Progress 03/01/23 to 02/29/24
Outputs
Target Audience:The knowledge gained through this project was shared with extension agents, the Florida grape growers association, muscadine products companies, grape breeders, and geneticists who are interested in enhancing the national grape industry and commercialization. Further, we have generated a training program that targets students in the local high schools and FAMU scientists and students to provide training in the areas of viticulture research (i.e., physiology, biology, computational biology, and others). The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identifying key transcription factor genes critical for the stenospermocarpy fruit-set program will not only cause a revolution in the muscadine grape industry but also in other diverse horticultural crops producing fleshy fruits. The objectives, technologies, and research outcomes have been discussed in several meetings with growers and grape breeders during the last-year grant: Grape Field Day (UF/IFAS), Citra, FL, May 18, 2023. The 2023 Florida State Horticultural Society (FSHS) Annual Conference, Daytona Beach, FL, June 10-13, 2023. North American Grape Breeders & Vitis-Muscadinia Alliance Conference (UA), Fayetteville, AR, October 3-5, 2023. Florida Wine and Grape Growers Association (FWGGA) 2023 Annual Conference, Deland, FL, January 11-12, 2024. Southeast Regional Fruit & Vegetable Conference 2024, Savannah, GA, January 12-13, 2024. Legislature visit to the Center for Viticulture, CAFS, FAMU that includes city/state representatives and industry representatives, Tallahassee, FL, January 23, 2024. Association of 1890 Research Directors (ARD) Research Symposium 2024, Nashville, TN, April 6-9, 2024 (two presentations). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We constantly strive to enhance the learning experience of our students in agreement with FAMU's motto of "Excellence with Caring" by improving the learning procedure and qualities. In 2021, we officially started a "Summer Biotech Academy" that targets a diverse pool of minority high school students and undergraduate FAMU students. The "Summer Biotech Academy" activities are sustained. The main goal of the program is to inspire students' awareness by exposing them to real-life examples and objectives, which develop communication, leadership, and management skills, encourage students to pursue advanced graduate degrees, expand students' intellectual knowledge and lab experiences and prepare them to better cope for a future workforce to empower and sustain the industry. During the life of the project, we have trained thirty-eight students (15 high school students and 23 FAMU undergraduate students). How have the results been disseminated to communities of interest?The knowledge gained through this project was shared with extension agents, the Florida grape growers association, muscadine products companies, grape breeders, biochemists, and geneticists who are interested in enhancing the national grape industry and commercialization. During the life of the project, we have produced 8 peer-reviewed publications and three extra major publications are in progress. Further, we have established two major database browsers, Sothern Grapes Genome/Transcriptome data and Sothern Grapes & Specialty Crops Breedomics that has been designed for long-term storage of all the genomic, transcriptomic, phenotypic, and metabolic data. Moreover, we have generated a training program (3 modules) that targets students in the local high schools and FAMU scientists and students to provide training in the areas of viticulture research (i.e., biotechnology, biology, computational biology, and others). Finally, we have been dynamically involved in enhancing the CAFS/FAMU mission of student recruitment via generating a website and a video highlighting the excellent FAMU capacities for the quality of education and research with outstanding examples of students who graduated under the CAFS programs. The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identifying key transcription factor genes critical for the stenospermocarpy fruit-set program will not only cause a revolution in the muscadine grape industry but also in other diverse horticultural crops producing fleshy fruits. The objectives, technologies, and research outcomes have been discussed in several meetings with growers and grape breeders during the life of the project (19 presentations in total) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Characterization of fruit-set programs among diverse grape genotypes. The objective has been performed among different grape species as planned in the proposal. Evaluate the changes in hormones accumulation profile throughout fruit ontogeny. The objective has been performed via quantifying the hormones of auxin (IAA), cytokinins (CKs), and abscisic acid (ABA) using Thompson Seedless cultivar and Thompson Seeded mutant vine. Evaluate the changes in transcriptome profile among grape genotypes exhibiting distinct fruit-set programs. The objective has been performed by generating a transcriptome data at early fruit-set stage using Thompson Seedless cultivar and Thompson Seeded mutant vine. Subcellular localization of identified key component genes. The objective has been performed as detailed in the reporting period. The main transcription factor associated with stenospermocarpy fruit set, which is annotated as AGL11 MADS gene, was localized within the nucleus compartment. We generated a mutated AGL11 version to mimic the isoform isolated from seedless grapes using site-directed mutagenesis approach. No changes detected in the localization of the protein due the mutation. Identify potential proteins that are able to interact with TFs strongly associated with the stenospermocarpy fruit-set program. The objective has been performed as detailed in the reporting period. A yeast library has been generated using RNA isolated from early fruit set stage. AGL11 transcription factor has been used as a bait to screen the library. The Y2H assay suggested that AGL11 is able to interact with 10 proteins, including 6 MADS box genes and genes encoding a tyrosine-protein phosphatase, a synaptotagmin, a UBP1-associated protein, and WAT1-related protein. The one to one assay confirmed the interaction quality of 9 out of 10 proteins. Further, we used DAP-seq strategy to identify potential downstream genes regulated by AGL11 TF and how the mutated version can change the interaction pattern. The experiment finished and we are in progress to confirm the DNA-protein interaction using Y1H assay. Evaluate the function of TFs associated with the stenospermocarpy fruit-set program using CRISPR/Cas9-gRNA ribonucleoproteins strategy. The objective has been performed. A total of 36 transformed muscadine events exhibiting nucleotides insertion/deletion were identified and they are growing under greenhouse conditions for the evaluation trials. Develop training modules in grape genomics and biotechnology. The objective has been performed, and the "Summer Biotech Academy" training program has been running since the summer of 2021.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Moniruzzaman M, Darwish AG, Gajjar P, Ismail A, Tsolova V, El-Kereamy A, El-Sharkawy I. (2023) Seedlessness trait and genome editing a review. International Journal of Molecular Sciences 24: 5660.
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Moniruzzaman M, El-Sharkawy I. Multiplex genome editing with CRISPR-spCas9 in muscadine grape. In Progress
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Ismail A, Park M, Gajjar P, Mahboob M, Moniruzzaman M, El-Sharkawy I. Genome-wide characterization of Muscadinia rotundifolia MADS-box transcription factors emphasizing their roles during berry development. In Progress
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Moniruzzaman M, El-Sharkawy I. The AGL11 and other MADS box genes synergistically coordinate embryo growth and seed formation. In Progress
|
Progress 03/01/19 to 02/12/24
Outputs
Target Audience:The knowledge gained through this project was shared with extension agents, the Florida grape growers association, muscadine products companies, grape breeders, and geneticists who are interested in enhancing the national grape industry and commercialization. Further, we have generated a training program that targets students in the students andscientists at FAMU as well aslocal high school studentsto provide training in the areas of viticulture/plant research (i.e., physiology, biology, computational biology, and others). The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identifying key transcription factor genes critical for the stenospermocarpy fruit-set program will not only cause a revolution in the muscadine grape industry but also in other diverse horticultural crops producing fleshy fruits. The objectives, technologies, and research outcomes have been discussed in several meetings during the project's life with growers and grape breeders (24 meetings): Grape Field Day at UF/IFAS (4 Presentations). The Florida State Horticultural Society (FSHS) Annual Conference (3 Presentations). North American Grape Breeders (2 Presentations). The Virtual Conference of the North Carolina Grape Growers Association (2 Presentations). Florida Wine and Grape Growers Association (FWGGA) Annual Conference (5 Presentations). Southeast Regional Fruit & Vegetable Conference (1 Presentation). Legislature visit to the Center for Viticulture, CAFS, FAMU that includes city/state representatives and industry representatives (2 Presentations). Association of 1890 Research Directors (ARD) Research Symposium (5 Presentations). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We constantly strive to enhance the learning experience of our students and trainees in agreement with FAMU's motto of "Excellence with Caring" by improving the learning procedure and qualities. In 2021, we officially started a "Summer Biotech Academy" that targets a diverse pool of minority high school students and undergraduate FAMU students. The "Summer Biotech Academy" activities are sustained. During the life of the project, we have trained thirty-eight students (15 high school students and 23 FAMU undergraduate students). Further, during the project life we recruited and trained 24 students and scientists, including 6 members in the field of Plant Science (4 undergraduate students, one graduate, and one post-doctorate), 2 post-doctorates in Molecular Biology, 7 post-doctorates in Genetics, and 9 members in Biotechnology (5 graduates and 4 post-doctorates). Our main goal is to inspire students'/trainees' awareness by exposing them to real-life examples and objectives, which develop communication, leadership, and management skills, encourage students to pursue advanced graduate degrees, expand students' intellectual knowledge and lab experiences, and prepare them to better cope for a future workforce to empower and sustain the industry. How have the results been disseminated to communities of interest?The knowledge gained through this project was shared with extension agents, the Florida grape growers association, muscadine products companies, grape breeders, and geneticists who are interested in enhancing the national grape industry and commercialization. During the life of the project, we have produced 8 peer-reviewed publications and three extra major publications are in progress. Further, we have established a major database browser, Sothern Grapes Genome/Transcriptome data that has been designed for long-term storage of all the genomic, transcriptomic, and phenotypic data. Moreover, we have generated a training program (3 modules) that targets students in the local high schools and FAMU scientists and students to provide training in the areas of viticulture/plant research (i.e., biology, computational biology, biotechnology, and others). Finally, we have been dynamically involved in enhancing the CAFS/FAMU mission of student recruitment via generating a website and a video highlighting the excellent FAMU capacities for the quality of education and research with outstanding examples of students who graduated under the CAFS programs. The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identifying key transcription factor genes critical for the stenospermocarpy fruit-set program will not only cause a revolution in the muscadine grape industry but also in other diverse horticultural crops producing fleshy fruits. The objectives, technologies, and research outcomes have been discussed in several meetings with growers and grape breeders during the life of the project (24 presentations in total). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Characterization of fruit-set programs among diverse grape genotypes. The objective has been performed among different grape species as planned in the proposal. Evaluate the changes in hormones accumulation profile throughout fruit ontogeny. The changes in plant hormones auxin (IAA), cytokinins (CKs), and abscisic acid (ABA) were quantified during different stages of early berry deveolpment using Vitis vinefera grapes, Thompson Seedless cultivar and Thompson Seeded mutant vine, exhibiting stenospermocary seedless and seeded fruit-set programs, respectively. Evaluate the changes in transcriptome profile among grape genotypes exhibiting distinct fruit-set programs. Total RNA was extracted from early fruit set stage of Thompson Seedless cultivar and Thompson Seeded mutant vine. Transcriptome profiling was assessed using RNA-seq strategy. The accumulation level of differentially expressed genes (DEGs) was assessed and confirmed using quantitative real-time PCR assay. Subcellular localization of identified key component genes. The main transcription factor associated with stenospermocarpy fruit set, which is annotated as AGL11 MADS gene was isolated from Thompson Seedless and Thompson Seeded mutant. Sequence analysis confirmed the occurrence of SNP mutations. The AGL11 genes from Thompson Seedless and Thompson Seeded were cloned in pGreen GFP vector. Further, the AGL11 from Thompson Seeded was subjected to site-directed mutagenesis to mimic seedless version. Subcellular localization assay indicated that AGL11 is always detected within the nucleus compartment. No changes in the localization of the protein due the mutation was visualized. Identify potential proteins that are able to interact with TFs strongly associated with the stenospermocarpy fruit-set program. A yeast library was generated using RNA isolated from early fruit-set stage. AGL11 transcription factor was used as a bait to screen the library. The Y2H assay suggested that AGL11 is able to interact with 10 proteins, including 6 MADS box genes and 4 genes encoding a tyrosine-protein phosphatase, a synaptotagmin, a UBP1-associated protein, and WAT1-related protein. The one to one assay confirmed the interaction quality of 9 out of 10 proteins. Further, we used DAP-seq strategy to identify potential downstream genes regulated by AGL11 TF and how the mutated AGL11 version associated with stenospermocarpy seedlessness can change the interaction pattern. The experiment finished and we are in progress to confirm the DNA-protein interaction using Y1H assay. Evaluate the function of TFs associated with the stenospermocarpy fruit-set program using CRISPR/Cas9-gRNA ribonucleoproteins strategy. The gene delivery system in grapes is the somatic embryos. Despite the existence of established protocol to generate somatic embryos from bunch grape genotypes, this protocol was not efficient for muscadine grapes. Accordingly, we have generated our own protocol to generate somatic embryos from muscadine grapes. Several CRISPR-Cas 9 constructs associated with AGL11 disruption either within N-terminal or C-terminal regions of the gene were generated and tested for efficacy. A total of 36 transformed muscadine events exhibiting nucleotides insertion/deletion were identified, confirmed by sequencing, and they are growing under growth chamber conditions for the evaluation trials. Develop training modules in grape genomics and biotechnology. The "Summer Biotech Academy" training program has been running since the summer of 2021. It was generated as a STEM-designed program in plant and biological sciences accessible for high school students and FAMU undergraduate students. During the life of the project, we have trained thirty-eight students (15 high school students and 23 FAMU undergraduate students). The developed training modules are as follows: Molecular Biology Summer Training - High School Students, Biochemistry Summer Training - FAMU Undergraduate Students, and Biotechnology Summer Training - FAMU Undergraduate Students.
Publications
|
Progress 03/01/22 to 02/28/23
Outputs
Target Audience:The knowledge gained through this project was shared with extension agents, the Florida grape grower's association, muscadine products companies, grape breeders, and geneticists who are interested in enhancing the national grape industry and commercialization. Further, we have generated a training program that targets students in the local high schools and FAMU students to provide training in the areas of viticulture research (i.e. physiology, biology, genomics, computational biology, and others). The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identifying key transcription factor genes critical for the stenospermocarpy fruit-set program will not only cause a revolution in the muscadine grape industry but also in other diverse horticultural crops producing fleshy fruits. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the first year grant: - The 20th Biannual Research Symposium - Association of 1890 Research Directors (ARD), Atlanta, GA, April 2 - 5, 2022 (two presentations). - The 2022 Florida State Horticultural Society (FSHS) Annual Conference, Sarasota, FL, June 5 - 7, 2022. - Florida Wine and Grape Growers Association (FWGGA) 2022 Annual Conference, Deland, FL, June 17 - 18, 2022. - Legislature visitto the Center for Viticulture, CAFS, FAMU that includes city/state representatives, industry representatives, and policymakers, Tallahassee, FL, February 8, 2023. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We constantly strive to enhance the learning experience of our students in agreement with FAMU's motto of "Excellence with Caring" by improving the learning procedure and qualities. In 2021, we officially started a "Summer Biotech Academy" that targets a diverse pool of minority high school students and undergraduate FAMU students. The main goal of the program is to inspire students' awareness by exposing them to real-life examples and objectives, which develop communication, leadership, and management skills, encourage students to pursue advanced graduate degrees, expand students' intellectual knowledge and lab experiences and prepare them to better cope for a future workforce to empower and sustain the industry. Thus far, we have trained twenty-six students (10 high school students and 16 FAMU undergraduate students). How have the results been disseminated to communities of interest?The objectives and research outcomes achieved have been already discussed in several meetings during the fourth year of the grant: The knowledge gained through this project was shared with extension agents, the Florida grape grower's association (FGGA), muscadine products companies, grape breeders, and geneticists who are interested in enhancing the national grape industry and commercialization. Further, we have generated a training program that targets students and teachers in the local high schools and FAMU scientists and students to provide training in the areas of viticulture research (i.e., biotechnology, biology, computational biology, and others). The current trend in food consumption demands liability in developing innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the last year: - The 20th Biannual Research Symposium - Association of 1890 Research Directors (ARD), Atlanta, GA, April 2 - 5, 2022 (two presentations). - The 2022 Florida State Horticultural Society (FSHS) Annual Conference, Sarasota, FL, June 5 - 7, 2022. - Florida Wine and Grape Growers Association (FWGGA) 2022 Annual Conference, Deland, FL, June 17 - 18, 2022. - Legislature visiting even to the Center for Viticulture, CAFS, FAMU that includes city, state representatives and agencies, industry representatives, and policymakers, Tallahassee, FL, February 8, 2023. What do you plan to do during the next reporting period to accomplish the goals?We almost finished the major project objectives. However, the ultimate critical objective of the project is still in progress, which is Objective 6: Evaluate the function of MrAGL11 associated with stenospermocarpy fruit-set, using CRISPR/Cas9-gRNA ribonucleoproteins strategy. During performing related work, several other publications will be generated.
Impacts
What was accomplished under these goals?
For the research objectives: 1. Characterization of fruit-set programs among diverse grape genotypes; the objective has been performed among different grape species as planned in the proposal. 2. Evaluate the changes in hormones accumulation profile throughout fruit ontogeny; the objective has been performed via quantifying the hormones of auxin (IAA), cytokinins (CKs), and abscisic acid (ABA). 3- Evaluate the changes in transcriptome profile among grape genotypes exhibiting distinct fruit-set programs. The objective has been performed as detailed in the reporting period. 4- Subcellular localization of identified key component genes. The objective has been performed as detailed in the reporting period. 5- Identify potential proteins that are able to interact with TFs strongly associated with the stenospermocarpy fruit-set program; the objective has been performed as detailed in the reporting period. 6- Evaluate the function of TFs associated with the stenospermocarpy fruit-set program using CRISPR/Cas9-gRNA ribonucleoproteins strategy. The objective is in progress. The muscadine embryos have been transformed with several CRISPR/Cas9 constructs, and we are waiting for the evaluation trials. 7- Develop training modules in grape genomics and biotechnology; the objective has been performed, and the "Summer Biotech Academy" training program has been running since the summer of 2021.
Publications
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:The knowledge gained through this project were shared with extension agents, Florida grape grower's association, muscadine products companies, grape breeders, and geneticists who are interested in enhancing national grape industry and commercialization. Further, we have generated a training program that targets students and teachers in the local high schools, and FAMU students to provide training in the areas of viticulture research (i.e. physiology, biology, genomics, computational biology, and others). Current trend in food consumption demands liability in the development of innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identification of key transcription factor genes critical for stenospermocarpy fruit-set program will not only cause a revolution in muscadine grape industry, but also in other diverse horticultural crops producing fleshy fruits. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the first year grant: - Florida Wine and Grape Growers Association (FWGGA) 2021 Annual Conference, Deland, FL. - The UF 2021 Virtual Grape Field Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The Virtual Conference of the North Carolina Grape Growers Association. - The Virtual Annual Meeting of the Florida State Horticultural Society. Changes/Problems:No problems during the reporting period. What opportunities for training and professional development has the project provided?We constantly strive to enhance the learning experience of our students in agreement with the FAMU's motto of "Excellence with Caring" by improving the learning procedure and qualities. Therefore, I have participated in multiple teaching enhancement programs at FAMU and became a Certified Online Instructor. In 2021, we have officially started a "Summer Biotech Academy" that targets a diverse pool of minority high school students and undergraduate FAMU students. The main goal of the program is to inspire students' awareness by exposing them to real-life examples and objectives, which develop communication, leadership, and management skills, encourage students to pursue advanced graduate degrees, expand students' intellectual knowledge and lab experiences, and prepare them to better cope for a future workforce to empower and sustain the industry. Thus far, we have trained eleven students (5 high school students and 6 FAMU undergraduate students). How have the results been disseminated to communities of interest?The objectives and research outcomes achieved, thus far, have been already discussed in several meetings with growers, grape products companies, and peer grape breeders during the second year of grant: The knowledge gained through this project was shared with extension agents, Florida grape grower's association, muscadine products companies, grape breeders, biochemists, and geneticists who are interested in enhancing national grape industry and commercialization. Further, we have generated a training program that targets students and teachers in the local high schools, and FAMU scientists and students to provide training in the areas of viticulture research (i.e. biotechnology, biology, computational biology, and others). Current trend in food consumption demands liability in the development of innovative foods with superior qualities to increase consumer acceptance and market value for sustaining production. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the first year grant: - Florida Wine and Grape Growers Association (FWGGA) 2021 Annual Conference, Deland, FL. - The UF 2021 Virtual Grape Field Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The Virtual Conference of the North Carolina Grape Growers Association. - The Virtual Annual Meeting of the Florida State Horticultural Society. What do you plan to do during the next reporting period to accomplish the goals?We almost finished the major project objectivs. However, two critical objectives are still in progress, which are Objective 5: Identify potential proteins that are able to interact with MrAGL11 strongly associated with stenospermocarpy fruit-set and Objective 6: Evaluate the function of MrAGL11 associated with stenospermocarpy fruit-set, using CRISPR/Cas9-gRNA ribonucleoproteins strategy. Both objectives are in progress. During performing related work, several other publications will be generated.
Impacts
What was accomplished under these goals?
For the research objectives: 1. Characterization of fruit-set programs among diverse grape genotypes; the objective has been performed among different grape species as planned in the proposal. 2. Evaluate the changes in hormones accumulation profile throughout fruit ontogeny; the objective has been performed via quantifying the hormones of auxin (IAA) and abscisic acid (ABA). 3- Evaluate the changes in transcriptome profile among grape genotypes exhibiting distinct fruit-set programs; the objective has been performed as detailed in the report period. 4- Subcellular localization of identified key component genes. ; the objective has been performed. 5- Identify potential proteins that are able to interact with TFs strongly associated with stenospermocarpy fruit-set; the objective is in progress. Large quantities of RNA are required to generate the libraries. 6- Evaluate the function of TFs associated with stenospermocarpy fruit-set, using CRISPR/Cas9-gRNA ribonucleoproteins strategy, the objective is in progress. The muscadine embryos has been transformed with several CRISPR/Cas9 constructs and we are in the selection stage. 7- Develop training modules in grape genomics and biotechnology; the objective has been performed and the "Summer Biotech Academy" training program is runing since summer 2021.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Campbell J, Sarkhosh A, Habibi F, Gajjar P, Ismail A, Tsolova V, El-Sharkawy I. (2021) Biometrics assessment of cluster- and berry-related traits of muscadine grape population. Plants 10: 1067.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Campbell J, Gajjar P, Ismail A, Habibi F, Darwish AG, Tsolova V, Sarkhosh A, El-Sharkawy I. (2021) Determination of fertility-related traits in muscadine grape population. Plants 10: 1175.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Park M, Sarkhosh A, Tsolova V, El-Sharkawy I. (2021) Diversification of LTR-retrotransposon repertoire by horizontal transfer in Vitis genomes. International Journal of Molecular Sciences, 22: 10446.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Park M, Vera D, Kambiranda D, Gajjar P, Cadle-Davidson L, Tsolova V, El-Sharkawy I. (2022) Chromosome-level genome sequence assembly and genome-wide association study of Muscadinia rotundifolia reveal the genetics of 12 berry-related traits. Horticulture Research 9: uhab011.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Ismail A, Gajjar P, Darwish A, Tsolova V, El-Sharkawy I. (2022) Genome-wide characterization of the MADS-box gene family in muscadine and assessment of its roles in flowering and fruit development. International Journal of Molecular Sciences.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Moniruzzaman M, Darwish A, Tsolova V, El-Sharkawy I. (2022) Stenospermocarpy: A potential trait to exploit in horticultural crops. Frontiers in Plant Science.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Jiovan Campbell. (2021) Characterization of large muscadine (Muscadinia rotundifolia Michx.) population for diverse phenological and reproductive performance traits.
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:The knowledge gained through this project will be shared with extension agents, Florida grape grower's association, muscadine products companies, grape breeders, biochemists, and geneticists who are interested in enhancing national grape industry and commercialization. Other target audiences will be city, county, state representatives and agencies, policymakers, as well as the general public. Further, we have generated a training program that targets students and teachers in the local high schools, and FAMU students to provide training in the areas of viticulture research (i.e. physiology, biochemistry, biology, genomics, computational biology, and others). Current trend in food consumption demands liability in the development of innovative foods with superior nutritional qualities to increase consumer acceptance and market value for sustaining production. Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identification of key transcription factor genes critical for stenospermocarpy fruit-set program will not only cause a revolution in muscadine grape industry, but also in other diverse horticultural crops producing fleshy fruits. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the first year grant: - Florida Wine and Grape Growers Association (FWGGA) 2020 Annual Conference, Deland, FL. - The University of Florida Muscadine Grape Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The Virtual Conference of the North Carolina Grape Growers Association. Changes/Problems:As it is already expected, there are several problem occurred during this period due to the spread of COVID-19 pandemic. This considerably affected the progression of our work at different levels. These problems have been started by early February 2020. Team members: During this year, we have lost the efforts of several team members either due to the COVID-19 infection or due closing of research site. All related research sites at Florida, California, and New York were affected. Work duties: Within this year, we planned to achieve objectives number 2 related to "Evaluate the changes in hormones accumulation profile throughout fruit ontogeny" and number 5 "Identify potential proteins that are able to interact with TFs strongly associated with stenospermocarpy fruit-set". These two objectives require the collection of flower/berry materials at different developmental stages to perform hormones extraction as well as large quantities of RNA extraction necessary for generating yeast libraries. In addition, the process for generating somatic embryos from muscadine grape was also affected. Despite all these delay, we hope to perform them with the third year of grant. In addition, we consumed significant amount of our time for the task related to meeting with growers and consumers as well as generating the training program for the "Summer Biotech Academy". We were obligated to change our strategies of interaction with the audience from face to face into virtual. This transition is still not in perfect shape; there are troubleshooting that we face. To overcome the problem related to interaction with high school students and teachers, we decided to generate descriptive videos about the project objective and several training modules that will be available on applicable websites at the FAMU. This will be available by fall 2021. What opportunities for training and professional development has the project provided?One MS-graduate students, a postdoctoral fellow, and two professionals were trained in samples collection and preparation, samples processing for DNA and RNA extraction, transcriptome analysis, molecular cloning and confocal microscopy imaging, and molecular analysis. They are very well trained in bioinformatic, histology, transcriptome, and genomic tools for sequence comparison among grape genotypes. How have the results been disseminated to communities of interest?The objectives and technologies used in this proposal have been already discussed in several meetings with growers, grape products companies, and peer grape breeders during the first year of grant: - Florida Wine and Grape Growers Association (FWGGA) 2020 Annual Conference, Deland, FL. - The University of Florida Muscadine Grape Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The Virtual Conference of the North Carolina Grape Growers Association. What do you plan to do during the next reporting period to accomplish the goals?During the next funding year, we will achieve research target 2 via evaluating the changes in hormones accumulation profile throughout fruit ontogenyand and 5 through identify potential proteins that are able to interact with TFs strongly associated with stenospermocarpy fruit-set.
Impacts
What was accomplished under these goals?
In relation to objective 4, during the first year grant and through the analysis of the Vitis vinifera grape cultivar Thompson Seedless and its sport Thompson Seeded, as well as other different seedless grape hybrids, we were able to identify VvAGL11 that belong to class D of MADS-box transcription factor gene family. Analysis of VvAGL11 mRNA sequence from different Vitis species allowed us to identify three critical nucleotide substitutions associated with stenospermocarpy seedless fruit-set, causing predicted changes in the amino acid residues Leu29 to Pro, Lys31 to Asn, or Arg197 to Leu, designated as VvAGL11-M1, -M2, and -M3, respectively. To gain a broader insight into VvAGL11 function, we investigated the localization compartment of VvAGL11-s isolated from seeded berries. In addition, the three mutated versions of VvAGL11 isolated from seedless berries, VvAGL11-M1, -M2, and -M3 associated with amino acids substitutions Leu29 to Pro, Lys31 to Asn, or Arg197 to Leu were independently generated using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA). The full-length coding sequences of VvAGL11-s and its three mutated versions were fused in frame with the GFP into the pGreenII vector using the BamHI site and expressed under the control of the 35S promoter. For protoplasts assay, the different constructs were transfected into protoplasts from suspension cultured tobacco BY-2 cells and analyzed for GFP fluorescence using confocal microscopy. All assays were repeated three times. Fluorescence microscopy revealed that the VvAGL11-s−GFP fusion was localized exclusively in the nucleus, which is consistent with its primary function as transcription regulator. Assessing the localization of the three mutated versions revealed that their proteins remained targeting the nucleus compartment, indicating that the generated mutations did not affect their potential function as transcription regulators. In relation to objective 6, we are in the process of producing somatic embryos from muscadine grape in order to generate seedless stenospermocarpy muscadines. In relation to objective 7, we have developed 4 modules that target high school students and undergraduate FAMU students. Due to COVID-19 restrictions, we currently try to convert these modules into online modules that will be uploaded into applicable FAMU website. The developed modules are as follow: 1- Principles of Molecular Biology tools. 2- Introduction to Plant Biochemistry. 3- Plant Genetics. 4- Plant Biotechnology, Principles and Methods.
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:The knowledge gained through this project will be shared with extension agents, Florida grape grower's association, grape breeders and geneticists interested to enhance national grape industry and commercialization. Other target audiences will be city, county, state representatives and agencies, policymakers, as well as the general public. Further, we have generated a training program that targets students and teachers in the local high schools, and FAMU students to provide training in the areas of viticulture research (i.e. physiology, biology, genomics, computational biology, and others). Stenospermocarpy fruit-set program could be only identified in particular Vitis vinifera grapes, producing relatively large berry size with traces of seeds. Identification of key transcription factor genes critical for stenospermocarpy fruit-set program will not only cause a revolution in muscadine grape industry, but also in other diverse horticultural crops producing fleshy fruits. The objectives and technologies used in this proposal have been already discussed in several meetings with growers and grape breeders during the first year grant: - Florida Wine and Grape Growers Association (FWGGA) 2020 Annual Conference, Deland, FL. - The 2019 North American Grape Breeders Conference, College of Agriculture, Mountain Grove, MO. - The University of Florida Muscadine Grape Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The 19th Biannual Research Symposium - Association of 1890 Research Directors (ARD), Jacksonville, FL. Changes/Problems:There are some minor changes occurred after receiving the grant; however, these changes does not considerably affect the project performance. This can be resumed as follow: 1- In relation to team members, two of our team member: a) Dr. Ashraf El-Kereamy has accepted a new position as Cooperative Extension Specialist and, accordingly, his sub-award has been changed to the University of California Riverside, Department of Botany and Plant Sciences in place of the University of California, ANR/Cooperative Extension Kern County. b) Dr. Sadanand A. Dhekney has accepted a new position as Associate Professor and, accordingly, his sub-award has been changed to the University of Maryland Eastern Shore, Department of Agriculture, Food and Resource Sciences in place of University of Wyoming, Sheridan Research and Extension Center. 2- In relation to transcriptome profiling, we have added a new RNA-seq technology that does not exist at commercial level during writing the grant. This technology, designated as PacBio Iso-Seq Sequel II system, helped us to accelerate and facilitate the identification of potential genes that can distinguish between the seeded and stenospermocarpy seedless fruit-set programs. The technology allowed us to detect differentially expressed genes and/or identify any potential SNPs or sequence variations within the codon region due to sequencing of large mRNA fragments with deep sequencing accuracy. However due to the high technology price ($5,200/sample), we were not able to use this with different grape genotypes. We have used only the most stable and accurate plant samples, including Thompson Seedless and its sport Thompson seeded. Other cheaper PCR and sequencing technologies have been used to determine the mutant frequency or mutant discovery in other grapevine genotypes. What opportunities for training and professional development has the project provided?Four undergraduate students, one MS-graduate student and a postdoctoral fellow were trained in samples collection and preparation, samples processing for histological, hormonal and molecular analysis. They are very well trained in bioinformatic and genomic tools for sequence comparison among grape genotypes. How have the results been disseminated to communities of interest?The objectives and technologies used in this proposal have been already discussed in several meetings with growers and peer grape breeders during the first year of grant: - Florida Wine and Grape Growers Association (FWGGA) 2020 Annual Conference, Deland, FL. - The 2019 North American Grape Breeders Conference, College of Agriculture, Mountain Grove, MO. - The University of Florida Muscadine Grape Day, UF/IFAS Plant Science Research and Education Unit, Citra, FL. - The 19th Biannual Research Symposium - Association of 1890 Research Directors (ARD), Jacksonville, FL. What do you plan to do during the next reporting period to accomplish the goals?The population resulted from crossing several stenospermocarpy seedless grapes (female parents) and seeded grapes (male parents) resulted in offsprings exhibiting several degrees of stenospermocarpy seedlessness. This suggested that the stenospermocarpy seedless trait is a quantitative character potentially coordinated by several genes. Accordingly, we are interested in characterizing other genes involved in the seed development pathway. In Arabidopsis, the class D of MADS-box transcription factor gene family consists of three genes, AtAGL11, AtSHP1 and AtSHP2. The three genes direct targets are VDD (VERDANDI), BAN (BANYULS / ANTHOCYANIDIN REDUCTASE), ABS (ARABIDOPSIS B SISTER), and EGL3 (ENHANCER OF GLABRA3) genes. We were able to isolate full-length genomic sequences of all these genes from seeded Vitis vinifera and Muscadinia rotundifolia grapes. Currently, this set of genes are under evaluation via performing: 1- Isolation of full-length genomic sequences from grape genotypes exhibiting stenospermocarpy and parthenocarpy fruit set program. The target is to detect any potential sequence variation associated with seedless fruit set program. Another important goal is to determine whether there is an overlap between the two seedless fruit set strategies (stenospermocarpy and parthenocarpy) or the stenospermocarpy occurred in an independent molecular pathway. 2- All these genes are under yeast two-hybrid assay in order to determine their protein-protein interaction behavior and potential mechanism of function. Further, we currently build several CRISPR/Cas9-gRNA constructs using MrAGL11 sequence. Our target is to change the highly conserved amino acid residues Leu29, Lys31, and Arg197 in MrAGL11 into Pro, Asn and Leu. Another construct will be built with a target to knockout MrAGL11 gene in muscadine grapes. We plan to start muscadine transformation in order to induce stenospermocarpy seedlessness by the spring of 2020. In terms of activities related to the development of "Summer Biotech Academy" to train local high school students and first year CAFS/FAMU students, we have communicated with local high schools within Leon County, including Leon High School, Lincoln High School and Lawton Chiles High School in order to select around 20 potential trainees/school. We also have selected 20 trainees from first year CAFS students to be included in the program. Further, we have developed three different training modules in grape genomics, biotechnology and biochemistry. We plan to start the activities of first year "Summer Biotech Academy" by first week of June 2020.
Impacts
What was accomplished under these goals?
To gain insight into the molecular mechanism underlying stenospermocarpy fruit-set program in Vitis vinifera grapes, berry fruit samples at different developmental stages were collected from several grape genotypes with different fruit set program, belonging to Vitis vinifera, Vitis hybrids and Muscadinia rotundifolia. These samples were subjected for RNA extraction and histological analysis, particularly during fruit-set timing. Thompson Seedless grape is a well-known commercial cultivar, displaying a stenospermocarpy fruit-set program. We were able to identify a sport (somatic mutant) from Thompson Seedless, designated as Thompson Seeded that exhibits seeded fruit-set program. Molecular characterization of Thompson Seedless grape and its Thompson Seeded mutant along with using a proper RNA-seq technology, designated as PacBio Iso-Seq Sequel II system allowed us to identify potential genes that can distinguish between the two different fruit-set programs. By using such technology, we were not only able to detect differentially expressed genes, but it also allowed us to identify any potential SNPs or sequence variations within the codon region due to sequencing of large mRNA fragments with high accuracy. Accordingly, we were able to identify a gene, nominated as VvAGL11, belonging to class D of MADS-box transcription factor gene family. In Arabidopsis, it has been proposed that this gene plays major role in seed morphogenesis and embryo development. At transcription level, VvAGL11 relative expression was barley detected throughout different developmental stages of the stenospermocarpy seedless grapes, whereas its transcript levels were significantly higher (~70-fold) in the seeds of seeded grapes at 2, 4, and 6 weeks after fruit set. Analysis of VvAGL11 mRNA nucleotide sequence in Thompson Seedless grape and its sport highlighted seven nucleotide substitutions, causing predicted substitutions in the amino acid residues Arg197 to Leu and Thr210 to Ala. Analysis of other Vitis hybrid grapes, exhibiting different degrees ofstenospermocarpy seedlessness allowed us to identify two other predicted substitutions in the amino acid residues Leu29 to Pro or Lys31 to Asn. AGL11 sequence comparison with its ortholog from different plant species suggested the critical substitution of the highly conserved amino acid residues Leu29, Lys31 and Arg197. In an attempt to unravel the structural organization of the VvAGL11 gene in Thompson Seedless and the seeded mutant grapevines, allelic variations were amplified by PCR with specific primers using genomic DNA extracted from the two genotypes. After allele specific isolation and sequencing, we identified two alleles for each genotype. The complete VvAGL11 allelic sequences constituted by their promoter, coding, and intragenic regions (eight exons and eight introns) were sequenced. Sequence analysis allowed us to identify two alleles for each cultivar. The two VvAGL11 alleles from Thompson Seeded showed 99% sequence identity to each other. In Thompson Seedless, one allele exhibited 99% identity with the two alleles from seeded sport; however, the second VvAGL11 allele presented a different group of polymorphisms when compared with all the other alleles. This allele was named as mVvAGL11 (mutated VvAGL11) and contains a group of exclusive polymorphisms composed of 28 INDELs (insertions/deletions) and 105 SNPs. Alignment of the four VvAGL11 alleles highlighted the SNPs detected in corresponding mRNA sequence (Arg197 to Leu and Thr210 to Ala). We also searched the sequences for MADS-box protein complex-binding sites (CArG-box sequences). We were able to identify 10 putative CArG-box sequences within the VvAGL11 gene structure, one located in the promoter region, eight in the second intron, and one in the 3'-UTR. In mVvAGL11 allele, a nucleotide modification within the seventh CArG-box resulted in the detection of only 9 putative CArG-box sequences. Given the nature of these substitutions, we predicted that the alleles holding Leu29, Lys31 and Arg197 residues would be functional and the allelic forms with the substitution Pro29, Asn31 or Leu197 would be inactive, or at best case scenario function differentially. Finally, we have isolated full-length AGL11 ortholog from Muscadinia rotundifolia seeded cultivar Nobel grape, designated as MrAGL11. Alignment of MrAGL11 nucleotide and amino acid sequences with different VvAGL11 from seeded grape sport indicated 98% and 99% sequence identity, respectively. Ten nucleotide substitutions were detected in MrAGL11, causing alteration in the amino acid residue Gly79 to Ala. However, all the amino acid residues Leu29, Lys31 and Arg197 associated with seeded fruit set program were conserved in muscadine sequence.
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