UNDERSTANDING PHYSIOLOGICAL AND GENETIC MECHANISMS OF TWO TYPES OF DROUGHT TOLERANT PEANUTS FOR FUTURE AND PRESENT ENVIRONMENTAL ADAPTATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030535
• Grant No.: 2023-67014-39607
• Cumulative Award Amt.: $649,372.00
• Proposal No.: 2022-10937
• Project Start Date: May 1, 2023
• Project End Date: Apr 30, 2027
• Grant Year: 2023
• Program Code: [A1152]- Physiology of Agricultural Plants

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
(N/A)

Non Technical Summary
The American Peanut council has identified drought as the top challenge facing the peanut industry as only 35% of growers in the U.S. have irrigation capabilities. For this reason, breeding for drought tolerance is paramount for the peanut industry. In previous work, we have identified cultivars that use two different strategies (water spenders and water savers) to maintain high yields under drought. However, the physiological and genetic mechanisms controlling these two strategies are not known. In other crops, these two mechanisms resulted in increased yields under specific environmental conditions. In this project, we will perform detailed physiological and RNA-sequencing experiments in peanuts under two rain-out shelter facilities to fully understand the physiological and genetic mechanisms of water-saver and water-spender drought-tolerant cultivars. In addition, we will use a RIL population derived from the cross of a water saver with a water spender line to find genomic regions (QTL) that relate to water saving or spending strategies and that can be used to breed for drought tolerance using marker-assisted selection. We will also use the data produced in the detailed physiological experiments to modify a peanut model that can predict the performance of each variable type under current and future environments to determine environments that favor one mechanism over another. With this approach, we will facilitate the selection of cultivars with different drought-tolerant strategies that with the help of crop modeling, can be tailored to yield more under different environments in the Southeast U.S.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1830	1020	70%
201	1830	1080	30%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1830 - Peanut;

Field Of Science
1080 - Genetics; 1020 - Physiology;

Keywords

drought tolerance

rna sequencing

crop modeling

peanut

water use efficiency

Goals / Objectives
The long-term goal of this project is to assist breeders in selecting cultivars with different mechanisms of drought tolerance so growers can produce high-yielding peanuts with reduced irrigation or under rainfed conditions and therefore increasing the sustainability of peanut farming. To achieve this objective, we need to better understand the physiology and genetics of "water saver" and "water spender" drought tolerant strategies and by using models, learn where these cultivars may perform better in the Southern United States. For this reason, the supporting objectives of this proposal are:Objective 1: Discover underlying physiological mechanisms of water spender and water saver cultivars.Objective 2: Model water saver and water spender drought tolerant mechanisms to find the best environments to maximize yield.Objective 3: Identifying QTL and probable genes underlying water saving and spender drought tolerant traits.

Project Methods
This study will leverage detailed physiological experiments, transcriptomic data, genomic QTL identification, and process modeling information from a select group of peanut cultivars and a bi-parental population to increase our understanding of mechanisms behind water saver and water spender drought tolerant strategies. First, detailed physiological measurements will be taken in a select number of cultivars in two rain-out shelter locations in Alabama and Georgia to be combined with modeling to identify the ideal locations to maximize long-term yield under different climate change scenarios using this drought tolerant strategies in the peanut belt. Second, a bi-parental population created from the cross of a water saver and water spender drought-tolerant peanuts will be genotyped by next-generation sequencing (NGS) and phenotyped to find the QTLs related to these two drought-tolerant strategies. RNA sequencing from the detailed physiological experiment and from the extremes in the bi-parental population experiments will help to hypothesize which genes may be involved in the control of these two drought-tolerant mechanisms.Objective 1: Discover subjacent physiological mechanisms of water spender and water saver cultivarsObjective 1, Task 1: Grow water-saver and water-spender peanut cultivars under two rain-out shelter environments.Two previously selected water savers (Line-8 and AU-16-28) and two water spender (PI502120 and AU-NPL 17) drought-tolerant cultivars, along with one drought-sensitive cultivar (AP-3) used as a check will be grown under two different rainout shelter environments on the first two years of this grant (Summer 2024and 2025).Objective 1, Task 2: Collect physiological data to understand the underlying mechanisms of water users and water spender drought tolerant cultivars.All the measurements will be performed in both locations (NPL and EV-Smith), therefore in this description, we will not separate between locations. All the measurements performed in Objective 1 will be used in the modeling efforts in Objective 2 and therefore will not be described in that section. To make this section of the approach clearer, the physiological measurements will be divided into 2 different groups:(1) Finding photosynthetic and transpiration limitations or advantages at leaf and canopy level between water user and water saver cultivars.We know that the water-saver cultivars selected for this experiment show half the stomatal conductance compared to water spenders cultivars when measured at midday, however, we do not know if these differences are maintained during the whole day or during the whole season. For this reason, diurnal gas exchange measurements using 4 sets of LI-6800 (LICOR Biosciences, Lincoln Nebraska) will be performed every month during the whole experiment and every two weeks during the drought period (Sanz-Saez et al., 2017; Soba et al., 2020).(2) Studying C partitioning between shoot and root can explain differences in water acquisition and usage between water spender and saver cultivars.Phenological dates including vegetative growth stage (V-stage), date of first flower and first pod, and end of pod addition will be measured to understand how C partitioning changes based on the development stage. Root growth characteristics will be measured during the entire growing season by installing 3 mini-rhizotron accession tubes per plot as performed by Rowland et al., (2015). Root images will be taken with a mini-rhizotron CI-602 (CID Biosciences, Camas, WA) every two weeks, and root length, diameter, and density will be estimated using RootSnap (CID Biosciences, Camas, WA). This data will be used for model calibration and evaluation. In order to estimate aboveground biomass growth during the season, the leaf area index will be measured using an Accu-PAR LP-80 on the same day of root characteristics measurements.Objective 1 Task 3: Comparing RNA expression of water saver peanut lines versus water spenders in response to drought.To find underlying genetic mechanisms of water saver and water spender drought tolerant genotypes, RNA-sequencing will be performed to find differentially expressed genes between cultivars and drought conditions. Leaf samples will be taken in new fully expanded leaves at midday two times after drought (approximately 5 and 10 days after drought initiation) has started in four replications per cultivar.We will sample the first time when the stomatal conductance is reduced a 30% compared to the control (5 days approximately) and the second time when the stomatal conductance is reduced a 60% compared to the control (10 days approximately).Objective 2: Model water saver and water spender drought tolerant mechanisms to find the best environments to maximize yield.Objective 2, Task 1: Modify the CSM-CROPGRO-Peanut model to include water saver and water spender traits in peanut.We will use the CSM-CROPGRO-Peanut model and data collected from the rainout shelters and irrigated controls to test the different hypotheses for water saver and water spender traits. Each of these traits can be controlled by changing model inputs. Each cultivar will be calibrated using one year of data from each location and evaluated using the second year of data from each location. In addition, yield plots (20 feet long, 4-row plots) with the 5 studied cultivars will be placed in two other locations in Alabama (Wiregrass Research and Extension Center, Headland and Gulf Coast Research and Extension Center, Fairhope), one in Georgia (Dawson), and one in New Mexico (Clovis) during year 2 and year 3 to calibrate and evaluate the yield response in different environments.Objective 2, Task 2: Perform spatial modeling to find the best environments to grow water-saver and water-spender cultivars.Spatial modeling will be used to simulate the performance of each cultivar across the peanut belt using 30 years of historical weather data and climate change data for 2050 and 2080.The model will be run for each of the five cultivars on a 1-km grid scale across the peanut belt to develop maps of performance using historical and future weather data.The end result will be maps that show areas where water savers and water spenders have advantages over traditional genetics.Objective 3: Identifying QTL and probable genes underlying water saving and spender drought tolerant traits.Objective 3 3, Task 1: Grow a water saver x water spender recombinant inbred line population under two rain-out shelter environments. Co-PI Chen is currently developing a RIL population from the cross of a water saver (AU16-28) and a water spender (AU-NPL-17) cultivars. By the time of starting this project, we will have an F6 RIL mapping population including 200 RILs. Two hundred F6 RILs, along with the parental lines (AU-NPL-17 and AU16-28) and one drought-sensitive cultivar (AP-3) used as a checkwill be grown under two different rainout shelter environments on the third and fourth years of this grant.Objective 3, Task 2: Phenotyping efforts to find QTLs for water users and water spender cultivars.Parental lines AU16-28 (water saver) and AU-NPL-17 (water spender), differ in their stomatal conductance and carbon isotope discrimination (Δ13C) under drought conditions (Zhang et al., 2022). Therefore, we will phenotype the RIL population resulting from their cross to phenotype at least these traits.Objective 3, Task 3: Find QTLs related to water saver and water spender drought tolerant mechanisms.Genotyping RIL population will be carried out by 3X Illumina sequencing. Plant leaf samples will be collected from greenhouse-grown plants during the third year of the project and stored at -80°C for DNA isolation. Libraries will be sequenced on two lanes of Illumina Novaseq to yield 750 Gb of raw sequence and about 3X coverage per line. Genome-wide SNPs will be called using Khufu (Korani et al., 2021).

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:There are several target audiences that are the focus of effort for the duration of this USDA NIFA Project. They include: Students: This includes student audiences through both teaching and research mentoring. Undergraduate and graduate students will be served by this project through direct involvement in research projects, data analysis and presentation of their research findings. Undergraduate and graduate students will also be served by this project through formal teaching at Auburn University through the "Crop Physiology" and "Principles of Plant Nutrition" courses taught annually. Students will also be directly involved in extension opportunities serving local farmers. Currently, the project is directly (funded by the grant) or indirectly (not supported by the grant but collaborating in the research) serving 3 PhD students. Breeders: Through collaboration with the peanut breeder at Auburn University in the Crop, Soil and Environmental Science Department, Dr. Charles Chen (Co-PI in this grant), we work together to directly select cultivars and breeding lines that will improve peanut drought tolerance. Broader scientific community: The findings from this work will be disseminated through conference presentations and open-access peer-reviewed publications. Additionally, genomic data will be deposited in public repositories (NCBI and Github). This will allow the larger scientific community access to our research findings and increase understanding and awareness of drought impacts on yield and physiological parameters. Last year, Dr. Sanz-Saez (PI) and Dr. Chen participated in several Alabama Extension events to disseminate information about this research, such as field days and the Alabama-Florida Peanut Show. Changes/Problems:We have not encountered any problems associated with our proposed aims. In the first year of experiments, we were able to run only one location. Depending on how this season turns and its results, we will decide if we need to run the detailed physiological measurements one year more or if we can start with the RIL phenotyping in the rain-out shelters in 2025. What opportunities for training and professional development has the project provided?Through the role of the PIs at Auburn University and the USDA-ARS National Peanut Laboratory, we have provided teaching and mentoring to the students involved in the project. As mentors (Sanz-Saez, Chen, Batchelor), we provide students with the skills and learning environment to further their own curiosity and research endeavors in peanut stress tolerance and breeding. PIs Sanz-Saez and Chen also teach courses focused on plant physiology and genetics and breeding, which are open to upper-level undergraduate students and graduate students at the university, and they use these experiments to teach students about drought tolerance and field research. Additionally, all PIs meet regularly to discuss project goals, recent progress, project hurdles, and next steps. This gives graduate students on the project an opportunity to network with all PIs on the proposal and practice their scientific communication skills. In addition, all students related with this grant will go to the 2024 APRES (American Peanut Research and Education Society) annual meeting in Oklahoma City (July 8-11) where they will learn about peanut breeding, agronomy and physiology and will present research related with this project. How have the results been disseminated to communities of interest?All data produced from this proposal will be publicly available through the use of data servers after publication, data repositories (i.e., GitHub), and federal data repositories (NCBI and SRA). Funds from this project will also be used to provide open access to all future publications to make them freely available to the public. The research conducted under this project (completed by the PIs, undergraduates, and graduate students) was presented at APRES annual meeting in 2023 and will be presented at APRES 2024 and at Auburn University Undergraduate Research Symposium (See in other products). In addition, in November 2024, students will travel to CSSA meeting to present data related to this project. What do you plan to do during the next reporting period to accomplish the goals?Regarding Objective 1, in 2024, we will perform detailed physiological experiments in both locations (Alabama and Georgia) also, including samplings for RNA sequencing. Regarding objective 2, next reporting period we will start modifying the crop models using data obtained in years 1nd 2 which would allow us to predict crop performance in the past and the future. ? Regarding objective 3, in 2024, we are increasing seed from the RIL population to perform replicated experiments in 2025.

Impacts
What was accomplished under these goals? Objective 1: Discover underlying physiological mechanisms of water spender and water saver cultivars. In this objective, we planned to perform field experiments at EV-Smith Research Station (Auburn University) and the National Peanut Laboratory (Dawson, Georgia) in 2023 to increase our understanding of water saver and water spenders drought tolerance mechanisms. However, as we got the news about the grant late in February 2023, the Dawson location was already devoted to another experiment, and we had to focus on the Auburn University Location (In 2024, we are performing the experiment in the two locations). In EV-Smith in 2023, the Rain-out Shelters used in this experiment were successfully moved from one field to another using a commercial tractor. This allowed us to do a proper crop rotation between years. The shelters were fixed into the new field and were prepared to be ready for planting and to implement drought during Mid-season drought (around Mid-July). This is quite a task because plastics need to be patched and all the instrumentation re-installed. Five peanut cultivars known for being water savers (AU16-28 and line-8), water spenders (AU-NPL-17 and PI505220), and drought-sensitive (AP-3) were planted in the rain-out shelter facility at EVS Smith Research Center on May 5th. Each cultivar was planted in a 2-row 30-foot-long plot following a complete randomized block design. Twenty days after planting, soil moisture sensors were placed at 3 depths (8,16, and 30 inches) below 3 cultivars in all the plots (AU16-28, AU-NPL-17, and PI505220). Drought started on July 17th and the first set of drought measurements started on July 24th with canopy photosynthesis and diurnal measurements. These measurements continued once a week until August 30th, when the drought was finished and the plants rewatered. Besides diurnal and canopy photosynthesis, CO2 curves were performed to test if the drought tolerance in some cultivars is given by a higher tolerance of rubisco to drought. Preliminary results indicate that plants under drought showed lower photosynthesis and stomatal conductance than under irrigation, which speaks of the success of the drought imposition treatment. We noticed that under irrigated and drought conditions, one of the water saver cultivars, AU-16-28, showed significant lower stomatal conductance than water spender cultivars AU-17 and PI505220, showing only a non-significant lower photosynthesis. This makes sense as AU16-28 has been described as a water saver. In addition, this underlines the importance of studying photosynthetic traits in the RIL population that is derived from AU16-28 and AU-17. In 2023, we observed that the cultivar AU16-28 showed a slight yield penalty compared to water spender cultivars, probably associated with the lower stomatal conductance observed. To give more information to build useful peanut models in objective 2, regular leaf area index measurements and biomass collections were performed during the season and onset of drought. This experiment has also been useful in finalizing the methodology to measure canopy photosynthesis in peanuts. We have realized that besides measuring the canopy photosynthesis in each plot, we need to harvest the aboveground biomass and measure again to account for the pod respiration in each plot as it interferes with the photosynthesis measurements. At the end of the drought, root crowns were excavated, and pictures were taken with a rhizovision system and are being analyzed for root characteristics such as angles, total root length, etc. Mini-rhizotron access tubes were not installed in 2023, as they arrived at Auburn University much later than the planting date due to delays from the company. Because of this delay, just before harvest, root cores were taken with a Giddins pneumatic probe to study how deep the peanut roots can reach in the soil and to study the root density at different depths. We took 2-inch diameter cores to a depth of 8 feet and started to look for roots in the deep end. We found that plants under irrigated conditions (mean of 45 inches deep) have shallower roots than plants grown under drought (mean of 67 inches deep). Although there is no significant effect between cultivars, one of the water spender cultivars (AU-17) showed the deepest roots under well water and drought conditions (maximum root depth of 81 inches). This preliminary data indicates that AU-17 is a water spender because of its deep roots. However, we think that the lack of differences between cultivars is due to the variability between subsamples for the root cores. Therefore, next season, we will take at least 5 subsamples per plot to ensure we can detect cultivar differences. Regarding yield, we found that drought significantly decreased yield from 4,516 to 3,441 lb/acre. Of all cultivars, PI505220 showed the highest yields; meanwhile, AU16-28 showed low yield but a low decrease in yield with drought. This could point to the capacity of AU16-28 to tolerate drought. In addition, in 2023, we collected leaf and root samples from the 5 peanut cultivars at different times during the drought (1, 2, 3, and 4 weeks after drought impositions). Samples are being extracted for RNA sequencing. In the year 2024, experiments were started at EV-Smith Research Station (Auburn University) on May 22nd and at the National Peanut Laboratory (Dawson, Georgia) on May 15th, 2024. Drought will start on July 15th at the National Peanut Laboratory and on July 22nd at EV-Smith Research Center coinciding with 60 Days after emergence, or mid pod filling. This year mini-rhizotron access tubes have been installed in both locations, and root measurements will be performed before drought, 4 weeks after drought, and at recovery. Physiological measurements are recorded regularly but will intensify to once per week once the drought starts. In conclusion, the goals of objective 1 are being fulfilled even though we did not have an extra location in 2023. We are taking extra physiological measurements to better understand peanut response to drought such as stomatal conductance, xylem vessel characteristics and root hair density. Objective 2: Model water saver and water spender drought tolerant mechanisms to find the best environments to maximize All physiological data will be used for the modeling objective during years 2-4. Dr. Sanz-Saez's PhD student, who is in charge of the physiological measurements, will start to being trained by Dr. Batchelor (Co-PI and modeler) on how to adapt the physiological parameters of the model to fit the physiological response of the different drought tolerant mechanisms to drought and irrigation. To have more yield data for this objective, five peanut cultivars known for being water savers (AU16-28 and line-8), water spenders (AU-NPL-17 and PI505220) and drought-sensitive (AP-3) have been planted at USDA-ARS, Cropping System Research Lab in Lubbock, Texas on April 30th, 2024 under irrigated and deficit irrigation (50% less water than irrigated) under the supervision of Dr. Naveen Puppala (External Collaborator). During the growing season, leaf stomatal conductance will be followed with an LI-600 to confirm water saver and water spender characteristics in this environment. Objective 3: Identifying QTL and probable genes underlying water saving and spender drought tolerant traits. In this objective, the main goal is to identify genomic markers of drought tolerance in a RIL population created by the cross of a water saver AU16-28 and a water spender AU-17. In the Summer of 2023, the F4 population was increased at Auburn University, and in the Fall of 2023, the F5 seeds were sent to Puerto Rico for seed increase. In the summer of 2024, the F6 population will be increased at Auburn University, with the idea of performing replicated trials in 2025.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sanz-Saez A, Zhang Q, Feng Y, Lamb M, Dang P, Chen C (2023). Oral. Biological Nitrogen Fixation Under Drought Depends on the Capacity of the Plant to Maintain a Good Water Status. American Peanut Research and Education Society, July 13, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rubin S, Hanif S, Sanz-Saez A, Heras J, Casado-Garc�a A (2024). Poster. Studying the effect of drought on peanuts stomatal characteristics: Development of an automatic detection method. Auburn University Undergraduate Research Symposium, March 26, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Deveau C, Hanif S, Sanz-Saez A, Rahman H, Rehman T, Hoffman L, Graff E (2024). Poster. Measuring Xylem Characteristics for Drought Tolerance in Peanuts. Auburn University Undergraduate Research Symposium, March 26, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sanz-Saez A, Zhang Q, Feng Y, Lamb M, Dang P, Chen C (2024). Poster. Biological Nitrogen Fixation Under Drought Depends on the Capacity of the Plant to Maintain a Good Water Status and not to intrinsic tolerance to drought of nitrogen fixation. Peanut Alabama-Florida Show, February 1, 2024.