1 Introduction

Chloroplast genomes, also known as plastomes, are essential components of plant cells, playing a crucial role in photosynthesis and other metabolic processes. Research on chloroplast genomes has significantly advanced our understanding of plant evolution, phylogenetics, and genetic diversity. Comparative genomic studies have revealed the structural organization, gene content, and evolutionary dynamics of chloroplast genomes across various plant species (Turmel et al., 2009; Fučíková et al., 2016). These studies have also identified mutation hotspots and sequence divergence patterns, which are critical for understanding the evolutionary history and adaptation mechanisms of plants (Wang et al., 2018; Liu et al., 2023).

Eucommia ulmoides, commonly known as the hardy rubber tree, is a Tertiary relic plant endemic to China with significant medicinal and industrial value. Despite its importance, the population genetics and genomic studies of E. ulmoides have been limited due to the scarcity of genomic data. Understanding the divergence patterns in the chloroplast genome of E. ulmoides is crucial for several reasons (Qing et al., 2021). It provides insights into the evolutionary history and genetic diversity of this species, which is essential for its conservation and sustainable utilization. Furthermore, identifying polymorphic regions and molecular markers can facilitate population genetics studies and breeding programs aimed at improving the medicinal and industrial traits of E. ulmoides (Wang et al., 2018; Du et al., 2023). Besides, studying the chloroplast genome can shed light on the phylogenetic relationships between E. ulmoides and other related species, enhancing our understanding of plant evolution (Zhong et al., 2022).

This study comprehensively analyzed the divergence patterns within the chloroplast genome of E. ulmoides. By comparing complete chloroplast genome sequences, it uncovered the structural organization and sequence variation of the chloroplast genome, including single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and other polymorphic regions that can serve as molecular markers for population genetics studies. Additionally, it explored the phylogenetic relationships between E. ulmoides and related species based on chloroplast genome data. This study not only supports conservation genomics and breeding programs for E. ulmoides but also deepens the understanding of plant evolution and genetic diversity. The findings provide valuable genomic resources and new insights into the evolutionary dynamics of the E. ulmoides chloroplast genome.

2 Structure of the Chloroplast Genome in Eucommia ulmoides

2.1 General characteristics of the chloroplast genome

The chloroplast genome of Eucommia ulmoides exhibits a typical quadripartite structure, which includes a large single-copy (LSC) region, a small single-copy (SSC) region, and two inverted repeat (IR) regions. The complete chloroplast genome length of E. ulmoides is approximately 163 586 base pairs (bp) (Zhu e al., 2020). The overall GC content of the chloroplast genome is 38.4%, with the LSC region being 86 773 bp, the SSC region 14 167 bp, and the IR regions each 31 323 bp. This structure is consistent with the general organization observed in many angiosperms.

2.2 Gene content and organization

The chloroplast genome of E. ulmoides contains a total of 135 genes, which include 89 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes (Zhu e al., 2020). The gene content and order are highly conserved, with no significant variations observed among different samples of E. ulmoides (Wang et al., 2018). The majority of the SNPs detected in the chloroplast genome are located in the gene regions, while most of the indels were found in the intergenic spacers. Additionally, all the putative coding-region-located SNPs identified were synonymous mutations, indicating a high level of conservation in the protein-coding regions.

2.3 Comparison with other plant species

When compared to other plant species, the chloroplast genome of E. ulmoides shows a high degree of structural similarity. For instance, the chloroplast genomes of various eucalypt species also exhibit a conserved gene content and order, with minimal length mutations in protein-coding genes (Bayly et al., 2013; Li et al., 2021). Similarly, the chloroplast genomes of green algae such as Pyramimonas and Monomastix display significant structural conservation, although they exhibit more variability in gene content and order compared to E. ulmoides (Figure 1) (Turmel et al., 2009; Liu et al., 2023).

Figure 1 Sequence coverages in the 15 ulvophycean chloroplast genomes compared in this study (Adopted from Turmel et al., 2017) Image caption: (a) Sizes of the SSC, IR and LSC regions. Red arrows indicate the direction of transcription of the rRNA operon in IR-containing genomes. Genomes lacking the IR are represented in grey. The names of the newly examined taxa are indicated in red. (b) Amounts of coding, intronic, intergenic and small repeated sequences (≥30bp). Note that intron-encoded genes were not considered as coding sequences but rather as intron sequences (Adopted from Turmel et al., 2017)

In terms of phylogenetic relationships, E. ulmoides is closely related to Aucuba japonica, as confirmed by chloroplast phylogenomic analyses. This relationship is consistent with the findings from other studies that have examined the chloroplast genomes of related species within the Garryales order. The conservation of the chloroplast genome structure and gene content across different species highlights the evolutionary stability of this organelle and its importance in plant biology. Overall, the chloroplast genome of E. ulmoides provides valuable insights into the genetic and evolutionary characteristics of this species, and it serves as a crucial resource for further studies on conservation genomics and population genetics (Wang et al., 2018; Zhu e al., 2020; Du et al., 2023).

3 Methods for Identifying Divergence Patterns

3.1 Data collection and sequence alignment

To identify divergence patterns in the chloroplast genome of Eucommia ulmoides, complete chloroplast genome sequences was first collected. One complete chloroplast genome was generated using the genome skimming approach, and it was compared to another available chloroplast genome of E. ulmoides (Wang et al., 2018; Zhu et al., 2020). The sequences were aligned using standard bioinformatics tools to ensure accurate comparison of genomic regions. This alignment process is crucial for identifying SNPs and indels across the genomes.

3.2 Phylogenetic analysis techniques

Phylogenetic analysis was conducted to understand the evolutionary relationships and divergence patterns within the chloroplast genomes. Multiple phylogenetic methods were employed, including parsimony analysis, which was sensitive to taxon sampling and could be computationally intensive with large datasets. Moreover, high-throughput sequencing data was used to analyze the phylogenetic signals of different genomic regions, including the complete chloroplast genome (Vargas et al., 2017; Qing et al., 2021). This approach helps in resolving phylogenetic incongruences that may arise due to hybridization and introgression events (Zhong et al., 2022).

3.3 Statistical methods for divergence detection

Statistical methods were applied to detect divergence patterns and identified mutation hotspots within the chloroplast genome. It identified heterogeneous sequence divergence patterns in different regions of the chloroplast genomes, with a significant number of SNPs located in gene regions and indels distributed in intergenic spacers (Wang et al., 2018). To further validate these findings, the D-statistic (ABBA-BABA test) was used to detect introgression and hybridization events, which could confound phylogenetic analyses (Vargas et al., 2017). These statistical tools are essential for accurately detecting and interpreting divergence patterns in the chloroplast genome. By integrating these methods, it’s able to identify and characterize the divergence patterns in the chloroplast genome of Eucommia ulmoides, providing valuable insights for future conservation genomics studies (Fučíková et al., 2016).

4 Divergence Patterns in Coding Regions

4.1 Identification of divergent genes

A comprehensive comparison of E. ulmoides chloroplast genome revealed significant sequence divergence patterns. Specifically, 59 out of 75 detected SNPs were located in gene regions, indicating a high level of genetic variability within these coding regions. Additionally, all 40 putative coding-region-located SNPs were identified as synonymous mutations, suggesting that these variations do not alter the amino acid sequences of the encoded proteins (Wang et al., 2018). This pattern of synonymous mutations highlights the presence of genetic diversity without immediate functional consequences on the protein level.

4.2 Functional implications of divergence

The functional implications of the identified divergence in the coding regions of the E. ulmoides chloroplast genome are multifaceted. Although the synonymous mutations do not change the protein sequences, they can still influence gene expression, stability of mRNA, and the efficiency of protein translation. These subtle changes can have downstream effects on the plant's physiological processes and adaptation mechanisms. For instance, the high expression of the ω-3 fatty acid desaturase coding gene (EU0103017) is crucial for the biosynthesis of α-linolenic acid, which is a key component in plant metabolic pathways (Du et al., 2023). Such functional implications underscore the importance of understanding genetic divergence in the context of the overall metabolic and adaptive strategies (Qing et al., 2021; Zhong et al., 2022).

4.3 Evolutionary significance of coding region divergence

The evolutionary significance of coding region divergence about the E. ulmoides chloroplast genome is profound. The presence of synonymous mutations suggests a mechanism of maintaining genetic diversity while preserving essential protein functions. This balance allows the plant to adapt to varying environmental conditions without compromising its core biological processes. Furthermore, the identification of polymorphic cpDNA fragments and the development of molecular markers provide valuable tools for studying the population genetics and evolutionary history of E. ulmoides (Wang et al., 2018). The evolutionary trajectory of E. ulmoides is also marked by whole-genome duplication events, which have contributed to its genetic complexity and adaptability (Du et al., 2023). These findings highlight the role of genetic divergence in shaping the evolutionary path and ecological success of E. ulmoides (Zhu et al., 2020).

The divergence patterns in the coding regions of the E. ulmoides chloroplast genome reveal a complex interplay of genetic variability, functional implications, and evolutionary significance. These insights not only enhance our understanding of the genetic architecture of E. ulmoides but also provide a foundation for future research on its conservation and utilization.

5 Divergence Patterns in Non-Coding Regions

5.1 Analysis of intergenic regions

Intergenic regions in the chloroplast genome of Eucommia ulmoides exhibit significant divergence patterns. A comprehensive comparison between two E. ulmoides chloroplast genomes revealed that most of the detected indels were distributed in the intergenic spacers, highlighting these regions as hotspots for structural variations (Wang et al., 2018). This pattern was consistent with findings in other plant species, where intergenic regions tend to show higher rates of indel mutations compared to coding regions (Yamane et al., 2006). The divergence in these regions is often driven by microstructural changes such as single nucleotide indels and tandem repeat indels, which are biased towards A/T-rich sequences.

5.2 Structural variations in non-coding DNA

Structural variations in non-coding DNA, particularly in intergenic regions, play a crucial role in the evolution of the chloroplast genome. In E. ulmoides, the variations in genome size were attributed to DNA repeat variations, which have been predominantly found in non-coding regions (Wang et al., 2018). These structural changes include insertions, deletions, and the integration of foreign DNA sequences, which could lead to genome rearrangements (Liu et al., 2023). The presence of pseudogenes and their faster evolutionary rate than coding regions further contribute to the structural diversity in non-coding DNA. Additionally, microsatellites or simple sequence repeats (SSRs) are abundant in non-coding regions and exhibit taxon-specific variations, influencing genome structure and function.

5.3 Role of non-coding regions in genome evolution

Non-coding regions play a pivotal role in the evolution of the chloroplast genome by acting as sites for adaptive evolution and selective constraint. In E. ulmoides, the high rate of indel mutations in intergenic regions suggests that these regions are under selective pressure, contributing to the overall genetic diversity of the species (Wang et al., 2018). Studies in other organisms, such as Drosophila, have shown that non-coding DNA can evolve under both purifying selection and positive selection, indicating its functional importance (Andolfatto, 2005). The adaptive evolution of non-coding regions is also evident in the integration of foreign DNA sequences, which can drive genome rearrangements and influence the evolutionary trajectory of the chloroplast genome (Liu et al., 2023). Overall, the divergence patterns in non-coding regions are crucial for understanding the mechanisms of genome evolution and the adaptive strategies of E. ulmoides.

6 Case Studies

6.1 Analysis of sex differentiation and divergence patterns in the Eucommia ulmoides genome

With the advancement of genomics technology, research on the genome of Eucommia ulmoides has gradually deepened, providing new insights into the mechanisms of sex differentiation and the biosynthesis of important metabolites. Du et al. (2023) successfully constructed a high-quality female Eucommia ulmoides genome using PacBio and Hi-C technologies and reassembled the male genome released in 2018. Comparative analysis of the male and female genomes revealed key genes and molecular mechanisms involved in sex differentiation and α-linolenic acid biosynthesis (Figure 2).

Figure 2 Internal anatomical structure and external morphological characteristics of male and female flower buds at different differentiation stages (Adopted from Du et al., 2023) Image caption: The image shows significant changes in the male and female flower buds of Eucommia ulmoides during four stages: inflorescence primordium formation, bract differentiation, pistil and stamen differentiation, and their morphological formation. Notably, during the pistil and stamen differentiation stage, the internal structures of the male and female flower buds begin to exhibit clear differences, with male buds forming stamen clusters and female buds forming pistils. This image illustrates the gradual morphological changes during the sex differentiation process in Eucommia ulmoides flower buds, providing direct morphological evidence for understanding the molecular mechanisms of sex differentiation (Adapted from Du et al., 2023)

The study identified several MADS-box genes closely related to floral organ development, such as EuAP3 and EuAG, which showed significant expression differences during the development of male flower buds, suggesting their important regulatory roles in the formation of male reproductive organs. Through gene function annotation, key enzyme genes related to α-linolenic acid synthesis, such as FAD7, were identified. The expression levels of these genes were significantly higher in fruits and leaves than those in other tissues, indicating that these are the main sites for the synthesis and accumulation of α-linolenic acid. The study not only provides new perspectives for the evolutionary analysis of Eucommia ulmoides but also deepens the understanding of its sex differentiation and α-linolenic acid accumulation mechanisms, laying a solid foundation for subsequent genetic improvements and cultivar development. It will accelerate the development of superior Eucommia ulmoides varieties, enhancing its value in both medicinal and economic fields.

6.2 Divergence in rbcL gene

The rbcL gene, which encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), is a critical component of the photosynthetic machinery in plants. In the chloroplast genome of Eucommia ulmoides, the rbcL gene has been identified as one of the regions exhibiting significant divergence. Comparative analysis with other species, such as those in the Helianthus genus, has shown that the rbcL gene can experience positive selection, particularly during transitions between annual and perennial life cycles (Azarin et al., 2021). This suggests that the rbcL gene in E. ulmoides may also be subject to evolutionary pressures that could influence its function and efficiency in photosynthesis (Bernabeu and Rosselló, 2021).

6.3 Inversions and repeats in the chloroplast genome

Inversions and repeats are common structural variations in chloroplast genomes that can lead to genome size variation and affect genome stability. In the chloroplast genome of Eucommia ulmoides, DNA repeat variations have been identified as a significant factor contributing to genome size differences between individuals (Wang et al., 2018; Abdullah et al., 2020). These variations can result in heterogeneous sequence divergence patterns, with a notable number of SNPs and indels distributed across different regions of the genome. Similar patterns have been observed in other plant species, such as the Helianthus genus, where SSRs in non-coding regions contribute to genome size variation (Azarin et al., 2021; Turudić et al., 2022).

7 Environmental and Evolutionary Factors Influencing Divergence

7.1 Impact of environmental changes on genome divergence

Environmental changes play a significant role in the divergence of the chloroplast genome in Eucommia ulmoides. The heterogeneous sequence divergence patterns observed in different regions of the E. ulmoides chloroplast genomes suggest that environmental pressures may influence the mutation rates and types in specific genomic regions. For instance, the majority of SNPs were found in gene regions, while indels were more common in intergenic spacers, indicating that different environmental factors might affect these regions differently (Wang et al., 2018; Zhang et al., 2020; Sugimoto et al., 2020). Additionally, the study of chloroplast genomes in other species, such as Ulva, has shown that environmental pressures could drive the compactness of genome organization and decrease overall GC content, which might also be applicable to E. ulmoides (Liu et al., 2023).

7.2 Co-evolution with other organelles

The co-evolution of chloroplasts with other organelles, particularly mitochondria, has been a crucial factor in the divergence of the chloroplast genome. Both chloroplasts and mitochondria originated from endosymbiotic events, and their genomes have undergone significant reduction and specialization over time. The episodic influx of prokaryotic genes into the eukaryotic lineage during major evolutionary transitions, such as the origin of chloroplasts and mitochondria, has contributed to the divergence of these organelles (Ku et al., 2015; Calderon and Strand, 2021). In land plants, despite sharing the same cell lineage and being dependent on the same nucleus for most of their gene products, chloroplast and mitochondrial genomes exhibit remarkably different tempos and patterns of evolutionary change, highlighting the complex interplay between these organelles (Tyszka et al., 2023).

7.3 Adaptive significance of chloroplast genome divergence

The divergence of the chloroplast genome in E. ulmoides has adaptive significance, particularly in terms of its medicinal and industrial value. The identification of polymorphic cpDNA fragments and cpSSR loci provides valuable molecular markers for population genetics studies, which can aid in the conservation and breeding of superior varieties of E. ulmoides (Wang et al., 2018). Furthermore, the adaptive significance of chloroplast genome divergence is evident in other species as well. For example, in Populus, comprehensive analyses of chloroplast genomes have revealed dynamic patterns of evolution that are crucial for understanding the adaptive strategies of the genus (Zhou et al., 2021). Similarly, the chloroplast genomes of green algae such as Pyramimonas and Monomastix have shown that major reductions in gene content and restructuring of the chloroplast genome occurred in conjunction with important changes in cell organization, indicating adaptive responses to environmental pressures (Turmel et al., 2009).

The divergence of the chloroplast genome in Eucommia ulmoides is influenced by environmental changes, co-evolution with other organelles, and adaptive significance. These factors collectively contribute to the unique genomic architecture and evolutionary trajectory of this important medicinal and industrial plant.

8 Implications for Conservation and Breeding

8.1 Conservation of genetic diversity in Eucommia ulmoides

The identification of heterogeneous sequence divergence patterns in the chloroplast genome of Eucommia ulmoides has significant implications for the conservation of this species. The discovery of 71 polymorphic cpDNA fragments, including 20 loci selected as potential molecular markers, provides valuable tools for population genetics studies. These markers can be used to assess genetic diversity within and between populations, which is crucial for developing effective conservation strategies. By understanding the genetic structure and diversity of E. ulmoides populations, conservationists can identify genetically distinct populations that may require targeted conservation efforts to preserve the overall genetic diversity of the species (Wang et al., 2018; Wang et al., 2023; Zhang et al., 2023).

8.2 Application of divergence patterns in breeding programs

The detailed analysis of the chloroplast genome has revealed specific regions with high levels of sequence divergence, including SNPs and indels. These divergence patterns can be leveraged in breeding programs to select for desirable traits. For instance, the identification of 40 putative coding-region-located SNPs, all of which are synonymous mutations, suggests that these regions can be targeted for marker-assisted selection without affecting the protein function. Additionally, the development of eight polymorphic cpSSR loci provides further molecular markers that can be used to track the inheritance of specific traits in breeding programs. This genomic information can facilitate the development of superior E. ulmoides varieties with enhanced medicinal and industrial properties (Wang and Zhang, 2017; Wang et al., 2018).

8.3 Potential for genetic improvement

The comprehensive genomic data, including the high-quality chromosome-level genome of both female and male E. ulmoides, offers a robust foundation for genetic improvement efforts. The identification of key genes involved in sex differentiation, such as EuAP3 and EuAG, and the high expression of the ω-3 fatty acid desaturase coding gene EU0103017, which is linked to high α-linolenic acid content, provides specific targets for genetic manipulation. By utilizing these genomic insights, breeders can develop E. ulmoides varieties with improved traits, such as higher α-linolenic acid content, which is valuable for its medicinal properties. Furthermore, the understanding of whole-genome duplication events and their impact on the genetic architecture of E. ulmoides can guide the selection of breeding strategies that maximize genetic gain while maintaining genetic diversity (Liu et al., 2022; Du et al., 2023).

The integration of chloroplast genome divergence patterns and comprehensive genomic data into conservation and breeding programs holds great promise for the preservation and enhancement of Eucommia ulmoides. These efforts will ensure the sustainable use of this valuable species for medicinal and industrial applications (Li et al., 2020; Deng et al., 2022).

9 Concluding Remarks

In this study, we conducted a comprehensive analysis of the chloroplast (cp) genome of Eucommia ulmoides, revealing significant insights into its structure and divergence patterns. Our findings indicate that the cp genome of E. ulmoides exhibits heterogeneous sequence divergence, with most SNPs located in gene regions and most indels in intergenic spacers. Notably, all coding-region-located SNPs were synonymous mutations, suggesting a conservation of protein function despite genetic variability. Additionally, we identified 71 polymorphic cpDNA fragments, with 20 loci proposed as potential molecular markers for future population genetics studies. These results contribute to a deeper understanding of the genetic diversity and evolutionary dynamics within the cp genome of E. ulmoides.

The insights gained from this study have several potential applications. In conservation genetics, the identified molecular markers can be utilized in efforts to monitor genetic diversity and manage breeding programs for E. ulmoides, an endangered species with significant medicinal and industrial value. The cp genome data can also aid in resolving phylogenetic relationships within the Garryales order, as demonstrated by the confirmed sister relationship between E. ulmoides and Aucuba japonica. Furthermore, understanding the cp genome structure and divergence can facilitate biotechnological applications, particularly in genetic engineering approaches aimed at enhancing desirable traits in E. ulmoides, such as increased production of bioactive compounds.

Future research should focus on several key areas to further elucidate the patterns and implications of chloroplast genome divergence in E. ulmoides. Expanded genomic comparisons, involving a broader range of E. ulmoides populations and related species, will provide a more comprehensive understanding of cp genome evolution and divergence. Functional studies are needed to investigate the consequences of identified SNPs and indels, particularly those in non-coding regions, to clarify their roles in gene regulation and adaptation. Additionally, exploring how environmental factors influence cp genome variation and expression can shed light on the adaptive mechanisms of E. ulmoides in different ecological contexts. Utilizing high-throughput sequencing technologies and advanced phylogenomic methods will enhance the resolution of evolutionary relationships and divergence times within the Garryales and other related taxa. By addressing these research directions, we can gain a more detailed and nuanced understanding of chloroplast genome divergence, which will ultimately contribute to the conservation and sustainable utilization of E. ulmoides.

Acknowledgments

The authors extend sincere thanks to two anonymous peer reviewers for their feedback on the manuscript.

Funding

This research was funded by a grant from the National Natural Science Foundation of China [31870285, 30660146]，Guizhou Academy of Agricultural Sciences Talent Special Project (No. 2023-02 and 2024-02), National High Tech nology Research and Development Program of China (“863” Program) grant number 2013AA102605-05, Talent Base for Germplasm Resources Utilization and Innovation of Characteristic Plant in Guizhou (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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