Phylogenetic analysis and development of molecular markers for five medicinal Alpinia species based on complete plastome sequences.

BACKGROUND: Alpinia species are widely used as medicinal herbs. To understand the taxonomic classification and plastome evolution of the medicinal Alpinia species and correctly identify medicinal products derived from Alpinia species, we systematically analyzed the plastome sequences from five Alpinia species. Four of the Alpinia species: Alpinia galanga (L.) Willd., Alpinia hainanensis K.Schum., Alpinia officinarum Hance, and Alpinia oxyphylla Miq., are listed in the Chinese pharmacopeia. The other one, Alpinia nigra (Gaertn.) Burtt, is well known for its medicinal values. RESULTS: The four Alpinia species: A. galanga, A. nigra, A. officinarum, and A. oxyphylla, were sequenced using the Next-generation sequencing technology. The plastomes were assembled using Novoplasty and annotated using CPGAVAS2. The sizes of the four plastomes range from 160,590 bp for A. galanga to 164,294 bp for A. nigra, and display a conserved quadripartite structure. Each of the plastomes encodes a total of 111 unique genes, including 79 protein-coding, 28 tRNA, and four rRNA genes. In addition, 293-296 SSRs were detected in the four plastomes, of which the majority are mononucleotides Adenine/Thymine and are found in the noncoding regions. The long repeat analysis shows all types of repeats are contained in the plastomes, of which palindromic repeats occur most frequently. The comparative genomic analyses revealed that the pair of the inverted repeats were less divergent than the single-copy region. Analysis of sequence divergence on protein-coding genes showed that two genes (accD and ycf1) had undergone positive selection. Phylogenetic analysis based on coding sequence of 77 shared plastome genes resolves the molecular phylogeny of 20 species from Zingiberaceae. In particular, molecular phylogeny of four sequenced Alpinia species (A. galanga, A. nigra, A. officinarum, and A. oxyphylla) based on the plastome and nuclear sequences showed congruency. Furthermore, a comparison of the four newly sequenced Alpinia plastomes and one previously reported Alpinia plastomes (accession number: NC_048461) reveals 59 highly divergent intergenic spacer regions. We developed and validated two molecular markers Alpp and Alpr, based on two regions: petN-psbM and psaJ-rpl33, respectively. The discrimination success rate was 100 % in validation experiments. CONCLUSIONS: The results from this study will be invaluable for ensuring the effective and safe uses of Alpinia medicinal products and for the exploration of novel Alpinia species to improve human health.

The complete chloroplast genome sequence of Alpinia oxyphylla Miq. and comparison analysis within the Zingiberaceae family.

Alpinia oxyphylla Miq. (A. oxyphylla) is an important edible and traditional herbal medicine. In this study, the complete chloroplast genome of A. oxyphylla was sequenced, analysed, and compared to five species in the Zingiberaceae family. The size of the A. oxyphylla chloroplast genome was 161351 bp, which consisted of a large single-copy (LSC, 87248 bp) and small single-copy (SSC, 16175 bp) region separated by a pair of inverted repeats (IRa and IRb, 28964 bp each). The genome encoded 132 unique genes, including 87 protein-coding genes, 37 tRNAs and four rRNAs. The GC content of the genome was 36.17%. A total of 53 simple sequence repeats (SSRs) and 80 long repeats were identified in the A. oxyphylla chloroplast genome. The chloroplast genome of A. oxyphylla shared the highest sequence similarity of >90% with the chloroplast genome of A. zerumbet, and six chloroplast genomes in the Zingiberaceae family were compared by using CGView Comparison Tool (CCT). According to the phylogenetic tree, the Zingiberaceae family is divided into two categories, which coincide with the classification of the characteristics of sun-like and shade-like in plants. Our results reveal the phototrophic component of NADH-dehydrogenase (ndhB and ndhC), photosystem II (psbZ) and ATP synthase (atpE, atpF) exhibit adaptive evolution under different environments, and the strength of light is an important trigger for the adaptations at the chloroplast level.

Complete Chloroplast Genome Sequences of Kaempferia Galanga and Kaempferia Elegans: Molecular Structures and Comparative Analysis.

Kaempferia galanga and Kaempferia elegans, which belong to the genus Kaempferia family Zingiberaceae, are used as valuable herbal medicine and ornamental plants, respectively. The chloroplast genomes have been used for molecular markers, species identification and phylogenetic studies. In this study, the complete chloroplast genome sequences of K. galanga and K. elegans are reported. Results show that the complete chloroplast genome of K. galanga is 163,811 bp long, having a quadripartite structure with large single copy (LSC) of 88,405 bp and a small single copy (SSC) of 15,812 bp separated by inverted repeats (IRs) of 29,797 bp. Similarly, the complete chloroplast genome of K. elegans is 163,555 bp long, having a quadripartite structure in which IRs of 29,773 bp length separates 88,020 bp of LSC and 15,989 bp of SSC. A total of 111 genes in K. galanga and 113 genes in K. elegans comprised 79 protein-coding genes and 4 ribosomal RNA (rRNA) genes, as well as 28 and 30 transfer RNA (tRNA) genes in K. galanga and K. elegans, respectively. The gene order, GC content and orientation of the two Kaempferia chloroplast genomes exhibited high similarity. The location and distribution of simple sequence repeats (SSRs) and long repeat sequences were determined. Eight highly variable regions between the two Kaempferia species were identified and 643 mutation events, including 536 single-nucleotide polymorphisms (SNPs) and 107 insertion/deletions (indels), were accurately located. Sequence divergences of the whole chloroplast genomes were calculated among related Zingiberaceae species. The phylogenetic analysis based on SNPs among eleven species strongly supported that K. galanga and K. elegans formed a cluster within Zingiberaceae. This study identified the unique characteristics of the entire K. galanga and K. elegans chloroplast genomes that contribute to our understanding of the chloroplast DNA evolution within Zingiberaceae species. It provides valuable information for phylogenetic analysis and species identification within genus Kaempferia.

[Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum].

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

[AFLP analysis of genetic diversity of Alpinia officinarum].

OBJECTIVE: To explore the genetic diversity and relationship of different Alpinia officinarum germplasm. METHOD: Amplified fragment length polymorphism (AFLP) markers were developed to analyze genetic polymorphism in A. officinarun from eight resources. The amplified fragments were used as primary matrix with NTSYSpc-2.11F software to analyze the similarity between the A. officinarum germplasm and to construct the genetic phylogenetic tree. RESULT: A total of 1,120 fragments were genotyped using AFLP with eight prime combinations. Analysis identified 1,044 polymorphic fragments, accounting for 92.57% of the total detected variation. Genetic phylogenetic tree analysis indicates that three categories can be divided among the eight resources of A. officinanrum. CONCLUSION: Significant polymorphism and genetic diversity can be observed among A. officinarum germplasm resources.

New Rev-export inhibitor from Alpinia galanga and structure-activity relationship.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, an HIV-1 viral regulatory protein, disclosed 1'-acetoxychavicol acetate (ACA, 1) as a new inhibitor for nuclear export of Rev from the roots of Alpinia galanga. Both analysis for mechanism of action with biotinylated probe (2) and several synthesized analogs established crucial portions in 1 for Rev-export inhibitory activity.