Location: Dale Bumpers National Rice Research Center
Project Number: 6028-21000-012-006-R
Project Type: Reimbursable Cooperative Agreement
Start Date: May 1, 2023
End Date: Apr 30, 2024
Objective:
1. Ascertain the genetic pathways for rice cold stress tolerance response mechanisms as a basis for gene discovery.
2. Fine-map new and previously reported cold tolerance quantitative trait loci (QTL) identified by mapping in genome-wide association mapping panels and biparental mapping populations to prioritize candidate genes for functional validation.
3. Identify and validate candidate genes associated with cold tolerant QTL using both lab and field evaluations across various genetic backgrounds to generate a small set of five to ten QTL and associated candidate genes for improving cold tolerance traits at germination and the early seedling stages through marker assisted breeding in U.S. rice cultivars.
Approach:
To ascertain pathways associated with cold tolerance, select three Wir11/Carolino164 backcross inbred lines (BILs) that had high Low Temperature Seedling Survivability (LTSS) scores, thus cold tolerant (CT) and three BILs with low LTSS scores from the BIL mapping population to identify differentially expressed genes (DEGs) following cold treatment vs control (warm) treatment and subsequently identify the genetic pathway associated with cold tolerance that differentiate the high vs low LTSS genetic extremes. Secondly, the lead researcher at Marquette University (MU) will identify homozygous overexpressed and knock-out transgenic lines from the lines developed at MU for the OsUGT90A1 gene which has a positive effect on membrane integrity and the OsUBC gene which has positive effect on seedling survivability, validate their CT phenotypes, isolate RNA and perform RNA-seq analyses to identify DEGs between wild type and transgenic plants, and identify specific genetic pathways modulated by the different CT genes.
To fine-map cold tolerance QTL, advance the four subpopulation-specific (indica, aus, temperate and tropical japonica) recombinant inbred line (RIL) populations under development to the F9 generation and evaluate the populations for LTSS and electrolyte leakage (EL); based on the RIL QTL mapping in the subspecific populations, selected RILs will be crossed for fine mapping, phenotyping for low temperature germinability (LTG) and antioxidant activity (AOX), and developing near isogenic lines (NILs); the Rice Diversity Panel 1 (RDP1) GWA mapping will be conducted using the recently available high density genotypes based on 3.5 million imputed markers, and crosses will be made between selected Wir11/Carolino164 BILs for fine-mapping and developing NILs. NILs will be used for gene confirmation, germplasm enhancement and marker-assisted selection to improve the chilling tolerance of elite rice lines.
To identify and validate candidate genes, 40 QTL associated with multiple traits evaluated and linked with multiple significant single nucleotide polymorphism (SNP) markers will be selected. The lead researcher at Marquette University will then conduct functional assays in transgenic plants to validate selected genes by overexpressing the gene(s) in a cold sensitive rice cultivar, knocking out the gene(s) in a cold tolerant rice cultivar, and developing transgenic plants (rice and Arabidopsis thaliana) using Agrobacterium-mediated transformation and/or CRISPR/Cas9 genome editing which will be phenotyped using the previously described physiological (EL, LTSS) and/or biochemical (AOX) assays; and validate cold tolerance in non-transgenic rice lines [selected Rice Minicore (RMC) or RDP1 accessions, RILs, NILs] in field studies near Milwaukee, Wisconsin.
