Plant Gene and Trait 2024, Vol.15, No.6, 285-294 http://genbreedpublisher.com/index.php/pgt 289 Figure 3 Maximum likelihood tree of MIKC MADS-box transcription factors in E. ulmoides, A. thaliana, S. lycopersicumand Oryza sativa(Adopted from Zhang et al., 2023) Image caption: MIKC* clade and 13 known MIKCC subgroups are indicated in different colors (Adopted from Zhang et al., 2023) 4.3 Validation of markers through genetic crosses Validation of the identified markers through genetic crosses is a critical step to confirm their reliability and accuracy. In the case of the ddRAD-seq identified marker MSL4, subsequent PCR amplification and Sanger sequencing were performed on additional individuals from a separate population, confirming its stability and repeatability in male individuals (Wang et al., 2020). Similarly, the SCAR marker derived from the AFLP marker was validated through its consistent presence in male plants across different populations (Wang et al., 2011). These validation steps ensure that the markers are not only specific to the sex of the plants but also reliable across different genetic backgrounds. 4.4 Field testing of marker reliability Field testing of the identified markers is essential to assess their practical applicability in real-world conditions. The ddRAD marker MSL4 has shown potential for rapid breeding practices and better commercial production by enabling early sex identification in seedlings (Wang et al., 2020; Liu et al., 2022). The SCAR markers developed from AFLP and RAPD techniques have also been suggested to facilitate future breeding programs by allowing early sexual identification, thus saving time and economic resources (Xu et al., 2004; Wang et al., 2011). These markers have been tested in various field conditions to ensure their reliability and effectiveness in different environmental settings, making them valuable tools for the breeding and cultivation of Eucommia ulmoides. By integrating these molecular techniques and validating the markers through genetic crosses and field testing, researchers have developed reliable methods for early sex determination in Eucommia ulmoides, which will significantly enhance breeding efficiency and commercial production (Ikten and Yılmaz, 2019; Priya et al., 2019; Bafeel and Bahieldin, 2020). 5 Case Study: Integrating Sex-Specific Markers in Breeding Programs 5.1 Strategic importance of marker-assisted breeding inEucommia ulmoides Marker-assisted breeding (MAB) is a powerful tool that can significantly enhance the efficiency and effectiveness of breeding programs, particularly for dioecious species like Eucommia ulmoides. The ability to identify the sex of plants at an early stage is crucial for optimizing breeding strategies and improving economic outcomes. Traditional methods of sex identification in E. ulmoides are time-consuming and unreliable, as they rely on morphological characteristics that only become apparent at later developmental stages (Jin et al., 2020; Liu et al.,
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