Progress 04/01/24 to 03/31/25
Outputs
Target Audience:The focus of this research is to develop seedless muscadine grapes for table use. Posters were presented at the conference, and presentations weredelivered at the departmental and international conference levels. Research articles were published in the peer-reviewed journals. Students, faculty, and scientists received training in biotechnological techniques. Changes/Problems:-Amplifying theAGL11genefor gene silencing in tomato plants from muscadine grapes proved to be a formidable challenge.Initial attempts to generate the amplicon were unsuccessful, necessitating multiple rounds of optimization and strategic primer redesign. -Compounding these difficulties, unexpected cold fronts in Tallahassee disrupted the experiment's timeline. The abrupt temperature shifts delayed full fruit set and significantly slowed fruit development, introducing an additional layer of complexity to an already intricate study. What opportunities for training and professional development has the project provided? Provided hands-on training to undergraduate and graduate students in molecular biology techniques, including agarose gel electrophoresis and bioinformatics analysis. Trained post-doctoral researchers and students in data interpretation, data analysis, report writing, and presentation skills for scientific conferences. How have the results been disseminated to communities of interest?The results of the project were presented through oral and poster presentations at departmental and international conferences, as well as publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?-Implement CRISPR-based editing in muscadine grapes using Agrobacterium-mediated transformation. -Validate gene editing results through sequencing of tissue culture-generated plants. -Continue hands-on training of students and faculty in CRISPR-Cas9 technology, enhancing their skills and career opportunities in Food, Agriculture, Natural Resources, and Human (FANH) sciences
Impacts
What was accomplished under these goals?
Identified the AGL11 gene responsible for stenospermocarpic seedlessness in muscadine grapes. Established a correlation between VroAGL11 expression levels and seed content (by weight) in muscadine and hybrid bunch/bunch grape cultivars. Characterized the VroAGL11 gene and its transcripts in both seeded and seedless muscadine grapes, providing insights into the genetic mechanisms of seedlessness. Further, to validate the role of VroAGL11 in seedlessness, we employed a heterologous tomato model system to validate the transformation process- Wedesigned primers to amplify the AGL11 gene from muscadine grape; however, initial attempts to produce the amplicon failed. After multiple optimizations and redesigns, we have successfully amplified the target gene and cloned it into the pFGC5941 expression vector for gene silencing.The recombinant constructs were verified through restriction digestion and PCR amplification.Transformation techniques were also optimized, and an Agrobacterium-mediated floral-dip transformation method was used employing Micro-Tom tomatoes as a model system.The transformed tomato plants (silenced)grew successfully and began fruiting alongside untreated controls.The silenced plants exhibited a range of phenotypes, from normal floral development to a variety of floral alterations in different degrees: thicker sepals and petals, laterally fused sepals, larger pistils, protruding stigma, retracted staminal cones, and larger but normal flowers, could result in a reduced seed set or abnormal seeds.Notably, the expression analysis of SlyAGL11atpost-anthesis (DPA) based on real-time PCR was performed in the transformedplant and control tomato plants. This expression decreased during anthesis in the transformed plant compared to a control plant. Students and researchers were trained in gene editing techniques. A hands-on workshop was organized to provide training to undergrad/graduate students on molecular biology and bioinformatic analysis.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Messeha, S.S., Agarwal, M., Gendy, S.G., Mehboob, S.B. and Soliman, K.F., 2024. The Anti-Obesogenic Effects of Muscadine Grapes through Ciliary Neurotrophic Factor Receptor (Cntfr) and Histamine Receptor H1 (Hrh1) Genes in 3T3-L1 Differentiated Mouse Cells. Nutrients, 16(12).
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Agarwal, M. and Sheikh, M.B., 2025. Isolation and Functional Characterization of Endophytic Bacteria from Muscadine Grape Berries: A Microbial Treasure Trove. Cells, 14(5), p.369.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Sheikh MB, Rahman MA, Ahmed IM, Agarwal M, Tushar Dhanani T. 2025 Transcriptome Profiling Reveal Molecular Evidence of Muscadine Grape cv. Noble During Berry Development.Plant and Animal Genome Conference, San Diego, CA, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
M. Agarwal*, A. Kaplan, and M. B. Sheikh. Muscadine Grape Bioactives: A Natural Source of Antimicrobial Power. ARD Research Symposium, 2024, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
M. Agarwal* and M. B. Sheikh. Muscadine Grape and Gut Health: Prebiotics and Probiotics in Harmony. ARD Research Symposium, 2024, USA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
T. Dhanani*, M. Agarwal, I. M. Ahmed and M. B. Sheikh. Unlocking the Nutraceutical Potential of Muscadine Grapes: Dynamic Evolution of Metabolites During Berry Development. ARD Research Symposium, 2024, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
T. Dhanani*, M. B. Sheikh, A. Kaplan, I. Ahmed and M. Agarwal. An Innovative Technique to Maximize the Recovery of Stilbenes for Enhancing Nutraceutical Properties of Muscadine Grape Extract. ARD Research Symposium, 2024, USA.
|
Progress 04/01/23 to 03/31/24
Outputs
Target Audience:The focus of this research is to develop knowledge, and technology and build capacity in cutting-edge gene and protein technologies for developing seedless muscadine grapes for table use and addressing critical issues impacting crop value, disease tolerance, nutritional and nutraceutical value to enhance market value, consumer acceptance, and industry income. These research activities are also helping identify the molecular and cellular mechanisms involved and identify the genes and gene products controlling seed development which help develop new seedless muscadine grape genotypes. Given the potential benefits of seedless muscadine genotypes the interested groups and audience for this research will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the outcome through increased consumption and sale of table use muscadine grape, increased market value and producer income, and establishment of new vineyards targeting the fresh fruit market. The project-related research activities also help build and strengthen functional genomics, omics technologies, and nutrigenomics research capability at FAMU for enhancing or altering the chemical constituents' content and characteristics of fruit and vegetable crops to improve their nutritional and health values for meeting the consumer/ stakeholder/clientele needs and demands as well as providing training venues for current and future students in cutting-edge technologies. The project has also invocated faculty innovation, productivity, and outreach activities and has naturally garnered their interest. In addition, the project has also instilled interest in scientists interested in learning about latest molecular and cellular technologies, and their application to modify the agronomic nutritional and nutraceutical traits of crops to promote healthy living. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. Hands-on training toundergraduate students in sample collection, sampleprocessing, DNA isolation, and RNA isolation. 2. The graduate student was provided training in experimental design, RNA extraction, purification, quantification, and related molecular techniques. 3. Two post-doctoral research associates were trained in phytochemical extraction from muscadine berries, method optimization for maximum phytochemical recovery from berry samples, RNA extraction, cDNA synthesis, Primer design, PCR technique, and bioinformatics, data analysis, scientific proposal writing, project management, report writing, and manuscript preparation. 4. Hands-on and virtual training was provided to international faculty on molecular and biochemical techniques. How have the results been disseminated to communities of interest?The objectives and results of the project were presented in a variety of formats, including verbal presentations at the departmental level, poster presentations at conferences, oral presentations at conferences, publication of a bulletin, and distribution at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Determine the expression pattern ofSlyAGL11, SlyVPE1,and SlyVPE2in tomato plants during flower and fruit development. 2 Validate the role of the muscadineVroAGL11gene for stenospermocarpic seedlessness in tomato plants through a complementation test. 3. Establishment of CRISPR-Cas9 gene editing system 4. Provide hands-on experiential learning to students and faculty on the CRISPR/Cas9 system to develop their expertise in this cutting-edge gene editing technology for enhancing career opportunities in FANH sciences.
Impacts
What was accomplished under these goals?
In our ongoing investigationtoobserve the transcriptional changes between seedless and seeded muscadine cultivars, high-throughput RNA sequencing was performed to identify genome-wide transcriptomic changes in 5 berry developmental stages of the commercially important muscadine wine grape, seeded cv "Noble" using Illumina NextSeq 550 paired-end sequencing technology according to the transcriptome analysis workflow. We obtained approximately 97.6 million clean and high-quality reads of 72-73 bp long after removing low-quality reads. Each berry developmental stage was represented by at least 17 million reads which is sufficient for quantitative analysis of gene expression. Then the clean reads were aligned with the Vitis vinifera genome as the reference genome using STAR alignment. The majority (93.9%) of clean reads were mapped to the Vitis vinifera genome, of which 89.7% were mapped to the exons (coding sequence, 76.4%, and 5'UTR+ 3'UTR, 13.3%) and 3.5-4.9% mapped within introns. We identified 2,606 (Pea vs Inf), 2,539 (PreV vs Inf), 3251 (Ver vs Inf), and 3,779 (Ripe vs V) totaling 12,175 differentially expressed genes (DEGs) between different berry development stages using DESeq2. GO and KEGG pathway enrichment analyses were conducted to annotate the functions of DEGs. All the DEGs in different stages of berry development were enriched and 270, 299, and 249 terms were annotated to three functional categories, viz., biological process (BP), cellular component (CC), and molecular function (MF) respectively. In the KEGG pathway enrichment analysis, these DEGs were enriched in 34 pathways, the most important pathway being cell wall development, lipid metabolism, starch-sugar metabolism, and the polyphenolic biosynthetic pathway. The expression profiles of the DEGs were determined by soft clustering analysis using the R package, Mfuzz. Genes were divided into eight clusters based on their expression modulation, representing the number of profiles indicated by the figure. Genes expressed in the early and late stages of berry development fell into clusters 1 and 2. Clusters 3 contained genes negatively modulated along the whole time course, clusters 4 and 6 contained genes modulated only during green and hard berry (PreV) interval, and clusters 5 and 8 contained genes positively and negatively modulated after veraison. Genes specifically induced at Pea, PreV, and Ver stages were grouped in clusters 7, 6, and 8 respectively. Our present study showed significant differences between seedless and seeded varieties and are more genes responsible for seed formation. Noble showed a higher expression of AGL11. Based on the current results, we are validating the role of VroAGL11 in seedlessness using a heterologous model system in Tomato. To validate tomato as an appropriate experimental model for comparisons of fruit and seed development with grapevine, the SlyAGL11 expression pattern during flower and fruit development is determined. For the target genes SlyAGL11, SlyVPE1,and SlyVPE2, the designed primers amplified fragments in the range of 100-200 bps. The primer is SlyAGL11-F, SlyAGL11-R, SlyVPE1-F, SlyVPE1-R, SlyVPE2-F and SlyVPE2-R. Furthermore, the genes involved in seed coat development, SlyVPE1 and SlyVPE2 in tomato are demonstrated as well in silenced tomato lines. During the early stages of seed development, VPE is involved in the death of limited cell layers to form the seed coat. This result reflects the hypothesis that VroVPE expression depends on VroAGL11 expression and that the partial dominance behavior of the VroAGL11 seedless allele is reflected in downstream genes. Taken together, these results demonstrate that seed coat-forming genes share spatial expression patterns with the D-class MADS-box genes VroAGL11 suggesting that VroAGL11 and SlyAGL11 participate in the positive regulation of seed coat-forming genes.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Characterization of Parthenocarpy-Related Gene/s Towards Development of Seedless Muscadine Grape Genotype.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Genome-wide Transcript Analysis of Muscadine Grape cv. Noble During Berry Development
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Differences in the Molecular and Cellular Components Content and Composition Between Hexose- and Sucrose-Accumulators and its Effect on Berry Sugar Content
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Differences in the Molecular and Cellular Components Between Leathery and Non-leathery Skinned Grape Genotypes
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Genetic Variation in Metabolites Content and Composition and its Effect on Bioactivity of Muscadine Grape Genotypes
|
Progress 04/01/22 to 03/31/23
Outputs
Target Audience:The focus of this research is to develop knowledge, technology and build capacity in cutting edge gene and protein technologies for developing seedless muscadine grape for table use and address critical issues impacting crop value, disease tolerance, nutritional and nutraceutic value to enhance market value, consumer acceptance and industry income. These research activities are also helping identify the molecular and cellular mechanisms involved and identify the genes and gene products controlling seed development which help develop new seedless muscadine grape genotypes. In view of the potential benefits of seedless muscadine genotypes the interested groups and audience for this research will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the outcome through increased consumption and sale of table use muscadine grape, increased market value and producer income and establishment of new vineyards targeting fresh fruit market. The project related research activities also are helping build and strengthen functional genomics, omics technologies and nutrigenomics research capability at FAMU for enhancing or altering chemical constituents' content and characteristics of fruit and vegetable crops to improve their nutritional and health values for meeting the consumer/ stakeholder/clientele needs and demands as well as providing training venue for current and future students in cutting edge technologies. The project has also invocated faculty innovation, productivity, and outreach activities and has naturally garnered their interest. In addition, the project has also instilled interest in scientists interested in learning about latest molecular and cellular technologies, and their application to modify the agronomic and nutritional and nutraceutic traits of crops to promote healthy living. Changes/Problems:The original principal investigator (PI) who was supposed to oversee the study has departed for a more promising academic job elsewhere, necessitating a new PI. It wasbe necessary to revise/initiate the official PI change procedure and make adjustments to the new PI's tasks and responsibilities. The new PI has taken over as leader. Addionally, a post doctoral fellow ( a plant molecular biologist)was recruited and has been trained to perform the tasks. What opportunities for training and professional development has the project provided?1.Hands-on and virtual training of undergraduate students in berry maturity determination,samplecollection and processing, phytochemicals extraction and quantification, RNA isolation andcharacterization techniques, instrumental use and other related biochemical techniques. 2. The graduate studentswere provided training inexperimental design, berry maturity determination, berry classification, sugars, phytochemicals isolation,purification and quantification, RNA extraction and quantification, and other related molecular and cellulartechniques, data collection and processing techniques to enhance their skills in cutting edge gene and protein techniques aswell as data processing and analysis tools for use in their thesis research. 3. Two post-doctoral research associates weretrained in varietal selection, berry developmental stage assessment, phytochemicals extraction, biochemical evaluation,sample processing, RNA extraction and quantification, electrophoretic techniques, Primer design, PCR technique,bioinformatics, other related molecular and cellular techniques,isolation and characterization of bioactivemolecules, instrumental analysis, bioinformatics and bioprocessing. These researchers were also trained in experimentaldesign, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, reportwriting and manuscript preparation. How have the results been disseminated to communities of interest?The primary goal of this research is to identify and characterize the gene/s encoding seedlessness trait in muscadine grape for use in developing non-GMO seedless muscadine grape genotype. Towards this goal the project workplan was developed to determine the seed developmental activity in maturing berry, conduct comparative genomic studies among various grape genotypes to identify the gene encoding seededness and seedlessness traits. These research activities have enabled identification of the gene associated with seededness in muscadine grape for the first time. Once its role in seed development is confirmed and used in the development of seedless muscadine grape using novel gene editing technology it will havetremendous impact on consumers, growers and the industry. In view of the expected benefits the beneficiaries and target audience for this research will be the growers, consumers, grape industry students, and other food processing industries who stand to gain significantly from the research outcome. Therefore, the project findings were propagated and shared with various groups including community groups, public, students, women, children, grape growers, grape industry personnel, biomedical scientists, academic community, USDA, collaborators and other 1890 and 1862 land-grant Universities, and outreach personnel using diverse educational and outreach activities including lectures, discussions employing diverse electronic and print media. Examples of these activities and conveyance means and media include presentations and publications in Journal articles, Conference presentations, Workshops, FloridaLegislature, Professional and scientific meetings, Social Media pages, Guest lectures, laboratory visits, grape festival,Extension outreach activity days, community events, etc. In addition, the research outcome was also communicated to thecommunity, consumers and growers at the Association of Research Directors of I890 Institutions Meeting and several othercommunity events to appraise and educate the interested groups. The project has also initiated discussions and potential forcollaborations with peers for furthering the studies. The research outcome will be also conveyed to the stakeholders throughfield demonstrations and workshops to train farmers, grape growers on varietal selection, application of appropriate culturalpractices for high quality grape production to safeguard product value and marketing strategies. The results obtained in the experimental studies will be published in peer reviewed scientific journals. The summary of interesting findings will also be published as popular articles in leading newspapers. The literature and educational materials will be made available to potential beneficiaries, consumers, growers, and industry at CVSF/FAMU website as documents, provide hands on training to students in molecular and cellular biology, proteomics, and transcriptomics. The interested faculty and students from biology, chemistry and pharmacy, and community groups will be trained in biotechnology, genetics, bioinformatics and other interactive courses focused on enhancing their skills and knowledge. Suitable propagation materials like posters, brochures and publications will be made with the help of extension specialist and used in dissemination of project outcome. What do you plan to do during the next reporting period to accomplish the goals?The purpose of this research is to create a variety of muscadine grape that does not produce seeds. Here, we identified a candidate gene for seed development. In addition, we used a number of biochemical assays to confirm that the seedless variety still had enough levels of other vital phytochemicals and secondary metabolites. We conducted a transcriptome study on cv. Noble. After that, we'll analyze the transcriptome of cv. "Fry Seedless" and compare it to a cultivar that does produce seeds. This will show if or not there is another gene that is differentially expressed throughout berry development and is substantially expressed in the seeded variety. Transcriptomic data for the cv "Noble" will be analyzed to completion. Using a heterologous system, such as Tomato/Arabidopsis, the muscadine VroAGL11 gene or any other putative target gene will be confirmed after analysis of the RNA seq data. In order to achieve this, we will use the freeze-thaw technique to introduce the wild (functional) and mutant coding area of the VroAGL11 gene into Agrobacterium tumefaciens strain EHA 105. After employing the floral dip approach to introduce the target T-DNA insertion into Arabidopsis stk mutant/seedless tomato plants, transformants will be tested for GFP fluorescence and confirmed by PCR. At least 5 siliques and/or 2 fruits per plant from ~10 positive T1 Arabidopsis/Tomato plants respectively will be utilized for the phenotypic investigation. VroAGL11 gene expression will be measured in T1 plants using quantitative polymerase chain reaction (qPCR). As a comparison, we propose to utilize wild Arabidopsis and/or wild seeded and seedless tomato plants. In all stages of the study, training for the students involved will be an essential component. To better prepare them for future careers in the FANH sciences, they will be given opportunity to get practical experience with state-of-the-art technologies like as gene and protein technologies, as well as instrumental analysis.
Impacts
What was accomplished under these goals?
The most desired attribute for generating the seedless table grape cultivar is seedlessness, However, due to male sterility, the sole accessible seedless muscadine genotype "Fry Seedless" cannot be utilized as a crossing parent in the breeding program.In this regard, previously we identified the AGL11 transcripts in muscadine grapes by qRT-PCR using gene-specific primers designed from the bunch grape VviAGL11 gene and validated by amplifying and sequencing the coding sequence (mRNA) from seeded and seedless muscadine cultivars. The highest expression ofAGL11gene at pea-size berry was found in seeded muscadine cultivar "Jane Bell" followed by "Noble" which was ~152- and ~130-fold higher and statistically highly significant than that of Fry seedless, respectively (Rahman et al., 2021). However, the lowest expression ofAGL11gene was found in seedless muscadine cultivar "Fry Seedless". Because the purpose of our research is to develop a seedless variety, it is critical to determine if the seedless variety does not impair the other components present in the berries that are vital from a health standpoint. We assessed total phenolics, antioxidant activity, anthocyanin content, total flavonoid content, stilbene content, and total sugar in seedless and seeded varieties for this purpose. To begin, we measured the content of total phenolics in the selected muscadine grapes cv. "Fry seedless, Fry, Late fry, Black Beauty, Black Fry, Diexie Red, Noble, Jene bell, Janet, and Cowart" harvested in at fully matured stage. The amount of phenolic content was observed to be highest in Fry seedless, followed by Black fry, Jene bell and lowest in Black beauty. This result denotes even inabsence of seeds, total phenolic content was high. Next, we measured the total flavonoid content. The significant difference in flavonoid content was also observed in different muscadine berries. Total flavonoid content in Diexie red variety showed highest followed by Fry seedless and Black beauty, while in Cowart and Janet showed lowest on the fresh weight basis. Further, total Anthocyanin content was measured.There was an increase in anthocyanin content, and a dramatic increase observed in Noble than other varieties. The results show that there is considerable variation among the grape cultivars examined in the anthocyanin content of the berry skin. In fully developed fruit of Noble and Black fry, there was an upsurge in anthocyanin content, and at the same time increase in the pools of non-colored phenolics was observed. It could be due to enzyme involved in anthocyanin biosynthesis, may compete more at later stages of berry development (Lee and Talcott, 2004). Later, Antioxidant activities were measured using DPPH activity. A positive correlation observed between total phenolics, anthocyanin content, and antioxidant activities. The Fry seedless, Black fry, Noble and Janet showed a high level of antioxidant potential at full maturity in DPPH assay, followed by Diexie red and Fry grape varieties. It indicates overall Fry seedless and Black Fry comparatively had the highest antioxidant potential by its ferric reducing ability during berry development and berry maturity stages. However, there was an inclined trend in antioxidant activities observed in all muscadine grape varieties. It may be due to the high accumulation of phenolic acids and various flavonoids at different developmental stages of grape berries (Ignat et al., 2011). The results provided evidence that mature fresh berries in the present study could be beneficial in the biomedical applications for reducing oxidative stress. This result was in accordance with the reports (Yilmaz et al., 2014; Cosmulescu et al., 2015; Panteli? et al., 2016). Fry seedless had the highest content of total phenolic compounds, and the strongest antioxidant capacity. Further, the test of Total Sugar (TS) content in all 10 muscadine grape varieties was found significantly increased (p ≤ 0.05) mature stage. The Fry seedless had the highest TS content than other varieties (Table 1). The increase in TS of all grape varieties was observed as an essential natural phenomenon in mature ripe berries. The maturity and ripening process involves a combination of both metabolism and catabolism activities or the interconversions of a series of carbohydrates. Therefore, the increase in TS content attributed due to the enzymatic conversion of higher polysaccharides into simple sugars (Tripathi et al., 1992). The results indicated that there is considerable variation among the grape varieties examined. The fruit compositional changes showed that the Fry seedless had the (P ≤ 0.05) highest TSS content at full maturity. It could be because of chemical and enzymatic changes of polysaccharides into sugars and degradation of natural acids present in grape (Harbertson et al., 2003). The phenylalanine metabolic pathway, is one of the best characterized metabolic pathways in plants. It produces a variety of secondary metabolites including flavonoids, lignins and taxon-specific compounds, such as the stilbenes in grapevine. Reduced content of stilbenoids can impact their potential value as nutraceuticals, an important attribute, which contributes toward health benefits and is mainly derived from the consumption of muscadine grapes and grape products. Here, to measure the stillbenes content, the whole berries was crushed in the liquid nitrogen and extract with methanol followed by homogenization. Samples were centrifuged and supernatant was subjected to the HPLC analysis to measure the stilbene content. The varieties showed variation in the composition of stilbenes tested. Compared to other varieties Cowart showed the highest contents of resveratrol, viniferin, pterostilbene and total stilbenes followed by Black fry, Fry, Jennebell, Fry seedless and Black beauty. Among the stilbene's resveratrol is the most studied and has been reported to be effective against various cancers, including breast, lung, colon, skin, prostate, ovarian, liver, oral cavities, thyroid, and leukemia (Elshear et al., 2018). Overall, the results show that the studied muscadine cultivars with seedless have mainly greater or equivalent levels ofsubstances such as phenolics, TS, and antioxidants, among other things. Moreover, to investigate the transcriptional changes, present between seedless and seeded variety and deuce the information about the more genes responsible for the seed formation we performed the whole RNA -sequencing of the selected cultivar. We chose the seeded cv "Noble" as it showed higher expression of AGL11, we performed RNA-sequencing of cv "Noble". Grape flower/berry samples were collected from muscadine cultivar 'Noble' at 5 different stages of berry development, namely, 1) inflorescence, 2) Pea-size berry pea, 3) Mature and Green berry, 4) Veriason stage, and 5) Harvest-ripe. Samples collected from 3 different clusters of 3 different canes of one plant makes one replication, thus we prepared 3 replications from three different plants located in three different rows of the vineyard. Principal Component Analysis (PCA) of the three replicates displayed strong correlations for the different stages of both cultivars, suggesting that the experiments had good reproducibility and reliability. Our quantitative transcriptome analysis (RNA-seq) of "Noble" muscadine cultivars was able to identify genes that may control berry phenotypes, such as the accumulation of high levels of antioxidants. The reference transcriptome developed in this study can be used by muscadine breeders to identify candidate genes for antioxidant biosynthesis, berry firmness, texture, skin thickness, seedlessness, and flavor/aroma.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identifying and characterizing parthenocarpic gene in muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Towards developing non-leathry muscadine table grape:identifying and characterizing gene associated with leathery skin in muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identification and characterization of molecular and cellular components to augument sugar metabolism for increasing sugar content of muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Development of value added functional foods from muscadine grapes
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Muscadine grape as a potential prebiotic and probiotic source to promote gut health
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Exploiting grape phytochemicals to promote healthy ageing
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Efficacy of muscadine grape phytochemicals to modulate prostate cancer progression
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Synergistic action of stilbenes has potent cytotoxic activity resveratrol alone
|
Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The focus of this research is to develop knowledge, technology and build capacity in cutting edge gene and protein technologies for developing seedless muscadine grape for table use and address critical issues impacting crop value, disease tolerance, nutritional and nutraceutic value to enhance market value, consumer acceptance and industry income. These research activities are also helping identify the molecular and cellular mechanisms involved and identify the genes and gene products controlling seed development which help develop new seedless muscadine grape genotypes. In view of the potential benefits of seedless muscadine genotypes the interested groups and audience for this research will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the outcome through increased consumption and sale of table use muscadine grape, increased market value and producer income and establishment of new vineyards targeting fresh fruit market. The project related research activities also are helping build and strengthen functional genomics, omics technologies and nutrigenomics research capability at FAMU for enhancing or altering chemical constituents' content and characteristics of fruit and vegetable crops to improve their nutritional and health values for meeting the consumer/ stakeholder/clientele needs and demands as well as providing training venue for current and future students in cutting edge technologies. The project has also invocated faculty innovation, productivity, and outreach activities and has naturally garnered their interest. In addition, the project has also instilled interest in scientists interested in learning about latest molecular and cellular technologies, and their application to modify the agronomic and nutritional and nutraceutic traits of crops to promote healthy living. Changes/Problems:The PI who was to manage and execute the project left the university for a more promising faculty position requiring change of PI. The formal PI change process and alteration in duties and responsibilities of the new PI has to be revised/initiated. The new PI has assumed the leadership and is effectively administering the project seamlessly. The project is on track and progressing accordingly. The pandemic and prescribed guidelines have limited options for student training in the lab, especially the experimentation requiring bench work. Since it was not possible to provide in person hands-on training in various sample collection and processing protocols, analytical techniques, instrumental analysis and data collection and analysis protocols using laboratory facilities, the PI has to resort to virtual training to support some of the training activities for students. The project personnel have successfully established collaborative research arrangements with scientists from collaborating institutions and worked on optimizing the experimental techniques to execute the prescribed experiments in a timely manner as well as provide student experiential learning to accomplish the project tasks for meeting project objectives. Presently we are executing the defined experimental workplan and hope to pursue the project tasks regularly. The collaborative arrangements and support from other researchers will help conduct project experiments successfully and complete the project tasks for meeting the objectives. What opportunities for training and professional development has the project provided?The pandemic and accompanying limitations on in person working and training allowed limited options for student training. However, some training activities were conducted successfully through virtual meetings. As the safety guidelines relaxed during the latter part of the year more access was permitted for in person student training and used. These training activities included: 1. Hands-on and virtual training of undergraduate students in berry maturity determination, classification, sample collection and processing, phytochemicals extraction and quantification, cell culture methods, protein and RNA isolation and characterization techniques, instrumental use and other related biochemical techniques. The student participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. The graduate students from agriculture and environmental sciences were provided training in experimental design, berry maturity determination, berry classification, sugars, amino acids and phytochemicals isolation, purification and quantification, RNA and protein extraction and quantification, and other related molecular and cellular techniques, data collection and processing techniques to enhance their skills in cutting edge gene and protein techniques as well as data processing and analysis tools for use in their thesis research. 3. Two post-doctoral research associates were trained in varietal selection, berry developmental stage assessment, phytochemicals extraction, biochemical evaluation, sample processing, RNA and protein extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, other related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, bioinformatics and bioprocessing. These researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation. How have the results been disseminated to communities of interest?The primary goal of this research is to identify and characterize the gene/s encoding seedlessness trait in muscadine grape for use in developing non-GMO seedless muscadine grape genotype. Towards this goal the project workplan was developed to determine the seed developmental activity in maturing berry, conduct comparative genomic studies among various grape genotypes to identify the gene encoding seededness and seedlessness traits. These research activities have enabled identification of the gene associated with seededness in muscadine grape for the first time. Once its role in seed development is confirmed and used in the development of seedless muscadine grape using novel gene editing technology it will have tremendous impact on consumers, growers and the industry. In view of the expected benefits the beneficiaries and target audience for this research will be the growers, consumers, grape industry students, and other food processing industries who stand to gain significantly from the research outcome. Therefore, the project findings were propagated and shared with various groups including community groups, public, students, women, children, grape growers, grape industry personnel, biomedical scientists, academic community, USDA, collaborators and other 1890 and 1862 land-grant Universities, and outreach personnel using diverse educational and outreach activities including lectures, discussions employing diverse electronic and print media. Examples of these activities and conveyance means and media include presentations and publications in Journal articles, Conference presentations, Florida Grape Grower Association meeting, Workshops, Florida Legislature, Professional and scientific meetings, Social Media pages, Guest lectures, laboratory visits, grape festival, Extension outreach activity days, community events, etc. In addition, the research outcome was also communicated to the community, consumers and growers at the Association of Research Directors of I890 Institutions Meeting and several other community events to appraise and educate the interested groups. The project has also initiated discussions and potential for collaborations with peers for furthering the studies. The research outcome will be also conveyed to the stakeholders through field demonstrations and workshops to train farmers, grape growers on varietal selection, application of appropriate cultural practices for high quality grape production to safeguard product value and marketing strategies. The results obtained in the experimental studies will be published in peer reviewed scientific journals. The summary of interesting findings will also be published as popular articles in leading newspapers. The literature and educational materials will be made available to potential beneficiaries, consumers, growers, and industry at CVSF/FAMU website as documents, provide hands on training to students in molecular and cellular biology, proteomics, and transcriptomics. The interested faculty and students from biology, chemistry and pharmacy, and community groups will be trained in biotechnology, genetics, bioinformatics and other interactive courses focused on enhancing their skills and knowledge. Suitable propagation materials like posters, brochures and publications will be made with the help of extension specialist and used in dissemination of project outcome. What do you plan to do during the next reporting period to accomplish the goals?The overall goal of this research is to develop a seedless muscadine grape cultivar for the fresh fruit market using cutting edge technologies and along the way build capacity and expertise in gene editing technology at CAFS/FAMU. In this regard studies on interrelationship between seed size, number and weight were completed. Recently VviAGL11 gene has been identified as master regulator of seed morphogenesis in Bunch grapes and is confirmed by silencing and overexpressing the VviAGL11 gene to decrease and increase the seed content in seeded and seedless Bunch grape cultivars, respectively. Towards this goal we have isolated and purified high quality RNA from different muscadine grape berry tissue and successfully detected presence of the AGL11 transcripts in muscadine grape berry using qRT-PCR and gene-specific primers designed from Bunch grape VviAGL11 gene and validated by amplifying and sequencing the coding sequence (mRNA) from seeded and seedless muscadine cultivars. Studies are in progress to further characterize the identified stenospermocarpic gene for seedlessness and its transcripts using the genomic DNA isolated from frozen berry tissue of selected seeded muscadine and seedless Bunch grape cultivars to determine its characteristics, role, extent of genetic variation, sequence information for exploring sequence differences at the genomic and transcriptomic levels. The copy number of the VroAGL11 in selected muscadine cultivars will be determined using Southern Blot in addition to the quantitative PCR on genomic DNA. The AGL11 gene and its transcripts (ORF) will be amplified from DNA and cDNA of selected grape cultivars respectively with gene-specific primers. PCR products will be purified and sequenced. The sequences of the AGL11 gene, ORFs and their deduced amino acid sequences will be analyzed to explore their sequence differences. Upon comparing and confirming AGL11 gene sequence with the known Bunch grape AGL11 gene sequence the role of muscadine VroAGL11 gene for stenospermocarpic seedlessness will be confirmed in a heterologous model system through complementation test. In this regard we will generate Arabidopsis/Tomato transgenic plants over expressing the muscadine VroAGL11 gene to confirm the competence of this gene in restoring the wild phenotype. Two constructs will be created harboring the wild (functional) and mutated coding region of VroAGL11 gene under 35S promoter and introduced to Agrobacterium tumefaciens strain EHA 105 by the freeze-thaw method. The Arabidopsis stk mutant/seedless tomato plants will then be transformed using the floral dip method and the transformants screened for GFP fluorescence and validated by PCR for the desired T-DNA insertion. At least 5 siliques and/or 2 fruits per plant from ~10 positive T1 Arabidopsis/Tomato plants respectively will be used for the phenotypic study. The relative expression of VroAGL11 gene will be quantified in T1 plants using qPCR. Wild Arabidopsis and/or wild seeded and seedless tomato plants will be used as controls. Student training will be an integral part of this project and continue in all phases of research. They will be provided hands-on experiential learning in cutting edge technologies to develop their expertise in various gene and protein technologies and instrumental analysis for enhancing their career opportunities in FANH sciences.
Impacts
What was accomplished under these goals?
To sustain the muscadine grape industry in Southeastern USA and meet the ever-increasing demand for table grapes in the fresh fruit market, the muscadine grape industry strives to develop seedless muscadine grape. Seedlessness can occur through two biological processes, namely, parthenocarpy and stenospermocarpy. Stenospermocarpic seedlessness does not occur or has not been reported so far in muscadine grapes. The only available seedless muscadine genotype 'Fry Seedless' is parthenocarpic and cannot be used as a crossing parent in the breeding program due to its male sterility. Studies to investigate the genetic basis of seedlessness found in certain grape species showed it to be controlled by a dominant allele named SEED DEVELOPMENT INHIBITOR (SDI) locus. A grape MADS-box transcription factor gene, AGAMOUS-LIKE11 (VviAGL11, Vv18s0041g01880) was identified in the SDI locus through in silico analysis and proposed as the SDI candidate gene for seedlessness in grape based on genetic linkage and putative homology with the Arabidopsis MADS-box transcription factor gene, AtAGL11 (At4g09960). The AtAGL11, an ortholog of VviAGL11, controls the ovule and seed development in Arabidopsis which is reflected in its mutant SEEDSTICK phenotype showing reduced number and size of seeds. The VviAGL11 gene found in Bunch grape consists of 8 exons spanning ~7.6 kb with a coding region of 672 bp (NCBI: KM401845 cv. Chardonnay). The VviAGL11 gene is highly expressed in floral and fruit tissues of Bunch grapes but repressed in roots, branches, leaves, buds, and tendrils. The reduced expression of the VviAGL11 gene was reported as the source or origin of stenospermocarpic seedlessness in 'Sultanina' and Sultanina-derived grape cultivars. The overexpression of the VviAGL11 gene in seedless grape cultivar produced small seeds in the seedless berries, while silencing of VviAGL11 gene in seeded Bunch grape cultivars induced the seedlessness by decreasing the seed content, confirming that VviAGL11 gene is the key master regulator of seed morphogenesis which can be manipulated for inducing seedlessness. However, the role of AGL11 gene for stenospermocarpic seedlessness has not been validated in muscadine grapes. This study is part of our overall aim to develop seedless muscadine table grape, through molecular understanding and divergence of the AGL11 orthologous gene in muscadine grapes (VroAGL11). This research is thus aimed at identifying and characterizing the VviAGL11 orthologous gene (VroAGL11) in native muscadine grape (Vitis rotundifolia) at the molecular level and analyzing its divergence from other plants. Ripe berries of muscadine, Florida hybrid Bunch, and Bunch grape cultivars grown at the FAMU Center for Viticulture and Small Fruit Research were used in this study. Twenty ripe berries (maturity stage EL 38) from 42 muscadine (Vitis rotundifolia), two Florida hybrid Bunch (Vitis spp) and one Bunch (Vitis vinifera) grape cultivars including seedless cultivars were randomly collected from at least three grapevines per cultivar during the season in June/July (Bunch and hybrid Bunch) and September (Muscadine) and the berry and seed characteristics studied. The mean berry weight (=BW/BN), seed number/berry (=SN/BN) and seed weight/berry (=SW/BN) and seed content of berry (=(SW/BW)*100) were measured, where BW=berry weight, BN=Berry number, SN=seed number, and SW=seed weight. The seed length and width of at least 12 seeds per cultivar were measured using electronic digital calipers. The results showed that the mean berry weight of muscadine grape varied from 2.5 to 15.9 g depending on the cultivar with the maximum weight of 15.9 g in Black Beauty and minimum weight of 2.5 g in Fry Seedless followed by muscadine cultivar Welder (3.3 g). However, the mean berry weight of Bunch/hybrid Bunch grapes were significantly less compared to muscadine grapes ranging between 1.6 to 2.0 g. The mean number of seeds in muscadine grape berries ranged between 2.3 and 4.9 per berry with the highest number of seeds in cv. Janet and the lowest in cv. Scuppernong. In Bunch/hybrid Bunch grapes, however, the mean seed number varied between 1.3 to 3.6 per berry. Muscadine grape berries contained seeds of different sizes and weights varying between 4.8 and 7.9 mm in length, 2.7 and 5.6 mm in width and 54 and 120 mg/seed depending on the cultivar. The longest and widest seeds were found in muscadine cultivars 'Ison' (7.9 mm) and 'Late Fry' (5.6 mm), respectively, but the highest mean seed weight was found in muscadine cultivar 'Jane Bell' (119.7 mg). On the other hand, the shortest and narrowest seeds were found in muscadine cultivar 'Welder' (4.8 mm) and 'Southern Home' (2.7 mm), respectively, and the lowest mean seed weight in cultivar, 'Pride' (54.1 mg). These data revealed that the mean berry weight is not correlated with the number of seeds/berry (r=0.1695, p=0.8661), seed length (r=0.3847, p=0.7023) and seed width (r=0.2891, p=7023), but there was a moderate positive relationship between mean berry weight and seed weight (r=0.6786) which is statistically nonsignificant (p=0.5010). However, highest percentage of seed (by weight) was found in muscadine grape cultivars 'Jane Bell' (7.2% of berry weight) and 'Welder' (6.5%) and the lowest in 'Supreme' (1.9%) and 'Black Beauty' (2.1%) cultivars. RNA Isolation and cDNA Synthesis: Tissue samples were randomly collected from young leaf (maturity stage EL 16), inflorescence (stage EL 23) and berries at different developmental stages viz. pea-size (~7 mm diameter, stage EL 31), green and mature (stage EL 33), veraison (stage EL 35) and harvest-ripe stage (stage EL 38) from three grapevines of selected seeded and seedless muscadine (cvs. Black Beauty, Black Fry, Dixie Red, Jane Bell, Noble, Fry, Fry Seedless), hybrid Bunch grape (cv. Blanc du Bois, BDB), and Bunch grape (cvs. Riesling (Vitis vinifera), Reliance Seedless (Vitis labrusca) cultivars representing different berry sizes and used in the molecular studies. All samples were collected between 9 AM and 9:30 AM (Muscadine: July-September, Bunch/hybrid Bunch: April-June) and frozen immediately in liquid N2 and stored at -80°C until further use. Total RNA was isolated from the frozen berries using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) following the manufacturer's instructions with some modification and treated with DNase using the TURBO DNA-free Kit (Invitrogen) to remove the genomic DNA. The quantity and quality, and integrity number (RIN) of RNAs was measured using a NanoDrop 2000c and Agilent Bioanalyzer 2100, respectively. The total RNA with RIN >8.0 was converted to cDNA using SuperScript III First-Strand synthesis system (Life Technologies) and oligo (dT) primer as per the manufacturer's protocol. Using the purified high-quality RNA from muscadine grape berry tissue and gene-specific primers designed from Bunch grape VviAGL11 gene we have detected the AGL11 transcripts in muscadine grape berry tissue. The identified VviAGL11 gene was further validated by amplifying and sequencing the coding sequence (mRNA) from seeded and seedless muscadine grape cultivars. The cDNA preparations from diverse muscadine grape genotypes are being used to analyze and characterize the muscadine grape AGL11 gene by subjecting it to more detailed molecular studies for determining its characteristics, role, extent of genetic variation, sequence information, etc.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Rahman MA, Balasubramani SP and Sheikh MB. 2021. Molecular characterization and phylogenetic analysis of MADS box gene AGL11 associated with stenospermocarpic seedlessness in muscadine grapes. Genes 12(2): 232. doi: 10.3390/genes12020232
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