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ARS Home » Southeast Area » Poplarville, Mississippi » Southern Horticultural Research » Research » Research Project #440391

Research Project: Establishing CRISPR/Cas9-mediated Targeted Mutagenesis Protocol to Develop Seedless Muscadine Grape

Location: Southern Horticultural Research

Project Number: 6062-21000-010-014-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Aug 1, 2021
End Date: Dec 31, 2023

Objective:
Muscadine grapes (Vitis rotundifolia) are grown commercially in the southeastern U.S. Muscadine grapes have resistance to Pierce’s disease, a limiting factor in the production of V. vinifera in the southeast of the U.S. The 2012 agricultural statistics show the top producing states of muscadine grapes for commercial use are North Carolina, Georgia, and Florida with an estimated 2600, 1700, and 1200 acres, respectively (USDA Agricultural Census 2012). Recent research shows concentrated muscadine extract is effective against breast and prostate cancer in animal models. Although the industry is relatively small, there is tremendous room for commercial growth, based on increasing consumers' interest in healthier diets and changing lifestyle. However, some fruit properties including thick skins and large seeds need improvement for successful market expansion. Seedlessness, is one of the most popular desired traits among muscadine consumers. Stenospermocarpy is the production of incomplete developed seeds in normal developed fruits. In V. vinifera, several studies have showed a genomic region on chromosome 18 to be associated with 90% of the phenotypic variance of seed lessness (Malabara et al 2017). Royo et al 2018, showed that a single point mutation resulting in arginine-197 to leucine substitution in the seed morphogenesis regulator gene (VviAGL11) was associated with Stenospermocarpy in V. vinifera. Stenospermocarpic seedlessness is a dominant trait and has been introduced into muscadine grape from V. vinifera through conventional breeding. However, the breeding process was granted a utility patent, so breeders are not allowed to introduce the gene through conventional breeding. With high heritability and the dominance of the seedless associated allele, VviAGL11 is an ideal target for genome editing. CRISPR (clustered, regularly interspaced, short palindromic repeats) is a natural defense mechanism in prokaryotes that utilizes the bacterial protein, Cas9, coupled with a site-specific engineered guide RNA (gRNA) to locate and cleave the proviral genome target sequence. Using genome editing, only a single base is replaced or deleted. RNA-guided genome editing using the CRISPR/Cas9 CRISPR system has been applied successfully in grape (Vitis vinifera). However, it has not been established whether this system can be applied to get biallelic mutations in muscadine grape. The objectives of this research are to (i) use the CRISPR/Cas9 system for generating precise genomic changes and knock out muscadine grape gene homologous to (VviAGL11); and (ii) determine whether the mutant plants have incomplete developed seeds. The outcome of this research will have broad utility and provide a foundation for developing improved muscadine grape with enhanced fruit quality.

Approach:
Targeted gene sequences homologous to (VviAGL11) will be identified from the muscadine grape reference genome and used to design sgRNAs. We have been working on a draft genome for muscadine grape clone US19-33. In addition, the recent muscadine grape published genome is available at http://www.grapegenomics.com/. Cooperator will design, assemble, and construct the appropriate CRISPR/Cas9 vectors. Upon validation by transient assays, the sgRNA plasmids will be transformed into muscadine grape “RubyCrisp” and clone US19-33 via agrobacteria. USDA ARS PI and collaborator will jointly evaluate the transformed plants for targeted mutagenesis with any changes in DNA sequences and trait observations.