Progress 01/15/24 to 01/14/25
Outputs
Target Audience:The goal of this project is to understand the genetics of Aluminum tolerance stress tolerance and to evaluate genes important to root growth under this toxicity treatment. The target audience for the project is primarily researchers in academia includingprofessors, graduate and undergraduate studentsalso extends to extension agents and farmers who are interested in especially for production of Cowpeas (Vigna unguiculata). Cowpeas, including varieties like crowder peas, black-eyed peas, and pink-eyed peas, are vital traditional crops in the southeastern United States. They generate the highest net income per acre compared to other grains and vegetables grown in the region. Graduate student presentations at Tennessee State University attract hundreds of students from across the University and over eighty from the College of Agriculture. This project, based at a 1890s University, offers PhD training to the first cohort of Plant Science doctoral program students and also offers plant breeding training to undergraduate students, a field typically taught at 1862 Land Grant Colleges of Agriculture and not many 1890s Land Grants. Changes/Problems:The GWAS trial for Al tolerance is on hold because conditions in the greenhouse have been too hot and variable for hydroponic testing of a large number of cowpea lines needed for association analysis. Our program was switched from a temporary hoophouse that had high humidity to a more permanent glass/polycarbonate greenhouse that replaced the ones we lost at TSU from a tornado that came through the AREC-Nashville center.The rebuilt greenhouse has a different heating and cooling system better suited to woody plants and difficult to use for herbaceous legumes. In any case, we have the panel of Southeastern American germplasm ready to test and have noted associations between origin in states and their soil types for tolerance in the mini-panel of 22 genotypes tested last year but we have not been able to design a greenhouse-based experiment that accommodates the 150+ lines we have tested in the field.Since the germplasm is new to phenotyping testing we believe that testing under optimal field conditions are a precursor to any testing for marginal soil adaptation.We have multiplied seed and characterized genotypes now for further work down the road at a higher Aluminum soil site / field site but would need a collaborator for that possibly at another 1890s land grant university such as Jackson State, Alabama A&M or within one of the UTIA stations (Crossville, Jackson, Knoxville, or Springfield).On a positive note, quality of the sequencing data for the American cowpeas has been excellent, with only one sample with sequence quality that shouldn't be used further down the pipeline. Therefore, analysis of our panel of Southeastern varieties should definitely be included for future plans justified by our need to understand well-adapted resources available in the United States.As most of the accessions lacked meaningful passport data in the GRIN database, our phenotyping efforts will be useful to database in the National Plant Germplasm system. What opportunities for training and professional development has the project provided?The PhD student for this project is Mr. Max C. Miller II, and he has conducted the phenotyping and genotyping goals with the help of a research associate, Dr. Hui Li who was with TSU until March of 2024.Max has trained various undergraduate students including Ms. Nadiya El'Tigani and Ms. Elana Bodude as Dean's scholars and Ms. Bronwen Swann as summer work aid student. Each has had a subproject of the main overall PhD project for their undergraduate laboratory training.Nadiya did seed scanning while Elana did pod scanning and Bronwen did in the field phenotypic evaluation in the case of summer work.Opportunities for professional development of the PhD student and undergraduates have included presenting and attending the following conferences: at 1) the 42nd TSU researchsymposium in 2024 in Nashville with all 4 students attending, 2) the National Association of Plant Breeders meeting in ST. Louis MO in July 2024 with Max presenting and 3) the Association of Research Directors 1890s meeting in Nashville with Max and Dr. Blair presenting.Blair and Miller also prepared for presentation of legume research accomplishments at the TSU-UTKAI Research Summit in February 2025. Summer internships were provided for two high school student, Ms. Kayla Banks and Mr. Christian Nobles, who were trained on using drones to collect phenotypic data over the black-eyed pea field with Max, the PhD student.The classes and flight supervision were provided by Dr. Anjin Chang an expert in this new area of phenomics to genomics. How have the results been disseminated to communities of interest?Articles and seminar abstracts have been the main way of disseminating to researchers in the field of plant breeding and physiology. Small Farm Expo was an important event to inform middle TN producers and consumers about black-eyed peas. We had a demonstration tent at the Nashville AREC in the field next to 192 entries of varied cowpeas planted by PhD student, Mr. Max Miller for his GWAS field experiment. Four tractor-pulled trolley rides came through with over 200 farmers and researchers interested in the field crops we were growing including the black-eyed peas. We used posters and pamphlet type hand outs to explain our work. We distributed a cowpea recipe book prepared on a different project so that consumers would know how to prepare this type of legume. We hope to have a databased of southeastern USA varieties nutritional value for the black-eyed peas studied in this project as part of a SARE fellowship that has supplemented Mr. Max Miller's work. To that end he completed nitric acid digestion of cowpea seed from 2023 field trials encompassing a total of ~550 samples to undergo ICP analysis (this is work toward the SARE student grant aimed at identifying southern pea germplasm with high micronutrient content). Seeds from 2024 are also ready to be crushed soon and digested to see year to year variability. Our program has also multiplied seed of popular standard types of cowpeas, such as pinkeye, black-eyed, crowder, to test in collaboration with Bush Bros. Inc "the Beautiful Bean " company with the goal of popularizing and re-discovering various types of cowpeas so that they don't all have to be a similar standard BEP. What do you plan to do during the next reporting period to accomplish the goals?We hope to have a database of southeastern USA varieties' genotype information.We will use this to test the potential of GWAS with the cowpea panel by evaluating the nutritional value for the black-eyed peas studied in this project. This is part of a SARE fellowship that has supplemented Mr. Max Miller's work.To that end he completed nitric acid digestion of cowpea seed from 2023 field trials encompassing a total of ~550 samples to undergo ICP analysis (this is work toward the SARE student grant aimed at identifying southern pea germplasm with high micronutrient content). Seeds from 2024 are also ready to be crushed soon and digested to see year to year variability. Our program has also multiplied seed of popular standard types of cowpeas, such as pinkeye, black-eyed, crowder, to test in collaboration with Bush Bros. Inc "the Beautiful Bean " company with the goal of popularizing and re-discovering various types of cowpeas so that they don't all have to be a similar standard BEPs.We are exploring how to conduct Al tolerance testing in the GWAS panel using soil collected from a low pH growing region for cowpeas but this has a lot of hurdles for set up as explained below for changes/problems encountered.
Impacts
What was accomplished under these goals?
Under goal 1 for phenotyping, a total of 22 cowpea genotypes mostly from the Southeastern United States were evaluated in a hydroponic system with three different treatments:control for pH (no Aluminum), acid solution (pH 4.5, no Aluminum) and toxicity treatment (pH 4.5 and 50 µM AlCl3). While the technique was good enough to distinguish extremes in the distribution, the hydroponics was impossible to use for 100+ genotypes. Therefore, Goal 2 of genome wide association QTL for Al tolerance per se was not possible yet, although we did field plant the genotypes and skim sequencing for 150 genotypes of cowpeas for SNP calling and to form a USA panel of black eyed and other type cowpeas.This was done by outsourcing to LGC genomics in Gainesville, FL.For Goal 3, the project's PhD student characterized the transcriptome of two cowpea varieties that exhibited significant differences in aluminum toxicity tolerance using RNA sequencing (RNAseq). The transcriptomes of the tolerant Mississippi Pinkeye 2 Purple Hull (MSP2PH) and sensitive White Acre (WA) were evaluated 6, 24, and 48 hours after treatment with 50µM AlCl3. Total RNA quality was high with all RNA integrity number (RIN) scores of all samples being above 9.0. The samples were sent to Vanderbilt University genomics center (VANTAGE) for cDNA library construction and sequencing.Using HiSeq Illumina sequencers, the total number of reads from each sample ranged from about 20,000,000 - 60,000,000. The transcript profiles of the samples clustered by variety, treatment, and time point after principal component analysis and hierarchical clustering indicating high sample consistency. A moderately strict threshold of 1 log-fold change (logFC) and a false-positive discovery rate of p ≤ 0.01 was used to identify differentially expressed genes (DEGs) for all comparisons. The total number of DEGs between plants of the same variety under different conditions at 6, 24, and 48 hours after treatment was 1806, 2067, and 1111 for MSP2PH and 2344, 2577, and 1918 for WA. The number of DEGs unique to the aluminum-toxic conditions that were not differentially expressed under control conditions between MSP2PH and WA at the 6, 24, and 48 hour time points were 390, 607, and 633, respectively. Additionally, 60 of these DEGs were identified across all three time points, and a total of 14 from this subset were up-regulated in MSP2PH. These 14 DEGs, which include trans-membrane and metal-binding proteins known for their involvement in metal toxicity tolerance in other species, are good candidate genes for future characterization and offer a deeper insight into the mechanisms of aluminum toxicity tolerance in cowpea. Goal 4 was training of undergraduate and graduate student and is described in greater detail in the next section for professional development but included one PhD student and three undergraduates.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Miller MC*, Blair MW* (2025) RNA sequencing approach to identifying genes associated with Aluminum toxicity tolerance in cowpeas. 47th Annual Tennessee State University-Wide Research Sponsored Programs Symposium March 24th 26th, 2025
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Miller MC*, Blair MW* (2025) Evaluation of summer grain legumes, especially black-eyed peas for acid soils and sustainable rotations in Tennessee. UT/TSU collaboration seminar. February 21st 2025, McMinnville TN (TSU Nursery Research Center)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Miller MC*, Li H, Blair MW* (2024) Screening and gene expression analysis of cowpeas and black-eyed peas for Aluminum tolerance. National Association of Plant Breeders / AFRI Plant Breeding meeting. St Louis MO, July 19-24
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Miller MC*, Blair MW* (2024) Screening and RNA Sequencing of Commercial Cowpea Cultivars under Aluminum Toxic Conditions. National Association of Plant Breeders / AFRI Plant Breeding meeting. St Louis MO, July 19-24
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The target audience for the project is primarily researchers in acedemia including other professors and many graduate students but also extends to extensionists and farmers especially for aluminum toxicity tolerance in Cowpeas (Vigna unguiculata). With many regional types from crowder peas to black-eyed or pink-eyed peas, cowpeasare some of the most important, traditional crops of the southeastern United States, being among the highest net income generating crops onper acre basisamong the grains and vegetables produced in this region. Like most legumes, cowpeas are sensitive to abiotic stresses especially aluminum toxicity found in acidic soils and are less likely to be limed because of the small-scale nature of production. Since testing of aluminum tolerance involves hydroponic screening this has been a good project to introduce the academic target audience of graduate students from other disciplies within Tennessee State University to this type of phenotyping. The graduate student presentations reach a large audience of several hundred other graduate students across the University and over eighty in the College of Agriculture here. The PI for this project has one of the only legume genomics/breeding programs in the region that is evaluating tolerances to aluminum in cowpeas. The strengtheningproject is set in an 1890s University and provides a PhD trianing for one of the initial cohorts of Plant Science doctoral program students at Tennessee State University. The projectalso provides training for undergraduate students from under-represented minorities in plant breeding field for which training is primarily given at 1862 Land Grant Colleges of Agriculture not at many 1890s Land Grants. Changes/Problems:We moved from a temporary greenhouse to a new greenhouse at AREC on Tennessee State University campus and are still adapting our germination and hydroponic systems to the new environment. GWAS will be conducted with a more complete nutrient solution for next experiment. Post doctoral research associateleft TSU during April 2024 so we will need to recruit a replacement. What opportunities for training and professional development has the project provided?The majority of the training so far has been for the PhD student (Mr. Max Miller) hired as a graduate research assistant on the project who is now finished with TSU classes and full time in research; and secondarily for a post-doctoral research associate (Dr. Hui Li) also at Tennessee State University. Both scientists have been involved in training of undergraduate dean scholars and part time or work-aid field and lab assistants (Mr. Baron Hill, Ms. Belevia Stovall, Ms. Bronwen Swann, Ms. Gabrielle Oliver and Ms. Nadiya El'Tigani). Thisgroup hasbeen recruited from TSU undergraduates and are all from under-represented backgrounds in science (first-generation or minority STEM). They area all either biotechnology, plant science or environmental science majors. How have the results been disseminated to communities of interest?Mr. Max Miller has made two presentations per year in both 2022-23 and 2023-24 academic years. For current year, these were presented at the TSU research symposium in March 2024and also in the Annual Research Directors Meeting in April 2024. We expect undergraduates to be presenting this year as well or to include sub-projects as seniorand/or honor's thesis topics. Mr. Max Miller and the PI Dr. Blair have been involved in a publication on GBS genotyping of cowpea, while the PI with previous TSU students, Dr. Xingbo Wu and Mr. Jesse Potts (current PhD in University of Florida) wrote another paper on KASP genotyping. In both the AFRI project was acknowledged. What do you plan to do during the next reporting period to accomplish the goals?Continue analyses of RNAseq results and skim-sequencing results. Complete the GWAS panel testing in the hydropoinic aluminum tolerance screening system during the greenhouse season. Correlate data from genotyping and phenotyping for QTL and gene identification.
Impacts
What was accomplished under these goals?
We have made progress towards the four goals of the project with the following accomplisments 1)Evaluate aluminum tolerance in world-wide cowpea germplasm. We have established a) the phenotyping method in hydroponic tanksand b) aluminum toxicity screening treatment level for evaluation of tolerance in cowpea germplasm. This includes192 entriesfrom the USDA collection obtained through the GRIN system with NPGS collaborators at Griffin GA as well as a smaller panel of 22 commercial checks from various seed company sources including Seedway, Southern Seed Exposure and WillhiteSeed Inc. Both sets of germplasm emphasized varietal releases in the United States and landraces from the Southeast, South and Southwest/Texas regions with other cowpeas from the Western Hemisphere for diversity. Multiplication of seed from the original set of USDA entries was done in a greenhouse to avoid outcrossing and later in the field in summer 2023 with follow up this summer. In terms of the Aluminum tolerance phenotyping method developed, the hydroponic system is a modification of the method we used previously for common bean and involved setting up shallower trays for earlier screening of seedlings but simultaneously increasing AlCl3+ to 50uM . We also simplified the treatment solution and included a 0 uM control at neutral pHas well as a low pH treatment to differentiate acid root zone reactions from aluminum stress reactions of the cowpea plants. Inaddition, we modified our seedling preparation method by germinating in sterilizedsphagnum moss, humidified with sterile ddH2O and filled into an autoclaved mason jar or later shallow non-welled, starter trayskept uncovered in a low airflow benchspace or greenhouse space, respectively. Before this, seed had been surface sterilized for germination and grew well without the contamination problems we saw previously while growing on petri dishes or germinating in wrapped paper towels soaking on plastic pipettes in large open-air beakers. Finally, our WinRhizo screening of the cowpea root systems weredone not only at 48 hours as previously done with common beans but rather at 0, 24 and 48 hours to distinguish growth rates and changes in other root parameters with data analysis still ongoing for the initial germplasm panel, and pending for the full GWAS panel. The former germplasm set was grown 4 times over two seasons from September to Februaryin the greenhouse hydroponic setup while the full 192 accession GWAS panel was multiplied in the field once so far in June to October. We believe that the results from the smaller panel of 22 genotypes will be reproducible in the larger panel of 192 genotypes using the same WinRhizo obtained traits. As opposed to field experiments where the cowpeas were grown to maturity, the greenhouse grown plants were only left to be 7 days old in total. This included 5 days pre-treatment (3 days germination, 2 days acclimation) followed by 2 day treatment for a total of 1 week of scanning at 0 hr., 24 hr and 48hr post treament. The trait from WinRhizo that was most significant was primary root length and growth rate. The worst genotypes in this respect were White Acre, Iron Clayand Texas Cream 8. The genotypes that stood out of the 22 variety panel were Mississippi Pinkeye II purple hull and Top Pick brown crowder. The first of these was chosen for further transcriptome work. 2. Conduct genome wide association QTL study of aluminum tolerance in cowpea: Because the germplasm is not of the core collection entirely, we found very little genotypic information for the entries and therefore grew them in the greenhouse for leaf tissue used for DNA extraction as well as seedmultiplication and stabilization. DNA for the 192 entries was successfully obtained and outsourced for skim sequencing at 5X genome coverage to the LGC releated facility in Jackshonville, FL. We are waiting on results from them but know that initial sequencing has gone well. 3. Validate gene discovery through transcriptome analysis of root meristems. The approach taken for this objective has been to conduct a RNAseq experiment on the best and worst genotypes in terms of Aluminum stress tolerance based on primary root growth rate from the commercial panel. The varieties White Acre and Mississippi Pinkeye II Purple Hull were found to be those worst and best genotypes, respectively, and in a separate experiment they were grown in hydroponic tanks with and without the Aluminum treatment for harvest of root tips into a tissue pool from 8 treated plants with 3 replicates each. Time points wereat 6, 24 and 48 hours after exposure to the 50 uM Aluminum with appropriate controls of 0 uM at same time points and same pH. RNA was extracted from same treatments and outsourced for cDNA library construction and sequencing at Vanderbilt Genome facility (VANTAGE). Data collected so far shows high RNA integrity numbers (RIN values) which indicated that the tissue source being rootsand the exposure to Aluminum is not affecting RNA extraction. 4. Train graduate and undergraduate students, especially from under-represented minorities in phenotyping and genotyping methods. The majority of the training so far has been for the PhD student (Mr. Max Miller) hired as a graduate research assistant on the project who is now finished with TSU classes and full time in research; and secondarily for a post-doctoral research associate (Dr. Hui Li) also at Tennessee State University. Both scientists have been involved in training of undergraduate dean scholars and part time or work-aid field and lab assistants (Mr. Baron Hill, Ms. Belevia Stovall, Ms. Bronwen Swann, Ms. Gabrielle Oliver and Ms. Nadiya El'Tigani). Thisgroup hasbeen recruited from TSU undergraduates and are all from under-represented backgrounds in science (first-generation or minority STEM). They area all either biotechnology, plant science or environmental science majors. Mr. Max Miller has made two presentations per year in both 2022-23 and 2023-24 academic years. We expect undergraduates to be presenting as well this year or to include sub-projects as seniorand/or honor's thesis topics
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Max Miller (presenter) Hui Li and Matthew W. Blair (advisor) Phenotypic Responses and Gene Expression of Southern Pea (Vigna unguiculata L. Walp.) to Aluminum Toxicity. Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, 37209. Presentation at Association for Research Directors 2014, Opryland Conference Center, Nashville TN, April 8, 2024
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Max Miller (presenter) Hui Li and Matthew W. Blair (advisor) Presentation. (2024). Screening and gene expression analysis of cowpeas and black-eyed peas for Aluminum tolerance. 46th Annual Tennessee State University-Wide Research Sponsored Programs Symposium March 25th 27th, 2024
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Potts J, Michael VN, Meru G, Wu X, Blair WM (2024) Dissecting the Genetic Diversity of USDA Cowpea Germplasm Collection Using Kompetitive Allele Specific PCR-Single Nucleotide Polymorphism Markers. SI: Genetics and Breeding of Legume Crops). Genes 2024, 15(3), 362; https://doi.org/10.3390/genes15030362
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wu X, Michael VN, L�pez LF, Cort�s AJ, Morris JB, Wang M, Tallury S, Miller MC, Blair MW (2024) Genetic Diversity and Genome-Wide Association in Cowpeas (Vigna unguiculata L. Walp). SI: Application of Molecular Marker Technology in Crop Breeding II), Agronomy 2024, 14(5), 961; https://doi.org/10.3390/agronomy14050961
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The goal of this project is to understand the genetics of Aluminum tolerance stress tolerance and to evaluate genes important to root growth under this toxicity treatment. As such, the project has a target audience geared towards other scientists and for training of students from 1890s land grant colleges. Researchers from other land grant and non-land grant research institutions that come in contact with our study of Aluminum tolerance are also target audiences. Other audiences are farm and commodity groups interested in cowpea production. For example we are in contact with Bush's beans Inc. which cans crowder and black eyed peas as two different types of cowpeas. Changes/Problems:Changes/Problems: A delay in phenotyping was caused by the COVID pandemic and by a tornado that destroyed the greenhouses we were using for hydroponic growth assays therefore we had to switch to a growth chamber and then hoop-house experiments. This has caused us to reduce the genome-wide association portion of the project (Activity 1 and 2) in favor of the transcriptomic and training parts (Activity 3 and 4). A project extension has been granted to make this a three year project. Genome-wide association will be conducted once improvements in the cowpea screening method are made which are underway as part of the PhD project of Mr. Max C. Miller II. RNAseq transcriptome analysis likewise has been started with new RNA samples. What opportunities for training and professional development has the project provided?We provided laboratory work opportunities for visiting students from New York (Cornell), North Carolina (Duke)and Georgia (Emory)universities. Illumina sequencing training was provided by that company to work on MiSeq and NextSeq machines for the graduate students and for one post-doctoral associate named Dr. Hui Li, who now has joined the project along with the more newly recruited student for the project, Mr. Max Miller, who arrived from Louisiana State Univ. in January 2022. Notably, a related dissertation with a chapter on Al tolerance in Andean common beans was completed by Dr. Daniel Demissie Ambachew in November 2021; so we are seamlessly building on that work. In addition, Dean's scholars Ms. Jewell Vickers, Ms. Chelsea Randle, and Ms. Kennedy Cooper were recruited at the beginning of the 2022 Spring semester, also Ms. Nadiya Eltigani for Spring 2023. In addition, we paid for work aid from Ms. Rachel Lawson. Four out of five of theundergraduate students are African American. Finally, a post-doctoral associate with experience in Al tolerance in tomatoes was hired on the grant to continue the work on cowpeas. How have the results been disseminated to communities of interest?Yes results have been disseminated at three conferences 1) TN Academy of Sciences (poster presentation by Mr. Max Miller); 2) Research Sponsored Programs symposium at Tennessee State University (poster and oral presentations from Mr. Max Miller with PI-Blair and Post-Doc: Hui Li); and 3) at the new crops conference for Southeastern Agriculture (NIFA project also under A1141, Plant Breeding section), where PI-Blairmade a presentation on March 16, 2022. This presentation was about the search for Al tolerance in legumes, specifically in the common bean from the dissertation work and publication of Dr. Daniel Demissie, PhD student in the same laboratory (TSU/Plant genomics) from Fall 2021. Expected graduation for Mr. Max Miller is in 2024. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete Objectives 1 and 2, with larger number of genotypes for Al tolerance phenotypic screening and SNP marker analysis either by re-sequencing, skimSeq or GBS methods of SNP discovery.Once putative genes for aluminum toxicity tolerance have been identified, these samples may be revisited for additional RT-qPCR using primers designed for these genes. With the improvement in germination, a larger panel of cowpea germplasm (192 accessions) will be utilized for a comprehensive genome-wide association study in the future.
Impacts
What was accomplished under these goals?
Objective 1:Initially, germination of cowpea was an issue. This has been much improved and a hoophouse / sphagnum moss method has been utilized to screen 22 commercial U.S. cowpea varieties for aluminum stress tolerance. The focus is on more commercial genotypes than raw landraces from other countries.This small panel showed a diverse yet consistent response to aluminum toxicity, particularly in relative primary root growth rate. The genotypes displayed a normal phenotypic distribution for both relative and outright root traits. Furthermore, it should be noted that some of the most tolerant varieties were either popular commercial varieties (Mississippi Pinkeye 2 Purple Hull, Top Pick Brown Crowder, Mississippi Purple Brown Crowder) or constituted the background for many modern lines (Mississippi Silver). Correlation of both relative traits and traits within each treatment were shown to vary, but there were no important traits that were negatively correlated. Variance components and heritability of all traits were calculated, and heritability of most traits was shown to generally be exceptionally high. These results indicate that most of the traits associated with aluminum toxicity tolerance can be targeted to improve tolerance overall, and it is unlikely that breeding for one trait reduces the quality of another that is important for aluminum toxicity tolerance. ? Objective 2: the GWAS portion of the study has not been started but plans are underway for genotyping of 150+ samples of U.S. germplasm of cowpeas. We also have a set of 192 adapted core collection germplasm that was genotyped by GBS with one publication distinguishing the vegetable, fodder, and grain types of cowpeas as sub-populations. Objective 3: Root tissues specifically from the root tips were collected from an experiment with the most resistant andsusceptible of the 24 genotypes grown under three treatments (low pH, high Al, low Al) and a first set of 24 RNA samples from that physiological study was sequenced for 24 hours of treatment effects.For insightful RNAseq analysis, the two lines with significantly different relative primary root growth rates after 24 and 48 hours were chosen for RNA extraction and sequencing. The 24 hour time point was used for sample collection and sequencing of RNA. Data has been generated and is currently in pipeline for analysis. In addition to the 24 hour time point, samples at 6 and 48 hours were also collected for RNA extraction. All time points will be used to determine expression profiles of stress-related genes using RT-qPCR. Objective 4: we have provided opportunities for training and professional development through the project. Training occurred for an M.S. student, Mr. David Hickok during the first year of the grant and for a PhD student, Mr. Max Miller during the second and third years. The first student has graduated and the second one is about to defend his proposal.Despite some challenges due to the COVID pandemic in virtual lessons, and quarantined lab work, Davidfinished his thesis on time and was able to make progress on the phenotypic and RNA extraction steps implicit in the goals of this project. David also is from an under-represented minority group, which is one of the groups of graduate and undergraduate students to train.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
SCREENING AND RESPONSE OF COMMERCIAL COWPEA [VIGNA UNGUICULATA (L.) WALP.] CULTIVARS TO LOW PH AND ALUMINUM TOXIC CONDITIONS. 45th ANNUAL TENNESSEE STATE UNIVERSITY-WIDE RESEARCH SPONSORED PROGRAMS SYMPOSIUM (2023), *Miller MCII, Li H, Blair MW. Graduate Poster. GP-GSA-05 Winner of 3rd prize for posters.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
SCREENING AND RESPONSE OF FIVE COMMERCIALLY AVAILABLE COWPEA VIGNA UNGUICULATA CULTIVARS TO LOW PH AND ALUMINUM TOXIC CONDITIONS. 32nd Meeting of the TENNESSEE ACADEMY OF SCIENCE. (2022) *Miller MCII, Li H, Blair MW.. Nashville TN. November 18th, 2022. Winner of 2nd prize for posters.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Ambachew DA+, Asfaw A, Blair MW* (2023). Genetic variability for root traits in a panel of Andean common beans grown under with and without Aluminum stress conditions. MDPI .Agronomy, 13(3), 619; https://doi.org/10.3390/agronomy13030619
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Pratap A, Kumar S, Polowick PL, Blair MW, Baum M (2022) Editorial : Accelerating Genetic Gains in Pulses. Division Plant Breeding, Frontiers in Plant Science 13:879377. https://doi.org/10.3389/fpls.2022.879377
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wu X+, Cortes AJ, Blair MW* (2022) Genetic differentiation of grain, fodder and pod vegetable type cowpeas (Vigna unguiculata L.) identified through genotyping-by-sequencing. Molecular Horticulture. 28 March 2022 https://molhort.biomedcentral.com/articles/10.1186/s43897-022-00028-x
- Type:
Book Chapters
Status:
Accepted
Year Published:
2024
Citation:
Blair MW*, Miller, M.C. II+, Yahaya, D+, Hickok D+, Wu, X+ (in press) Allele Mining in Cowpea (Vigna unguiculata) Sub-species and close relatives. In C. Kole (Ed.) Allele Mining in Pulses. Springer Inc.
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Mahesha HS. Keerthi MC, Shivakumar KV, Bhargavi HA, Saini RP, Manjunatha L, Hickok D+, Blair MW* (2022). Development of Biotic Stress Resistant Cowpea. pp 213251 In: Kole, C. (eds) Genomic Design Biotic Stress Resistant Pulse Crops. Springer, Cham. 19 March 2022 https://doi.org/10.1007/978-3-030-91043-3_4
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Veeranagappa P, Blair MW*, B.Manu B, Hickok D+, Prasad G, Naveena NL, Manjunath L, Tripathi K (2022) Advanced Breeding Strategies for Abiotic Stress Tolerance in Cowpea. pp 115144. In Chitta Kole (ed) Genomic Designing of Abiotic Stress Resistance Pulse Crops. Springer Publishing Inc., Cham. 24 March 2022: https://doi.org/10.1007/978-3-030-91039-6_4
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
SCREENING AND GENE EXPRESSION ANALYSIS OF COWPEAS AND BLACK-EYED PEAS FOR ALUMINUM TOLERANCE. 45th ANNUAL TENNESSEE STATE UNIVERSITY-WIDE RESEARCH SPONSORED PROGRAMS SYMPOSIUM (2023), *Miller MCII, Li H, Blair MW. Graduate Agricultural Sciences III. GO-AGNS-19. March 27th 30th, 2023.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The goal of this project is tounderstand the genetics of this abiotic stress tolerance and to evaluate QTL and genes important to root growth under aluminum treatment. As such, the project has a target audience geared towards other scientists and for training of students from 1890s land grant colleges. Researchers from other land grant and non-land grant research institutions that come in contact with our study of Aluminum tolerance are also target audiences. Changes/Problems:Delay in phenotyping was caused by the COVID pandemic and by a tornado that destroyed the greenhouses we were using for hydroponic growth assays therefore we had to switch to growth chamber experiments. This may cause us to reduce the genome wide association portion of the project (Activity 1 and 2) in favor of the transcriptomic and training parts (Activity 3 and 4). A project extension will be requested due to the unforeseen changes and delays due to the natural disaster of the tornado and pandemic closures. What opportunities for training and professional development has the project provided?Training occurred for an M.S. student, Mr. David Hickok during the first year of the grant. He was in the second year of training having received a graduate research assistantship from the department and college in Fall 2019 with expected graduation in Summer 2021. Despite some challenges due to the COVID pandemic in virtual lessons, and quarantined lab work, he finished his thesis on time and was able to make progress on the phenotypic and RNA extraction steps implicit in the goals of this project. David also is from an under-represented minority group, which is one of the groups of graduate and undergraduate students to train. We provided laboratory work opportunities for visiting students from New York (Cornell) and North Carolina (Duke) universities. Illumina sequencing training was provided by that company to work on MiSeq and NextSeq machines for the graduate students and for one post-doctoral associate named Dr. Hui Li, who now has joined the project along with one newly recruited student for the project, Mr. Max Miller, who arrived from Louisiana State Univ. in January 2022. Notably, a related dissertation with a chapter on Al tolerance in Andean common beans was completed by Dr. Daniel Demissie Ambachew in November 2021; so we are seamlessly building on that work. In addition, Dean's scholars Ms. Jewell Vickers, Ms. Chelsea Randle, and Ms. Kennedy Cooper were recruited at the beginning of the 2022 Spring semester and all three undergraduate students are African American. Finally, a post-doctoral associate with experience in Al tolerance in tomatoes was hired on the grant to continue the work on cowpeas. How have the results been disseminated to communities of interest?Yes, at the new crops conference for Southeastern Agriculture (NIFA project also under A1141, Plant Breeding section), we made a presentation on March 16, 2022. This presentation was about the search for Al tolerance in legumes, specifically in common bean from the dissertation work and publication of Dr. Daniel Demissie, PhD student in the same laboratory (TSU/Plant genomics) from Fall 2021. What do you plan to do during the next reporting period to accomplish the goals?Genome wide association will be conducted once improvements in the cowpea screening method are made. RNAseq transcriptome analysis likewise will be carried out with new RNA samples. A project extension will be requested due to delays caused by COVID pandemic.
Impacts
What was accomplished under these goals?
A preliminary abiotic stress tolerance test was established for six North American grain-type cowpea varieties (Vigna unguiculataspp. unguiculata) in a hydroponic tank with full nutritional supplementation and transplant of two-week-old seedlings. The varieties included Queen Anne (QA), Dixie Lee (DL), Quick Pick (QP), Top Pick (TP), Top Pick Brown Crowder (TPBC) and Mississippi Silver (MS). The seeds were germinated in Petri dishes with autoclaved paper towels for one week and then transferred into control and Al (20 uM) stress conditions. The varieties were sourced from Seedway, and Southern Exposure Seed Exchange two companies selling cowpeas commercially. Origins were from Arkansas, Georgia, Louisiana, Mississippi, and Virginia and represented different growth types from determinant and bush to indeterminate semi-bush. Damping-off was observed to be a problem in rolled paper towels used in other legumes (soybean, mung bean, or common bean) compared to the petri dish method of germination using either sand or circular paper inserts. After initial screening, QA and DL were selected for two experiments each with four imaging rounds. The hydroponic chamber consisted of 9 x 12-inch trays with 3.5 inch depth and a 3 L capacity for the solutions. Foam spacers were used to allow seedlings to float in the trays and the solutions were aerated with agitation and oxygen bubbles. Aluminum chloride was added to the stress treatment but both types of solutions were maintained at pH 4.5. WinRhizo software was used to evaluate scanned images of seedlings as they grow into plants for 8 root traits (length, surface area, projected area, volume, average diameter, number of forks, tips, and crosses). Analysis of variance was conducted for experiment 1 (balanced, 5 repetitions) and experiment 2 (unbalanced, QA>DL). In addition to scanned root images, biological samples were collected of lateral roots, taproots (100 mg each), and solution exudates (1.5 mL Eppendorf tube). Both types of samples were flash-frozen in liquid nitrogen for RNA extraction and metabolomic evaluation. RNA was extracted with a RapidPure plant kit eluted into 45 uL of solution and frozen at -80C. RNA quality was measured with a Nanodrop and then a Bioanalyzer to measure nucleic acid concentration and determine banding pattern and integrity scores (RIN values). The best samples were sent for sequencing at Novogene and results are pending.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Buitrago-Bitar MA, Cort�s AJ, L�pez-Hern�ndez F, Londo�o-Caicedo JM, Mu�oz-Florez JE, Mu�oz LC, Blair MW* (2021) Allele diversity at abiotic stress responsive candidate genes in the drought tolerant bean species Phaseolus acutifolius A. Gray. MDPI Genes, 12, 556. https://doi.org/10.3390/genes12040556
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2022
Citation:
Veeranagappa P, B.Manu B, Prasad G, Naveena NL, Manjunath L, Tripathi K, Hickok D, Blair MW (accepted) Abiotic Stress Tolerance In Cowpea In Chitta Kole (ed) Genomics of Abiotic Stress Tolerance in Legumes. Springer
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