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ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Plant Physiology and Genetics Research » Research » Research Project #434838

Research Project: Enhancing Abiotic Stress Tolerance of Cotton, Oilseeds, and Other Industrial and Biofuel Crops Using High Throughput Phenotyping and Other Genetic Approaches

Location: Plant Physiology and Genetics Research

2021 Annual Report

The objectives of the plan concentrate on utilizing advanced phenomic and genomic approaches to genetically improve cotton, oilseed crops, bioenergy and industrial crops and expand their use for food, feed, fuel, and fiber production for United States agricultural sectors and global use. To reach that goal our specific objectives are: Objective 1: Use existing and newly developed field-based phenotyping methods to evaluate cotton, oilseeds, and other industrial and biofuel crops, and utilize the results to enable effective use of high-througput phenotyping (HTP) methodology for crop genetic improvement and management. Sub-objective 1A: Field-based evaluation of cotton using high-throughput phenotyping and conventional methods for germplasm improvement and crop management. Sub-objective 1B: Field-based phenotypic evaluations for biofuel crop camelina using high-throughput and traditional phenotyping technologies for traits related to drought stress. Sub-objective 1C: Use high-throughput and traditional phenotyping strategies to identify soybean germplasm with abiotic stress tolerance traits. Sub-objective 1D: Phenotypic characterization of USDA guayule collection under abiotic stress conditions and Arizona growing conditions using traditional and high-throughput phenotyping technologies. Objective 2: Utilize various new and conventional genetic approaches to identify genes and associated molecular markers conditioning abiotic stress tolerance in arid environments, and determine relationships with important agronomic traits. Sub-objective 2A: Identify molecular markers associated with genes involved in temporal patterns with abiotic stress tolerance and agronomic traits in cotton using high-throughput phenotyping. Sub-objective 2B: Identifying alleles/genes and associated molecular markers conditioning yield and abiotic stress tolerance and related traits in bioenergy crop, camelina. Sub-objective 2C: Identify genes/alleles and associated molecular markers conditioning yield and abiotic stress tolerance in soybean.

The objectives of the plan will be carried out using various high through-put phenotyping (HTP) approaches used to identify and improve cotton, camelina, soybean and guayule crops with increased tolerance to abiotic stress and stable productivity. For each crop, a genetic population/diversity panel will be planted under well-watered (WW) and water-limited treatments, based on agronomic recommendations of each crop, in replicated design over several years. The HTP data will be collected on a weekly basis throughout the growing season using HTP platforms that use electronic sensors to measure crop height, canopy multi-spectral reflectances and canopy temperature. In addition to HTP measurements, morphological, physiological and agronomic traits including plant height, lodging score, and flowering date will be collected during the growing season. At physiological maturities, plots will be harvested and seed/lint yield will be determined. Oil and leaf wax contents and compositions will be quantified using standard gas chromatography analysis. For guayule, rubber and resin will be determined using an Ion chromatography system. Traits will be analyzed using MIXED model in statistical analysis software (SAS) software, where water treatments, different environments and accessions will be considered as fixed effects and replicates will be the random effect. Differences among lines within each water treatment will be determined with a Bonferroni adjustment for multiplicity test. G×E interaction analysis will be conducted for recorded traits where water treatments, replicates, environments, and accessions will be considered as random effects. Quantitative trait loci (QTL)/alleles/genes associated with complex traits like heat and drought stress tolerances will also be identified. Cotton recombinant inbred line (RIL) population and camelina and soybean diversity panels will be genotyped using Genotyping-by-Sequencing technology. Genome-Wide Association Studies (GWAS) and QTL analyses will be used to identify molecular markers that are associated with and controlling the dynamic changes in plant growth under stress conditions, crop productivity traits and stability and oil and wax content and quality (Objective 1). Best linear unbiased predictors (BLUPs) of each phenotypic trait will be determined using mixed model of SAS software. GWAS analyses will be conducted using the trait analysis by association, evolution and linkage (TASSEL) package. To find the best model that is able to detect the associations between phenotypic traits and single nucleotide polymorphism (SNP) markers, and reduce the number of false-positive associations, the Mixed Linear Models (MLM) approach of TASSEL will be used. Candidate genes from multiple GWAS analyses will be identified from genomic intervals in the reference genome assemblies. In cotton, QTL analyses will be conducted using the inclusive composite interval mapping (ICIM) program.

Progress Report
In support of Objective 1, we evaluated aerial High-throughput phenotyping (HTP) platforms for effective use of HTP methodology for crop genetic improvement and management. Both a manned aerial vehicle (MAV) and an Unmanned aerial vehicle (UAV) equipped with a multispectral camera and Real-time kinematic positioning (RTK) Global Positioning System (GPS) were deployed to analyze images for phenotypic metrics. Aerial images were acquired in soybean, guayule, and cotton fields in Maricopa, Arizona, with ground truth (GT) measurements using a hand-held normalized difference vegetation index sensor. The images were processed for plot-level metrics through radiometric calibration, segmentation, masking, and gridding using custom-developed Image Mapping and Analytics for Phenotyping (IMAP) software. We achieved closely matching metrics between two platforms and vegetation indexes highly corrected with the GT data. In support of Sub-objective 1A, the first cotton trial (1.1) was comprised of a population developed by ARS researchers in College Station, Texas, and Florence, South Carolina, first received in 2016 and evaluated for yield and fiber quality traits in multi-year, multi-location trials. The field evaluations were completed in 2019. The yield and fiber quality data from all three locations was processed in 2020 and identified five germplasms adapted to multiple locations for release. The germplasm release notice # P.0061.20 was submitted in September 2020 and accepted in December 2020. A journal article detailing the released germplasm was published in the Journal of Plant Registration. The second cotton trial evaluated, (1.2) is comprised of selections from the Regional Breeders Testing Network (RBTN) population(s) and the North Carolina cotton association mapping population. These selections were made from trials that were run between 2016 – 2019 for variation in leaf chlorophyll content, chlorophyll fluorescence, gas exchange, and biomass including leaf area. These traits are components of radiation use efficiency (RUE). High-throughput phenotyping methods have been developed to measure leaf chlorophyll content and chlorophyll fluorescence and are undergoing further validation during the 2021 field season. Results continue to indicate that cotton has high leaf chlorophyll content and high light interception but is inefficient at photosynthesis. To support Sub-objective 1B, a camelina spring diversity panel consisting of 250 accessions, plus ten commercial varieties, were planted in an alpha-lattice design, with three replications, under field conditions of Maricopa, Arizona, over two years. The genotypes were planted under well-irrigated and reduced-irrigated trials. The HTP data were collected on a weekly basis throughout the growing season. In addition to HTP measurements, traditional morphological and physiological traits including flowering time and plant height were collected. At physiological maturity, plots were harvested for seed yield and seed weight determination. Seed samples were collected for oil content and fatty acids and glucosinolate compositions using near-infrared spectroscopy (NIRS). Statistical analyses over environments and years indicated significant effects for genotypes, environments, but no Genotype x Environment (GxE) effects were observed for the studied traits. In support of Sub-objective 1C, cuticular leaf wax accumulation was studied as one of the mechanisms for drought stress tolerance in soybean. The total waxes and wax classes including, alcohols, triterpenoids, aldehydes, alkanes, fatty acids, and wax esters were extracted from leaf samples of 200 diverse soybean genotypes grown under well-irrigated and reduced-irrigated conditions at Maricopa, Arizona. The preliminary results indicated the wide phenotypic diversity in total waxes and constitutions and their relation to abiotic stress responses among soybean genotypes. Under stress conditions, there was an increase in accumulation of waxes. In support of Sub-objective 1D, a new phenotypic trial was started, including USDA guayule accessions and Mariola wild accessions. Mariola is the closest relative to guayule and had been used to transfer cold tolerance traits to guayule to expand its planting regions up to Colorado. Both guayule and Mariola genotypes are grown in field trials at Maricopa, Arizona, under two irrigation regimes (well-irrigation vs. reduced irrigation). Morphological, resin, rubber, and HTP-related traits were measured. Results showed wide phenotypic variations among genotypes and irrigation levels (stress conditions). Drought stress affected rubber and resin content. The phenotypic stabilities varied among studied genotypes, and that could indicate the different genetic bases controlling drought tolerance among guayule genotypes. These genetic bases could regulate different stress tolerance mechanisms. As expected, rubber content was lower in Mariola compared to guayule. Under stress conditions. Mariola produced resin content comparable to guayule, but resin composition in both could be different. HTP traits, including canopy temperature and vegetation indexes, were collected. Results showed that guayule has high canopy temperature values under stress conditions. Still, under stress conditions, some genotypes were more stable (less change in temperature) than others. Guayule genotypes showed a decrease in vegetation indexes under stress, where the reduction percentages were not fixed among genotypes, and some genotypes exhibited minimal reduction in vegetation indexes. Results suggested that guayule and Mariola populations could have drought tolerance mechanisms and are good candidates for future genetic improvement programs. In support of Sub-objective 2A, germplasm from the Regional Breeders Testing Network (RBTN) population in 2017 was identified with reduced canopy temperatures under low soil moisture and with increased leaf wax; both traits are associated with drought tolerance and was crossed with a line known to have heat adaptation traits to develop a reciprocal recombinant inbred line mapping population. In 2019 and again in 2020, five bolls from a single plant for each line were harvested for the next year. This year (2021) the F5:F6 seed was planted in a replicated randomized complete block design and the lines will be evaluated for leaf chlorophyll content using the developed field-based high-throughput phenotyping method, chlorophyll fluorescence, and leaf area. To support Objective 2B, genome-wide association studies (GWAS) analyses were conducted using genome-by-sequencing technology (GBS) genotyping and phenotypic data for traits of interests in a two-year multi-location field trials planted at Maricopa, Arizona. Phenotypic data were collected for 15 different fatty acids, 10 fatty acid ratio, and four agronomic traits and salt stress tolerance. GWAS analyses identified 57 single nucleotide polymorphics (SNPs) significantly associated with palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, oil content, protein content, plant height and flowering date. Of the 57 identified SNPs, six SNPs shared associations between different traits, indicating putative genes related to fatty acid biosynthesis and transfer. Saline soil is one of the abiotic stresses that affect camelina seed germination where it reduces optimal canopy stand and reduces final seed yield. GWAS analyses identified 19 SNPs that are significantly associated with camelina seed germination rate and seedling dry weight under salt stress. The significant SNPs were directly or indirectly related to phosphatase metabolism, signaling transductions, and cell membrane activities that affected by salt stress. The putative trait-associated SNPs can be used to unveil candidate genes and molecular mechanisms underlying secondary metabolite biosynthesis, plant growth and development, and genetically dissect important agronomic traits and to be used as molecular makers in breeding programs.

1. Shuttle breeding collaboration releases five upland cotton germplasm adapted to multiple environments. Cotton fiber quality is an important characteristic for United States producers who compete to sell cotton in an international market. When purchasing bulk cotton, textile mills have certain “premiums” established for differing fiber characteristics, like length and strength, that improve production of yarn and woven fabrics. Unfortunately, fiber quality characteristics are affected by many factors including the environment and management practices. ARS researchers in Maricopa, Arizona, College Station, Texas, and Florence, South Carolina, developed, grew, characterized, and selected germplasm from a breeding population at each location for the last four years. These lines provide public and private breeders with resources that broaden the genetic base while concurrently improving fiber quality and yield performance in upland cotton and have been broadly adapted across the United States.

2. Identification of candidate genes involved in drought stress tolerance in soybean. Drought causes significant soybean yield losses each year in rain-fed production systems of many regions. Genetic improvement of soybean for drought tolerance is a cost-effective approach to stabilize yield under rain-fed management. Identifying genes controlling drought tolerance traits such as slow canopy wilting will increase soybean productivity under drought stress conditions. ARS researchers in Maricopa, Arizona, Stoneville, Mississippi, and Columbia, Missouri, in collaboration with researchers from the University of Arkansas and University of Missouri, designed a multi-state trial to assay 200 diverse maturity group VI soybean accessions under irrigated and rain-fed conditions to confirm and identify molecular markers and candidate genes related to slow canopy wilting traits. Among the 183 identified candidate genes, 57 single nucleotide polymorphic (SNP) markers were collocated within genes coding for proteins with biological functions involved in plant stress responses. The confirmed genomic regions may be an important resource for pyramiding favorable alleles and as candidates for genomic selection aimed at enhancing soybean drought tolerance.

3. Characterization of biofuel traits in guayule. The byproducts from guayule shrubs are suitable for conversion to be bio-oil and bio-char via the pyrolysis process, and thus have the potential to serve as a new biofuel resource. ARS researchers in Maricopa, Arizona, and Wyndmoor, Pennsylvania, analyzed the pyrolysis byproducts production in guayule genotypes. Guayule pyrolysis byproducts were significantly affected by guayule genotypes and irrigation levels. The observed phenotypic variations could lay a foundation for genetic improvement of guayule byproduct production in future breeding programs to release superior biofuel breeding lines.

4. Identification of candidate genes involved in salt stress tolerance in camelina. Camelina is an important renewable oilseed crop for biofuel and feedstock that can reduce the reliance on petroleum-derived oils and decrease greenhouse gases and waste solids which result from petroleum-derived oil consumption. Carmelina molecular mechanisms affected by salinity stress are yet unknown. ARS researchers in Maricopa, Arizona, screened a camelina diversity panel that were germinated under high levels of salt stress. Significant trait-associated molecular markers were identified to be related to salt resistance in camelina. These molecular markers are located on the putative candidate genes controlling plant root development and related to salt stress resistance. These identified markers could provide a foundation for future molecular breeding efforts aiming to improve salt tolerance in camelina.

5. A novel trap crop is identified for a cotton agroecosystem. The western tarnished plant bug (lygus) is a major pest of cotton in the United States that causes substantial yield loss if populations are not properly managed. Improvement of current integrated pest management (IPM) approaches could include the use of trap crops that uses “attractive” plants to lure pests away from the cash crop. Researchers in Maricopa, Arizona, identified Vernonia, an annual plant native to eastern Africa, as a potential trap crop for lygus, and another pest, the cotton flea hopper. Vernonia also provided a summer habitat for a wide range of pollinators not typically seen in cotton monoculture practices. The identification of Vernonia as a trap crop provides novel IPM approaches in cotton that may help growers reduce pest management costs.

Review Publications
Boehm Jr., J., Abdel-Haleem, H.A., Schapaugh Jr., W., Rainey, K., Pantalone, V.R., Shannon, G., Klein, J., Carter Jr, T.E., Cardinal, A.J., Shipe, E.R., Gillen, A.M., Chen, P., Smith, J.R., Weaver, D.B., Boerma, R., Li, Z. 2019. Genetic improvement of US soybean in maturity groups V, VI, and VII. Crop Science. 59(5):1838-1852.
Campbell, B.T., Hinze, L.L., Thompson, A.L., Jones, M., Jones, D. 2021. Registration of PD 20170048, PD 20170049, PD 20170050, PD 20170053, and PD 20170054 germplasm lines of cotton. Journal of Plant Registrations.
Chamarthi, S.K., Kaler, A.S., Abdel-Haleem, H.A., Fritschi, F.B., Gillman, J.D., Ray, J.D., Smith, J.R., Dhanapal, A.P., King, C.A., Purcell, L.C. 2021. Identification and confirmation of loci associated with canopy wilting in soybean using genome wide association mapping. Frontiers in Plant Science. 12. Article 698116.
Hagler, J.R., Thompson, A.L., Machtley, S.A., Miles, C.T. 2021. Arthropod demography, distribution, and dispersion in a novel trap-cropped cotton agroecosystem. Journal of Insect Science. 21(1). Article 20.
Luo, Z., Szczepanek, A.E., Abdel-Haleem, H.A. 2020. Genome-wide association study (GWAS) analysis of camelina seedling germination under salt stress condition. Agronomy. 10(9). Article 1444.
Luo, Z., Mullen, C.A., Abdel-Haleem, H.A. 2020. Pyrolysis GC/MS analysis of improved guayule genotypes. Industrial Crops and Products. 155. Article 112810.