IJH_2024v14n3

International Journal of Horticulture 2024, Vol.14, No.3 http://hortherbpublisher.com/index.php/ijh © 2024 HortHerb Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Publisher HortHerb Publisher Edited by Editorial Team of International Journal of Horticulture Email: edit@ijh.hortherbpublisher.com Website: http://hortherbpublisher.com/index.php/ijh Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada International Journal of Horticulture (ISSN 1927-5803) is an open access, peer reviewed journal published online by HortHerb Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all aspects of horticultural and its relative science, containing horticultural products, protection; agronomic, entomology, plant pathology, plant nutrition, breeding, post harvest physiology, and biotechnology, are also welcomed; as well as including the tropical fruits, vegetables, ornamentals and industrial crops grown in the open and under protection. HortHerb Publisher is an international Open Access publisher specializing in horticulture, herbal sciences, and tea-related research registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. All the articles published in International Journal of Horticulture 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. HortHerb Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.

International Journal of Horticulture (online), 2024, Vol. 14, No.3 ISSN 1927-5803 http://hortherbpublisher.com/index.php/ijh © 2024 HortHerb Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Harnessing Genetic Populations in Plant Breeding: Innovative Strategies for Construction and Application Xuanjun Fang International Journal of Horticulture, 2024, Vol. 14, No. 3, 110-116 Genomic Advances in Cucurbitaceae: Implications for Crop Improvement and Breeding Xuewen Xu, Xiaodong Yang, Xuehao Chen International Journal of Horticulture, 2024, Vol. 14, No. 3, 117-126 Efficacy of Home-made and Commercial Trapping Baits for the Management of Fruit Flies in Mandarin Orchards Prashanna Acharya, Nirajan Acharya, Karishma Bhusal, Binita Lamsal, Riya Pradhan, Shashi Pandey, Madhav Prasad Lamsal, Jiban Shrestha International Journal of Horticulture, 2024, Vol. 14, No. 3, 127-134 Effect of Various Mulching Methods on Growth and Yield Parameters of Potato (Solanum tuberosum) Varieties in Achham, Nepal Sujan Ghimire, Pooja Bhusal, Ashok Rijal, Nirajan Acharya, Praju Ghimire International Journal of Horticulture, 2024, Vol. 14, No. 3, 135-141 Developing Citrus Germplasm Resistant to Asian Citrus Psyllid Using CRISPR/Cas9 Gene Editing Technology: Recent Advances and Challenges Xi Wang, Yiwei Li, Fuping Liu, Wenbin Dong, Liyu Liang, Dongkui Chen, Hongli Li, Huihong Liao International Journal of Horticulture, 2024, Vol. 14, No. 3, 142-155 Dragon Fruit Farming in Nepal: A Comprehensive Review Arati Chapai, Kiran Prasad Upadhayaya, Susma Adhikari, Kiran Thapa International Journal of Horticulture, 2024, Vol. 14, No. 3, 156-162 Varietal Performance on Flowering of Different Varieties of Mango (Mangifera indica) at Sarlahi, Nepal Kiran Thapa, Rupesh Chaudhary, Pratiksha Sharma, Sunil Kumar Chaudhary, Poojan Adhikari, Pawan Pyakurel, Arati Chapai International Journal of Horticulture, 2024, Vol. 14, No. 3, 163-168

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 110 Invited Review Open Access Harnessing Genetic Populations in Plant Breeding: Innovative Strategies for Construction and Application Xuanjun Fang1,2 1 Hainan Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025, Hainan, China 2 Hainan Provincial Key Laboratory of Crop Molecular Breeding, Sanya, 572025, Hainan, China Corresponding email: james.xj.fang@qq.com International Journal of Horticulture, 2024, Vol.14, No.3 doi: 10.5376/ijh.2024.14.0012 Received: 03 Dec., 2023 Accepted: 10 Jan., 2024 Published: 01 May, 2024 Copyright © 2024 Fang, 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: Fang X.J., 2024, Harnessing genetic populations in plant breeding: innovative strategies for construction and application, International Journal of Horticulture, 14(3): 110-116 (doi: 10.5376/ijh.2024.14.0012) Abstract This study explores innovative strategies for utilizing genetic populations in plant breeding to enhance crop performance and adaptability. We review the foundational concepts of population and quantitative genetics in the context of modern breeding techniques and discuss the application of evolutionary plant breeding for developing resilient crop varieties suited to changing environmental conditions. The integration of advanced genetic tools, such as whole-genome strategies and marker-assisted selection, is emphasized for its role in improving breeding efficiency. Additionally, we explore the emerging role of plant microbiomes in breeding, highlighting how symbiotic relationships enhance plant fitness and genetic diversity. The study also addresses the importance of conserving genetic diversity during breeding processes and presents case studies that demonstrate the successful application of these strategies worldwide. The potential of these approaches to significantly impact global agriculture, particularly in enhancing food security and sustainability, is discussed. We advocate for the integration of these innovative genetic tools with conventional breeding practices to meet global food demands. Keywords Plant breeding; Genetic populations; Evolutionary plant breeding; Marker-assisted selection; Multi-parent advanced generation inter-cross (MAGIC) populations; Plant microbiomes Introduction Plant breeding has historically leveraged the natural genetic variability within plant populations to cultivate crops that are higher yielding, more resistant to pests and diseases, and better adapted to various environmental conditions. The genetic diversity found within these populations is a fundamental resource for achieving these improvements, as it provides the raw material for selection and adaptation processes. By understanding and harnessing genetic populations, breeders can direct the evolution of crop species more effectively and efficiently (Jain, 1992). The significance of genetic populations in plant breeding cannot be overstated. These populations are reservoirs of genetic variations that breeders use to introduce new and beneficial traits into crops. These traits can enhance crop productivity, improve nutritional quality, and increase resistance to environmental stresses such as drought, salinity, and pests (Ellstrand, 1992). Moreover, the strategic manipulation of genetic populations can lead to innovations in plant breeding methods, facilitating the development of varieties that are well-suited to sustainable agriculture practices. This study aims to explore innovative strategies for constructing and applying genetic populations to improve crop yields, disease resistance, and environmental adaptability. We intend to delve into recent advancements in genetic techniques, such as whole-genome strategies and the use of multi-parent populations, to better understand their potential in enhancing the efficiency and effectiveness of plant breeding. Additionally, the paper will investigate the integration of microbiomes into breeding strategies, which offers a novel approach to increasing genetic variability and enhancing plant fitness. By addressing these areas, the paper seeks to contribute to the

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 111 development of new breeding strategies that not only meet the demands of global food security but also align with the principles of sustainability and conservation of genetic diversity. 1 Review of Genetic Foundations in Plant Breeding 1.1 Basic principles of population and quantitative genetics in plant breeding The application of population and quantitative genetics forms the bedrock of modern plant breeding strategies. Jain (1992) elucidates that the essence of plant breeding lies in the manipulation of genetic variation through deliberate selection and breeding cycles. These cycles involve the selection of phenotypically superior plants, followed by their cross-breeding to combine desirable traits, and subsequently selecting the best progeny over successive generations. This process depends critically on the principles of population genetics, which deals with the frequencies of alleles and genotypes in a breeding population and how these frequencies change over time under the influence of forces like selection, mutation, and genetic drift. Quantitative genetics, on the other hand, focuses on traits influenced by multiple genes, known as quantitative trait loci (QTLs). Jain (1992) discusses the importance of understanding polygenic traits, which are controlled by several to many genes and are often significantly influenced by the environment. The breeding methods vary depending on the breeding system of the plant species—whether outbreeding, inbreeding, or asexual—and the specific objectives of the breeding program. Each method leverages these genetic principles to optimize the creation of new, desirable plant varieties that can contribute to increased agricultural productivity and sustainability (Jain, 1992). 1.2 The role of genetic diversity and gene flow in plant populations Gene flow, the movement of genes between populations, plays a crucial role in maintaining genetic diversity within plant populations. Ellstrand (1992) emphasizes that gene flow is one of the key mechanisms by which genetic variation can be introduced into a plant population, thus counteracting the effects of natural selection and genetic drift which might otherwise lead to a reduction in genetic diversity. High genetic diversity within a plant population is advantageous as it provides a broader base for natural and artificial selection, increasing the population's resilience to environmental stresses and diseases. According to Ellstrand (1992), gene flow in plants is mediated by various vectors, including wind, water, animals, and human activities, and can occur over varying distances, depending on the species and the ecosystem. The level of gene flow significantly impacts the genetic structure of plant populations, influencing their evolutionary potential and adaptability to changing environments. In the context of plant breeding, managing gene flow can be critical for developing new varieties that are both productive and adapted to local conditions. Enhanced understanding and manipulation of gene flow and genetic diversity are thus vital for the effective breeding of crops capable of meeting the demands of global food security (Ellstrand, 1992). 2 Evolutionary Plant Breeding 2.1 Concept and application of evolutionary breeding to enhance resilience under changing climates Evolutionary plant breeding is a dynamic approach that harnesses the natural genetic diversity within crop populations to adapt to local environmental conditions and changing climates. This method involves creating crop populations with high genetic diversity and subjecting them to the forces of natural selection through cycles of sowing and re-sowing. Plants that are most suited to the prevailing conditions thrive and contribute more to the genetic makeup of subsequent generations. Over time, this process leads to the evolution of crop populations that are more resilient to environmental stresses, including climate variability. Döring and colleagues highlight the resilience of evolving plant populations as a major advantage, particularly in the face of global climate change. This approach not only enhances crop adaptability but also reduces the reliance on chemical inputs, contributing to sustainable agriculture practices (Döring et al., 2011). 2.2 Case studies demonstrating the success of evolutionary breeding in cereals The success of evolutionary plant breeding is particularly evident in cereals, where diverse genetic populations have been developed and tested across various environmental conditions. For example, studies have shown that

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 112 wheat and barley populations derived through evolutionary breeding exhibit enhanced performance and greater disease resistance compared to conventionally bred varieties. These populations are better able to exploit local soil and climatic conditions, resulting in improved yield stability across different seasons and regions. Such case studies underscore the potential of evolutionary breeding as a robust strategy to increase the genetic fitness of cereals, thereby ensuring food security in an era of climate change. The concept and application of evolutionary breeding, as discussed by Döring et al. (2011), provide a solid foundation for implementing this approach in global agricultural practices. By continuing to study and apply these principles, plant breeders can better equip crops to handle the unpredictable stresses associated with global climate variations. 3 Advanced Genetic Techniques 3.1 Whole-genome strategies for marker-assisted breeding and their impact on plant breeding efficiency The adoption of whole-genome strategies for marker-assisted breeding has significantly revolutionized plant breeding by enhancing efficiency and precision. Xu et al. (2012) elaborate on how molecular breeding for complex traits in crops necessitates an understanding and manipulation of myriad factors that influence plant growth and stress responses. These strategies employ full genome sequencing and genome-wide molecular markers to address various genomic and environmental factors comprehensively. This holistic approach is crucial in the effective application of genetic resources and breeding materials, optimizing the selection for desirable traits through a detailed understanding of specific genomic regions, genes/alleles, haplotypes, and their phenotypic contributions (Xu et al., 2012). 3.2 The development and use of multi-parent populations (MAGIC) for genetic analysis and selection Multi-parent advanced generation inter-cross (MAGIC) populations represent a cutting-edge approach in genetic analysis and selection, offering a robust tool for the dissection of complex traits. Arrones et al. (2020) discuss the construction of MAGIC populations, which involves the intermingling and recombination of genomes from multiple founder parents. This results in a set of recombinant inbred lines that display a lack of genetic structure and high genetic and phenotypic diversity. The strength of MAGIC populations lies in their ability to combine significant levels of genetic recombination, thus providing a powerful resource for the genetic analysis of quantitative traits and the selection of elite breeding material. These populations have proven particularly valuable in crop species where complex traits are a focus, allowing breeders to achieve more targeted and efficient selection outcomes (Arrones et al., 2020). The development and implementation of these advanced genetic techniques are crucial for the future of plant breeding, enabling researchers and breeders to enhance crop varieties with greater precision and efficiency, ultimately contributing to sustainable agricultural practices and food security. 4 Integrating Microbiomes in Plant Breeding 4.1 The potential of microbiomes in enhancing genetic variability and plant fitness Recent research highlights the significant role of microbiomes in enhancing the genetic variability and fitness of plant populations. Gopal and Gupta (2016) emphasize that plants, though stationary, have developed intricate relationships with microbial communities to counter various biotic and abiotic stresses. These symbiotic relationships not only bolster plant resilience but also introduce a critical source of genetic variability. Microbiomes, particularly those in the rhizosphere and phyllosphere, play pivotal roles in nutrient uptake, disease resistance, and stress tolerance, contributing to the overall adaptability and health of plants. By harnessing these microbial interactions, plant breeders can exploit an untapped reservoir of genetic diversity to enhance crop performance under diverse environmental conditions (Gopal and Gupta, 2016). 4.2 Strategies for incorporating microbiome selection into traditional plant breeding frameworks To integrate microbiomes effectively into plant breeding, it is essential to develop strategies that consider both the plant and its associated microbial communities as a single holistic unit. One approach is the use of microbial inoculants as a breeding tool, where specific beneficial microbes are introduced to the plant during critical growth

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 113 phases to enhance performance traits such as yield, drought tolerance, and nutrient utilization. Another strategy involves the selective breeding of plants that naturally attract beneficial microbes, thus selecting for traits that promote a beneficial microbiome. This could involve genomic selection where markers associated with beneficial microbiome traits are targeted. Additionally, advanced techniques such as synthetic community (SynCom) analysis can be employed (Marin et al., 2021). This involves constructing specific microbial communities in the lab and testing their impact on plant phenotypes under controlled conditions. Successful communities can then be applied in breeding programs to ensure that crop varieties are optimized not only for their genetic traits but also for their ability to harness beneficial microbial functions. Incorporating microbiome data into decision-making tools for breeders can further refine this integration. By understanding the microbiome's influence on plant phenotypes, breeders can make more informed selections that consider the microbiome as an extension of the plant's phenotype (Martins et al., 2023). By adopting these strategies, plant breeding can evolve to not only select for optimal genetic traits but also for an optimal microbiome, leading to robust, resilient crops that are well-adapted to their growing conditions and capable of meeting the challenges posed by a changing global climate. 5 Conservation of Genetic Diversity 5.1 The importance of maintaining genetic diversity within cultivated plant populations Maintaining genetic diversity within cultivated plant populations is crucial for several reasons. Genetic diversity is the foundation of plant adaptability to varying environmental conditions and resistance to pests and diseases. It allows plant populations to evolve over time, enhancing their resilience and ensuring stability in yield across different environments. Gray (1996) highlights that genetic diversity in plant populations correlates strongly with environmental variability, pointing to natural selection's role in shaping this diversity. By preserving a wide range of genetic variation, breeders can ensure that crops are capable of adapting to future changes in climate or agricultural practices, thus supporting sustainable agriculture (Gray, 1996). 5.2 Methods for conserving genetic resources during breeding processes Conserving genetic resources during the breeding process involves several strategies aimed at maximizing genetic variation and minimizing the loss of genetic traits. These methods include: In situ and ex situ conservation: In situ conservation involves protecting plants in their natural habitats, allowing them to evolve under natural conditions and pressures. Ex situ conservation, such as seed banks and botanical gardens, preserves genetic material outside its natural habitat, providing a backup for lost genetic diversity in the wild. Use of diverse breeding lines and wild relatives: Incorporating a wide range of genetic material, including wild relatives of cultivated plants, can introduce beneficial traits that enhance crop resilience and productivity. These wild strains often contain alleles that confer resistance to diseases and environmental stresses not present in cultivated varieties. Genomic selection and marker-assisted breeding: These modern techniques allow breeders to identify and select for genetic traits that promote diversity within the breeding population. By using markers linked to desirable genetic traits, breeders can efficiently incorporate these traits into new cultivars without compromising the genetic base of the population. Managed gene flow: Intentionally introducing genes from one population to another can help maintain or increase genetic diversity. This method is particularly useful in small or isolated populations at risk of inbreeding depression.

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 114 Participatory breeding programs: Engaging local communities in the breeding process helps preserve and utilize traditional knowledge and local crop varieties, which are often well-adapted to specific environmental conditions and cultural preferences. By implementing these strategies, plant breeders can conserve genetic resources effectively, ensuring that agricultural systems remain productive and sustainable. This conservation is not only a technical challenge but also a fundamental aspect of modern agriculture's ecological and ethical dimensions. 6 Case Studies and Applications 6.1 Examples of successful applications of innovative genetic population strategies in global agriculture Innovative genetic population strategies have demonstrated significant success across various agricultural contexts globally. One prominent example involves the use of evolutionary plant breeding in cereals, which has proven effective under diverse and changing environmental conditions. Döring et al. (2011) discussed how crop populations with high genetic diversity, subjected to natural selection, show enhanced adaptation capabilities to local growing conditions over successive generations, benefiting crop resilience and yield stability in the face of climatic variability (Döring et al., 2011). Another example is the development of Multi-parent Advanced Generation Inter-Cross (MAGIC) populations, which have been applied in various crops to create a highly recombined genetic mosaic that combines multiple founder genomes. This approach has facilitated the genetic dissection of complex traits and accelerated the breeding of elite cultivars with desirable characteristics. Arrones et al. (2020) highlighted the significant impact of MAGIC populations in breeding programs, especially in cereals, where they have enabled the integration of desirable traits from multiple parents into new, high-performing lines (Arrones et al., 2020). 6.2 Impact assessment of these strategies on crop productivity and sustainability The impact of innovative genetic population strategies on crop productivity and sustainability has been profound. The evolutionary breeding approach, as discussed by Döring et al. (2011), not only enhances genetic diversity within crop populations but also improves their overall fitness and adaptability, leading to sustainable crop production systems that are better equipped to withstand environmental stresses (Döring et al., 2011). MAGIC populations, on the other hand, have contributed to increased genetic gains by combining the benefits of high genetic diversity and minimal population structure. This strategy has allowed breeders to effectively map quantitative trait loci and select for traits that contribute to yield stability and stress resilience, thus enhancing both productivity and sustainability in agricultural systems. The use of MAGIC populations in crops like wheat and rice has shown promising results in improving yield under various environmental conditions while maintaining high genetic diversity (Arrones et al., 2020). These case studies exemplify how innovative genetic population strategies are pivotal in not only enhancing crop yields but also in promoting sustainable agricultural practices that are crucial for meeting the growing global food demand in a changing climate. 7 Future Directions and Challenges 7.1 Emerging Technologies and their potential impact on plant breeding Emerging technologies in plant breeding, particularly those involving genomic tools and bioinformatics, are poised to significantly advance our capacity to harness genetic populations. One of the most promising areas is the use of Multi-parent Advanced Generation Inter-Cross (MAGIC) populations, which integrate the genomes of multiple founder parents to enhance genetic recombination and diversity. This approach not only increases the resolution of genetic mapping but also enhances the selection of traits in breeding programs (Arrones et al., 2020). Furthermore, the integration of machine learning algorithms with genomic data is set to revolutionize plant breeding by predicting phenotypic outcomes from genetic data, thus expediting the breeding cycles and enhancing the precision of selection (Scott et al., 2020).

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 115 7.2 Policy and ethical considerations in the deployment of genetic population strategies The deployment of innovative genetic population strategies in plant breeding raises several policy and ethical considerations. First, there is a need for policies that ensure equitable access to genetic resources, which can help prevent the monopolization of genetic materials by a few corporations. This is crucial for maintaining genetic diversity and ensuring that the benefits of plant breeding innovations are shared widely (Louwaars, 2018). Additionally, the ethical implications of genetic modification technologies, such as CRISPR/Cas systems, must be considered. These technologies can drive significant advancements in plant breeding but also raise concerns about potential off-target effects and long-term impacts on ecosystems. Policies need to be in place to govern the safe use of these technologies, ensuring that they do not harm human health or the environment (Cowling, 2013). These future directions highlight the dynamic intersection of technology, policy, and ethics in the field of plant breeding. As technologies evolve, so too must the regulatory frameworks that ensure these tools are used responsibly and equitably to benefit global agriculture and food security. 8 Concluding Remarks In conclusion, the exploration of genetic populations in plant breeding presents a dynamic avenue for enhancing agricultural productivity and sustainability. Through the implementation of evolutionary plant breeding, breeders can develop crops more resilient to environmental changes. The integration of whole-genome strategies and marker-assisted selection further illustrates how technological advancements can revolutionize breeding practices, improving the selection efficiency and genetic diversity of crops. The novel application of microbiomes in plant breeding highlights an emerging strategy that taps into microbial genetics to boost plant health and adaptability. To truly harness the potential of these innovative strategies, there is a pressing need for the integration of these advanced genetic tools with traditional breeding practices. This synthesis not only promises to propel crop improvement forward but also serves as a fundamental strategy to combat the pressing global food security challenges posed by a rapidly growing population and changing climate conditions. The collaborative efforts of geneticists, breeders, and policymakers are essential to advance these technologies from research to field applications, ensuring that the genetic potential of plant populations is fully realized for future generations. 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. References Arrones A., Vilanova S., Plazas M., Mangino G., Pascual L., Díez M., Prohens J., and Gramazio P., 2020, The dawn of the age of multi-parent MAGIC populations in plant breeding: novel powerful next-generation resources for genetic analysis and selection of recombinant elite material, Biology, 9(8): 229. https://doi.org/10.3390/biology9080229 Cowling W., 2013, Sustainable plant breeding, Plant Breeding, 132(1): 1-9. https://doi.org/10.1111/PBR.12026 Döring T., Knapp S., Kovács G., Murphy K., and Wolfe M., 2011, Evolutionary plant breeding in cereals-into a new era, Sustainability, 3(10): 1944-1971. https://doi.org/10.3390/SU3101944 Ellstrand N., 1992, Gene flow among seed plant populations, New Forests, 6, pp. 241-256. https://doi.org/10.1007/BF00120647 Gopal M., and Gupta A., 2016, Microbiome selection could spur next-generation plant breeding strategies, Frontiers in microbiology, 7: 209912. https://doi.org/10.3389/fmicb.2016.01971 Gray A., 1996, Genetic diversity and its conservation in natural populations of plants, Biodiversity Letters, 3(3): 71-80. https://doi.org/10.2307/2999720 Jain S.K., 1992, Population management in new plant breeding approaches, In: Jain, S.K., Botsford, L.W. (eds) Applied Population Biology, Monographiae Biologicae, 67: 121-147. Springer, Dordrecht. https://doi.org/10.1007/978-0-585-32911-6_6 Louwaars N., 2018, Plant breeding and diversity: A troubled relationship?, Euphytica, 214(7): 114. https://doi.org/10.1007/s10681-018-2192-5

International Journal of Horticulture, 2024, Vol.14, No.3, 110-116 http://hortherbpublisher.com/index.php/ijh 116 Marín O., González B., and Poupin M., 2021, From microbial dynamics to functionality in the rhizosphere: a systematic review of the opportunities with synthetic microbial communities, Frontiers in Plant Science, 12: 650609. https://doi.org/10.3389/fpls.2021.650609 Martins S., Pasche J., Silva H., Selten G., Savastano N., Abreu L., Bais H., Garrett K., Kraisitudomsook N., Pieterse C., and Cernava T., 2023, The use of synthetic microbial communities to improve plant health, Phytopathology®, 113(8): 1369-1379. https://doi.org/10.1094/PHYTO-01-23-0016-IA Scott M., Ladejobi O., Amer S., Bentley A., Biernaskie J., Boden S., Clark M., Dell’Acqua M., Dixon L., Filippi C., Fradgley N., Gardner K., Mackay I., O’Sullivan D., Percival-Alwyn L., Roorkiwal M., Singh R., Thudi M., Varshney R., Venturini L., Whan A., Cockram J., and Mott R., 2020, Multi-parent populations in crops: a toolbox integrating genomics and genetic mapping with breeding, Heredity, 125: 396-416. https://doi.org/10.1038/s41437-020-0336-6 Xu Y., Lu Y., Xie C., Gao S., Wan J., and Prasanna B., 2012, Whole-genome strategies for marker-assisted plant breeding, Molecular Breeding, 29: 833-854. https://doi.org/10.1007/s11032-012-9699-6 Disclaimer/Publisher’s Note The statements, opinions, and data contained in all publications are solely those of the individual authors and contributors and do not represent the views of the publishing house and/or its editors. The publisher and/or its editors disclaim all responsibility for any harm or damage to persons or property that may result from the application of ideas, methods, instructions, or products discussed in the content. Publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

International Journal of Horticulture, 2024, Vol.14, No.3, 117-126 http://hortherbpublisher.com/index.php/ijh 117 Invited Review Open Access Genomic Advances in Cucurbitaceae: Implications for Crop Improvement and Breeding Xuewen Xu, Xiaodong Yang, Xuehao Chen School of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou, 225009, Zhejiang, China Corresponding email: xhchen@yzu.edu.cn International Journal of Horticulture, 2024, Vol.14, No.3 doi: 10.5376/ijh.2024.14.0013 Received: 17 Feb., 2024 Accepted: 30 Apr., 2024 Published: 10 May, 2024 Copyright © 2024 Xu 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: Xu X.W., Yang X.D., and Chen X.H., 2024, Genomic advances in Cucurbitaceae: implications for crop improvement and breeding, International Journal of Horticulture, 14(3): 117-126 (doi: 10.5376/ijh.2024.14.0013) Abstract The Cucurbitaceae family, encompassing a wide array of economically and nutritionally significant crops, has been the focus of extensive genomic research aimed at enhancing breeding and crop improvement. Recent advancements in sequencing technologies and bioinformatics have led to the sequencing of genomes from various Cucurbitaceae species, providing valuable insights into gene identification, genome evolution, and genetic variation. This has opened new avenues for molecular breeding, leveraging genetic transformation and gene editing technologies, including CRISPR/Cas9, to overcome the limitations of conventional breeding methods. The integration of next-generation sequencing (NGS) and omics approaches has furthered our understanding of complex traits, such as disease resistance and fruit quality, and has facilitated the development of high-density genetic maps and the identification of functional genes. Additionally, the construction of genetic and cytogenetic maps has been instrumental in revealing the genomic structure of cucurbit crops, aiding in the alignment of linkage groups with chromosomes and enhancing marker-assisted selection. The exploration of genetic diversity through the analysis of wild Cucurbitaceae species using cytogenetic mapping has also contributed to the phylogenetic understanding and breeding resource development. With the accumulation of genomic resources and the advent of high-throughput genotyping methods, new strategies such as genome-wide association studies (GWAS) and the use of multi-parent populations have emerged, leading to the discovery of quantitative trait loci (QTL) for key agronomic traits. The synergy of these genomic tools and their implications for breeding is poised to revolutionize the improvement of Cucurbitaceae crops, ensuring food security and meeting the demands of a growing population. Keywords Cucurbitaceae; Genomic sequencing; Genetic transformation; Gene editing; Genetic linkage map; Cytogenetic mapping; Marker-assisted selection; Genome-wide association studies; Quantitative trait loci Introduction The Cucurbitaceae family, encompassing a wide array of economically significant and diverse crops, is integral to global agriculture and human nutrition. This family includes species such as cucumbers, melons, squashes, and pumpkins, which are not only staples in diets worldwide but also hold cultural and medicinal value (Grumet et al., 2017). The genetic diversity within this family is remarkable, with genome sequences available for multiple species across different tribes, providing a rich resource for understanding evolutionary relationships and agronomic traits (Ma et al., 2022). In the realm of agriculture, genomic research has become a cornerstone for crop improvement and breeding programs. The advent of next-generation sequencing (NGS) and omics technologies has revolutionized our approach to plant breeding, allowing for a more profound understanding of the genotype-phenotype relationship, particularly for complex traits (Pawełkowicz et al., 2016). These advancements have facilitated the identification of functional genes, the development of molecular markers, and the construction of high-density genetic linkage maps, which are crucial for marker-assisted selection (MAS) in breeding (Ren et al., 2009; Fukino and Kawazu, 2016). Moreover, the recent progress in genetic transformation and gene editing technologies, such as CRISPR/Cas9, has opened new avenues for enhancing cucurbit crops' genetic diversity and overcoming the limitations of conventional breeding methods (Feng et al., 2023).

International Journal of Horticulture, 2024, Vol.14, No.3, 117-126 http://hortherbpublisher.com/index.php/ijh 118 This study is to examine the impact of these recent genomic advances on the breeding and crop improvement of Cucurbitaceae species. By integrating the wealth of genomic tools and resources now available, including draft genome sequences and high-throughput genotyping methods, we aim to provide a comprehensive overview of how these technologies are shaping the future of cucurbit breeding. We will explore the implications of genomic tools for dissecting complex traits, enhancing selection efficiency, and ultimately contributing to the development of superior cucurbit cultivars with improved yield, quality, and stress tolerance (Phan and Sim, 2017). This study will encompass the latest developments in genomic research and their practical applications in cucurbit breeding, offering insights into the potential of these technologies to meet the challenges of food security and agricultural sustainability in the 21st century. 1 Genomic Tools and Technologies in Cucurbitaceae Research 1.1 Description of genomic tools (e.g., high-throughput sequencing, CRISPR/Cas9 gene editing) The advent of genomic tools has revolutionized the field of plant breeding and genetics, particularly within the Cucurbitaceae family. High-throughput sequencing (HTS) technologies, such as next-generation sequencing (NGS), have enabled the rapid and cost-effective generation of large volumes of genomic data. These technologies facilitate the study of genotype-phenotype relationships, especially for complex traits, by allowing the discovery of new genes, regulatory sequences, and the development of extensive collections of molecular markers (Pawełkowicz et al., 2016). CRISPR/Cas9 gene editing has emerged as a powerful tool for creating targeted mutations, enabling the study of gene function and the development of crops with desirable traits. In cucurbits, CRISPR/Cas9 has been used to create mutants with compact plant architecture, which is beneficial for high-density planting and mechanical harvesting (Xin et al., 2022). 1.2 Advances in genotyping and sequencing technologies applied to Cucurbitaceae The Cucurbitaceae family has seen significant advances in genotyping and sequencing technologies. The sequencing of 18 different cucurbit species genomes has provided insights into gene identification, genome evolution, and genetic variation (Ma et al., 2022). The development of highly polymorphic simple sequence repeat (SSR) markers from whole genome shotgun sequencing has led to the construction of high-density genetic linkage maps, facilitating whole genome sequencing and molecular breeding in cucurbits like cucumber (Ren et al., 2009). Additionally, the sequencing of the mitochondrial genomes of species such as Citrullus lanatus (watermelon) and Cucurbita pepo (zucchini) has provided insights into the evolution of genome size and the content of RNA editing (Alverson et al., 2010). 1.3 Role of bioinformatics in genomic research: from data collection to analysis Bioinformatics plays a crucial role in genomic research by managing and analyzing the vast amounts of data generated by HTS and other genomic technologies. The Cucurbit Genomics Database (CuGenDB) serves as a central portal for the storage, mining, analysis, integration, and dissemination of large-scale genomic and genetic datasets for cucurbits (Zheng et al., 2018). This database includes genome sequences, ESTs, genetic maps, transcriptome profiles, and sequence annotations, as well as tools for comparative genomic analysis such as synteny blocks and homologous gene pairs between different cucurbit species. The development of tools like 'SyntenyViewer' and the 'RNA-Seq' module within CuGenDB has greatly facilitated the visualization and analysis of genomic data, aiding researchers in the cucurbit breeding community (Zheng et al., 2018). In conclusion, the integration of genomic tools and technologies, such as HTS, CRISPR/Cas9, and bioinformatics platforms, has significantly advanced the research and breeding of Cucurbitaceae crops. These advancements have not only enhanced our understanding of the genetic basis of important agronomic traits but have also provided the means for targeted crop improvement (Ren et al., 2009; Alverson et al., 2010; Pawełkowicz et al., 2016; Zheng et al., 2018; Ma et al., 2022; Xin et al., 2022).

International Journal of Horticulture, 2024, Vol.14, No.3, 117-126 http://hortherbpublisher.com/index.php/ijh 119 2 Genomic Discoveries in Cucurbitaceae 2.1 Key genomic features and architectures discovered in different Cucurbitaceae species Recent studies have shed light on the genetic architecture of fruit size and shape variation in cucurbits, revealing a complex network of quantitative trait loci (QTL) and candidate genes. In cucumbers, melons, and watermelons, over 150 consensus QTLs for fruit size, shape, and weight have been identified, and a genome-wide survey has pinpointed 253 homologs of key fruit size/weight-related genes (Pan et al., 2019). These discoveries underscore the structural and functional conservation of fruit size/shape gene homologs across cucurbits, exemplified by genes such as CsSUN25-26-27a and CsTRM5 in cucumber, and CmOFP1a in melon (Pan et al., 2019). Additionally, the identification of 142 metal-tolerance proteins (MTPs) across eight Cucurbitaceae species highlights the importance of these transporters in plant metal tolerance and ion homeostasis (Jiang et al., 2021). The evolutionary analysis of these MTPs, which are under purifying selection, provides a basis for understanding ion transport functions and mechanisms in Cucurbitaceae (Jiang et al., 2021). 2.2 Comparative genomics: insights from comparing Cucurbitaceae genomes with other plant families Comparative genomics has been instrumental in understanding the evolution of mitochondrial genome size within the Cucurbitaceae family. The sequencing of the mitochondrial genomes of Citrullus lanatus and Cucurbita pepo revealed significant size variation, which is attributed to the accumulation of chloroplast sequences and short repeated sequences (Alverson et al., 2010). This variation is decoupled from mutation rate and RNA editing frequency, suggesting independent evolutionary pathways for these genomic features (Alverson et al., 2010). Furthermore, a multi-locus chloroplast phylogeny has provided insights into the character evolution and classification within the family, correlating well with flower characters such as the number of free styles and fusion of filaments and/or anthers (Kocyan et al., 2007). 2.3 Functional genomics: gene discoveries related to traits such as disease resistance, fruit quality, and stress tolerance The functional genomics landscape of Cucurbitaceae has expanded with the identification of calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) in six species. These kinases play crucial roles in plant growth, development, and stress response (Wei et al., 2019). Expression studies in watermelon have revealed genes that are induced by salinity and maintain high expression levels in male flowers, suggesting their potential roles in stress tolerance and reproductive development (Wei et al., 2019). Additionally, the complete chloroplast genome sequences of ten Cucurbitaceae species have been described, identifying genes under selection that are involved in chloroplast protein synthesis, gene transcription, energy transformation, and plant development (Zhang et al., 2018). These findings, along with the discovery of a large number of SSRs and SNPs in Cucurbita pepo, provide valuable resources for breeding programs aimed at improving disease resistance, fruit quality, and stress tolerance in cucurbits (Guo et al., 2020). 3 Applications of Genomic Research in Breeding 3.1 Marker-assisted selection (MAS): how genomic markers are used to accelerate breeding processes Marker-assisted selection (MAS) is a process where molecular markers are used to assist in the selection of desirable traits in crop improvement. In the context of Cucurbitaceae breeding, MAS has been significantly advanced by the development of high-throughput genotyping technologies and the identification of single nucleotide polymorphisms (SNPs). These advancements have facilitated the discovery of key genes and molecular markers linked to important traits in vegetables, including those in the Cucurbitaceae family (Mohan et al., 1997; He et al., 2014; Hao et al., 2019). Genotyping-by-sequencing (GBS) is one such technique that combines molecular marker discovery and genotyping, proving to be a cost-effective MAS tool in plant breeding (Figure 1) (He et al., 2014). 3.2 Genomic selection: principles and case studies in Cucurbitaceae Genomic selection (GS) is a breeding method that uses genome-wide marker data to predict the breeding values of individuals in a population. This approach is particularly useful for traits that are influenced by many genes, each

International Journal of Horticulture, 2024, Vol.14, No.3, 117-126 http://hortherbpublisher.com/index.php/ijh 120 with a small effect. GS has been shown to increase genetic gain per unit time and cost, and its early empirical and simulation results are promising for Cucurbitaceae crops (Heffner et al., 2009; Jannink et al., 2010; Heslot et al., 2015). The success of GS relies on the use of all available marker information to predict phenotypes, which allows for the capture of more variation due to small-effect quantitative trait loci (QTL) (Goddard and Hayes, 2007; Heffner et al., 2009). However, careful consideration of resource allocation and the cost-benefit balance of using markers is necessary for the effective implementation of GS (Heslot et al., 2015). Figure 1 Schematic steps of the genotyping-by-sequencing (GBS) protocol for plant breeding (Adopted from He et al., 2014) Image caption: Panel (A): tissue is obtained from any plant species as depicted here a young triticale plant; Panel (B): ground leaf tissues for DNA isolation, quantification and normalization. At this step it is important to prevent any cross-contamination among samples; Panel (C): DNA digestion with restriction enzymes; Panel (D): ligations of adaptors (ADP) including a bar coding (BC) region in adapter 1 in random PstI-MseI restricted DNA fragments; Panel (E): representation of different amplified DNA fragments with different bar codes from different biological samples/lines. These fragments represent the GSB library; Panel (F): analysis of sequences from library on a NGS sequencer; Panel (G): bioinformatic analysis of NGS sequencing data; Panel (H): possible application of GBS results (Adopted from He et al., 2014) 3.3 Genetic engineering and editing: targeted modifications to improve crop traits Genetic engineering and gene editing technologies, such as CRISPR/Cas9, have opened new avenues for targeted modifications of crop genomes to improve traits. In Cucurbitaceae, these technologies have been used to overcome the limitations of conventional breeding methods, such as the narrow genetic bases and low variation rates of these crops (Feng et al., 2023). Recent progress in genetic transformation and gene editing in cucurbits has led to improvements in genetic transformation efficiency and the application of gene editing for trait

International Journal of Horticulture, 2024, Vol.14, No.3, 117-126 http://hortherbpublisher.com/index.php/ijh 121 enhancement (Figure 2) (Feng et al., 2023). The integration of genomic and variomic information has the potential to rapidly increase breeding efficiency in cucurbit crops (Hao et al., 2019). Figure 2 Illustration of editing genes in cucurbit crops (Adopted from Feng et al., 2023) Image caption: This diagram illustrates the CRISPR/Cas9 gene editing process adopted for cucurbit crops. The system introduces engineered endonucleases into the plant via genetic transformation, enabling precise and efficient modification of specific nucleotide sequences within the genome. Key steps include the design of the CRISPR system, introduction into the target plant, and the eventual elimination of carrier fragments through hybridization. Successful applications in various cucurbit species demonstrate the technology's adaptability and effectiveness in enhancing desirable crop traits (Adapted from Feng et al., 2023) The implementation of CRISPR/Cas9 technology in cucurbit crops marks a significant advancement in agricultural biotechnology, offering a powerful tool for precise genomic manipulation. This approach not only accelerates the process of crop improvement but also allows for the targeted editing of genes with high specificity and minimal off-target effects. By enabling specific genetic modifications, CRISPR technology facilitates the study of gene functions and the development of cucurbit varieties with improved traits, such as increased resistance to pathogens or enhanced nutritional profiles. In conclusion, the application of genomic research in Cucurbitaceae breeding, through MAS, GS, and genetic engineering and editing, is revolutionizing the way breeders approach crop improvement. These genomic tools enable more precise and efficient selection, ultimately accelerating the development of improved crop varieties to meet the demands of a growing global population. 4 Case Studies 4.1 Successful implementation of genomic advances in Cucurbitaceae crop improvement The Cucurbitaceae family, encompassing crops such as cucumber, melon, watermelon, squash, and pumpkin, has seen significant advancements in genomic research that have facilitated crop improvement and breeding efforts. The development of high-density genetic linkage maps, as reported in the cucumber genome, has been instrumental in enhancing the resolution of recombination breakpoints and has enabled the integration of gene and trait knowledge across cucurbits (Ren et al., 2009). The advent of next-generation sequencing (NGS) and omics technologies has further revolutionized Cucurbitaceae breeding by allowing the discovery of new genes and regulatory sequences, thereby aiding in the understanding of complex traits such as disease resistance, cold tolerance, and fruit quality (Pawełkowicz et al., 2016).

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