From fibers, to flowering, to metabolites: unlocking hemp (Cannabis sativa L.) potential with the guidance of novel discoveries and tools.

Cannabis sativa L. is an ancient crop whose agricultural adoption has been interrupted to prevent the use of marijuana as psychoactive drug. Nevertheless, hemp - the Cannabis sativa type with low concentrations of intoxicating Δ9-tetrahydrocannabinoid - is experiencing resurged interest thanks to loosened cultivation restrictions and its potential as multipurpose bio-based crop. In fact, hemp has valuable applications, including production of medicines from its non-intoxicating cannabinoids, food, medical, and industrial uses of its seed oil rich in poly-unsaturated fatty acids, and production of fibers for textiles and industry from its stems. Recently, several hemp genomic and genetic resources have been developed, allowing for a significant expansion of the genetic knowledge on major hemp traits, as cannabinoids, oil, and fibers synthesis, and regulation of flowering and sex determination. Still, hemp is an under-improved crop, whose advancement will depend on the ability to expand and collectively use the novel resources available in light of the fast advancements in bioinformatics and plant phenotyping technologies. This review discusses on the current genetic and genomic knowledge on the most important hemp traits, and provides a perspective on how to further expand such knowledge and tackle hemp improvement with the most up-to-date tools for plant and hemp research.

Exploring the alpha-gliadin locus: the 33-mer peptide with six overlapping coeliac disease epitopes in Triticum aestivum is derived from a subgroup of Aegilops tauschii.

Most alpha-gliadin genes of the Gli-D2 locus on the D genome of hexaploid bread wheat (Triticum aestivum) encode for proteins with epitopes that can trigger coeliac disease (CD), and several contain a 33-mer peptide with six partly overlapping copies of three epitopes, which is regarded as a remarkably potent T-cell stimulator. To increase genetic diversity in the D genome, synthetic hexaploid wheat lines are being made by hybridising accessions of Triticum turgidum (AB genome) and Aegilops tauschii (the progenitor of the D genome). The diversity of alpha-gliadins in A. tauschii has not been studied extensively. We analysed the alpha-gliadin transcriptome of 51 A. tauschii accessions representative of the diversity in A. tauschii. We extracted RNA from developing seeds and performed 454 amplicon sequencing of the first part of the alpha-gliadin genes. The expression profile of allelic variants of the alpha-gliadins was different between accessions, and also between accessions of the Western and Eastern clades of A. tauschii. Generally, both clades expressed many allelic variants not found in bread wheat. In contrast to earlier studies, we detected the 33-mer peptide in some A. tauschii accessions, indicating that it was introduced along with the D genome into bread wheat. In these accessions, transcripts with the 33-mer peptide were present at lower frequencies than in bread wheat varieties. In most A. tauschii accessions, however, the alpha-gliadins do not contain the epitope, and this may be exploited, through synthetic hexaploid wheats, to breed bread wheat varieties with fewer or no coeliac disease epitopes.

Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica.

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.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing.

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.

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.

Expansion of the gamma-gliadin gene family in Aegilops and Triticum.

BACKGROUND: The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. RESULTS: We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. CONCLUSIONS: We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.

Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation.

BACKGROUND: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. RESULTS: A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. CONCLUSIONS: Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).

Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease.

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of 'low CD toxic' as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD.

Genetic Architecture of Flowering Time and Sex Determination in Hemp (Cannabis sativa L.): A Genome-Wide Association Study.

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.

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.

Genetic Variability of Morphological, Flowering, and Biomass Quality Traits in Hemp (Cannabis sativa L.).

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.

Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci.

BACKGROUND: Alpha-gliadins form a multigene protein family encoded by multiple alpha-gliadin (Gli-2) genes at three genomic loci, Gli-A2, Gli-B2 and Gli-D2, respectively located on the homoeologous wheat chromosomes 6AS, 6BS, and 6DS. These proteins contain a number of important celiac disease (CD)-immunogenic domains. The alpha-gliadins expressed from the Gli-B2 locus harbour fewer conserved CD-epitopes than those from Gli-A2, whereas the Gli-D2 gliadins have the highest CD-immunogenic potential. In order to detect differences in the highly CD-immunogenic alpha-gliadin fraction we determined the relative expression level from the homoeologous Gli-2 loci in various tetraploid and hexaploid wheat genotypes by using a quantitative pyrosequencing method and by analyzing expressed sequence tag (EST) sequences. RESULTS: We detected large differences in relative expression levels of alpha-gliadin genes from the three homoeologous loci among wheat genotypes, both as relative numbers of expressed sequence tag (EST) sequences from specific varieties and when using a quantitative pyrosequencing assay specific for Gli-A2 genes. The relative Gli-A2 expression level in a tetraploid durum wheat cultivar ('Probstdorfer Pandur') was 41%. In genotypes derived from landraces, the Gli-A2 frequency varied between 12% and 58%. In some advanced hexaploid bread wheat cultivars the genes from locus Gli-B2 were hardly expressed (e.g., less than 5% in 'Lavett') but in others they made up more than 40% (e.g., in 'Baldus'). CONCLUSION: Here, we have shown that large differences exist in relative expression levels of alpha-gliadins from the homoeologous Gli-2 loci among wheat genotypes. Since the homoelogous genes differ in the amount of conserved CD-epitopes, screening for differential expression from the homoeologous Gli-2 loci can be employed for the pre-selection of wheat varieties in the search for varieties with very low CD-immunogenic potential. Pyrosequencing is a method that can be employed for such a 'gene family-specific quantitative transcriptome profiling'.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines.

BACKGROUND: Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD). RESULTS: The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the alpha-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the omega-gliadin, gamma-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties. CONCLUSION: The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties.

The Complex Interactions Between Flowering Behavior and Fiber Quality in Hemp.

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.

Latitudinal Adaptation and Genetic Insights Into the Origins of Cannabis sativa L.

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.

Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes.

BACKGROUND: Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the alpha-gliadins contain several peptides that are associated to the disease. RESULTS: We obtained 230 distinct alpha-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All alpha-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, alpha-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-alpha9 and glia-alpha20, but never the intact epitopes glia-alpha and glia-alpha2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific. CONCLUSION: Our analysis shows that alpha-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxicity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa).

An octaploid (Fragaria x ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria x ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways.

Screening for recombinants of Crambe abyssynica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration.

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.

A gene encoding a polygalacturonase-inhibiting protein (PGIP) shows developmental regulation and pathogen-induced expression in strawberry.

• Polygalacturonase-inhibiting proteins (PGIPs) have been demonstrated to play a role in host defence in several plants. • The PGIP now cloned from strawberry (Fragaria × ananassa) showed a high degree of homology to other fruit PGIPs. The gene expression of strawberry PGIP was monitored in healthy leaves, flowers and fruit at different maturity stages. PGIP transcript levels were also analysed following fruit inoculation with the fungal pathogen Botrytis cinerea in strawberry cultivars displaying variation in susceptibility. • Healthy mature berries showed the highest constitutive PGIP gene expression levels compared with leaves, flowers and immature fruit, indicating that the gene is developmentally regulated. Among the cultivars studied ('Elsanta', 'Korona', 'Polka', 'Senga sengana', 'Tenira'), 'Polka' had the highest constitutive expression level of PGIP. After inoculation with B. cinerea, all five cultivars displayed a significant induction of PGIP gene expression, but the differences between them were not statistically significant. • The high induction of the PGIP gene after inoculation with B. cinerea indicates that PGIP has a role in defence of strawberry.