SUMMARY Legume nodule formation is induced by rhizobia secreted Nod factors (NFs). It has been shown that NF receptors also accumulate in the apex of Medicago truncatula nodules. However, the NF signaling induced transcriptional changes in there have never been studied. Here, we studied this by using NF signaling mutant TE7, a weak allele of IPD3, blocked in rhizobial release. Nodule apices were isolated with laser microdissection and used for transcriptional analysis. We identified 1655 NF signaling controlled genes in nodule apex. By comparing this with the transcriptome data from VAMP721d&e RNAi nodule apices, we identified a subset of 445 genes whose expression depends on NF signaling and rhizobial release. Further, we compared the set of genes controlled by NF signaling in nodule apices with that controlled in root epidermis, and these showed only a small overlap. NIN is induced by NF signaling both in the root epidermis and in the nodule. By overexpression of NIN in TE7 and knock down of NIN in wildtype nodules we showed that NF signaling controlled rhizobial release depends on NIN. NF signaling controls a distinct set of genes in nodules, the function of which depends at least in part on NIN.

The root of Taraxacum koksaghyz (Tk) represents a promising alternative for sustainable natural rubber (NR) production. It addresses current challenges in NR supply faced by the current world NR producer, the rubber tree (Hevea brasiliensis). Such challenges are pathogen vulnerability, sustainability issues, and economic pressure. Moreover, the fossil resource dependency of synthetic rubber (SR) production is of concern. Co-extracted from the roots is inulin, a fructan polymer with great potential as a renewable feedstock for green chemistry. The dual output of NR and inulin positions Tk as a multipurpose crop. Its use would support both the development of a biobased economy and reduce reliance on fossil resources for SR production. This review evaluates past accomplishments and recent advances in molecular breeding, omics-driven crop improvement, and bioprocessing technologies. Developments aim to enhance rubber and inulin yields from Tk, and focus on insights into the interplay between the metabolisms of both NR and inulin. We discuss the optimization of the entire production chain, from seed to extraction, including agronomic practices and genetic modification/genome editing. Also, the possible valorization of other plant components in industrial and commercial applications is outlined. Collectively, these developments contribute to the productivity and economic viability of Tk as a multipurpose crop. Key scientific and technological accomplishments are outlined. We propose strategies to accelerate the adoption of Tk as a viable, sustainable source of NR and inulin-based green chemicals. Future research directions are defined to target the remaining challenges in upscaling cultivation and integrating Tk into sustainable agricultural systems.

We present the first reference genome of the highly heterozygous autotetraploid Allium porrum (leek). Combining long‐read sequencing with single‐nucleotide polymorphism (SNP)‐array screening of two experimental F1 populations, we generated a genetic map with 11,429 SNP markers across eight linkage groups and a chromosome‐scale assembly of A. porrum (leek) totaling 15.2 Gbp in size. The high quality of the reference genome is substantiated by 97.2% BUSCO completeness and a mapping rate of 96% for full‐length transcripts. The linkage map exposes the recombination landscape of leek and confirms that crossovers are predominantly proximal, located to the centromeres, contrasting with distal recombination landscapes observed in other Allium species. Comparative genomics reveals structural rearrangements between A. porrum and its relatives ( Allium fistulosum , Allium sativum , and Allium cepa ), suggesting a closer genomic relationship to A. sativum . Our annotated high‐quality reference genome delivers crucial insights into the leek genome structure, recombination landscape, and evolutionary relationships within the Allium genus, with implications for species compatibility in breeding programs, facilitating marker‐assisted selection and genetic improvement in leek.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

SUMMARYThe allopolyploid okra (Abelmoschus esculentus) unveiled telomeric repeats flanking distal gene‐rich regions and short interstitial TTTAGGG telomeric repeats, possibly representing hallmarks of chromosomal speciation. Ribosomal RNA (rRNA) genes organize into 5S clusters, distinct from the 18S–5.8S–28S units, indicating an S‐type rRNA gene arrangement. The assembly, in line with cytogenetic and cytometry observations, identifies 65 chromosomes and a 1.45 Gb genome size estimate in a haploid sibling. The lack of aberrant meiotic configurations implies limited to no recombination among sub‐genomes. k‐mer distribution analysis reveals 75% has a diploid nature and 15% heterozygosity. The configurations of Benchmarking Universal Single‐Copy Ortholog (BUSCO), k‐mer, and repeat clustering point to the presence of at least two sub‐genomes one with 30 and the other with 35 chromosomes, indicating the allopolyploid nature of the okra genome. Over 130 000 putative genes, derived from mapped IsoSeq data and transcriptome data from public okra accessions, exhibit a low genetic diversity of one single nucleotide polymorphisms per 2.1 kbp. The genes are predominantly located at the distal chromosome ends, declining toward central scaffold domains. Long terminal repeat retrotransposons prevail in central domains, consistent with the observed pericentromeric heterochromatin and distal euchromatin. Disparities in paralogous gene counts suggest potential sub‐genome differentiation implying possible sub‐genome dominance. Amino acid query sequences of putative genes facilitated phenol biosynthesis pathway annotation. Comparison with manually curated reference KEGG pathways from related Malvaceae species reveals the genetic basis for putative enzyme coding genes that likely enable metabolic reactions involved in the biosynthesis of dietary and therapeutic compounds in okra.

Tomato is the most consumed vegetable in the world. Increasing its natural resistance and resilience is key for ensuring food security within a changing climate. Plant breeders improve those traits by generating crosses of cultivated tomatoes with their wild relatives. Specific allele introgression relying on meiotic recombination, is hampered by structural divergence between parental genomes. However, previous studies of interspecific tomato hybridization focused in single cross or lacked resolution due to prohibitive sequencing costs of large segregating populations. Here, we used pooled-pollen sequencing to reveal unprecedented details of recombination patterns in five interspecific tomato hybrids. We detected hybrid-specific recombination coldspots that underscore the influence of structural divergence in shaping recombination landscape. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. We also found gene complexes associated with metabolic processes, stress resistance and domestication syndrome traits, revealing undesired consequences of recombination suppression to phenotypes. Finally, we demonstrate that by using resequencing data of wild and domesticated tomato populations, we can screen for alternative parental genomes to overcome recombination barriers. Overall, our results will allow breeders better informed decisions on generating disease-resistant and climate-resilient tomato. ### Competing Interest Statement The authors have declared no competing interest.

Wholemeal flours from blends of bread wheat, emmer and spelt were processed into bread using yeast-based and sourdough fermentation. The bread wheat flour contained significantly higher concentrations of total dietary fibre and fructans than the spelt and emmer flours, the latter having the lowest contents. Breadmaking using sourdough and yeast systems resulted in changes in composition from flour to dough to bread including increases in organic acids and mannitol in the sourdough system and increases in amino acids and sugars (released by hydrolysis of proteins and starch, respectively) in both processing systems. The concentrations of fructans and raffinose (the major endogenous FODMAPs) were reduced by yeast and sourdough fermentation, with yeast having the greater effect. Both systems resulted in greater increases in sugars and glycerol in emmer than in bread wheat and spelt, but the significance of these differences for human health has not been established.

Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks.

During the 20th century, the economic position of oats (Avena sativa L.) decreased strongly in favour of higher yielding crops including winter wheat and maize. Presently, oat represents only ~1.3% of the total world grain production, and its production system is fragmented. Nonetheless, current interest is growing because of recent knowledge on its potential benefits in food, feed and agriculture. This perspective will serve as a further impetus, with special focus on the recently valued advantages of oats in human food and health. Five approved European Food Safety Authority (EFSA) health claims apply to oats. Four relate to the oat-specific soluble fibres, the beta-glucans, and concern the maintenance and reduction of blood cholesterol, better blood glucose balance and increased faecal bulk. The fifth claim concerns the high content of unsaturated fatty acids, especially present in the endosperm, which reduces the risks of heart and vascular diseases. Furthermore, oat starch has a low glycemic index, which is favourable for weight control. Oat-specific polyphenols and avenanthramides have antioxidant and anti-inflammatory properties. Thus, oats can contribute significantly to the presently recommended whole-grain diet. Next to globulins, oats contain a small fraction of prolamin storage proteins, called 'avenins', but at a much lower quantity than gluten proteins in wheat, barley and rye. Oat avenins do not contain any of the known coeliac disease epitopes from gluten of wheat, barley and rye. Long-term food studies confirm the safety of oats for coeliac disease patients and the positive health effects of oat products in a gluten-free diet. These effects are general and independent of oat varieties. In the EU (since 2009), the USA (since 2013) and Canada (since 2015) oat products may be sold as gluten-free provided that any gluten contamination level is below 20ppm. Oats are, however, generally not gluten-free when produced in a conventional production chain, because of regular contamination with wheat, barley or rye. Therefore, establishing a separate gluten-free oat production chain requires controlling all steps in the chain; the strict conditions will be discussed. Genomic tools, including a single nucleotide polymorphism (SNP) marker array and a dense genetic map, have recently been developed and will support marker-assisted breeding. In 2015, the Oat Global initiative emerged enabling a world-wide cooperation starting with a data sharing facility on genotypic, metabolic and phenotypic characteristics. Further, the EU project TRAFOON (Traditional Food Networks) facilitated the transfer of knowledge to small- and medium-sized enterprises (SMEs) to stimulate innovations in oat production, processing, products and marketing, among others with regard to gluten-free. Finally, with focus on counteracting market fragmentation of the global oat market and production chains, interactive innovation strategies between customers (consumers) and companies through co-creation are discussed.

Sprouting induces activation and de novo synthesis of hydrolytic enzymes that make nutrients available for plant growth and development. Consumption of sprouted grains is suggested to be beneficial for human health. Positive consumer perceptions about sprouted cereals have resulted in new food and beverage product launches. However, because there is no generally accepted definition of "sprouting," it is unclear when grains are to be called sprouted. Moreover, guidelines about how much sprouted grain material food products should contain to exert health benefits are currently lacking. Accordingly, there is no regulatory base to develop appropriate food labeling for "sprouted foods." This review describes the nutritional and technological properties of sprouted grains in relation to processing conditions and provides guidelines to optimize sprouting practices in order to maximize nutritive value. Relatively long sprouting times (3 to 5 days) and/or high processing temperatures (25 to 35 °C) are needed to maximize the de novo synthesis and/or release of plant bioactive compounds. Nutrient compositional changes resulting from sprouting are often associated with health benefits. However, supportive data from clinical studies are very scarce, and at present it is impossible to draw any conclusion on health benefits of sprouted cereals. Finally, grains sprouted under the above-mentioned conditions are generally unfit for use in traditional food processing and it is challenging to use sprouted grains as ingredients without compromising their nutrient content. The present review provides a basis for better defining what "sprouting" is, and to help further research and development efforts in this field as well as future food regulations development.

A strict gluten-free diet is currently the only treatment for the 1-2% of the world population who suffer from coeliac disease (CD). However, due to the presence of wheat and wheat derivatives in many food products, avoiding gluten consumption is difficult. Gluten-free products, made without wheat, barley or rye, typically require the inclusion of numerous additives, resulting in products that are often less healthy than gluten-based equivalents. Here, we present and discuss two broad approaches to decrease wheat gluten immunogenicity for CD patients. The first approach is based on food processing strategies, which aim to remove gliadins or all gluten from edible products. We find that several of the candidate food processing techniques to produce low gluten-immunogenic products from wheat already exist. The second approach focuses on wheat breeding strategies to remove immunogenic epitopes from the gluten proteins, while maintaining their food-processing properties. A combination of breeding strategies, including mutation breeding and possibly genome editing, will be necessary to produce coeliac-safe wheat. Individuals suffering from CD and people genetically susceptible who may develop CD after prolonged gluten consumption would benefit from reduced CD-immunogenic wheat. Although the production of healthy and less CD-toxic wheat varieties and food products will be challenging, increasing global demand may require these issues to be addressed in the near future by food processing and cereal breeding companies.

In this article wheat is described in its historical context, as a part of the Paleolithic diet and Neolithic “food package.” The health-promoting roles of wheat in the prevention of modern chronic diseases are described, as well as several wheat-related disorders. Celiac disease has particularly high negative economic and health effects. Several breeding strategies aimed at the reduction or elimination of celiac disease immunogenicity in wheat, such as selection, reconstitution, chemical- and irradiation-induced mutation, and the application of advanced techniques (including genetic modification and CRISPR-Cas9 [clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9]), have been elaborated and evaluated.

The use of oats in the human diet has decreased over the past 70 years. This is an unfortunate development from the perspective of human health because oats have a high nutritional value and contain many compounds, including β-glucan, polyphenols, vitamins, and unsaturated fatty acids that are able to maintain or may even improve consumer’s health. In addition, oats fit into a gluten-free diet of celiac disease patients because they lack the T-cell stimulating epitopes from wheat, rye, and barley. We focused on the presence of health-related compounds in oats and how their levels vary among varieties in response to the type of soil. Ten oat varieties were grown in the Netherlands in sandy and clay soil and were analyzed for the presence and concentration of healthy compounds (β-glucan, fatty acids, vitamin E, and antioxidant activity), avenin composition, total protein and starch content, and agronomical characteristics. Principal component analysis showed that genetic background influenced the levels of all analyzed components. Protein, starch, β-glucan, and antioxidants were also affected by the type of soil. The obtained results showed that this kind of analysis can be used to profile oat varieties in general and enables the selection of specific varieties with specific compound characteristics.

Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the R5-ELISA is the approved method to detect if food products can be labeled gluten-free. Because the R5-ELISA detects gluten in general, there is a demand for an improved detection method that quantifies specifically CD-epitopes. Therefore, we developed a new method for detection and quantification of CD-epitopes, based on liquid chromatography (LC) coupled to mass spectrometry (MS) in multiple reaction monitoring (MRM) mode. This method enables targeted label free comparative analysis of the gluten proteins present in different wheat varieties and species, and in wheat-based food products. We have tested our method by analyzing several wheat varieties that vary in CD-epitope content, as was shown before using immunoblotting and specific monoclonal antibodies. The results showed that a modern bread wheat variety Toronto contained the highest amounts of CD immunogenic peptides compared with the older bread wheat variety Minaret and the tetraploid wheat variety Dibillik Sinde. Our developed method can detect quantitatively and simultaneously multiple specific CD-epitopes in a high throughput manner.

Gluten meten in gehydrolyseerde en gefermenteerde voedingsmiddelen – zoals pils – is lastig. De door de Codex Alimentarius gevalideerde test onderschat het gehalte gluten in deze producten. Een uitgebreide LC-MS/MS-analyse geeft gedetailleerde informatie over de aanwezige coeliakie-stimulerende gluten in pils. Met deze gegevens is een geschikte test te ontwikkelen.

Cereals, especially wheat, may cause several food-related diseases, of which gluten intolerance (coeliac disease, CD) is the best defined: specific immunogenic epitopes, nine amino acid-long peptide sequences, have been identified from various gluten proteins. These may activate T cells, causing inflammation of the small intestine and a wide variety of other symptoms. Here, we review several breeding-related strategies aiming at reduction or elimination of such epitopes from wheat, including variety selection, re-synthesis of hexaploids, deletion of specific chromosomal fragments, RNA-interference, mutagenesis and genome editing using CRISPR/Cas9. The related issue of genetic modification (GM) is discussed. These strategies should lead to wheat food products to be used in gluten-free diets for diagnosed CD individuals and/or to strongly reduce the burden from immunogenic gluten to the non-diagnosed CD population.

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.

Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the α-gliadins. It has been shown that α-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from α-gliadins. We have analyzed over 3,000 expressed α-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived α-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the α-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations α-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.

A significant increase has been observed in the prevalence of coeliac disease (CD) during the last decades. Increased consumption of wheat and in particular gluten is considered one of the major causes. CD is a food-related disease caused by certain gluten peptides from wheat, rye, and barley containing T-cell stimulating epitopes. The use of screening protocols in wheat breeding detecting the absence of CD-epitopes (e. g. Glia-alpha 9 and Glia-alpha 20) can result in the reduction of CD-epitopes in commercial wheat and wheat based products. This approach will lead to decreased exposure, especially in many CD-patients with silent or latent CD that have not yet been diagnosed. In the long-term, this may lead to a large-scale reduction of the incidence of CD and a significant improvement of the quality of life of CD-patients.