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.     

Nondestructive assessment of protein content in single seeds of rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy

The potential of near-infrared reflectance spectroscopy (NIRS) to detectwithin-plant differences for seed protein content was investigated. Fourhundred and fifty-one single seeds were scanned by NIRS using a specialadapter. After non-destructive NIRS scanning, the seeds were analysed forprotein content by the Dumas combustion method and a calibrationequation was developed. A validation set of 117 additional seeds fromthree individual plants from the cultivars Bristol, Lirajet and Maplus wasanalysed for protein content both by NIRS and combustion. The coefficientof determination between NIRS and combustion values in the validation setwas 0.94, with a standard error of performance (SEP) of 0.77% and aratio of the SEP to the standard deviation (SD) of the validation set of0.28. The coefficient of variation (CV) for seed protein content inindividual plants, as determined by the combustion method, was 11.7%for Bristol, 8.9% for Lirajet, and 9.5% for Maplus. The comparison ofsuch variation with the standard error (SE) of NIRS analysis, defined as thecombination of the SE of the combustion method and the SEP of NIRScalibration equation, revealed that the maximum explainable variance withinindividual plants that can be detected using NIRS analysis of proteincontent in single seeds was 0.86 for Bristol, 0.83 for Lirajet, and 0.85 forMaplus. These results demonstrated that NIRS is a powerful tool fornon-destructive assessment of within-plant variation for seed protein contentin rapeseed.     

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.     

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.     

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.     

Development of novel clubroot resistant rapeseed lines (Brassica napus L.) effective against Japanese field isolates by marker assisted selection

Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F₁ Chinese cabbage cultivars. We produced the resynthesized clubroot resistant Brassica napus harboring two resistant loci, Crr1 and Crr2, by interspecific crossing and developed resistant rapeseed lines for southern and northern regions by marker-assisted selection and backcrossing. We improved the DNA marker for erucic acid content to remove linkage drag between Crr1 and high erucic acid content and successfully selected lines with clubroot resistance and zero erucic acid for northern regions. A novel line, ‘Tohoku No. 106’, suitable for southern regions showed stable resistance against all six isolates and high performance in infested fields. We conclude that Crr1 and Crr2 are important genes for CR rapeseed breeding and marker-assisted selection is effective in improving clubroot resistance.     

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.     

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.     

Analysis of total glucosinolate content and individual glucosinolates in Brassica spp. by near-infrared reflectance spectroscopy

This study was conducted to test the applicability of near-infrared reflectance spectroscopy (NIRS) for estimating the total glucosinolate (GSL) content in samples of intact seed from a wide range of Brassica species, and to develop calibration equations to estimate simultaneously the percentage of individual GSLs. A total of 290 samples from 15 different Brassica species were scanned by NIRS and analysed for glucosinolate content by high-pressure liquid chromatography (HPLC). A calibration equation for total GSL content was developed using 270 samples of 14 species in a range between 6 and 193μmol/g seed, resulting in an r² of 0.99 in calibration and cross-validation, and 0.95 in independent validation with 20 samples of Brassica rapa, a species not represented in the calibration. Furthermore, calibration equations to estimate the relative amount (mol/mol) of progoitrin, sinigrin, and gluconapin were successfully developed (r² > 0.85 in cross-validation) and validated with samples from species not included in the calibration. It was also possible to discriminate between entries with high and low values of glucoiberin, 4-hydroxyglucobrassicin and glucoerucin.     

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。     

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。     

Introgression of genes into cultivated Brassica napus through resynthesis of B. napus via ovule culture and the accompanying change in fatty acid composition

Fifteen lines of Brassica napus were resynthesized via ovule culture through 24 interspecific crosses between four Brassica oleracea and three Brassica campestris accessions. The degree of success in the interspecific crosses was significantly influenced by maternal genotypes. The interspecific hybrid production rate (HPR) varied with combinations from 0 to 76.9%, with a mean HPR of 24.7% for the crosses with B. campestris as the female parent and 6.9% for the crosses with B. oleracea as female parent. Twenty‐four crosses between seven natural and six resynthesized B. napus gave, on average, 10.3 seeds per pod, and ranged from 1.2 to 22.0 seeds per pod, depending on genotypes of both parents. Resynthesized lines of B. napus showed high erucic acid content and variable content of linolenic acid, ranging from 3.4% to 9.9%. The fatty acid composition in hybrid seeds between natural and resynthesized B. napus was dominated by the embryo genotypes; an additive mode was shown for erucic acid and positive over‐dominance for linolenic acid content.     

国内外甘蓝型油菜种质SSR标记遗传多样性分析

用SSR分子标记对国内外48份甘蓝型油菜品种进行遗传多样性分析,结果表明：筛选出的45对引物共扩增出326个位点,多态位点281个,多态性比率为86.2%;平均每对引物扩增的条带数和多态性条带数分别为7.2和6.2。多态性信息含量（PIC）在0.374~0.856,平均为0.699。遗传相似系数在0.48~0.79之间,参试材料差异较大;以0.51为阈值将48份参试材料划分为冬性、半冬性和春性三大类群,三大类群间相对独立又有一定程度的渗透,说明材料之间存在不同程度的亲缘关系。主成分分析与聚类分析结果一致。说明SSR标记能够较全面地反应种质材料的遗传多样性,能够为种质的保存提供帮助,同时可以用来分析育种材料的遗传多样性,对育种工作有重要的参考意义。     

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。     

Inheritance of long chain fatty acid content in rapeseed (Brassica napus L.)

A large industrial oil market has recently developed for high erucic acid (>500 g kg⁻¹) rape (Brassica napus L.) cultivars. This research was conducted to: (i) determine genetic effects for five fatty acids, (ii) determine if maternal effects influence fatty acid content of progeny, and (iii) estimate correlations among fatty acid contents in hybrid progeny. Lines with very high erucic acid content and very low erucic acid content were used to develop eight generations to estimate additive, dominance, and epistatic effects for fatty acid content using Generation Means Analyses. Mean oleic, linoleic, linolenic, eicosenoic and erucic acid content differed among generations and additive genetic effects were important for control of all five fatty acids, contributing from 84% to 97% of the total sums of squares for each fatty acid. Epistasis was observed in the inheritance of eicosenoic acid. Maternal effects were not detected.     

Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts

A protocol for Agrobacterium tumefaciens‐mediated transformation of Brassica napus mesophyll protoplasts is described. A strain with a neomycin phosphotransferase (nptII) gene and a KCS gene under control of a napin promoter was used at co‐cultivation. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR of the nptII gene and NAP/KCS expression cassette, and Southern blot analysis. Seeds of the transformants showed a changed fatty acid profile: two transformants had a higher erucic acid level and differed significantly from that of B. napus. Genetic analysis of the progeny revealed that the kanamycin resistance introduced was inherited in a Mendelian fashion.     

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.     

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.     

Fatty Acid Composition of Oil from Erucic Acid-Free Summer Rape Seed (Brassica napus L.) in Relation to Nitrogen Nutrition and Raceme Position

A greenhouse study was conducted to determine the effect of nitrogen supply (30, 100 or 170 ppm N) and raceme position on the fatty acid composition of oil extracted from erucic acid-free summer rape seed ( Brassica napus cv. Callypso ). The seven fatty acids analyzed for include palmitic, palmitolcic, stearic, oleic, linoleic, linolemc, and eicosenoic acids; of which oleic (59.54–64.84 %) and palmitoleic (0.36–0.4 %) acids were the highest and lowest levels respectively. Generally, N nutrition influenced fatty acid pattern only to a little extent. Palmitic, palmitoleic and stearic acid levels were increased by 170 ppm N, depending on raceme position, but oleic and linolenic acids were unaffected. Similarly, 170 ppm N produced the highest fatty acid levels in seeds on the lower portions of racemes, with the exception of oleic acid. This was also true in the case of the upper portions of racemes, except that 30 ppm N produced the highest levels of oleic and linoleic acids in rape seeds. Under the optimum N supply level (i.e. 100 ppm N), position of raceme on the rape plant did not greatly influence the levels of different fatty acids in lipids.