Non Technical Summary
A common feature of plant response to stress is that, when plants are subjected to repeated stress, their progeny may perform better under the stress than their parents. This implies that plants can "store and recollect" memories of past stress, a phenomenon known as plant memory response. Memory response can be beneficial or detrimental. In crop production systems, memory response has been demonstrated in plant defense against environmental stress, pathogens, and herbivory. However, we are not aware of any research on how memory response affects crop-weed interaction. If exposing crops to multiple generations of weed competition elicits a beneficial memory response, it is expected the progeny may be more competitive with weeds than the parents. This has the potential for the development of more competitive crop cultivars and thus, contributing to sustainable intensification. In this research-only seed grant application, we propose greenhouse and laboratory studies to evaluate the growth, developmental, and genetic basis of wheat response to multigenerational exposure to weed pressure. Our specific objectives are: (i) assess growth and developmental changes in wheat in response to multigeneration weed competition, (ii) evaluate hormonal expression among five generations of competition and the parent seed, and (iii) assess epigenetic patterns and gene expression in spring wheat response to multigenerational weed competition. Spring wheat has been exposed to broadleaf and grassy weed competition for five generations. Seeds from the five generations along with the original seed (parent seed) will be grown in the greenhouse at the same time to assess differences in growth, development, hormones, gene expression as well as DNA methylation. Results from this project will provide foundational knowledge on the growth, developmental, and genetic basis of wheat memory response to weed competition. This will provide useful information for developing competitive crop varieties for improved weed competitiveness and yield.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
213	2300	1140	50%
201	1549	1080	50%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 213 - Weeds Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other; 2300 - Weeds;

Field Of Science
1140 - Weed science; 1080 - Genetics;

Goals / Objectives
The long-term goal of this project is to understand the phenotypic and genetic basis of wheat response to multigenerational exposure to weed pressure to provide foundational knowledge for breeding competitive crops. The specific objectives are:1. Assess growth and phenotypic plasticity in wheat in response to multigenerational weed competition.2. Evaluate hormonal expression among the generations.3. Assess epigenetic patterns and gene expression in spring wheat response to weed competition.3a) RNA-Sequencing to evaluate gene expression differences among the generations.3b) Whole Genome Bisulphite Sequencing to assess DNA methylation differences among the generations.

Project Methods
Greenhouse studies were initiated in 2021 at the University of Idaho Kimberly Research and Extension Center to evaluate the response of spring wheat to repeated exposure to weed competition. There were four treatments arranged in a completely randomized design with 15 replications. The four treatments composed of one wheat seed grown in the center of the pot without any surrounding plants (wheat-only), one wheat seed grown in the center of the pot surrounded by eight kochia weeds (wheat-kochia), one wheat seed grown in the center of the pot surrounded by eight Italian ryegrass weeds (wheat-Italian ryegrass), and one wheat seed grown in the center of the pot surrounded by eight other wheat plants (wheat-wheat).One spring wheat ('UI Cookie') was planted in the center of a 3 L pot filled with potting soil and surrounded with either wheat, Italian ryegrass, kochia, or no plants. There were 8 surrounding plants to maximize competition and ensure plants were not too crowded to prevent seed production. Wheat in the center of the pot was grown to maturity. At heading, wheat in the center of each pot was covered with pollination bags to prevent cross-pollination or gene flow.At maturity, the number of seed heads was counted, and plants were harvested to measure biomass, seed number, and seed weight per plant. Seeds from the first generation were used to plant the second generation. That is, seeds from the wheat-only treatment were planted in the wheat-only treatment in the second generation, seeds from the wheat-wheat treatment were used to plant the wheat-wheat treatment in the second generation, etc. This was done to ensure that plants were exposed to the same form of stress/competition in each generation. The process was repeated to obtain the third, fourth, and fifth generations. Plants were sprinkler-irrigated as needed but no fertilizer was applied to maximize competition for growth resources. The original seed (G0), as well as seeds from generations 1 to 5 (G1 to G5), were saved for a common garden experiment.The common garden experiment will consist of 84 treatments; the five generations (G1 to G5) by four competition levels from each generation (as described above), in addition to the G0. These 21 treatment combinations will be subjected to the four competition levels in the common garden experiment to give a total of 84 treatments. Each of the 84 treatments will be replicated 14 times. This crossed design will enable us to evaluate how wheat that went through single or multiple generations of competition performs in the absence or presence of competition.Given the potential change in biomass allocation due to the treatments, we will conduct three destructive samplings (3 replicates at each sampling) at 20, 40, and 60 days after planting to measure relative growth rate (RGR), number of leaves, number of tillers, leaf area, and stem biomass. Photosynthesis/gas exchange and leaf greenness (SPAD) will be measured before each destructive sampling. At maturity, 5 replicates of each treatment will be harvested to measure stem biomass, number of seed heads, number of seeds, and seed weight.Extraction and purification of the phytohormones will be done on plants from common garden experiments. A sample of 1 g fresh leaf from each treatment will be ground to powder in liquid nitrogen and aliquoted (50 mg each) into 2 mL microcentrifuge tubes and stored at -80 . Samples will be extracted with 1 mL of -20 MTBE/MeOH extraction buffer, vortexed, and put on a shaker for 30 min at 4 and sonicated for 15 min on an ice-cooled bath sonicator. About 0.5 mL of 0.1% HCl acidified water will be added and vortexed for 1 min. Samples will again be put on a shaker for 30 min at 4 followed by centrifugation at 13,000 rpm for 10 min at 4 . 1 mL of the supernatant will be transferred into a 1.5 mL microcentrifuge tube and dried down at room temperature. The dried pellets will be resuspended in 100 μL MeOH:water (1:1, v:v) solution to be used for UPLC-MS/MS hormonal analysis.RNA-seq analysis will be performed using TruSeq method to compare gene expression differences in the 5 generations and G0. Leaf samples from the 5-week-old plants will be isolated and immediately put into liquid nitrogen. RNA will be extracted using a Qiagen-RNeasy Mini kit following the manufacturer's instructions. Sequencing libraries will be prepared using the TruSeq RNA Sample Preparation kit v2. mRNA will be purified using poly-T oligo-attached magnetic beads, cDNA will be synthesized after fragmentation, followed by A-tailing, adaptor ligation, and PCR amplification. Libraries will be checked for quality followed by deep sequencing on a single lane of Illumina HiSeq 2000 to generate 75 bp pair-end reads.For each treatment (wheat only, wheat-kochia, wheat-Italian ryegrass, and wheat-wheat), DNA methylome analysis will be performed on the 5th generation and the parental lines (G0) using FASTmC method to assess genome-wide methylation level.Phenotypic plasticity data will be analyzed using linear mixed-effects ANOVA in R statistical language. Generation and competition treatments would be considered fixed effects, and block and year would be considered random effects. Repeated measurement variables like leaf and tiller counts will be analyzed using repeated measures ANOVA. RNA seq data will be trimmed and processed for quality using rnaseq2 then mapped using Kallisto. Sample normalization and differential gene expression (DEG) will be performed using DESeq2. Whole-genome bisulfite sequencing (WGBS) will be trimmed using TrimmGalore, and Bismark and bwa-meth/MeyhylDackel toaligned to the genome. PCR duplicate will be removed, and methylation site will be called using nf-core/methylseq. We will utilize multivariate analytical procedures including cluster, principal components, and discriminant analysis to assess any differences in competitive features among the generations and competition treatments.