Plant Gene and Trait 2024, Vol.15, No.6, 285-294 http://genbreedpublisher.com/index.php/pgt 292 7.4 Study limitations and areas for future research Despite the progress made, there are limitations to the current studies on sex-specific markers in E. ulmoides. One limitation is the focus on male-specific markers, with fewer studies identifying female-specific markers (Wang et al., 2020; Liu et al., 2020). Future research should aim to identify and validate female-specific markers to provide a more comprehensive understanding of sex determination. Additionally, while several markers have been identified, their functional roles in sex determination remain to be fully elucidated. Further studies should investigate the regulatory pathways and interactions of these markers to uncover the molecular mechanisms driving sex differentiation (Wang et al., 2017; Zhang et al., 2023). Expanding the research to include a broader range of genetic and environmental factors influencing sex determination will also be crucial for developing robust and reliable markers for practical applications in breeding and conservation programs. 8 Concluding Remarks In this study, we identified sex-specific markers in Eucommia ulmoides, a dioecious species with significant industrial and medicinal value. Utilizing various molecular techniques, we discovered several male-specific markers, including a 479 bp ddRAD marker (MSL4), a 247 bp SCAR marker derived from a 350 bp AFLP marker, and a 569 bp RAPD marker linked to pistillate plants. Additionally, genome-wide analyses revealed key genes involved in sex differentiation, such as EuAP3 and EuAG, and identified differentially expressed genes (DEGs) between male and female individuals. These findings collectively enhance our understanding of the genetic mechanisms underlying sex determination in E. ulmoides. The identification of sex-specific markers has profound implications for the breeding and conservation of E. ulmoides. The ability to determine the sex of seedlings at an early stage using markers like MSL4 and SCARmr can significantly streamline breeding programs by allowing for the selection of desired sexes, thereby optimizing resource allocation and improving economic yields. Furthermore, these markers can aid in the conservation of genetic diversity by ensuring balanced sex ratios in cultivated populations, which is crucial for maintaining the reproductive viability of this species. Future research in molecular plant breeding of E. ulmoides should focus on several key areas. First, expanding the identification of sex-specific markers to include female-specific loci will provide a more comprehensive toolkit for sex determination. Second, further functional characterization of the identified DEGs and their roles in sex differentiation will deepen our understanding of the underlying genetic pathways. In addition, integrating advanced genomic technologies, such as CRISPR/Cas9, could facilitate targeted manipulation of sex-determining genes, offering new avenues for breeding and genetic improvement. Exploring the ecological and evolutionary implications of sex differentiation in E. ulmoides will provide insights into the adaptive significance of dioecy in this species and inform conservation strategies. Acknowledgments We are grateful to Dr S. Singh for critically reading the manuscript and providing valuable feedback that improved the clarity of the text. We also thank the anonymous reviewers for their insightful comments and suggestions that greatly improved the manuscript. Funding This study was funded by the Guizhou Academy of Agricultural Sciences Talent Special Project (No. 2023-02 and 2024-02); National High Technology Research and Development Program of China (“863” Program) [grant number 2013AA102605-05] and Talent Base for Germplasm Resources Utilization and Innovation of Characteristic Plant in Guizhou (grant number RCJD2018-14). Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
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