Haplotype-Based Genome-Wide Association Analysis Using Exome Capture Assay and Digital Phenotyping Identifies Genetic Loci Underlying Salt Tolerance Mechanisms in Wheat.

Soil salinity can impose substantial stress on plant growth and cause significant yield losses. Crop varieties tolerant to salinity stress are needed to sustain yields in saline soils. This requires effective genotyping and phenotyping of germplasm pools to identify novel genes and QTL conferring salt tolerance that can be utilised in crop breeding schemes. We investigated a globally diverse collection of 580 wheat accessions for their growth response to salinity using automated digital phenotyping performed under controlled environmental conditions. The results show that digitally collected plant traits, including digital shoot growth rate and digital senescence rate, can be used as proxy traits for selecting salinity-tolerant accessions. A haplotype-based genome-wide association study was conducted using 58,502 linkage disequilibrium-based haplotype blocks derived from 883,300 genome-wide SNPs and identified 95 QTL for salinity tolerance component traits, of which 54 were novel and 41 overlapped with previously reported QTL. Gene ontology analysis identified a suite of candidate genes for salinity tolerance, some of which are already known to play a role in stress tolerance in other plant species. This study identified wheat accessions that utilise different tolerance mechanisms and which can be used in future studies to investigate the genetic and genic basis of salinity tolerance. Our results suggest salinity tolerance has not arisen from or been bred into accessions from specific regions or groups. Rather, they suggest salinity tolerance is widespread, with small-effect genetic variants contributing to different levels of tolerance in diverse, locally adapted germplasm.

An image dataset of diverse safflower (Carthamus tinctorius L.) genotypes for salt response phenotyping.

This article describes a dataset of high-resolution visible-spectrum images of safflower (Carthamus tinctorius L.) plants obtained from a LemnaTec Scanalyser automated phenomics platform along with the associated image analysis output and manually acquired biomass data. This series contains 1832 images of 200 diverse safflower genotypes, acquired at the Plant Phenomics Victoria, Horsham, Victoria, Australia. Two Prosilica GT RGB (red-green-blue) cameras were used to generate 6576 × 4384 pixel portable network graphic (PNG) images. Safflower genotypes were either subjected to a salt treatment (250 mM NaCl) or grown as a control (0 mM NaCl) and imaged daily from 15 to 36 days after sowing. Each snapshot consists of four images collected at a point in time; one of which is taken from above (top-view) and the remainder from the side at either 0°, 120° or 240°. The dataset also includes analysis output quantifying traits and describing phenotypes, as well as manually collected biomass and leaf ion content data. The usage of the dataset is already demonstrated in Thoday-Kennedy et al. (2021) [1]. This dataset describes the early growth differences of diverse safflower genotypes and identified genotypes tolerant or susceptible to salinity stress. This dataset provides detailed image analysis parameters for phenotyping a large population of safflower that can be used for the training of image-based trait identification pipelines for a wide range of crop species.

Digital Phenotyping to Delineate Salinity Response in Safflower Genotypes.

Salinity is a major contributing factor to the degradation of arable land, and reductions in crop growth and yield. To overcome these limitations, the breeding of crop varieties with improved salt tolerance is needed. This requires effective and high-throughput phenotyping to optimize germplasm enhancement. Safflower (Carthamus tinctorius L.), is an underappreciated but highly versatile oilseed crop, capable of growing in saline and arid environments. To develop an effective and rapid phenotyping protocol to differentiate salt responses in safflower genotypes, experiments were conducted in the automated imaging facility at Plant Phenomics Victoria, Horsham, focussing on digital phenotyping at early vegetative growth. The initial experiment, at 0, 125, 250, and 350 mM sodium chloride (NaCl), showed that 250 mM NaCl was optimum to differentiate salt sensitive and tolerant genotypes. Phenotyping of a diverse set of 200 safflower genotypes using the developed protocol defined four classes of salt tolerance or sensitivity, based on biomass and ion accumulation. Salt tolerance in safflower was dependent on the exclusion of Na+ from shoot tissue and the maintenance of K+ uptake. Salinity response identified in glasshouse experiments showed some consistency with the performance of representatively selected genotypes tested under sodic field conditions. Overall, our results suggest that digital phenotyping can be an effective high-throughput approach in identifying candidate genotypes for salt tolerance in safflower.

High-throughput phenotyping to dissect genotypic differences in safflower for drought tolerance.

Drought is one of the most severe and unpredictable abiotic stresses, occurring at any growth stage and affecting crop yields worldwide. Therefore, it is essential to develop drought tolerant varieties to ensure sustainable crop production in an ever-changing climate. High-throughput digital phenotyping technologies in tandem with robust screening methods enable precise and faster selection of genotypes for breeding. To investigate the use of digital imaging to reliably phenotype for drought tolerance, a genetically diverse safflower population was screened under different drought stresses at Agriculture Victoria's high-throughput, automated phenotyping platform, Plant Phenomics Victoria, Horsham. In the first experiment, four treatments, control (90% field capacity; FC), 40% FC at initial branching, 40% FC at flowering and 50% FC at initial branching and flowering, were applied to assess the performance of four safflower genotypes. Based on these results, drought stress using 50% FC at initial branching and flowering stages was chosen to further screen 200 diverse safflower genotypes. Measured plant traits and dry biomass showed high correlations with derived digital traits including estimated shoot biomass, convex hull area, caliper length and minimum area rectangle, indicating the viability of using digital traits as proxy measures for plant growth. Estimated shoot biomass showed close association having moderately high correlation with drought indices yield index, stress tolerance index, geometric mean productivity, and mean productivity. Diverse genotypes were classified into four clusters of drought tolerance based on their performance (seed yield and digitally estimated shoot biomass) under stress. Overall, results show that rapid and precise image-based, high-throughput phenotyping in controlled environments can be used to effectively differentiate response to drought stress in a large numbers of safflower genotypes.

High-throughput phenotyping using digital and hyperspectral imaging-derived biomarkers for genotypic nitrogen response.

The development of crop varieties with higher nitrogen use efficiency is crucial for sustainable crop production. Combining high-throughput genotyping and phenotyping will expedite the discovery of novel alleles for breeding crop varieties with higher nitrogen use efficiency. Digital and hyperspectral imaging techniques can efficiently evaluate the growth, biophysical, and biochemical performance of plant populations by quantifying canopy reflectance response. Here, these techniques were used to derive automated phenotyping of indicator biomarkers, biomass and chlorophyll levels, corresponding to different nitrogen levels. A detailed description of digital and hyperspectral imaging and the associated challenges and required considerations are provided, with application to delineate the nitrogen response in wheat. Computational approaches for spectrum calibration and rectification, plant area detection, and derivation of vegetation index analysis are presented. We developed a novel vegetation index with higher precision to estimate chlorophyll levels, underpinned by an image-processing algorithm that effectively removed background spectra. Digital shoot biomass and growth parameters were derived, enabling the efficient phenotyping of wheat plants at the vegetative stage, obviating the need for phenotyping until maturity. Overall, our results suggest value in the integration of high-throughput digital and spectral phenomics for rapid screening of large wheat populations for nitrogen response.

A Robust Automated Image-Based Phenotyping Method for Rapid Vegetative Screening of Wheat Germplasm for Nitrogen Use Efficiency.

Nitrogen use efficiency (NUE) in crops is generally low, with more than 60% of applied nitrogen (N) being lost to the environment, which increases production costs and affects ecosystems and human habitats. To overcome these issues, the breeding of crop varieties with improved NUE is needed, requiring efficient phenotyping methods along with molecular and genetic approaches. To develop an effective phenotypic screening method, experiments on wheat varieties under various N levels were conducted in the automated phenotyping platform at Plant Phenomics Victoria, Horsham. The results from the initial experiment showed that two relative N levels-5 mM and 20 mM, designated as low and optimum N, respectively-were ideal to screen a diverse range of wheat germplasm for NUE on the automated imaging phenotyping platform. In the second experiment, estimated plant parameters such as shoot biomass and top-view area, derived from digital images, showed high correlations with phenotypic traits such as shoot biomass and leaf area seven weeks after sowing, indicating that they could be used as surrogate measures of the latter. Plant growth analysis confirmed that the estimated plant parameters from the vegetative linear growth phase determined by the "broken-stick" model could effectively differentiate the performance of wheat varieties for NUE. Based on this study, vegetative phenotypic screens should focus on selecting wheat varieties under low N conditions, which were highly correlated with biomass and grain yield at harvest. Analysis indicated a relationship between controlled and field conditions for the same varieties, suggesting that greenhouse screens could be used to prioritise a higher value germplasm for subsequent field studies. Overall, our results showed that this phenotypic screening method is highly applicable and can be applied for the identification of N-efficient wheat germplasm at the vegetative growth phase.