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Dalbergia odorifera T. Chen (Fabaceae) is a woody tree species indigenous to Hainan Island in China. Due to its high medicinal and commercial value, this tree species has been planted over 3500 ha2 in southern China. There is an urgent need for improvement of the D. odorifera germplasm, however, limited information on germplasm collection, conservation, and assessment of genetic resources is available. Therefore, we have built a database of 251 individuals collected across the whole of southern China, which included 42 wild trees and 210 cultivated trees, with the following objectives. (1) Evaluate genetic diversity and population structure of the database using 19 microsatellite markers and (2) develop a core collection for improvement and breeding programs. Totally, the 19 microsatellite markers harbored 77 alleles across the database with the polymorphic information content (PIC) ranging from 0.03 to 0.66. Medium genetic diversity level was inferred by Nei’s gene diversity (0.38), Shannon’s information index (0.65), and observed (0.33) and expected heterozygosity (0.38). Structure analysis showed that four was the optimum cluster size using the model-based Bayesian procedure, and the 251 D. odorifera individuals were grouped into five populations including four pure ones (RP1-4) and one mixed one (MIX) based on their maximum membership coefficients. Among these populations, the expected heterozygosity varied from 0.30 (RP3) to 0.38 (RP4). Analysis of molecular variance (AMOVA) showed 11% genetic variation existed among populations, and moderate population differentiation was inferred by the matrix of pairwise Fst (genetic differentiation among populations), which was in the range of 0.031 to 0.095. Moreover, a core collection of 31 D. odorifera individuals including six wild and 25 cultivated trees was developed, which was only 12.4% of the database but conserved the whole genetic diversity. The results of this study provided additional insight into the genetic structure of the large D. odorifera germplasm, and the core collection will be useful for the efficient and sustainable utilization of genetic resources, as well as efficient improvement in breeding programs.

T. Chen, known as fragrant rosewood, is a rare and endangered tree species. Studies have shown that plant growth regulators can effectively promote heartwood formation. This study aimed to investigate the effects of ethephon (ETH) on heartwood formation and the influence of ethephon and hydrogen peroxide (H O ) on the physiological characteristics in . branches underwent treatment with 2.5% plant growth regulators, including ETH, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), H O , and inhibitors such as ascorbic acid (AsA) to inhibit H O synthesis, and (S) -trans 2-amino-4 - (2-aminoethoxy) -3-butene (AVG) to inhibit ethylene synthesis. After a 14-day period, we conducted an analysis to evaluate the impact of these plant growth regulators on elongation distance, vessel occlusion percentage, and trans-nerol content. Additionally, the effects of ETH and H O on endogenous plant hormones, H O content, soluble protein content, and enzyme activity were investigated within 0-48 h of treatment. After treatment with ETH for 14 days, the extension distance of the heartwood material was 15 cm, while the trans-nerolol content was 15 times that of the ABA group. ETH and H O promoted endogenous ethylene synthesis; Ethylene content peaked at 6 and 18 h. The peak ethylene content in the ETH group was 68.07%, 12.89%, and 20.87% higher than the initial value of the H O group and ddH O group, respectively, and 29.64% higher than that in the AVG group. The soluble protein content and activity of related enzymes were significantly increased following ETH treatment. ETH exhibited the most impact on heartwood formation while not hindering tree growth. This treatment effectively triggered the production of endogenous ethylene in plants and enhanced the activity of essential enzymes involved in heartwood formation. These findings serve as a valuable reference for future investigations into heartwood formation.

Dalbergia odorifera T. Chen (Fabaceae) is a semi-deciduous tree species indigenous to Hainan Island in China. Due to its precious heartwood “Hualimu (Chinese)” and Chinese medicinal components “Jiangxiang”, D. odorifera is seriously threatened of long-term overexploitation and has been listed on the IUCN (International Union for Conservation of Nature’s) red list since 1998. Therefore, the elucidation of its genetic diversity is imperative for conservation and breeding purposes. In this study, we evaluated the genetic diversity of 42 wild D. odorifera trees from seven populations covering its whole native distribution. In total, 19 SSR (simple sequence repeat) markers harbored 54 alleles across the 42 samples, and the medium genetic diversity level was inferred by Nei’s gene diversity (0.36), observed (0.28) and expected heterozygosity (0.37). Among the seven wild populations, the expected heterozygosity (He) varied from 0.31 (HNQS) to 0.40 (HNCJ). The analysis of molecular variance (AMOVA) showed that only 3% genetic variation existed among populations. Moderate population differentiations among the investigated populations were indicated by pairwise Fst (0.042–0.115). Structure analysis suggested two clusters for the 42 samples. Moreover, the seven populations were clearly distinguished into two clusters from both the principal coordinate analysis (PCoA) and neighbor-joining (NJ) analysis. Populations from Haikou city (HNHK), Baisha autonomous county (HNBS), Ledong autonomous county (HNLD), and Dongfang city (HNDF) comprised cluster I, while cluster II comprised the populations from Wenchang city and Sansha city (HNQS), Changjiang autonomous county (HNCJ), and Wuzhisan city (HNWZS). The findings of this study provide a preliminary genetic basis for the conservation, management, and restoration of this endemic species.

Dalbergia tonkinensis is very similar to Dalbergia odorifera in material, texture, and other macroscopic characteristics, and a comparison of the commonalities and differences in the chemical composition of the extractives may help to distinguish the two. However, the chemical composition of Dalbergia tonkinensis heartwood is unknown. The total flavonoid content of Dalbergia tonkinensis heartwood was determined to range between 49.67 to 101.95 mg, which indicated that Dalbergia tonkinensis is as equally rich in flavonoids as Dalbergia odorifera. Thirty-one flavonoids were identified via ultra-performance liquid chromatography-mass spectrometry in Dalbergia tonkinensis. Among them, the contents of 15 medicinal active flavonoids with were determined, and the liquiritigenin, naringenin, formononetin, pinocembrin, and biochanin A contents were found to be high. The 12 volatile compositions of Dalbergia tonkinensis heartwood identified via gas chromatography-mass spectrometry were very similar to those of Dalbergia odorifera and were dominated by trans-nerolidol, caryophyllene oxide, and eudesmol. The relationship between the chemical composition of Dalbergia tonkinensis and Dalbergia odorifera heartwood extracts was determined via a principal component analysis, and the results indicated that there was no significant difference in chemical composition between the two. This suggested that Dalbergia tonkinensis could be used as a potential substitute for Dalbergia odorifera.

Dalbergia odorifera, a traditional Chinese medicine, has been used to treat cardio- and cerebrovascular diseases in China for thousands of years. Flavonoids are major active compounds in D. odorifera. In this paper, a rapid and sensitive ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method was developed and validated for simultaneous determination of 17 flavonoids in D. odorifera. Quantification was performed by multiple reaction monitoring using electrospray ionization in negative ion mode. Under the optimum conditions, calibration curves for the 17 analytes displayed good linearity (r2 > 0.9980). The intra- and inter-day precisions (relative standard deviations) were lower than 5.0%. The limit of quantitation ranged from 0.256 to 18.840 ng/mL. The mean recovery range at three spiked concentrations was 94.18–101.97%. The validated approach was successfully applied to 18 samples of D. odorifera. Large variation was observed for the contents of the 17 analytes. Sativanone and 3′-O-methylviolanone were the dominant compounds. The fragmentation behaviors of six flavonoids were investigated using UPLC with quadrupole time-of-flight tandem mass spectrometry. In negative ion electrospray ionization mass spectrometry, all the flavonoids yielded prominent [M − H]− ions. Fragments for losses of CH3, CO, and CO2 were observed in the mass spectra. Formononetin, liquiritigenin, isoliquiritigenin, sativanone, and alpinetin underwent retro-Diels–Alder fragmentations. The proposed method will be helpful for quality control of D. odorifera.

Background Dalbergia odorifera is an economically and culturally important species in the Fabaceae because of the high-quality lumber and traditional Chinese medicines made from this plant, however, overexploitation has increased the scarcity of D. odorifera . Given the rarity and the multiple uses of this species, it is important to expand the genomic resources for utilizing in applications such as tracking illegal logging, determining effective population size of wild stands, delineating pedigrees in marker assisted breeding programs, and resolving gene networks in functional genomics studies. Even the nuclear and chloroplast genomes have been published for D. odorifera , the complete mitochondrial genome has not been assembled or assessed for sequence transfer to other genomic compartments until now. Such work is essential in understanding structural and functional genome evolution in a lineage (Fabaceae) with frequent intergenomic sequence transfers. Results We integrated Illumina short-reads and PacBio CLR long-reads to assemble and annotate the complete mitochondrial genome of D. odorifera . The mitochondrial genome was organized as a single circular structure of 435 Kb in length containing 33 protein coding genes, 4 rRNA and 17 tRNA genes. Nearly 4.0% (17,386 bp) of the genome was annotated as repetitive DNA. From the sequence transfer analysis, it was found that 114 Kb of DNA originating from the mitochondrial genome has been transferred to the nuclear genome, with most of the transfer events having taken place relatively recently. The high frequency of sequence transfers from the mitochondria to the nuclear genome was similar to that of sequence transfer from the chloroplast to the nuclear genome. Conclusion For the first-time, the complete mitochondrial genome of D. odorifera was assembled in this study, which will provide a baseline resource in understanding genomic evolution in the highly specious Fabaceae. In particular, the assessment of intergenomic sequence transfer suggests that transfers have been common and recent indicating a possible role in environmental adaptation as has been found in other lineages. The high turnover rate of genomic colinearly and large differences in mitochondrial genome size found in the comparative analyses herein providing evidence for the rapid evolution of mitochondrial genome structure compared to chloroplasts in Faboideae. While phylogenetic analyses using functional genes indicate that mitochondrial genes are very slowly evolving compared to chloroplast genes.

The heartwood of Dalbergia odorifera T. Chen has garnered significant attraction due to its high medicinal, aromatic and timber values; however, its formation mechanism remains unexplored. This study utilized the sapwood (N-B), transition zone (N-T), and heartwood (N-H) of the xylem of 15-year-old, naturally heartwood-forming D. odorifera to observe the nuclei of parenchyma cells, revealing that no living cells were specialized in synthesizing the secondary metabolites of heartwood in the N-H. Additionally, analysis of gene expression patterns across different compartments indicated that differentially expressed genes (DEGs) involved in the synthesis of secondary metabolites of heartwood were primarily up-regulated in the N-T, suggesting that the pattern of heartwood formation in D. odorifera follows the Type-I (Robinia-Type) model, wherein secondary metabolites are synthesized in situ in the ray parenchyma cells of the N-T, followed by programmed cell death (PCD) leading to heartwood formation. Furthermore, DEGs related to ethylene biosynthesis and signaling pathways were up-regulated in the N-T, suggesting that ethylene signaling may play a critical role in regulating the heartwood formation process of D. odorifera. Treatment of suspension cells with polyethylene glycol (PEG) and an ethylene synthesis inhibitor (AVG) further confirmed that ethylene acts as a key signaling molecule in the formation of heartwood-like material in D. odorifera. This study provides initial insights into the molecular mechanisms underlying heartwood formation in D. odorifera and offers a foundation for developing heartwood formation and promotion technologies.

Salvia miltiorrhiza - Dalbergia odorifera coupled-herbs (SMDOCH) has been used to treat coronary heart disease (CHD) for thousands of years, but its unclear bioactive components and mechanisms greatly limit its clinical application. In this study, for the first time, we used network pharmacology to elucidate the mechanisms of action of SMDOCH on CHD. We collected 270 SMDOCH-related targets from 74 bioactive components and 375 CHD-related targets, with 58 overlapping common targets. Next, we performed enrichment analysis for common-target network and protein-protein interaction (PPI) network. The results showed that SMDOCH affected CHD mainly through 10 significant signaling pathways in three biological processes: ‘vascular endothelial function regulation’, ‘inflammatory response’, and ‘lipid metabolism’. Six pathways belonged to the ‘vascular endothelial function regulation’ model, which primarily regulated hormone (renin, angiotensin, oestrogen) activity, and included three key upstream pathways that influence vascular endothelial function, namely KEGG:04933, KEGG:05418, and KEGG:04066. Three pathways, namely KEGG:04668, KEGG:04064, and KEGG:04620, belonged to the ‘inflammatory response’ model. One pathway (KEGG:04920) belonged to the ‘lipid metabolism’ model. To some extent, this study revealed the potential bioactive components and pharmacological mechanisms of SMDOCH on CHD, and provided a new direction for the development of new drugs for the treatment of CHD.

To systematically analyze the gene function of Dalbergia odorifera , the seedlings of D. odorifera were treated with low-temperature stress for 6 h. Total RNA was extracted from a mixture of seedling roots, stems, and leaves, and a low-temperature-induced D. odorifera yeast cDNA expression library was constructed. The library volume was 1.032 × 10 8 CFU, and the PCR (Polymerase Chain Reaction) identification of the library bacterial fluid showed that the amplification was around 1000 bp, with a single randomly distributed band, indicating that the library had been recombinantly inserted into the pYES2 vector. The GO (Gene Ontology) analysis showed that the library genes were mainly involved in metabolic and stress signaling pathways. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that the genes were primarily related to energy and metabolic pathways. Twenty-one genes were screened or obtained at -20°C for low-temperature tolerance. In addition, the organ expression profiles of the candidate genes were analyzed based on RNA-seq data, and the expression profiles of the candidate genes under low-temperature stress were also examined. The construction of the yeast library provides genetic resources for the analysis of the mechanism of low-temperature tolerance of D. odorifera , which is important for comprehending and utilizing the genetic resources of D. odorifera .

Background Dalbergia odorifera is a precious tree species with unique economic and medicinal values, which is difficult to distinguish from Dalbergia tonkinensis by traditional identification methods such as morphological characteristics and wood structure characteristics. It has been demonstrated that the identification of tree species can be effectively achieved using DNA barcoding, but there is a lack of study of the combined sequences used as DNA barcodes in the two tree species. In this study, 10 single sequences and 4 combined sequences were selected for analysis, and the identification effect of each sequence was evaluated by the distance-based method, BLAST-based search, character-based method, and tree-based method. Results Among the single sequences and the combined sequences, the interspecies distance of trnH-psbA and ITS2 +  trnH-psbA was greater than the intraspecies distance, and there was no overlap in their frequency distribution plots. The results of the Wilcoxon signed-rank test for the interspecies distance of each sequence showed that the interspecies differences of the single sequences except trnL-trnF , trnH-psbA , and ycf3 were significantly smaller than those of the combined sequences. The results of BLAST analysis showed that trnH-psbA could accurately identify D. odorifera and D. tonkinensis at the species level. In the character-based method, single sequences of trnL-trnF , trnH-psbA with all the combined sequences can be used for the identification of D. odorifera and D. tonkinensis . In addition, the neighbor-joining (NJ) trees constructed based on trnH-psbA and ITS2 +  trnH-psbA were able to cluster D. odorifera and D. tonkinensis on two clades. Conclusions The results showed that the character-based method with the BLOG algorithm was the most effective among all the evaluation methods, and the combined sequences can improve the ability to identify tree species compared with single sequences. Finally, the trnH-psbA and ITS2 +  trnH-psbA were proposed as DNA barcodes to identify D. odorifera and D. tonkinensis .