Phenotyping Tools Research Articles

Sunflower-like self-sustainable plant-wearable sensing probe.

Powering and communicating with wearable devices on bio-interfaces is challenging due to strict weight, size, and resource constraints. This study presents a sunflower-like plant-wearable sensing device that harnesses solar energy, achieving complete energy self-sustainability for long-term monitoring of plant sap flow, a crucial indicator of plant health. It features foldable solar panels along with all essential flexible electronic components, resulting in a compact system that is lightweight enough for small plants. To tackle the low-energy density of solar power, we developed an ultralow-energy light communication mechanism inspired by fireflies. Together with unmanned aerial vehicles and deep learning algorithms, this approach enables efficient data retrieval from multiple devices across large agricultural fields. With its simple deployment, it shows great potential as a low-cost plant phenotyping tool. We believe our energy and communication solution for wearable devices can be extended to similar resource-limited and challenging scenarios, leading to exciting applications.

Spatial analysis with unoccupied aircraft systems data in wheat breeding yield trials

AbstractAn important aspect of reliable cultivar development is good field trial evaluations. Unoccupied aircraft systems (UAS also known as drones or UAVs) are a popular high‐throughput phenotyping tool that has been used to successfully evaluate plant stress and other canopy characteristics in a field. In precision agriculture applications, UAS imagery has been used to identify spatial variability in field settings. Here, we use UAS spectral imagery to improve field trial spatial analysis, better control spatial variability, and reduce errors for more reliable selections. UAS imagery data were collected across 47 breeding trials planted in an augmented complete block design (ACBD) or alpha‐lattice replicated designs from 2020 through 2023. Trials were evaluated using three spatial analysis strategies: linear models incorporating block effect, row‐column effect, or 2D splines. UAS‐derived spectral reflectance indices (SRI) were combined with each model as covariates. Modeling strategies were used across all trials and evaluated for autocorrelation, model fitness, and coefficient of variation (CV). Akaike information criterion (AIC) was used to assess model fitness. For spatial analysis trials, SRIs significantly lowered model AIC by an average of 38.4 for alpha‐lattice trials and 69.1 for ACBD trials. CV scores were also lowered when SRIs were utilized, with average CV values being 2.6 lower for alpha‐lattice and 2.1 for ACBD trials. This study highlights the potential for SRIs to improve the analyses of field breeding trials despite extreme environmental variables and climate conditions, improving experiment reliability and changing the way breeders plan and implement breeding experiments.

Using Multi-Sensor Data Fusion Techniques and Machine Learning Algorithms for Improving UAV-Based Yield Prediction of Oilseed Rape

Accurate and timely prediction of oilseed rape yield is crucial in precision agriculture and field remote sensing. We explored the feasibility and potential for predicting oilseed rape yield through the utilization of a UAV-based platform equipped with RGB and multispectral cameras. Genetic algorithm–partial least square was employed and evaluated for effective wavelength (EW) or vegetation index (VI) selection. Additionally, different machine learning algorithms, i.e., multiple linear regression (MLR), partial least squares regression (PLSR), least squares support vector machine (LS-SVM), back propagation neural network (BPNN), extreme learning machine (ELM), and radial basis function neural network (RBFNN), were developed and compared. With multi-source data fusion by combining vegetation indices (color and narrow-band VIs), robust prediction models of yield in oilseed rape were built. The performance of prediction models using the combination of VIs (RBFNN: Rpre = 0.8143, RMSEP = 171.9 kg/hm2) from multiple sensors manifested better results than those using only narrow-band VIs (BPNN: Rpre = 0.7655, RMSEP = 188.3 kg/hm2) from a multispectral camera. The best models for yield prediction were found by applying BPNN (Rpre = 0.8114, RMSEP = 172.6 kg/hm2) built from optimal EWs and ELM (Rpre = 0.8118, RMSEP = 170.9 kg/hm2) using optimal VIs. Taken together, the findings conclusively illustrate the potential of UAV-based RGB and multispectral images for the timely and non-invasive prediction of oilseed rape yield. This study also highlights that a lightweight UAV equipped with dual-image-frame snapshot cameras holds promise as a valuable tool for high-throughput plant phenotyping and advanced breeding programs within the realm of precision agriculture.

GestaltMatcher Database - A global reference for facial phenotypic variability in rare human diseases.

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.

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.

A portable multi-taxa phenotyping device to retrieve physiological performance traits

Organismal phenotyping to identify fitness traits is transforming our understanding of adaptive responses and ecological interactions of species within changing environments. Here we present a portable Multi-Taxa Phenotyping (MTP) system that can retrieve a suite of metabolic and photophysiological parameter across light, temperature, and/or chemical gradients, using real time bio-optical (oxygen and chlorophyll a fluorescence) measurements. The MTP system integrates three well-established technologies for the first time: an imaging Pulse Amplitude Modulated (PAM) chlorophyll a fluorometer, custom-designed well plates equipped with optical oxygen sensors, and a thermocycler. We demonstrate the ability of the MTP system to distinguish phenotypic performance characteristics of diverse aquatic taxa spanning corals, mangroves and algae based on metabolic parameters and Photosystem II dynamics, in a high-throughput capacity and accounting for interactions of different environmental gradients on performance. Extracted metrics from the MTP system can not only provide information on the performance of aquatic taxa exposed to differing environmental gradients, but also provide predicted phenotypic responses of key aquatic organisms to environmental change. Further work validating how rapid phenotyping tools such as the MTP system predict phenotypic responses to long term environmental changes in situ are urgently required to best inform how these tools can support management efforts.

Self-supervised learning of wrist-worn daily living accelerometer data improves the automated detection of gait in older adults

Progressive gait impairment is common among aging adults. Remote phenotyping of gait during daily living has the potential to quantify gait alterations and evaluate the effects of interventions that may prevent disability in the aging population. Here, we developed ElderNet, a self-supervised learning model for gait detection from wrist-worn accelerometer data. Validation involved two diverse cohorts, including over 1000 participants without gait labels, as well as 83 participants with labeled data: older adults with Parkinson's disease, proximal femoral fracture, chronic obstructive pulmonary disease, congestive heart failure, and healthy adults. ElderNet presented high accuracy (96.43 ± 2.27), specificity (98.87 ± 2.15), recall (82.32 ± 11.37), precision (86.69 ± 17.61), and F1 score (82.92 ± 13.39). The suggested method yielded superior performance compared to two state-of-the-art gait detection algorithms, with improved accuracy and F1 score (p < 0.05). In an initial evaluation of construct validity, ElderNet identified differences in estimated daily walking durations across cohorts with different clinical characteristics, such as mobility disability (p < 0.001) and parkinsonism (p < 0.001). The proposed self-supervised method has the potential to serve as a valuable tool for remote phenotyping of gait function during daily living in aging adults, even among those with gait impairments.

A screen for cell envelope stress uncovers an inhibitor of prolipoprotein diacylglyceryl transferase, Lgt, in Escherichia coli

The increasing prevalence of antibiotic resistance demands the discovery of antibacterial chemical scaffolds with unique mechanisms of action. Phenotypic screening approaches, such as the use of reporters for bacterial cell stress, offer promise to identify compounds while providing strong hypotheses for follow-on mechanism of action studies. From a collection of ∼1,800 Escherichia coli GFP transcriptional reporter strains, we identified a reporter that is highly induced by cell envelope stress-pProm rcsA -GFP. After characterizing pProm rcsA -GFP induction, we assessed a collection of bioactive small molecules for reporter induction, identifying 24 compounds of interest. Spontaneous suppressors to one compound in particular, MAC-0452936, mapped to the gene encoding the essential prolipoprotein diacylglyceryl transferase, lgt. Lgt inhibition by MAC-0452936 inhibition was confirmed through genetic, phenotypic, and biochemical approaches. The oxime ester, MAC-0452936, represents a useful small molecule inhibitor of Lgt and highlights the potential of using pProm rcsA -GFP as a phenotypic screening tool.

Quantitative sensory testing and chronic pain syndromes: a cross-sectional study from TwinsUK

ObjectiveThe chronic pain syndromes (CPS) include syndromes such as chronic widespread pain (CWP), dry eye disease (DED) and irritable bowel syndrome (IBS). Highly prevalent and lacking pathognomonic biomarkers, the CPS...

Automated multi-scale computational pathotyping (AMSCP) of inflamed synovial tissue

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research.

CMR to characterize myocardial structure and function in heart failure with preserved left ventricular ejection fraction.

Despite remarkable progress in therapeutic drugs, morbidity, and mortality for heart failure (HF) remains high in developed countries. HF with preserved ejection fraction (HFpEF) now accounts for around half of all HF cases. It is a heterogeneous disease, with multiple aetiologies, and as such poses a significant diagnostic challenge. Cardiac magnetic resonance (CMR) has become a valuable non-invasive modality to assess cardiac morphology and function, but beyond that, the multi-parametric nature of CMR allows novel approaches to characterize haemodynamics and with magnetic resonance spectroscopy (MRS), the study of metabolism. Furthermore, exercise CMR, when combined with lung water imaging provides an in-depth understanding of the underlying pathophysiological and mechanistic processes in HFpEF. Thus, CMR provides a comprehensive phenotyping tool for HFpEF, which points towards a targeted and personalized therapy with improved diagnostics and prevention.

Eye-tracking as an objective phenotyping tool to identify associations between metal exposure and social cognition: A pilot study

Eye-tracking as an objective phenotyping tool to identify associations between metal exposure and social cognition: A pilot study

Using Dynamic Laser Speckle Imaging for Plant Breeding: A Case Study of Water Stress in Sunflowers.

This study focuses on the promising use of biospeckle technology to detect water stress in plants, a complex physiological mechanism. This involves monitoring the temporal activity of biospeckle pattern to study the occurrence of stress within the leaf. The effects of water stress in plants can involve physical and biochemical changes. Some of these changes may alter the optical scattering properties of leaves. The present study therefore proposes to test the potential of a biospeckle measurement to observe the temporal evolution in different varieties of sunflower plants under water stress. An experiment applying controlled water stress with osmotic shock using polyethylene glycol 6000 (PEG) was conducted on two sunflower varieties: one sensitive, and the other more tolerant to water stress. Temporal monitoring of biospeckle activity in these plants was performed using the average value of difference (AVD) indicator. Results indicate that AVD highlights the difference in biospeckle activity between day and night, with lower activity at night for both varieties. The addition of PEG entailed a gradual decrease in values throughout the experiment, particularly for the sensitive variety. The results obtained are consistent with the behaviour of the varieties submitted to water stress. Indeed, a few days after the introduction of PEG, a stronger decrease in AVD indicator values was observed for the sensitive variety than for the resistant variety. This study highlights the dynamics of biospeckle activity for different sunflower varieties undergoing water stress and can be considered as a promising phenotyping tool.

ASPEN study case: Real time in situ apples detection and characterization

Due to an increasing demand for food and pressures on our ecosystem, mechanisation and automation in agriculture has been proposed as one of the main solutions to the problems associated with overpopulation given today's life standards. To encourage the use of new technologies and bridge the gap between plant and computer science, here we validate an open-source pipeline capable of predicting real time in situ fruit characteristics, specifically in this case for apples. Using Agroscope's phenotyping tool (ASPEN), we achieve an average precision at intercept over union of 50 % of 0.75 when using YOLOv8 - m as the object detection algorithm, and with thanks to the use of multiple sensors, we find an average diameter error of 4.4 mm in the task of apple size determination. Our research demonstrates that although the pipeline tends to underestimate the actual fruit size, size estimation cannot only be used to determine the size of apples per scan, but also to track temporal apple size distribution in 4 different varieties. This research supports ASPEN in potentially replacing traditional field measurements, also suggesting that other traits could also be digitally measured for standard orchard phenotyping, either for scientific or agricultural output goals. Finally, we make publicly available a new dataset of more than 600 images (Agroscope_apple dataset) and a pre-trained model based on YOLOv8 and specifically trained for the in-situ apple detection task. By doing so, we hope to increase the accessibility and use of new technologies in the field of agriculture.

Exploring actigraphy as a digital phenotyping measure: A study on differentiating psychomotor agitation and retardation in depression.

Psychomotor activity stands out as a crucial symptom in characterizing behaviors associated with depression. This study aims to explore the potential of actigraphy as a tool for digital phenotyping in characterizing symptoms of psychomotor agitation and retardation, which are clinically challenging dimensions to capture, in patients diagnosed with major depressive episode (MDE) according to DSM-5 criteria. We compared rest-activity circadian rhythm biomarkers measured by the Motion Watch 8 actigraphy between 58 (78.4%) patients with MDE and psychomotor retardation (PMR), and 16 (21.6%) patients with MDE and psychomotor agitation (PMA), according to DSM-5 criteria. Actigraphy allowed to objectively report PMA through heightened activity over a 24-h period, while PMR manifests as reduced activity during the most active 10 h. Lower rest-activity rhythm (RAR) amplitude in PMR was accompanied by increased irregularities in intra- and inter-day rhythms. Interestingly, actigraphy emerges as an objective tool to measure the characteristics of the active and rest periods, free from the confounding effects of sleep disturbances. Indeed, no differences in sleep disturbances were identified between patients exhibiting psychomotor agitation and those displaying PMR. Digital phenotyping through actigraphy may aid in distinguishing psychomotor retardation and psychomotor agitation allowing for a more precise characterization of the depression phenotype. When integrated with clinical assessment, measurements from actigraphy could offer additional insights into activity rhythms alongside subjective assessments and hold the potential to augment existing clinical decision-making processes in psychiatry.

Development of a deep-learning phenotyping tool for analyzing image-based strawberry phenotypes.

In strawberry farming, phenotypic traits (such as crown diameter, petiole length, plant height, flower, leaf, and fruit size) measurement is essential as it serves as a decision-making tool for plant monitoring and management. To date, strawberry plant phenotyping has relied on traditional approaches. In this study, an image-based Strawberry Phenotyping Tool (SPT) was developed using two deep-learning (DL) architectures, namely "YOLOv4" and "U-net" integrated into a single system. We aimed to create the most suitable DL-based tool with enhanced robustness to facilitate digital strawberry plant phenotyping directly at the natural scene or indirectly using captured and stored images. Our SPT was developed primarily through two steps (subsequently called versions) using image data with different backgrounds captured with simple smartphone cameras. The two versions (V1 and V2) were developed using the same DL networks but differed by the amount of image data and annotation method used during their development. For V1, 7,116 images were annotated using the single-target non-labeling method, whereas for V2, 7,850 images were annotated using the multitarget labeling method. The results of the held-out dataset revealed that the developed SPT facilitates strawberry phenotype measurements. By increasing the dataset size combined with multitarget labeling annotation, the detection accuracy of our system changed from 60.24% in V1 to 82.28% in V2. During the validation process, the system was evaluated using 70 images per phenotype and their corresponding actual values. The correlation coefficients and detection frequencies were higher for V2 than for V1, confirming the superiority of V2. Furthermore, an image-based regression model was developed to predict the fresh weight of strawberries based on the fruit size (R2 = 0.92). The results demonstrate the efficiency of our system in recognizing the aforementioned six strawberry phenotypic traits regardless of the complex scenario of the environment of the strawberry plant. This tool could help farmers and researchers make accurate and efficient decisions related to strawberry plant management, possibly causing increased productivity and yield potential.

Glycogen Quantification and Gender Identification in Di-, Tri-, and Tetraploid Crassostrea gigas Using Portable Near-Infrared Spectroscopy.

Near-infrared spectroscopy (NIR) has become an essential tool for non-destructive analysis in various fields, including aquaculture. This study presents a pioneering application of portable NIR spectrometers to analyze glycogen content in the gonadal tissues of the Pacific oyster (Crassostrea gigas), marking the first instance of developing quantitative models for glycogen in tetraploid C. gigas. The research also provides a comparative analysis with models for diploid and triploid oysters, underscoring the innovative use of portable NIR technology in aquaculture. Two portable NIR spectrometers were employed: the Micro NIR 1700 (908-1676 nm) and the Micro PHAZIR RX (1624-2460 nm). Near-infrared spectra were acquired from the gonadal tissues of diploid, triploid, and tetraploid C. gigas. Quantitative models for glycogen content were developed and validated using cross-validation methods. Additionally, qualitative models for different ploidies and genders were established. For the Micro NIR 1700, the cross-validation correlation coefficients (Rcv) and cross-validation relative predictive errors (RPDcv) for glycogen were 0.949 and 3.191 for diploids, 0.915 and 2.498 for triploids, and 0.902 and 2.310 for tetraploids. The Micro PHAZIR RX achieved Rcv and RPDcv values of 0.781 and 2.240 for diploids, 0.839 and 2.504 for triploids, and 0.717 and 1.851 for tetraploids. The Micro NIR 1700 demonstrated superior quantitative performance, with RPD values exceeding 2, indicating its effectiveness in predicting glycogen content across different ploidy levels. Qualitative models showed a performance index of 91.6 for diploid and 95 for tetraploid genders using the Micro NIR 1700, while the Micro PHAZIR RX achieved correct identification rates of 99.79% and 100% for diploid and tetraploid genders, respectively. However, differentiation of ploidies was less successful with both instruments. This study's originality lies in establishing the first quantitative models for glycogen content in tetraploid C. gigas using portable NIR spectrometers, highlighting the significant advancements in non-destructive glycogen analysis. The applicability of these findings is substantial for oyster breeding programs focused on enhancing meat quality traits. These models provide a valuable phenotyping tool for selecting oysters with optimal glycogen content, demonstrating the practical utility of portable NIR technology in aquaculture.

Dieback of royal poinciana (Delonix regia) trees induced by Alternaria tenuissima and its impact on photochemical efficiency of photosystem II

In Saudi Arabia, poinciana (Delonix regia) trees are afflicted by dieback disease, manifesting symptoms that can lead to complete defoliation and, ultimately, the demise of trees in advanced stages. To investigate the dieback symptoms, samples were collected from branches at the transition zone. Identification of fungal isolates involved the assessment of colony and spore morphology, as well as PCR amplification and sequencing of the internal transcribed spacer (ITS) gene regions. The predominant fungus isolated was Alternaria tenuissima. Chlorophyll a fluorescence quenching and polyphasic fluorescence induction kinetics were conducted to assess the photochemical and nonphotochemical processes of Photosystem II (PSII) in stem barks affected by dieback. The dieback disease significantly reduced chlorophyll content, potentially hindering PSII performance in terms of maximum photochemical efficiency, quantum yield, electron flux, and photoprotection. These findings suggest that compromised photochemistry and photoprotection render poinciana trees inoculated by dieback less efficient in utilizing light energy and more susceptible to photoinhibition. Additionally, this study implies the use of non-invasive chlorophyll fluorescence techniques as a phenotyping tool for assessing photoprotective and photoinhibitory responses of stems to dieback stress.

Linguistic style as a digital marker for depression severity: An ambulatory assessment pilot study in patients with depressive disorder undergoing sleep deprivation therapy.

Digital phenotyping and monitoring tools are the most promising approaches to automatically detect upcoming depressive episodes. Especially, linguistic style has been seen as a potential behavioral marker of depression, as cross-sectional studies showed, for example, less frequent use of positive emotion words, intensified use of negative emotion words, and more self-references in patients with depression compared to healthy controls. However, longitudinal studies are sparse and therefore it remains unclear whether within-person fluctuations in depression severity are associated with individuals' linguistic style. To capture affective states and concomitant speech samples longitudinally, we used an ambulatory assessment approach sampling multiple times a day via smartphones in patients diagnosed with depressive disorder undergoing sleep deprivation therapy. This intervention promises a rapid change of affective symptoms within a short period of time, assuring sufficient variability in depressive symptoms. We extracted word categories from the transcribed speech samples using the Linguistic Inquiry and Word Count. Our analyses revealed that more pleasant affective momentary states (lower reported depression severity, lower negative affective state, higher positive affective state, (positive) valence, energetic arousal and calmness) are mirrored in the use of less negative emotion words and more positive emotion words. We conclude that a patient's linguistic style, especially the use of positive and negative emotion words, is associated with self-reported affective states and thus is a promising feature for speech-based automated monitoring and prediction of upcoming episodes, ultimately leading to better patient care.

Fourier‐transform mid‐infrared spectroscopy for in vitro protein digestibility measurement of pulse crops

AbstractThe standard method of estimating in vitro protein digestibility, the protein digestibility corrected amino acid score (PDCAAS) assay, does not support the expected workflow of a pulse breeding program. This is mainly due to its low‐throughput design and long processing time (∼16–24 h) per sample. Fourier‐transform mid‐infrared (FT‐MIR) spectroscopy has been developed as a high‐throughput phenotyping tool to estimate protein digestibility in pulses. The mid‐infrared region representing the amide I band (1756.81–1586.27 cm−1) was utilized to perform chemometric modeling with partial least squares regression (PLSR) to estimate in vitro protein digestibility in dry pea (Pisum sativum L.), lentil (Lens culinaris Medik.), and chickpea (Cicer arietinum L.) flours. The root mean square error of predictions of the developed PLSR models for dry pea, lentil, and chickpea were 0.00039, 0.00024, and 0.00017, respectively. Phenotyping with the FT‐MIR approach is more rapid than with the PDCAAS assay and estimates protein digestibility from the flour of a single seed in a shorter time (∼1–2 min). The FT‐MIR approach is robust as the spectroscopic data are consistent and chemically fingerprint this nutritional trait. Accordingly, FT‐MIR can resolve the phenotypic bottleneck of pulse breeding related to in vitro protein digestibility measurements by enabling a high‐throughput phenotyping workflow.