Performing Department
(N/A)
Non Technical Summary
Rice production is impacted by both daytime and nighttime heat stress. However, high nighttime temperature (HNT) has a more detrimental effect on rice yield and quality and is considered an immediate threat as the average nighttime temperature is rising rapidly worldwide, including in the U.S. Several studies have categorically linked HNT during reproductive stages to low grain yield and quality. The normal nighttime temperature for the rice growing regions in the U.S. is considered to be ~22 oC, but the average nighttime temperature has been increasing over the last decades, especially in California and Arkansas rice growing regions. The HNT of 28 oC during the flowering stage could lead to significant yield declines and HNT of 32 oC is devastating, resulting in up to 90% loss in the grain yield. Significant increase in percentage chalk were also observed in rice grains under HNT. which is an undesirable charactersitics that impacts grain appearance, cooking quality, and palatability leading to the reduction of the market value of US rice in the export marketOur ultimate goal is to provide plant breeders and rice producers in the U.S. a rice germplasm and/or variety that is tolerantt to HNT particularly on traits such as spikelet fertility and percentage chalk that influences grain yield and quality respectively. We will identify genes or quantitative trait loci (QTL) in the DNA of US rice germplasm as well as N22, an exotic rice that has a known tolerance to HNT using genomewide association studies (GWAS) and QTL mapping approach. We will tag those identified genes or QTL and deveop DNA markers. These DNA markers for HNT tolerance will be used in the breeding program to identify lines/varieties that can be tested and advanced in the breeding pipeline. These lines will then be screened in controlled growth chamber for HNT tolerance as well as tested for all other agronomic and grain quality characteristicss across different locations and several years in Arkansas before releasing as a germplasm and/or variety. Additionaly, Our gene editing appraoch will help us understand the mechanism associated with a well-known chalk QTL and could reveal pathways and genes controlling grain yield and chalkiness. The transcriptomic, proteomic, and metabolomic results will also aid in the development of HNT tolerant elite rice lines and will contribute to the genetic resources that could be used in public breeding programs or released as cultivars, These datasets will be made available to the rice research community through public repositories.
Animal Health Component
0%
Research Effort Categories
Basic
30%
Applied
35%
Developmental
35%
Goals / Objectives
Goal: The goal of the project is to mitigate the effects of hight night-time temperature (HNT) stress in rice yield and quality through identification of useful genes, understand the mechanisms, and develop pre-breeding rice lines with HNT tolerance.Objective1: Identify candidate genes associated with high nighttime temperature (HNT) tolerance traits using genome-wide association studies (GWAS) on the 500 US rice diversity panel.Objective 2 Identify genomic regions of the non-adapted rice line N22 in the biparental population of 310 Diamond x N22 RILs associated with HNT tolerance by QTL mapping approach.Objective 3: Dissect the molecular and biochemical basis of HNT tolerance in vpp5 suppression lines using transcriptomics, proteomics, and metabolomics, and validate vpp5 mutation in a modern cultivar through CRISPR/Cas9-mediated gene editing.Objective 4: Develop adapted HNT tolerant rice breeding lines by incorporating markers associated with candidate genes identified in Objectives 1, 2, and 3 into a marker- assisted selection pipeline.
Project Methods
Obj. 1: Identify candidate genes associated with high nighttime temperature tolerance traits using genome-wide association studies (GWAS) on the 500 Arkansas diverse rice panel.Efforts: Two samples of each line will be raised in greenhouses in pots of 4×4×10 in (length x width x depth). Day and night temperatures will be maintained at 86.0-89.6 °F (30-32 °C) and 73.4-78.8 °F (23- 26 °C), respectively. Natural light will be supplemented with metal halide at a minimum of 13-hour day length. Upon reaching the R2 growth stage, all pots will be removed from the greenhouses to be placed in our walk-in climate-controlled chambers. One rep of each cultivar will be randomly placed in each growth chamber in two batches. Set temperatures during the day in the growth chambers will gradually increase from 82.4 °F (28 °C) at 6:00 AM and to 91.4°F (33°C) at 12:00 PM. At night, it will gradually decline to a low of 82.4°F (28 °C) for HNT treatment and 73.4°F (23 °C) for control treatment. Additionally, we will genotype the rice diversity panel using the Diversity Array Technology (DArT) seq platform.Evaluation: Our project will focus on two major traits: spikelet fertility and percentage chalk. However, other important agronomic traits and grain quality will also be measured, including maturity, height, milling yield (head and total rice), panicle length, seed weight, and grain dimensions. All phenotypic data will be analyzed using a linear mixed model with ASReml-R. We will identify genotype-to-phenotype associations by running univariate and multivariate GWAS. Traits will be considered for multivariate GWAS if they have a correlation above 0.5. To identify the candidate genes, we will do a combination of literature review and a genome annotation pipeline in the Rice Genome Annotation (Osa1) Release 7.Obj. 2: Discover genomic regions in rice cultivar N22 associated with high nighttime temperature tolerance by QTL mapping approach of Diamond x N22 RILsEfforts: We will gather all phenotypic data in the Diamond x N22 RIL population. Data includes percentage chalk (% chalk), spikelet fertility (SF), height, stalk diameter, number of tillers, days to R2, days to R5, flag leaf length, panicle length, number of panicle branches, presence of awns, chlorophyll content, and head rice yield.Evaluation: Phenotypic data will be analyzed using a linear mixed model implemented in the ASReml-R package. QTLs will be mapped using inclusive composite interval mapping by QTL IciMapping Version 4.2 (Meng et al., 2015). The physical positions will be retrieved through Gramene and the map will be generated using JMP genomics Ver. 9.Obj. 3: Dissect the molecular and biochemical basis of HNT tolerance in vpp5 suppression lines using transcriptomics, proteomics, and metabolomics, and validate vpp5 mutation in modern cultivars through gene editing.'Omics' analysis towards dissecting the mechanism.Efforts: Eighty plants each of vpp5 and the tissue culture derived WT will be grown in the greenhouse, and at the R2 stage equally distributed into two growth chambers: The control and HNT chamber. On transcriptome analysis by RNA-seq. RNA will be isolated and will be submitted to the service provider for RNA-seq and bioinformatics analysis. On proteome analysis, total protein will be extracted from 14 and 21 DAF tissues. This will be submitted to IDeA National Resource for Quantitative Proteomics at UAMS for Global Proteomics using DIA design for analysis. For metabolome analysis, we will extract metabolites from 14 and 21 DAF caryopses (n=6) of each genotype/treatment.Evaluation: The significantly different proteins or metabolites in vpp5 line under normal or HNT conditions at 7 or 14 DAF stages will be correlated with genes using PMN or STRING databases, respectively. The identified genes will be searched in the transcriptome of the vpp5 line at 7, 14, and 21 DAF to draw a correlation between genes and proteins/metabolites.Spikelet fertility, grain quality, and imagingEfforts: The number of filled grains will be determined manually on the first emerged panicle on 5 different plants of each genotype grown in HNT and control chambers. The physiologically mature grains harvested at ~40 DAF and dried for 2 weeks at room temperature will be used for WinSEEDLETM analysis with 150 grains for each genotype/treatment to determine the percentage of chalkiness and the average chalk size per grain.Evaluation: Chalky grains from each set (genotype/treatment) will be subjected to scanning electron micrography (SEM) to develop granule images at 2000× magnification. This analysis will point to the mechanism related to starch biosynthesis, degradation, or other (e.g., an abundance of small-size protein bodies).CRISPR-mediated targeted mutagenesis of VPP5 in elite riceEfforts: Using the protocols developed for Arkansas rice tissue culture and transformation (Srivastava & Zhao, 2017), rice cultivar Diamond and RoyJ will be subjected to genetic transformation using Chalk5-CRISPR/Cas9 vector, pJU46 (Srivastava & Zhao, 2017). Embryogenic callus from the mature seeds will be used for tissue culture and transformation. The regenerated shoots will be transferred to rooted in hormone-free media, and the resulting plants will be transferred to the greenhouse for seed set.Evaluation: The plant lines will be analyzed by PCR-sequencing for the presence of insertions-deletions (indels) at the target sites. The amplicons will be sequenced, and the resulting sequences will be aligned with the reference sequence to determine mutations in eachplant line. Expression analysis will be conducted using total RNA from 7 - 21 DAF caryopses of the lines developed in this project. The mature seeds from the homozygous edited plants and non-transgenic controls will be used for determining the effect of HNT on spikelet fertility and grain chalk.Obj 4: Develop adapted HNT tolerance rice breeding lines by incorporating markers associated with candidate genes identified in objectives 1, 2, and 3 into a marker-assisted selection pipeline.Efforts: On testing and validation of HNT markers in early and advanced rice lines, we will employ a combination of pedigree and marker assisted selection (MAS) using KASP markers. A core subset of the populations in our rice breeding program will be used to validate and test the utility of QTLs and genes discovered from QTL mapping, transcriptomics, and GWAS. These have one or both parents exhibited HNT tolerance in one or both traits of interest (SF and % chalk) based on preliminary studies. The advanced F5 populations will be planted in panicle rows in Stuttgart, AR. The lines will be visually selected for appearance, such as the standard height of 35-40 inches, upright leaves, long panicles, early maturing, glabrous leaves, non-lodging/thick straw, absence, or short awns. Additionally, several lines in the F4 generations will be planted in rows. Depending on uniformity and plant type, we will select from the F4 rows F5 lines with tolerant and susceptible alleles as control. All lines in F5:6 generations in 2025, 2026, and 2027 will be planted in a preliminary yield trial and screened under HNT in the growth chambers simulating normal and high nighttime temperature conditions.Evaluation: Data for spikelet fertility, chalk, and other yield-related traits will be evaluated. Samples will be genotyped using KASP markers and correlation analysis will be implemented to determine the accuracy of markers in predicting tolerance to HNT for spikelet fertility and chalk. Depending on the performance against check varieties, HNT tolerant lines can be advanced to Arkansas Variety Advancement Trial and in Uniform Regional Rice Nursery as part of the breeding and testing pipeline before being released as a variety. The lines will also be recycled back into the breeding program as parents of new HNT-tolerant varieties.