Identification and characterization of functionally relevant SSR markers in natural Dalbergia odorifera populations.

BACKGROUND: Dalbergia odorifera is a rare and precious rosewood specie, which is valued for its amber tones, abstract figural patterns, and impermeability to water and insects. However, the information on genetic diversity and marker-assisted selection breeding of D. odorifera is still limited. Simple sequence repeat (SSR) markers are an ideal tool for genetic diversity analysis and marker-assisted molecular breeding for complex traits. RESULTS: Here, we have developed SSR markers within candidate genes and used them to explore the genetic diversity among D. odorifera germplasm resources. A total of 635 SSR loci were identified. The proportions of mono-, di- and tri-nucleotide repeat motifs were 52.28%, 22.99% and 21.42%, respectively. From these, a total of 114 SSR primers were synthesized, of which 24 SSR markers displayed polymorphism (polymorphic information content (PIC) > 0.25). Subsequently, these polymorphic markers were used for the genetic diversity analysis of 106 D. odorifera individuals from 11 natural populations. According to the genetic diversity analysis of D. odorifera natural populations, the average observed heterozygosity (Ho) was 0.500, the average expected heterozygosity (He) was 0.524, and the average Shannon's information index (I) was 0.946. These indicated that the natural populations had moderate genetic diversity. AMOVA analysis showed that 5% of the total variation was within the individuals of a population, whereas 95% of the variation was among the individuals of the populations, indicating a high degree of genetic variation between populations. On the basis of their genetic structures, these populations could be divided into four groups. CONCLUSIONS: Our study provides important experimental resources for genetic studies and assists in the program of molecular breeding of D. odorifera wood formation.

Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera.

BACKGROUND: Alternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio's SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera. RESULTS: We identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA's role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis. CONCLUSIONS: This study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Dalbergia odorifera undergoes massive molecular shifts in response to waterlogging combined with salinity.

Field and greenhouse studies attempting to describe the molecular responses of plant species under waterlogging (WL) combined with salinity (ST) are almost nonexistent. We integrated transcriptional, metabolic, and physiological responses involving several crucial transcripts and common differentially expressed genes and metabolites in fragrant rosewood (Dalbergia odorifera) leaflets to dissect plant-specific molecular responses and patterns under WL combined with ST (SWL). We discovered that the synergistic pattern of the transcriptional response of fragrant rosewood under SWL was exclusively characterized by the number of regulated transcripts. The response patterns under SWL based on transcriptome and metabolome regulation statuses revealed different patterns (additive, dominant, neutral, minor, unilateral, and antagonistic) of transcripts or metabolites that were commonly regulated or expressed uniquely under SWL. Under SWL, the synergistic transcriptional response of several functional gene subsets was positively associated with several metabolomic and physiological responses related to the shutdown of the photosynthetic apparatus and the extensive degradation of starch into saccharides through α-amylase, ß-amylase, and α-glucosidase or plastoglobuli accumulation. The dissimilarity between the regulation status and number of transcripts in plants under combined stresses led to nonsynergistic responses in several physiological and phytohormonal traits. As inferred from the impressive synergistic transcriptional response to morpho-physiological changes, combined stresses exhibited a gradually decreasing effect on the changes observed at the molecular level compared to those in the morphological one. Here, by characterizing the molecular responses and patterns of plant species under SWL, our study considerably improves our understanding of the molecular mechanisms underlying combined stress.

The DNA barcode identification of Dalbergia odorifera T. Chen and Dalbergia tonkinensis Prain.

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.

De novo transcriptome assembly of Dalbergia sissoo Roxb. (Fabaceae) under Botryodiplodia theobromae-induced dieback disease.

Dalbergia sissoo Roxb. (Shisham) is a timber-producing species of economic, cultural, and medicinal importance in the Indian subcontinent. In the past few decades, Shisham's dieback disease caused by the fungus Botryodiplodia theobromae has become an evolving issue in the subcontinent endangering its survival. To gain insights into this issue, a standard transcriptome assembly was deployed to assess the response of D. sissoo at the transcriptomic level under the stress of B. theobromae infection. For RNA isolation, the control and infected leaf tissue samples were taken from 1-year-old greenhouse-grown D. sissoo plants after 20 days of stem-base spore inoculation. cDNA synthesis was performed from these freshly isolated RNA samples that were then sent for sequencing. About 18.14 Gb (Giga base) of data was generated using the BGISEQ-500 sequencing platform. In terms of Unigenes, 513,821 were identified after a combined assembly of all samples and then filtering the abundance. The total length of Unigenes, their average length, N50, and GC-content were 310,523,693 bp, 604 bp, 1,101 bp, and 39.95% respectively. The Unigenes were annotated using 7 functional databases i.e., 200,355 (NR: 38.99%), 164,973 (NT: 32.11%), 123,733 (Swissprot: 24.08%), 142,580 (KOG: 27.75%), 139,588 (KEGG: 27.17%), 99,752 (GO: 19.41%), and 137,281 (InterPro: 26.72%). Furthermore, the Transdecoder detected 115,762 CDS. In terms of SSR (Simple Sequence Repeat) markers, 62,863 of them were distributed on 51,508 Unigenes and on the predicted 4673 TF (Transcription Factor) coding Unigenes. A total of 16,018 up- and 19,530 down-regulated Differentially Expressed Genes (DEGs) were also identified. Moreover, the Plant Resistance Genes (PRGs) had a count of 9230. We are hopeful that in the future, these identified Unigenes, SSR markers, DEGs and PRGs will provide the prerequisites for managing Shisham dieback disease, its breeding, and in tree improvement programs.

Function of the R2R3-MYB Transcription Factors in Dalbergia odorifera and Their Relationship with Heartwood Formation.

R2R3-MYB transcription factors (TFs) form one of the most important TF families involved in regulating various physiological functions in plants. The heartwood of Dalbergia odorifera is a kind of high-grade mahogany and valuable herbal medicine with wide application. However, the role of R2R3-MYB genes in the growth and development of D. odorifera, especially their relevance to heartwood formation, has not been revealed. A total of 126 R2R3-MYBs were screened from the D. odorifera genome and named DodMYB1-126 based on their location on 10 chromosomes. The collinearity results showed that purification selection was the main driving force for the evolution of the R2R3-MYB TFs family, and whole genome/fragment replication event was the main form for expanding the R2R3-MYB family, generating a divergence of gene structure and function. Comparative phylogenetic analysis classified the R2R3-MYB TFs into 33 subfamilies. S3-7,10,12-13,21 and N4-7 were extensively involved in the metabolic process; S9,13,16-19,24-25 and N1-3,8 were associated with the growth and development of D. odorifera. Based on the differential transcriptional expression levels of R2R3-MYBs in different tissues, DodMYB32, DodMYB55, and DodMYB89 were tentatively screened for involvement in the regulatory process of heartwood. Further studies have shown that the DodMYB89, localized in the nucleus, has transcriptional activation activity and is involved in regulating the biosynthesis of the secondary metabolites of heartwood by activating the promoters of the structural genes DodI2'H and DodCOMT. This study aimed to comprehensively analyze the functions of the R2R3-MYB TFs and screen for candidate genes that might be involved in heartwood formation of D. odorifera.

Comparative Analysis of Codon Usage Patterns in Nuclear and Chloroplast Genome of Dalbergia (Fabaceae).

The Dalbergia plants are widely distributed across more than 130 tropical and subtropical countries and have significant economic and medicinal value. Codon usage bias (CUB) is a critical feature for studying gene function and evolution, which can provide a better understanding of biological gene regulation. In this study, we comprehensively analyzed the CUB patterns of the nuclear genome, chloroplast genome, and gene expression, as well as systematic evolution of Dalbergia species. Our results showed that the synonymous and optimal codons in the coding regions of both nuclear and chloroplast genome of Dalbergia preferred ending with A/U at the third codon base. Natural selection was the primary factor affecting the CUB features. Furthermore, in highly expressed genes of Dalbergia odorifera, we found that genes with stronger CUB exhibited higher expression levels, and these highly expressed genes tended to favor the use of G/C-ending codons. In addition, the branching patterns of the protein-coding sequences and the chloroplast genome sequences were very similar in the systematic tree, and different with the cluster from the CUB of the chloroplast genome. This study highlights the CUB patterns and features of Dalbergia species in different genomes, explores the correlation between CUB preferences and gene expression, and further investigates the systematic evolution of Dalbergia, providing new insights into codon biology and the evolution of Dalbergia plants.

Characterization of the complete chloroplast genome of Dalbergia congesta (Fabaceae), an endangered legume endemic to the Nilgiri Hills of Tamil Nadu, India.

Reference-guided de novo assembly of the Dalbergia congesta chloroplast genome was carried out using whole-genome sequencing data. The newly generated chloroplast genome size had a total length of 156,048 bp and a GC content of 36.1%. The plastome showed the classical quadripartite structure with two inverted repeats regions (IRs; each 25,715 bp) separating the large single-copy region (LSC; 85,456 bp) from the small single-copy region (SSC; 19,162 bp). The plastid genome contained 111 unique genes, including 77 protein-coding genes (CDS), 30 tRNAs, and 4 rRNAs. The phylogenomic analyses based on whole chloroplast genome sequences recovered Dalbergia as a distinct clade of the Papilionoideae, with Dalbergia congesta having a sister relationship to a clade comprising D. fusca and D. cultrata. The newly available plastome sequence will facilitate future genetic and conservational research aiming to protect this economically important but highly threatened legume species.

Characterization of the complete chloroplast genome sequences of six Dalbergia species and its comparative analysis in the subfamily of Papilionoideae (Fabaceae).

Dalbergia spp. are numerous and widely distributed in pantropical areas in Asia, Africa and America, and most of the species have important economic and ecological value as precious timber. In this study, we determined and characterized six complete chloroplast genomes of Dalbergia species (Dalbergia obtusifolia, D. hupeana, D. mimosoides, D. sissoo, D. hancei, D. balansae), which displayed the typical quadripartite structure of angiosperms. The sizes of the genomes ranged from 155,698 bp (D. hancei) to 156,419 bp (D. obtusifolia). The complete chloroplast genomes of Dalbergia include 37 tRNA genes, eight rRNA genes and 84 protein-coding genes. We analysed the sequence diversity of Dalberigia chloroplast genomes coupled with previous reports. The results showed 12 noncoding regions (rps16-accD, trnR-UCU-trnG-UCC, ndhE-ndhG, trnG-UCC-psbZ, rps8-rpl14, trnP-UGG-psaJ, ndhH-rps15, trnQ-UUG-rps16, trnS-GCU-psbI, rps12-clpP, psbA-trnK-UUU, trnK-UUU-intron), and four coding regions (rps16, ycf1, rps15 and ndhF) showed many nucleotide variations that could be used as potential molecular markers. Based on a site-specific model, we analysed the selective pressure of chloroplast genes in Dalbergia species. Twenty-two genes with positively selected sites were detected, involving the photosynthetic system (ndhC, adhD, ndhF, petB, psaA, psaB, psbB, psbC, psbK and rbcL), self-replication category of genes (rpoA, rpoC2, rps3, rps12 and rps18) and others (accD, ccsA, cemA, clpP, matK, ycf1 and ycf2). Additionally, we identified potential RNA editing sites that were relatively conserved in the genus Dalbergia. Furthermore, the comparative analysis of cp genomes of Dalbergieae species indicated that the boundary of IRs/SSC was highly variable, which resulted in the size variation of cp genomes. Finally, phylogenetic analysis showed an inferred phylogenetic tree of Papilionoideae species with high bootstrap support and suggested that Amorpheae was the sister of the clade Dalbergieae. Moreover, three genera of the Pterocarpus clade showed a nested evolutionary relationship. These complete cp genomes provided valuable information for understanding the genetic variation and phylogenetic relationship of Dalbergia species with their relatives.

A target capture approach for phylogenomic analyses at multiple evolutionary timescales in rosewoods (Dalbergia spp.) and the legume family (Fabaceae).

Understanding the genetic changes associated with the evolution of biological diversity is of fundamental interest to molecular ecologists. The assessment of genetic variation at hundreds or thousands of unlinked genetic loci forms a sound basis to address questions ranging from micro- to macroevolutionary timescales, and is now possible thanks to advances in sequencing technology. Major difficulties are associated with (i) the lack of genomic resources for many taxa, especially from tropical biodiversity hotspots; (ii) scaling the numbers of individuals analysed and loci sequenced; and (iii) building tools for reproducible bioinformatic analyses of such data sets. To address these challenges, we developed target capture probes for genomic studies of the highly diverse, pantropically distributed and economically significant rosewoods (Dalbergia spp.), explored the performance of an overlapping probe set for target capture across the legume family (Fabaceae), and built the general purpose bioinformatic pipeline CaptureAl. Phylogenomic analyses of Malagasy Dalbergia species yielded highly resolved and well supported hypotheses of evolutionary relationships. Population genomic analyses identified differences between closely related species and revealed the existence of a potentially new species, suggesting that the diversity of Malagasy Dalbergia species has been underestimated. Analyses at the family level corroborated previous findings by the recovery of monophyletic subfamilies and many well-known clades, as well as high levels of gene tree discordance, especially near the root of the family. The new genomic and bioinformatic resources, including the Fabaceae1005 and Dalbergia2396 probe sets, will hopefully advance systematics and ecological genetics research in legumes, and promote conservation of the highly diverse and endangered Dalbergia rosewoods.

A complete mitochondrial genome for fragrant Chinese rosewood (Dalbergia odorifera, Fabaceae) with comparative analyses of genome structure and intergenomic sequence transfers.

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.

Evaluating the genetic diversity in two tropical leguminous trees, Dalbergia cochinchinensis and D. nigrescens, in lowland forests in Cambodia and Thailand using MIG-seq.

It is vital to measure the levels of genetic diversity and differentiation between populations in a species to understand the current genetic structure and evolution of the species. Here, MIG-seq (multiplexed inter-simple sequence repeat genotyping by sequencing) was employed to assess the genetic variation in two tropical leguminous tree species, Dalbergia cochinchinensis and D. nigrescens, in Cambodia and Thailand. Sequence data for 255-618 loci, each with an approximate length of 100 bp, were obtained, and the nucleotide diversity, Tajima's D and FST were computed. The estimates calculated from the data obtained by MIG-seq were compared to those obtained by Sanger sequencing of nine nuclear coding genes in D. cochinchinensis in our previous study. The nucleotide diversity at the MIG-seq loci was slightly higher than that at silent sites in the coding loci, whereas the FST values at the MIG-seq loci were generally lower than those at the coding loci, although the differences were not significant. Moreover, nucleotide diversities within populations of the two species were similar to each other, at approximately 0.005. Three and four population clusters were genetically recognized in D. cochinchinensis and D. nigrescens, respectively. Although the populations were differentiated from each other, the levels of differentiation among them, as measured by FST, were higher in D. cochinchinensis than in D. nigrescens. This indicates higher levels of gene flow between the populations in the latter species. We recommend using MIG-seq for quick surveys of genetic variation because it is cost-effective and results in smaller variance in the estimates of population genetic parameters.

Reference transcriptomes and comparative analyses of six species in the threatened rosewood genus Dalbergia.

Dalbergia is a pantropical genus with more than 250 species, many of which are highly threatened due to overexploitation for their rosewood timber, along with general deforestation. Many Dalbergia species have received international attention for conservation, but the lack of genomic resources for Dalbergia hinders evolutionary studies and conservation applications, which are important for adaptive management. This study produced the first reference transcriptomes for 6 Dalbergia species with different geographical origins and predicted ~ 32 to 49 K unique genes. We showed the utility of these transcriptomes by phylogenomic analyses with other Fabaceae species, estimating the divergence time of extant Dalbergia species to ~ 14.78 MYA. We detected over-representation in 13 Pfam terms including HSP, ALDH and ubiquitin families in Dalbergia. We also compared the gene families of geographically co-occurring D. cochinchinensis and D. oliveri and observed that more genes underwent positive selection and there were more diverged disease resistance proteins in the more widely distributed D. oliveri, consistent with reports that it occupies a wider ecological niche and has higher genetic diversity. We anticipate that the reference transcriptomes will facilitate future population genomics and gene-environment association studies on Dalbergia, as well as contributing to the genomic database where plants, particularly threatened ones, are currently underrepresented.

The chromosome-level draft genome of Dalbergia odorifera.

BACKGROUND: Dalbergia odorifera T. Chen (Fabaceae) is an International Union for Conservation of Nature red-listed tree. This tree is of high medicinal and commercial value owing to its officinal, insect-proof, durable heartwood. However, there is a lack of genome reference, which has hindered development of studies on the heartwood formation. FINDINGS: We presented the first chromosome-scale genome assembly of D. odorifera obtained on the basis of Illumina paired-end sequencing, Pacific Biosciences single-molecule real-time sequencing, 10x Genomics linked reads, and Hi-C technology. We assembled 97.68% of the 653.45 Mb D. odorifera genome with scaffold N50 and contig sizes of 56.16 and 5.92 Mb, respectively. Ten super-scaffolds corresponding to the 10 chromosomes were assembled, with the longest scaffold reaching 79.61 Mb. Repetitive elements account for 54.17% of the genome, and 30,310 protein-coding genes were predicted from the genome, of which ∼92.6% were functionally annotated. The phylogenetic tree showed that D. odorifera diverged from the ancestor of Arabidopsis thaliana and Populus trichocarpa and then separated from Glycine max and Cajanus cajan. CONCLUSIONS: We sequence and reveal the first chromosome-level de novo genome of D. odorifera. These studies provide valuable genomic resources for the research of heartwood formation in D. odorifera and other timber trees. The high-quality assembled genome can also be used as reference for comparative genomics analysis and future population genetic studies of D. odorifera.

Molecular Mechanism Underlying Mechanical Wounding-Induced Flavonoid Accumulation in Dalbergia odorifera T. Chen, an Endangered Tree That Produces Chinese Rosewood.

Dalbergia odorifera, a critically endangered tree species, produces heartwood containing a vast variety of flavonoids. This heartwood, also known as Chinese rosewood, has high economic and medicinal value, but its formation takes several decades. In this study, we showed that discolored wood induced by pruning displays similar color, structure, and flavonoids content to those of natural heartwood, suggesting that wounding is an efficient method for inducing flavonoid production in D. odorifera. Transcriptome analysis was performed to investigate the mechanism underlying wounding-induced flavonoids production in D. odorifera heartwood. Wounding upregulated the expression of 90 unigenes, which covered 19 gene families of the phenylpropanoid and flavonoid pathways, including PAL, C4H, 4CL, CHS, CHI, 6DCS, F3'5'H, F3H, FMO, GT, PMAT, CHOMT, IFS, HI4'OMT, HID, IOMT, I2'H, IFR, and I3'H. Furthermore, 47 upregulated unigenes were mapped to the biosynthesis pathways for five signal molecules (ET, JA, ABA, ROS, and SA). Exogenous application of these signal molecules resulted in the accumulation of flavonoids in cell suspensions of D. odorifera, supporting their role in wounding-induced flavonoid production. Insights from this study will help develop new methods for rapidly inducing the formation of heartwood with enhanced medicinal value.

Genetic Diversity and Population Structure Analysis of DalbergiaOdorifera Germplasm and Development of a Core Collection Using Microsatellite Markers.

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.

Selection and Validation of Reference Genes for Gene Expression Studies by RT-PCR in Dalbergia odorifera.

Perennial tree Dalbergia odorifera T. Chen could form the precious heartwood used to produce chinese traditional medicine, rosewood furniture and fragrances. However the formation of heartwood is time-consuming and low efficient, leading to the severe destruction of its wild resources. Thus, it is urgent to study the molecular mechanism of heartwood formation in D. odorifera. But till now, there is no report about the reference gene selection in this species. In this study, the expression stability of nine candidate reference genes were evaluated across different tissues and stems treated by wound and chemical stimulators. Four algorithms were applied to obtain the robust genes. The results support HIS2, GAPDH, and CYP to be the most stable reference genes in samples under different wound treatments while DNAj was the least stable. In different tissues, HIS2, UBQ, and RPL were the most stable reference genes while DNAj was the least stable. The selected reference genes were validated through the normalization of the qRT-PCR data of six heartwood related genes in terpene biosynthesis pathway and ethylene signal pathway. The results showed that their expression levels were accurate when they were normalized by the most stable reference gene HIS2, or by the combination of the two or three most stable reference genes. These results demonstrated that these selected reference genes are reliable.

Characterization of the complete chloroplast genome sequence of Dalbergia species and its phylogenetic implications.

The pantropical plant genus Dalbergia comprises approximately 250 species, most of which have a high economic and ecological value. However, these species are among the most threatened due to illegal logging and the timber trade. To enforce protective legislation and ensure effective conservation of Dalbergia species, the identity of wood being traded must be accurately validated. For the rapid and accurate identification of Dalbergia species and assessment of phylogenetic relationships, it would be highly desirable to develop more effective DNA barcodes for these species. In this study, we sequenced and compared the chloroplast genomes of nine species of Dalbergia. We found that these chloroplast genomes were conserved with respect to genome size, structure, and gene content and showed low sequence divergence. We identified eight mutation hotspots, namely, six intergenic spacer regions (trnL-trnT, atpA-trnG, rps16-accD, petG-psaJ, ndhF-trnL, and ndhG-ndhI) and two coding regions (ycf1a and ycf1b), as candidate DNA barcodes for Dalbergia. Phylogenetic analyses based on whole chloroplast genome data provided the best resolution of Dalbergia, and phylogenetic analysis of the Fabaceae showed that Dalbergia was sister to Arachis. Based on comparison of chloroplast genomes, we identified a set of highly variable markers that can be developed as specific DNA barcodes.

Multiple mutations in the aglycone binding pocket to convert the substrate specificity of dalcochinase to linamarase.

Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are ß-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol. So, the space provided by the I185A mutation appeared to be a prerequisite for accommodation of the aglycone moiety containing three substituents at the carbinol carbon. However, quantitative analysis of the energy parameters revealed mostly antagonistic interactions between these mutations. In addition, the N189F mutant showed a potential for use in enzymatic synthesis of alkyl glucosides via transglucosylation and reverse hydrolysis reactions. Thus, substitution of only 2-3 key residues in the aglycone binding pocket of dalcochinase could convert its specificities to that of linamarase, as well as to be suitable for any chosen hydrolytic or synthetic applications.

DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species.

MAIN CONCLUSION: ITS2+ trnH - psbA was the best combination of DNA barcode to resolve the Dalbergia wood species studied. We demonstrate the feasibility of building a DNA barcode reference database using xylarium wood specimens. The increase in illegal logging and timber trade of CITES-listed tropical species necessitates the development of unambiguous identification methods at the species level. For these methods to be fully functional and deployable for law enforcement, they must work using wood or wood products. DNA barcoding of wood has been promoted as a promising tool for species identification; however, the main barrier to extensive application of DNA barcoding to wood is the lack of a comprehensive and reliable DNA reference library of barcodes from wood. In this study, xylarium wood specimens of nine Dalbergia species were selected from the Wood Collection of the Chinese Academy of Forestry and DNA was then extracted from them for further PCR amplification of eight potential DNA barcode sequences (ITS2, matK, trnL, trnH-psbA, trnV-trnM1, trnV-trnM2, trnC-petN, and trnS-trnG). The barcodes were tested singly and in combination for species-level discrimination ability by tree-based [neighbor-joining (NJ)] and distance-based (TaxonDNA) methods. We found that the discrimination ability of DNA barcodes in combination was higher than any single DNA marker among the Dalbergia species studied, with the best two-marker combination of ITS2+trnH-psbA analyzed with NJ trees performing the best (100% accuracy). These barcodes are relatively short regions (<350 bp) and amplification reactions were performed with high success (≥90%) using wood as the source material, a necessary factor to apply DNA barcoding to timber trade. The present results demonstrate the feasibility of using vouchered xylarium specimens to build DNA barcoding reference databases.