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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 #424770

Research Project: Genetic Improvement and Phenotyping of Cotton, Bioenergy and Other Industrial Crops

Location: Plant Physiology and Genetics Research

2016 Annual Report


Objectives
1. Develop genotyping-by-sequencing methods for diverse cotton, oilseed, and industrial crop germplasm, and map genetic markers for economically and agronomically important traits in these crops. 1.1 Genotyping-by-sequencing of cotton. 1.2 Genotyping-by-sequencing of oilseed rape. 1.3 Genetic and phenotypic characterization of new guayule germplasm. 2. Develop novel phenotyping approaches for quantitative genetic analysis of drought and heat tolerance traits in cotton and oilseed crops. 2.1 High-throughput phenotyping of traits related to drought and heat tolerance in cotton. 2.2 High-throughput phenotyping of traits related to drought and heat tolerance in oilseed rape. 3. Identify molecular markers associated with stress tolerance traits in cotton and oilseed crops. 3.1 Genome-wide association studies and marker-trait validation in cotton. 3.2 Genome-wide association studies and marker-trait validation in oilseed rape.


Approach
The three objectives of the plan will be carried out using various field-based instrumentation and modern approaches in plant breeding and genetics, and will be focused on the three important crops; cotton, oilseed rape, and guayule. These approaches are targeted toward the creation of genotyping-by-sequencing marker maps, development of novel phenotyping tools for quantitative genetic analysis of heat and drought tolerance traits, and identification of molecular markers associated with stress tolerance, quality, and yield related traits. The experiments will apply translational genomics approaches, leveraging statistical genetics and genomics for dissection of quantitative traits and the utilization of rapid, high-throughput phenotyping.


Progress Report
The primary goal for Objective 1 is to develop genomic resources for studying a variety of important crop species including cotton and various oilseeds. To develop resources for studying cotton, oilseeds, and guayule, a genomics lab was established. The lab is now fully equipped to handle “genotyping by sequencing” (GBS) library preparation and is being set-up to establish protocols for RNA library preparation for transcriptomics. A small diversity panel of Brassica napus was used to validate the lab’s work flow and protocol efficiency. The GBS libraries were sent to the ARS sequencing lab in Stoneville, Mississippi, for analysis. A portion of the sequencing data has come back and is being used to validate the sequence-processing pipeline for this lab. To characterize the genetic diversity of guayule, scientists in Maricopa, Arizona, collaborated with scientists at Cornell University and West Virginia University to determine portions of the genome sequences for the publically available USDA guayule collection (56 lines), then compared the sequences to characterize the evolutionary history and population structure of the plants. The results were consistent with a history of breeding selections from two distinct groups of wild guayule plants. To further characterize the germplasm available for guayule breeding programs, the USDA guayule collection and wild types collected from North Mexican and Southern Texas deserts were grown in replicated field trials in Maricopa, Arizona, under two irrigation regimes: well-watered and reduced water treatments. Morphological traits including plant height, canopy volume and perimeter, main branches number, stem thickness and leaf traits were measured. Initial results revealed significant variation in growth vigor among the guayule accessions under favorable growth conditions. The growth vigor of guayule accessions was suppressed in response to drought stress compared to those grown under favorable conditions. The phenotypic variation among guayule accessions and the variable response to the water stress will be helpful in identifying parental candidates for guayule breeding programs to eventually increase genetic gains of growth vigor, and consequently enhance rubber and latex yield and production. To identify additional factors that might be important for improving heat and drought stress tolerance of guayule, the content and composition of leaf waxes in guayule were determined. Six guayule lines were grown in a replicated experiment under greenhouse conditions with two different irrigation levels; well-irrigated and dry treatments. Extraction and analysis of waxes by gas chromatography-mass spectrometry (GC-MS) revealed variation in total wax content and composition among the studied lines within each water stress treatment as well as between treatments for each line. These results suggest that wax content might play a role in differences in tolerance to abiotic stress (drought tolerance) in guayule, and could be a potential target for crop improvement through breeding. Research conducted in support of Objective 2 focuses on the development of field-based, high-throughput phenotyping methods that can be applied for the study of cotton, guayule, and other crops under heat and drought conditions. In collaboration with scientists in the Water Management Unit and the University of Arizona, research has focused on developing a core set of proximal sensors for field data collection. The core sensors capture crop height, canopy temperature, and multi-spectral reflectance indices including normalized difference vegetation index and leaf chlorophyll index. To validate field-based high-throughput phenotyping (FB-HTP) measurements collected with a tractor, hand measurements for crop height, canopy temperature, and leaf chlorophyll content have been assessed in a Regional Breeders Testing Network (RBTN) cotton population with 35 entries (fourth year). Using these data, a HTP processing pipeline has been established, data storage and management protocols validated, and tractor-based measurements assessed for accuracy. Initial results indicate that when cotton plants are small, there is a great deal of ground interference that the sensors detect, and this problem is alleviated as the plants grow and biomass increases. We are working on solutions to increase the accuracy of tractor measurements when plants are small. The primary goal for Objective 2.1 is to characterize the phenotypic basis for variation in heat and drought stress traits in cotton. This is the first year of a cotton field trial with eight lines provided by collaborators from Arkansas, South Carolina, the USDA Cotton Germplasm repository in Texas, and commercial checks. The trial has two planting dates and four water treatments with three replicates per water treatment. Plant growth was monitored weekly by hand measurements and included soil moisture, height, growth stage, leaf chlorophyll content, flowering date, photosynthetic yield, and pollen sterility. Complementary data on plant height, canopy temperature, and spectral reflectance were recorded using the HTP tractor. At harvest, the plots will be assessed for seed and fiber quality. The planting dates will allow us to establish a baseline for traits heavily influenced by heat stress as opposed to traits influenced by water stress. Understanding these differences will allow us to identify better targets for genetic mapping and plant breeding. A small subset of cotton lines (five) from a previous trial has also been assessed for the effect of water stress on fiber wax compounds in collaboration with scientists from Cornell University. Fiber wax is an important trait for spinning and dye quality of cotton fiber. Using GC-MS, 53 compounds were identified in extracted cotton fiber wax. Statistical analyses indicated that genotype had a significant impact on the concentration of all but six compounds. The water treatment had a significant effect on 11 compounds. Three compounds initially identified as unknown have now been potentially identified as long chain monoacylglycerols (MAGs), which are not widely observed in plants. The MAGs, along with the 11 treatment-significant compounds, are of interest to further understand how they affect fiber quality and processing. To identify potential oilseed crops that are suitable the arid Southwest, a set of high-market-value oilseed elite cultivars (49) including Brassica napus, B. carianta, B. juncea, B. rapa, Sinapis alba, and Camelina sativa were grown in Maricopa, Arizona, during the fall season under drought stress and non-stressed field conditions, in replicated experiments. The plots were harvested, and measured traits included seed yield, flowering date, plant height, and seed quality traits such as oil content and fatty acid composition. The experiments will be repeated in the coming fall, and will also be used to develop FB-HTP methods for crop analysis in response to drought stress. The primary goal for Objective 3 is to identify regions of plant genomes that are associated with important agricultural traits, including stress tolerance, in crops such as cotton and oilseeds. Cottonseed oil and protein are important by-products of cotton processing that can further increase the market value for agricultural producers. To identify cotton genomic regions associated with these seed quality traits, a nuclear magnetic resonance (NMR) machine has been calibrated for whole cottonseed oil content. We are in the process of calibrating the machine for whole cottonseed protein using 2015 RBTN seed samples. A cotton association mapping panel, developed by North Carolina State University, has been fully genotyped using GBS techniques and single nucleotide polymorphisms (SNPs) identified. Seeds from this panel, grown in 2015 and 2016, will be assessed for seed oil and protein content using our calibrated NMR. Statistical analyses will identify cotton genomic regions associated with these traits, providing valuable molecular markers to assist in breeding efforts to improve these traits. The goal of Objective 3.1 is to phenotype cotton populations arising from selected superior lines cultivated under drought and heat stress conditions. Cotton populations developed by ARS collaborators in Texas and South Carolina have been received. First-year Maricopa field trials are being conducted to assess these lines for heat stress using both hand-held and tractor-based measurements described above. After harvest, yield and fiber quality will be assessed. These lines will continue to be evaluated at the Maricopa, Texas, and South Carolina locations. The expected outcome of this work is to release germplasm that is adapted for growth in multiple as well as single locations.


Accomplishments
1. Characterization of the genetic diversity in Brassica napus, a valuable oilseed crop used for food, feed, and biofuel purposes. To expand the resources available for breeding of B. napus, ARS scientists in Maricopa, Arizona, Peoria, Illinois, Morris, Minnesota, Sidney, Montana, Mandan, North Dakota, Temple, Texas, Ames, Iowa, Akron, Colorado, Pendleton, Oregon, and scientists from Idaho State University, and Cornell University, collaborated to collect and genetically characterize a global population of Brassica napus plants. Eight hundred Brassica lines were genotyped using genotyping by sequencing (GBS) technology. Comparison of the DNA sequences revealed three distinct and diverse groups distinguished by growth habit and geographical origin including spring, winter-European and winter-Asian subgroups. Each subgroup had a different historical evolutionary path, which was also reflected in the traits and genes that each subgroup possessed. This work provides insight to the diversity of plants available within the B. napus population and also provides single nucleotide polymorphism (SNP) markers that can be used to identify genes and genomic regions that are associated with traits of interest. This work will thus be of greatest interest to those scientists interested in improving the agronomic performance of B. napus using genome-assisted techniques.


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Review Publications
Gazave, E., Tassone, E.E., Ilut, D.C., Wingerson, M., Datema, M., Witsenboer, H., Davis, J.B., Grant, D.M., Dyer, J.M., Jenks, M.A., Brown, J., Gore, M.A. 2016. Population genomic analysis reveals differential evolutionary histories and patterns of diversity across subgenomes and subpopulations of Brassica napus L. Frontiers in Plant Science. 7:525. doi: 10.3389/fpls.2016.00525.
Ilut, D.C., Sanchez, P.L., Costich, D.E., Friebe, B., Coffelt, T.A., Dyer, J.M., Jenks, M.A., Gore, M.A. 2015. Genomic diversity and phylogenetic relationships in the genus Parthenium (Asteraceae). Industrial Crops and Products. 76:920-929.
Tassone, E.E., Lipka, A.E., Tomasi, P., Lohrey, G.T., Qian, W., Dyer, J.M., Gore, M.A., Jenks, M.A. 2015. Chemical variation for leaf cuticular waxes and their levels revealed in a diverse panel of Brassica napus L. Industrial Crops and Products. 79:77-83.