Source: TENNESSEE STATE UNIVERSITY submitted to NRP
GERMPLASM ENHANCEMENT IN SOYBEAN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
EVANS-ALLEN
Reporting Frequency
Annual
Accession No.
1017807
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2018
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental Sciences
Non Technical Summary
Genetic variation is one of the necessities in plant breeding. This variation can be expanded by screening collections of soybean landraces and its related wild species from around the world. It is also possible to generate such genetic variation through induced mutation. The objective of this study is developing an EMS mutagenic soybean population using recently released cultivar "JTN-5203" adapted to the southern states. This population will also be screened for herbicide tolerance mutants.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2021820108050%
2011820108150%
Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Screening the EMS mutant soybean population for herbicide resistance genes using Tilling by sequencing approach.Targeted gene editing in soybean for herbicide resistance genesDeveloping new genome editing lines using CRISPR/Cas9 system Training students in plant molecular breeding and molecular genetics. Students will gain knowledge and experience in greenhouse research and management, plant molecular breeding and genetics
Project Methods
1.1.Screening the mutant population using Tilling-by-sequencing approach (TbyS)Various reverse genetic techniques have been utilized to identify mutations in genes with known sequences. The function of these genes can then be characterized by comparing the phenotypic response of these mutant plants in comparison with the wild-type parent. Previously, mutation screenings were based on mismatch-specific endonucleases for SNPs detection. However, with the advancements in NGS technologies and reduced cost of sequencing, new protocols have been developed to detect rare SNPs using multidimensional DNA pooling and sequencing techniques (24; 25;26). In this method, the target gene will be amplified from this DNA pool and after barcoding, they will be sent to genomic center (Genewiz, South Plainfield, NJ) for library prep and sequencing (Supplementary Figure 6). We have already extracted DNA from 6,400 individual mutants (5913 reached to full maturity) and after normalization of each sample to 100ng/ul, pooled the samples in a two-dimensional pooling strategy. Each target gene will be amplified using proof-reading enzymes such as Takara ExTaq HS polymerase and will be prepared for sequencing using Illumina HiSeq 2500.1.2.Screening for novel mutants in genes conveying herbicide resistanceAcetolactate synthase (ALS) is an enzyme controlled by ALS gene and is essential for plant growth. ALS is involved in the biosynthesis of branched-chain amino acids including isoleucine, valine, and leucine. Speci?c mutations within ALS (acetolactate synthase) gene(s) can contribute to resistance for SU herbicides. In Camelina (Camelina sativa L.), researchers discovered ALS herbicide resistance mutants in the EMS mutant population (27). Similarly in soybean, ALS resistance mutants can be recovered after screening for mutation in ALS genes (Glyma01g09920.1, Glyma04g37270.1, Glyma06g17790.1, Glyma13g31470.1 and Glyma15g07860.1). We will use Tilling by sequencing approach as discussed earlier to pull out individuals carrying any potential mutation in these ALS genes. With the current high throughput Illumina sequencing systems (HiSeq 2500 with 450-500 Gb of 2x150 bp data/lane), the generated data will give us enough coverage to identify mutations in different genes. We then study the effect of such mutations in herbicide resistance behavior of such individuals.1.3.Utilizing CRISPR/Cas9 genome editing system to induce targeted mutations in genes conferring herbicide resistanceFor designing and constructing CRISPR/Cas9, we will follow the Stupar's lab protocol from the University of Minnesota (http://stuparlab.cfans.umn.edu/protocols/crisprcas9-glycine-max/). We will use E. coli DH5-α strain for cloning and two vectors for CRISPR/Cas9 including pBlu gRNA and Cas9 MDC123. After completion of CRISPR/Cas9 construct, we will transform the vector to the soybean via Agrobacterium-mediated transformation. We will use Agrobacterium tumefaciens EHA105 strain, and soybean cultivar Williams 82 as plant material following modified protocol of soybean whole-plant transformation from half-seed explant (http://stuparlab.cfans.umn.edu/protocols/soybean-whole-plant-transformation-half-seed-explant/).

Progress 10/01/18 to 09/30/21

Outputs
Target Audience:Soybean farmers with interest in soybean lines with improved seed composition quality, reduced raffinose content and improved fatty acid profile Students enrolled in the graduate and undergraduate program that are becoming familiar with better cultural practices in soybean Soybean breeders interested in improving soybean seed quality Poultry and animal feed sector with interest in soybean seeds with reduced raffinose content and increased amino acid content Changes/Problems:Loss of greenhouses due to the tornado damage in March 2020 has caused a delay in the greenhouse work. New hoophouses are being built and will be used in the following seasons. Use of nanoparticles as novel antimicrobial agents was not part of the initial project, however, as we used them for DNA delivery into the plant cells, we also noticed that they carry antimicrobial properties and are also able to deliver DNA into bacterial cells. There was no impact on overall goals and objectives of the entire proj What opportunities for training and professional development has the project provided? This project had a combination of field, greenhouse and laboratory experiments and students were trained in multiple aspects of molecular plant breeding, genome editing techniques and nano-delivery tools. The students gained hands on training in basic molecular techniques such as DNA extraction, bacterial culture, PCR, Primer design and plant transformation. They also became familiar with the most novel technologies such as CRISPR/Cas9 genome editing techniques, use of nanoparticles as novel nanocarriers in the cells and their use as antimicrobial agents. Over the three years of this project we were able to train and graduate five students including both undergraduate and graduate students. One undergraduate student is enrolled in the PhD program at Texas A&M, two master students joined the University of Minnesota for pursuing their PhD degrees while the other master student enrolled into the PhD program here at Tennessee State University. One PhD student who graduated from this lab in 2020 has joined USDA-NRCS as a Natural Resource Specialist. How have the results been disseminated to communities of interest?The results have been presented in the Tennessee Soybean Promotion board this year. Presentation to the scientific audience at the Tennessee Academy of Science Data was presented at the ARD Research Symposium and in the annual Research Symposium at Tennessee State University. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Performed crosses between high oleic mutant lines to increase the oleic acid content Optimized and Established hairy root transformation in soybean for evaluation of the CRISPR CAS9 vector. CRISPR/Cas9 vector was also developed in this project using specific promoters from soybean that drive the expression of Cas9 and gRNA in transgenic plants. We also inserted a GFP reporter gene in this vector for ease of tracing it in positive transgenic events. Used Carbon dots as novel DNA delivery system in the cells. This system has shown promise in bacterial transformation, but it still requires optimization in DNA delivery in to the plant cells. Trained five graduate and undergraduate students in plant molecular breeding, molecular genetics and use of nanomaterials as antimicrobial or nano carrier agents. Students gained knowledge and experience in greenhouse research and management, plant molecular breeding and genetics. Advanced soybean crosses to F2 generation after initial crosses in the previous year. The soybean seeds will be harvested in November for seed trait analysis. Conducted Tilling by sequencing approach for screening EMS mutant soybean lines. Screened the soybean mutant population for anti-nutritional raffinose contents and identified mutants with reduced raffinose contents. These mutants are a valuable resource in breeding new soybean lines. We are characterizing these mutants and planning to identify the genetic mechanisms involved in reducing raffinose contents in these mutants.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Pandey, Anju; Devkota, Asmita; Yadegari, Zeinab; Dumenyo, Korsi; Taheri, Ali. Antibacterial Properties of Citric Acid/b-Alanine Carbon Dots against Gram-Negative Bacteria. Nanomaterials,11,8,2012,2021
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Ravi, R., Taheri, A., Khandekar, D., & Millas, R. (2019). Rapid Profiling of Soybean Aromatic Compounds Using Electronic Nose. Biosensors, 9(2), 66
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Adeleke, E; Dzantor, E; Taheri, A. Inoculation and amendment strategies influence switchgrass establishment in degraded soil. Ecological Indicators,121,107068,2020
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Oral presentation on Carbon dots as a novel tool for delivery of foreign DNA and microbial transformation. At Tennessee Academy of Science Annual Meeting on November 21, 2020.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Oral Presentation on Carbon dots as a novel tool for delivery of foreign DNA and microbial transformation. at 43rd Annual Tennessee State University - Wide Research symposium, March 22-26, 2021.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Poster presentation on Manipulation of multiple genes in Glyceollin synthesis pathway in soybean using CRISPR/Cas9. at 43rd Annual Tennessee State University - Wide Research symposium, March 22-26, 2021.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Poster presentation on CRISPR/Cas9 mediated genome editing of the genes involved in soybean root hairs development at 43rd Annual Tennessee State University - Wide Research symposium, March 22-26, 2021.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2021 Citation: Asmita Devkota. Antimicrobial and nanocarrier properties of carbon dots (Cds) synthesized from different materials. 2021
  • Type: Theses/Dissertations Status: Accepted Year Published: 2021 Citation: Anju Pandey. Dual functional carbon quantum dots: novel tools for DNA delivery in bacterial transformation and as antimicrobial agents against gram-negative bacteria. 2021
  • Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Ekundayo Adeleke. effects of organic amendments and commercial mycorrhizal fungi on switchgrass biomass production in degraded soils. 2020
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taheri, A., M.J. Espina, C M S. Ahmed, A.F. Bernardini, E. Adeleke, Z. Yadegari, P. Arelli, and V. Pantalone. Germplasm Enhancement in Soybean Using Chemical Mutagenesis and Tilling-By-Sequencing Technologies. 2019 ARD Research Symposium. Jacksonville, FL
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Millas, R and Taheri, A. Development and characterization of soybean with improved fatty acid profile through mutation breeding and genome editing. 2019. Graduate Thesis Defense. College of Agriculture. Tennessee State University. Nashville, TN
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taheri A. Presentation for 120 high school students from LEAD Public Schools. Sep 2019. College of Agriculture, TSU
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taheri, A. Screening Soybean Mutant Population for Heart Healthy Germplasm. 2019 ARD Research Symposium. Jacksonville, FL
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Taheri, A. Rapid Profiling of Volatile Compounds in Soybean using Electronic Nose. 2019 ARD Research Symposium. Jacksonville, FL
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Adeleke, E.; Millas, R.; McNeal, W.; Faris, J.; Taheri, A. Variation Analysis of Root System Development in Wheat Seedlings Using Root Phenotyping System. Agronomy 2020, 10, 206. https://doi.org/10.3390/agronomy10020206
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Millas, R., Espina, M. J., Ahmed, C. S., Bernardini, A., Adeleke, E., Yadegari, Z., ... & Taheri, A. (2020). Detection of novel allelic variations in soybean mutant population using Tilling by Sequencing. BioRxiv, 711440 doi: https://doi.org/10.1101/711440
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Reneth Millas, Mary Espina, CM Sabbir Ahmed, Angelina Bernardini, Ekundayo Adeleke, Zeinab Yadegari, Korsi Dumenyo, Ali Taheri. 2020. Novel fatty acid alleles in soybean mutant population using forward genetics approach


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Soybean Farmers High school students and general public visiting the research field and labs. Graduate and undergraduate students participating in research symposiums. Scintific community and participants Changes/Problems:We had power outage which lasted almost two weeks which delayed our progress and were not able to conduct our experiments properly. Due to tornado damage in the last fall, our entire greenhouse area dn farms were damaged and we were unable to carry out any greenhouse work. This tornado, also caused power outage in the support building of the greenhouses where we were storing our mutant collection (6400) inside the freezers. We are still evaluating the extent of this damage on the viability of the seeds stored in those freezers. A small subset of this collection was planted at the University of Tennessee research station, but neighther of them germinated.We will plant new batched of these mutants and re-evaluate the damage extent. Due to Covid-19, we had a couple of weeks shutdown to the campus and were not able to access our laboratories. After that, we were able to get back to the lab wth following proper social distancing and safetprotocols. What opportunities for training and professional development has the project provided?This project has a combination of field, greenhouse and laboratory experiments and students are trainind in multiple aspects of molecular plant breeding and genome editing techniques. The students gained hands on training in basic molecular techniques such as DNA extraction, bacterial culture, PCR, Primer design and plant transformation. They also become familiar with most novel technologies such as CRISPR/Cas9 genome editing techniques How have the results been disseminated to communities of interest?The results has been presented in the Tennessee Soybean Promotion board in this year. Due to COVID, scientific conferences has been eaither cancelled or limited to virtua presentation via zoom. What do you plan to do during the next reporting period to accomplish the goals?Advance the soybean crossed to the next generation and evaluate the effect of double mutations in the crossed we made in summer 2020. Deliver the CRISPR constructs to the soybean using hairy root transformation and evaluate their efficiency in proper editing of the target genes Mentor Graduate students involved in the project and help them graduate and appy for PhD programs, Finalize sunmission of the manuscripts and address the reviewers comments

Impacts
What was accomplished under these goals? 1- Conducted tilling by sequencing on the mutant population and analyzed the data for identification of novel allelic variations in target genes. 2- Constructed a new CRISPR/CAS9 vector that carries GFP reported gene for tracing the expression pattern of the CAS9 construct 3- Established hairy root transformation as a faster approach in confimation of CRISPR CAS9/gRNA efficiency 4- Training two graduate students invovled in this project 5- Perforemed crossed between different soybean mutants in the field so that we can evaluate the additive effect of different mutations in traits of interest

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Millas, R., Espina, M. J., Ahmed, C. S., Bernardini, A., Adeleke, E., Yadegari, Z., ... & Taheri, A. (2020). Detection of novel allelic variations in soybean mutant population using Tilling by Sequencing. BioRxiv, 711440 doi: https://doi.org/10.1101/711440 Reneth Millas, Mary Espina, CM Sabbir Ahmed, Angelina Bernardini, Ekundayo Adeleke, Zeinab Yadegari, Korsi Dumenyo, Ali Taheri. 2020. Novel fatty acid alleles in soybean mutant population using forward genetics approach


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Soybean Farmers High school students and general publicvisiting the research field and labs Graduate andundergraduate students participating in research symposiums Scintific community and participants Changes/Problems:We had twopower outages at the TSU campus which lasted more than 3-4 days each time and our deep freezers didnt recover after the outage. This outagenegativaly affected our samples in the deep freezerincluding 6400 DNA sample collection and also bacterial cultures (carrying CRISPR/CAS9 vectors). We have started preparing new bacterial cultures and evaluating the extent of damage to the DNA samples. What opportunities for training and professional development has the project provided?So far, three graduate studentshave been involved in this project that one of them completed her MsC degree successfully and is currentlyat the PhD program at the Universityof Minnesota. Two other master students have joined this program recently and are gaining knowledge in genome editting and sequencing techniques used in this project. Students have prepared and presented their scientific findings at the scientific conferences. How have the results been disseminated to communities of interest?Research outcome of this project has beenshared with Tennessee soybean growers every year in their annual meetings. Students has also presented their findings from this project at the TSU annual research symposium and also atthe 2019 HBCUAssociation of Research Directors (ARD). A peer reviewed paper has been published from this work at the Biosensor journal. Ravi, R., Taheri, A., Khandekar, D., & Millas, R. (2019). Rapid Profiling of Soybean Aromatic Compounds Using Electronic Nose. Biosensors, 9(2), 66 What do you plan to do during the next reporting period to accomplish the goals?Continue vector construction for the CRISPR/CAS9 genome editting and optimizing the protocols. Evaluating PCR primers for herbicide resistance genes in TILLING approach. Presenting the scientific findings at the umcoming symposiums such as biannual soybean research meeting and ASPB.

Impacts
What was accomplished under these goals? Primers were designed for specific genes involved in resisiatnce to ALS herbicide which will be used for screening the mutant population. CRISPR/CAS9 vectors were obtained from Addgene and gRNA was designed for the target genes. One master student worked on this project and finished her degree and now is at the University of Minnesota pursuing her PhD in plant sciences.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Papers: Ravi, R., Taheri, A., Khandekar, D., & Millas, R. (2019). Rapid Profiling of Soybean Aromatic Compounds Using Electronic Nose. Biosensors, 9(2), 66 Presentations: Taheri, A., M.J. Espina, C M S. Ahmed, A.F. Bernardini, E. Adeleke, Z. Yadegari, P. Arelli, and V. Pantalone. Germplasm Enhancement in Soybean Using Chemical Mutagenesis and Tilling-By-Sequencing Technologies. 2019 ARD Research Symposium. Jacksonville, FL Millas, R and Taheri, A. Development and characterization of soybean with improved fatty acid profile through mutation breeding and genome editing. 2019. Graduate Thesis Defense. College of Agriculture. Tennessee State University. Nashville, TN Taheri A. Presentation for 120 high school students from LEAD Public Schools. Sep 2019. College of Agriculture, TSU Taheri, A. Screening Soybean Mutant Population for Heart Healthy Germplasm. 2019 ARD Research Symposium. Jacksonville, FL Taheri, A. Rapid Profiling of Volatile Compounds in Soybean using Electronic Nose. 2019 ARD Research Symposium. Jacksonville, FL