Plant Gene and Trait 2025, Vol.16, No.1, 23-31 http://genbreedpublisher.com/index.php/pgt 25 Figure 1 Karyotypic analysis of haploid plants generated through parthenogenesis under high-temperature treatments (48 °C for 6 h and 54 °C for 4 h) inE. ulmoides (Adopted from Li et al., 2020) Image caption: a Somatic chromosome number of the haploids (2n = x = 17). b Ploidy levels obtained from 3-week-old first leaf samples from haploid plants by flow cytometric analysis. c Ploidy levels obtained from 3-week-old first leaf samples from a mixture of haploid and diploid plants by flow cytometric analysis. d A haploid plant (left) and diploid plant (right) of E. ulmoides (Adopted from Li et al., 2020) Li et al. (2020) treated Eucommia ulmoides at two different high temperatures, namely heating at 48°C for 6 hours and 54°C for 4 hours, to induce parthenogenesis. The results showed that this method could indeed enable Eucommia ulmoides to form haploids. Through karyotype analysis, they found that the somatic chromosome number of these haploids was 2n = x = 17. They analyzed the samples with three-week-old leaves using a flow cytometer and concluded that haploid plants have clear ploidy characteristics. Plants with haploid and diploid mixtures have different levels of ploidy. High-temperature treatment is an effective method that can be used to obtain Eucommia haploid plants, which is helpful for breeding and genetic research. 3.3 Applications of chloroplast markers Chloroplast markers are helpful for understanding the genetic diversity, population distribution and evolutionary process of Eucommia ulmoides. The cpSSR loci and SNP markers identified in the relevant research can be used in conservation genomics to formulate plans for protecting this endangered plant. These markers are also very useful in phylogenetic research. Scientists have also discovered through the analysis of chloroplast genomes that Eucommia ulmoides and Aucuba japonica in Japan are “sister species”. Liu et al. (2022) established a high-density genetic map using SNP markers, which is helpful for identifying QTLS related to growth traits. 4 Key Applications of Chloroplast Molecular Markers 4.1 Genetic diversity and population structure 4.1.1 Assessing genetic variation Dong et al. (2012) identified 71 polymorphic chloroplast DNA fragments in their early studies, among which 20 sites were selected as molecular markers that might be used in subsequent studies. Scientists have also developed more polymorphic chloroplast SSR markers (cpSSR), making the assessment of genetic differences more accurate. Firetti et al. (2017) hold that these markers are beneficial for researchers to conduct a more detailed analysis of the genetic diversity of Eucommia ulmoides and to understand its evolutionary process and environmental adaptability. 4.1.2 Population differentiation In their 2015 study, Yu et al. used ISSR and SRAP markers and found that there were significant genetic differences among Eucommia ulmoides populations in different regions (Figure 2). The results of molecular variation analysis (AMOVA) show that most genetic variations occur within the same population, indicating that local genetic diversity is also very important. Li et al. (2019) demonstrated in their study that these research results indicated that chloroplast markers were helpful for identifying different genetic populations and understanding the genetic structure of Eucommia ulmoides populations.
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