Genetic analysis on oil content in rapeseed (Brassica napus L.)

High oil content is one of the most important characteristics of rapeseed (Brassica napus L.) breeding. In order to understand the genetic basis of seed oil content, a series of reciprocal crosses between rapeseed parents with high oil content (53110, 61616 and 6F313), medium-oil content (Zhongshuang 9) and low oil content (51070 and 93275) were conducted. It was found that the oil content of F₁ hybrid seeds in rapeseed was mainly controlled by the maternal genotype. The maternal effect value of oil content was estimated to be 0.86. The pollen parent had a xenia effect on oil content, estimated to be 0.14 which changed the mean value by 1.86 percent. The inheritance of oil content was studied in a set of 8 × 8 diallel crosses of different varieties. The results indicated that the inheritance of oil content could be explained by an additive-dominant-epistasis model. Although the dominant and additive effects played major roles and accounted for more than 70% of the total variance, there was also a small epistatic effect. The broad and narrow sense heritability of oil content was 83.88 and 36.94%, respectively. Based on the oil content differences between the reciprocal crosses in the same offspring generation (F₁ and F₂) in rapeseed, it could be concluded that there were significant cytoplasmic effects on oil content. In this study, two lines with significantly cytoplasmic effects, either positive or negative, were selected.     

Genetic and functional analysis of tocopherol biosynthesis pathway genes from rapeseed (Brassica napus L.)

Brassica napus L. is one of the most important oilseed crops in the world. Improving oil quality by increasing vitamin E content is a major target of rapeseed breeding. Vitamin E compounds, or tocopherols, are lipid‐soluble antioxidants that are essential nutrients for mammals. In this study, we report the characterization of rapeseed orthologs of the Arabidopsis tocopherol genes VTE1, VTE2 and PDS1. For each gene, at least two homologous sequences were found, and their expression was analysed in different tissues from three rapeseed genotypes. Genetic complementation experiments demonstrated that BnaX.VTE1.b and BnaA.VTE2.b homologs are capable of recovering seed γ‐tocopherol in Arabidopsis mutants. Overexpression of the genes in Arabidopsis Col‐0 shifted the seed tocopherol composition towards higher α‐tocopherol without increasing the total tocopherol content. To address the functionality of BnPDS1 sequences, we performed overexpression tests in Escherichia coli and enzymatic activity analyses. Overall, our results show that the identified sequences from rapeseed are functional orthologs of the Arabidopsis VTE genes and thus have considerable potential as molecular markers for selecting rapeseed with improved seed oil quality.     

甘蓝型黄籽油菜产量性状的遗传分析

利用加性－显性－母体的遗传模型，分析了7个不同遗传来源甘蓝型黄籽油菜品系所配制的完全双列杂交资料。结果表明，产量性状主要受加性和显性效应共同控制，环境对产量性状有一定的影响而母体效应影响很小。基因效应分析发现y4单株粒重有极显著的加性效应，y3×y4单株粒重有极显著的显性效应。     

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.     

Genetic and genotype × environment interaction effects analysis for erucic acid content in rapeseed (Brassica napus L.)

Genetic main effects and genotype × environment (GE) interaction effects for erucic acid content (EAC) in rapeseed(Brassica napus L.) were analyzed for two year data by using the genetic models and corresponding statistical approaches for quantitative traits of diploid plant seeds. Eight parents were included in adiallel mating design in two environments. It was found that the embryo, cytoplasmic and maternal main effects and their GE interaction effects could simultaneously affect the performance of EAC, especially for the cytoplasmic and maternal effects. Since the amount of genetic main effects from the expression of genes in different genetic systems accounted for about 64.1%of the total genetic effects, EAC of rapeseed was mainly controlled by genetic main effects. The total narrow-sense heritability for EAC was 83.6% with the general heritability being 51.9% and the interaction heritability being 31.7%. It was suggested by the predicted genetic effects, that Tower and Youcai 601 were better than other parents for reducing EAC in rapeseed quality breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potential of genomic selection in rapeseed (Brassica napus L.) breeding

Genomic selection employs genome‐wide marker data to predict genomic breeding values. In this study, a population consisting of 391 lines of elite winter oilseed rape derived from nine families was used to evaluate the prospects of genomic selection in rapeseed breeding. All lines have been phenotyped for six morphological, quality‐ and yield‐related traits and genotyped with genome‐wide SNP markers. We used ridge regression best linear unbiased prediction in combination with cross‐validation and obtained medium to high prediction accuracies for the studied traits. Our results illustrate that among‐family variance contributes to the prediction accuracy and can lead to an overestimation of the prospects of genomic selection within single segregating families. We also tested a scenario where estimation of effects was carried out without individuals from the family in which breeding values were predicted, which yielded lower but nevertheless attractive prediction accuracies. Taken together, our results suggest that genomic selection can be a valuable genomic approach for complex agronomic traits towards a knowledge‐based breeding in rapeseed.     

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 mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

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.     

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.     

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。     

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.     

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.     

Selection for fast germination in rapeseed (Brassica napus L. and B. Campestris L.)

Summary Eleven populations of Brassica napus L. and twelve populations of B. campestris L. were subjected to three cycles of selection for fast germination at 2°C and at 25°C. The seeds from the selected populations, and unselected control populations grown in the same environment as the selected populations, were examined for germination behaviour at 2°C and 25°C, and for growth behaviour at 10°C. The populations in both species responded differently in terms of germination behaviour to selection for fast germination. In most of the populations that did respond positively to selection, selection practised at 2°C was superior to selection at 25°C in improving percent germination at 2°C, and was as good as the selection at 25°C in improving germination rate at the higher temperature. Selection for fast germination had no effect on growth characteristics of B. napus and B. campestris populations grown at 10°C. Thus, selection for fast germination at one low temperature may lead to improvement in germination characteristics over a range of temperatures, but will not necessarily lead to improved growth performance of the selected populations.     

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.     

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。     

Prediction of heterosis using QTLs for yield traits in rapeseed (Brassica napus L.)

Three double low (erucic acid and glucosinolates) self-incompatible lines and 22 varieties from different origins were selected to produce 66 hybrids according to a NC II mating design. Field experiments for identification of hybrid performance and heterosis were conducted in two successive rapeseed growing seasons in Wuhan, China. After heterosis identifications, SI-1300 and Eagle were chosen to construct an F₂ segregating population. One hundred and eighty four F_2:3 lines were planted at Wuhan and Jingmen to test yield traits. F₂ plants and the 25 parents were analyzed using simultaneously AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat) markers. A total of 270 and 718 polymorphic loci were detected in the F₂ population and among the 25 parental lines, respectively. Of the 718 polymorphic loci, 178 were significantly correlated to yield traits. With the use of one-way ANOVA, 84 common QTLs were detected for 12 traits at two trial locations. Although the genetic distances based on general/specific heterozygosities and single-locus QTLs showed significant correlations with hybrid performance and heterosis for some yield traits, the determination coefficients were low. The results suggested that neither heterozygosities nor QTLs for yield traits were suitable to predict hybrid performance and heterosis in Brassica napus.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

甘蓝型黄籽油菜遗传分类及遗传多样性研究

黄籽油菜的遗传研究是实现油菜籽粒高含油量且饼粕低纤维素、低单宁、低色素等育种目标的重要途径。为了对不同来源的黄籽油菜进行遗传分类和多样性研究,对黄籽材料进行遗传等位性测验及聚类分析。结果表明,经过遗传测验,按照相互杂交F1、F2粒色是否分离将11份黄籽材料分为5组,‘油研10’、CZV55、E718、Arm为一组,为一般显性遗传;Q33、D615为一组,为不完全显性遗传;2006C、X2006、740C为一组;Polo为一组;HY15为一组,后3组为一般隐性遗传。但是,2006C、‘油研10’等的黄籽性状的遗传根据杂交测验亲本的不同而呈现显性或隐性的变化。依据SSR标记检测结果进行聚类分析,将35份黄籽材料分为甘蓝型油菜、白菜型、芥菜型三大组。甘蓝型油菜又可再分为8个亚组,各自代表材料有2006C和740C、油研系统、Q33;法国Ramiro、陕西的GQ4、源自加拿大的Arm、prof;源自波兰的Polo等。不同材料按照系谱关系、育种单位、地理来源聚集在一起。研究结果为黄籽油菜杂种优势利用奠定理论基础。     

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.