Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Genetic Architecture of Flowering Time and Sex Determination in Hemp (Cannabis sativa L.) : A Genome-Wide Association Study
    Petit, Jordi ; Salentijn, Elma M.J. ; Paulo, Maria João ; Denneboom, Christel ; Trindade, Luisa M. - \ 2020
    Frontiers in Plant Science 11 (2020). - ISSN 1664-462X
    Cannabis sativa - flowering time - GWAS - hemp - plant breeding - QTL - sex determination

    Flowering time and sex determination in hemp (Cannabis sativa L.) strongly influence fiber quality and seed production of this crop. The control of these traits is paramount for the breeding of new cultivars. Yet, little is known about the genetics underlying such complex traits and a better understanding requires in depth knowledge of the molecular mechanisms responsible for these traits. In this report, the genetic architecture of flowering time and sex determination in hemp was studied using a Genome-Wide Association Studies (GWAS) approach. Association studies were performed on a panel of 123 hemp accessions, tested in three contrasting environments, using a set of 600 K SNP markers. Altogether, eight QTLs were identified across environments; six for flowering time traits and two for sex determination. These QTLs covered genomic regions with 33 transcripts predicted to be involved in flowering and sex determination as well as a microRNA, miR156. Genes related to perception and transduction of light and transcription factors well-known to regulate flowering were identified in QTLs for flowering time traits. Transcription factors and genes involved in regulating the balance of phytohormones, specially auxins and gibberellic acid, were identified in QTLs for sex determination. Sex determination QTLs were associated with the development of male flowers in female plants and thus with the stability of sex determination in monecious plants. The present study elucidates relevant knowledge on the genetic mechanisms of flowering and sex determination traits in hemp, and provides new tools for hemp breeding.

    Elucidating the Genetic Architecture of Fiber Quality in Hemp (Cannabis sativa L.) Using a Genome-Wide Association Study
    Petit, Jordi ; Salentijn, Elma M.J. ; Paulo, Maria João ; Denneboom, Christel ; Loo, Eibertus N. van; Trindade, Luisa M. - \ 2020
    Frontiers in Genetics Livestock Genomics 11 (2020). - ISSN 1664-8021
    Cannabis sativa - cell wall composition - fiber quality - GWAS - hemp - QTL - RAD-sequencing
    Hemp (Cannabis sativa L.) is a bast-fiber crop with a great potential in the emerging bio-based economy. Yet, hemp breeding for fiber quality is restricted and that is mainly due to the limited knowledge of the genetic architecture of its fiber quality. A panel of 123 hemp accessions, with large phenotypic variability, was used to study the genetic basis of seven cell wall and bast fiber traits relevant to fiber quality. These traits showed large genetic variance components and high values of broad sense heritability in this hemp panel, as concluded from the phenotypic evaluation across three test locations with contrasting environments. The hemp panel was genotyped using restriction site associated DNA sequencing (RAD-seq). Subsequently, a large set (> 600,000) of selected genome-wide single nucleotide polymorphism (SNP) markers was used for a genome-wide association study (GWAS) approach to get insights into quantitative trait loci (QTLs) controlling fiber quality traits. In absence of a complete hemp genome sequence, identification of QTLs was based on the following characteristics: (i) association level to traits, (ii) fraction of explained trait variance, (iii) collinearity between QTLs, and (iv) detection across different environments. Using this approach, 16 QTLs were identified across locations for different fiber quality traits, including contents of glucose, glucuronic acid, mannose, xylose, lignin, and bast fiber content. Among them, six were found across the three environments. The genetic markers composing the QTLs that are common across locations are valuable tools to develop novel genotypes of hemp with improved fiber quality. Underneath the QTLs, 12 candidate genes were identified which are likely to be involved in the biosynthesis and modification of monosaccharides, polysaccharides, and lignin. These candidate genes were suggested to play an important role in determining fiber quality in hemp. This study provides new insights into the genetic architecture of fiber traits, identifies QTLs and candidate genes that form the basis for molecular breeding for high fiber quality hemp cultivars.
    Dataset for the annotations of the transcriptome of the hemp cultivar C. sativa ‘Finola’ (finola1) and the marihuana cultivar C. sativa ‘Purple Kush’
    Petit Pedro, Jordi ; Salentijn, Elma ; Loo, Robert van; Caldas Paulo, Joao ; Denneboom, Christel ; Trindade, Luisa - \ 2020
    Wageningen University & Research
    GWAS - Cannabis sativa - hemp - fibre quality - cell wall composition - RAD-sequencing - QTL - sex determination - flowering time - plant breeding
    This dataset contains the annotations of the two transcriptomes of the hemp cultivar C. sativa ‘Finola’ (finola1) and the marihuana cultivar C. sativa ‘Purple Kush’, aligned to the C. sativa ‘Purple Kush’ (canSat3 version GAC 000230575.1) assembly from the C. sativa Genome Browser Gateway (van Bakel et al., 2011). The annotations were performed using Local Blast and the data is provided in gff files. Gff means General Feature Format and it's a simple tab-delimited text file for describing genomic features. The files are ready to be uploaded in a genomic viewer.
    Dataset of the RADseq data and genotyping from a panel of 123 hemp accessions
    Petit Pedro, Jordi ; Salentijn, Elma ; Loo, Robert van; Caldas Paulo, Joao ; Denneboom, Christel ; Trindade, Luisa - \ 2020
    Wageningen University and Research
    GWAS - Cannabis sativa - hemp - fibre quality - cell wall composition - RAD-sequencing - QTL - sex determination - flowering time - plant breeding
    This dataset contains the raw RADseq data and genotyping data from a panel of 123 hemp (Cannabis sativa L.) accessions. For extra information about the RAD sequencing data analysis and the SNP marker selection, read the materials and methods of the paper 'Elucidating the genetic architecture of fibre quality in hemp (Cannabis sativa L.) using the Genome-Wide Association Study'.
    Genetic Variability of Morphological, Flowering, and Biomass Quality Traits in Hemp (Cannabis sativa L.)
    Petit, Jordi ; Salentijn, Elma M.J. ; Paulo, Maria João ; Thouminot, Claire ; Dinter, Bert Jan van; Magagnini, Gianmaria ; Gusovius, Hans Jörg ; Tang, Kailei ; Amaducci, Stefano ; Wang, Shaoliang ; Uhrlaub, Birgit ; Müssig, Jörg ; Trindade, Luisa M. - \ 2020
    Frontiers in Plant Science 11 (2020). - ISSN 1664-462X
    Cannabis sativa - cell wall composition - fiber quality - flowering time - genetic variability - genotype-by-environment (G×E) interactions - hemp - sex determination

    Hemp (Cannabis sativa L.) is a bast-fiber crop well-known for the great potential to produce sustainable fibers. Nevertheless, hemp fiber quality is a complex trait, and little is known about the phenotypic variability and heritability of fiber quality traits in hemp. The aim of this study is to gain insights into the variability in fiber quality within the hemp germplasm and to estimate the genetic components, environmental components, and genotype-by-environment (G×E) interactions on fiber quality traits in hemp. To investigate these parameters, a panel of 123 hemp accessions was phenotyped for 28 traits relevant to fiber quality at three locations in Europe, corresponding to climates of northern, central, and southern Europe. In general, hemp cultivated in northern latitudes showed a larger plant vigor while earlier flowering was characteristic of plants cultivated in southern latitudes. Extensive variability between accessions was observed for all traits. Most cell wall components (contents of monosaccharides derived from cellulose and hemicellulose; and lignin content), bast fiber content, and flowering traits revealed large genetic components with low G×E interactions and high broad-sense heritability values, making these traits suitable to maximize the genetic gains of fiber quality. In contrast, contents of pectin-related monosaccharides, most agronomic traits, and several fiber traits (fineness and decortication efficiency) showed low genetic components with large G×E interactions affecting the rankings across locations. These results suggest that pectin, agronomic traits, and fiber traits are unsuitable targets in breeding programs of hemp, as their large G×E interactions might lead to unexpected phenotypes in untested locations. Furthermore, all environmental effects on the 28 traits were statistically significant, suggesting a strong adaptive behavior of fiber quality in hemp to specific environments. The high variability in fiber quality observed in the hemp panel, the broad range in heritability, and adaptability among all traits prescribe positive prospects for the development of new hemp cultivars of excellent fiber quality.

    The complex interactions between flowering behavior and fiber quality in hemp
    Salentijn, Elma M.J. ; Petit, Jordi ; Trindade, Luisa M. - \ 2019
    Frontiers in Plant Science 10 (2019). - ISSN 1664-462X
    Cannabis sativa - Fiber development - Flowering-time - Hemp - Phenology - Sex determination - Short-day plant

    Hemp, Cannabis sativa L., is a sustainable multipurpose fiber crop with high nutrient and water use efficiency and with biomass of excellent quality for textile fibers and construction materials. The yield and quality of hemp biomass are largely determined by the genetic background of the hemp cultivar but are also strongly affected by environmental factors, such as temperature and photoperiod. Hemp is a facultative short-day plant, characterized by a strong adaptation to photoperiod and a great influence of environmental factors on important agronomic traits such as “flowering-time” and “sex determination.” This sensitivity of hemp can cause a considerable degree of heterogeneity, leading to unforeseen yield reductions. Fiber quality for instance is influenced by the developmental stage of hemp at harvest. Also, male and female plants differ in stature and produce fibers with different properties and quality. Next to these causes, there is evidence for specific genotypic variation in fiber quality among hemp accessions. Before improved hemp cultivars can be developed, with specific flowering-times and fiber qualities, and adapted to different geographical regions, a better understanding of the molecular mechanisms controlling important phenological traits such as “flowering-time” and “sex determination” in relation to fiber quality in hemp is required. It is well known that genetic factors play a major role in the outcome of both phenological traits, but the major molecular factors involved in this mechanism are not characterized in hemp. Genome sequences and transcriptome data are available but their analysis mainly focused on the cannabinoid pathway for medical purposes. Herein, we review the current knowledge of phenotypic and genetic data available for “flowering-time,” “sex determination,” and “fiber quality” in short-day and dioecious crops, respectively, and compare them with the situation in hemp. A picture emerges for several controlling key genes, for which natural genetic variation may lead to desired flowering behavior, including examples of pleiotropic effects on yield quality and on carbon partitioning. Finally, we discuss the prospects for using this knowledge for the molecular breeding of this sustainable crop via a candidate gene approach.

    Green Plant Biotechnology at work
    Bovy, A.G. ; Heusden, A.W. van; Kreike, C.M. ; Peer, A.F. van; Salentijn, E.M.J. ; Schouten, H.J. ; Smulders, M.J.M. ; Wiel, C.C.M. van de - \ 2019
    Wageningen : Groen Kennisnet
    The pages below contain the topics, questions, and instruction of a course on Plant Biotechnology and Molecular Plant Breeding, which is taken by students at the Universities of Applied Sciences in the Netherlands who specialize in Green Biotechnology. In the course they learn about the use of molecular techniques for applications in plant breeding companies and in related research in The Netherlands.
    Latitudinal adaptation and genetic insights into the origins of cannabis sativa L.
    Zhang, Qingying ; Chen, Xuan ; Guo, Hongyan ; Trindade, Luisa M. ; Salentijn, Elma M.J. ; Guo, Rong ; Guo, Mengbi ; Xu, Yanping ; Yang, Ming - \ 2018
    Frontiers in Plant Science 871 (2018). - ISSN 1664-462X
    Cannabaceae - CpDNA - Genetic diversity - Industrial hemp - Phylogeography

    Cannabis is one of the most important industrial crops distributed worldwide. However, the phylogeographic structure and domestication knowledge of this crop remains poorly understood. In this study, sequence variations of five chloroplast DNA (cpDNA) regions were investigated to address these questions. For the 645 individuals from 52 Cannabis accessions sampled (25 wild populations and 27 domesticated populations or cultivars), three haplogroups (Haplogroup H, M, L) were identified and these lineages exhibited distinct high-middle-low latitudinal gradients distribution pattern. This pattern can most likely be explained as a consequence of climatic heterogeneity and geographical isolation. Therefore, we examined the correlations between genetic distances and geographical distances, and tested whether the climatic factors are correlated with the cpDNA haplogroup frequencies of populations. The “isolation-by-distance” models were detected for the phylogeographic structure, and the day-length was found to be the most important factor (among 20 BioClim factors) that influenced the population structures. Considering the distinctive phylogeographic structures and no reproductive isolation among members of these lineages, we recommend that Cannabis be recognized as a monotypic genus typified by Cannabis sativa L., containing three subspecies: subsp. sativa, subsp. Indica, and subsp. ruderalis. Within each haplogroup which possesses a relatively independent distribution region, the wild and domesticated populations shared the most common haplotypes, indicating that there are multiregional origins for the domesticated crop. Contrast to the prevalent Central-Asia-Origin hypothesis of C. saltiva, molecular evidence reveals for the first time that the low latitude haplogroup (Haplogroup L) is the earliest divergent lineage, implying that Cannabis is probably originated in low latitude region.

    Genetically engineering Crambe abyssinica- A potentially high-value oil crop for salt land improvement
    Qi, W. ; Tinnenbroek-Capel, I.E.M. ; Salentijn, E.M.J. ; Zhang, Zhao ; Huang, Bangquan ; Cheng, Jihua ; Shao, Hongbo ; Visser, R.G.F. ; Krens, F.A. ; Loo, E.N. van - \ 2018
    Land Degradation and Development 29 (2018)4. - ISSN 1085-3278 - p. 1096 - 1106.
    Crambe abyssinica (crambe) is a new industrial oil crop that can grow on saline soil and tolerates salty water irrigation. Genetically engineered crambe in which the seed‐oil composition is manipulated for more erucic acid and less polyunsaturated fatty acid (PUFA) would be highly beneficial to industry. In this research, lysophosphatidic acid acyltransferase 2 RNA interference (CaLPAT2‐RNAi) was introduced into the crambe genome to manipulate its oil composition. The result showed in comparison with wild type, CaLPAT2‐RNAi could significantly reduce linoleic and linolenic acid content, simultaneously increasing erucic acid content. Systematic metabolism engineering was then carried out to further study CaLPAT2‐RNAi, combined with the overexpression of Brassica napus fatty acid elongase (BnFAE), Limnanthes douglasii LPAT (LdLPAT), and RNAi of endogenous fatty acid desaturase 2 (CaFAD2‐RNAi). Oil composition analysis on the tranformants' seeds showed that (a) with CaFAD2‐RNAi, PUFA content could be dramatically decreased, in comparison with BnFAE + LdLPAT + CaFAD2‐RNAi, and BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi seeds showed lower linolenic acid content; (b) BnFAE + LdLPAT + CaFAD2‐RNAi + CaLPAT2‐RNAi could increase the erucic acid content in crambe seed oil from less than 66.6% to 71.6%, whereas the highest erucic acid content of BnFAE + LdLPAT + CaFAD2‐RNAi was 79.2%; (c) although the four‐gene combination could not increase the erucic acid content of seed oil to a higher level than the others, it led to increased carbon resource deposited into C22:1 and C18:1 moieties and lower PUFA. Summarily, the present research indicates that suppression of LPAT2 is a new, promising strategy for seed‐oil biosynthesis pathway engineering, which would increase the value of crambe oil.
    New developments in fiber hemp (Cannabis sativa L.) breeding
    Salentijn, E.M.J. ; Zhang, Qingying ; Amaducci, Stefano ; Yang, Ming ; Trindade, L.M. - \ 2015
    Industrial Crops and Products 68 (2015). - ISSN 0926-6690 - p. 32 - 41.
    Breeding - Fiber quality - Genetics - Hemp

    Fiber hemp (Cannabis sativa L.) is a sustainable and high yielding industrial crop that can help to meet the high global demand for fibers. Hemp can be grown for fiber, seeds, and/or for dual purpose in a wide range of geographic zones and climates. Currently the main hemp producing regions in the world are China, Europe, and Canada. The number of new cultivars developed for each of these regions has gradually increased, with each region producing its own typical hemp cultivars for different purposes. In this article, the state of the art of fiber hemp breeding programs in Europe, China, and Canada are reviewed. The breeding strategies and tools used in the breeding of hemp cultivars are discussed. We also provide an overview of genetic diversity in hemp for different traits. In addition, the current knowledge of the main breeding goals for fiber hemp, which are an improvement of fiber quality and fiber yield, breeding for specific cannabinoid profiles, control of flowering behavior, male flowering control, and breeding of cultivars for specific environments are evaluated. Lastly, we discuss the inestimable value of next generation technologies to breed new hemp cultivars that are suitable for a biobased economy.

    Screening for recombinants of Crambe abyssinica after transformation by the PMF1 marker-free vector based on chemical selection and meristematic regeneration
    Qi, W. ; Tinnenbroek-Capel, I.E.M. ; Salentijn, E.M.J. ; Schaart, J.G. ; Cheng, J. ; Denneboom, C. ; Zhang, Z. ; Zhang, X. ; Zhao, H. ; Visser, R.G.F. ; Huang Bangquan, ; Loo, E.N. van; Krens, F.A. - \ 2015
    Scientific Reports 5 (2015). - ISSN 2045-2322 - 15 p.
    The T-DNA region of pMF1 vector of marker-free system developed by Wageningen UR, has Recombinase R-LBD gene fusion and nptII and codA gene fusion between two recombination sites. After transformation applying dexamethasone (DEX) can activate the recombinase to remove the T-DNA fragment between recombination sites. The recombinant ought to be selected on 5-fluorocytocine (5-FC) because of codA converting 5-FC into 5-fluorouracil the toxic. A PMF1 vector was transformed into hexaploid species Crambe abyssinica. Two independent transformants were chosen for DEX-induced recombination and later 5-FC selection. In contrast to earlier pMF1 experiments, the strategy of stepwise selection based on meristematic regeneration was engaged. After a long period of 5-FC selection, recombinants were obtained successfully, but most of the survivors were wildtype and non-recombinant. The results revealed when applying the PMF1 marker-free system on C. abyssinica, 1) Increasing in the DEX concentration did not correspondingly enhance the success of recombination; 2) both of the DEX-induced recombination and 5-FC negative selection were apparently insufficient which was leading to the extremely high frequency in chimerism occurring for recombinant and non-recombinant cells in tissues; 3) the strategy of stepwise selection based on meristem tissue regeneration was crucial for successfully isolating the recombinant germplasm from the chimera.
    Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica
    Cheng, J. ; Salentijn, E.M.J. ; Huang Bangquan, ; Denneboom, C. ; Dechesne, A.C. ; Krens, F.A. ; Visser, R.G.F. ; Loo, E.N. van - \ 2015
    Plant Biotechnology Journal 13 (2015)4. - ISSN 1467-7644 - p. 471 - 481.
    induced point mutations - crop improvement - reverse genetics - oleic-acid - functional genomics - fad2 gene - discovery - arabidopsis - populations - cloning
    Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.
    Quantitation of coeliac toxicity in wheat using genomics and proteomics
    Gilissen, L.J.W.J. ; Salentijn, E.M.J. ; Broeck, H.C. van den; Cordewener, J.H.G. ; America, T.H.P. ; Schaart, J.G. ; Meer, I.M. van der; Smulders, M.J.M. - \ 2013
    In: Proceedings of the 27th meeting of the working group on prolamin analysis and toxicity. - - p. 45 - 51.
    Several tests are currently marketed for measuring the amount of gluten in food products and to determine whether products are gluten-free. Of these tests, the Codex Alimentarius approved the R-Biopharm R5 ELISA as the gluten detection standard. This test is based on recognition by a monoclonal antibody (mAb) of five amino acidlong peptide sequences, which are abundantly present in the gliadin proteins of wheat gluten. Another mAb-based test recognises specific peptide sequences of six amino acids (G12 ELISA, Romer Labs). Both tests enable estimating the total amount of gluten (gluten = gliadin x 2) in a wheat product. As the number and composition of coeliac disease (CD) epitopes vary between gliadins and glutenins, among varieties, and between wheat, rye and barley, there is no direct relationship between the estimated gluten content and the presence of CD epitopes. Many research groups have raised epitope-specific T cell clones (TCCs) from patient biopsies that can be used for detection of specific CD-immunogenic gluten epitopes. Such CD epitopes are specific nine amino acid-long peptide sequences rich in prolamin (P) and glutamin (Q) residues. Recently, a list of internationally agreed CD epitopes has been published [1]. Unfortunately, T cell-based tests are mostly qualitative, indicating the presence or absence of a particular epitope, and they are unable to quantitate the overall CD-immunogencity of a given wheat variety. Proper quantitation of CD epitopes is relevant because the amount of non-CDimmunogenic gluten proteins can differ among wheat varieties and genomes (ploidy levels) [2], and the CD-immunogenicity of individual epitopes can be different according to the patient’s sensitivity profile [3,4]. Already a single amino acid substitution in a T cell epitope, especially from proline (P) to serine (S), may abolish T cell binding and thus eliminate the epitope’s CD-immunogenicity [5]. Therefore, gluten detection in the context of coeliac disease should be in line with the internationally agreed list of CD-relevant epitopes. To overcome the shortcomings of mAb-based and T cell-based tests, new approaches are now under development, especially based on genomic and proteomic analysis, aiming at the identification of the profile of CD-immunogenic epitopes of individual wheat species and varieties (for breeding and selection), and at quantitation of CD epitope-containing gluten proteins or fragments in foods (for food diagnostics).
    Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplican sequencing
    Salentijn, E.M.J. ; Esselink, D.G. ; Goryunova, S.V. ; Meer, I.M. van der; Gilissen, L.J.W.J. ; Smulders, M.J.M. - \ 2013
    BMC Genomics 14 (2013). - ISSN 1471-2164 - 16 p.
    gliadin gene family - t-cell response - alpha-gliadin - bread wheat - snp discovery - tissue transglutaminase - aegilops-tauschii - coding loci - triticum - expression
    Background - Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. Results - A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. Conclusions - The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.
    Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica using RNAi-mediated gene silencing
    Cheng, J. ; Zhu, L. ; Salentijn, E.M.J. ; Huang, B. ; Gruber, J. ; Dechesne, A.C. ; Krens, F.A. ; Qi, W. ; Visser, R.G.F. ; Loo, E.N. van - \ 2013
    BMC Plant Biology 13 (2013). - ISSN 1471-2229
    high oleic-acid - erucic-acid - brassica-napus - fad2 gene - fae1 gene - carrier protein - expression - suppression - metabolism - mutations
    Background Crambe abyssinica produces high erucic acid (C22:1, 55-60 %) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. Results The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5 %) and PUFA reduction (by 8.6% to 10.2 %). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P <0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. Conclusions CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.
    Isolation and characterization of the omega-6 fatty acid desaturase (FAD2) gene family in the allohexaploid oil seed crop Crambe abyssinica Hochst
    Cheng, J. ; Salentijn, E.M.J. ; Huang Bangquan, ; Krens, F.A. ; Dechesne, A.C. ; Visser, R.G.F. ; Loo, E.N. van - \ 2013
    Molecular Breeding 32 (2013)3. - ISSN 1380-3743 - p. 517 - 531.
    brassica-napus l. - high oleic-acid - erucic-acid - expression - genome - biosynthesis - suppression - stability - evolution - varieties
    Crambe (Crambe abyssinica Hochst ex. R. E. Fries) is an ideal crop for industrial oil production because of its high erucic acid content (C22:1, approx. 60 %) in its seed oil. The value of crambe oil can be improved by increasing C22:1 content or reducing polyunsaturated fatty acids (PUFA). The FAD2 gene plays a critical role in PUFA biosynthesis. To identify targets for breeding, we characterized FAD2 in crambe for copy number and expression profile. Seven copies of FAD2 were detected in allohexaploid crambe. Three genes (CaFAD2-C1, -C2 and -C3) were expressed in multiple tissues, including root, seedling, leaf, flower, bud and developing seeds. In developing seeds, the expression of these genes was upregulated with CaFAD2-C3, being expressed predominantly with a peak at 20 days after pollination. This gene is therefore a promising candidate gene for determining PUFA levels in seed oil. Four other FAD2 genes were considered to be “pseudo-genes” as they harbour internal stop codons and were not expressed. Among the six crambe varieties with consistent variation in oil composition, no nucleotide polymorphisms were found in the CaFAD2-C1, -C2 and -C3 genes. In seeds at 30 days after pollination, statistically significant expression level polymorphisms for only one gene, CaFAD2-C2, was found among the varieties. However, although significantly different, the difference in expression was small and did not explain the variation in oil composition. Given the absence of genetic variation in CaFAD2 genes in crambe breeding lines, breeding for high erucic acid content calls for a molecular breeding approach whereby mutations are chemically induced to increase the genetic variation.
    Avenin diversity analysis of the genus Avena (oat). Relevance for people with celiac disease
    Londono, D.M. ; Westende, W.P.C. van 't; Goryunova, S.V. ; Salentijn, E.M.J. ; Broeck, H.C. van den; Meer, I.M. van der; Visser, R.G.F. ; Gilissen, L.J.W.J. ; Smulders, M.J.M. - \ 2013
    Journal of Cereal Science 58 (2013)1. - ISSN 0733-5210 - p. 170 - 177.
    gluten-free diet - t-cell epitopes - natural variation - oats - wheat - varieties - prolamins - gliadins - toxicity - proteins
    Oat is widely consumed by people with celiac disease (CD). Its safety has been disputed because two peptides from oat avenins can be recognized as T cell epitopes by some CD patients. Differential signals of gluten-specific monoclonal antibodies and in-vitro T cells to oat varieties have suggested the existence of differences in immunogenicity. We aimed to clarify the nature of such responses by cloning avenin genes from 13 Avena species. A single oat plant contained up to 10 avenin genes. Avenin proteins clustered in four groups of which two contained the two avenin CD epitopes. All Avena species examined harbored avenins of these two groups, and as a consequence all contained avenins with the two avenin-specific epitopes, which makes it very unlikely to find oat cultivars that are devoid of these sequences. The established gluten epitopes from wheat, rye and barley were not present in oat avenins; some variants with two and three amino acid substitutions occurred, but they were predicted not to resist proteolysis in the gastro-intestinal tract. Perfect recognition sites of antibodies R5 and G12 were also not present in avenins. Thus, their signals to oat should not be interpreted as differences in immunogenicity for CD patients.
    Oat avenins do not contain coeliac disease epitopes known from wheat, rye and barley
    Londono, D.M. ; Westende, W.P.C. van 't; Goryunova, S.V. ; Salentijn, E.M.J. ; Broeck, H.C. van den; Meer, I.M. van der; Visser, R.G.F. ; Gilissen, L.J.W.J. ; Smulders, M.J.M. - \ 2012
    Food-related strategies towards reduction of gluten intolerance and gluten sensitivity
    Gilissen, L.J.W.J. ; Broeck, H.C. van den; Londono, D.M. ; Salentijn, E.M.J. ; Koning, F. ; Meer, I.M. van der; Smulders, M.J.M. - \ 2012
    In: Proceedings of the 25th Meeting Working Group on Prolamin Analysis and Toxicity (PWG), Felbach, Germany, September 29 - October 2, 2012. - - p. 29 - 35.
    Assessing the CD-toxic potential of wheat varieties by deep 454 transcript sequencing
    Salentijn, E.M.J. ; Esselink, G. ; Goryunova, S.V. ; Gilissen, L.J.W.J. ; Smulders, M.J.M. - \ 2012
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