Agriculture and Food Systems - Theses

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    Advancing Genomics Resources and Phenotyping Methods to Improve Salt Tolerance in Lentil
    Dissanayake Ralalage, Ruwani Prasangika Dissanayake ( 2021)
    Pulses, also known as grain legumes, are members of the family Leguminosae and are grown for their edible seeds, containing high amounts of proteins and fibre. The work performed in this thesis is focused on lentil (Lens sp.), which is a self-pollinating, diploid, cool-season grain legume. Lentil production is constrained by multiple biotic and abiotic stresses that reduce growth and grain yield. The development of lentil varieties/cultivars with improved characteristics, including better yield, adaptation, and resistance to biotic and abiotic stresses, is a priority for international breeding programs. Therefore, the thesis investigated advanced genomic and phenomic approaches to characterize lentil germplasm for breeding purposes. Cultivated lentil (Lens culinaris Medik.) has a relatively narrow genetic base. Therefore, characterization of genetic diversity and genomic differentiation of wild gene pools is essential to identify any favorable alleles/genes that can be introduced into elite germplasm. A total of 467 wild and cultivated lentil accessions originating from multiple geographical locations were assessed for understanding genetic and allelic variations using transcriptome sequencing. An enriched single nucleotide polymorphic (SNP) resource (c. 422,101) has been delivered to lentil breeders for mining diverse genotypes for hybridization in future research and breeding. Understanding the relationship between lentil accessions and their geographical origins is also vital for identifying favorable alleles/traits that can be introgressed into the lentil germplasm. However, a weak correlation was observed between the lentil accessions, except for some accessions belong to L. culinaris and L. ervoides. Therefore, the study proposed that identifying lentil accessions with wide genetic distance variations within the same gene pool is more promising for selecting lentil accessions for breeding purposes, which also avoids crossing barriers between different gene pools. Lentil accessions that belong to L. culinaris, L. ervoides and L. nigricans were shown broad genetic distance boundaries. Therefore, these accessions with specific agronomic traits can be used to widen the lentil germplasm for breeding purposes. The genomic differentiation in each lentil species/subspecies was also analyzed using the allele-frequency-based analysis. The major genomic differentiation was observed on Chromosome 1 (Chr1; c. 1.0 Mbp), and results implied that L. nigricans was distantly related to other lentil species/subspecies. A total of five candidate genes were identified on c. 1.0 Mbp physical distance; however, the functionality of these genes in relation to wild and cultivated lentil species/subspecies still needs to be understood. One of the major abiotic stresses affecting gross profit and yield stability in Australian lentil cultivation is soil salinity. Identification of salt-tolerant varieties is the most viable and long-term option to maintain lentil productivity. However, this requires reliable and efficient screening methods. Salt tolerance assessment in lentil is currently based on morpho-physiological characterization and visual score ratings, which are often time-consuming, labor-intense and error-prone. Therefore, a novel high-throughput phenotyping (HTP) approach based on an image-based screen was developed using the LemnaTec 3D scanalyzer system to circumvent the limitations faced by current methods and accelerate the identification of the salt-tolerant varieties. The optimal salt concentration (100 mmol) and growth stages that distinguish salt tolerance levels were identified. Among the multiple phenotypic traits measured, area and color parameters were identified as the most informative traits for salt tolerance in lentil. The significant correlation observed between traditional and image-based screens (r = 0.55; p < 0.0001) demonstrated the accuracy of the developed protocol for salt tolerance in lentil, thereby can replace the conventional phenotyping approach. In addition to the phenotypic approaches, the understanding of the genetic basis of salt tolerance in lentil is important to develop salt-tolerant varieties. Recently, genome-wide association studies (GWAS) have been identified as a powerful tool to dissect the genetic basis of many phenotypic traits in diverse germplasm. Advances in resequencing approaches such as genotyping-by-sequencing (GBS) methods have also enabled the generation of a panel of SNP markers for large genome species like lentil. Two GBS approaches, targeted-capture (tGBS) and transcriptome-based sequencing (GBS-t), were tested to generate high-confidence SNP markers for association study. Among them, tGBS delivered the highest number of SNP markers with uniform distribution across the genome. Genomic regions for salt tolerance in lentil were identified on Chromosome 2 as well as on Chromosome 4. A high-affinity potassium transporter (HKT) gene was identified as the most possible candidate gene for salt tolerance in lentil. Mineral composition analysis performed on salt-treated and control lentil accessions has also been identified; Na+ ions absorbed by tolerant lentil accessions actively re-translocated them into roots or hold within the roots, supporting the candidate gene identified through GWAS. Pedigree analysis performed on salt-tolerant lentil genotypes identified two lentil accessions, ILL7685 and ILL1719, that could have been potential sources of allele contribution to salt tolerance in the lentil population. Overall, the study enriched the genomic and phenomic resources associated with lentil, thereby assisting future lentil research and breeding.