Tree Genetics and Molecular Breeding 2024, Vol.14 http://genbreedpublisher.com/index.php/tgmb © 2024 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Tree Genetics and Molecular Breeding 2024, Vol.14 http://genbreedpublisher.com/index.php/tgmb © 2024 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. GenBreed Publisher is an international Open Access publisher specializing in tree genetics and molecular breeding, trees genetic diversity and conservation genetics registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher GenBreed Publisher Editedby Editorial Team of Tree Genetics and Molecular Breeding Email: edit@tgmb.genbreedpublisher.com Website: http://genbreedpublisher.com/index.php/tgmb Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Tree Genetics and Molecular Breeding (ISSN 1927-5781) is an open access, peer reviewed journal published online by GenBreed Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all aspects of tree genetics and molecular breeding, include studies in tree genetics and molecular breeding, include studies in crop/fruit/forest/ornamental/horticultural trees genetic diversity, conservation genetics, molecular genetics, evolutionary genetics, population genetics, physiology, biochemistry, transgene, genetic rule analysis, QTL analysis, vitro propagation; fruit/forest/ornamental/horticultural trees breeding studies and advanced breeding technologies. All the articles published in Tree Genetics and Molecular Breeding are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. GenBreed Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Tree Genetics and Molecular Breeding (online), 2024, Vol. 14 ISSN 1927-5781 http://genbreedpublisher.com/index.php/tgmb © 2024 GenBreed Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher Latest Content CRISPR/Cas9-Mediated Trait Improvement in Kiwifruit: Current Progress and Future Directions Wenfang Wang Tree Genetics and Molecular Breeding, 2024, Vol. 14, No. 6, 269-276 Wild Tea Species as a Genetic Resource for Future Breeding Programs Jie Huang, Xiazhen Huang, Meifang Li Tree Genetics and Molecular Breeding, 2024, Vol. 14, No. 6, 277-285 Current Status and Advances in Loquat Genomics: From Genome Mapping to Molecular Breeding Jianquan Li Tree Genetics and Molecular Breeding, 2024, Vol. 14, No. 6, 286-294 Meta-analysis of Genetic Markers for Yield and Quality Traits in Dragon Fruit Zhongmei Hong, Wenzhong Huang Tree Genetics and Molecular Breeding, 2024, Vol. 14, No. 6, 295-303 Case Study on Precision Viticulture: Implementing Technology for High Yields and Sustainability Shaomin Yang, Xingzhu Feng, Dandan Huang Tree Genetics and Molecular Breeding, 2024, Vol. 14, No. 6, 304-312
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 269 Feature Review Open Access CRISPR/Cas9-Mediated Trait Improvement in Kiwifruit: Current Progress and Future Directions Wenfang Wang Institute of Life Sciences, Jiyang Colloge of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: wenfang.wang@jicat.org Tree Genetics and Molecular Breeding, 2024, Vol.14, No.6 doi: 10.5376/tgmb.2024.14.0026 Received: 03 Oct., 2024 Accepted: 08 Nov., 2024 Published: 15 Nov., 2024 Copyright © 2024 Wang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang W.F., 2024, CRISPR/Cas9-mediated trait improvement in kiwifruit: current progress and future directions, Tree Genetics and Molecular Breeding, 14(6): 269-276 (doi: 10.5376/tgmb.2024.14.0026) Abstract Kiwifruit is a globally significant economic and nutritional crop, but its breeding faces numerous challenges, including the need for improvements in yield, quality, and stress resistance traits. In recent years, the application of CRISPR/Cas9 technology in crop genetic improvement has demonstrated immense potential, providing a novel technical pathway for optimizing kiwifruit traits. This review systematically summarizes the research progress on key traits and their genetic basis in kiwifruit, with a focus on the application of CRISPR/Cas9 technology in kiwifruit breeding, including functional studies of key trait-related genes and case studies on trait improvement through gene editing. The study explores the technical bottlenecks of CRISPR/Cas9 technology in kiwifruit, such as off-target effects, editing efficiency, and genetic stability, and summarizes methods for improving editing efficiency and the prospects of applying novel Cas variants. Additionally, this review integrates the latest achievements in multi-omics studies, elucidating the role of genomics, transcriptomics, and epigenomics data in precision gene editing and proposing strategies for integrating gene editing with traditional breeding approaches. This review provides a comprehensive theoretical foundation for the research and application of CRISPR/Cas9 technology in kiwifruit, offering practical guidance for developing high-quality kiwifruit varieties with enhanced market competitiveness. Keywords Kiwifruit; CRISPR/Cas9; Gene editing; Trait improvement; Multi-omics integration; Breeding strategy 1 Introduction Kiwifruit, belonging to the Actinidia genus, is a crop of significant economic and nutritional value globally. Originating from China, it has become a staple in countries like New Zealand, Italy, and Chile due to its rich content of vitamins C, E, and K, which contribute to its high nutritional profile (Zhou et al., 2020). The expansion of kiwifruit cultivation has been driven by the development of new cultivars and improved agricultural practices, making it a vital component of the global fruit market. Despite its economic importance, kiwifruit breeding faces several challenges. Traditional breeding methods are often time-consuming and inefficient, particularly in addressing traits such as disease resistance, fruit quality, and plant architecture (Fizikova et al., 2021). The long juvenile phase and dioecious nature of kiwifruit further complicate breeding efforts, necessitating innovative approaches to accelerate the development of improved varieties. CRISPR/Cas9 technology offers a promising solution for precise genetic improvement in kiwifruit. This genome-editing tool allows for targeted mutagenesis, enabling the modification of specific genes to enhance desirable traits such as reduced plant dormancy, improved fruit quality, and altered plant architecture (Nazir et al., 2024). The technology’s ability to create transgene-free modifications also addresses regulatory concerns, facilitating broader acceptance and application in crop improvement. This study explored the use of CRISPR / Cas 9 technology for improving the key kiwi traits (e. g., flowering time, plant structure, and fruit quality). By using this advanced genome editing tool, studies overcome the limitations of traditional breeding methods and accelerate the development of high quality kiwi varieties. The results will provide important contributions to the field of improvement of fruit crops and provide insights into the integration of modern genetic technologies in breeding programs.
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 270 2 Key Traits of Kiwifruit and Their Genetic Basis 2.1 Overview of key traits in kiwifruit Kiwifruit, belonging to the genus Actinidia, is valued for several key traits including yield, fruit quality, and disease resistance. Yield is influenced by factors such as flowering duration and fruit weight, which have shown high heritability, indicating potential for genetic improvement through selection. Fruit quality is characterized by attributes like vitamin C content, soluble solids, and fruit weight, with significant genetic variation observed among different kiwifruit germplasms. Disease resistance, although less frequently highlighted, is crucial for maintaining healthy crops and ensuring consistent yield and quality (Scaglione et al., 2015). 2.2 Advances in kiwifruit genomics and the identification of regulatory genes Recent advances in kiwifruit genomics have significantly enhanced our understanding of its genetic makeup. The draft genome of Actinidia chinensis has been sequenced, revealing important insights into its genetic structure, including ancient hexaploidization and recent whole-genome duplication events that have contributed to the diversification of genes regulating key traits like vitamin C and flavonoid metabolism (Liao et al., 2019). Genome-wide association studies (GWAS) have identified SNP markers associated with important traits, facilitating molecular breeding efforts4. Additionally, transcriptome analyses have identified key genes involved in anthocyanin biosynthesis, which are crucial for fruit coloration (Figure 1) (Liao et al., 2021a). Figure 1 Analysis of key regulatory genes for key metabolites in kiwifruit (Adopted from Liao et al., 2021a) Image caption: Test materials (A) ‘Ganlv 1’ and ‘Ganlv 2’ had similar botanical traits and flowering stages; (B) Simple sequence repetition (SSR) technology was used for genetic similarity coefficient analysis, and the genetic similarity coefficient value of test materials was less than 0.08, the bar at the bottom is the bar of genetic similarity coefficient, the solid circle represents the female germplasm, the hollow circle represents the male germplasm, and ‘Ganlv 2’ was marked in red; (C) Fruit developmental stages and sampling time points of ‘Ganlv 2’ and ‘Ganlv 1’, the early mature germplasm ‘Ganlv 2’ had reached the commercial harvested standard at S7 (Adopted from Liao et al., 2021a) 2.3 Impact of genomic complexity on genetic improvement of kiwifruit The genomic complexity of kiwifruit, characterized by polyploidy and extensive genetic diversity, presents both challenges and opportunities for genetic improvement. The presence of multiple whole-genome duplication events complicates the genetic landscape, making it challenging to pinpoint specific genes responsible for desirable traits (Liao et al., 2021b). However, this complexity also provides a rich genetic resource for breeding programs, allowing for the incorporation of diverse traits through techniques like marker-assisted selection and interspecific hybridization. The development of linkage maps and identification of sex-determining chromosomes further aid in the efficient selection and breeding of kiwifruit varieties (Lee et al., 2020).
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 271 3 Application of CRISPR/Cas9 Technology in Kiwifruit Improvement 3.1 Overview of CRISPR/Cas9 principles and applications CRISPR/Cas9 technology is a revolutionary tool for genome editing that allows for precise modifications at specific genomic loci. It involves the use of a guide RNA (gRNA) to direct the Cas9 protein to a specific DNA sequence, where it introduces double-strand breaks. This system has been widely adopted in various crops, including kiwifruit, due to its simplicity, efficiency, and versatility (Varkonyi-Gasic et al., 2018). The technology has been applied to improve agronomic traits, enhance nutritional value, and develop disease-resistant varieties in fruit crops. 3.2 Functional validation and editing of key trait-related genes using CRISPR In kiwifruit, CRISPR/Cas9 has been used to target and edit genes associated with important traits. For instance, the AcPDS gene, involved in carotenoid biosynthesis, was successfully edited using a paired-sgRNA/Cas9 system, resulting in a high mutagenesis frequency and the induction of an albino phenotype in kiwifruit plantlets (Keul et al., 2022). Additionally, the AcBFT2 gene, which regulates plant dormancy, was targeted to produce an evergrowing phenotype without affecting flowering, demonstrating the potential for climate adaptation (Herath et al., 2022). 3.3 Case studies: improving fruit quality in kiwifruit through CRISPR technology CRISPR/Cas9 has been instrumental in enhancing fruit quality traits in kiwifruit. By targeting genes involved in fruit ripening and bioactive compound synthesis, researchers have been able to modify fruit texture, color, and nutritional content. For example, the manipulation of genes like Lycopene desaturase (PDS) and Pectate lyases (PL) has shown promise in improving fruit quality and extending shelf life (Zhou et al., 2020). These advancements highlight the potential of CRISPR technology to meet consumer demands for high-quality fruit. 3.4 Gene editing research on disease resistance and stress tolerance traits Research on CRISPR/Cas9 in kiwifruit has also focused on enhancing disease resistance and stress tolerance. By editing genes associated with these traits, such as those involved in plant architecture and flowering time, kiwifruit can be made more resilient to environmental stresses and pathogens. This approach not only improves crop yield and quality but also reduces the need for chemical inputs, contributing to sustainable agriculture (Wang et al., 2018). 4 Technical Optimization and Application Challenges 4.1 Key technical challenges in applying CRISPR to kiwifruit Applying CRISPR/Cas9 technology to kiwifruit presents several technical challenges. One major issue is the optimization of the CRISPR system for high efficiency in this specific species. The editing capability can vary significantly depending on the combination of synthetic guide RNA (sgRNA) and Cas9 protein expression devices used. For instance, the development of a paired-sgRNA/Cas9 system has shown promise in increasing mutagenesis frequency, but it requires careful optimization to achieve desired results in kiwifruit (Liu et al., 2021). Additionally, the delivery of CRISPR constructs into kiwifruit cells and ensuring stable integration and expression remain significant hurdles (Wang et al., 2024). 4.2 Off-target effects and issues with gene editing efficiency Off-target effects are a critical concern in CRISPR/Cas9 applications, as unintended edits can lead to undesirable traits or affect plant health. In kiwifruit, the specificity of the CRISPR system must be finely tuned to minimize these effects. The efficiency of gene editing is also variable, with some systems like the polycistronic tRNA-sgRNA cassette (PTG) showing higher efficiency compared to traditional CRISPR/Cas9 systems (Xu et al., 2020). However, achieving consistent and high editing efficiency across different genetic backgrounds and target sites remains a challenge. 4.3 Genetic stability and heritability after kiwifruit gene editing Ensuring genetic stability and heritability of edited traits in kiwifruit is crucial for the practical application of CRISPR technology. Edited traits must be stably inherited across generations to be useful in breeding programs.
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 272 Studies have shown that while CRISPR/Cas9 can induce desired mutations, the stability and heritability of these changes can vary, necessitating further research to understand and control these aspects. The potential for large chromosomal deletions, as observed in some CRISPR applications, also raises concerns about genetic stability (Kaur et al., 2020). 4.4 Methods to improve editing efficiency and applications of novel cas variants To improve editing efficiency, researchers are exploring various strategies, including the use of novel Cas variants and advanced delivery methods. The PTG/Cas9 system, for example, has demonstrated a tenfold increase in mutagenesis frequency compared to traditional systems, suggesting that alternative sgRNA expression devices can significantly enhance efficiency (Wan et al., 2021). Additionally, novel Cas variants with improved specificity and reduced off-target effects are being developed, which could further enhance the precision and applicability of CRISPR in kiwifruit. Heat treatment and other environmental manipulations have also been proposed to increase editing efficiency. 5 Role of Multi-omics Integration in Kiwifruit Trait Improvement 5.1 Integration of genomics, transcriptomics, and epigenomics data The integration of genomics, transcriptomics, and epigenomics data is pivotal in understanding the complex biological processes that govern kiwifruit traits. Genomics provides the foundational genetic information, while transcriptomics offers insights into gene expression patterns under various conditions. Epigenomics adds another layer by revealing modifications that affect gene activity without altering the DNA sequence (Yang et al., 2021). Together, these omics approaches enable a comprehensive understanding of the regulatory mechanisms involved in kiwifruit development and trait expression. The integration of these datasets can lead to the identification of key regulatory genes and pathways that are crucial for trait improvement (Chao et al., 2023). 5.2 Construction and application of regulatory networks for key traits Constructing regulatory networks involves mapping the interactions between genes, proteins, and metabolites that influence key traits in kiwifruit. By utilizing multi-omics data, researchers can build detailed models that depict these interactions, providing insights into the molecular basis of traits such as fruit ripening, flavor, and nutritional content (Mahmood et al., 2022). These networks can be used to predict how changes in one part of the network might affect the overall phenotype, thus guiding targeted interventions for trait enhancement. The application of such networks is crucial for developing new kiwifruit varieties with improved qualities (Figure 2) (Shu et al., 2023). Figure 2 Summary of metabolome and transcriptome datasets for the construction of kiwi metabolic regulatory network (Adopted from Shu et al., 2023)
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 273 5.3 Data-driven precision gene editing and trait optimization Data-driven approaches in precision gene editing, such as CRISPR/Cas9, are revolutionizing kiwifruit trait optimization. By leveraging multi-omics data, researchers can identify specific genes or regulatory elements that are prime targets for editing to achieve desired traits. This precision allows for the modification of specific traits without affecting other important characteristics, leading to more efficient breeding programs. The integration of omics data ensures that gene editing is informed by a comprehensive understanding of the genetic and molecular landscape, thereby enhancing the success rate of trait improvement efforts1 (Tian et al., 2021). 6 Future Directions for CRISPR/Cas9 in Kiwifruit Breeding 6.1 Integration of gene editing with traditional breeding strategies The integration of CRISPR/Cas9 with traditional breeding strategies offers a promising avenue for enhancing kiwifruit breeding programs. By combining the precision of gene editing with the genetic diversity available in traditional breeding, it is possible to accelerate the development of superior kiwifruit varieties. This approach can help incorporate beneficial traits more efficiently, such as disease resistance and improved fruit quality, while maintaining the genetic diversity necessary for long-term crop resilience (Sardar, 2023). 6.2 Development of gene-editing platforms for multi-trait improvement Advancements in CRISPR/Cas9 technology have led to the development of platforms capable of targeting multiple traits simultaneously. This multiplex genome editing approach can significantly enhance the efficiency of breeding programs by allowing the simultaneous modification of several genes associated with desirable traits, such as yield, stress resistance, and fruit quality. The use of optimized sgRNA/Cas9 systems has already shown promise in achieving high-efficiency editing in kiwifruit, paving the way for multi-trait improvement (Pimentel and Fortes, 2020). 6.3 Potential of gene-editing technology to enhance breeding efficiency and economic benefits CRISPR/Cas9 technology holds the potential to greatly enhance breeding efficiency by reducing the time and resources required to develop new kiwifruit cultivars (Ahmad et al., 2020). The ability to precisely edit genes associated with key agronomic traits can lead to faster development of varieties that meet market demands and environmental challenges. This can result in significant economic benefits by increasing yield, reducing losses due to pests and diseases, and improving fruit quality, thereby enhancing the competitiveness of kiwifruit in the global market. 6.4 Expanding applications of crispr/cas9 in kiwifruit quality enhancement and stress resistance improvement The application of CRISPR/Cas9 in kiwifruit is expanding beyond basic trait improvement to include enhancements in fruit quality and stress resistance. By targeting genes involved in nutrient content, flavor, and shelf life, CRISPR/Cas9 can help develop kiwifruit varieties that offer superior quality. Additionally, editing genes related to stress responses can improve the plant's resilience to biotic and abiotic stresses, ensuring stable production under varying environmental conditions (De Mori et al., 2020). 7 Data Integration and Collaborative Mechanisms 7.1 Importance of data sharing in kiwifruit genomic research Data sharing is crucial in kiwifruit genomic research as it facilitates the rapid dissemination of findings and accelerates the pace of discovery. The CRISPR/Cas9 system has been applied to various fruit crops, including kiwifruit, to improve traits such as disease resistance and fruit quality (Liu et al., 2023). Sharing genomic data allows researchers to build upon each other's work, reducing redundancy and fostering innovation. It also enables the integration of diverse datasets, which can lead to more comprehensive insights into the genetic basis of important traits. 7.2 Role of international collaboration in the development and application of gene-editing technology International collaboration plays a pivotal role in advancing gene-editing technologies like CRISPR/Cas9. By pooling resources and expertise, researchers can overcome technical challenges and accelerate the development of
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 274 new applications. For instance, the optimization of CRISPR/Cas9 systems for specific crops, such as kiwifruit, benefits from collaborative efforts that bring together diverse scientific perspectives and methodologies. Such collaborations also facilitate the exchange of knowledge and technology, which is essential for the global advancement of agricultural biotechnology (Ho et al., 2020). 7.3 Necessity of building an open-access kiwifruit genetic resource database An open-access kiwifruit genetic resource database is essential for supporting ongoing research and development efforts. This database would serve as a centralized repository for genetic information, including gene sequences and phenotypic data, which are critical for CRISPR/Cas9-mediated trait improvement (Arora and Narula, 2017). By providing researchers with easy access to comprehensive genetic resources, such a database would enhance the efficiency of breeding programs and enable more precise gene-editing interventions. It would also promote transparency and reproducibility in research, fostering a collaborative scientific community (Huang, 2024). 8 Concluding Remarks CRISPR/Cas9 technology has significantly advanced the genetic improvement of kiwifruit by enabling precise genome editing. This technology has been successfully applied to modify key genes associated with important traits such as disease resistance, plant architecture, and fruit quality. For instance, the use of CRISPR/Cas9 has led to the development of kiwifruit with reduced susceptibility to diseases and improved fruit yield and quality. The optimization of CRISPR/Cas9 systems, such as the paired-sgRNA/Cas9 system, has further enhanced editing efficiency, allowing for more effective trait improvement in kiwifruit. The future of gene-editing research in kiwifruit looks promising, with potential advancements in transgene-free genome editing and the de novo domestication of wild relatives. These approaches could lead to the development of new kiwifruit cultivars with enhanced traits such as increased resistance to environmental stressors and improved nutritional content. Additionally, the integration of CRISPR/Cas9 with other emerging technologies, such as base editing, could further expand the scope of genetic modifications possible in kiwifruit. Future breeding strategies may focus on multiplex genome editing to simultaneously target multiple traits, thereby accelerating the development of superior kiwifruit varieties. This study underscores the transformative impact of CRISPR/Cas9 technology on the genetic improvement of kiwifruit, offering a powerful tool for addressing key challenges in kiwifruit cultivation. By enabling precise and efficient genetic modifications, CRISPR/Cas9 facilitates the development of kiwifruit varieties with enhanced agronomic traits, which can lead to increased productivity and economic benefits for the kiwifruit industry. The advancements in gene-editing techniques also hold the potential to improve the sustainability of kiwifruit production by reducing reliance on chemical inputs and enhancing resilience to climate change. Overall, this study highlights the critical role of CRISPR/Cas9 in driving innovation and growth in the kiwifruit sector. Acknowledgments The author thanks the scientific research projects and funding institutions for funding this study and providing the necessary resource support for the research. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Ahmad S., Wei X., Sheng Z., Hu P., and Tang S., 2020, CRISPR/Cas9 for development of disease resistance in plants: recent progress, limitations and future prospects, Briefings in Functional Genomics, 19(1): 26-39. https://doi.org/10.1093/bfgp/elz041 PMid:31915817
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Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 277-285 http://genbreedpublisher.com/index.php/tgmb 277 Feature Review Open Access Wild Tea Species as a Genetic Resource for Future Breeding Programs Jie Huang, Xiazhen Huang, Meifang Li Tropical Medicinal Plant Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China Corresponding email: meifang.li@hitar.org Tree Genetics and Molecular Breeding, 2024, Vol.14, No.6 doi: 10.5376/tgmb.2024.14.0027 Received: 11 Oct., 2024 Accepted: 18 Nov., 2024 Published: 25 Nov., 2024 Copyright © 2024 Huang et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang J., Huang X.Z., and Li M.F., 2024, Wild tea species as a genetic resource for future breeding programs, Tree Genetics and Molecular Breeding, 14(6): 277-285 (doi: 10.5376/tgmb.2024.14.0027) Abstract This study reviews the significance and potential utilization of wild tea species in tea breeding programs. Wild tea species provide rich genetic diversity that supports the genetic improvement of cultivated tea. These wild relatives possess critical traits such as resistance to pests and diseases, tolerance to abiotic stresses, and quality enhancement, which are key to improving tea yield, quality, and adaptability to environmental changes. The study also explores the application of modern breeding technologies, including genome sequencing, genome-wide association studies (GWAS), and marker-assisted selection (MAS), as well as strategies for balancing the development and conservation of wild tea species. Future research and international collaboration will enhance the efficient utilization of wild tea resources, thereby promoting the sustainable development of the tea industry. Keywords Wild tea species; Genetic diversity; Tea breeding; Pest and disease resistance; Abiotic stress tolerance 1 Introduction Tea cultivation holds significant economic importance globally, as it is one of the most widely consumed beverages, produced from the leaves of the tea plant, Camellia sinensis. This evergreen crop is cultivated in over 50 countries, with China and India being the largest producers (Meegahakumbura et al., 2018; Xia et al., 2020). The economic value of tea is not only due to its widespread consumption but also its cultural significance and health benefits, which have driven the demand for high-quality tea products (Xia et al., 2020). The tea industry relies heavily on the genetic diversity of tea plants to improve yield, quality, and resistance to environmental stresses (Li et al., 2023). However, tea breeding faces several challenges, primarily due to the limited genetic base resulting from conventional breeding methods, which are slow and often constrained by low cross-compatibility and genetic drag (Mukhopadhyay et al., 2015). The genetic improvement of tea is further complicated by its large and complex genome, which poses difficulties in genetic studies and breeding programs (Zhang et al., 2015). To overcome these challenges, there is a pressing need for greater genetic diversity to enhance breeding efforts and develop tea varieties that can withstand biotic and abiotic stresses while maintaining high quality (Chen et al., 2019; Niu et al., 2019). Wild tea species represent an untapped reservoir of genetic diversity that could be crucial for future breeding programs. These wild relatives of cultivated tea, such as Camellia taliensis, possess valuable traits like abiotic tolerance and biotic resistance, which are not present in cultivated varieties. The genetic resources found in wild tea species can provide insights into the domestication and evolutionary history of tea plants, offering potential for the discovery of genes associated with important traits such as stress resistance and flavor. By leveraging the genetic diversity of wild tea species, breeders can develop new tea cultivars with improved characteristics, ensuring the sustainability and economic viability of the tea industry in the face of changing environmental conditions. 2 Diversity and Distribution of Wild Tea Species 2.1 Taxonomy and classification of wild tea species Wild tea species, primarily belonging to the genus Camellia, are classified under the section Thea. This section includes Camellia sinensis, the most widely cultivated tea plant, and its wild relatives. The classification within
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 277-285 http://genbreedpublisher.com/index.php/tgmb 278 this section has been challenging due to reliance on macromorphological features, which can be variable. However, recent studies have utilized foliar sclereids, which are stable anatomical features, to address taxonomic issues. These sclereids exhibit a wide diversity and can be categorized into 12 types, providing a reliable basis for classification and identification of both wild and cultivated tea species (Zhang et al., 2009). 2.2 Geographic distribution and ecological niches Wild tea species are predominantly found in subtropical regions, with significant populations in China, particularly in Yunnan and Guizhou provinces. These regions provide diverse ecological niches, ranging from lowland subtropical forests to high-altitude mountainous areas. The distribution of wild tea is influenced by various environmental factors, including temperature, precipitation, and soil pH. For instance, Camellia taliensis, a wild tea species, is distributed from the west and southwest of Yunnan province to northern Myanmar, thriving in diverse habitats that contribute to its genetic diversity (Rao et al., 2018). The subtropical forests of China, rich in plant species, serve as crucial habitats for these wild tea species, supporting their conservation and utilization. 2.3 Genetic diversity in wild tea compared to cultivated varieties Wild tea species exhibit significant genetic diversity, which is crucial for breeding programs aimed at improving cultivated varieties. Studies using molecular markers such as EST-SSR and SNPs have revealed high levels of genetic diversity in wild tea populations. For example, Camellia taliensis populations in Qianjiazhai show high genetic diversity at the species level, with substantial gene flow among populations at different altitudes (Rao et al., 2018; Wang et al., 2023). In contrast, cultivated tea varieties, while also diverse, often show less genetic variation compared to their wild counterparts. This is evident in the genetic analysis of Camellia sinensis populations, where cultivated types exhibit a higher level of genetic diversity than pure wild types, but less than ancient landraces and admixed wild types (Niu et al., 2019). The genetic diversity in wild tea species provides a valuable resource for breeding programs, offering traits that can enhance disease resistance, stress tolerance, and other desirable characteristics in cultivated tea plants (Huang, 2024). 3 Genetic Traits in Wild Tea Species 3.1 Disease and pest resistance Wild tea species possess significant genetic traits that contribute to disease and pest resistance, making them valuable resources for breeding programs. For instance, certain genotypes of Camellia sinensis, such as Cd19 and Cd289, have demonstrated strong resistance to the tea green leafhopper, Empoasca onukii, under field conditions, which is a major pest in East Asia (Yorozuya et al., 2021). The genetic diversity found in wild tea species, such as those from the Guizhou plateau, also provides a rich pool of alleles that can be harnessed to develop resistant cultivars (Niu et al., 2019). These genetic resources are crucial for enhancing the resilience of tea plants against biotic stresses. 3.2 Abiotic stress tolerance Wild tea species are known for their ability to tolerate a range of abiotic stresses, including drought, salinity, and temperature extremes. Camellia taliensis, a wild relative of the cultivated tea tree, exhibits a remarkable expansion of late embryogenesis abundant (LEA) genes, which are associated with stronger stress resistance compared to cultivated varieties (Zhang et al., 2015). This genetic trait is particularly valuable for breeding programs aimed at developing tea plants that can withstand the challenges posed by climate change and other environmental stresses (Singh and Abhilash, 2018). 3.3 Quality-related traits The genetic diversity in wild tea species also extends to quality-related traits such as flavor, aroma, and phytochemical content. Genome-wide association studies have identified candidate genes involved in flavonoid biosynthesis, such as CsANR, CsF3’5’H, and CsMYB5, which play a crucial role in the production of catechins, key bioactive compounds in tea (Zhang et al., 2020). Additionally, genes associated with terpene biosynthesis, which contribute to tea aroma, have been significantly amplified in the tea plant genome through recent tandem duplications (Figure 1) (Xia et al., 2020). These findings highlight the potential of wild tea species to enhance the quality attributes of cultivated tea.
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 277-285 http://genbreedpublisher.com/index.php/tgmb 279 Figure 1 Landscape of the tea plant genome: transcription factors (Adopted from Xia et al., 2020) Image caption: (a), Gene density (b), intact LTR-RTs from the copia (c) and gypsy families (d), GC content (e), SSR density (f), and differentially expressed genes between leaf and root tissues (g) are shown. The inner circle represents the collinear blocks identified in the tea plant genome. (B) LAI evaluation of the genome assemblies of two tea plant varieties and seven other plant species, CSS, Camellia sinensis var. sinensis; CSA, C. sinensis var. assamica; OSA, Oryza sativa ssp. japonica; ZMA, Zea mays; CCA, Coffea canephora; TCA, Theobroma cacao;ACH, Actinidia chinensis;ATH, Arabidopsis thaliana; VVI, Vitis vinifera. * CSS indicates the previous assembly of C. sinensis var. sinensis (Wei et al., 2018). (C) Estimated times of insertion for intact LTR-RTs (≤4 MYA) of the tea plant, rice, and maize. (D) Distribution of the distance of tea plant LTR-RTs to protein-coding genes. Only distances≤10 kb are plotted. (E) Repeat content in introns of the tea plant and other three representative plants. (F) Expression levels of duplicated genes with (orange color) or without (green color) intronic TE insertions in eight tissues of tea plant: roots (RT), stems (ST), apical buds (BD), young leaves (YL), mature leaves (ML), old leaves (OL), flowers (FL) and fruits (FR). P**<0.01; P*<0.05. (G) Proportion of TEs in intronic regions of protein-coding genes and pseudogenes of the tea plant (Adopted from Xia et al., 2020) 4 Tools and Technologies for Utilizing Wild Tea Species in Breeding 4.1 Advances in genomic sequencing and annotation for tea Recent advancements in genomic sequencing have significantly enhanced our understanding of tea plant genetics. The assembly of high-quality reference genomes for wild tea species, such as the ancient tea tree, has provided valuable insights into the genetic makeup and evolutionary history of tea plants (Zhang et al., 2020). These genomic resources facilitate the identification of candidate genes associated with important traits like flavonoid biosynthesis, which are crucial for tea quality. Additionally, the development of comprehensive transcriptome datasets for wild relatives of tea, such as Camellia taliensis, has enabled the identification of genes involved in stress response and tea quality, further supporting breeding efforts (Zhang et al., 2015). 4.2 Application of genome-wide association studies (GWAS) Genome-wide association studies (GWAS) have become a pivotal tool in tea breeding, allowing researchers to link specific genetic variations with desirable traits. For instance, GWAS has been employed to identify SNPs associated with quality-related metabolites in tea, such as catechins and caffeine, which are essential for
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 277-285 http://genbreedpublisher.com/index.php/tgmb 280 improving tea quality through breeding (Yamashita et al., 2020). Moreover, GWAS has been used to study traits like the timing of spring bud flush, providing markers for marker-assisted selection (MAS) in breeding programs (Wang et al., 2019). These studies leverage large-scale SNP data to uncover genetic markers that can be used to enhance tea plant traits. 4.3 Molecular breeding techniques Molecular breeding techniques, including marker-assisted selection (MAS), have been instrumental in accelerating tea breeding programs. The development of molecular markers, such as unigene-derived microsatellite markers, has facilitated genetic analysis and gene mapping in tea (Sharma et al., 2009). These markers allow for the efficient selection of desirable traits, such as yield, quality, and resistance, by providing a genetic basis for breeding decisions (Li et al., 2023). The integration of genomic data with MAS enables breeders to make informed selections, thereby improving the efficiency and effectiveness of breeding programs. 4.4 Emerging technologies Emerging technologies like CRISPR-Cas9 and epigenetic studies hold great promise for the future of tea breeding. CRISPR-Cas9 offers precise genome editing capabilities, allowing for the targeted modification of genes associated with important traits, potentially overcoming limitations of traditional breeding methods (Mukhopadhyay et al., 2015). Additionally, understanding epigenetic modifications in tea plants can provide insights into gene expression regulation and stress responses, offering new avenues for enhancing tea plant resilience and quality (Wang et al., 2019). These technologies represent the frontier of genetic improvement in tea, providing innovative tools for developing superior tea cultivars. 5 Case Study: Resistance of Wild Tea Species to Blister Blight 5.1 Background on tea blight and its impact on cultivation Tea is one of the most widely consumed beverages, made from the tender leaves of the tea plant. Various biotic and abiotic factors are directly related to tea yield. Among the biotic factors, the most destructive is blister blight, caused by the obligate parasitic fungus Exobasidium vexans Massee. The pathogen invades the tender leaves of tea plants, directly impacting the economic growth of tea-producing countries due to the significant export value of tea. Numerous studies have identified the symptoms, epidemiology, and control strategies of this pathogen. Traditionally, control measures have relied on copper-based fungicides, but these approaches are not long-term sustainable solutions due to environmental concerns and the potential development of resistance in pathogens (Figure 2) (Sen et al., 2020). Therefore, identifying genetic resistance in tea species is crucial for sustainable disease management. Figure 2 Symptoms of blister blight in tea (Adopted from Sen et al., 2020) Image caption: A: Infected leaves in tea bush; B: Various degree of infection; C: Typical symptoms of infection (Adopted from Sen et al., 2020)
Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 277-285 http://genbreedpublisher.com/index.php/tgmb 281 5.2 Identification of resistance genes in a specific wild tea species Research has identified several genetic markers and genes associated with resistance to blister blight in tea plants. Notably, the transcription factor CsWRKY14 has been isolated from Camellia sinensis and shown to play a critical role in mediating resistance through the salicylic acid (SA) signaling pathway (Liu et al., 2021). This gene is more highly expressed in resistant cultivars, suggesting its potential as a genetic resource for breeding programs. Additionally, a functional molecular marker, EST-SSR073, has been identified, which is associated with blister blight resistance, facilitating marker-assisted selection in breeding programs (Karunarathna et al., 2020). 5.3 Experimental breeding trials integrating resistance genes into cultivated tea Experimental breeding trials have focused on integrating these resistance genes into cultivated tea varieties. The use of marker-assisted selection (MAS) has been pivotal in these efforts, allowing for the precise incorporation of resistance traits into new cultivars. For instance, the EST-SSR073 marker has been used to track and select for resistance alleles in breeding populations, significantly expediting the breeding process (Karunarathna et al., 2020). These trials aim to produce cultivars that maintain high resistance levels while preserving desirable agronomic traits. 5.4 Analysis of outcomes and implications for future breeding The outcomes of these breeding trials have been promising, with new tea cultivars exhibiting enhanced resistance to blister blight. The integration of resistance genes like CsWRKY14 has not only improved disease resistance but also contributed to better stress tolerance overall (Zhang et al., 2015; Liu et al., 2021). These advancements underscore the potential of wild tea species as genetic resources for breeding programs. The success of these trials highlights the importance of continued exploration of wild tea genetic diversity to discover additional resistance genes, which could be crucial for developing resilient tea cultivars in the face of evolving pathogen threats. This approach not only ensures sustainable tea production but also reduces reliance on chemical fungicides, aligning with environmental conservation goals. 6 Challenges and Opportunities 6.1 Ethical and conservation concerns in using wild species The utilization of wild tea species as genetic resources for breeding programs presents significant ethical and conservation challenges. Wild tea species, such as Camellia taliensis, are crucial for maintaining biodiversity and ecological balance in their native habitats (Zhang et al., 2015). The exploitation of these species for genetic improvement must be balanced with conservation efforts to prevent the depletion of natural populations. Ethical considerations also arise in the context of bioprospecting, where the genetic resources of wild species are used without adequate benefit-sharing with local communities or countries of origin (Mukhopadhyay et al., 2015). Ensuring that conservation strategies are in place and that ethical guidelines are followed is essential to sustainably harness the genetic potential of wild tea species. 6.2 Bridging the gap between research and practical applications There is a notable gap between the extensive research conducted on wild tea species and their practical application in breeding programs. While genomic and transcriptomic studies have identified valuable genetic traits in wild species, such as stress resistance and quality improvement (Zhang et al., 2015; Zhang et al., 2022), translating these findings into practical breeding strategies remains a challenge. The complexity of tea plant genomes and the long breeding cycles further complicate the integration of research into practice (Mukhopadhyay et al., 2015; Lubanga et al., 2020). Bridging this gap requires the development of efficient genomic selection strategies and the application of biotechnological tools to accelerate breeding processes and enhance the genetic gain in tea breeding programs (Lubanga et al., 2020). 6.3 Potential for global collaboration in wild tea resource utilization The global nature of tea cultivation and consumption presents a unique opportunity for international collaboration in the utilization of wild tea resources. Collaborative efforts can facilitate the sharing of genetic resources, research findings, and breeding technologies across countries, enhancing the genetic diversity and resilience of tea cultivars worldwide (Xia et al., 2020). Such collaborations can also promote the development of standardized
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