Frontiers in Plant Science

BackgroundPollinosis, or hay fever, is the most prevalent allergic disorder. Assessing the impact of real-world pollen exposure on symptoms remains challenging due to the extensive efforts required at the patient level. ObjectivesWe explore the potential of wastewater-based epidemiology (WBE) to investigate the impact of exposure to specific pollen taxa on symptoms and to assess self-medication patterns of second-generation antihistamines at a population-scale. MethodsIn Zurich (Switzerland), 279 wastewater samples were collected from 2021-2023. Each sample represents a 24-hour period with excreta from approximately 471,000 individuals. Eleven antihistamine markers were analyzed in the samples using liquid chromatography high-resolution mass spectrometry. The relationship between antihistamine loads in wastewater and concentrations of airborne pollen (47 taxa) was investigated using multivariate linear regression analysis. ResultsThe loads of three second-generation antihistamines in wastewater showed strong day-to-day variation correlating with airborne pollen patterns. About 50% of the annual fexofenadine consumption was linked to acute pollen exposure, 20% to baseline consumption during pollen season, and 30% was independent of pollen. Alder, birch, grasses, hornbeam, plane, and plantain explained most of the variance in consumption (R2 = 0.82), with grass pollen alone causing a quarter of the annual consumption. Increased fexofenadine consumption during periods without elevated concentrations of common allergenic pollen suggests the presence of additional triggers for allergy symptoms, potentially yew pollen. ConclusionsOur study demonstrates that WBE can effectively capture substantial day-to-day variation in antihistamine consumption caused by pollen exposure symptoms. As such, WBE is an objective, cost-effective, and questionnaire-independent method for investigating pollinosis at a population-scale.

HaHB11 is a sunflower transcription factor previously described as conferring improved yield to maize hybrids and lines. Here we report that transgenic HaHB11 maize lines exhibited a better performance funder waterlogging, both in greenhouse and field trials carried out during three growth cycles. One of these trials was particularly affected by a strong storm during flowering, causing severe defoliation. Controlled defoliation assays indicated that the transgenic genotypes were able to set more grains than controls. Hybrids were generated by crossing B73 HaHB11 lines with the contrasting Mo17 lines and tested in the field. These hybrids exhibited the same beneficial traits as the parental lines when compared with their respective controls. Waterlogging tolerance coursed via the root architecture improvement, including more xylem vessels, reduced tissue damage, less superoxide accumulation, and altered carbohydrate metabolism compared to controls. Multivariate analyses corroborated the robustness of the differential traits observed. Furthermore, canopy spectral reflectance data, computing 29 vegetation indices associated with biomass, chlorophyll, and abiotic stress, helped to identify genotypes as well as their growing conditions. Altogether the results reported here indicate that this sunflower gene constitutes a suitable tool to improve maize plants for environments prone to waterlogging and/or wind defoliation. One sentence summaryPhenotyping and big data analyses indicate that the transcription factor HaHB11 confers waterlogging and defoliation tolerance, and increased yield to maize lines and hybrids in all tested conditions.

Hairy-root transformation is widely used to generate transgenic plant roots for genetic functional characterisation studies. However, transformation efficiency can be limited, largely due to the use of binary vectors. Here, we report on the development of novel integrative vectors that significantly increase the transformation efficiency of hairy roots. This includes pHGUS7, for promoter::reporter visualisation studies, and pHOG13, for genetic insertion and overexpression studies. These vectors have been designed to simplify cloning workflows, enhance the selection of positively transformed Agrobacterium colonies, and increase the transformation efficiency and ease of selection of genetically modified hairy roots. To demonstrate the efficacy of the new vectors, Too Much Love (TML) encoding genes acting in the Autoregulation Of Nodulation (AON) pathway of soybean were investigated. Both constructs provided significantly higher transformation rates than the binary vector control, often resulting in >70% of the roots being transformed. Overexpression of each individual TML encoding gene (GmTML1a, GmTML1b and GmTML2) using pHOG13 resulted in a significant reduction in nodule number, demonstrating the role of all three in inhibiting nodule organogenesis. Moreover, reporter-fusions with the promoter of each TML encoding gene using pHGUS7 revealed that each exhibits a unique pattern of expression in nodules, with GmTML1b displaying considerably stronger expression than GmTML1a or GmTML2. Taken together, these results demonstrate the utility and efficiency of the new pHOG13 and pHGUS7 integrative vectors in hairy-root transformation, and improve our understanding of the critical TML-encoding genes in soybean nodulation control.

The development of genetically modified (GM) or gene edited (GE) turfgrass requires transformation systems that are both efficient and broadly applicable across genotypes. However, traditional Agrobacterium-mediated callus culture methods remain limited by low transformation efficiency, extended culture durations, and strong genotype dependence. In this study, we compare a modified classical callus culture protocol with an approach that incorporates the developmental regulator genes WUSCHEL2 (wus2) and BABY BOOM (bbm), along with an inducible Cre recombinase and a dual luciferase assay to test variable promoter strengths in perennial ryegrass (Lolium perenne L.). We show that traditional protocols failed to regenerate plants, despite successful callus formation and transgene expression. In contrast, the developmental regulator system enabled efficient callus induction and plant regeneration independent of genotype. This optimized protocol significantly reduces the time and genotype constraints of perennial ryegrass transformation, offering a practical platform for advanced genetic engineering applications of an important turf and forage grass.

The spring barley cultivar Golden Promise (GP) is the major reference genotype for transformation due to its high transformability and availability of a reference genome. However, GP is characterized by a long generation cycle and stress susceptibility under non-optimal growth conditions because it carries a mutation at the floral inducer Photoperiod-H1 (Ppd-H1). Previously, we showed that a GP introgression line, Golden Promise-fast (GP-fast), generated by introducing the wild-type Ppd-H1 allele from the winter barley cultivar Igri, exhibits early flowering and improved stress resilience. In this study, we generated a fast-cycling genotype, Golden Promise-rapid (GP-rapid), isogenic to GP with high transformation efficiency. We conducted two backcrosses of GP-fast to reduce the residual Igri genome. The resulting genotype contains only a single introgression of approximately 0.6 Mbp at the Ppd-H1 locus on chromosome 2H. Under speed breeding conditions, its generation time was reduced to 63 days (25% shorter than GPs 84 days). Parallel transformation of GP, GP-fast, and GP-rapid using CRISPR/Cas9-mediated genome editing of Ppd-H1 revealed high regeneration and transformation efficiencies of GP-rapid, comparable to GP. Overall, we report on the development of a fast-cycling GP isogenic line as a research tool for efficient generation of transgenic and gene-edited barley plants. HighlightsA new fast-cycling barley genotype, GP-rapid, reduces generation time by 25% while retaining high transformation efficiency, advancing functional genomic studies in barley.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the fungal powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the powdery mildew disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calcium-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT LW/RR mutant variants in comparison to the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Fungal pathogens pose a major threat to Cannabis sativa production, requiring safe and effective management procedures to control disease. Chitin and chitosan are natural molecules that elicit plant defense responses. Investigation of their effects on C. sativa will advance understanding of plant responses towards elicitors and provide a potential pathway to enhance plant resistance against diseases. Plants were grown in the in vitro Root-TRAPR system and treated with colloidal chitin and chitosan. Plant morphology was monitored, then plant tissues and exudates were collected for enzymatic activity assays, phytohormone quantification, qPCR analysis and proteomics profiling. Chitosan treatments showed increased total chitinase activity and expression of pathogenesis-related (PR) genes by 3-5 times in the root tissues. In the exudates, total peroxidase and chitinase activities and levels of defense proteins such as PR protein 1 and endochitinase 2 were increased. Shoot development was unaffected, but root development was inhibited after chitosan exposure. No significant effects on plant defense were observed upon chitin treatment. These results indicate that colloidal chitosan significantly promoted production and secretion of plant defense proteins in C. sativa root system and could be used as a potential elicitor, particularly in hydroponic scenarios to manage crop diseases. HighlightChitosan induces defense protein productions and secretions in the root tissues and exudates of C. sativa, offering a potential pathway to enhance plant resistance against fungal attack.

Neonicotinoid seed treatments protect maize during early growth but can induce phytotoxicity that intensifies during storage. Despite recognized genotypic variation in tolerance, standardized phenotyping methods are lacking. We evaluated nine commercial maize hybrids under three seed treatments (control, one neonicotinoid [1N], and two neonicotinoids [2N]) across two storage periods (0 and 6 months at 25 {degrees}C) using germination, accelerated aging, and cold tests. A Seed Treatment Tolerance Index (STTI) was analyzed through hierarchical clustering, principal component analysis, and multivariate analysis of variance. Results showed a significant triple interaction among genotype, seed treatment, and storage. Hybrids from female line A maintained STTI above 0.95, while female C hybrids showed germination reductions up to 48 percentage points and vigor losses up to 90 percentage points under 2N after six months. Tolerance was associated with hydrogen peroxide regulation by catalase and ascorbate peroxidase. The STTI proved a reliable tool for classifying genotypic tolerance, with direct applications for breeding programs and seed industry logistics.

BackgroundOver the last decade, progress in DNA sequencing technologies and assembly methods allowed plant scientists to move beyond the use of model organisms and work directly on the genomes of the major crops. Forage grass research can also benefit from this revolution, enabling progress in population genetic studies, functional biology, and genomics-assisted breeding. Due to its large genome size and high repeat content, so far only incomplete and fragmented assemblies are available for the grasses of the Lolium and Festuca species complex. FindingsHere, we report a highly contiguous draft assembly of Italian ryegrass (L. multiflorum Lam.), spanning 4.5 Gb and with a N50 of 3 Mb, containing ~70,000 gene models. Thanks to its relatedness to barley, 78% of the assembly was anchored on seven pseudomolecules. The high heterozygosity of the plant allowed obtaining a diploid assembly - i.e. across 95% of the assembly, both alleles were assembled on separate sequences. This feature allowed unraveling a very high amount of intergenic sequence variation between allelic sequences. ConclusionsWe present a nearly complete genome assembly of a genotype used in contemporary Swiss forage grass breeding programs. This work shows how genomic research has improved, allowing to decode the genetic code of large, complex, and heterozygous plants. It also allows the functional characterization of the ryegrass gene repertoire and the large-scale development of molecular markers. Furthermore, it paves the road for a reference-based characterization and exploitation of the genetic variation within the Lolium and Festuca species complex.

Maize (Zea mays) is the most widely produced crop in the world, and conventional production requires significant amounts of synthetic nitrogen fertilizer, which has negative economic and environmental consequences. Maize landraces from Oaxaca, Mexico, can acquire nitrogen from nitrogen-fixing bacteria that live in a mucilage secreted by aerial nodal roots. The development of these nodal roots is a characteristic traditionally associated with the juvenile vegetative stage of maize plants. However, mature Oaxacan landraces develop many more nodes with aerial roots than commercial maize varieties. Our study shows that Oaxacan landraces develop aerial roots during both the juvenile and adult vegetative phases and even during early flowering under greenhouse and field conditions. Surprisingly, the development of these roots was only minimally affected by soil nitrogen and ambient humidity. These findings are an important first step in developing maize varieties that can reduce fertilizer needs in maize production across different environmental conditions.

Premise of the ResearchPlants remain underrepresented among species with sequenced mitochondrial genomes (mitogenomes), due to the difficulty in assembly with short-read technology. Invasive species lag behind crops and other economically important species in this respect, representing a lack of tools for management and land conservation efforts. MethodologyThe mitogenome of Microstegium vimineum, one of the most damaging invasive plant species in North America, was sequenced and analyzed using long-read data, providing a resource for biologists and managers. We conducted analyses of genome content, phylogenomic analyses among grasses and relatives based on mitochondrial coding regions, and an analysis of mitochondrial single nucleotide polymorphism in this invasive grass species. Pivotal ResultsThe assembly is 478,010 bp in length and characterized by two large, inverted repeats, and a large, direct repeat. However, the genome could not be circularized, arguing against a "master circle" structure. Long-read assemblies with data subsets revealed several alternative genomic conformations, predominantly associated with large repeats. Plastid-like sequences comprise 2.4% of the genome, with further evidence of Class I and Class II transposable element-like sequences. Phylogenetic analysis placed M. vimineum with other Microstegium species, excluding M. nudum, but with weak support. Analysis of polymorphic sites across 112 accessions of M. vimineum from the native and invasive ranges revealed a complex invasion history. ConclusionsWe present an in-depth analysis of mitogenome structure, content, phylogenetic relationships, and range-wide genomic variation in M. vimineums invasive US range. The mitogenome of M. vimineum is typical of other andropogonoid grasses, yet mitochondrial sequence variation across the invasive and native ranges is extensive. Our findings suggest multiple introductions to the US over the last century, with subsequent spread, secondary contact, long-distance dispersal, and possibly post-invasion selection on awn phenotypes. Efforts to produce genomic resources for invasive species, including sequenced mitochondrial genomes, will continue to provide tools for their effective management, and to help predict and prevent future invasions.

Previous studies determined that maize mutants in miR394-regulated genes, ZmLCR1 and ZmLCR2, are more tolerant than wild-type seedlings to prolonged periods of drought. In order to evaluate the effect of these mutations in a genetic background more similar to that of maize commercialization, in this work we evaluate growth of double mutant hybrid plants in W22/B73 genetic background and also evaluated plant fitness, flowering and yield in experimental plots under two watering regimes, and compared the nutritional content of wild-type and mutant hybrids. Our results show that mutant hybrid seedlings exhibit improved physiology under normal watering conditions as well as in drought conditions, exhibiting an increase in epicuticular wax content, unaltered membrane damage in drought and lower ROS production, supporting higher survival after severe drought for double mutant hybrid seedlings. We also established that the hybrid mutants grown in typical agricultural conditions do not show differences in flowering time or in physiological and nutritional aspects, but they present a higher yield in comparison to wild-type W22/B73 hybrids, as determined by higher ear weight and number of kernels per ear in mutant hybrids, when grown in field rainfed conditions. HighlightsO_LIDouble mutants in miR394-regulated genes, ZmLCR1 and ZmLCR2, show enhanced drought tolerance in hybrid maize seedlings, with improved physiological traits under both normal and stress conditions. C_LIO_LIMutant hybrids exhibit increased epicuticular wax accumulation and reduced ROS production, supporting greater survival during prolonged drought stress. C_LIO_LIField-grown mutant hybrids in a W22/B73 background maintain normal flowering time and nutritional composition, indicating no agronomic penalties from the mutations. C_LIO_LIYield is significantly higher in mutant hybrids compared to wild-type controls, as shown by increased ear weight and kernel number under rainfed field conditions. C_LIO_LIThese findings highlight ZmLCR1 and ZmLCR2 as valuable targets for breeding drought-tolerant, high-yielding maize cultivars suited to production environments. C_LI

Priming activity of plant-based allelochemicals is advanced research nowadays meaning a high potential in sustainable agriculture. The ELICE16INDURES(R) (RIMPH LTD, Hungary) plant conditioner of CO2 botanical extracts is rich in plant-active ingredients such as phenolic compounds, alkaloids, and flavonoids formulated in small multilamellar liposomes. This product was investigated in autumn barley (Hordeum vulgare). Field experiments of ELICE16INDURES showed augmented NDVI values interconnected with higher photosynthetic activity and yield increase. Background of the better vitality of plants was investigated by whole genomic gene expression profiling and showed an enhanced response to wounding, jasmonic acid, oxidative detoxification, and chloroplast activity. Among top50 differentially expressed genes the TIFY domain protein TIFY11B and RHOMBOID-like protein 2 related to JA signaling were up-regulated in field-collected samples. Phytotron experiments of barley were set up to validate and evaluate the transcriptomic effect of ELICE16INDURES. Well-studied priming active agents such as salicylic acid and beta-aminobutyric acid were compared with ELICE16INDURES and confirmed as priming inducer material with positive regulation of TIFY11B, TIFY3B, TIFY9, TIF10A, and RHOMBOID like protein 2 by using NGS GEx and RT-qPCR methods. One-sentence summaryELICE16INDURES(R) is a plant conditioner agent with a high amount of allelochemicals encapsulated into small multilamellar liposomes and found as an immune priming activator tested in H. vulgare field and phytotron cultures.

Chitosan oligosaccharides are the main degradation products from chitosan or chitin and have been reported to induce resistance to diseases in herbaceous plants like cucumber and Arabidopsis. Concomitantly, pine wilt disease is a devastating disease of conifer tree species. Here, we hypothesized that chitosan oligosaccharides induce plant resistance gene (PRG) expression in the woody plant Masson pine, Pinus massoniana. Chitosan oligosaccharides were inoculated into P. massoniana seedlings and the BGISEQ-500 platform was used to generate transcriptomes from chitosan oligosaccharide-treated P. massoniana and control seedlings. A total of 501 differentially expressed genes (DEGs) were identified by comparing the treatment and control groups. A total of 251 (50.1%) DEGs were up-regulated in the treatment relative to the control seedlings and 250 (49.9%) were down-regulated. Inoculation of chitosan oligosaccharide induced the expression of 31 PRGs in P. massoniana seedlings and the relative expression levels of six of the PRGs were verified by RT-qPCR. This is the first study to demonstrate that chitosan oligosaccharide induces the expression of PRGs in a tree species. These results provide important insights into the function of chitosan oligosaccharides and further the prospects of developing a chitosan oligosaccharide-based immune inducer for controlling pine wilt disease.

Maize (Zea mays L.) is a globally important crop for food, feed, and industrial uses. Modern breeding increasingly targets traits beyond yield, including stress tolerance, nutritional quality, and pest resistance. Progress toward these goals is constrained by the narrow genetic diversity of commercial varieties, a consequence of the repeated use of a limited number of inbred lines. Maize landraces therefore represent valuable reservoirs of genetic and phenotypic diversity. Northern Argentina is one of the southernmost regions of maize landrace cultivation and comprises two main centers of diversity: Northeastern Argentina (NEA; <2000 m.a.s.l.) and Northwestern Argentina (NWA; >2000 m.a.s.l.). Despite their potential relevance, phenotypic characterization of these landraces remains limited, particularly for biochemical traits, which, although less visible, play key roles in biomass accumulation, defense against pathogens and herbivores, tolerance to environmental stress, and quality attributes such as flavor. Here, we evaluated 17 phenotypic traits, including morphological traits, leaf biochemical compounds (such as pigments, carbohydrates, and phenolics), and salt stress tolerance, in 19 maize landrace accessions from Northern Argentina. Substantial variation was detected across all traits, both within and among accessions, indicating that each accession harbors a distinct phenotypic profile. While no significant differences were observed between regions, redundancy analysis revealed associations between phenotypic variation and collection-site altitude. These findings highlight the value of Argentine maize landraces as sources of biochemical and stress-related traits and support their conservation and use in breeding programs aimed at broadening the genetic base of cultivated maize. Main ConclusionMaize landraces from Southern South America show high within and between accession phenotypic variability, while differences between regions of origin are associated with altitude.

Indoor vertical farming (VF) offers several practical advantages for the cultivation of plant protein bio-factories, including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved in recent years toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environment control and space use optimization in VF systems. Here, we used a CRISPR-Cas9 gene editing approach to generate mutant lines of transient protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype compared to the so-called LAB strain commonly used for protein production. Our strategy consisted of altering apical dominance by suppressing the biosynthesis of strigolactone, a negative regulator of axillary bud outgrowth-promoting cytokinins. Strigolactone-depleted lines were generated by knocking-down the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the metabolic pathway leading to strigolactone synthesis. Knocking-down the genes of either enzyme had no impact on the overall growth rate of the plant but drastically influenced its leaf proteome, auxin/cytokinin ratio and overall architecture. More specifically, the {Delta}CCD mutants exhibited altered glycolytic and malate-processing enzyme fluxes driving the production of pyruvate and cytokinins in leaf tissue, an axillary growth-oriented development pattern and, most importantly, a spatial footprint reduced by 45% to 50% compared to the LAB strain. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as here illustrated for the model protein GFP and for rituximab, a chimeric monoclonal antibody of confirmed clinical value in humans. Our data demonstrate the usefulness of {Delta}CCD7 and {Delta}CCD8 knockout leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines well suited to VF systems intended for biopharmaceutical production.

Napier grass (Cenchrus purpureus syn. Pennisetum purpureum), a perennial C4 forage and bioenergy crop, exhibits strong drought resilience, yet the integrative mechanisms underlying this tolerance remain incompletely understood. This study examined physiological, hydraulic, and metabolic responses of four Napier grass cultivars under PEG-induced osmotic stress and progressive soil water deficit. Drought significantly increased the root-to-shoot ratio, indicating preferential biomass allocation to roots, which supported maintenance of shoot growth and tissue water status. All cultivars showed an approximate twofold increase in water-use efficiency (WUE) under water deficit, with cv2 and cv7 displaying superior performance. Upregulation of aquaporin genes (PIP2;2 and PIP2;3) suggested active hydraulic regulation that sustained carbon assimilation under reduced transpiration. Metabolic profiling revealed pronounced root-centered osmotic adjustment, including accumulation of galactinol, myo-inositol, raffinose family oligosaccharides, proline, and several amino acids. Enhanced expression of the galactinol synthase gene confirmed activation of raffinose biosynthesis pathways. Genotypic variation highlighted cv2 as particularly drought resilient. Rapid post-stress regrowth further underscored the importance of perennial root persistence. In conclusion, drought tolerance in Napier grass arises from coordinated hydraulic resilience, osmotic adjustment, and C4 photosynthetic efficiency, supporting its suitability for forage and bioenergy production in water-limited environments. SignificantThis study shows drought tolerance in Napier grass relies on root-driven hydraulic and metabolic regulation with efficient water-use efficiency, rather than avoidance, and that PEG responses predict field performance.

1Sparse testcrossing is an effective strategy for increasing both short- and long-term genetic gain in hybrid breeding programs. Maize hybrid breeding programs aim to develop new hybrid varieties by crossing genetically distinct parents from different heterotic pools, exploiting heterosis for improved performance. The programs typically consist of two main components: population improvement and product development. The population improvement component aims to enhance the heterotic pools through reciprocal recurrent selection based on general combining ability (GCA). However, especially in the early stages of testing, evaluating large numbers of hybrid combinations to estimate GCA is impractical due to considerable logistical challenges and costs. Therefore, breeders often evaluate the initial population of selection candidates using only a single tester to narrow down the candidate pool before further evaluation. Using a single tester, however, may not adequately represent the heterotic pool, leading to inaccurate GCA estimates and suboptimal selection decisions. To address this, we propose sparse testcrossing for early-stage testing, where subsets of candidate genotypes are testcrossed with different testers, connected through a genomic relationship matrix. We conducted stochastic simulations to compare various sparse testcrossing designs with a conventional testcross strategy using a single tester over 15 cycles of reciprocal recurrent genomic selection. Our results show that using 3-5 testers, sparsely distributed among full-sibs, sparse testcrossing offers breeders a practical balance between simple testcross designs, resource efficiency, and increased prediction accuracy for GCA, ultimately resulting in increased rates of genetic gain. Key messageSparse testcrossing with 3-5 testers enhances genetic gain in hybrid breeding programs, offering a practical balance of simple testcross designs, resource efficiency, and increased prediction accuracy for general combining ability.

Phenotyping high-biomass perennial crops is laborious and the rate of genetic gain in perennial crop breeding programs is typically low. So, it is especially important to identify methods that produce efficiency gains in the breeding process. Miscanthus is a C4 perennial grass with favorable characteristics for producing biomass as a feedstock for biofuels and diverse biobased products. Increasing biomass yield will increase profitability and environmental benefits, so is a key target for Miscanthus breeding. In addition, the identification of well-adapted genotypes across a wide range of environmental conditions requires the establishment of multi-environment trials (METs). Sparse testing is a genomic prediction-based strategy that reduces the phenotyping costs in METs by selecting a subset of genotypes to evaluate in a subset of environments and then predicts the performance of the unobserved genotype-environment combinations. A Miscanthus sacchariflorus (MSA) population comprising 336 genotypes observed across three environments was analyzed. Three prediction models considering main effects (environments, genotypes, genomic) and interaction effects (genotype-by-environment; GxE interaction) were implemented for forecasting dry biomass yield (YDY), total culm (TCM), average internode length (AIL), and culm node number (CNN). Multiple calibration sets based on different compositions and sizes were considered to evaluate performance in terms of the predictive ability (PA) and the mean square error (MSE) for a fixed testing set size. The training set size ranged from 52 to 112 to predict a fixed set of 224 unobserved genotypes across all three environments. The results showed that the model accounting for GxE interaction presented the highest PA and the lowest MSE for CNN (PA: [~]0.77, MSE: [~]0.5) and YDY (PA: [~]0.70, MSE: [~]1.3) while for TCM and AIL these ranged from [~]0.28 to 0.41 and [~]1.3 to 4.3, respectively. Overall, varying training sets and allocation strategies did not affect PA and MSE, with 52 non-overlapping and 0 overlapping genotypes per environment as the optimal cost-effective allocation framework. This suggests that implementing sparse testing designs could significantly reduce phenotyping costs by fivefold, without compromising PA in breeding programs for perennial crops such as Miscanthus.

Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one of the main pasture grasses for poorly drained soils in the tropics. However, genetic and genomic resources for this species are lacking due to its genomic complexity, including high heterozygosity, evidence of segmental allopolyploidy, and reproduction by apomixis. These complexities hinder the application of marker-assisted selection (MAS) in breeding programs. Here, we developed the highest-density linkage map currently available for the hexaploid tropical forage grass U. humidicola. This map was constructed using a biparental F1 population generated from a cross between the female parent H031 (CIAT 26146), the only known sexual genotype for the species, and the apomictic male parent H016 (BRS cv. Tupi). The linkage analysis included 4,873 single nucleotide polymorphism (SNP) markers with allele dosage information. It allowed mapping of the apospory locus and phenotype to linkage group 3, in a region syntenic with chromosome 3 of Urochloa ruziziensis and chromosome 1 of Setaria italica. We also identified hexaploid haplotypes for all individuals, assessed the meiotic configuration, and estimated the level of preferential pairing in parents during the meiotic process, which revealed the autopolyploid origin of sexual H031 in contrast to H016, which presented allopolyploid behavior in preferential pairing analysis. These results provide new information regarding the genetic organization, mode of reproduction, and allopolyploid origin of U. humidicola, potential SNPs markers associated to apomixes for MAS and resources for research on polyploids and tropical forage grasses. Key messageWe present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and species origin, aiding polyploid and tropical forage research.