Location: Crop Production and Pest Control Research
2019 Annual Report
Objectives
Objective 1: Identify genetic loci involved in seed oil, protein, and carbohydrate content using forward and reverse genetic approaches, and create genetic combinations that serve as new variability for selection in breeding programs.
Subobjective 1A: Identification of genes implicated in control of seed protein/oil levels.
Subobjective 1B: Alteration of carbohydrate partitioning in soybean seeds.
Subobjective 1C: Combining genes identified by this project to form a basis for improved soybean germplasm.
Objective 2: Evaluate newly identified sources of resistance against Phytophthora sojae, identify candidate Avr gene(s) in the pathogen that are recognized by the new resistance, and design strategies to maintain resistance.
Objective 3: Characterize population structure of Fusarium virguliforme, determine the role of root endophyte community in SDS (soybean Sudden Death Syndrome) resistance, identify key endophytes that can be used for development of new control strategies, and create a SDS genome-wide molecular marker database as a resource for research.
Objective 4: Construct, coordinate, and publish the results of a relevant regional and national variety testing program for soybean that provides timely phenotypic information useful for selection.
Approach
Objective 1: Genes regulating carbohydrate, protein, and fatty acid levels of soybean seeds will be identified using a combination of forward and reverse genetic approaches. Mutants will be evaluated by NIR, GC, and HPLC analysis for multiple aspects of seed composition. Conventional and sequence-enabled mapping techniques will be used to determine gene positions. qRT-PCR will be used to measure the expression of candidate genes during seed development. The best candidate genes will be validated through transformation assays to determine gene impact on seed phenotype.
Objective 2: The performance of resistance genes, including several recently identified, will be evaluated against a large collection of Phytophthora sojae isolates. The best gene pyramiding combination will be recommended for soybean breeding efforts. Genomes and transcriptomes of a subset of isolates will be sequenced to determine how P. sojae evades gene-mediated host resistance, and to identify candidates for uncharacterized Avr genes.
Objective 3: A genome-wide informative microsatellite marker database will be constructed for Fusarium virguliforme using a comparative genomics approach. Identified microsatellite markers will be used to investigate global population structure of F. virguliforme. Prokaryotic and eukaryotic root endobiome of soybean lines susceptible or highly resistant to sudden death syndrome will be characterized using culture and high-throughput sequencing approaches. Endophytic groups associated with SDS resistance will be identified.
Objective 4: Public soybean breeders submit their soybean breeding lines for evaluation of agronomic performance, disease resistance and quality traits. Entries are separated by maturity group and assigned to either the ‘Preliminary Tests’ or the ‘Uniform Tests’. Seeds of each entry, along with those of the standard reference varieties, are packaged and distributed to collaborators throughout the U.S. and Canada for evaluation. In addition, entries will be evaluated at multiple locations in Indiana. Harvested seeds will also be tested for quality traits. Collaborators submit performance data from their locations to ARS after harvest. This data is compiled and analyzed by this research group following established protocols. The results will be published in an annual report book and online.
Progress Report
Objective 1. From the 2018 field season, we obtained data from 90 distinct mutant lines identified in previous years on the basis of abnormal protein or oil levels. New data was compared to historical data to determine multi-year patterns and overall stability of the traits. We obtained and profiled 50 BC1 lines from a subset of these mutants, and used this data to determine if each mutation has a dominant or recessive effect on seed composition. We obtained 15 different F2 mapping populations where segregation of seed protein or oil levels was observed, and extracted DNA samples from these populations for initial mapping of these traits, which is expected to be completed in August of 2019. For the 2019 field season, an additional 11 mapping populations were planted, as well as the F2 progeny of 8 complementation crosses (crosses to known high protein germplasm). We also planted control populations to determine what is the best parent to use for genetic mapping of protein traits isolated in our Williams-82 background. In the 2019 field season we will focus on obtaining additional complementation, mapping, and back crosses.
We profiled expression of several seed sucrose biosynthesis enzymes during seed development, and we chose candidates for transgenic manipulation. We initiated discussion with the plant transformation facility at the University of Wisconsin, but there is a waiting list for soybean transformation.
The third goal (Objective 1c) is to obtain soybean where the high oleic acid trait is combined with ultra-low RFO trait (HO-ulRFO – a 4-gene stack), and a high oleic-low linolenic ultra-low RFO soybean (HOLL-ulRFO – a 6-gene stack) using our own alleles for these traits. The future goal is to evaluate these lines at multiple locations for seed composition (in 2020 and 2021). In the 2019 field season we will make a cross to obtain the 6-gene stack, meanwhile we will increase seed for the HO-ulRFO lines and evaluate composition in our local environment.
Objective 2. Over 600 Phytophthora sojae isolates have been pathotyped using differentials carrying Rps 1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6,7, and 8. A subset (170 isolates) have been pathotyped with differentials carrying Rps UN1, UN2 and 11. All differentials were seed-increased in 2018.
Using a comparative genomics approach, a high-quality, genome-wide microsatellite database was created for P. sojae. This database includes 157 polymorphic microsatellite loci, with 20 loci experimentally validated.
Objective 3. Primers were designed for a list of 29 polymorphic microsatellite loci of Fusarium virguliforme identified through the comparative genomics approach. Experimental validation confirmed that 17 loci were indeed polymorphic and the primers could amplify in all isolates tested. These primers and loci were used to genotype 93 isolates of Fusarium virguliforme from the United States and Argentina.
Objective 4. The ARS scientist in West Lafayette, Indiana, organized the 2018 Northern Uniform Soybean Tests and is organizing the 2019 tests. In 2018 tests, 448 soybean breeding lines and checks in maturity groups 00 to IV were evaluated and the tests were conducted at 46 sites in 10 Midwest states in the United States and two provinces in Canada. These soybean lines were evaluated for yield and other agronomic characteristics including seed quality, lodging, and shattering, etc. They were also evaluated for disease resistance including soybean cyst nematode and Phytophthora root and stem rot. Soybean lines bred for seed quality traits (oil and protein content, amino acid, high oleic acid) were also evaluated for their target traits. These breeding lines were submitted by public breeders in the Unites States and Canada. The ARS scientist in West Lafayette, Indiana, directly evaluated lines at three sites in Indiana, and evaluated all lines for resistance to Phytophthora root rot under greenhouse conditions. We collected data from participants, analyzed the data, and published the results in the book “THE UNIFORM SOYBEAN TESTS NORTHERN REGION 2018”. The hard copy was delivered to participants and interested stakeholders. The electronic copy of the book is freely available online. The 2019 tests are underway.
Accomplishments
1. Publication of the book “THE UNIFORM SOYBEAN TESTS NORTHERN REGION 2018”. The ARS lab in West Lafayette, Indiana, organizes the Northern Uniform Soybean Tests yearly. The tests evaluate soybean breeding lines for agronomic performance, disease resistance and seed quality traits in northern states in the United States and provinces in Canada. The soybean lines are those developed by public breeders in the United States and Canada that are suitable for planting in these regions. In addition to performing tests at multiple sites in Indiana, the ARS lab in West Lafayette, Indiana, collected data from participants, analyzed the data, and published the 2018 test results in this book. The hard copy was delivered to participants and interested stakeholders. The electronic copy of the book is freely available online. This book is used as primary evidence by breeders when making the decision to further advance their lines or release their lines to the public.
2. Publication of a high-quality, genome-wide, informative microsatellite database for Phytophthora sojae, the soybean root and stem rot pathogen. The database was created using a comparative genomics approach. It includes 157 polymorphic loci, with 20 loci experimentally validated. This database is a valuable tool for studying the population genetics and dynamics of Phytophthora sojae. The publication would allow tracking and prediction of new sources of disease.
Review Publications
Cai, G., Myers, K., Fry, W.E., Hillman, B.I. 2018. PiRV-2, a novel RNA virus from Phytophthora infestans, does not belong to any known virus groups. Archives of Virology. 164: 567-572. https://doi.org/10.1007/s00705-018-4050-0.
Cai, G., Fleury, T.J., Zhang, N. 2019. Comparative genomics approach to build a genome-wide database of high-quality, informative microsatellite markers: application on Phytophthora sojae, a soybean pathogen. Scientific Reports. 9:7969. https://doi.org/10.1038/s41598-019-44411-z.
