Location: Grain Legume Genetics Physiology Research
Project Number: 2090-21000-038-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Aug 31, 2026
Objective:
Overall Objective: Dissect the effects of the growing environment on the functional and nutritional quality of peas and develop foundational knowledge of the associated genes expressed in developing seeds. The supporting objectives are:
1. Assess the genetic diversity of yellow pea lines for nutritional quality as well as protein/starch purity and functional properties.
2. Determine environmental variables affecting nutritional quality of pea seed and functional properties of pea protein and starch.
3. Investigate expression patterns of pea protein and starch biosynthesis genes during seed development as affected by water and heat stresses.
Approach:
Obj1: Pea yield trials will be planted at two locations in Washington and two in Montana using a randomized complete block design with three reps. Trials are maintained using conventional practices typical for each area and are harvested at physiological maturity. Throughout the growing season, agronomic data is collected on the performance of the entries and, at harvest, weight of the seeds per plot is recorded. 200g subsamples of clean seed will be taken from each plot. Protein concentration will be quantified using an FP828p protein analyzer. The starch concentrations of the entries will be determined with Megazyme starch assay kits. An alkaline extraction and isoelectric precipitation method will be used to prepare protein isolates. The functional properties measured for protein isolates include solubility and foaming properties and water and oil holding capacities. For the starch-rich fraction, pasting property analysis using Rapid Visco Analyzer will be conducted. The gel firmness will be assessed using a TA-XT2i texture analyzer. Differential scanning calorimetry (DSC) will be used to evaluate the thermal properties of the starch fraction.
Obj 2: Data collected from Obj. 1 will be leveraged to explore effects of weather variables on quality traits. Weather data will be obtained from AgWeatherNet (https://weather.wsu.edu) and MT Mesonet (https://mesonet.climate.umt.edu). Soil physical and chemical characteristics will be determined from soil samples taken at field trial sites. Separate samples will be taken at 0-20cm and 20-65cm and sent to a commercial lab for analysis.
Obj 3: Four growth chambers and three genotypes, selected from pasting profile data from a preliminary study, will be used. Two chambers will be assigned a high temperature treatment (>30oC) and two an optimum temperature treatment (27oC). Within growth chambers, two moisture treatments will be applied (non-stressed and stressed). In each growth chamber, 10 pots will be assigned each to moisture stress and non-stress treatments. Prior to anthesis, all chambers will have optimum temperature and watering. Moisture and temperature stress will be introduced at anthesis. Soil moisture data will be collected using ECH20 10HS Large Volume Moisture Sensor. At anthesis, flowers will be tagged and pod samples will be taken 4 days after anthesis (DAA), 7DAA, 14DAA, 21DAA, and at physiological maturity in replicates of three and stored at -80°C. At physiological maturity, all pods remaining after sampling for expression analysis will be harvested in bulk and used to determine seed composition and functional properties of protein and starch. Three rounds of growth chamber experiments will be conducted; in each round one of the three pea genotypes will be evaluated.
Genes involved in protein and starch biosynthesis will be selected for gene expression analysis based on literature survey. Primers are designed based on sequences accessed from the pea genome v.1a (Kreplak et al., 2019). Eurofins Genomics will synthesize the primers. RNA will be extracted from samples and expression of selected genes will be evaluated by quantitative reverse transcription PCR using Bio-Rad CF.
