Chaenomelin, a New Phenolic Glycoside, and Anti-Helicobacter pylori Phenolic Compounds from the Leaves of Salix chaenomeloides.

Salix chaenomeloides Kimura, commonly known as pussy willow, is a deciduous shrub and tree belonging to the Salicaceae family. The genus Salix spp. has been known as a healing herb for the treatment of fever, inflammation, and pain relief. The current study aimed to investigate the potential bioactive natural products from S. chaenomeloides leaves and evaluate their antibacterial activity against Helicobacter pylori. A phytochemical investigation of the ethanol (EtOH) extract of S. chaenomeloides leaves led to the isolation of 13 phenolic compounds (1-13) from the ethyl acetate (EtOAc) fraction, which showed antibacterial activity against H. pylori strain 51. The chemical structure of a new phenolic glycoside, chaenomelin (1), was established by a detailed analysis of 1D and 2D (1H-1H correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC)) nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectroscopy (HR-ESIMS), and chemical reactions. The other known compounds were identified as 5-O-trans-p-coumaroyl quinic acid methyl ester (2), tremulacin (3), citrusin C (4), benzyl 3-O-ß-d-glucopyranosyl-7-hydroxybenzoate (5), tremuloidin (6), 1-[O-ß-d-glucopyranosyl(1→2)-ß-d-glucopyranosyl]oxy-2-phenol (7), arbutin cinnamate (8), tremulacinol (9), catechol (10), 4-hydroxybenzaldehyde (11), kaempferol 3-rutinoside (12), and narcissin (13), based on the comparison of their NMR spectra with the reported data and liquid chromatography/mass spectrometry (LC/MS) analysis. The isolated compounds were evaluated for antibacterial activity against H. pylori strain 51. Among the isolates, 1-[O-ß-d-glucopyranosyl(1→2)-ß-d-glucopyranosyl]oxy-2-phenol (7) and arbutin cinnamate (8) exhibited antibacterial activity against H. pylori strain 51, with inhibitions of 31.4% and 33.9%, respectively, at a final concentration of 100 µM. These results were comparable to that of quercetin (38.4% inhibition), which served as a positive control. Generally, these findings highlight the potential of the active compounds 7 and 8 as antibacterial agents against H. pylori.

Assessment of the Genetic Diversity and Structure of the Korean Endemic Freshwater Fish Microphysogobio longidorsalis (Gobioninae) Using Microsatellite Markers: A First Glance from Population Genetics.

Microphysogobio longidorsalis is endemic to South Korea and inhabits small areas of the Namhangang, Bukhangang, and Imjingang Rivers in the Hangang River water system. Endemic species usually are more vulnerable than species with a wide distribution. Notably, there is a lack of basic conservation data for M. longidorsalis. We analyzed 19 microsatellite loci in six populations of M. longidorsalis in South Korea to characterize their population structure and genetic diversity. The genetic diversity of the microsatellites was 0.741-0.779, which is lower than that of other freshwater fishes. The pairwise genetic differentiation of microsatellite (FST) values ranged from 0.007 to 0.041, suggesting low genetic differentiation between the populations. The Jojongicheon stream population (CP) had an effective population size of <100. Therefore, conservation efforts are required to prevent inbreeding depression in M. longidorsalis. Discriminant analysis of principal components showed that the Hangang River water system would be a single management unit (MU). Our findings provide fundamental genetic insights for the formulation of conservation strategies for M. longidorsalis.

Genome assembly and microsatellite marker development using Illumina and PacBio sequencing in Persicaria maackiana (Polygonaceae) from Korea.

BACKGROUND: Persicaria maackiana (Regel) is a potential medicinal plant that exerts anti-diabetic effects. However, the lack of genomic information on P. maackiana hinders research at the molecular level. OBJECTIVE: Herein, we aimed to construct a draft genome assembly and obtain comprehensive genomic information on P. maackiana using high-throughput sequencing tools PacBio Sequel II and Illumina. METHODS: Persicaria maackiana samples from three natural populations in Gaecheon, Gichi, and Uiryeong reservoirs in South Korea were used to generate genomic DNA libraries, perform genome de novo assembly, gene ontology analysis, phylogenetic tree analysis, genotyping, and identify microsatellite markers. RESULTS: The assembled P. maackiana genome yielded 32,179 contigs. Assessment of assembly integrity revealed 1503 (93.12%) complete Benchmarking Universal Single-Copy Orthologs. A total of 64,712 protein-coding genes were predicted and annotated successfully in the protein database. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs, 13,778 genes were annotated into 18 categories. Genes that activated AMPK were identified in the KEGG pathway. A total of 316,992 microsatellite loci were identified, and primers targeting the flanking regions were developed for 292,059 microsatellite loci. Of these, 150 primer sets were randomly selected for amplification, and 30 of these primer sets were identified as polymorphic. These primers amplified 3-9 alleles. The mean observed and expected heterozygosity were 0.189 and 0.593, respectively. Polymorphism information content values of the markers were 0.361-0.754. CONCLUSION: Collectively, our study provides a valuable resource for future comparative genomics, phylogeny, and population studies of P. maackiana.

Global Natural Products Social (GNPS)-Based Molecular-Networking-Guided Isolation of Phenolic Compounds from Ginkgo biloba Fruits and the Identification of Estrogenic Phenolic Glycosides.

Ginkgo biloba L. stands as one of the oldest living tree species, exhibiting a diverse range of biological activities, including antioxidant, neuroprotective, anti-inflammatory, and cardiovascular activities. As part of our ongoing discovery of novel bioactive components from natural sources, we directed our focus toward the investigation of potential bioactive compounds from G. biloba fruit. The profiles of its chemical compounds were examined using a Global Natural Products Social (GNPS)-based molecular networking analysis. Guided by this, we successfully isolated and characterized 11 compounds from G. biloba fruit, including (E)-coniferin (1), syringin (2), 4-hydroxybenzoic acid 4-O-ß-D-glucopyranoside (3), vanillic acid 4-O-ß-D-glucopyranoside (4), syringic acid 4-O-ß-D-glucopyranoside (5), (E)-ferulic acid 4-O-ß-D-glucoside (6), (E)-sinapic acid 4-O-ß-D-glucopyranoside (7), (1'R,2'S,5'R,8'S,2'Z,4'E)-dihydrophaseic acid 3'-O-ß-D-glucopyranoside (8), eucomic acid (9), rutin (10), and laricitrin 3-rutinoside (11). The structural identification was validated through a comprehensive analysis involving nuclear magnetic resonance (NMR) spectroscopic data and LC/MS analyses. All isolated compounds were evaluated using an E-screen assay for their estrogen-like effects in MCF-7 cells. As a result, compounds 2, 3, 4, 8, and 9 promoted cell proliferation in MCF-7 cells, and these effects were mitigated by the ER antagonist, ICI 182,780. In particular, cell proliferation increased most significantly to 140.9 ± 6.5% after treatment with 100 µM of compound 2. The mechanism underlying the estrogen-like effect of syringin (2) was evaluated using a Western blot analysis to determine the expression of estrogen receptor α (ERα). We found that syringin (2) induced an increase in the phosphorylation of ERα. Overall, these experimental results suggest that syringin (2) can potentially aid the control of estrogenic activity during menopause.

Genome Assembly and Microsatellite Marker Development Using Illumina and PacBio Sequencing in the Carex pumila (Cyperaceae) from Korea.

This study is the first to report the characterization of Carex pumila genomic information. Assembly of the genome generated a draft of C. pumila based on PacBio Sequel II and Illumina paired-end sequencing, which was assembled from 2941 contigs with an estimated genome size of 0.346 Gb. The estimate of repeats in the genome was 31.0%, and heterozygosity ranged from 0.426 to 0.441%. The integrity evaluation of the assembly revealed 1481 complete benchmarked universal single-copy orthologs (BUSCO) (91.76%), indicating the high quality of the draft assembly. A total of 23,402 protein-coding genes were successfully predicted and annotated in the protein database. UpsetR plots showed that 7481 orthogroups were shared by all species. The phylogenetic tree showed that C. pumila is a close but distant relative of Ananas comosus. C. pumila had greater contraction (3154) than expansion (392). Among the extended gene families, aquaporins have been found to be enriched. Primers for microsatellite markers determined 30 polymorphic markers out of 100. The average number of alleles amplified by these 30 polymorphic markers was 4 to 12, with an average polymorphism information content (PIC) value of 0.660. In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of C. pumila.

Phytochemical Investigation of Equisetum arvense and Evaluation of Their Anti-Inflammatory Potential in TNFα/INFγ-Stimulated Keratinocytes.

Equisetum arvense L. (Equisetaceae), widely known as 'horsetail', is a perennial plant found extensively across Asia. Extracts of E. arvense have been used in traditional medicine, particularly for the treatment of inflammatory disorders. This study aimed to determine the phytochemical compounds in E. arvense ethanolic extract and their anti-inflammatory properties. Subsequently, we isolated and identified nine secondary metabolites, including kaempferol 3,7-di-O-ß-D-glucopyranoside (1), icariside B2 (2), (Z)-3-hexenyl ß-D-glucopyranoside (3), luteolin 5-O-ß-D-glucopyranoside (4), 4-O-ß-D-glucopyranosyl caffeic acid (5), clemastanin B (6), 4-O-caffeoylshikimic acid (7), (7S,8S)-threo-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-ß-D-glucopyranoside (8), and 3-O-caffeoylshikimic acid (9). The chemical structures of the isolated compounds (1-9) were elucidated using HR-ESI-MS data, NMR spectra, and ECD data. Next, the anti-inflammatory effects of the isolates were evaluated in tumor necrosis factor (TNF)α/interferon (IFN)γ-induced HaCaT, a human keratinocyte cell line. Among the isolates, compound 3 showed the highest inhibitory effect on the expression of pro-inflammatory chemokines, followed by compounds 6 and 8. Correspondingly, the preceding isolates inhibited TNFα/IFNγ-induced activation of pro-inflammatory transcription factors, signal transducer and activator of transcription 1, and nuclear factor-κB. Collectively, E. arvense could be employed for the development of prophylactic or therapeutic agents for improving dermatitis.

Genetic Structure and Genetic Diversity of the Endemic Korean Aucha Perch, Coreoperca herzi (Centropomidae), in Korea.

The Korean endemic aucha perch, Coreoperca herzi, belongs to the family Centropomidae. Thus far, studies on C. herzi have focused on mitochondrial genomes, egg development, and early life history, while studies on their genetic diversity or genetic structure are lacking. We investigated these aspects in this study using mitochondrial DNA data. Haplotypes were divided into the Hangang River, Nakdonggang River, Geumgang River, and southwest region water system populations. A translocated population, the Yangyang Namdaechun Stream, was confirmed to have originated from the Hangang River water system population based on haplotype distribution and genetic structure results. The FST of the mitochondrial DNA indicated distinct genetic differentiation in the Hangang, Nakdonggang, Geumgang, and southwest regions. According to COI and analyses, the analysis of molecular variance revealed a higher variance in the four water system groups (98.41%) than in the southwest region water system versus the Hangang River water system (80.27%) groups. This study presents basic data for conservation by providing extensive information on the genetic diversity, genetic structure, and translocation population of C. herzi.

Complete Chloroplast Genome Determination of Ranunculus sceleratus from Republic of Korea (Ranunculaceae) and Comparative Chloroplast Genomes of the Members of the Ranunculus Genus.

Ranunculus sceleratus (family: Ranunculaceae) is a medicinally and economically important plant; however, gaps in taxonomic and species identification limit its practical applicability. This study aimed to sequence the chloroplast genome of R. sceleratus from Republic of Korea. Chloroplast sequences were compared and analyzed among Ranunculus species. The chloroplast genome was assembled from Illumina HiSeq 2500 sequencing raw data. The genome was 156,329 bp and had a typical quadripartite structure comprising a small single-copy region, a large single-copy region, and two inverted repeats. Fifty-three simple sequence repeats were identified in the four quadrant structural regions. The region between the ndhC and trnV-UAC genes could be useful as a genetic marker to distinguish between R. sceleratus populations from Republic of Korea and China. The Ranunculus species formed a single lineage. To differentiate between Ranunculus species, we identified 16 hotspot regions and confirmed their potential using specific barcodes based on phylogenetic tree and BLAST-based analyses. The ndhE, ndhF, rpl23, atpF, rps4, and rpoA genes had a high posterior probability of codon sites in positive selection, while the amino acid site varied between Ranunculus species and other genera. Comparison of the Ranunculus genomes provides useful information regarding species identification and evolution that could guide future phylogenetic analyses.

Bioactive Phytochemicals from Salix pseudolasiogyne Twigs: Anti-Adipogenic Effect of 2'-O-Acetylsalicortin in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin (1) and 2'-O-acetylsalicortin (2). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin (1) in twigs of S. pseudolasiogyne. Here, we found that the isolated compounds, oregonin (1) and 2'-O-acetylsalicortin (2), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'-O-acetylsalicortin (2), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'-O-acetylsalicortin (2)-treated 3T3-L1 cells. Taken together, these results indicate that 2'-O-acetylsalicortin (2), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Anti-Adipogenic Effects of Salicortin from the Twigs of Weeping Willow (Salix pseudolasiogyne) in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae), the "weeping willow," has been used in traditional Korean medicine to treat pain and fever due to its high concentrations of salicylic acid and salicin. The present study investigated bioactive compounds from S. pseudolasiogyne twigs to discover bioactive natural products. Phytochemical investigation of the ethanol (EtOH) extract of S. pseudolasiogyne twigs followed by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of two salicin derivatives, salicortinol and salicortin, the structures of which were determined by interpretation of their NMR spectra and data from the LC/MS analysis. To the best of our knowledge, this is the first report of salicortinol isolated from S. pseudolasiogyne. The isolated compounds were evaluated for their anti-adipogenic effects in 3T3-L1 cells. Both salicortinol and salicortin were found to significantly inhibit adipocyte differentiation in 3T3-L1 cells. In particular, salicortin exhibited a strong inhibitory effect on lipid accumulation. Furthermore, salicortin inhibited the expression of lipogenic and adipogenic transcription factors, including FASN, FABP4, C/EBPα, C/EBPß, and PPARγ, without inducing cytotoxicity. These results suggest that salicortin could be a potential therapeutic compound for the prevention or treatment of metabolic disorders such as obesity.

The complete chloroplast genome of Persicaria maackiana (Regel) Nakai ex T. Mori (Polygonaceae) in Korea.

Persicaria maackiana (Regel) Nakai ex T. Mori (1922), a species of the Polygonaceae family, is an annual plant widely distributed in Northeast Asia. We aimed to sequence the complete chloroplast genome of P. maackiana using Illumina HiSeq paired-end sequencing. The chloroplast genome was determined to be 160,635 bp. The complete chloroplast genome contained 129 genes, including 84 protein-coding genes, 37 tRNA, and eight rRNA genes. Phylogenetic analysis of the chloroplast genome sequences of 15 Polygonaceae plants revealed that P. maackiana was most closely related to P. perfoliata. Our findings might be useful for future phylogenetic studies of Polygonaceae.

Phytochemical Investigation of Marker Compounds from Indigenous Korean Salix Species and Their Antimicrobial Effects.

Salix species, including willow trees, are distributed in the temperate regions of Asian countries, including South Korea. Willow trees are used to treat pain and inflammatory diseases. Due to the medicinal properties of willow trees, pharmacological studies of other Salix spp. have gained attention; however, only a few studies have investigated the phytochemicals of these species. As part of our ongoing natural product research to identify bioactive phytochemicals and elucidate their chemical structures from natural resources, we investigated the marker compounds from indigenous Korean Salix species, namely, Salix triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida. The ethanolic extract of each Salix sp. was investigated using high-performance liquid chromatography combined with thin-layer chromatography and liquid chromatography−mass spectrometry-based analysis, and marker compounds of each Salix sp. were isolated. The chemical structures of the marker compounds (1−8), 3-(4-hydroxyphenyl)propyl ß-D-glucopyranoside (1), 2-O-acetylsalicin (2), 1-O-p-coumaroyl glucoside (3), picein (4), isograndidentatin B (5), 2'-O-acetylsalicortin (6), dihydromyricetin (7), and salicin (8) were elucidated via nuclear magnetic resonance spectroscopy and high-resolution liquid chromatography−mass spectrometry using ultrahigh-performance liquid chromatography coupled with a G6545B Q-TOF MS system with a dual electrospray ionization source. The identified marker compounds 1−8 were examined for their antimicrobial effects against plant pathogenic fungi and bacteria. Dihydromyricetin (7) exhibited antibacterial activity against Staphylococcus aureus, inducing 32.4% inhibition at a final concentration of 125 µg/mL with an MIC50 value of 250 µg/mL. Overall, this study isolated the marker compounds of S. triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida and identified the anti-Staphylococcus aureus bacterial compound dihydromyricetin.

Complete mitochondrial genome sequence of Squalidus chankanensis tsuchigae (Cypriniformes: Cyprinidae).

We determined the complete mitochondrial genome sequence of Squalidus chankanensis tsuchigae (Cypriniformes: Cyprinidae). It is 16,603 bp long, with 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNA genes, and 1 control region, which is 927 bp long, located between tRNApro and tRNAphe. The overall base composition is as follows: 29.98% A, 16.86% G, 25.44% T, and 27.72% C, with a slight AT bias. These results provide necessary data for phylogenetic studies on Squalidus species.

The complete mitochondrial genome of Liparis ochotensis and a preliminary phylogenetic analysis.

Liparis ochotensis is a snailfish commonly confused with similar fish species because of unclear morphological characteristics. Moreover, molecular genetic studies have not been conducted for snailfish in Korea. Here, we report the complete mitogenome sequence of L. ochotensis, obtained via long PCR using universal primers for the fish mitogenome. The L. ochotensis mitogenome is 17,522 bp long, comprising 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. A neighbour-joining phylogenetic tree based on CO1 sequences depicted a close relationship with Liparis gibbus. The complete mitogenome is a valuable resource to classify and conserve L. ochotensis.

The complete mitochondrial genome of Liparis tessellatus and its phylogenetic analysis.

Liparis tessellatus is a cubed snailfish that inhabits the northwestern region of the Pacific Ocean. The family Liparidae is difficult to distinguish morphologically due to the typical body color and shape variation, which are used interchangeably due to the differences in local dialects. Therefore, we determined the complete mitochondrial genome sequence of L. tesellatus. The mitochondrial genome length of L. tesellatus was determined as 17,221 bp, which consisted of 22 tRNA genes, two rRNA genes, 13 protein-coding genes (PCGs), and a control region (D-loop). The base composition was as follows: 28.6% of A, 29.5% of T, 26.5% of C, and 15.4% of G. The phylogenetic analysis revealed that L. tesellatus clustered together with different species of the genus Liparis. Thus, the complete mitochondrial genome sequence provided herein would further help in understanding the evolution of Liparis species.

The complete mitochondrial genome sequence and phylogenetic analysis of Squalidus multimaculatus.

This study reports the complete mitochondrial genome of Squalidus multimaculatus. The length of the mitochondrial genome of S. multimaculatus is 16,597 bp, including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. The overall base composition of A, G, T, and C is 28%, 18.8%, 25%, and 28.2%, respectively. A phylogenetic analysis by the neighbour-joining method showed a close relationship between S. multimaculatus and S. japonicus coreanus. We believe these results will provide essential data for phylogeographic studies of the genus Squalidus.

Identification of microsatellite markers and their application in yellow catfish (Pseudobagrus fulvidraco Richardson, 1846) population genetics of Korea.

Microsatellite markers from a fresh water yellow catfish, Pseudobagrus fulvidraco, were developed by whole-genome sequencing in the Ion S5 system. Of the 40 chosen sets of microsatellite markers, with tetra-repeat and penta-repeat motifs, from a total 19,743 sequence, only 13 markers were successfully applied in 78 individual fish sampled to detect genomic variability from four natural populations of Korea. On an average, the number of alleles per marker was 6.7. The observed heterozygosity varied from 0.048 to 0.810. Twelve microsatellite markers conformed to Hardy-Weinberg equilibrium and none exhibited significant linkage disequilibrium. In yellow catfish, genetic differentiation among four natural populations was further supported by FST (P < 0.05) and STRUCTURE analysis. The microsatellite markers identified could facilitate genetic diversity and population structure studies and thus aid in conservation of the yellow catfish.

Genetic diversity and structure of an endangered medicinal herb: implications for conservation.

Human-driven habitat fragmentation leads to spatial isolation of endangered plant species increasing extinction risk. Understanding genetic variability and population structure of rare and isolated plant species is of great importance for assessing extinction risk and setting up conservation plans. Aconitum austrokoreense, an endangered and endemic species in Korea, is a perennial herb commonly used for medicinal purposes. We used five nuclear microsatellites and one chloroplast marker to investigate genetic diversity and population structure for 479 individuals of A. austrokoreense from seven populations throughout South Korea. A multivariate approach, discriminant analysis of principal components analysis, revealed broad-scale spatial patterns of A. austrokoreense populations across three major mountains that were composed of seven genetically distinct subgroups. High pairwise FST values (mean FST = 0.35; highest FST = 0.55) suggested significant differentiation among populations. Overall within population genetic variation was low. Based on Mantel test, there was significant correlation between geographical and genetic distances indicating pattern of isolation by distance. Our results suggest that A. austrokoreense populations may have undergone recent population bottlenecks. Given the limited dispersal ability of the species and ongoing habitat fragmentation, population isolation may further be exacerbated leading to increased extinction risk.

The complete mitochondrial genome of Gadus chalcogramma and phylogenetic analysis.

Here, we report the complete mitochondrial genome sequence of Alaska Pollock, Gadus chalcogramma. The genome sequence was obtained via long PCR reactions using universal primer sets for the fish mitochondrial genome. The total length of mitochondrial genome was 16,571 bp, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region (D-loop). Except for ND6 and eight tRNA genes, all of the other mitochondrial genes were encoded on the heavy strand. The NJ tree of the combined 13 protein-coding gene sequences of Gadus chalcogramma possesses a relatively closer relationship with the Okhotsk Sea Pollock (Gadus chalcogrammus). Our complete mitochondrial genome will be valuable resources for phylogeny, genetics, and conservation of the Gadus chalcogramma in Korea.

The complete mitochondrial genome of long-tailed whiskered bat, Myotis frater (Myotis, Vespertilionidae).

Here, we report the complete mitogenome of Myotis frater with the GenBank accession number MH177276 as a first step to elucidate genetic characteristics of this species. Its mitogenome was 17,089 bp long and consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a control region. The gene order and composition of M. frater was similar to that of most other vertebrates. The base composition of the 13 PCG in descending order was A (33.8%), C (22.7%), T (30.4%), and G (13.1%), with an AT content of 64.2%. Four overlapping regions in ATP8/ATP6, ATP6/COX3, ND4L/ND4, and ND5/ND6, among the 13 PCGs were found. The 935 bp long control region is located between tRNA-Pro and tRNA-Phe with 4 ATTACATAATACATTATATGTATAATCGTACATTAAATTAACTCCCACATGAATATTAAGCATGTCCATACTAATATTAAT-repeat at 5' region and 45 ACGCAT-repeat at 3' terminus. Phylogenetic analysis suggested that M. frater is most closely related to M. bechsteinii (KX757757), it was supported by 100% bootstrap under both ML and NJ tree.