Integrating Hyperspectral Reflectance-Based Phenotyping and SSR Marker-Based Genotyping for Assessing the Salt Tolerance of Wheat Genotypes under Real Field Conditions.

Wheat breeding programs are currently focusing on using non-destructive and cost-effective hyperspectral sensing tools to expeditiously and accurately phenotype large collections of genotypes. This approach is expected to accelerate the development of the abiotic stress tolerance of genotypes in breeding programs. This study aimed to assess salt tolerance in wheat genotypes using non-destructive canopy spectral reflectance measurements as an alternative to direct laborious and time-consuming phenological selection criteria. Eight wheat genotypes and sixteen F8 RILs were tested under 150 mM NaCl in real field conditions for two years. Fourteen spectral reflectance indices (SRIs) were calculated from the spectral data, including vegetation SRIs and water SRIs. The effectiveness of these indices in assessing salt tolerance was compared with four morpho-physiological traits using genetic parameters, SSR markers, the Mantel test, hierarchical clustering heatmaps, stepwise multiple linear regression, and principal component analysis (PCA). The results showed significant differences (p ≤ 0.001) among RILs/cultivars for both traits and SRIs. The heritability, genetic gain, and genotypic and phenotypic coefficients of variability for most SRIs were comparable to those of measured traits. The SRIs effectively differentiated between salt-tolerant and sensitive genotypes and exhibited strong correlations with SSR markers (R2 = 0.56-0.89), similar to the measured traits and allelic data of 34 SSRs. A strong correlation (r = 0.27, p < 0.0001) was found between the similarity coefficients of SRIs and SSR data, which was higher than that between measured traits and SSR data (r = 0.20, p < 0.0003) based on the Mantel test. The PCA indicated that all vegetation SRIs and most water SRIs were grouped with measured traits in a positive direction and effectively identified the salt-tolerant RILs/cultivars. The PLSR models, which were based on all SRIs, accurately and robustly estimated the various morpho-physiological traits compared to using individual SRIs. The study suggests that various SRIs can be integrated with PLSR in wheat breeding programs as a cost-effective and non-destructive tool for phenotyping and screening large wheat populations for salt tolerance in a short time frame. This approach can replace the need for traditional morpho-physiological traits and accelerate the development of salt-tolerant wheat genotypes.

Multi-class plant hormone HILIC-MS/MS analysis coupled with high-throughput phenotyping to investigate plant-environment interactions.

Plant hormones are chemical signals governing almost every aspect of a plant's life cycle and responses to environmental cues. They are enmeshed within complex signaling networks that can only be deciphered by using broad-scale analytical methods to capture information about several plant hormone classes simultaneously. Methods used for this purpose are all based on reversed-phase (RP) liquid chromatography and mass spectrometric detection. Hydrophilic interaction chromatography (HILIC) is an alternative chromatographic method that performs well in analyses of biological samples. We therefore developed and validated a HILIC method for broad-scale plant hormone analysis including a rapid sample preparation procedure; moreover, derivatization or fractionation is not required. The method enables plant hormone screening focused on polar and moderately polar analytes including cytokinins, auxins, jasmonates, abscisic acid and its metabolites, salicylates, indoleamines (melatonin), and 1-aminocyclopropane-1-carboxylic acid (ACC), for a total of 45 analytes. Importantly, the major pitfalls of ACC analysis have been addressed. Furthermore, HILIC provides orthogonal selectivity to conventional RP methods and displays greater sensitivity, resulting in lower limits of quantification. However, it is less robust, so procedures to increase its reproducibility were established. The method's potential is demonstrated in a case study by employing an approach combining hormonal analysis with phenomics to examine responses of three Arabidopsis ecotypes toward three abiotic stress treatments: salinity, low nutrient availability, and their combination. The case study showcases the value of the simultaneous determination of several plant hormone classes coupled with phenomics data when unraveling processes involving complex cross-talk under diverse plant-environment interactions.

Evaluating waterlogging stress response and recovery in barley (Hordeum vulgare L.): an image-based phenotyping approach.

Waterlogging is expected to become a more prominent yield restricting stress for barley as rainfall frequency is increasing in many regions due to climate change. The duration of waterlogging events in the field is highly variable throughout the season, and this variation is also observed in experimental waterlogging studies. Such variety of protocols make intricate physiological responses challenging to assess and quantify. To assess barley waterlogging tolerance in controlled conditions, we present an optimal duration and setup of simulated waterlogging stress using image-based phenotyping. Six protocols durations, 5, 10, and 14 days of stress with and without seven days of recovery, were tested. To quantify the physiological effects of waterlogging on growth and greenness, we used top down and side view RGB (Red-Green-Blue) images. These images were taken daily throughout each of the protocols using the PSI PlantScreen™ imaging platform. Two genotypes of two-row spring barley, grown in glasshouse conditions, were subjected to each of the six protocols, with stress being imposed at the three-leaf stage. Shoot biomass and root imaging data were analysed to determine the optimal stress protocol duration, as well as to quantify the growth and morphometric changes of barley in response to waterlogging stress. Our time-series results show a significant growth reduction and alteration of greenness, allowing us to determine an optimal protocol duration of 14 days of stress and seven days of recovery for controlled conditions. Moreover, to confirm the reproducibility of this protocol, we conducted the same experiment in a different facility equipped with RGB and chlorophyll fluorescence imaging sensors. Our results demonstrate that the selected protocol enables the assessment of genotypic differences, which allow us to further determine tolerance responses in a glasshouse environment. Altogether, this work presents a new and reproducible image-based protocol to assess early stage waterlogging tolerance, empowering a precise quantification of waterlogging stress relevant markers such as greenness, Fv/Fm and growth rates.

Complementary approaches to dissect late leaf rust resistance in an interspecific raspberry population.

Over the last ten years, global raspberry production has increased by 47.89%, based mainly on the red raspberry species (Rubus idaeus). However, the black raspberry (Rubus occidentalis), although less consumed, is resistant to one of the most important diseases for the crop, the late leaf rust caused by Acculeastrum americanum fungus. In this context, genetic resistance is the most sustainable way to control the disease, mainly because there are no registered fungicides for late leaf rust in Brazil. Therefore, the aim was to understand the genetic architecture that controls resistance to late leaf rust in raspberries. For that, we used an interspecific multi-parental population using the species mentioned above as parents, two different statistical approaches to associate the phenotypes with markers (GWAS and copula graphical models), and two phenotyping methodologies from the first to the seventeenth Day After Inoculation (D.A.I.) (high-throughput phenotyping with a multispectral camera and traditional phenotyping by disease severity scores). Our findings indicate that a locus of higher effect, at position 13.3 Mb on chromosome five, possibly controls late leaf rust resistance, as both GWAS and the network suggested the same marker. Of the twelve genes flanking its region, four were possible receptors, three were likely defense executors, one gene was likely part of signaling cascades, and four were classified as non-defense related. Although the network and GWAS indicated the same higher effect genomic region, the network identified other different candidate regions, potentially complementing the genetic control comprehension.

Prediction accuracy and repeatability of UAV based biomass estimation in wheat variety trials as affected by variable type, modelling strategy and sampling location.

BACKGROUND: This study explores the use of Unmanned Aerial Vehicles (UAVs) for estimating wheat biomass, focusing on the impact of phenotyping and analytical protocols in the context of late-stage variety selection programs. It emphasizes the importance of variable selection, model specificity, and sampling location within the experimental plot in predicting biomass, aiming to refine UAV-based estimation techniques for enhanced selection accuracy and throughput in variety testing programs. RESULTS: The research uncovered that integrating geometric and spectral traits led to an increase in prediction accuracy, whilst a recursive feature elimination (RFE) based variable selection workflowled to slight reductions in accuracy with the benefit of increased interpretability. Models, tailored to specific experiments were more accurate than those modelling all experiments together, while models trained for broad-growth stages did not significantly increase accuracy. The comparison between a permanent and a precise region of interest (ROI) within the plot showed negligible differences in biomass prediction accuracy, indicating the robustness of the approach across different sampling locations within the plot. Significant differences in the within-season repeatability (w2) of biomass predictions across different experiments highlighted the need for further investigation into the optimal timing of measurement for prediction. CONCLUSIONS: The study highlights the promising potential of UAV technology in biomass prediction for wheat at a small plot scale. It suggests that the accuracy of biomass predictions can be significantly improved through optimizing analytical and modelling protocols (i.e., variable selection, algorithm selection, stage-specific model development). Future work should focus on exploring the applicability of these findings under a wider variety of conditions and from a more diverse set of genotypes.

Rice breeding for low input agriculture.

A low-input-based farming system can reduce the adverse effects of modern agriculture through proper utilization of natural resources. Modern varieties often need to improve in low-input settings since they are not adapted to these systems. In addition, rice is one of the most widely cultivated crops worldwide. Enhancing rice performance under a low input system will significantly reduce the environmental concerns related to rice cultivation. Traits that help rice to maintain yield performance under minimum inputs like seedling vigor, appropriate root architecture for nutrient use efficiency should be incorporated into varieties for low input systems through integrated breeding approaches. Genes or QTLs controlling nutrient uptake, nutrient assimilation, nutrient remobilization, and root morphology need to be properly incorporated into the rice breeding pipeline. Also, genes/QTLs controlling suitable rice cultivars for sustainable farming. Since several variables influence performance under low input conditions, conventional breeding techniques make it challenging to work on many traits. However, recent advances in omics technologies have created enormous opportunities for rapidly improving multiple characteristics. This review highlights current research on features pertinent to low-input agriculture and provides an overview of alternative genomics-based breeding strategies for enhancing genetic gain in rice suitable for low-input farming practices.

Integrity of xylan backbone affects plant responses to drought.

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

Deciphering salt stress responses in Solanum pimpinellifolium through high-throughput phenotyping.

Soil salinity is a major environmental stressor affecting agricultural productivity worldwide. Understanding plant responses to salt stress is crucial for developing resilient crop varieties. Wild relatives of cultivated crops, such as wild tomato, Solanum pimpinellifolium, can serve as a useful resource to further expand the resilience potential of the cultivated germplasm, S. lycopersicum. In this study, we employed high-throughput phenotyping in the greenhouse and field conditions to explore salt stress responses of a S. pimpinellifolium diversity panel. Our study revealed extensive phenotypic variations in response to salt stress, with traits such as transpiration rate, shoot mass, and ion accumulation showing significant correlations with plant performance. We found that while transpiration was a key determinant of plant performance in the greenhouse, shoot mass strongly correlated with yield under field conditions. Conversely, ion accumulation was the least influential factor under greenhouse conditions. Through a Genome Wide Association Study, we identified candidate genes not previously associated with salt stress, highlighting the power of high-throughput phenotyping in uncovering novel aspects of plant stress responses. This study contributes to our understanding of salt stress tolerance in S. pimpinellifolium and lays the groundwork for further investigations into the genetic basis of these traits, ultimately informing breeding efforts for salinity tolerance in tomato and other crops.

High-throughput seed quality analysis in faba bean: leveraging Near-InfraRed spectroscopy (NIRS) data and statistical methods.

Near-infrared spectroscopy (NIRS) provides a high-throughput phenotyping technique to assist breeding for improved faba bean seed quality. We combined chemical analysis of protein, oil content (and composition) with NIRS through chemometrics, employing Partial Least Squares (PLS), Elastic Net (EN), Memory-based Learning (MBL), and Bayes B (BB) as prediction models. Protein was the most reliably predicted trait (R2 = 0.96-0.98) across field trials, followed by oil (R2 = 0.82-0.86) and oleic acid (R2 = 0.31-0.68). Samples for training the models were selected using K-means clustering. The optimal statistical approach for prediction was compound-specific: PLS for protein (Root Mean Squared Error - RMSE = 0.46), BB for oil (RMSE = 0.067), and EN for oleic acid content (RMSE = 2.83). Reduced training set simulations revealed different effects on prediction accuracy depending on the model and compound. Several NIR regions were pinpointed as highly informative for the compounds, using the shrinkage and variable selection capabilities of EN and BB.

Investigating the water availability hypothesis of pot binding: small pots and infrequent irrigation confound the effects of drought stress in potato (Solanum tuberosum L.).

To maximise the throughput of novel, high-throughput phenotyping platforms, many researchers have utilised smaller pot sizes to increase the number of biological replicates that can be grown in spatially limited controlled environments. This may confound plant development through a process known as "pot binding", particularly in larger species including potato (Solanum tuberosum), and under water-restricted conditions. We aimed to investigate the water availability hypothesis of pot binding, which predicts that small pots have insufficient water holding capacities to prevent drought stress between irrigation periods, in potato. Two cultivars of potato were grown in small (5 L) and large (20 L) pots, were kept under polytunnel conditions, and were subjected to three irrigation frequencies: every other day, daily, and twice daily. Plants were phenotyped with two Phenospex PlantEye F500s and canopy and tuber fresh mass and dry matter were measured. Increasing irrigation frequency from every other day to daily was associated with a significant increase in fresh tuber yield, but only in large pots. This suggests a similar level of drought stress occurred between these treatments in the small pots, supporting the water availability hypothesis of pot binding. Further increasing irrigation frequency to twice daily was still not sufficient to increase yields in small pots but it caused an insignificant increase in yield in the larger pots, suggesting some pot binding may be occurring in large pots under daily irrigation. Canopy temperatures were significantly higher under each irrigation frequency in the small pots compared to large pots, which strongly supports the water availability hypothesis as higher canopy temperatures are a reliable indicator of drought stress in potato. Digital phenotyping was found to be less accurate for larger plants, probably due to a higher degree of self-shading. The research demonstrates the need to define the optimum pot size and irrigation protocols required to completely prevent pot binding and ensure drought treatments are not inadvertently applied to control plants.