Research Objective
The project aimed to identify DNA markers, also known as single-nucleotide polymorphisms (SNPs), linked to water-use efficiency (WUE) and seed micronutrient content, such as iron (Fe) and zinc (Zn), in pea. These markers can be used by breeders for marker-assisted selection to develop pea varieties with improved WUE and enhanced nutritional quality. The results apply to Saskatchewan and Western Canadian production systems.
A genome-wide association study (GWAS) was used to identify SNPs linked with targeted traits. A composite collection of global pea germplasm and breeding lines, termed the GWAS-2 pea panel, was grown in replicated trials in five locations during 2020 and 2021. The seed samples were analyzed to estimate WUE using carbon isotope discrimination (δ¹³C) and nitrogen isotope composition (δ¹⁵N), which are indicators of WUE and nitrogen use, respectively. The seed samples were also analyzed to measure over 20 mineral nutrients. The GWAS-2 panel was previously genotyped using an Axiom® 90K SNP array developed by Thermofisher Scientific, United States. The phenotypic and genotypic information were used to identify trait-linked markers in multi-locus association mapping. SNPs that showed strong statistical significance, stable associations across environments, and meaningful phenotypic variation explained were converted to breeder-friendly Kompetitive Allele Specific polymerase (KASP) chain reaction (PCR) assays. Candidate genes flanking the SNPs were identified by searching the pea reference genome annotation of cv. Cameor.
Direct measurement of WUE/drought tolerance under field conditions requires tedious methods and is impractical for large breeding programs. In addition, there have been limited genetic tools available to improve WUE in pea, and key nutritional traits such as iron and zinc have lacked efficient selection methods. This project addresses these gaps by developing reliable DNA markers that allow breeders to select for these traits early in the breeding process.
The project aims to develop future pea cultivars with improved drought adaptation and enhanced nutritional value for Saskatchewan production systems.
| Objective | Final Outcomes |
|---|---|
| Rapid determination of WUE of a pea diversity panel (GWAS-2). | Identified significant variation in WUE among pea lines using δ¹³C. |
| Determination of micronutrient profile of a pea diversity panel (GWAS-2). | Measured 20+ micronutrients across environments. Identified strong genetic control for key nutrients such as zinc, magnesium, phosphorus, and calcium. |
| Compare the δ¹³C results between mature seed vs. vegetative tissue in selected accessions. | Seed δ¹³C showed moderate correlation with whole plant measurements, confirming that seed-based testing is practical for breeding programs. |
| Association mapping of micronutrients and WUE of pea. | Identified multiple SNP markers for WUE, nitrogen use, iron, and zinc. |
| Conversion of trait-associated markers to breeder-friendly KASP assays. | Converted selected SNPs into breeder-friendly KASP assays to enable rapid screening in breeding pipelines. |
Seed samples of 233 accessions of the GWAS-2 pea panel grown in replicated trials across five environments in 2020 and 2021 were analyzed for δ¹³C and δ¹⁵N as indicators of WUE and nitrogen use efficiency, respectively, along with micronutrient concentrations using mass spectrometry-based ionomics analyses.
Twenty-eight mineral elements, including barium, sodium, magnesium, phosphorus, sulphur, calcium, manganese, iron, cobalt, nickel, zinc, cadmium, lanthanum, and other trace elements, were quantified, providing a comprehensive assessment of seed nutrient composition.
The δ¹³C values ranged from -27.6 to -24.4%, indicating variation in physiological WUE among accessions. Seed iron and zinc concentrations ranged from 36.6 to 67.0 μg g⁻¹ and 28.8 to 61.0 μg g⁻¹, respectively, across environments.
Several traits, such as WUE and seed zinc concentration, showed high heritability (H2 = 0.84 to 0.86), meaning they can be improved through breeding. The GWAS-2 panel had previously been genotyped using the Axiom® 90K SNP (single nucleotide polymorphism) array, and 21,321 high-quality SNP markers were used for genome-wide association analysis.
Several significant marker-trait associations were identified for WUE, δ¹⁵N, and mineral nutrient accumulation. Key markers identified include Chr6LG2_264389013 and Chr5LG3_566189037 for δ¹³C (WUE), Chr1LG6_268443216 for δ¹⁵N, Chr3LG5_193905441 for seed iron, and Chr5LG3_186437816 for seed zinc. Candidate genes associated with carboxylic acid metabolism and Zn ion binding were identified near these markers.
These results demonstrate that WUE and micronutrient accumulation in pea are influenced by identifiable genomic regions, providing useful tools for marker-assisted selection to develop improved pea cultivars for Saskatchewan production systems.
Next steps include:
- Validate markers in breeding populations.
- Integrate markers into early-generation selection.
- Combine WUE and nutrient traits with yield testing.
Marker-assisted selection for WUE will enable breeders to select pea lines with better performance under dry conditions, greater yield stability under climate variability, and enhanced nutritional value (e.g., iron and zinc). The use of these tools will accelerate the development and delivery of improved pea cultivars to Saskatchewan farmers.