Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): With increasing global climate change challenges, there is an increasing demand for new clean bioenergy resources such as developing new crops, and genetically improving current crops to increase their tolerance to environmental changes and to sustain the agricultural sectors in semi- arid regions. The overall goal of this research project is to investigate genetic variations in bioenergy and industrial crops such as camelina, guayule, sorghum, and soybean to identify candidate genes controlling abiotic stress tolerance traits and identify new germplasm that can be used to develop superior cultivars with high yield and stable productivity to meet those challenges. The specific objectives of the project are: Objective 1: Conduct research to identify alleles, candidate genes, and molecular markers for drought and/or heat tolerance of oilseed and biomass crops in semi-arid field conditions and determine association of abiotic stress tolerance with agronomic performance and biofuel traits such as biomass yield and conversion. Sub-objective 1.A: Identify alleles/genes and associated molecular markers controlling oil content and composition, and related abiotic stress tolerance traits in camelina. Sub-objective 1.B: Identify alleles/genes and associated molecular markers conditioning seed quality and composition and related stress tolerance traits in soybean. Sub-objective 1.C: Identify alleles/genes and associated molecular markers conditioning biofuel production, biomass yield, and traits related to abiotic stress tolerance in bioenergy sorghum. Sub-objective 1.D: Screen USDA guayule germplasm collection to discover biofuel-related traits, determine their variation in pyrolysis production, and associations with abiotic stress tolerance. Objective 2: Conduct research to determine and effectively utilize bioinformatics and other genomic processing pipelines, such as transcriptomics and metabolomics, to enhance genetic improvement and trait enhancement of food, industrial, and biofuel crops. Sub-objective 2.A: Characterize the genetic mechanisms governing wax content and composition in soybean growing under abiotic stress conditions. Sub-objective 2.B: Explore guayule resin pathway(s) and related candidate genes using multi-omics approaches. Approach (from AD-416): This project establishes a sustainable agricultural system for semi-arid regions using new and established crops for biofuel and industrial purposes. The approaches will explore genetic variation in bioenergy and industrial crops to discover abiotic stress tolerance traits and identify genes/alleles controlling those traits. Objective 1 will focus on identifying candidate genes for abiotic stress tolerance using developed populations (camelina) and diversity panels (soybean and sorghum) planted under stress/non-stress conditions. Oil content and composition, biofuel and related abiotic stress tolerance traits will be collected using traditional and high throughput phenotyping platforms and analyzed using MIXED model. G�E interaction analyses will be conducted across irrigation levels for recorded traits. In Camelina, Quantitative Trait Loci (QTL) associated with recorded traits and stability parameters will be conducted. Candidate genes under QTL interval will be identified using SNP position on the camelina reference genome. GWAS analyses will be conducted for soybean and sorghum diversity panels. Candidate genes from multiple GWAS analyses will be identified from genomic intervals in the soybean and sorghum reference genome assemblies. Guayule will be planted under stress/non-stress conditions and traits related to abiotic stress tolerance will be collected. Rubber and resin, pyrolysis and biofuel related traits will be determined. Objective 2 will focus on identifying candidate genes using multi-omics approaches, such as transcriptomics and metabolomics, to characterize genetic mechanisms governing wax content in soybean and resin in guayule. For transcriptome analyses, RNA will be harvested from soybean and guayule plants planted under stress/non-stress conditions and cDNA libraries will be sequenced. Following the structured pipeline, the paired-end clean reads will be aligned and mapped. Genes, with an adjusted p value, will be declared as differentially expressed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) will be used to annotate genes to biological/metabolic pathway. For metabolomic approach, tissues from guayule will be harvested and resins will be extracted, purified and analyzed using liquid chromatography�mass spectrometry. Raw data will be processed to identify unknow compounds, search chemical databases for putative candidates, and annotate spectra with predicted fragmentation. Principal component analysis will be used to identify abundant metabolites and putative biomarkers responsible for differences among resin content in guayule genotypes and irrigation treatments. To interpret the metabolite�s function, pathway enrichments for detected metabolites will be calculated using KEGG database. This report documents progress for project 2020-21410-008-000D, �Developing Biofuels and New Industrial Crops for Sustainable Semi-arid Agricultural Systems�, which began in June 2023. In support of Objective 1, research focused on identifying alleles and candidate genes involved in abiotic stress tolerance in oilseed and industrial crops growing under semi-arid field conditions. Under sub- objective 1A, a Recombinant Inbred Lines (RIL) population was developed and advanced to an F5 generation. The population and checks were planted in an alpha lattice design with three replications under well-watered and water-limited conditions in Maricopa, Arizona. Field based high throughput phenotyping (HTP) data were collected using established protocols for canopy temperature and vegetation indexes at lab on weekly bases throughout the growing season. In addition to HTP measurements, traditional morphological traits, including plant height, were measured. At physiological maturity, plots were harvested for seed traits related to seed yield and seed weight. Seed samples are being analyzed for oil content and fatty acid compositions and glucosinolates using Near- Infrared Spectroscopy (NIRS). Preliminarily results showed the wide variations among the inbred lines as well their responses to stress conditions. Based on the data collected some genotypes showed high stability when grown under stressed conditions. In support of Sub-objective 1B, 100 advanced soybean lines bred for drought tolerance were planted at Maricopa, Arizona, in a split split- plot completely randomized design with three replications. The irrigation levels (well-irrigated and limited irrigation) were the main plot, maturity groups (MG) were the sub-plot, and the sub-plots were the soybean lines. Canoy wilting, traits related to soybean drought tolerance, HTP related traits and oil and fatty acids data were collected and analyzed. Results showed significant effect of drought stress on maturity groups and genotypes within each maturity groups as expressed by studied traits. The plants in the irrigated trial were cooler than those in water stressed trial by 10 degrees celsius. Data as well showed the effects of drought stress on seed compositions and quality. Based on the results, tolerant genotypes within each MG were selected to be used as checks in further experiments to identify candidate genes and alleles related to soybean drought stress tolerance. A diversity panel consists of 350 soybean accessions plus check varieties are planted in Maricopa under well-irrigated and reduced-irrigation conditions. Initial observations revealed variations in soybean growth due to stress conditions, and the reduction in growth vigor in response to drought stress. Canopy wilting (CW) trait and other traits are being recorded at both trials. Comprehensive statistical analysis over irrigation treatments and genotypes will be carried out. Sub-objective 1D focuses on exploring the biofuel related traits and characteristics in guayule. Field trials including USDA guayule accessions and wild relative, mariola, are grown at Maricopa, Arizona, under two irrigation regimes (well-irrigation versus reduced irrigation). Mariola is the closest relative to guayule and had been used to transfer cold tolerance traits to guayule to potentially expand its production zones up to northern regions. Morphological and established HTP-related traits were collected and analyzed. Initial results revealed the variations in those traits among guayule accessions under stress and non- stress conditions. The growth vigor of guayule accessions was decreased in response to drought stress (reduced irrigation) compared to those grown under well-watered conditions. Beside those traits, biofuel related traits such as lignin, hemicellulose, and cellulose were estimated and analyzed. Results showed wide phenotypic variation in lignin, cellulose and hemicellulose contents among genotypes and irrigation treatments. Bades on these results, some genotypes showed stability for growing under different irrigation regimes. Objective 2 focuses on using omics approach to identify molecular mechanisms controlling the abiotic stress tolerance in food, industrial, and biofuel crops. In support of Sub-objective 2A, ARS researchers selected two soybean genotypes with significant differences in leaf wax contents to conduct a growth chamber experiment to study the effects of high temperatures, drought stresses and their combinations on wax accumulation and the mechanisms controlling it. Chlorophyll content and photosynthetic efficiency were also estimated via leaf level fluorescence traits. Genotypes were planted in three replicates each in a split-split plot design with two levels of temperature as the main-plot and two levels of irrigation (drought stress) as the subplot. Analysis of variance showed significant effects of soybean genotypes and treatments on leaf wax accumulation and physiological traits. Total leaf wax accumulation was affected by drought and high temperature stresses, but it was not affected by a combination of those stressors. In general, total leaf wax was increased in response to drought stress but decreased in response to high heat. The leaf wax classes and components accumulations, as well, were affected by drought, high heat and their interactions. For example, free fatty acids and primary alcohols waxes production were increased under high temperature stress and increased under drought stress. In contrary, the accumulation of alkanes, aldehydes and wax esters were increased under drought stress and decreased under high heat stress. Preliminary results indicated significant differences in chlorophyll content and photosynthetic efficiency traits between genotypes as well among stress treatments. Leasf tissues were harvested for RNA extractions from soybean genotypes that were planted under different stress levels of drought and temperature and their combinations to study their effects on altering gene expression. Transcriptome analyses of the data showed differential gene expression levels in response to the abiotic stresses. Around 6981, 4715 and 8785 genes were differentially expressed in response to high temperatures and drought together, respectively. Gene Ontology analyses annotated expressed genes into biological processes such as response to temperature stimulus, response to heat, response to salt stress, response to osmotic stress and photosynthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated expressed genes to biological and metabolic pathways that could be involved in stress responses and tolerance such as fatty acid biosynthesis, fatty acid degradation, cutin, suberine and wax biosynthesis, galactose metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, adenosine triphosphate binding cassette (ABC) transporters and flavonoid biosynthesis. In support of Sub-objective 2B, ARS researchers explored the molecular mechanisms involved in resin accumulation and abiotic stress tolerance in guayule. Several guayule accessions with wide phenotypic diversity in resin accumulations and response to stress tolerance were identified. Three accessions with the highest/lowest resin content are being used to explore those mechanisms. The experiment is initiated to include the three guayule genotypes under controlled conditions to apply drought stress at two levels. During the experiment, morphological and physiological data related to stress tolerance will be collected. Artificial Intelligence (AI)/Machine Learning (ML) Neither artificial intelligence (AI) nor Machine Learning (ML) methods were used for this project during FY 2024. ACCOMPLISHMENTS 01 Phenotypic diversity in leaf wax in the natural population of Carinata. Brassica carinata has received considerable attention as a renewable biofuel crop for semi-arid zones. Increasing accumulated leaf cuticular wax in plants is one of the strategies to reduce nonstomatal water loss and thus increase crop tolerance to drought. ARS researchers at Maricopa, Arizona, extended the understanding of phenotypic variations in cuticular wax content and composition in Carinata using leaf waxes extracted and quantified from Carinata accessions. These accessions represent a diversity panel with a wide range in total leaf wax contents, wax classes and its components. High heritability values of these waxes suggested the high likelihood of selection for these waxes during early generations of Carinata breeding programs. Identifying leaf waxes traits in the Carinata will underpin future analysis of the wax biosynthetic pathways, help to identify candidate genes controlling these traits, and enable the development of molecular markers for molecular breeding programs aimed at increasing drought tolerance of Carinata. 02 The synergetic effects of drought and high heat stresses on Canola leaf wax accumulation. Plants are facing multiple challenges to grow under unpredictable events of climate change such as drought and high heat stresses. Breeding strategies to enhance stresses tolerance to individual stressors as well as combinations of stressors are required to cope with those effects. ARS researchers at Maricopa, Arizona, studied the effects of drought and heat stresses on accumulation of leaf waxes on Brassica napus, an important oilseed crop, to understand the mechanisms controlling leaf cuticular wax production in response to stress. A combination of drought and heat stresses proved to have synergetic effects on specific brassica leaf waxes indicating the possibility to breed for both drought and heat tolerances in Brassica napus. Specific waxes showed abundance on the leaves of brassica plants grown under a combination of drought and heat stresses, suggesting them as potential candidates for improving the stress tolerance against those stressors. 03 Genetic diversity of global sesame natural population. The rapid advancements in next generation sequencing technologies reduce the cost, time, and efforts to develop and utilize high-throughput genotyping pipelines. Using next generation sequencing technology, the ARS researchers at Maricopa, Arizona, in collaboration with scientists from private sector genotyped a diversity panel of sesame accessions with 24K single polymorphism markers to explore their genetic diversity and population structure. The high-quality Single Nucleotide Polymorphism (SNP) markers are providing sufficient marker information for further genetic and breeding studies. Population structure and phylogenetic tree analyses identified two distinct subpopulations originated from two different geographical regions. This information can be used in future studies for allele/gene identification using genome-wide association analysis studies (GWAS), and ultimately providing a tool to enhance genetic gain in sesame breeding programs using innovative breeding methodologies such as marker assisted selection and genomic selection.
Impacts
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Publications
- Selim, M.E., ELShamey, E.E., ELkholy, N.A., Abdelrahman, M., Abo-Marzoka, E.A., Elgamal, W.H., Elsayed, M.A., Attia, K.A., Ghazy, A., Abdel-Haleem, H.A. 2024. Genetic determinants of weed control in rice (Oryza sativa L.) using allelopathy approach. Cereal Research Communications. https://doi. org/10.1007/s42976-024-00506-6.
- Tomasi, P., Luo, Z., Abdel-Haleem, H.A. 2024. Drought and high heat stresses modify Brassica napus L. leaf cuticular wax. Plant Stress. 13. Article 100513. https://doi.org/10.1016/j.stress.2024.100513.
- Seay, D., Szczepanek, A.E., De La Fuente, G., Votava, E., Abdel-Haleem, H. A. 2024. Genetic diversity and population structure of a large USDA sesame collection. Plants. 13(13). Article 1765. https://doi.org/10.3390/ plants13131765.
- Tomasi, P., Abdel-Haleem, H.A. 2023. Phenotypic diversity in leaf cuticular waxes in Brassica carinata accessions. Plants. 12(21). Article 3716. https://doi.org/10.3390/plants12213716.
- Ghazy, A., Ali, M., Ibrahim, E., Sallam, M., Al Ateeq, T., Al-Ashkar, I., Motawei, M., Abdel-Haleem, H.A., Al-Doss, A. 2024. Characterization of improved barley germplasm under desert environments using agro- morphological and SSR markers. Agronomy. 14(8). Article 1716. https://doi. org/10.3390/agronomy14081716.
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