Section Paeonia Research Articles

Characterization of phytochemicals in the roots of wild herbaceous peonies from China and screening for medicinal resources

Paeonia Radix Rubra (PRR) is a very common traditional Chinese medicine (TCM). The roots of Paeonia lactiflora and Paeonia anomala subsp. veitchii are used for the production of PRR. However, other species of section Paeonia in China are also used to produce PRR. The roots of section Paeonia from 20 populations of seven species and two subspecies in China were analyzed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF- MS). A total of 21 metabolites were identified, including nine monoterpene glycosides, seven tannins, three phenols, one paeonol and one flavonoid. There were significant differences in the composition and content of metabolites among different populations. The relative contents of monoterpene glycosides and tannins were generally higher in most samples. Cluster analysis showed that 20 populations could be divided into four groups. Among them, the populations of P. lactiflora and Paeonia mairei were clustered together in one group. The content of paeoniflorin in P. lactiflora was high (>22.20 mg g−1, dry weight, the same below), and the content of lactiflorin in P. mairei (>17.50 mg g−1) was significantly higher than in other species (<2.30 mg g−1). The monoterpene glycoside content in P. mairei (>51.60 mg g−1) was significantly higher than in other species (<43.40 mg g−1), suggesting that it could be useful medicinal germplasm for the development of monoterpene glycosides. In addition, some populations of Paeonia emodi, Paeonia sterniana and P. mairei may also be used as potential germplasm resources for use in PRR. Genetic and environmental factors resulted in differences in the composition and content of metabolites in different populations of the same species. Therefore, it is necessary to carefully consider the origin of Paeonia germplasm when selecting medicinal materials to be used as resources for the production of PRR.

New insights into Paeoniaceae used as medicinal plants in China

Paeoniaceae is an abundant germplasm resource with significant medicinal values in China, the principal medicinal components of which include paeoniflorin and paeonol. These compounds are typically obtained from air-dried root samples, which the use of freeze-drying as an alternative method has not been tested. Additionally, the presence of these two compounds in various wild Paeoniaceae species has not been previously explored, nor have the differences between various plant organs been fully evaluated. Here, freeze-drying and air-drying methods were compared to assess the changes in paeoniflorin and paeonol in root samples using ultra-performance liquid chromatography-mass spectrometer. The contents of these compounds in the roots, leaves, stems, and petals were then tested in freeze-dried materials. We also quantitatively detected the paeoniflorin and paeonol contents in the roots of 14 species collected from 20 natural habitats. Results indicated that the paeoniflorin content decreased under air-drying in comparison to freeze-drying, while the opposite trend was observed for paeonol. Our findings also demonstrated that the root xylem of species in Section Moutan, particularly Paeonia ostii, contains considerable paeonol and paeoniflorin and should thus be fully utilized as a medicinal resource. Furthermore, paeonol was mainly detected in the roots, while paeoniflorin was widely distributed in different organs; the highest content was in the leaf at the budding stage, suggesting that the leaves should be developed as a new paeoniflorin resource. Paeoniflorin contents were also found to be higher at earlier development stages. Based on the standards of the Chinese Pharmacopoeia, five species of Section Moutan and six species of Section Paeonia could be used as potential traditional Chinese medicinal resources. These findings of this study enhance our understanding of these two medicinal compounds and provide a foundation for the further development and utilization of Paeoniaceae as medicinal plant resources.

Identification of Floral Fragrances and Analysis of Fragrance Patterns in Herbaceous Peony Cultivars

Thirty herbaceous peony (section Paeonia of the genus Paeonia) cultivars were divided into four groups (no fragrance, light fragrance, medium fragrance, or intense fragrance) based on their sensory evaluation scores. Using dynamic headspace sampling (DHS) and automatic thermal desorption–gas chromatography/mass spectrometry (ATD-GC/MS), 130 volatile organic components were detected in these 30 cultivars and a total of 72 compounds were identified as scent components. The main compounds were phenylethyl alcohol, β-caryophyllene, linalool, (R)-citronellol, and nerol. Selecting α-pinene as the standard, the volatile components of these cultivars were quantitatively analyzed. By combining the sensory evaluation scores and the results of quantitative analysis, we found that ‘Going Bananas’, ‘Cream Delight’, ‘Zhu Sha Pan’, ‘Qiao Ling’, ‘Duchess de Nemours’, and ‘Yang Fei Chu Yu’ displayed an intense fragrance and, thus, had relatively high commercial value for the flower fragrance industry. ‘Red Magic’, ‘Joker’, ‘Fairy Princess’, ‘Lovely Rose’, ‘Carina’, and ‘Etched Salmon’ were excluded from the hierarchical cluster of aromatic compounds and the analysis of fragrance patterns because of the low amount of fragrance they released and poor sensory evaluation results. Based on a cluster analysis, assessment of the major aromatic compounds, and the results of sensory evaluation, the remaining 24 cultivars were divided into five fragrance patterns for the first time: woody scent [cluster I (major fragrance β-caryophyllene)], fruity scent [cluster II (phenylethyl alcohol)], lily scent [cluster III (linalool)], rose scent {cluster IV [(R)-citronellol]}, and an orange blossom scent [cluster V (nerol)].

Microsatellite Marker Development in Peony using Next Generation Sequencing

Peonies (Paeonia), the grand garden perennial of spring and early summer, are economically important to the international cut flower market. Herbaceous peonies (Paeonia section Paeonia), tree peonies (Paeonia section Moutan), and intersectional crosses between the two types (Itoh Paeonia hybrids) are of interest to gardeners, growers, and nursery producers. Thousands of peony cultivars exist and identity is traditionally determined by experienced horticulturists knowledgeable in plant and bloom characteristics. With DNA extraction possible during any time of the year, molecular markers can provide genotype identity confirmation for dormant roots or mature post-bloom plants. The primary objective of our research was to rapidly and inexpensively develop microsatellite markers in a range of Paeonia species using barcoded Illumina libraries. A secondary objective was to apply these simple sequence repeat (SSR) markers to fingerprint 93 accessions that include tree, intersectional, and herbaceous peonies. We used 21 primers to distinguish cultivars and their close relatives. Also from our sequence information, greater than 9000 primers were designed and are made available.

English

Paeonia is a phylogenetically and taxonomically complex group. In this study, two chloroplast coding region of the matK and rbcL genes, two intergenic spacers, 5’ trnK-matKand matK-3’ trnK, the ribosomal RNA 18S and 5S genes, and the nuclear ribosomal DNA ITS gene were sequenced to study phylogenetic relationships of four Paeonia species. In the matK coding region, most nucleotide variations pointed at the divergence betweenPaeonia suffruticosa (MO) and other three species, strongly supporting the phylogenetic evolution of section Paeonia and section Moutan in the Paeonia genus; the variable sites ofPaeonia lactiflora CHA1 accession compared to CHA accession were all identical toPaeonia obovata SHAN accession, indicating that CHA1 might be a hybrid result of CHA and SHAN. Another chloroplast coding region, rbcL showed lower variation rate and less phylogenetic information than the matK region in all four Paeonia species investigated in this study, while the 5S rRNA gene and nrDNA ITS region showed higher variation rate. However, combined phylogenetic relationship of all four peony species is more clearer than any single-locus one. This study would provide more sequence sources of peony species and help further understanding of the phylogenetic relationship. Key words: Deoxyribonucleic acid (DNA) barcoding, matK, nuclear ribosomaldeoxyribonucleic acid internal transcribed spacer (nrDNA ITS), Paeonia, phylogeny, ribulose-bisphosphate carboxylase (rbcL), 5S ribosomal ribonucleic acid (rRNA).

Interspecific hybridization in the genus Paeonia (Paeoniaceae): Polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids

The ITS1-5.8S rDNA-ITS2 regions of 33 accessions belonging to 16 species and five natural and garden interspecific hybrids of the genus Paeonia L. were sequenced. Chromatograms of the peony hybrids demonstrated the presence of the signals, corresponding to two different nucleotides at the positions differing in the parents, indicating that in the hybrids, no rDNA isogenization usually occurred, and they preserved rDNA of both parents. Analysis of these polymorphic sites (PS) showed that P. x majkoae was interspecific hybrid between P. tenuifolia and P. caucasica. The ITS of P. hybrida differs from ITS of P. x majkoae in 19 mutations. Because of this, P. x majkoae is definitely not synonymous to P. hybrida. Comparative analysis of ITS 1-5.8S rDNA-ITS2 showed that species diversity in section Paeonia was based on recombination as a result of intraspecific hybridization of three haplotype families. Specifically, haplotypes A, typical of the P. tenuifolia and P. anomala genomes, haplotypes B, typical of P. mlokosewitschii and P. obovata, and haplotypes of family C, currently represented in rDNA of diploid and tetraploid forms of some Caucasian and Mediterranean species. The ITS regions many diploid peonies contain no dimorphic sites, while P. oreogeton, P. cambessedesii, P. rhodia, and P. daurica carry from ten to 17 PS, and supposed to be the interspecific hybrids. Most of the tetraploid peonies contain from six to 18 PS in the ITS regions. These are alloploids with one of the parental genomes similar to that of P. mlokosewitschii (B1), or P. obovata (B3). The second parental genome in P. banatica, P. peregrina, and P. russii is represented by the genome, close to that of P. tenuifolia (A). P. macrophylla, P. mascula, P. coriacea, P. wittmanniana, and P. tomentosa carry genome of series B and genome of series C, which slightly resembles genome A.

Genetic diversity and phylogenetic analysis of genus Paeonia based on nuclear ribosomal DNA ITS sequence

The genus Paeonia belongs to the family Paeoniaceae having significant medicinal and ornamental importance. The present investigation was undertaken with an aim to understand phylogenetic relationships of three Paeonia species (P. lactiflora, P. obovata, and P. suffruticosa) that are widely distributed in China, Korea, and Japan, using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence and to compare the phylogeny results with investigations reported earlier using existed sequences of the same species. The size variation obtained among sequenced nrDNA ITS region was narrow and ranged from 722 to 726 bp. The highest interspecific genetic distance (GD) was found between P. lactiflora and P. suffruticosa or P. obovata. The phylogram obtained using our nrDNA ITS sequences showed non-congruence with previous hypothesis of the phylogeny between section Paeonia and section Moutan of genus Paeonia. This result was supported by the phylogenetic relations showed in the phylogram constructed with existed sequences in NCBI. The present study suggested that P. obovata belonging to section Paeonia was phylogenetically closer to P. suffruticosa representing section Moutan of genus Paeonia than P. lactiflora belonging to section Paeonia. The main reason of the paraphyly of section Paeonia is thought to be nucleotide additivity directly caused by origin hybridization. This study provides more sequence sources of genus Paeonia, and will help for further studies in intraspecies population, and their phylogentic analysis and molecular evolution.

Phylogenetic Utility of the Glycerol-3-Phosphate Acyltransferase Gene: Evolution and Implications in Paeonia (Paeoniaceae)

The nuclear-encoded chloroplast-expressed glycerol-3-phosphate acyltransferase (GPAT) gene has been found to be single-copy in a number of angiosperm families. In this study we investigated the phylogenetic utility of the GPAT gene at the interspecific level using the genus Paeonia (Paeoniaceae) as an example. An approximately 2.3- to 2.6-kb fragment of the GPAT gene, containing a large intron of more than 2 kb, was amplified, cloned, and sequenced from 19 accessions representing 13 Paeonia species. The GPAT gene phylogeny inferred by parsimony analysis supported interspecific relationships that were previously unresolved, suggesting that large introns of low-copy nuclear genes are particularly informative in the resolution of close relationships at low taxonomic levels. Whereas the GPAT phylogeny is largely congruent with the previous phylogenetic hypothesis of Paeonia, it shows a significant discordance involving the paraphyly of section Paeonia. Given evidence of an ancient duplication and the subsequent silencing of one GPAT locus in P. anomala, this discordance is most likely the result of paralogy. Two distinct genomic clones containing partial GPAT genes were isolated from P. anomala. The GPAT sequence from one clone corresponded to the functional copy of the gene, and the second genomic clone was determined to contain a GPAT pseudogene. The insertion of a retrotransposon in an intron of this pseudogene may have been responsible for the silencing of this GPAT locus in P. anomala. This study suggests that, although it is unlikely that universal nuclear gene markers free from paralogy are usually available, low-copy nuclear genes can be very useful in plant phylogenetic reconstruction, especially at low taxonomic levels, as long as the evolutionary dynamics of the genes are carefully examined.

Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S-5.8S-26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.