1 Introduction

Eucommia ulmoides, also known as hardy rubber tree, is of great value in both industrial and medicinal fields (Wang et al., 2019). It is the sole member of the Eucommulaceae family and holds an important position in traditional Chinese medicine, especially being very common in the treatment of orthopedic diseases (Zhu et al., 2020). Eucommia ulmoides can also produce rubber, and this unique ability has attracted the attention of many scientists and enterprises (Li et al., 2020). However, Eucommia ulmoides is dioecious, that is to say, each tree is either male or female. This characteristic has brought a lot of troubles to breeding and cultivation (Wang et al., 2019).

The structure of the chloroplast genome is relatively stable, which is very helpful for studying the genetic relationship and classification among plants (Yu et al., 2015). The research conducted by Zhang et al. in 2023 demonstrated that the chloroplast genome of Eucommia ulmoides has been fully sequenced, revealing a typical tetrad structure with 135 genes, containing a wealth of useful genetic information. These data can be used to develop molecular markers, which are of great value for the breeding and conservation of Eucommia ulmoides. Meng et al. in the same year, that is, in 2023, believed that the chloroplast genome is involved in photosynthesis and other important metabolic processes and is a crucial research object in plant physiology and biochemistry studies.

This study analyzed the molecular markers obtained from the chloroplast genome of Eucommia ulmoides. By summarizing existing research, it understood the role of these genetic markers in breeding, species conservation, and in-depth study of the specific traits of Eucommia ulmoides. It emphasized their importance in the entire field of plant genomics and their application potential in enhancing the industrial and medicinal value of Eucommia ulmoides. This study aims to provide a foundation for the subsequent scientific research and development of Eucommia ulmoides.

2 Chloroplast Genome Structure in Eucommia ulmoides

2.1 General features

The chloroplast genome of Eucommia ulmoides has a typical four-segment structure, which is divided into a large single-copy (LSC) region, a small single-copy region (SSC), and two inverted repeat (IR) regions. The total length of its genome is approximately 163 586 base pairs (Wang et al., 2018). Liu et al. (2022) found that the GC content of the entire genome was 38.4%. From the perspective of base composition, adenine (A) and thymine (T) each account for 30.8%, while cytosine (C) and guanine (G) each account for 19.2%. This structure is similar to that of most other angiosperms, indicating that the chloroplast genome of Eucommia ulmoides has strong conservation.

2.2 Gene content and organization

The chloroplast genome of Eucommia ulmoides contains 135 genes, among which 89 are protein-coding genes, 38 are transfer RNA (tRNA) genes, and 8 are ribosomal RNA (rRNA) genes. The quantity and arrangement of these genes are relatively stable, and most of them are related to photosynthesis, transcription and translation. Some SSRs and SNPs were also found in the chloroplast genome of Eucommia ulmoides. These variant regions are very helpful for studying genetic diversity and developing molecular markers (Zhang et al., 2016). Zhong et al. demonstrated in their 2022 study that their presence also indicates that there are some mutation hotspots and potential genetic variations in Eucommia ulmoides.

2.3 Comparison with other plant species

The phylogenetic study by Jin et al. (2022) indicates that Eucommia ulmoides is closely related to Aucuba japonica, both belonging to the Oleocales order. This is also supported by the similarity of their chloroplast genomes. Li et al. (2014a) found that the genome of Eucommia ulmoides also exhibited some interesting difference patterns. Most SNPS occurred in gene regions, while insertions and deletions (Indels) were mainly concentrated in the blank areas between genes. This pattern also shows a similar phenomenon in other angiosperms, indicating that the chloroplast genomes of different plants have been relatively conserved during the evolutionary process. The chloroplast genome of Eucommia ulmoides has a typical quad structure, rich genes and obvious genetic variations.

3 Molecular Markers from Chloroplast Genomes

3.1 Types of molecular markers

3.1.1 Simple sequence repeats (SSRs)

Simple sequence repeats (also known as microsatellites) are short and repetitive DNA sequences that usually have a high degree of diversity. Researchers have identified and developed some SSR markers in the chloroplast genome of Eucommia ulmoides that can be used as molecular markers. Jin et al. (2020) developed eight polymorphic chloroplast SSR (cpSSR) loci, which can be used to study the population genetics of Eucommia ulmoides.

3.1.2 Single nucleotide polymorphisms (SNPs)

Single nucleotide polymorphism (SNPs) refers to the change of a certain base in a DNA sequence. Many such SNPs have been discovered in the chloroplast genome of Eucommia ulmoides. Meng et al. identified 75 SNPs in their 2023 study, among which 59 were located in genetic regions. Among the 40 SNPs presumed to belong to the coding region, all are synonymous mutations, indicating that they do not change the structure of the protein.

3.1.3 Insertions and deletions (Indels)

Sometimes, DNA sequences may undergo some changes due to the addition or deletion of certain bases, which are known as insertions and deletions (Indels). Zhong et al. (2022) identified 80 such Indels in the chloroplast genome of Eucommia ulmoides, with the majority occurring in the regions between genes. These Indels can be used as “molecular markers”, which are helpful for phylogenetic research and analyzing the relationships among different populations.

3.2 Development and identification of markers

Researchers conducted a comprehensive genomic analysis to develop and identify molecular markers in the chloroplast genome of Eucommia ulmoides. They obtained the complete chloroplast genome by screening methods and then compared different samples to identify the regions with differences. In their 2020 study, Li et al. discovered 71 mutated chloroplast DNA fragments and selected 20 loci from them for population genetic analysis. High-throughput sequencing technologies such as PacBio and Hi-C make genome assembly more accurate and make it easier to find reliable markers (Figure 1).

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 is 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.

Yu et al. (2015) analyzed the population structure of Eucommia ulmoides using the STRUCTURE program and two molecular markers, ISSR and SRAP, and found that there were obvious genetic groups within the population of Eucommia ulmoides, indicating that it has a high genetic diversity. The ISSR marking shows a complex population structure with multiple small subpopulations. The SRAP markers more clearly show the differentiation among the main genetic populations, indicating that combining different types of molecular markers can provide a more comprehensive understanding of the genetic situation of Eucommia ulmoides. This study emphasizes that the use of multiple molecular markers can more accurately analyze the population structure and genetic diversity of plants.

4.1.3 Phylogeographic studies

Chloroplast DNA is single-parent inheritance with a slow rate of change and is very useful in systematic geography research. Hu et al. (2019) hold that this type of marking is useful for understanding the historical changes and evolutionary process of a species in space. In the study of Eucommia ulmoides, the analysis of chloroplast genomes confirmed that it is a “sister species” to Aucuba japonica in Japan, providing new clues for understanding the systematic geographical history of Eucommia ulmoides. Moghaddam et al. (2021) demonstrated that such research could help clarify how Eucommia ulmoides was previously distributed and possibly migrated.

4.2 Phylogenetic and evolutionary studies

In 2023, Du et al. discovered the genetic relationship between Eucommia ulmoides and other plants through complete chloroplast genome analysis, and it is clustered in the same branch as Aucuba japonica in Japan. Some chloroplast regions with particularly large variations have also been identified, which is beneficial for studying the relationships between species with relatively close classifications. Walker et al. (2014) demonstrated in their early research that these markers laid the foundation for studying the evolutionary process and genetic ties of Eucommia ulmoides in a larger plant phylogenetic context.

4.3 Conservation genetics

Yu et al. (2015) obtained data on the genetic diversity and population structure of Eucommia ulmoides through chloroplast molecular markers. This information is useful for identifying different genetic populations and regions with high genetic diversity, and can also provide a basis for conservation efforts. These data can also be used to establish a “core germplasm bank” to preserve the most crucial genetic resources. Chloroplast labeling can also be used to guide breeding work and improve the genetic stability of Eucommia ulmoides populations. Zhou et al. (2021) demonstrated that chloroplast molecular markers provide significant data support and research tools for studying the genetic differences, phylogeny, and geographical history of Eucommia ulmoides.

5 Methodologies for Analyzing Chloroplast Markers

5.1 DNA extraction and amplification

To analyze chloroplast markers, high-quality DNA should be extracted first. The modified CTAB method is very effective for Eucommia ulmoides and can extract genomic DNA with high purity and good integrity, facilitating subsequent experiments (Dong et al., 2012). After extracting the DNA, amplification is the next step. Researchers usually use polymerase chain reaction (PCR) to amplify certain regions of the chloroplast genome. In some experiments, especially when the initial amount of DNA is not large, a technique called “multi-primer loop amplification” is also used to enrich chloroplast DNA (Atherton et al., 2010). This method can not only increase the quantity of chloroplast DNA, but also improve its quality, which is beneficial to the subsequent high-throughput sequencing.

5.2 Sequencing techniques

High-throughput sequencing platforms such as Illumina Genome Analyzer II (GAII) have been successfully applied in the sequencing of chloroplast DNA (Wang et al., 2010). This method can conduct comprehensive sequencing of chloroplast genomes. SNPs and Indels were identified from it (Figure 3) (Wang et al., 2018). There is also a technique called “genomic screening” (shallow sequencing of the entire genome), which can also obtain the complete chloroplast genome sequence. This method is highly efficient, not costly, and can also provide sufficient data for subsequent comparative genomic analysis.

Wang et al. (2018) discovered that the chloroplast genomic structure of Eucommia ulmoides was very stable. They compared the chloroplast genome sequence (labeled as KU204775.1) obtained from the GenBank database with the newly sequenced data using the MAFFT program and found that the sequences of the two were very similar, indicating that the internal variations of the chloroplast genome of Eucommia ulmoides are few and the structural changes are not significant. They also conducted further analysis using MAUVE software and found that the overall structure of the genome and the genetic content remained almost unchanged, proving its conservation. These results indicate that the chloroplast genome of Eucommia ulmoides is highly stable, which is of great significance for studying its evolutionary relationships, phylogenetic process and genetic protection. This stability also provides a good foundation for the development of molecular markers, which can be used in the research of species identification and genetic diversity.

5.3 Data analysis and interpretation

Researchers obtained a large amount of sequence data through high-throughput sequencing, de novo assembled it, reconstructed the complete chloroplast genome, and used comparative genomic analysis to identify variations like SNPs and Indels. The research of Wang et al. (2011) found that most SNPS in Eucommia ulmoides occurred in gene regions, and Indels were mostly concentrated in the regions between genes. Systematic genomic analysis has also been used to study evolutionary relationships, such as confirming that Eucommia ulmoides and Aucuba japonica in Japan are “sister species”. Scientists have also identified regions with particularly large variations in chloroplasts and used these regions to design primers to amplify specific target sequences, which is crucial for DNA barcoding and phylogenetic research.

6 Challenges and Limitations

6.1 Technical challenges

Liu and Zhu (2022) hold that the chloroplast genome is rather complex and of considerable scale. Only by using relatively advanced sequencing technologies and bioinformatics tools can it be accurately assembled and analyzed. New technologies such as PacBio and Hi-C have been used to complete the high-quality de novo assembly of the haploid genome of Eucommia ulmoides, with much better results than earlier studies. However, not all teams have the ability to acquire these advanced technologies, and the costs required prevent some research from being carried out in depth. How to identify and verify the regions with relatively large variations in the chloroplast genome is another difficulty. Some studies have identified variant sites suitable for phylogenetic analysis and DNA barcoding, but this process is time-consuming and laborious, requiring a large number of genomic alignings. In order to enable these variant sites to be smoothly amplified and sequenced in different species or populations, it becomes very necessary to develop specialized primers, which also increases a lot of technical difficulties (Yu et al., 2015).

6.2 Intraspecific variation

If the genetic differences within the same species are too large, the recognition of markers will become unstable and difficult to unify. A study analyzed the hybrid population of Eucommia ulmoides using ISSR and SRAP markers and found that its genetic diversity was very high, showing strong polymorphism, indicating that there was a large variation among individuals of Eucommia ulmoides. This difference may affect the repeatability and accuracy of molecular markers, making it difficult to draw clear conclusions when analyzing population relationships and evolutionary processes. There are also synonymous mutations and variations in repetitive regions in the chloroplast genome, which will also increase the complexity of the analysis. Jin et al. (2020) conducted a detailed comparison of the chloroplast genomes of two Eucommia ulmoides and found that their sequence differences were uneven. Most SNPS occurred in the gene regions, while Indels were mainly concentrated in the intergene regions.

6.3 Cross-species comparability

In their study in 2011, Nock et al. demonstrated that the evolution rate of chloroplast genes is relatively slow, making it less sensitive when studying the phylogenetic relationships of closely related species. Although some highly variable loci have been identified, the performance of these loci varies greatly among different species, which will affect their reliability in comparative studies. The chloroplast genomes of different species also have considerable differences in structure and sequence, which hinders the development of universal molecular markers. Xu et al. (2004) found in their early research between Eucommia ulmoides and its “sister species” - Aucuba japonica in Japan that there were significant differences in their chloroplast genomes, which led to the fact that the markers between the two were not necessarily interoperable. It becomes necessary to develop specific markers for a certain species or to identify areas that are relatively conservative and universal among multiple species.

7 Advances and Future Directions

7.1 Technological innovations

Zhang et al. (2016) have obtained complete chloroplast genomic data through genomic screening and next-generation sequencing (NGS), revealing its sequence differences and mutation hotspots. High-throughput sequencing platforms like Roche 454 FLX+ and PacBio are helpful for better isolation and analysis of SSRS and SNPS. This information is very important for studying the genetic diversity of Eucommia ulmoides and conducting marker-assisted breeding. Liu et al. (2022) hold that the high-density genetic maps developed using genotyping sequencing (GBS) provide an important basis for quantitative trait locus (QTL) analysis and are beneficial for identifying genetic markers related to growth traits.

7.2 Emerging applications

Meng et al. demonstrated in their 2023 study that the molecular markers found in the chloroplast genome of Eucommia ulmoides are significant in conservation genomics, as they can be used to assess genetic diversity and population structure. Wang et al. (2011) also developed gender-related markers such as AFLP and SCAR in the early stage. These markers enable researchers to determine the gender of plants when they are very young and select individuals with ideal traits in advance during breeding. Qun (2004) also combined metabolomics with molecular markers to identify different chemical types and their corresponding genetic markers, which is conducive to enhancing the medicinal value and nutritional components of Eucommia ulmoides leaves.

7.3 Research opportunities

There are still many aspects worthy of further in-depth research on the chloroplast genome of Eucommia ulmoides (Taberlet et al., 1991). Yu et al. (2015) believe that future research can pay more attention to expanding the genetic linkage map and refining the QTL analysis, which is helpful to identify more growth-related traits and the genetic mechanisms behind them. The molecular markers that have been discovered still need further research to see their specific roles in metabolic processes and plants' responses to external stress. With the help of new bioinformatics tools and machine learning methods, it is also possible to analyze large amounts of genomic data more effectively, helping to make more accurate predictions and discoveries. To truly exert the role of these molecular markers, collaboration among geneticists, conservation biologists and breeding experts is indispensable (Li et al., 2014b).

8 Concluding Remarks

The study of the chloroplast genome of Eucommia ulmoides has provided a lot of understanding of genetic structure and brought new opportunities for the development of molecular markers. The complete chloroplast genome of Eucommia ulmoides has been sequenced. It has a typical tetrad structure, with a total length of 163 586 bp and a GC content of 38.4%. Researchers identified 71 polymorphic chloroplast DNA fragments in comparative genomic analysis, among which 20 sites were selected to be used as potential markers in population genetics studies. Eight polymorphic chloroplast SSR markers (cpSSR) were also developed, further enriching the tools for genetic analysis. The kinship between Eucommia ulmoides and Aucuba japonica in Japan has also confirmed , which makes the understanding of its evolutionary background clearer.

Molecular markers developed from the chloroplast genome of Eucommia ulmoides have helped researchers understand the genetic diversity and population structure of the species. These markers can also be applied to breeding work to screen out plants with ideal traits, thereby enhancing the yield and quality of Eucommia ulmoides in both medicinal and industrial applications. The high-density genetic maps established through these markers and the growth-related QTLS identified indicate that they play an important role in understanding the genetic mechanisms of key phenotypic traits.

Future research should expand the genetic resources and genomic data of Eucommia ulmoides more. Researchers can comprehensively understand the genetic variation of Eucommia ulmoides by sequencing the chloroplast genomes from different regions, or combine the chloroplast data with the nuclear genome and mitochondrial genome to gain a more complete understanding of the genetic composition of Eucommia ulmoides. The molecular markers and candidate genes that have been identified should also continue to undergo functional research to clarify their roles in plant growth, development and response to environmental stress. Moreover, these molecular markers need to be truly applied in breeding to enhance the economic value and ecological adaptability of Eucommia ulmoides. By deeply leveraging the information provided by the chloroplast genome, scientists are expected to achieve greater breakthroughs in the protection, breeding and utilization of Eucommia ulmoides.

Acknowledgments

The author would like to express his gratitude to the two anonymous peer reviewers for their critical assessment and constructive suggestions on our manuscript.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Atherton R., McComish B., Shepherd L., Berry L., Albert N., and Lockhart P., 2010, Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform, Plant Methods, 6: 22.

https://doi.org/10.1186/1746-4811-6-22

Dong W., Liu J., Yu J., Wang L., and Zhou S., 2012, Highly variable chloroplast markers for evaluating plant phylogeny at low taxonomic levels and for DNA barcoding, PLoS One, 7(4): e35071.

https://doi.org/10.1371/journal.pone.0035071

Du Q., Wu Z., Liu P., Qing J., He F., Du L., Sun Z., Zhu L., Zheng H., Sun Z., Yang L., Wang L., and Du H., 2023, The chromosome-level genome of Eucommia ulmoides provides insights into sex differentiation and α-linolenic acid biosynthesis, Frontiers in Plant Science, 14: 1118363.

https://doi.org/10.3389/fpls.2023.1118363

Firetti F., Zuntini A., Gaiarsa J., Oliveira R., Lohmann L., and Sluys M., 2017, Complete chloroplast genome sequences contribute to plant species delimitation: a case study of the Anemopaegma species complex, American Journal of Botany, 104(10): 1493-1509.

https://doi.org/10.3732/ajb.1700302

Hu Y., Wang X., Zhang X.X., Zhou W., Chen X.Y., and Hu X.S., 2019, Advancing phylogeography with chloroplast DNA markers, Biodiversity Science, 27(2): 219-234.

https://doi.org/10.17520/biods.2018319

Jin C., Li Z., Li Y., Wang S., Li L., and Liu M., 2020, Update of genetic linkage map and QTL analysis for growth traits in Eucommia ulmoides Oliver, Forests, 11(3): 311.

https://doi.org/10.3390/f11030311

Li X., Tan W., Sun J., Du J., Zheng C., Tian X., Zheng M., Xiang B., and Wang Y., 2019, Comparison of four complete chloroplast genomes of medicinal and ornamental Meconopsis species: genome organization and species discrimination, Scientific Reports, 9: 10567.

https://doi.org/10.1038/s41598-019-47008-8

Li Y., Wang D., Li Z., Wei J., Jin C., and Liu M., 2014a, A molecular genetic linkage map of Eucommia ulmoides and quantitative trait loci (QTL) analysis for growth traits, International Journal of Molecular Sciences, 15: 2053-2074.

https://doi.org/10.3390/ijms15022053

Li Y., Wang S., Li Z., Jin C., and Liu M., 2014b, Genetic diversity and relationships among Chinese Eucommia ulmoides cultivars revealed by sequence-related amplified polymorphism, amplified fragment length polymorphism, and inter-simple sequence repeat markers, Genetics and Molecular Research, 13(4): 8704-8713.

https://doi.org/10.4238/2014.October.27.11

Li Y., Wei H., Yang J., Du K., Li J., Zhang Y., Qiu T., Liu Z., Ren Y., Song L., and Kang X., 2020, High-quality de novo assembly of the Eucommia ulmoides haploid genome provides new insights into evolution and rubber biosynthesis, Horticulture Research, 7: 183.

https://doi.org/10.1038/s41438-020-00406-w

Liu C., Wang L., Lu W., Zhong J., Du H., Liu P., Du Q., Du L., and Qing J., 2022, Construction of SNP-based high-density genetic map using genotyping by sequencing (GBS) and QTL analysis of growth traits in Eucommia ulmoides Oliver, Forests, 13(9): 1479.

https://doi.org/10.3390/f13091479

Liu W., and Zhu C., 2022, Activity evaluation of Eucommia ulmoides extracts for urinary system disease treatment and phylogenetic analysis, Journal of Biobased Materials and Bioenergy, 16(6): 830-835.

https://doi.org/10.1166/jbmb.2022.2238

Meng Y., Du Q., Du H., Wang Q., Wang L., Du L., and Liu P., 2023, Analysis of chemotypes and their markers in leaves of core collections of Eucommia ulmoides using metabolomics, Frontiers in Plant Science, 13: 1029907.

https://doi.org/10.3389/fpls.2022.1029907

Moghaddam M., Ohta A., Shimizu M., Terauchi R., and Kazempour-Osaloo S., 2021, The complete chloroplast genome of Onobrychis gaubae (Fabaceae-Papilionoideae): comparative analysis with related IR-lacking clade species, BMC Plant Biology, 22: 75.

https://doi.org/10.1186/s12870-022-03465-4

Nock C., Waters D., Edwards M., Bowen S., Rice N., Cordeiro G., and Henry R., 2011, Chloroplast genome sequences from total DNA for plant identification, Plant Biotechnology Journal, 9(3): 328-333.

https://doi.org/10.1111/j.1467-7652.2010.00558.x

Qun Z., 2004, Sequence analysis of chloroplast maturase K gene of Chinese medical plant Eucommia ulmoides, Ecologic Science, 23(2): 141-143.

Taberlet P., Gielly L., Pautou G., and Bouvet J., 1991, Universal primers for amplification of three non-coding regions of chloroplast DNA, Plant Molecular Biology, 17: 1105-1109.

https://doi.org/10.1007/BF00037152

Walker J., Zanis M., and Emery N., 2014, Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae), American Journal of Botany, 101(4): 722-729.

https://doi.org/10.3732/ajb.1400049

Wang C., Tang L., He J., Li J., and Wang Y., 2019, Ethnobotany, phytochemistry and pharmacological properties of Eucommia ulmoides: a review, The American Journal of Chinese Medicine, 47(2): 259-300.

https://doi.org/10.1142/S0192415X19500137

Wang D., Li Y., and Li Z., 2011, Identification of a male-specific amplified fragment length polymorphism (AFLP) and a sequence characterized amplified region (SCAR) marker in Eucommia ulmoides Oliv., International Journal of Molecular Sciences, 12(1): 857-864.

https://doi.org/10.3390/ijms12010857

Wang D.W., Li Y., Zhou W., and Li Z.Q., 2010, Establishment and optimization of AFLP reaction system in Eucommia ulmoides Oliv., Journal of Northwest A&F University, 38: 88-94.

Wang W., Chen S., and Zhang X., 2018, Whole-genome comparison reveals heterogeneous divergence and mutation hotspots in chloroplast genome of Eucommia ulmoides Oliver, International Journal of Molecular Sciences, 19(4): 1037.

https://doi.org/10.3390/ijms19041037

Xu W., Wang B., and Cui K., 2004, RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv., Euphytica, 136: 233-238.

https://doi.org/10.1023/B:EUPH.0000032741.99885.c4

Yu J., Wang Y., Peng L., Ru M., and Liang Z., 2015, Genetic diversity and population structure of Eucommia ulmoides Oliver, an endangered medicinal plant in China, Genetics and Molecular Research, 14(1): 2471-2483.

https://doi.org/10.4238/2015.March.30.5

Zhang W., Li Y., Zhao J., Wu C., Ye S., and Yuan W., 2016, Isolation and characterization of microsatellite markers for Eucommia ulmoides (Eucommiaceae), an endangered tree, using next-generation sequencing, Genetics and Molecular Research, 15: 10.4238.

Zhang X., Wang X., Pan L., Guo W., Li Y., and Wang W., 2023, Genome-wide identification and expression analysis of MADS-box transcription factors reveal their involvement in sex determination of hardy rubber tree (Eucommia ulmoides oliv.), Frontiers in Genetics, 14: 1138703.

https://doi.org/10.3389/fgene.2023.1138703

Zhong J., Qing J., Wang Q., Liu C., Du H., Liu P., Du Q., Du L., and Wang L., 2022, Genome-wide identification and expression analyses of the 4-coumarate: CoA ligase (4CL) gene family in Eucommia ulmoides, Forests, 13(8): 1253.

https://doi.org/10.3390/f13081253

Zhou J., Zhang S., Wang J., Shen H., Ai B., Gao W., Zhang C., Fei Q., Yuan D., Wu Z., Tembrock L., Li S., Gu C., and Liao X., 2021, Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution, Scientific Reports, 11: 9471.

https://doi.org/10.1038/s41598-021-88160-4

Zhu B., Jiang J., Zheng Y., Li S., Zhan Z., Ye P., and Cao T., 2020, Characterization of the chloroplast genome of Eucommia ulmoides, a traditional Chinese medicine plant in China, Mitochondrial DNA Part B, 5(2): 1705-1706.

https://doi.org/10.1080/23802359.2020.1748539