Identification and phylogenetic analysis of five Crataegus species (Rosaceae) based on complete chloroplast genomes.

MAIN CONCLUSION: The chloroplast genomes of the five Crataegus species were shown to have a conserved genome structure. Complete chloroplast genome sequences were more suitable than highly variable regions for the identification and phylogenetic analysis of Crataegus species. Hawthorn, which is commonly used as a traditional Chinese medicine, is one of the most popular sour fruits and has high economic value. Crataegus pinnatifida var. pinnatifida and C. pinnatifida var. major are frequently adulterated with other Crataegus species on the herbal medicine market. However, most Crataegus plants are difficult to identify using traditional morphological methods. Here, we compared five Crataegus chloroplast (CP) genomes comprising two newly sequenced (i.e., C. pinnatifida var. pinnatifida and C. pinnatifida var. major) and three previously published CP genomes. The CP genomes of the five Crataegus species had a conserved genome structure, gene content and codon usage. The total length of the CP genomes was 159,654-159,865 bp. A total of 129-130 genes, including 84-85 protein-coding genes, 37 tRNA genes and 8 rRNA genes, were annotated. Bioinformatics analysis revealed 96-103 simple sequence repeats (SSRs) and 48-70 long repeats in the five CP genomes. Combining the results of mVISTA and nucleotide diversity, five highly variable regions were screened for species identification and relationship studies. Maximum likelihood trees were constructed on the basis of complete CP genome sequences and highly variable regions. The results showed that the former had higher discriminatory power for Crataegus species, indicating that the complete CP genome could be used as a super-barcode to accurately authenticate the five Crataegus species.

Molecular structure and phylogenetic analyses of the complete chloroplast genomes of three original species of Pyrrosiae Folium.

Pyrrosia petiolosa, Pyrrosia lingua and Pyrrosia sheareri are recorded as original plants of Pyrrosiae Folium (PF) and commonly used as Chinese herbal medicines. Due to the similar morphological features of PF and its adulterants, common DNA barcodes cannot accurately distinguish PF species. Knowledge of the chloroplast (cp) genome is widely used in species identification, molecular marker and phylogenetic analyses. Herein, we determined the complete cp genomes of three original species of PF via high-throughput sequencing technologies. The three cp genomes exhibited a typical quadripartite structure with sizes ranging from 158 165 to 163 026 bp. The cp genomes of P. petiolosa and P. lingua encoded 130 genes, whilst that of P. sheareri encoded 131 genes. The complete cp genomes were compared, and five highly divergent regions of petA-psbJ, matK-rps16, ndhC-trnM, psbM-petN and psaC-ndhE were screened as potential DNA barcodes for identification of Pyrrosia genus species. The phylogenetic tree we obtained indicated that P. petiolosa and P. lingua are clustered in a single clade and, thus, share a close relationship. This study provides invaluable information for further studies on the species identification, taxonomy and phylogeny of Pyrrosia genus species.

Genome-wide identification of abscisic acid (ABA) receptor pyrabactin resistance 1-like protein (PYL) family members and expression analysis of PYL genes in response to different concentrations of ABA stress in Glycyrrhiza uralensis.

As abscisic acid (ABA) receptor, the pyrabactin resistance 1-like (PYR/PYL) protein (named PYL for simplicity) plays an important part to unveil the signal transduction of ABA and its regulatory mechanisms. Glycyrrhiza uralensis, a drought-tolerant medicinal plant, is a good model for the mechanism analysis of ABA response and active compound biosynthesis. However, knowledge about PYL family in G. uralensis remains largely unknown. Here, 10 PYLs were identified in G. uralensis genome. Characterization analysis indicated that PYLs in G. uralensis (GuPYLs) are relatively conserved. Phylogenetic analysis showed that GuPYL1-3 belongs to subfamily I, GuPYL4-6 and GuPYL10 belong to subfamily II and GuPYL7-9 belongs to subfamily III. In addition, transcriptome data presented various expression levels of GuPYLs under different exogenous ABA stresses. The expression pattern of GuPYLs was verified by Quantitative real-time polymerase chain reaction (qRT-PCR). The study proved that GuPYL4, GuPYL5, GuPYL8 and GuPYL9 genes are significantly up-regulated by ABA stress and the response process is dynamic. This study paves the way for elucidating the regulation mechanism of ABA signal to secondary metabolites and improving the cultivation and quality of G. uralensis using agricultural strategies.

Complete chloroplast genome sequence of the medicinal plant Arctium lappa.

Arctium lappa, commonly called burdock, has a long medicinal and edible history. It has recently gained increasing attention because of its economic value. In this study, we obtained the complete chloroplast genome of A. lappa by Illumina Hiseq. The complete chloroplast genome of A. lappa is a typical circular structure with 152 708 bp in length. The GC content in the whole chloroplast genome of A. lappa is 37.7%. A total of 37 tRNA genes, 8 rRNA genes, and 87 protein-coding genes were successfully annotated. And the chloroplast genome contains 113 unique genes, 19 of which are duplicated in the inverted repeat. The distribution of 39 simple sequence repeats was analysed, and most of them are in the large single-copy (LSC) sequence. An inversion comprising 16 genes was found in the LSC region, which is 26 283 bp long. We performed multiple sequence alignments using 72 common protein-coding genes of 29 species and constructed a Maximum Parsimony (MP) tree. The MP phylogenetic result shows that A. lappa grouped together with Carthamus tinctorius, Centaurea diffusa, and Saussurea involucrata. The chloroplast genome of A. lappa is a valuable resource for further studies in Asteraceae.

Gene Losses and Variations in Chloroplast Genome of Parasitic Plant Macrosolen and Phylogenetic Relationships within Santalales.

Macrosolen plants are parasitic shrubs, several of which are important medicinal plants, that are used as folk medicine in some provinces of China. However, reports on Macrosolen are limited. In this study, the complete chloroplast genome sequences of Macrosolen cochinchinensis, Macrosolen tricolor and Macrosolen bibracteolatus are reported. The chloroplast genomes were sequenced by Illumina HiSeq X. The length of the chloroplast genomes ranged from 129,570 bp (M. cochinchinensis) to 126,621 bp (M. tricolor), with a total of 113 genes, including 35 tRNA, eight rRNA, 68 protein-coding genes, and two pseudogenes (ycf1 and rpl2). The simple sequence repeats are mainly comprised of A/T mononucleotide repeats. Comparative genome analyses of the three species detected the most divergent regions in the non-coding spacers. Phylogenetic analyses using maximum parsimony and maximum likelihood strongly supported the idea that Loranthaceae and Viscaceae are monophyletic clades. The data obtained in this study are beneficial for further investigations of Macrosolen in respect to evolution and molecular identification.

Comparative and Phylogenetic Analyses of Ginger (Zingiber officinale) in the Family Zingiberaceae Based on the Complete Chloroplast Genome.

Zingiber officinale, commonly known as ginger, is an important plant of the family Zingiberaceae and is widely used as an herbal medicine and condiment. The lack of chloroplast genomic information hinders molecular research and phylogenetic analysis on ginger. We introduced the complete chloroplast genome of Z. officinale and identified its phylogenetic position in Zingiberaceae. The chloroplast genome of Z. officinale is 162,621 bp with a four-part circular structure and 36.1% GC content. All 113 unique genes were annotated. A total of 78 simple sequence repeats (SSRs) and 42 long repeat sequences, which are potential areas for species authentication, were found. Comparative analysis revealed some highly variable regions, including rps16-trnQ-UUG, atpH-atpI, trnT-UGU-trnL-UAA, ycf1, and psaC-ndhE. Moreover, the small single-copy (SSC) region was the most variable region in all four shared regions, indicating that it may be undergoing rapid nucleotide substitution in the family Zingiberaceae. Phylogenetic analysis based on all available chloroplasts of Zingiberales in the National Center for Biotechnology Information indicated that Zingiber is a sister branch to Kaempferia species. The availability of the Z. officinale chloroplast genome provided invaluable data for species-level authentication and phylogenetic analysis and can thus benefit further investigations on species in the family Zingiberaceae.

Identification and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Ephedra Herbs Containing Ephedrine.

Ephedrae Herba and Ephedrae Radix et Rhizoma (Mahuang) have been used as Chinese herbal medicines. Ephedra plants mainly live in deserts and have good governance of desertification. Despite their important medicinal and environmental protection value, dietary supplements containing ephedrine from Ephedra species may threaten the health of people. Morphological resemblance amongst species causes difficulty in identifying the original species of Ephedra herbs. Chloroplast (CP) genome shows good prospects in identification and phylogenetic analysis. This study introduced the structures of the CP genomes of three Ephedra species and analysed their phylogenetic relationships. Three complete CP genomes of Ephedra showed four-part annular structures, namely, two single-copy regions and two inverted repeat regions. The entire CP genomes of three Ephedra species in terms of size were 109,550 bp (E. sinica), 109,667 bp (E. intermedia), and 109,558 bp (E. equisetina). Each CP genome of the three Ephedra species encoded 118 genes, including 73 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. Eleven high-variation regions were screened through mVISTA to be potential specific DNA barcodes for identifying Ephedra species. Maximum likelihood and maximum parsimony trees showed that CP genomes could be used to identify Ephedra species. The Ephedra species had a close phylogenetic relationship with Gnetum species and Welwitschia mirabilis. This research provided valuable information for the identification and phylogenetic analysis of gymnosperms and drug safety of Ephedra.

Identification of Ligularia Herbs Using the Complete Chloroplast Genome as a Super-Barcode.

More than 30 Ligularia Cass. (Asteraceae) species have long been used in folk medicine in China. Morphological features and common DNA regions are both not ideal to identify Ligularia species. As some Ligularia species contain pyrrolizidine alkaloids, which are hazardous to human and animal health and are involved in metabolic toxification in the liver, it is important to find a better way to distinguish these species. Here, we report complete chloroplast (CP) genomes of six Ligularia species, L. intermedia, L. jaluensis, L. mongolica, L. hodgsonii, L. veitchiana, and L. fischeri, obtained through high-throughput Illumina sequencing technology. These CP genomes showed typical circular tetramerous structure and their sizes range from 151,118 to 151,253 bp. The GC content of each CP genome is 37.5%. Every CP genome contains 134 genes, including 87 protein-coding genes, 37 tRNA genes, eight rRNA genes, and two pseudogenes (ycf1 and rps19). From the mVISTA, there were no potential coding or non-coding regions to distinguish these six Ligularia species, but the maximum likelihood tree of the six Ligularia species and other related species showed that the whole CP genome can be used as a super-barcode to identify these six Ligularia species. This study provides invaluable data for species identification, allowing for future studies on phylogenetic evolution and safe medical applications of Ligularia.

Complete Chloroplast Genomes of Papaver rhoeas and Papaver orientale: Molecular Structures, Comparative Analysis, and Phylogenetic Analysis.

Papaver rhoeas L. and P. orientale L., which belong to the family Papaveraceae, are used as ornamental and medicinal plants. The chloroplast genome has been used for molecular markers, evolutionary biology, and barcoding identification. In this study, the complete chloroplast genome sequences of P. rhoeas and P. orientale are reported. Results show that the complete chloroplast genomes of P. rhoeas and P. orientale have typical quadripartite structures, which are comprised of circular 152,905 and 152,799-bp-long molecules, respectively. A total of 130 genes were identified in each genome, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence divergence analysis of four species from Papaveraceae indicated that the most divergent regions are found in the non-coding spacers with minimal differences among three Papaver species. These differences include the ycf1 gene and intergenic regions, such as rpoB-trnC, trnD-trnT, petA-psbJ, psbE-petL, and ccsA-ndhD. These regions are hypervariable regions, which can be used as specific DNA barcodes. This finding suggested that the chloroplast genome could be used as a powerful tool to resolve the phylogenetic positions and relationships of Papaveraceae. These results offer valuable information for future research in the identification of Papaver species and will benefit further investigations of these species.

Molecular Structure and Phylogenetic Analyses of Complete Chloroplast Genomes of Two Aristolochia Medicinal Species.

The family Aristolochiaceae, comprising about 600 species of eight genera, is a unique plant family containing aristolochic acids (AAs). The complete chloroplast genome sequences of Aristolochia debilis and Aristolochia contorta are reported here. The results show that the complete chloroplast genomes of A. debilis and A. contorta comprise circular 159,793 and 160,576 bp-long molecules, respectively and have typical quadripartite structures. The GC contents of both species were 38.3% each. A total of 131 genes were identified in each genome including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene (ycf1). The simple-sequence repeat sequences mainly comprise A/T mononucletide repeats. Phylogenetic analyses using maximum parsimony (MP) revealed that A. debilis and A. contorta had a close phylogenetic relationship with species of the family Piperaceae, as well as Laurales and Magnoliales. The data obtained in this study will be beneficial for further investigations on A. debilis and A. contorta from the aspect of evolution, and chloroplast genetic engineering.