International Journal of Horticulture, 2024, Vol.14, No.6, 438-450 http://hortherbpublisher.com/index.php/ijh 446 6.3 Adaptive strategies of Eucommia ulmoides Eucommia ulmoides has developed several adaptive strategies to cope with varying environmental conditions, which in turn influence its sex determination mechanisms. The species exhibits significant phenotypic variation within and among populations, which is a key adaptive trait. This variation allows E. ulmoides to thrive in diverse climatic and geographic conditions (Wang et al., 2023). Additionally, the genetic mechanisms underlying sex determination in E. ulmoides involve complex interactions between multiple genes and environmental factors. For instance, the expression of certain MADS-box transcription factors, which are involved in floral organ development, is differentially regulated in male and female plants, suggesting a sophisticated genetic control of sex differentiation that can respond to environmental cues (Zhang et al., 2023). These adaptive strategies enable E. ulmoides to maintain balanced sex ratios and reproductive success across different habitats. 7 Molecular and Genetic Tools in Sex Determination Research 7.1 Advances in molecular markers for sex determination Recent advancements in molecular markers have significantly enhanced our understanding of sex determination in dioecious plants, including Eucommia ulmoides. Techniques such as Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) markers have been employed to identify sex-specific markers. For instance, a 569 bp RAPD marker linked to sex determination in E. ulmoides was identified and subsequently converted into a SCAR marker, which can be used to screen plants for gender before they reach reproductive maturity, thus saving time and resources (Xu et al., 2004). Additionally, double-digest restriction site-associated DNA sequencing (ddRAD-seq) has been utilized to screen sex-linked molecular markers, leading to the identification of a male-specific locus, MSL4, which is highly conserved and can reliably distinguish male from female seedlings (Wang et al., 2020). These molecular markers are invaluable for early sex identification and breeding programs. 7.2 Application of CRISPR/Cas9 in sex determination studies The CRISPR/Cas9 genome editing technology has revolutionized genetic research, including studies on sex determination in plants. Although specific applications of CRISPR/Cas9 in E. ulmoides have not been extensively reported, the technology holds great potential for manipulating sex-determining genes. For example, CRISPR/Cas9 can be used to knock out or modify genes involved in sex differentiation pathways, such as the MADS-box genes, which play a crucial role in the development of unisexual flowers in E. ulmoides (Zhang et al., 2023). By targeting these genes, researchers can potentially control the sex of the plants, which would be highly beneficial for breeding and commercial production. 7.3 Future directions for genetic research Future research in the genetic mechanisms of sex determination in dioecious plants like E. ulmoides should focus on several key areas. There is a need for comprehensive genome-wide association studies (GWAS) to identify additional sex-linked genes and regulatory elements. The high-quality chromosome-level genome assemblies of both female and male E. ulmoides provide a valuable resource for such studies. Integrating transcriptome analysis with advanced bioinformatics tools can help elucidate the complex regulatory networks involved in sex differentiation. For instance, genes such as EuAP3 and EuAG have been identified as key regulators in E. ulmoides, and further functional studies are needed to understand their roles (Du et al., 2023). Furthermore, the application of CRISPR/Cas9 and other gene-editing technologies should be expanded to validate the functions of candidate sex-determining genes and to develop new strategies for sex control in breeding programs. By addressing these areas, researchers can gain deeper insights into the molecular basis of sex determination and improve the efficiency of breeding dioecious plants. The integration of advanced molecular markers, genome editing technologies, and comprehensive genetic studies will pave the way for significant advancements in understanding and manipulating sex determination in dioecious plants like Eucommia ulmoides.
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