Molecular Pathogens 2024, Vol.15, No.1, 40-49 http://microbescipublisher.com/index.php/mp 46 RNAi is based on two main strategies: master-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS). In HIGS, transgenic plants are engineered to express double-stranded RNA (dsRNA) against pathogen genes. These dsRNAs are recognized and cleaved into small interfering RNAs (siRNAs) within plant cells by the Dicer protein, which downregulates the expression of the target gene. In SIGS, dsRNA is sprayed directly on the surface of plants and pathogens. These molecules can be taken up by both cells and, depending on the delivery method used, the dsRNA can be processed by the RNAi machinery of the plant and/or the pathogen, resulting in downregulation of virulence genes and reducing the harmful effects of the pathogen. 8 Challenges and Future Directions 8.1 Technical and practical challenges The integration of genomic approaches in grapevine breeding programs faces several technical and practical challenges. One significant issue is the complexity of grapevine genomes, which complicates the identification and manipulation of resistance genes. For instance, while marker-assisted selection has been successful in identifying major resistance genes, these genes are often vulnerable to breakdown due to rapid changes in pathogen races. The genetic architecture of resistance is shifting towards a more complex model involving multiple minor genes, making breeding for durable resistance more challenging. Another practical challenge is the need for efficient gene transfer procedures and the optimization of biotechnological techniques, such as gene overexpression and gene silencing, to ensure that modifications do not introduce undesired traits. The development of new resistant varieties is a long-term and costly process, requiring significant resources and time (Merdinoglu et al., 2018). 8.2 Ethical and regulatory considerations The use of genomic technologies in grapevine breeding also raises ethical and regulatory concerns. The deployment of genetically modified organisms (GMOs) in agriculture is subject to strict regulations in many countries, which can hinder the adoption of new biotechnological methods (Pirrello et al., 2022). There are also societal concerns regarding the safety and environmental impact of GMOs, which need to be addressed through transparent communication and rigorous safety assessments. The potential for resistance breakdown, as observed with the emergence of resistance-breaking isolates in grapevine downy mildew, highlights the need for careful management and monitoring of resistant varieties to prevent the spread of virulent pathogen strains. 8.3 Future trends in genomic breeding Looking forward, several trends are expected to shape the future of genomic breeding in grapevines. One promising direction is the use of CRISPR/Cas9 technology for precise genome editing, which has already shown success in enhancing resistance to powdery mildew by targeting susceptibility genes. Another trend is the shift from marker-assisted selection to genomic selection, which involves whole-genome prediction models and can improve the accuracy and efficiency of breeding for complex traits such as disease resistance (Poland and Rutkoski, 2016). Additionally, the integration of biotechnological strategies, such as RNA interference (RNAi) and the pyramiding of multiple resistance genes, can provide a more holistic approach to disease management (Agurto et al., 2017; Capriotti et al., 2020). The continued exploration and characterization of existing germplasm will be crucial for developing new varieties that can perform well under diverse environmental conditions and meet market demands (Gaspero and Cattonaro, 2010). By addressing these challenges and leveraging emerging technologies, grapevine breeding programs can enhance disease resistance and contribute to sustainable viticulture practices. 9 Concluding Remarks The application of genomic approaches in grapevine breeding programs has shown significant promise in enhancing disease resistance. The use of CRISPR/Cas9-mediated mutagenesis has been effective in inducing targeted mutations in grapevine genes, such as VvMLO3, resulting in enhanced resistance to powdery mildew. The availability of the grapevine reference genome has facilitated the use of marker-assisted selection, allowing
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