High-resolution image dataset for the automatic classification of phenological stage and identification of racemes in Urochloa spp. hybrids.

Urochloa grasses are widely used forages in the Neotropics and are gaining importance in other regions due to their role in meeting the increasing global demand for sustainable agricultural practices. High-throughput phenotyping (HTP) is important for accelerating Urochloa breeding programs focused on improving forage and seed yield. While RGB imaging has been used for HTP of vegetative traits, the assessment of phenological stages and seed yield using image analysis remains unexplored in this genus. This work presents a dataset of 2,400 high-resolution RGB images of 200 Urochloa hybrid genotypes, captured over seven months and covering both vegetative and reproductive stages. Images were manually labelled as vegetative or reproductive, and a subset of 255 reproductive stage images were annotated to identify 22,340 individual racemes. This dataset enables the development of machine learning and deep learning models for automated phenological stage classification and raceme identification, facilitating HTP and accelerated breeding of Urochloa spp. hybrids with high seed yield potential.

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

Over the last 10 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 multiparental population using the species mentioned above as parents, 2 different statistical approaches to associate the phenotypes with markers [GWAS (genome-wide association studies) and copula graphical models], and 2 phenotyping methodologies from the first to the 17th day after inoculation (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 5, possibly controls late leaf rust resistance, as both GWAS and the network suggested the same marker. Of the 12 genes flanking its region, 4 were possible receptors, 3 were likely defense executors, 1 gene was likely part of signaling cascades, and 4 were classified as nondefense 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.

Multispectral and thermal infrared data, visual scores for severity of common rust symptoms, and genotypic single nucleotide polymorphism data of three F2-derived biparental doubled-haploid maize populations.

Three F2-derived biparental doubled haploid (DH) maize populations were generated for genetic mapping of resistance to common rust. Each of the three populations has the same susceptible parent, but a different resistance donor parent. Population 1 and 3 consist of 320 lines each, population 2 consists of 260 lines. The DH lines were evaluated for their susceptibility to common rust in two years and with two replications in each year. For phenotyping, a visual score (VS) for susceptibility was assigned. Additionally, unmanned aerial vehicle (UAV) derived multispectral and thermal infrared data was recorded and combined in different vegetation indices ("remote sensing", RS). The DH lines were genotyped with the DarTseq method, to obtain data on single nucleotide polymorphisms (SNPs). After quality control, 9051 markers remained. Missing values were "imputed" by the empirical mean of the marker scores of the respective locus. We used the data for comparison of genome-wide association studies and genomic prediction when based on different phenotyping methods, that is either VS or RS data. The data may be interesting for reuse for instance for benchmarking genomic prediction models, for phytopathological studies addressing common rust, or for specifications of vegetation indices.

Satellite imagery for high-throughput phenotyping in breeding plots.

Advances in breeding efforts to increase the rate of genetic gains and enhance crop resilience to climate change have been limited by the procedure and costs of phenotyping methods. The recent rapid development of sensors, image-processing technology, and data-analysis has provided opportunities for multiple scales phenotyping methods and systems, including satellite imagery. Among these platforms, satellite imagery may represent one of the ultimate approaches to remotely monitor trials and nurseries planted in multiple locations while standardizing protocols and reducing costs. However, the deployment of satellite-based phenotyping in breeding trials has largely been limited by low spatial resolution of satellite images. The advent of a new generation of high-resolution satellites may finally overcome these limitations. The SkySat constellation started offering multispectral images at a 0.5 m resolution since 2020. In this communication we present a case study on the use of time series SkySat images to estimate NDVI from wheat and maize breeding plots encompassing different sizes and spacing. We evaluated the reliability of the calculated NDVI and tested its capacity to detect seasonal changes and genotypic differences. We discuss the advantages, limitations, and perspectives of this approach for high-throughput phenotyping in breeding programs.

Reflectance Spectroscopy for the Classification and Prediction of Pigments in Agronomic Crops.

Reflectance spectroscopy, in combination with machine learning and artificial intelligence algorithms, is an effective method for classifying and predicting pigments and phenotyping in agronomic crops. This study aims to use hyperspectral data to develop a robust and precise method for the simultaneous evaluation of pigments, such as chlorophylls, carotenoids, anthocyanins, and flavonoids, in six agronomic crops: corn, sugarcane, coffee, canola, wheat, and tobacco. Our results demonstrate high classification accuracy and precision, with principal component analyses (PCAs)-linked clustering and a kappa coefficient analysis yielding results ranging from 92 to 100% in the ultraviolet-visible (UV-VIS) to near-infrared (NIR) to shortwave infrared (SWIR) bands. Predictive models based on partial least squares regression (PLSR) achieved R2 values ranging from 0.77 to 0.89 and ratio of performance to deviation (RPD) values over 2.1 for each pigment in C3 and C4 plants. The integration of pigment phenotyping methods with fifteen vegetation indices further improved accuracy, achieving values ranging from 60 to 100% across different full or range wavelength bands. The most responsive wavelengths were selected based on a cluster heatmap, ß-loadings, weighted coefficients, and hyperspectral vegetation index (HVI) algorithms, thereby reinforcing the effectiveness of the generated models. Consequently, hyperspectral reflectance can serve as a rapid, precise, and accurate tool for evaluating agronomic crops, offering a promising alternative for monitoring and classification in integrated farming systems and traditional field production. It provides a non-destructive technique for the simultaneous evaluation of pigments in the most important agronomic plants.

Chile Pepper (Capsicum) Breeding and Improvement in the "Multi-Omics" Era.

Chile pepper (Capsicum spp.) is a major culinary, medicinal, and economic crop in most areas of the world. For more than hundreds of years, chile peppers have "defined" the state of New Mexico, USA. The official state question, "Red or Green?" refers to the preference for either red or the green stage of chile pepper, respectively, reflects the value of these important commodities. The presence of major diseases, low yields, decreased acreages, and costs associated with manual labor limit production in all growing regions of the world. The New Mexico State University (NMSU) Chile Pepper Breeding Program continues to serve as a key player in the development of improved chile pepper varieties for growers and in discoveries that assist plant breeders worldwide. Among the traits of interest for genetic improvement include yield, disease resistance, flavor, and mechanical harvestability. While progress has been made, the use of conventional breeding approaches has yet to fully address producer and consumer demand for these traits in available cultivars. Recent developments in "multi-omics," that is, the simultaneous application of multiple omics approaches to study biological systems, have allowed the genetic dissection of important phenotypes. Given the current needs and production constraints, and the availability of multi-omics tools, it would be relevant to examine the application of these approaches in chile pepper breeding and improvement. In this review, we summarize the major developments in chile pepper breeding and present novel tools that can be implemented to facilitate genetic improvement. In the future, chile pepper improvement is anticipated to be more data and multi-omics driven as more advanced genetics, breeding, and phenotyping tools are developed.

Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus).

BACKGROUND: Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. RESULTS: To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. CONCLUSIONS: This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

Convolutional Neural Networks to Estimate Dry Matter Yield in a Guineagrass Breeding Program Using UAV Remote Sensing.

Forage dry matter is the main source of nutrients in the diet of ruminant animals. Thus, this trait is evaluated in most forage breeding programs with the objective of increasing the yield. Novel solutions combining unmanned aerial vehicles (UAVs) and computer vision are crucial to increase the efficiency of forage breeding programs, to support high-throughput phenotyping (HTP), aiming to estimate parameters correlated to important traits. The main goal of this study was to propose a convolutional neural network (CNN) approach using UAV-RGB imagery to estimate dry matter yield traits in a guineagrass breeding program. For this, an experiment composed of 330 plots of full-sib families and checks conducted at Embrapa Beef Cattle, Brazil, was used. The image dataset was composed of images obtained with an RGB sensor embedded in a Phantom 4 PRO. The traits leaf dry matter yield (LDMY) and total dry matter yield (TDMY) were obtained by conventional agronomic methodology and considered as the ground-truth data. Different CNN architectures were analyzed, such as AlexNet, ResNeXt50, DarkNet53, and two networks proposed recently for related tasks named MaCNN and LF-CNN. Pretrained AlexNet and ResNeXt50 architectures were also studied. Ten-fold cross-validation was used for training and testing the model. Estimates of DMY traits by each CNN architecture were considered as new HTP traits to compare with real traits. Pearson correlation coefficient r between real and HTP traits ranged from 0.62 to 0.79 for LDMY and from 0.60 to 0.76 for TDMY; root square mean error (RSME) ranged from 286.24 to 366.93 kg·ha-1 for LDMY and from 413.07 to 506.56 kg·ha-1 for TDMY. All the CNNs generated heritable HTP traits, except LF-CNN for LDMY and AlexNet for TDMY. Genetic correlations between real and HTP traits were high but varied according to the CNN architecture. HTP trait from ResNeXt50 pretrained achieved the best results for indirect selection regardless of the dry matter trait. This demonstrates that CNNs with remote sensing data are highly promising for HTP for dry matter yield traits in forage breeding programs.

High-resolution spectral information enables phenotyping of leaf epicuticular wax in wheat.

BACKGROUND: Epicuticular wax (EW) is the first line of defense in plants for protection against biotic and abiotic factors in the environment. In wheat, EW is associated with resilience to heat and drought stress, however, the current limitations on phenotyping EW restrict the integration of this secondary trait into wheat breeding pipelines. In this study we evaluated the use of light reflectance as a proxy for EW load and developed an efficient indirect method for the selection of genotypes with high EW density. RESULTS: Cuticular waxes affect the light that is reflected, absorbed and transmitted by plants. The narrow spectral regions statistically associated with EW overlap with bands linked to photosynthetic radiation (500 nm), carotenoid absorbance (400 nm) and water content (~ 900 nm) in plants. The narrow spectral indices developed predicted 65% (EWI-13) and 44% (EWI-1) of the variation in this trait utilizing single-leaf reflectance. However, the normalized difference indices EWI-4 and EWI-9 improved the phenotyping efficiency with canopy reflectance across all field experimental trials. Indirect selection for EW with EWI-4 and EWI-9 led to a selection efficiency of 70% compared to phenotyping with the chemical method. The regression model EWM-7 integrated eight narrow wavelengths and accurately predicted 71% of the variation in the EW load (mg·dm-2) with leaf reflectance, but under field conditions, a single-wavelength model consistently estimated EW with an average RMSE of 1.24 mg·dm-2 utilizing ground and aerial canopy reflectance. CONCLUSIONS: Overall, the indices EWI-1, EWI-13 and the model EWM-7 are reliable tools for indirect selection for EW based on leaf reflectance, and the indices EWI-4, EWI-9 and the model EWM-1 are reliable for selection based on canopy reflectance. However, further research is needed to define how the background effects and geometry of the canopy impact the accuracy of these phenotyping methods.

High-throughput sleep phenotyping produces robust and heritable traits in Diversity Outbred mice and their founder strains.

STUDY OBJECTIVES: This study describes high-throughput phenotyping strategies for sleep and circadian behavior in mice, including examinations of robustness, reliability, and heritability among Diversity Outbred (DO) mice and their eight founder strains. METHODS: We performed high-throughput sleep and circadian phenotyping in male mice from the DO population (n = 338) and their eight founder strains: A/J (n = 6), C57BL/6J (n = 14), 129S1/SvlmJ (n = 6), NOD/LtJ (n = 6), NZO/H1LtJ (n = 6), CAST/EiJ (n = 8), PWK/PhJ (n = 8), and WSB/EiJ (n = 6). Using infrared beam break systems, we defined sleep as at least 40 s of continuous inactivity and quantified sleep-wake amounts and bout characteristics. We developed assays to measure sleep latency in a new environment and during a modified Murine Multiple Sleep Latency Test, and estimated circadian period from wheel-running experiments. For each trait, broad-sense heritability (proportion of variability explained by all genetic factors) was derived in founder strains, while narrow-sense heritability (proportion of variability explained by additive genetic effects) was calculated in DO mice. RESULTS: Phenotypes were robust to different inactivity durations to define sleep. Differences across founder strains and moderate/high broad-sense heritability were observed for most traits. There was large phenotypic variability among DO mice, and phenotypes were reliable, although estimates of heritability were lower than in founder mice. This likely reflects important nonadditive genetic effects. CONCLUSIONS: A high-throughput phenotyping strategy in mice, based primarily on monitoring of activity patterns, provides reliable and heritable estimates of sleep and circadian traits. This approach is suitable for discovery analyses in DO mice, where genetic factors explain some proportion of phenotypic variation.

Hyperspectral Reflectance-Derived Relationship Matrices for Genomic Prediction of Grain Yield in Wheat.

Hyperspectral reflectance phenotyping and genomic selection are two emerging technologies that have the potential to increase plant breeding efficiency by improving prediction accuracy for grain yield. Hyperspectral cameras quantify canopy reflectance across a wide range of wavelengths that are associated with numerous biophysical and biochemical processes in plants. Genomic selection models utilize genome-wide marker or pedigree information to predict the genetic values of breeding lines. In this study, we propose a multi-kernel GBLUP approach to genomic selection that uses genomic marker-, pedigree-, and hyperspectral reflectance-derived relationship matrices to model the genetic main effects and genotype × environment (G × E) interactions across environments within a bread wheat (Triticum aestivum L.) breeding program. We utilized an airplane equipped with a hyperspectral camera to phenotype five differentially managed treatments of the yield trials conducted by the Bread Wheat Improvement Program of the International Maize and Wheat Improvement Center (CIMMYT) at Ciudad Obregón, México over four breeding cycles. We observed that single-kernel models using hyperspectral reflectance-derived relationship matrices performed similarly or superior to marker- and pedigree-based genomic selection models when predicting within and across environments. Multi-kernel models combining marker/pedigree information with hyperspectral reflectance phentoypes had the highest prediction accuracies; however, improvements in accuracy over marker- and pedigree-based models were marginal when correcting for days to heading. Our results demonstrate the potential of using hyperspectral imaging to predict grain yield within a multi-environment context and also support further studies on the integration of hyperspectral reflectance phenotyping into breeding programs.

Assessing Wheat Traits by Spectral Reflectance: Do We Really Need to Focus on Predicted Trait-Values or Directly Identify the Elite Genotypes Group?

Phenotyping, via remote and proximal sensing techniques, of the agronomic and physiological traits associated with yield potential and drought adaptation could contribute to improvements in breeding programs. In the present study, 384 genotypes of wheat (Triticum aestivum L.) were tested under fully irrigated (FI) and water stress (WS) conditions. The following traits were evaluated and assessed via spectral reflectance: Grain yield (GY), spikes per square meter (SM2), kernels per spike (KPS), thousand-kernel weight (TKW), chlorophyll content (SPAD), stem water soluble carbohydrate concentration and content (WSC and WSCC, respectively), carbon isotope discrimination (Δ13C), and leaf area index (LAI). The performances of spectral reflectance indices (SRIs), four regression algorithms (PCR, PLSR, ridge regression RR, and SVR), and three classification methods (PCA-LDA, PLS-DA, and kNN) were evaluated for the prediction of each trait. For the classification approaches, two classes were established for each trait: The lower 80% of the trait variability range (Class 1) and the remaining 20% (Class 2 or elite genotypes). Both the SRIs and regression methods performed better when data from FI and WS were combined. The traits that were best estimated by SRIs and regression methods were GY and Δ13C. For most traits and conditions, the estimations provided by RR and SVR were the same, or better than, those provided by the SRIs. PLS-DA showed the best performance among the categorical methods and, unlike the SRI and regression models, most traits were relatively well-classified within a specific hydric condition (FI or WS), proving that classification approach is an effective tool to be explored in future studies related to genotype selection.

A Direct Comparison of Remote Sensing Approaches for High-Throughput Phenotyping in Plant Breeding.

Remote sensing (RS) of plant canopies permits non-intrusive, high-throughput monitoring of plant physiological characteristics. This study compared three RS approaches using a low flying UAV (unmanned aerial vehicle), with that of proximal sensing, and satellite-based imagery. Two physiological traits were considered, canopy temperature (CT) and a vegetation index (NDVI), to determine the most viable approaches for large scale crop genetic improvement. The UAV-based platform achieves plot-level resolution while measuring several hundred plots in one mission via high-resolution thermal and multispectral imagery measured at altitudes of 30-100 m. The satellite measures multispectral imagery from an altitude of 770 km. Information was compared with proximal measurements using IR thermometers and an NDVI sensor at a distance of 0.5-1 m above plots. For robust comparisons, CT and NDVI were assessed on panels of elite cultivars under irrigated and drought conditions, in different thermal regimes, and on un-adapted genetic resources under water deficit. Correlations between airborne data and yield/biomass at maturity were generally higher than equivalent proximal correlations. NDVI was derived from high-resolution satellite imagery for only larger sized plots (8.5 × 2.4 m) due to restricted pixel density. Results support use of UAV-based RS techniques for high-throughput phenotyping for both precision and efficiency.

Chlorophyll, anthocyanin, and gas exchange changes assessed by spectroradiometry in Fragaria chiloensis under salt stress.

Chlorophyll and anthocyanin contents provide a valuable indicator of the status of a plant's physiology, but to be more widely utilized it needs to be assessed easily and non-destructively. This is particularly evident in terms of assessing and exploiting germplasm for plant-breeding programs. We report, for the first time, experiments with Fragaria chiloensis (L.) Duch. and the estimation of the effects of response to salinity stress (0, 30, and 60 mmol NaCl/L) in terms of these pigments content and gas exchange. It is shown that both pigments (which interestingly, themselves show a high correlation) give a good indication of stress response. Both pigments can be accurately predicted using spectral reflectance indices (SRI); however, the accuracy of the predictions was slightly improved using multilinear regression analysis models and genetic algorithm analysis. Specifically for chlorophyll content, unlike other species, the use of published SRI gave better indications of stress response than Normalized Difference Vegetation Index. The effect of salt on gas exchange is only evident at the highest concentration and some SRI gave better prediction performance than the known Photochemical Reflectance Index. This information will therefore be useful for identifying tolerant genotypes to salt stress for incorporation in breeding programs.