Location: Sugarbeet Research
Project Number: 3060-21000-045-019-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Aug 31, 2025
Objective:
1. Identify germplasm lines with lower respiration rate, higher root firmness and cell wall has lower water-soluble pectin content, and then use the identified germplasm as genetic source for breeding.
2. Detect genomic regions associated with respiration rate, root firmness and cell wall components.
3. Develop markers linked to target genomic regions for marker-assisted selection to develop new pre-breeding lines toward reducing post-harvest sucrose loss.
Approach:
Line purification and phenotype evaluation (FY23-FY24): In the 2023 growing season, the selected 300 lines will be grown in a field in Fargo, ND, and any plants bolting in the field will be removed. During harvesting, no more than 10 uniform roots from each line that are white and with characteristic sugarbeet root shape will be selected for seed production. Plants from 10 roots of each line will be bagged together during pollination time. Seeds harvested from these plants will be planted in the field in 2024 at two locations (Fargo and Prosper, ND) with two replications in each location. Roots from each replication will be used for evaluating storage respiration rate, root firmness, and cell wall pectin composition. Meanwhile, 10 roots from each line will be selected for seed reproduction, providing a second round of purification to further reduce genetic variation within each line.
2. Phenotypic evaluations and genotyping (FY24-FY25): all lines will be grown in fields at two locations (Fargo and Prosper, ND) with two replications in each location. For genotyping, about 3 g leaf tissue will be collected from 30 plants (0.1 g per plant) of each line at the 4-leaf stage to extract DNA for genotype by sequencing (GBS) to identify SNPs among lines. Roots from each location will be harvest and used for evaluating storage respiration rate, root firmness, and cell wall pectin analysis.
3. For each trait, a best linear unbiased prediction (BLUP) will be calculated for each line based on data collected from previous years. GWAS will be conducted using all BLUPs and genotypic data. PCR-based markers will be developed from target genomic regions.
4. Root respiration rate will be measured using the procedure described in Fugate et al. (2022). Measuring root resistance to penetration and root firmness will be performed as indicated in Kleuker & Hoffmann (2022).
5. Root cell wall pectin content measurements will utilize the methods of Deng et al. (2005) and Chen et al. (2015) with modification. Brei will be lyophilized from each line. Approximately 0.1 g of this material will be extracted with boiling 80% ethanol to remove soluble sugars and inactivate enzymes. The samples will then be washed twice with water, chloroform–ethanol (2:1), and acetone, respectively, to remove starch. The remaining insoluble material will be resuspended in 50 mM sodium acetate buffer (pH 6.5) with shaking for 6 h, and water-soluble pectin (WSP) in the supernatant will be obtained by centrifuging sample at 4 °C. The pellet from centrifugation will be resuspended in 50 mM sodium acetate buffer (pH 6.5) containing 50 mM EDTA and shaken for 6 h to extract chelator soluble pectin (CSP) by collecting the supernatant after centrifugation. The remained pellet will be resuspended in 50 mM Na2CO3 containing 2 mM EDTA. After shaking for 6 h and centrifugation at 4 °C, sodium carbonate soluble pectin (SSP) will be collected from the supernatant. The concentration of pectin in each fraction will be measured using the m-hydroxydiphenyl method (Paul & Jerome, 1982) with galacturonic acid as a standard.
