Homoeologous non-reciprocal translocation explains a major QTL for seed lignin content in oilseed rape (Brassica napus L.).

KEY MESSAGE: A homoeologous non-reciprocal translocation was identified in the major QTL for seed lignin content in the low lignin line SGDH14. The lignin biosynthetic gene PAL4 was deleted. Oilseed rape is a major oil crop and a valuable protein source for animal and human nutrition. Lignin is a non-digestible, major component of the seed coat with negative effect on sensory quality, bioavailability and usage of oilseed rape's protein. Hence, seed lignin reduction is of economic and nutritional importance. In this study, the major QTL for reduced lignin content found on chromosome C05 in the DH population SGDH14 x Express 617 was further examined. SGDH14 had lower seed lignin content than Express 617. Harvested seeds from a F2 population of the same cross were additionally field tested and used for seed quality analysis. The F2 population showed a bimodal distribution for seed lignin content. F2 plants with low lignin content had thinner seed coats compared to high lignin lines. Both groups showed a dark seed colour with a slightly lighter colour in the low lignin group indicating that a low lignin content is not necessarily associated with yellow seed colour. Mapping of genomic long-reads from SGDH14 against the Express 617 genome assembly revealed a homoeologous non-reciprocal translocation (HNRT) in the confidence interval of the major QTL for lignin content. A homologous A05 region is duplicated and replaced the C05 region in SGDH14. As consequence several genes located in the C05 region were lost in SGDH14. Thus, a HNRT was identified in the major QTL region for reduced lignin content in the low lignin line SGDH14. The most promising candidate gene related to lignin biosynthesis on C05, PAL4, was deleted.

Agronomic and Metabolomic Side-Effects of a Divergent Selection for Indol-3-Ylmethylglucosinolate Content in Kale (Brassica oleracea var. acephala).

Brassica oleracea var. acephala (kale) is a cruciferous vegetable widely cultivated for its leaves and flower buds in Europe and a food of global interest as a "superfood". Brassica crops accumulate phytochemicals called glucosinolates (GSLs) which play an important role in plant defense against biotic stresses. Studies carried out to date suggest that GSLs may have a role in the adaptation of plants to different environments, but direct evidence is lacking. We grew two kale populations divergently selected for high and low indol-3-ylmethylGSL (IM) content (H-IM and L-IM, respectively) in different environments and analyzed agronomic parameters, GSL profiles and metabolomic profile. We found a significant increase in fresh and dry foliar weight in H-IM kale populations compared to L-IM in addition to a greater accumulation of total GSLs, indole GSLs and, specifically, IM and 1-methoxyindol-3-ylmethylGSL (1MeOIM). Metabolomic analysis revealed a significant different concentration of 44 metabolites in H-IM kale populations compared to L-IM. According to tentative peak identification from MS interpretation, 80% were phenolics, including flavonoids (kaempferol, quercetin and anthocyanin derivates, including acyl flavonoids), chlorogenic acids (esters of hydroxycinnamic acids and quinic acid), hydroxycinnamic acids (ferulic acid and p-coumaric acid) and coumarins. H-IM kale populations could be more tolerant to diverse environmental conditions, possibly due to GSLs and the associated metabolites with predicted antioxidant potential.

Mapping QTL controlling agronomic traits in a doubled haploid population of winter oilseed rape (Brassica napus L.).

Identification of superior alleles for agronomic traits in genetic resources of oilseed rape (Brassica napus L.) would be useful for improving the performance of locally adapted cultivars in Iran. The objective of the present work was to analyse the genetic variation and inheritance of important agronomic traits in a doubled haploid population derived from a cross between two German oilseed rape cultivars, Sansibar and Oase. Field experiments were performed in 2016-2017 with 200 doubled haploid lines and the parental genotypes applying an alpha-lattice design with two replicates. Phenological traits were recorded during the cultivation period and at maturity, seed yield, yield components and seed quality traits were determined. Significant genetic variation was found in most of the traits and heritabilities ranged from medium (48.5%) for days to end of flowering to high (92.6%) for oil content. A molecular marker linkage map was used to map 36 QTL for different traits on 17 linkage groups. Between three and four QTL were identified for each seed yield, seed weight, oil and protein content. Some of the plant material and positive QTL alleles identified for agronomic traits may be useful for improving those characters in locally adapted cultivars in Iran.

A major QTL on chromosome C05 significantly reduces acid detergent lignin (ADL) content and increases seed oil and protein content in oilseed rape (Brassica napus L.).

KEY MESSAGE: A reduction in acid detergent lignin content in oilseed rape resulted in an increase in seed oil and protein content. Worldwide increasing demand for vegetable oil and protein requires continuous breeding efforts to enhance the yield of oil and protein crop species. The oil-extracted meal of oilseed rape is currently mainly used for feeding livestock, but efforts are undertaken to use the oilseed rape protein in food production. One limiting factor is the high lignin content of black-seeded oilseed rape that negatively affects digestibility and sensory quality of food products compared to soybean. Breeding attempts to develop yellow seeded oilseed rape with reduced lignin content have not yet resulted in competitive cultivars. The objective of this work was to investigate the inheritance of seed quality in a DH population derived from the cross of the high oil lines SGDH14 and cv. Express. The DH population of 139 lines was tested in field experiments in 14 environments in north-west Europe. Seeds harvested from open pollinated plants were used for extensive seed quality analysis. A molecular marker map based on the Illumina Infinium 60 K Brassica SNP chip was used to map QTL. Amongst others, one major QTL for acid detergent lignin content, explaining 81% of the phenotypic variance, was identified on chromosome C05. Lines with reduced lignin content nevertheless did not show a yellowish appearance, but showed a reduced seed hull content. The position of the QTL co-located with QTL for oil and protein content of the defatted meal with opposite additive effects, suggesting that the reduction in lignin content resulted in an increase in oil and protein content.

Effects of intensive and alternative production systems on the technological and quality parameters of rapeseed seed (Brassica napus L. 'Siska').

BACKGROUND: Organic production is widely assumed to affect quality and nutritional parameters of arable crops. A field experiment was started in 2009 following integrated (INT), organic (ORG) and biodynamic (BD) production standards for wheat, maize and rapeseed production. With resort to standard analytical procedures and chemometrics, compositional parameters of Brassica napus L. 'Siska' seed (water, protein, oil and glucosinolate content) by non-destructive near-infrared spectroscopy (NIRS), and fatty acid composition determined by gas chromatography, were assessed. RESULTS: The BD and ORG production systems positively influenced oleic fatty acid and oil content in comparison to INT, which had higher levels of protein and water content (19.8% and 4.7%, respectively), as well as linolenic (3.8%), gadoleic (12.6%) and hexadecadienoic (15.5%) fatty acids. Increases in the listed parameters were linked to mineral fertilizer application in the INT production system. With principal component analysis (PCA) the INT samples were clearly differentiated. The PCA results were influenced by differences in linolenic, gadoleic and behenic fatty acid levels as well as protein and water content. CONCLUSION: Because quality parameters in rapeseed are increased in alternative production systems, production with an understanding of the soil processes and yield formation presents an opportunity for ORG and BD farmers. © 2016 Society of Chemical Industry.

Genetic variation and inheritance of phytosterol and oil content in a doubled haploid population derived from the winter oilseed rape Sansibar × Oase cross.

KEY MESSAGE: Identification of QTL for phytosterol content, oil content, fatty acids content, protein content of defatted meal, and seed weight by multiple interval mapping in a Brassica napus DH population. Phytosterols are minor seed constituents in oilseed rape which have recently drawn wide-interest from the food and nutrition industry due to their health benefit in lowering LDL cholesterol in humans. To understand the genetic basis of phytosterol content and its relationship with other seed quality traits in oilseed rape, QTL mapping was performed in a segregating DH population derived from the cross of two winter oilseed rape varieties, Sansibar and Oase, termed SODH population. Both parental lines are of canola quality which differ in phytosterol and oil content in seed. A genetic map was constructed for SODH population based on a total of 1638 markers organized in 23 linkage groups and covering a map length of 2350 cM with a mean marker interval of 2.0 cM. The SODH population and the parental lines were cultivated at six environments in Europe and were phenotyped for phytosterol content, oil content, fatty acids content, protein content of the defatted meal, and seed weight. Multiple interval mapping identified between one and six QTL for nine phytosterol traits, between two and six QTL for four fatty acids, five QTL for oil content, four QTL for protein content of defatted meal, and three QTL for seed weight. Colocalizations of QTL for different traits were more frequently observed than individual isolated QTL. Major QTL (R (2) ≥ 25%) were all located in the A genome, and the possible candidate genes were investigated by physical localization of the QTL to the reference genome sequence of Brassica rapa.

Glucosinolate profile and distribution among plant tissues and phenological stages of field-grown horseradish.

Profile and distribution of glucosinolates (GLS) were detected in plant tissues of horseradish at different developmental stages: beginning of vegetative re-growth, flowering and silique formation. The GLS profile varied widely in the different tissues: we identified 17 GLS in roots and sprouts, one of which was not previously characterized in horseradish, i.e. the 2(S)-hydroxy-2-phenylethyl-GLS (glucobarbarin) and/or 2(R)-hydroxy-2-phenylethyl-GLS (epiglucobarbarin), 11 already found in the roots, including the putative 2-methylsulfonyl-oxo-ethyl-GLS, and 5 previously recognized only in the sprouts. Fifteen of those GLS were also identified in young and cauline leaves, 12 in the mature leaves and 13 in the inflorescences. No difference in GLS profile was observed in plant among the phenological stages. Differences in concentrations of GLS, quantified as desulfated, were found in plant. At the beginning of vegetative re-growth, sprouts while showing the same profile of the roots were much richer in GLS having the highest total GLS concentrations (117.5 and 7.7µmolg(-1) dry weight in sprouts and roots, respectively). During flowering and silique forming stages, the roots still maintained lower amount of total GLS (7.4µmolg(-1) of dry weight, on average) with respect to the epigeous tissues, in which mature and young leaves showed the highest total concentrations (70.5 and 73.8µmolg(-1) of dry weight on average, respectively). Regardless of the phenological stages, the aliphatic GLS were always predominant in all tissues (95%) followed by indolic (2.6%) and benzenic (2.4%) GLS. Sinigrin contributed more than 90% of the total GLS concentration. Aliphatic GLS concentrations were much higher in the epigeous tissues, particularly in the mature and young leaves, while benzenic and indolic GLS concentrations were higher in the roots. Through the phenological stages, GLS concentration increased in young and mature leaves and decreased in cauline leaves and inflorescences, while it remained constant over time in roots.

Mapping of QTL for the seed storage proteins cruciferin and napin in a winter oilseed rape doubled haploid population and their inheritance in relation to other seed traits.

KEY MESSAGE: Cruciferin (cru) and napin (nap) were negatively correlated and the cru/nap ratio was closely negative correlated with glucosinolate content indicating a link between the two biosynthetic pathways. Canola-type oilseed rape (Brassica napus L.) is an economically important oilseed crop in temperate zones. Apart from the oil, the canola protein shows potential as a value-added food and nutraceutical ingredient. The two major storage protein groups occurring in oilseed rape are the 2 S napins and 12 S cruciferins. The aim of the present study was to analyse the genetic variation and the inheritance of napin and cruciferin content of the seed protein in the winter oilseed rape doubled haploid population Express 617 × R53 and to determine correlations to other seed traits. Seed samples were obtained from field experiments performed in 2 years at two locations with two replicates in Germany. A previously developed molecular marker map of the DH population was used to map quantitative trait loci (QTL) of the relevant traits. The results indicated highly significant effects of the year and the genotype on napin and cruciferin content as well as on the ratio of cruciferin to napin. Heritabilities were comparatively high with 0.79 for napin and 0.77 for cruciferin. Napin and cruciferin showed a significant negative correlation (-0.36**) and a close negative correlation of the cru/nap ratio to glucosinolate content was observed (-0.81**). Three QTL for napin and two QTL for cruciferin were detected, together explaining 47 and 35 % of the phenotypic variance. A major QTL for glucosinolate content was detected on linkage group N19 whose confidence interval overlapped with QTL for napin and cruciferin content. Results indicate a relationship between seed protein composition and glucosinolate content.

Mapping of QTL for seed dormancy in a winter oilseed rape doubled haploid population.

Following winter oilseed rape cultivation, considerable numbers of volunteer oilseed rape plants may occur in subsequent years in following crops. The appearance of volunteer oilseed rape plants is based on the capability of the seeds to become secondary dormant and to survive in this stage for many years in the soil. Genetic reduction of secondary seed dormancy in oilseed rape could provide a means to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The objective of the present study was to analyse the inheritance of primary and secondary seed dormancy in a winter oilseed rape doubled haploid population derived from the cross Express 617 × R53 and to study correlations to other seed traits. Field experiments were performed in Germany for 2 years at two locations with two replicates. Seeds harvested from open pollinated plants were used for all analyses, including a laboratory test for seed dormancy. A previously developed molecular marker map of the doubled haploid population was used to map QTL of the relevant traits. For primary, secondary and total seed dormancy, the results showed significant effects of the genotypes and their interactions, with years and locations. Two, four and five QTL were detected for primary, secondary and total seed dormancy which explained 19, 35 and 42 % of the phenotypic variance, respectively. Results show that secondary seed dormancy is a heritable trait and that selection for low secondary seed dormancy is possible.

A new near-infrared reflectance spectroscopy method for high-throughput analysis of oleic acid and linolenic acid content of single seeds in oilseed rape (Brassica napus L.).

The development of oilseed rape cultivars with a high content of oleic acid (18:1) and a low content of linolenic acid (18:3) in the seed oil is an important breeding aim. Oil of this quality is increasingly being sought by the food and the oleochemical industry. Since the oil quality is determined by the genotype of the seed, a selection can be performed among single seeds of segregating populations. For this purpose a high-throughput Near-Infrared Reflectance Spectroscopy (NIRS) method using an automated sample presentation unit for single seeds of oilseed rape and a spectrometer equipped with a photodiode array detector was developed. Single-seed analyses have been accomplished with a throughput of up to 800 seeds per hour. Seeds from segregating populations of different origin were analyzed by NIRS and gas chromatography. Calibration equations were developed and validated applying the Modified Partial Least Square regression (MPLS) and LOCAL procedure. In three independent validations, standard errors of prediction corrected for bias between 2.7% and 3.7% for oleic acid and 1.2% and 1.8% for linolenic acid were determined using MPLS. Similar results were obtained applying the LOCAL procedure. The results show that the new high-throughput method can be applied to predict the oleic acid and linolenic acid content of single seeds of oilseed rape.

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.).

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F(2) plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F(2) plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F(2) plants were confirmed in the next generation by analysing F(4) seeds harvested from five F(3) plants per selected F(2) plant. F(3) lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.

QTL for phytosterol and sinapate ester content in Brassica napus L. collocate with the two erucic acid genes.

Improving oil and protein quality for food and feed purposes is an important goal in rapeseed (Brassica napus L.) breeding programs. Rapeseed contains phytosterols, used to enrich food products, and sinapate esters, which are limiting the utilization of rapeseed proteins in the feed industry. Increasing the phytosterol content of oil and lowering sinapate ester content of meal could increase the value of the oilseed rape crop. The objective of the present study was to identify quantitative trait loci (QTL) for phytosterol and sinapate ester content in a winter rapeseed population of 148 doubled haploid lines, previously found to have a large variation for these two traits. This population also segregated for the two erucic acid genes. A close negative correlation was found between erucic acid and phytosterol content (Spearman's rank correlation, r(s) = -0.80**). For total phytosterol content, three QTL were detected, explaining 60% of the genetic variance. The two QTL with the strongest additive effects were mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. For sinapate ester content four QTL were detected, explaining 53% of the genetic variance. Again, a close negative correlation was found between erucic acid and sinapate ester content (r(s) = -0.66**) and the QTL with the strongest additive effects mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. The results suggests, that there is a pleiotropic effect of the two erucic acid genes on phytosterol and sinapate ester content; the effect of the alleles for low erucic acid content is to increase phytosterol and sinapate ester content. Possible reasons for this are discussed based on known biosynthetic pathways.

Resveratrol glucoside (Piceid) synthesis in seeds of transgenic oilseed rape (Brassica napus L.).

Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 microg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 microg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L.

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.

Photorespiratory NH(4)(+) production in leaves of wild-type and glutamine synthetase 2 antisense oilseed rape.

Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15 degrees C and 25 degrees C increased photorespiratory NH(4)(+) production from 0.7 to 3.5 micromol m(-2) s(-1). Despite the 5-fold increase in the rate of NH(4)(+) production, the NH(4)(+) concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH(4)(+) production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH(4)(+) concentrations than wild-type plants and no overall correlation between GS activity and tissue NH(4)(+) concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14 degrees C to 27 degrees C or to a trifold increase in the O(2) to CO(2) ratio did not show any change in steady-state leaf tissue NH(4)(+) concentration or in NH(3) emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH(4)(+) in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH(3) fluxes.