Plant Gene and Trait 2024, Vol.15, No.6, 285-294 http://genbreedpublisher.com/index.php/pgt 288 ♀ ♂ Figure 2 Male (♂) and female (♀) individuals of Eucommia ulmoides distinguished by flowers (Zhao et al., unpublished data) 3.4 Validation processes in controlled and natural conditions Validation of identified markers is essential to confirm their reliability and applicability in different conditions. In the male-specific SCAR marker was validated for early sexual identification (Čerenak et al., 2019), which is crucial for breeding programs. The pistillate-specific SCAR marker developed in was confirmed through Southern blotting (Xu et al., 2004), ensuring its exclusiveness to pistillate plants. In the male-specific locus MSL4 was validated through PCR and Sanger sequencing on a separate population (Oh et al., 2023), demonstrating its stability and repeatability. These validation processes ensure that the identified markers are reliable and can be used in both controlled and natural conditions. By integrating these approaches, researchers can effectively identify and validate sex-specific markers in Eucommia ulmoides, facilitating breeding programs and improving economic cropping practices (Iglesias-Andreu and Favián-Vega, 2021; Amjad et al., 2022). 4 Identification of Candidate Genes and Marker Development inEucommia ulmoides 4.1 Selection of candidate genes for sex determination The identification of sex-specific markers in Eucommia ulmoides has been a significant focus due to the dioecious nature of the species, which complicates early sex determination. Various molecular techniques have been employed to identify candidate genes linked to sex determination. For instance, double-digest restriction site-associated DNA sequencing (ddRAD-seq) was utilized to screen for sex-linked molecular markers, resulting in the identification of five candidate male-specific loci, with one ideal sex-linked locus, MSL4, being highly conserved in male individuals (Krueger-Hadfield et al., 2020; Wang et al., 2020). Additionally, Amplified Fragment Length Polymorphism (AFLP) and Sequence Characterized Amplified Region (SCAR) markers have been developed, with a 350 bp male-specific AFLP marker being converted into a 247 bp SCAR marker for early sexual identification (Qing et al., 2021). Random Amplified Polymorphic DNA (RAPD) techniques have also identified a 569 bp pistillate-specific SCAR marker, SCARmr, which is exclusive to female plants (Xu et al., 2004). Furthermore, genome-wide analyses have revealed differentially expressed MADS-box transcription factors between male and female flowers, suggesting their involvement in sex determination (Figure 3) (Zhang et al., 2023). 4.2 Development of primers and optimization of PCR protocols The development of reliable primers and optimization of PCR protocols are crucial for the effective use of identified markers. For instance, the SCAR marker derived from the 350 bp AFLP marker was successfully amplified using specific primers, facilitating early sex identification (Fang et al., 2019). Similarly, primers were synthesized for the 569 bp pistillate-specific SCAR marker, SCARmr, enabling its use in screening for gender before reproductive maturity (Wu et al., 2022). The optimization of PCR conditions, such as the concentration of DNA template, primers, and Taq polymerase, has been performed to ensure high stability and repeatability of the reactions (Huang et al., 2013). These optimized protocols are essential for the consistent amplification of sex-specific markers across different samples and conditions (Zhao et al., 2021).
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