Plant Biotechnology Journal

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

Terpenes constitute the largest and most structurally diverse class of plant secondary metabolites, with critical roles in plant-environment interactions and broad industrial applications. Although nuclear genome engineering of terpene pathways has been extensively explored, chloroplast genome engineering remains largely undeveloped, with all reported studies restricted to the model plant Nicotiana. Here we report successful chloroplast genome engineering for diterpene production in the crop plant potato (Solanum tuberosum). First we identified the trnT/trnL plastomic locus as optimal for minimizing integration-associated growth penalties. Insertion of a bifunctional diterpene synthase gene into this plastomic site yielded transplastomic plants with successful diterpene production, but with reduced growth. The co-expression of a geranylgeranyl diphosphate synthase gene to enhance precursor supply restored normal growth while elevating diterpene accumulation. Transplastomic plants were otherwise agronomically comparable to wild-type. This work expands chloroplast engineering as a viable strategy for terpene pathway engineering in crop improvement and high-value terpene production.

Buckwheat (Fagopyrum spp.) is a climate-resilient pseudocereal, yet its global adoption is constrained by the distylous self-incompatibility of common buckwheat (F. esculentum) and grain bitterness of Tartary buckwheat (F. tataricum). While the S-locus early flowering 3 (FeS-ELF3) gene has been identified as a key regulator of self-compatibility, a stable genetic transformation of F. esculentum has not yet been developed. In this study, we developed an Agrobacterium-mediated transformation protocol of F. esculentum (27% transformation efficiency) and applied it to the agronomically relevant Panda cultivar. Inactivation of the FeS-ELF3 gene using the CRISPR/Cas9 system yielded self-compatible lines with long-homostylous flowers. fes-elf3 mutants showed a distinct architectural shift: mutant plants were shorter and had shorter inflorescences than wild-type plants. Notably, these traits did not compromise yield, as the mutants produced a similar number of seeds per plant in the greenhouse. In F. tataricum, we targeted the rutin-degrading enzyme (FtRDE2), which was suspected to be a driver of grain bitterness by hydrolysing rutin into the bitter quercetin. Metabolic profiling of seeds of two ftrde2 mutant lines revealed significantly lower quercetin levels in both. Analysis of enzyme extracts confirmed the loss of rutinosidase activity; the mutant samples maintained stable rutin concentrations without the characteristic increase in quercetin observed in the control. Furthermore, organoleptic sensory evaluation of flour demonstrated that respondents identified the control as significantly more bitter than the flour from the ftrde2 mutants. These precise edits show proof-of-concept of overcoming domestication barriers: self-incompatibility and palatability, establishing a framework for rapid improvement of buckwheat.

Plants produce specialized metabolites that function in plant defense and as attractants to pollinators and symbionts. One class of specialized metabolites are terpenoids that are synthesized from universal C5 building blocks via activities including terpene synthases, cytochromes P450, and glycosyl transferases. Some terpenes are highly valued for their use as insect repellants, fragrances, antimicrobial compounds, low calorie sweeteners, flavors, and medicines. Low abundance in target tissues, present in complex mixtures, as well as challenging extraction logistics are barriers to economic sustainable production of these compounds from their native species. While heterologous expression of terpenoid biosynthetic genes is feasible, the potential derivation of the products into conjugates via endogenous cytochromes P450 and glycosyl transferases limits this approach. In this project, we used multiplex gene editing technologies to overcome these challenges by creating novel tomato chassis with altered terpenoid biosynthetic capacity in fruit. Excluding central metabolic genes to minimalize impacts on growth and development, we selected 23 known and potential terpene-related genes expressed specifically in the fruit for gene editing. Fruit production and metabolic profiles of three chassis lines with alterations in the major classes of fruit specialized metabolites indicate loss of these genes is tolerated for fruit production. These combinatorial knockouts also showed modulation of native carbon reallocation toward endogenous sinks beneficial for a biosynthetic chassis. Establishing metabolite-modified fruit chassis demonstrates efficient combinatorial editing of entire branches of plant specialized metabolism, facilitating engineering of heterologous terpenes of industrial interest in tomato fruit.

Polyploid genome assembly presents unique challenges due to extensive heterozygosity and complex haplotype structure. We report a haplotype-resolved, chromosome-scale assembly of Regen-SY27x, a genotype of autotetraploid alfalfa (Medicago sativa), which is widely used for genetic modification because of its excellent regenerative capacity in tissue culture. Using PacBio HiFi long reads, Omni-C scaffolding, and linkage map guided phasing, we generated a 3.2 GB assembly comprising four haplotypes with high contiguity and completeness. Kmer-based validation confirmed accurate haplotype separation, while linkage map integration and dotplot analysis identified and corrected chimeric scaffolds. Gene annotation yielded 221,688 protein-coding genes, with more than 99% assigned to pseudochromosomes. Repetitive elements accounted for 62.7% of the genome, dominated by long terminal repeat retrotransposons and a high fraction of Helitrons. The spatial enrichment of Helitrons within gene-dense distal chromosome arms underscores their pivotal role as key drivers of genomic innovation and gene family expansion. We identified 3,696 nucleotide-binding leucine-rich repeat R genes, with Toll/interleukin-1 receptor-like and Rx-type subclasses forming large tandem clusters across haplotypes. Comparative analyses revealed strong macrosyntenic conservation among Regen-SY27x and the publicly available Chinese alfalfa genomes but extensive structural variation both within Regen-SY27x haplotypes and between Regen-SY27x and the Chinese genotypes with tens of thousands of duplications, inversions, and translocations detected. These results demonstrate that a single autotetraploid individual captures extensive structural diversity, but individuals from different populations vary greatly. The Regen-SY27x assembly provides a foundational genomic resource for investigating polyploid genome evolution and identifying genetic variation relevant to biological and agronomic improvement in alfalfa. Article SummaryThis study presents the first chromosome-scale, haplotype-resolved genome assembly of the US alfalfa germplasm, Regen-SY27x, a key alfalfa genotype used widely for genetic engineering. We integrated HiFi long reads, Omni-CTM scaffolding, and linkage map-guided phasing to reconstruct all four haplotypes of this complex autotetraploid. Our results identified 221,688 protein-coding genes and reveal immense intra-individual structural variations dominated by small duplications. This high-quality reference serves as a foundational tool for the alfalfa community, enabling researchers to link complex structural diversity with agronomic traits and further enhance the biotechnological potential of this essential forage crop.

Sex pheromones are eco-friendly agents to control pest insects. Yet, the cost of synthetic pheromone production has restricted their use essentially to high value crops. Plant-based bioproduction of pheromone compounds offers a promising and sustainable alternative, opening up for the deployment of pheromone-mediated pest control also in row crops. The rice leaffolder Cnaphalocrocis medinalis uses C18 pheromone compounds and is a major rice pest in South China. We demonstrate that the fatty acid precursor of the main C18 pheromone component of C. medinalis, (Z)-13-octadecenal, can be biotechnologically produced using the oilseed crop Camelina sativa. First, we used deuterium-labelling to establish an elongation-based pathway for pheromone precursor formation from hexadecanoic (palmitic) acid via desaturation to (Z)-11-hexadecenoic acid and elongation to (Z)-13-octadecenoic acid. Using transient expression in Nicotiana benthamiana, we identified a minimal pathway consisting of a {Delta}11 desaturase (CmedDES1), an elongase (CmedELO1), and a fatty acyl reductase (CmedFAR2) for the heterologous biosynthesis of long-chain pheromone monounsaturated C18 precursors in planta. Co-expression of the insect desaturases CmedDES1 or CsupYPAQ with the Camelina elongase CsaKCS1 in Nicotiana benthamiana resulted in higher production of Z13-18 products compared to co-expression with insect elongases. We finally engineered Camelina sativa seeds through seed-specific co-expression of the Z11-16-forming insect desaturase CsupYPAQ and CsaKCS1 elongase, enabling the accumulation of C18 fatty acid pheromone precursors. Our study paves the way for sustainable production of the sex pheromone of C. medinalis and other C18 monounsaturated pheromone precursors for crop protection.

Budding yeast Saccharomyces cerevisiae is a workhorse chassis for producing added food and agricultural compounds. However, building multi-enzymatic pathways for these chemicals often requires iterative genomic integration, underscoring the need for efficient, rapid genome-editing tools that can reliably target transcriptionally active chromosomal regions. In this study, to accelerate strain construction, we established a genome-editing toolkit to rapidly engineer eight loci, highly expressed hot-spots, but nonessential genomic sites suitable for stable pathway assembly. Our approach integrates three key design features: (i) selectable markers to enable rapid screening of edited cells, (ii) extended homology arms that leverage the yeast homology-directed repair machinery for robust genomic integration, and (iii) co-delivery of Cas9 and guide RNAs to promote efficient double-stranded DNA breaks at specific integration sites. The sequence independence of FASTOP relies on the release of integration cassettes from integrative vectors, mediated by restriction digestion at two flanking multiple-cutting sites in the integration module to minimize the risk of introducing sequence errors during PCR amplification of the integration cassettes. Following the introduction of a fluorescent reporter cassette, we observed high integration efficiencies across the target sites. We then integrated the biosynthetic pathway of plant-derived flavonoid naringenin into the hot-spots of the yeast genome using the FASTOP toolkit. Our results demonstrated that upon expressing the five essential genes in simple shake flask culture, naringenin production reached 505.7 mg/L, representing a significant (69-fold) increase over previously reported titers for comparable minimal heterologous pathways in S. cerevisiae. Together, the FATSOP toolkit provides a user-friendly platform for reliably modifying hot-spot loci to rapidly construct multi-enzymatic metabolic pathways in S. cerevisiae, while achieving high production levels for high-value food-relevant metabolites.

Aegilops species are the closest wild relatives of wheat and an important reservoir of genetic diversity for its improvement. Despite their potential, many Aegilops genomes remain poorly characterized. Here we present high-quality assemblies of 18 diploid, tetraploid, and hexaploid Aegilops genomes, which, along with the previously published genomes, complete the production of reference assemblies for all 25 genomes in this genus. Assembly sizes ranged from 5.24 Gb in diploids to 12.65 Gb in hexaploids, with scaffold N50 values up to 749.2 Mb. Gene annotation identified 53,035-156,779 protein-coding genes, of which 21,865-60,490 were classified as high-confidence. Orthogroup-based pangenome analysis across the 25 Aegilops genomes identified 80,521 orthogroups, including 15,809 core, 61,735 dispensable, and 2,977 species-specific orthogroups, highlighting substantial gene content variation among genomes. Phylogenetic analysis of 63 Triticum and Aegilops genomes/subgenomes based on near single-copy orthologs defines the phylogenetic relationships within the Triticum/Aegilops complex and confirms diploid progenitors of polyploid lineages. Ae. mutica (T) and Ae. speltoides (S) belong to the B lineage while the remaining Sitopsis grouped within the D lineage. Structural variation analyses using diploid progenitors as references revealed extensive large-scale rearrangements following polyploidization, emphasizing the dynamics of their evolution. Transposable element (TE) annotation further highlighted subgenome-specific TE expansions and contractions, providing insights into the mechanisms shaping genome structure after polyploidization. Collectively, these genomic resources provide a comprehensive framework for exploring Aegilops diversity, understanding polyploid evolution, and accelerating wheat improvement.

Haploid induction is a key component of doubled haploid technology and an increasingly valuable tool for plant breeding, genome editing, and clonal seed production. While in planta haploid induction through haploid inducer lines offers significant advantages over in vitro approaches, its application remains limited in many crop species. Previously, disruption of the sperm cell-expressed KOKOPELLI (KPL) gene was shown to induce maternal haploids in Arabidopsis thaliana. Here, we report the creation of novel haploid inducer lines in two globally important crops, maize (Zea mays), a major staple food crop, and tomato (Solanum lycopersicum), a widely cultivated vegetable crop. Using targeted genome editing, we generated mutations in KPL orthologs and demonstrated that loss of KPL function confers haploid induction capacity, enabling the production of haploid seedlings in both species. These findings establish KPL as a conserved target to trigger haploid induction and expand the genetic toolbox available for haploid inducer development in crop species.

Tomato wild relatives are valuable genetic resources for trait discovery and understanding the genetic basis of fruit metabolism and quality. Yet, only a fraction of naturally occurring variation has been exploited. Here, we performed metabolite profiling of two large Backcross Inbred Line populations derived from crosses between the wild species S. pennellii accession LA5240 (Lost) and cultivated genotypes LEA (determinate) and TOP (indeterminate), including [~]1400 and [~]500 lines, respectively. High-resolution mapping identified enormous metabolic quantitative trait loci (mQTL), including a new locus on chromosome 12 associated with fruit sucrose accumulation that harbours INVERTASE INHIBITOR 3 (SlINVINH3) protein. Comparative analysis indicated that SlINVINH3 is highly expressed in wild S. pennellii 0716 fruit, whereas a six-amino acid deletion is present in its coding sequence compared with S.pennellii LA5240 and S. lycopersicum. We further demonstrated that in SlINVINH3-overexpressing tomato plants, only the S. pennellii LA5240 allele led to increased sucrose, accompanied by reduced fructose and glucose levels. Furthermore, the large population size enabled us to assess the epistatic interactions, with approximately 40% of interactions being more-than-additive and 60% less-than-additive. Our results demonstrate the power of permanent exotic populations to reveal hidden metabolic diversity and provide an approach for improving fruit quality through targeted breeding and metabolic engineering.

Viroids are plant infectious agents that threaten agricultural production. Current viroid detection methods rely on RT-PCR-based assays, which require specialized laboratory equipment and can sometimes produce false-negative results or non-specific amplification due to the high sequence conservation among closely related viroid species. CRISPR-based diagnostics, particularly Cas12-based systems for DNA detection (DETECTR) and Cas13a-based systems (SHERLOCK) for RNA detection, have emerged as powerful tools for nucleic acid diagnostics. However, most existing workflows still rely on target amplification and, in the case of Cas13a systems, require additional in vitro transcription steps, limiting their simplicity and direct applicability for plant diagnostics. Here, we developed a direct amplification-free Cas13a-based detection platform for viroids using potato spindle tuber viroid (PSTVd) as a model. We optimized CRISPR RNA (crRNA) design, identified inhibitory effects of plant total RNA on readout signal, and employed simplified viroid RNA enrichment workflows enabling robust detection in plant samples. The system further supported both PSTVd-specific and broad-spectrum pospiviroid (genus Pospiviroid) detection and was successfully extended to avocado sunblotch viroid (family Avsunviroidae), demonstrating its adaptability across distinct viroid families. Together, these results establish a practical and modular Cas13a-based platform, not only for viroid diagnostics, but also for broader applications in RNA-derived plant pathogen detection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/736049v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1d04170org.highwire.dtl.DTLVardef@1783aa3org.highwire.dtl.DTLVardef@51baa7org.highwire.dtl.DTLVardef@1b542b9_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementA simplified RNA enrichment workflow combined with CRISPR-Cas13a enables direct, amplification-free detection of plant viroids. The assay supports early and reliable diagnosis across different tomato varieties and provides a practical strategy for improving molecular detection of plant pathogens.

Celiac disease (CeD) is an immune-mediated condition triggered by wheat gluten in genetically predisposed individuals. The immune reaction in people with CeD is driven by particular gluten amino acid sequences, or immunogenic epitopes. Some of these epitopes elicit strong immune responses in the majority of CeD patients and are designated as immunodominant epitopes. Previous research has shown correlations between the amount of immunogenic wheat epitopes consumed and the onset of CeD, suggesting that reducing wheat immunogenic epitopes may reduce CeD incidence at the population level. Gluten consists of gliadins and glutenins, with gliadins having the majority of the immunodominant epitopes and glutenins playing a major role in dough strength and breadmaking quality (BMQ). This study used radiation-induced deletions, chemical mutagenesis, and natural variation in wheat (Triticum aestivum) to generate genetic stocks with reduced immunogenic epitope content. Most lines were developed in the wheat cultivar Summit, for which we produced a full genome assembly and annotation. We used exome capture to characterize these deletions and identify prolamins located within and outside the deletions. We combined different deletions and developed molecular markers to facilitate their deployment. For chromosome arms 1BS and 1DS, we generated two alternative lines: one lacking immunogenic epitopes for the development of CeD-safe genetic stocks for research purposes, and another retaining selected glutenins for breeding commercial lines with reduced immunogenicity and adequate BMQ. By making these non-transgenic genetic stocks publicly available, we aim to accelerate the development of wheat varieties with reduced immunogenicity and, eventually, a fully CeD-safe wheat.

The ability to insert, delete, or modify genetic information is crucial for mechanistic studies and biotechnological applications. However, efficient genetic transformation remains a major bottleneck for research in maize and many other crops. Here, we report an optimized Agrobacterium-mediated transformation platform based on systematic reconstruction of tissue culture handling in the maize inbred line A188. Refinement of callus induction, selection, and regeneration substantially improved recovery of transgenic plantlets. To distinguish independent T-DNA insertion events, we developed TAFLP (T-DNA Amplified Fragment Length Polymorphism), a simple and inexpensive assay that amplifies T-DNA flanking sequences and can be performed using standard laboratory equipment. Our enhanced transformation pipeline was also applicable to the inbred line B104 as well as to hygromycin and G418 selection systems, demonstrating broad utility of our method. We validated the platform for CRISPR/Cas9 mutagenesis and reporter line generation. Using this approach, we isolated new loss-of-function alleles of MAC1 and ACOZ1 and generated reporter lines for analysis of meiotic protein dynamics. Together, these results provide a broadly applicable framework for improving maize transformation efficiency and recovering independent transgenic and genome-edited events.

Targeted epigenetic modifications of specific gene regulatory regions have the potential to confer beneficial traits for crop improvement. Two recently developed CRISPR/Cas9-based epigenome editing tools were tested in transgenic cassava to target cytosine methylation to the promoter region of MeSWEET10a, a necessary gene for infection by the Cassava bacterial blight pathogen, Xanthomonas phaseoli pv. manihotis. The two systems leverage unique methyltransferases, and each induced distinct DNA methylation profiles at the targeted site, decreased effector-triggered MeSWEET10a expression, and attenuated water-soaking symptoms in inoculated leaves. Further, DNA methylation was simultaneously targeted, in addition to MeSWEET10a, to two susceptibility genes for Cassava brown streak virus. Relative levels of de novo DNA methylation at the three loci were inversely correlated with DNA methylation-antagonizing H3K4me3 marks. Finally, an initial assessment of DNA methylation after one generation indicated specific inheritance of CpG methylation that was unstable in the absence of the methyltransferase systems.

Auxin is a key phytohormone that regulates all aspects of plant growth, development, and environmental responses, making the precise analysis of its distribution and signaling essential for understanding plant adaptation and physiological processes. However, despite the agricultural importance of oilseed rape (Brassica napus), the lack of robust, species-specific molecular tools limits detailed studies of hormone signaling in this crop. Here, we developed and characterized reporter systems for the sensitive visualization and quantification of auxin distribution and signaling in B. napus. The DR5cc auxin signaling reporter and a novel synthetic auxin-responsive reporter, BIP3, assembled from promoter fragments of three oilseed rape IAA genes, were generated to drive GUS expression. In hairy roots, both reporters showed auxin-responsive expression in the root apical meristem that became broader after auxin treatment. In transgenic seedlings, flowers at anthesis, and 12-day-old embryos, DR5cc exhibited a more defined expression pattern than BIP3. To monitor real-time auxin dynamics under abiotic stress, DR5cc fluorescent reporters were employed in hairy roots. Mannitol and NaCl treatments induced a time-dependent increase in fluorescence, peaking at 6-12 h before returning to basal levels after 24 h. Furthermore, dual-reporter assays enabled simultaneous monitoring of auxin and cytokinin signaling, revealing distinct hormone-specific spatial responses in hairy roots. Finally, we established a quantitative DII (qDII) reporter system using degron domains from B. napus Aux/IAA proteins, providing a high-resolution quantitative readout of auxin depletion. Together, these reporter systems enable spatial, temporal, and quantitative analyses of auxin dynamics during development and stress adaptation in oilseed rape.

Legumes include some of the worlds most significant crop species, such as cowpea (Vigna unguiculata), a subsistence crop widely grown in sub-Saharan Africa. Despite their importance, legume crop improvement is hindered by a lack of high-resolution expression data, particularly for reproductive tissues and cell types. Here, we report on VigExp, a tool for visualising cowpea gene expression datasets. We demonstrate its utility across a range of vegetative and reproductive cell types of varieties IT97K-499-35 and IT86D-1010, which exhibit 93.75% protein sequence conservation and are amenable to stable transformation. This includes previously published transcriptomes of vegetative, floral and seed tissues, combined with developmentally staged male and female reproductive tissues. Also integrated are novel transcriptomes of laser-captured cell types covering reproductive development from meiosis to early embryo formation post-fertilisation. Spatial expression patterns and transcript levels can be visualised through an electronic fluorescent pictograph (eFP) browser. Validated by RT-qPCR, in situ hybridisation, transgenic, and CRISPR gene editing analyses, the predictive accuracy of VigExp matches prior cowpea functional study observations. Critical genes for nodule development and regulation were also identified and their expression patterns established in cowpea. Novel reference genes, constitutively expressed gene promoters for visualization makers/gene-editing, and tissue and cell specific gene promoters for targeting these regions, are identified. The A-type cyclin, VuTAM2, was also identified, with a critical role in male meiosis established. Collectively, VigExp represents an adaptable and updatable resource to support crop improvement in cowpea and other legumes, which are often highly syntenic with respect to genome composition.

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.

Common wheat (Triticum aestivum L.) is a cornerstone of global food security. However, the standard reference genome, IWGSC RefSeq v2.1, is derived from wheat cultivar Chinese Spring, a historically important lineage that is not representative of modern cultivated wheat. In contrast, Pavon 76 is a CIMMYT Green Revolution wheat cultivar grown worldwide and is present in the pedigrees of numerous elite wheats, as well as in the genetic background of over 600 cytogenetic stocks used in studies ranging from recombination to male sterility. As no Pavon 76 reference has been reported, interpretation of these Pavon 76-derived stocks has relied on the genetically distant IWGSC RefSeq v2.1. To address this gap, we generated PacBio HiFi reads and Hi-C read pairs to assemble Pavon 76s genome. The final assembly comprises the expected 21 chromosomes, spans 15 Gb, and has an N50 of 710 Mb. Annotation identified 167,201 genes, of which 89.5% were functionally annotated. Comparative sequence and gene synteny analysis between Pavon 76 and IWGSC RefSeq v2.1 revealed significant structural divergence, establishing that IWGSC RefSeq v2.1 is not fully representative of modern wheats. This robust Pavon 76 assembly establishes the first chromosome-scale de novo assembly of a CIMMYT-derived wheat and provides a foundation for future genetic and genomic studies.

Flowering time strongly influences crop adaptation, plant architecture, generation turnover, and breeding efficiency, but the functional organization of florigen genes remains poorly resolved in many polyploid orphan crops. Quinoa (Chenopodium quinoa) is a climate-resilient allotetraploid crop with extensive variation in flowering behavior, and genome analyses have identified multiple FLOWERING LOCUS T (FT)-like homologs. However, genome sequence and expression information alone cannot determine which homologs provide effective florigenic output in planta. Here, we combined apple latent spherical virus-mediated overexpression (VOX) and virus-induced gene silencing (VIGS) in quinoa with heterologous expression in Arabidopsis thaliana, domain-swapping analyses, and cross-germplasm validation to functionally dissect quinoa FT activity. Although several CqFT homologs were transcriptionally induced during the floral transition, their functional outputs were markedly unequal. CqFT1A and CqFT1B-1 acted as the major florigenic activators: overexpression of either gene induced rapid and synchronized flowering, whereas CqFT1-VIGS delayed flowering. In contrast, CqFT2A and CqFT2B retained only weak flowering-promoting activity, whereas CqFT1B-2 showed no detectable promotive effect under the conditions tested, revealing a clear functional hierarchy among transcriptionally induced CqFT homologs. Domain-swapping analyses showed that C-terminal variation contributes to, but does not fully explain, functional divergence among CqFT homologs. In late-flowering highland lines, elevated FT input accelerated flowering, induced coordinated floral transition, and shortened the time to viable seed production. These findings identify CqFT1A and CqFT1B-1 as the major florigenic activators in quinoa and establish a functional genomics framework for resolving and modulating flowering-time control in polyploid orphan crops.

Durum wheat (Triticum turgidum subsp. durum) is a globally important crop for pasta and couscous production. Chili and Mahmoudi are historically significant Tunisian landraces valued for exceptional grain quality, high protein content, and adaptation to arid Mediterranean climates. Yet no high-quality reference genome assemblies were available for either variety before this work. We assembled both genomes using publicly available PacBio HiFi long reads and Illumina Hi-C proximity ligation data deposited under NCBI BioProject PRJNA1420514. HiFi reads were assembled with hifiasm v0.25.0 in primary mode, and Hi-C scaffolding was performed with YAHS v1.2a.2 after read alignment with BWA-MEM. Assembly quality was assessed with QUAST v5.3.0 and BUSCO v5.8.0 (embryophyta_odb10 lineage). The Chili assembly spans 10.84 Gbp across 3,472 scaffolds with a scaffold N50 of 844.9 Mbp and BUSCO completeness of 99.4%. The Mahmoudi assembly spans 10.70 Gbp across 3,258 scaffolds with a scaffold N50 of 2,072 Mbp and BUSCO completeness of 99.3%. Merqury v1.3 confirmed high base accuracy (QV 68.0 for Chili, QV 68.3 for Mahmoudi) and k-mer completeness (>98% for both). Independent validation with wfmash confirmed 98.6% mean alignment identity across 11,172 chromosome-to-reference alignments. Post-assembly characterization of GC profiling, centromere architecture, ribosomal DNA arrays, and structural variation revealed extensive genome-level detail. Both assemblies substantially exceed the contiguity of existing durum wheat references and represent the first chromosome-scale-contiguity genomic resources for North African durum wheat landraces. The Mahmoudi accession carried a putative 2B-3B homeologous fusion on chromosome 3B (4,710 Mbp), a structural novelty in an ancient Tunisian landrace. Assembly and Pseudomolecules available from Zenodo (10.5281/zenodo.20366290). The workflow was executed reproducibly on the public Galaxy Europe platform, demonstrating that reference-quality plant genome assembly is achievable without local HPC infrastructure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/727644v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@159c9d4org.highwire.dtl.DTLVardef@1d1a0deorg.highwire.dtl.DTLVardef@19853dborg.highwire.dtl.DTLVardef@1a96018_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst chromosome-scale-contiguity assemblies for Chili and Mahmoudi landraces C_LIO_LIChili genome: 10.84 Gbp, scaffold N50 844.9 Mbp, BUSCO 99.4% C_LIO_LIMahmoudi genome: 10.70 Gbp, scaffold N50 2,072 Mbp, BUSCO 99.3% C_LIO_LIMerqury QV ~68 confirms base accuracy, >98% k-mer completeness C_LIO_LI>140-fold scaffold N50 improvement over Svevo v1 reference C_LI