DNA methylation regulates the secondary metabolism of saponins to improve the adaptability of Eleutherococcus senticosus during drought stress.

Plant growth and development can be significantly impacted by drought stress. Plants will adjust the synthesis and accumulation of secondary metabolites to improve survival in times of water constraint. Simultaneously, drought stress can lead to modifications in the DNA methylation status of plants, and these modifications can directly impact gene expression and product synthesis by changing the DNA methylation status of functional genes involved in secondary metabolite synthesis. However, further research is needed to fully understand the extent to which DNA methylation modifies the content of secondary metabolites to mediate plants' responses to drought stress, as well as the underlying mechanisms involved. Our study found that in Eleutherococcus senticosus (E. senticosus), moderate water deprivation significantly decreased DNA methylation levels throughout the genome and at the promoters of EsFPS, EsSS, and EsSE. Transcription factors like EsMYB-r1, previously inhibited by DNA methylation, can re-bind to the EsFPS promotor region following DNA demethylation. This process promotes gene expression and, ultimately, saponin synthesis and accumulation. The increased saponin levels in E. senticosus acted as antioxidants, enhancing the plant's adaptability to drought stress.

[Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus].

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Genome-wide identification of MBD gene family members in Eleutherococcus senticosus with their expression motifs under drought stress and DNA demethylation.

BACKGROUND: Methyl-binding domain (MBD) is a class of methyl-CpG-binding domain proteins that affects the regulation of gene expression through epigenetic modifications. MBD genes are not only inseparable from DNA methylation but have also been identified and validated in various plants. Although MBD is involved in a group of physiological processes and stress regulation in these plants, MBD genes in Eleutherococcus senticosus remain largely unknown. RESULTS: Twenty EsMBD genes were identified in E. senticosus. Among the 24 chromosomes of E. senticosus, EsMBD genes were unevenly distributed on 12 chromosomes, and only one tandem repeat gene existed. Collinearity analysis showed that the fragment duplication was the main motif for EsMBD gene expansion. As the species of Araliaceae evolved, MBD genes also evolved and gradually exhibited different functional differentiation. Furthermore, cis-acting element analysis showed that there were numerous cis-acting elements in the EsMBD promoter region, among which light response elements and anaerobic induction elements were dominant. The expression motif analysis revealed that 60% of the EsMBDs were up-regulated in the 30% water content group. CONCLUSIONS: By comparing the transcriptome data of different saponin contents of E. senticosus and integrating them with the outcomes of molecular docking analysis, we hypothesized that EsMBD2 and EsMBD5 jointly affect the secondary metabolic processes of E. senticosus saponins by binding to methylated CpG under conditions of drought stress. The results of this study laid the foundation for subsequent research on the E. senticosus and MBD genes.

Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Chromosomal-scale genome assembly of Eleutherococcus senticosus provides insights into chromosome evolution in Araliaceae.

Siberian ginseng (Eleutherococcus senticosus, also known as ciwujia) belongs to the Araliaceae family, which contains more than 1,500 species in 41 genera with diverse chromosome numbers and genome sizes. General consensus posits that ancient whole-genome duplication events and rapid evolutionary radiation are the driving forces for this variation in genome properties. In an attempt to generate more genomic information for the Araliaceae family, we report a 1.30 Gb high-quality draft genome assembly (contig N50 = 309.43 kb) of E. senticosus via PacBio long reads and Hi-C chromatin interaction maps. We found that transposable elements accounted for 72.82% of the genome and a total of 36,372 protein-coding genes were predicted. Comparative analyses of the E. senticosus, Panax notoginseng and Daucus carota genomes revealed a burst expansion of Tekay chromoviral elements in Araliaceae after its divergence with Apiaceae. We also found that E. senticosus underwent a lineage-specific whole-genome duplication event Es-α and a whole-genome duplication event Araliaceae-ß that was probably shared by all Araliaceae species. Even though the rediploidization of the E. senticosus genome is evident, pathway analyses show that these two whole-genome duplication events may have contributed to the adaptation of E. senticosus to a cold environment. Taken together, the high-quality genome assembly of E. senticosus provides a valuable genomic resource for future research on the evolution of Araliaceae.

Isolation and Functional Characterization of New Terpene Synthase Genes from Traditional Edible Plants.

Terpene synthase (TPS) genes were isolated and functionally characterized from three traditional edible plants, Acanthopanax sciadophylloides ("Koshiabura") and Acanthopanax sieboldianus ("Himeukogi"), belonging to the family Araliaceae, and Curcuma zedoaria (zedoary, "Gajutsu"), belonging to the family Zingiberaceae. These plants emit characteristic fragrances and are used for traditional foods and folk medicines. From their fragrant tissues, i.e., sprouts of Araliaceae plants and developing rhizomes of zedoary, total RNAs were extracted and reverse transcribed. The resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. From the contig sequences obtained, full-length Tps genes were amplified by PCR with newly synthesized primer sets. The isolated full-length genes were introduced into engineered Escherichia coli cells, which can utilize acetoacetate to synthesize farnesyl diphosphate, the substrate for TPSs, through the mevalonate pathway. TPS products synthesized in the transformed E. coli cells were analysed by gas chromatography-mass spectrometry, nuclear magnetic resonance, and optical rotation. Consequently, the isolated Tps genes were found to encode ß-caryophyllene synthase, germacrene D synthase, linalool/(3S)-(+)-nerolidol synthase, ß-eudesmol synthase, and germacrene B synthase. These results lead us to expect that some of the effective ingredients in folk medicines are volatile terpenes and that intake of traditional foods including these edible plants would have some positive effects on our health.

The Identification of Araliaceae Species by ITS2 Genetic Barcoding and Pollen Morphology.

The genetic barcode ITS2 (ITS: internal transcribed spacer) and pollen morphology were used for the identification of the pharmacologically valuable wild Araliaceae species Panax ginseng, Oplopanax elatus, Aralia elata, Aralia continentalis, Eleutherococcus senticosus, and Eleutherococcus sessiliflorus inhabiting the natural forests of Primorye, Russia. The ITS2 locus successfully identified all six species, which supports the use of ITS2 as a standard barcode for medicinal plants. However, the ITS2 locus was insufficient for intra-specific discrimination in these species, neither within Primorye nor from other world representatives within GenBank. Araliaceae pollen was confirmed to undergo size-reducing metamorphosis. The final morphotypes were species-specific for each of the six species but could not discriminate intra-species geographic localities within Primorye. The morphologies of the final pollen morphotypes from homologous species inhabiting other parts of the world are not yet known. Therefore, whether pollen is applicable for Araliaceae intra-species discrimination between Primorye and other world localities could not be established. Based on these findings, we propose that the ITS2 genetic barcode and the final pollen morphotypes are suitable for the identification of Araliaceae species. However, further studies will be needed to determine the suitability of genetic and pollen traits for Araliaceae geographic authentication.

Authentication of Eleutherococcus and Rhodiola herbal supplement products in the United Kingdom.

Siberian ginseng (Eleutherococcus senticosus, Araliaceae) and roseroot (Rhodiola rosea, Rosaceae) are popular herbal supplements which have been shown to improve resilience to conditions such as stress and exhaustion. Using DNA barcoding methods we tested 25 Siberian ginseng and 14 roseroot products which are widely available to UK customers to test whether the herbal ingredient stated on the label is also in the product. All Siberian ginseng supplements contained E. senticosus, however, 36% also contained an Eleutherococcus species other than E. senticosus. In three out of the 13 roseroot products which produced amplifiable DNA, we could only retrieve sequences matching alfalfa (declared on the product label) and fenugreek (not declared). In the other 10 supplements Rhodiola was detected but only five matched the target species R. rosea. As DNA can get severely degraded during the manufacturing process we did not take the absence of Rhodiola DNA as proof for a compromised product. Contamination could explain the presence of non-target species such as fenugreek but is unlikely to be account for the detection of congeneric Rhodiola species in roseroot preparations. Our results therefore suggest that the substitution or mixing of the target medicinal ingredient in these two popular supplements with other species is common.

The complete chloroplast genome of Eleutherococcus gracilistylus (W.W.Sm.) S.Y.Hu (Araliaceae).

Eleutherococcus gracilistylus is a plant species that is close to E. senticosus, a famous medicinal plant called Siberian ginseng. The complete chloroplast genome sequence of the E. gracilistylus was determined by de novo assembly using whole genome next generation sequences. The chloroplast genome of E. gracilistylus was 156 770 bp long and showed distinct four partite structures such as a large single copy region of 86 729 bp, a small single copy region of 18 175 bp, and a pair of inverted repeat regions of 25 933 bp. The overall GC contents of the genome sequence were 36.8%. The chloroplast genome of E. gracilistylus contains 79 protein-coding sequences, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis with the reported chloroplast genomes confirmed close taxonomical relationship of E. gracilistylus with E. senticosus.

Transcriptome profiling and digital gene expression by deep sequencing in early somatic embryogenesis of endangered medicinal Eleutherococcus senticosus Maxim.

Somatic embryogenesis (SE) has been studied as a model system to understand molecular events in physiology, biochemistry, and cytology during plant embryo development. In particular, it is exceedingly difficult to access the morphological and early regulatory events in zygotic embryos. To understand the molecular mechanisms regulating early SE in Eleutherococcus senticosus Maxim., we used high-throughput RNA-Seq technology to investigate its transcriptome. We obtained 58,327,688 reads, which were assembled into 75,803 unique unigenes. To better understand their functions, the unigenes were annotated using the Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. Digital gene expression libraries revealed differences in gene expression profiles at different developmental stages (embryogenic callus, yellow embryogenic callus, global embryo). We obtained a sequencing depth of >5.6 million tags per sample and identified many differentially expressed genes at various stages of SE. The initiation of SE affected gene expression in many KEGG pathways, but predominantly that in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. This information on the changes in the multiple pathways related to SE induction in E. senticosus Maxim. embryogenic tissue will contribute to a more comprehensive understanding of the mechanisms involved in early SE. Additionally, the differentially expressed genes may act as molecular markers and could play very important roles in the early stage of SE. The results are a comprehensive molecular biology resource for investigating SE of E. senticosus Maxim.

[Establishment of prokaryotic expression and optimization ox expression conditions of Eleutherococcus senticosus P450 gene].

According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.

[Expression profiles analysis of two member of squaleneepoxidase gene family from Eleutherococcus senticosus].

In order to find the characteristics of two members of gene family of squaleneexpoxidase (SE) , a quantitative real time PCR method was developed to analyze the expression of Eleutherococcus senticosus SE1 and SE2 gene from different growth periods and in different organs. The result indicated that all the expression of SE2 more than SE1 in the whole growth period and organs of E. senticosus. And in the whole growth period, expression of SE1 showed a low-high-low characteristic. Both expression of SE2 and growth period showed the same trend. The lowest content of the expression was in the roots. SE1 expression have been improved more than SE2 when treated with MeJA. The expression of E. senticosus SE1 and saponins content had significantly positive correlation (P < 0.05) and the correlation coefficients was 0. 858, while the correlation was not significant for SE2. That indicated that SE1 played a key enzyme gene in the biosynthesis of triterpenoidsaponins

[Genetic variation of the relict species Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem. (Araliaceae) in Primorsky Krai].

Based on the analysis of 17 genes encoding the allozyme diversity of 12 enzyme systems, data were obtained on the genetic variation of a relict of the Tertiary flora, a valuable medicinal plant Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem. (Araliaceae) in the Russian area of its habitat. Indicators of polymorphism for populations had rather high values on average (P95 = 42.4%, A = 1.55, H(o) = 0.211, and H(e) = 0.168), which are comparable with the known data for populations of A. sessiliflorus from the peninsula of Korea. The level of genetic diversity and its distribution among populations reflects the interaction of several factors, among which the most important are the historical past of the species, genetic drift, and the plasticity of the reproduction system. The obtained data can serve as a basis for the conservation of genetic resources of Far Eastern Araliaceae species.

Large-scale plantlet conversion and ex vitro transplantation efficiency of Siberian ginseng by bioreactor culture.

To achieve large-scale low-cost ex vitro acclimatization of Siberian ginseng plants, heart- and torpedo-shaped secondary somatic embryos (SEs) induced from germinated SEs on agar medium were collected and then inoculated to 10-l bubble column bioreactor, respectively. For plantlet conversion, inoculation of torpedo-shaped secondary SEs was more effective than heart-shaped SEs. TS2 (culture of torpedo-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets had a higher root number and leaf number and larger leaf area than did HS3 (culture of heart-shaped SEs in a bioreactor with a 3-week subculture interval) and HS2 (culture of heart-shaped SEs in a bioreactor with a 2-week subculture interval) plantlets. Of these converted plants, TS2 plantlets had higher survival rate (83.7%) and growth characteristics after transplantation in a simple shed covered with a 50% sunshade net only for 6 months. TS2 plantlets also showed significantly lower H2O2 content and significantly increased superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) expression levels as compared to HS2 plants when exposure to ex vitro conditions.

[Analysis on codon usage of chloroplast genome of Eleutherococcus senticosus].

OBJECTIVE: To analyze the codon usage of chloroplast genome and the influencing factor in Eleutherococcus senticosus. METHOD: Codon of 52 genes, which were selected from the chloroplast genome sequence of E. senticosus, was multivariate statistical and correspondence analyzed using CodonW and SPSS software. RESULT: GC content at the three position of codons by turns was 46.46%, 38.26%, 29.88%, whereas GC1 and GC2 had a significant correlation coefficient (P < 0.01). The correlation coefficient with GC12, and GC3 was 0.205 and was not significant correlated. There were 30 codons which relative synonymous codon usage was greater than 1 and 29 codons end with A and T. In the corresponding analysis, the first axis shows 10.35% variation. And there was significant correlation coefficient between ENC and GC3. The correlation coefficients with GC3 and ENC were -0.288 and 0.353, respectively. We defined 16 codons from 16 amino acids as the major preference codons in chloroplast genome of E. senticosus. CONCLUSION: The third positions for all codon are preferred to ending with A and T. The codon usage bias is formed under effect of mutation and selection, as well as other factors. But the selection will have a far greater impact than others.

Callose deposition is required for somatic embryogenesis in plasmolyzed Eleutherococcus senticosus zygotic embryos.

Dynamic changes in callose content, which is deposited as a plant defense response to physiological changes, were analyzed during somatic embryogenesis in Eleutherococcus senticosus zygotic embryos plasmolyzed in 1.0 M mannitol. During plasmolysis, callose deposition was clearly observed inside the plasma membrane of zygotic embryo epidermal cells using confocal laser scanning microscopy. The callose content of zygotic embryos gradually increased between 0 and 12 h plasmolysis and remained stable after 24 h plasmolysis. During eight weeks induction of somatic embryogenesis, the callose content of explants plasmolyzed for 12 h was slightly higher than explants plasmolyzed for 6 or 24 h, with the largest differences observed after 6 weeks culture, which coincided with the maximum callose content and highest number of globular somatic embryos. The highest frequency of somatic embryo formation was observed in explants plasmolyzed for 12 h. The somatic embryo induction rate and number of somatic embryos per explant were markedly different in zygotic embryos pretreated with plasmolysis alone (78.0%, 43 embryos per explant) and those pretreated with plasmolysis and the callose synthase inhibitor 2-deoxy-d-glucose (11.5%, 8 embryos per explant). This study indicates that callose production is required for somatic embryogenesis in plasmolyzed explants.

[Cloning of Eleutherococcus senticosus calmodulin gene and effect of endophytic fungus on expression amount of gene].

OBJECTIVE: To clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene. METHOD: The CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR. RESULT: The full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control. CONCLUSION: The CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.

[Molecular cloning of farnesyl diphosphate synthase from Eleutherococcus senticosus and its bioinformatics and expression analysis].

OBJECTIVE: To clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene. METHOD: The FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR. RESULT: The full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05). CONCLUSION: The FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

[Cloning and sequence analysis on cDNA of squalene epoxidase gene in Eleutherococcus senticosus].

OBJECTIVE: To clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus. METHOD: Total RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy. RESULT: Sequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices. CONCLUSION: The two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

The complete chloroplast DNA sequence of Eleutherococcus senticosus (Araliaceae); comparative evolutionary analyses with other three asterids.

This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or intron regions. These medium size repeats may contribute to developing a cp genome-specific gene introduction vector because the region may use for specific recombination sites.