markers identified in this study should be useful tools for the early generation selection of high oleic and low linolenic acid genotypes in oilseed rape breeding programmes.     

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.     

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。     

A mutant of Brassica napus with increased palmitic acid content

An induced mutant from European winter oilseed rape with increased palmitic acid content was phenotypically characterized and genetically analysed. The mutant showed a palmitic acid content of 9.2% compared with 4.5% in the parental cultivar. The oleic acid content decreased from 61.6% to 44.2%, whereas the linoleic and linolenic acid contents increased. The mutant plants grew poorly and their seed oil content was only 31.2% compared with 42.8% in the parental cultivar. The inheritance of the mutant was oligogenic and determined by at least four genes. In the F₂ generation, palmitic acid content was negatively correlated with oil content. This mutant may be useful to improve understanding of the genetic regulation of storage lipid synthesis, but has no immediate value for oilseed rape improvement.     

A comparison of traditional and haploid-derived breeding populations of oilseed rape (Brassica napus L.) for fatty acid composition of the seed oil

Summary Microspore embryogenesis technology allows plant breeders to efficiently generate homozygous micros-pore-derived breeding populations of oilseed rape (Brassica napus L.) without traditional generations of inbreeding. This study was conducted to compare the frequency distribution of microspore-derived population and single seed descent populations with respect to fatty acids of seed oil. Both microspore-derived populations and single seed descent populations were produced from each of three crosses made between selected parents containing contrasting amount of erucic, oleic, linoleic and linolenic acids. The fatty acid content of F₃ plants derived lines (F₅ seed) developed by single seed descent was compared to that of microspore-derived populations. The means, ranges and distribution pattern of seed fatty acid contents were similar in both populations for each fatty acid studied, although a few heterozygous lines were observed in the single seed descent populations. The results indicated that microspore-derived population form random, homozygous F₁ plant derived gametic arrays for all fatty acids evaluated. Selection for altered fatty acid composition in microspore-derived and single seed descent homozygous populations should be equally efficient, in the absence of linkage of traits investigated.     

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。     

甘蓝型油菜高油酸材料的遗传分析

为探索甘蓝型油菜突变自交系高油酸性状的遗传模式,以应用于油菜高油酸育种工作。本研究以低油酸亲本‘扬6026’（P1）和甘蓝型油菜高油酸突变自交系‘HO05’（P2）为材料,通过气相色谱单粒法分析对该组合的杂交后代F1、RF1、B1、RB1、B2、RB2、F2和RF2的高油酸性状的进行数量性状遗传分析。试验分析结果表明：亲本的油酸含量比较稳定,为加性-显性遗传模式,并且加性主效基因作用明显,是由于基因遗传差异作用的,高油酸性状由2对主效基因控制高油酸性状,狭义遗传力hN2为63.16%,另外的36.84%是由环境的变化引起的;低油酸亲本（P1）和高油酸亲本（P2）正反交试验表明,高油酸的遗传没有细胞质遗传效应。