Development and Application of Image-Based High-Throughput Phenotyping Methodology for Salt Tolerance in Lentils

Abstract

Soil salinity is a major abiotic stress in Australian lentil-producing areas. It is therefore imperative to identify genetic variation for salt tolerance in order to develop lentil varieties suitable for saline soils. Conventional screening methods include the manual assessment of stress symptoms, which can be very laborious, time-consuming, and error-prone. Recent advances in image-based high-throughput phenotyping (HTP) technologies have provided unparalleled opportunities to screen plants for a range of stresses, such as salt toxicity. The current study describes the development and application of an HTP method for salt toxicity screening in lentils. In a pilot study, six lentil genotypes were evaluated to determine the optimal salt level and the growth stage for distinguishing lentil genotypes using red–green–blue (RGB) images on a LemnaTec Scanalyzer 3D phenomics platform. The optimized protocol was then applied to screen 276 accessions that were also assessed earlier in a conventional phenotypic screen. Detailed phenotypic trait assessments, including plant growth and green/non-green color pixels, were made and correlated to the conventional screen (r = 0.55; p < 0.0001). These findings demonstrated the improved efficacy of an image-based phenotyping approach that is high-throughput, efficient, and better suited to modern breeding programs.

Application of Genomics to Understand Salt Tolerance in Lentil

“…A wider distribution was observed for salt tolerance score (2.87-7.53), with a mean value of 5.29. For shoot dry mass, a narrow (4.56-5.76) level of distribution was observed with a mean value of 4.93 [40]. To establish the image-based phenotypic assay, a pilot study comprising six lentil genotypes (ILL2024, CIPAL1522 (tolerant); PBA Bolt, PBA Hurricane (moderately tolerant); PBA Ace, and PBA Jumbo2 (intolerant)) with known variation to salt stress was performed using a LemnaTec Scanalyzer 3D (LemnaTec GmbH, Aachen, Germany) plant phenomics platform at Plant Phenomics Victoria (PPV), Bundoora, VIC, Australia (Experiment 2).…”

“…All of the traits collected from the traditional phenotypic screen and image-based phenomics screen were normally distributed. Among these traits, traditional phenotypic scores and phenomics color pixels (from the image-based screen) were moderately correlated (r = 0.55; p < 0.0001) [40].…”

“…The phenotypic data used has previously been described in Dissanayake et al [40] and also provided in Table S1. To summarize, the study included salt stress screening data from 276 lentil accessions selected from the Pulse Breeding Australia (PBA) lentil breeding program.…”

“…The formation of the blocks was performed using the confidence intervals method [58], which defined the blocks based on 95% confidence intervals of the D' values, classifying as strong linkage disequilibrium (LD). The haplotypic variations in tolerant and intolerant accessions (salt tolerance classes identified from the previous study [40], Table S1) were further evaluated based on significant SNPs using TASSEL (version 5.2.48) [49]. Regions identified from marker-trait associations were also assessed using the gene-finding format (GFF) file of CDC Redberry (v2.0) [39] to examine any potential causative genes for salt tolerance in lentil.…”

“…The phenotypic data was attained through conventional and high-throughput phenotyping screens [40]. Two GBS methods, tGBS [41] and GBS-t [42], were used to obtain the genomewide SNP markers for GWAS analysis.…”