Molecular Pathogens 2024, Vol.15, No.5, 219-226 http://microbescipublisher.com/index.php/mp 224 6.3 Evolution of pathogen resistance Pathogens are constantly evolving, which can lead to the breakdown of resistance in previously resistant strawberry cultivars. The evolution of resistance-breaking pathogen strains is a significant concern, as it can render existing resistance genes ineffective. This challenge is exemplified by the resistance to Verticillium wilt, where genetic gains in breeding for resistance have been negligible over the past 60 years, partly due to the evolution of the pathogen (Pincot et al., 2020; Feldmann et al., 2023). The genetic background of the host plant can also influence the evolution of resistance-breaking isolates, complicating the breeding process. 6.4 Challenges in breeding for broad-spectrum resistance Breeding for broad-spectrum resistance, which is effective against multiple pathogens, is particularly challenging. The complexity of host-pathogen interactions and the need to combine multiple resistance genes make this a difficult task. For instance, the development of resistance to Botrytis cinerea requires the identification of genotypes that combine resistance with desirable horticultural traits, a goal that has yet to be achieved (Bestfleisch et al., 2015). The identification and utilization of diverse resistance genes from underutilized gene bank resources can enhance the genetic variance and improve the accuracy of genomic selection for broad-spectrum resistance (Jiménez et al., 2022). 6.5 Regulatory and economic constraints Regulatory and economic constraints also pose significant challenges in disease-resistant strawberry breeding. The development and deployment of genetically modified or gene-edited crops face stringent regulatory hurdles, which can delay the introduction of new resistant cultivars. Moreover, the economic viability of breeding programs depends on the balance between the costs of developing resistant cultivars and the potential economic benefits. The use of advanced genomic and bioinformatics tools, such as synthetic biology-assisted intragenesis strategies, can accelerate genetic gains and reduce the reliance on pesticides, but these approaches require substantial investment and regulatory approval (Súnico et al., 2021). 7 Concluding Remarks Recent advancements in disease-resistant strawberry breeding have been significantly propelled by the advent of CRISPR/Cas9 genome-editing technology. This method has demonstrated remarkable precision and efficiency in creating targeted gene modifications, surpassing traditional breeding techniques in speed and accuracy. The application of CRISPR/Cas9 has enabled the development of transgene-free disease-resistant crops, which is crucial for addressing regulatory and public acceptance issues. Additionally, the integration of genomic selection and genome-wide association studies (GWAS) has provided deeper insights into the genetic basis of disease resistance, facilitating the identification of key resistance loci and enhancing the accuracy of breeding programs. These technological advancements have opened new avenues for the rapid development of disease-resistant strawberry cultivars, promising to mitigate the impact of pathogens and improve crop yields. Future research should focus on expanding the genetic diversity of breeding populations by incorporating underutilized gene bank resources, which can introduce rare and favorable alleles into modern cultivars. Efforts should be made to refine CRISPR/Cas9 techniques to minimize off-target effects and enhance editing specificity, possibly through the use of shorter single-guide RNAs and dual Cas9 nickases. It is also essential to develop robust delivery systems for CRISPR/Cas9 components that avoid the integration of foreign DNA, thereby addressing concerns related to genetically modified organisms (GMOs). Integrating genomic selection with traditional breeding methods can accelerate the development of disease-resistant cultivars by enabling early selection without extensive phenotyping. Collaborative efforts between researchers, breeders, and regulatory bodies will be crucial to streamline the adoption of these advanced breeding techniques and ensure their successful implementation in commercial strawberry production. The future of disease-resistant strawberry breeding looks promising, with CRISPR/Cas9 technology at the forefront of this transformation. The ability to make precise genetic modifications rapidly and efficiently offers unprecedented opportunities to develop cultivars that can withstand biotic and abiotic stresses. As the field of genome editing continues to evolve, it is anticipated that these technologies will play a pivotal role in sustainable agricultural practices, ensuring food security and environmental protection. By harnessing the full potential of
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