Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction.

In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

Unpredictable Chemical Diversity of Essential Oils in Cinnamomum burmanni (Lauraceae) Living Collections: Beyond Maternally Inherited Phylogenetic Relationships.

The genus Cinnamomum encompasses diverse species with various applications, particularly in traditional medicine and spice production. This study focuses on Cinnamomum burmanni, specifically on a high-D-borneol-content chemotype, known as the Meipian Tree, in Guangdong Province, South China. This research explores essential oil diversity, chemotypes, and chloroplast genomic diversity among 28 C. burmanni samples collected from botanical gardens. Essential oils were analyzed, and chemotypes classified using GC-MS and statistical methods. Plastome assembly and phylogenetic analysis were conducted to reveal genetic relationships. Results showed distinct chemotypes, including eucalyptol and borneol types, with notable variations in essential oil composition. The chloroplast genome exhibited conserved features, with phylogenetic analysis revealing three major clades. Borneol-rich individuals in clade II suggested a potential maternal inheritance pattern. However, phylogenetic signals revealed that the composition of essential oils is weakly correlated with plastome phylogeny. The study underscores the importance of botanical gardens in preserving genetic and chemical diversity, offering insights for sustainable resource utilization and selective breeding of high-yield mother plants of C. burmanni.

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 µg/mL) and bactericidal concentration (2.0 µg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2-2×MIC) and times (0-24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens.

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.

Essential Oil Chemotypes and Genetic Variability of Cinnamomum verum Leaf Samples Commercialized and Cultivated in the Amazon.

Cinnamomum verum (Lauraceae), also known as "true cinnamon" or "Ceylon cinnamon" has been widely used in traditional folk medicine and cuisine for a long time. The systematics of C. verum presents some difficulties due to genetic variation and morphological similarity between other Cinnamomum species. The present work aimed to find chemical and molecular markers of C. verum samples from the Amazon region of Brazil. The leaf EOs and the genetic material (DNA) were extracted from samples cultivated and commercial samples. The chemical composition of the essential oils from samples of C. verum cultivated (Cve1-Cve5) and commercial (Cve6-c-Cv9-c) was grouped by multivariate statistical analysis of Principal Component Analysis (PCA). The major compounds were rich in benzenoids and phenylpropanoids, such as eugenol (0.7-91.0%), benzyl benzoate (0.28-76.51%), (E)-cinnamyl acetate (0.36-32.1%), and (E)-cinnamaldehyde (1.0-19.73%). DNA barcodes were developed for phylogenetic analysis using the chloroplastic regions of the matK and rbcL genes, and psbA-trnH intergenic spacer. The psbA-trnH sequences provided greater diversity of nucleotides, and matK confirmed the identity of C. verum. The combination of DNA barcode and volatile profile was found to be an important tool for the discrimination of C. verum varieties and to examine the authenticity of industrial sources.

Transcriptome and metabolome reveal the accumulation of secondary metabolites in different varieties of Cinnamomum longepaniculatum.

BACKGROUND: Cinnamomum longepaniculatum (Gamble) N. Chao ex H. W. Li, whose leaves produce essential oils, is a traditional Chinese medicine and economically important tree species. In our study, two C. longepaniculatum varieties that have significantly different essential oil contents and leaf phenotypes were selected as the materials to investigate secondary metabolism. RESULT: The essential oil content and leaf phenotypes were different between the two varieties. When the results of both transcriptome and metabolomic analyses were combined, it was found that the differences were related to phenylalanine metabolic pathways, particularly the metabolism of flavonoids and terpenoids. The transcriptome results based on KEGG pathway enrichment analysis showed that pathways involving phenylpropanoids, tryptophan biosynthesis and terpenoids significantly differed between the two varieties; 11 DEGs (2 upregulated and 9 downregulated) were associated with the biosynthesis of other secondary metabolites, and 12 DEGs (2 upregulated and 10 downregulated) were related to the metabolism of terpenoids and polyketides. Through further analysis of the leaves, we detected 196 metabolites in C. longepaniculatum. The abundance of 49 (26 downregulated and 23 upregulated) metabolites differed between the two varieties, which is likely related to the differences in the accumulation of these metabolites. We identified 12 flavonoids, 8 terpenoids and 8 alkaloids and identified 4 kinds of PMFs from the leaves of C. longepaniculatum. CONCLUSIONS: The combined results of transcriptome and metabolomic analyses revealed a strong correlation between metabolite contents and gene expression. We speculate that light leads to differences in the secondary metabolism and phenotypes of leaves of different varieties of C. longepaniculatum. This research provides data for secondary metabolite studies and lays a solid foundation for breeding ideal C. longepaniculatum plants.

Transcriptome analysis of Cinnamomum migao seed germination in medicinal plants of Southwest China.

BACKGROUND: Cinnamomum migao is an endangered evergreen woody plant species endemic to China. Its fruit is used as a traditional medicine by the Miao nationality of China and has a high commercial value. However, its seed germination rate is extremely low under natural and artificial conditions. As the foundation of plant propagation, seed germination involves a series of physiological, cellular, and molecular changes; however, the molecular events and systematic changes occurring during C. migao seed germination remain unclear. RESULTS: In this study, combined with the changes in physiological indexes and transcription levels, we revealed the regulation characteristics of cell structures, storage substances, and antioxidant capacity during seed germination. Electron microscopy analysis revealed that abundant smooth and full oil bodies were present in the cotyledons of the seeds. With seed germination, oil bodies and other substances gradually degraded to supply energy; this was consistent with the content of storage substances. In parallel to electron microscopy and physiological analyses, transcriptome analysis showed that 80-90 % of differentially expressed genes (DEGs) appeared after seed imbibition, reflecting important development and physiological changes. The unigenes involved in material metabolism (glycerolipid metabolism, fatty acid degradation, and starch and sucrose metabolism) and energy supply pathways (pentose phosphate pathway, glycolysis pathway, pyruvate metabolism, tricarboxylic acid cycle, and oxidative phosphorylation) were differentially expressed in the four germination stages. Among these DEGs, a small number of genes in the energy supply pathway at the initial stage of germination maintained high level of expression to maintain seed vigor and germination ability. Genes involved in lipid metabolism were firstly activated at a large scale in the LK (seed coat fissure) stage, and then genes involved in carbohydrates (CHO) metabolism were activated, which had their own species specificity. CONCLUSIONS: Our study revealed the transcriptional levels of genes and the sequence of their corresponding metabolic pathways during seed germination. The changes in cell structure and physiological indexes also confirmed these events. Our findings provide a foundation for determining the molecular mechanisms underlying seed germination.

Universal barcoding regions, rbcL, matK and trnH-psbA do not discriminate Cinnamomum species in Sri Lanka.

The genus Cinnamomum consists of about 250 species spread globally. Out of these, C. verum (C. zeylanicum), also known as true cinnamon or Ceylon cinnamon, has gained worldwide attention due to its culinary uses and medicinal values. Sri Lanka is the largest true cinnamon producer in the world and accounts for about 80-90% of global production. Other than the cultivated species, Sri Lankan natural vegetation is home to seven endemic wild species of the genus Cinnamomum. While these are underutilized, proper identification and characterization are essential steps in any sustainable conservation and utilization strategies. Currently, species identification is purely based on morphological traits, and intraspecific diversity has made it more challenging. In this study, all the eight Cinnamomum species found in Sri Lanka, C. capparu-coronde, C. citriodorum C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum, C. sinharajaense, and C. verum were collected in triplicates and identified using typical morphological traits. DNA extracted with the same collection was assessed with universal barcoding regions, rbcL, matK, and trnH-psbA. While no intraspecific sequence differences were observed in C. citriodorum, C. rivulorum, and C. verum, the others had polymorphic sites in one, two, or all regions assessed. Interestingly, two individuals of C. sinharajaense had identical barcodes to the cultivated species C. verum, while the other one had one variable cite in matK region and three cites in trnH-psbA reigon. Further, one C. dubium and one C. capparu-coronde accession each had identical, rbcL, and trnH-psbA sequences while those had only a single nucleotide variation observed in matK region. Overall, the phylogeny of Cinnamomum species found in Sri Lanka could not be completely resolved with DNA barcoding regions studied.

Identification of hybridization and introgression between Cinnamomum kanehirae Hayata and C. camphora (L.) Presl using genotyping-by-sequencing.

Cinnamomum kanehirae Hayata and C. camphora (L.) Presl are important tree species in eastern Asia. The wood of C. kanehirae is in increasing demand for culturing Antrodia cinnamomea, a medicinal fungus that naturally grows inside the trunk of C. kanehirae. Putative hybrids between C. kanehirae and C. camphora were previously reported but with no scientific evidence, leading to confusion or misplanting. First, to identify the female parent of putative hybrids, the maternal inheritance InDel (insertion/deletion) markers were developed by using low-coverage sequencing. SNPs were developed by using genotyping-by-sequencing (GBS) approach in C. kanehirae, C. camphora and putative hybrids. The results indicated that the female parent of the studied hybrids was C. camphora. Eight hundred and forty of the 529,006 high-density SNPs were selected and used for analysis. Hybrids were classified as F1 (C. kanehirae × C. camphora), F2 and backcrosses. Hybridization has occurred in the human-developed area of eastern and southwestern Taiwan, and the introgression was bidirectional. For producing pure wood, buffering zones should be established around seed orchards to avoid cross-species pollination and to preserve the genetic purity of C. kanehirae. The DNA markers developed in this study will also be valuable for further wood identification, breeding and evolutionary research.

Full-Length Transcriptome Sequencing and Different Chemotype Expression Profile Analysis of Genes Related to Monoterpenoid Biosynthesis in Cinnamomum porrectum.

Leaves of C. porrectum are rich in essential oils containing monoterpenes, sesquiterpenes and aromatic compounds, but the molecular mechanism of terpenoid biosynthesis in C. porrectum is still unclear. In this paper, the differences in the contents and compositions of terpenoids among three chemotypes were analyzed using gas chromatography mass spectrometry (GC/MS). Furthermore, the differential expression of gene transcripts in the leaf tissues of the three C. porrectum chemotypes were analyzed through a comparison of full-length transcriptomes and expression profiles. The essential oil of the three C. porrectum chemotypes leaves was mainly composed of monoterpenes. In the full-length transcriptome of C. porrectum, 104,062 transcripts with 306,337,921 total bp, an average length of 2944 bp, and an N50 length of 5449 bp, were obtained and 94025 transcripts were annotated. In the eucalyptol and linalool chemotype, the camphor and eucalyptol chemotype, and the camphor and linalool chemotype comparison groups, 21, 22 and 18 terpene synthase (TPS) unigenes were identified respectively. Three monoterpene synthase genes, CpTPS3, CpTPS5 and CpTPS9, were upregulated in the eucalyptol chemotype compared to the linalool chemotype and camphor chemotype. CpTPS1 was upregulated in the camphor chemotype compared to the linalool chemotype and the eucalyptol chemotype. CpTPS4 was upregulated in the linalool chemotype compared to the camphor chemotype and the eucalyptol chemotype. Different unigenes had different expression levels among the three chemotypes, but the unigene expression levels of the 2-C-methyl-D-erythritol 4phosphate (MEP) pathway were generally higher than those of the mevalonate acid (MVA) pathway. Quantitative reverse transcription PCR(qRT-PCR) further validated these expression levels. The present study provides new clues for the functional exploration of the terpenoid synthesis mechanism and key genes in different chemotypes of C. porrectum.

Transcriptome analysis of Cinnamomum longepaniculatum by high-throughput sequencing

Background: Cinnamomum longepaniculatum is an important commercial crop and the main source of volatile terpenoids. The biosynthesis of key bioactive metabolites of C. longepaniculatum is not well understood because of the lack of available genomic and transcriptomic information. To address this issue, we performed transcriptome sequencing of C. longepaniculatum leaves to identify factors involved in terpenoid metabolite biosynthesis. Results: Transcriptome sequencing of C. longepaniculatum leaves generated over 56 million raw reads. The transcriptome was assembled using the Trinity software and yielded 82,061 unigenes with an average length of 879.43 bp and N50 value of 1387 bp. Furthermore, Benchmarking Universal Single-Copy Orthologs analysis indicated that our assembly is 91% complete. The unigenes were used to query the nonredundant database depending on sequence similarity; 42,809 unigenes were homologous to known genes in different species, with an annotation rate of 42.87%. The transcript abundance and Gene Ontology analyses revealed that numerous unigenes were associated with metabolism, while others were annotated in functional categories including transcription, signal transduction, and secondary metabolism. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 19,260 unigenes were involved in 385 metabolic pathways, with 233 unigenes found to be involved in terpenoid metabolism. Moreover, 23,463 simple sequence repeats were identified using the microsatellite identification tool. Conclusion: This is the first detailed transcriptome analysis of C. longepaniculatum. The findings provide insights into the molecular basis of terpenoid biosynthesis and a reference for future studies on the genetics and breeding of C. longepaniculatum.

Origins and evolution of cinnamon and camphor: A phylogenetic and historical biogeographical analysis of the Cinnamomum group (Lauraceae).

Tropical and subtropical amphi-Pacific disjunction is among the most fascinating distribution patterns, but received little attention. Here we use the fossil-rich Cinnamomum group, a primarily tropical and subtropical Asian lineage with some species distributed in Neotropics, Australasia and Africa to shed light upon this disjunction pattern. Phylogenetic and biogeographic analyses were carried out using sequences of three nuclear loci from 94 Cinnamomum group and 13 outgroup samples. Results show that although there are three clades within a monophyletic Cinnamomum group, Cinnamomum and previously recognized subdivisions within this genus were all rejected as natural groups. The Cinnamomum group appears to have originated in the widespread boreotropical paleoflora of Laurasia during the early Eocene (ca. 55Ma). The formation and breakup of the boreotropics seems to have then played a key role in the formation of intercontinental disjunctions within the Cinnamomum group. The first cooling interval (50-48Ma) in the late early Eocene resulted in a floristic discontinuity between Eurasia and North America causing the tropical and subtropical amphi-Pacific disjunction. The second cooling interval in the mid-Eocene (42-38Ma) resulted in the fragmentation of the boreotropics within Eurasia, leading to an African-Asian disjunction. Multiple dispersal events from North into South America occurred from the early Eocene to late Miocene and a single migration event from Asia into Australia appears to have occurred in the early Miocene.

Identification of novel acyl-ACP thioesterase gene ClFATB1 from Cinnamomum longepaniculatum.

A putative fatty acyl-acyl carrier protein (acyl-ACP) thioesterase (thioesterase) full-length cDNA sequence named as ClFATB1 was obtained from the seed cDNA library of Cinnamomum longepaniculatum by the SMART-RACE method. The novel gene encodes a protein of 382 amino acid residues with close homology to fatty acid thioesterase type B (FATB) enzymes of other plants, with two essential residues (His285 and Cys320) for thioesterase catalytic activity. The gene was transcribed in all tissues of C. longepaniculatum, the highest being in seeds. Recombinant ClFATB1 in Escherichia coli had higher specific activities against saturated 16:0- and 18:0-ACPs than on unsaturated 18:1-ACP. Overexpression of ClFATB1 in transgenic tobaccos upregulated thioesterase activities of crude proteins against 16:0-ACP and 18:0-ACP by 20.3 and 5.7%, respectively, and resulted in an increase in the contents of palmitic and stearic acids by 15.4 and 10.5%, respectively. However, ectopic expression of this gene decreased the substrate specificities of crude proteins to unsaturated 18:1-ACP by 12.7% in transgenic tobacco and lowered the contents of oleic, linoleic, and linolenic acids in transgenic leaves. So ClFATB1 would potentially upregulate the synthesis of saturated fatty acids and downregulate unsaturated ones in the fatty acid synthesis pathway of plants.

Development of microsatellite markers for Cinnamomum camphora (Lauraceae).

PREMISE OF THE STUDY: Cinnamomum camphora is an evergreen tree distributed in southern Japan, Taiwan, and southeastern China. Because of its vast utilization and cultivation, the natural distribution area of this species has been controversial. METHODS AND RESULTS: I isolated and characterized 22 microsatellite loci in C. camphora. Levels of polymorphism were evaluated in 104 adult trees from three populations in Japan: Meiji Jingu (Shinto Shrine), Kajiya Plantation, and Manazuru Peninsula. The mean number of alleles per locus ranged from 4.1 to 8.0 among populations. The mean observed and expected heterozygosities per population ranged from 0.53 to 0.60 and 0.55 to 0.68, respectively. CONCLUSIONS: All of 22 loci showed a clear and strong single band for each allele, and revealed a useful degree of polymorphism. The microsatellite markers described here will be useful to study the history, population dynamics, mating system, and genetic structure of C. camphora.

[Researches on relationship between genetic differentiation and chemical variation of Cinnamomum migao].

OBJECTIVE: To study the relationship between the genetic diversity and chemical variation of Cinnamomum migao. METHOD: ISSR marker technique was used to research the genetic structure of 9 population, GC-MS was used to analyze the main ingredients of the volatile oil in C. migao. RESULT: The analysis on the main ingredients of the volatile oil showed that there were significant or extremely significant differences in 9 populations. The minimum variation index of population was Yunnan Funing and the maximum variation index of population was Guangxi Yueye. ISSR marker analysis showed that the average of polymorphic loci percentage (P) was 42.41%, expected heterozygosity (H) was 0.181 0, Shannon's information index (I) was 0.293 8, the Nei's genetic diversity (H(s)) in the group was 0.188 9, genetic differentiation index (G(st)) was 2.269 1. The relationship between the genetic diversity and chemical variation showed that there was no significant correlation between the main ingredients of the volatile oil and 4 indexes of genetic structure of C. migao. CONCLUSION: The genetic diversity of C. migao was relatively high at the population levels, while it is low within the population levels, the relationship between chemical variation and genetic diversity was not obvious, that may indicate that other factors causes the chemical variation of C. migao.

Historical spatial range expansion and a very recent bottleneck of Cinnamomum kanehirae Hay. (Lauraceae) in Taiwan inferred from nuclear genes.

BACKGROUND: Species in the varied geographic topology of Taiwan underwent obvious demographic changes during glacial periods. Cinnamomum kanehirae has been exploited for timber and to obtain medicinal fungi for the past 100 years. Understanding anthropogenic factors influencing the demography of this species after the last glacial maximum (LGM) is critically important for the conservation of this species. RESULTS: Populations of C. kanehirae were classified into four geographic regions: northwestern (NW), west-central (WC), southwestern (SW), and southeastern (SE). In total, 113 individuals from 19 localities were sampled, and variations in the chalcone synthase gene (Chs) intron and leafy (Lfy) intron-2 sequences of nuclear DNA were examined in order to assess phylogeographic patterns, the timescales of demographic and evolutionary events, and recent anthropogenic effects. In total, 210 Chs and 170 Lfy sequences, which respectively constituted 36 and 35 haplotypes, were used for the analyses. Estimates of the migration rate (M) through time revealed a pattern of frequent gene flow during previous and the present interglacials. The isolation-by-distance test showed that there generally was no significant correlation between genetic and geographic distances. The level of among-region genetic differentiation was significant when comparing eastern to western populations. However, no significant among-region genetic differentiation was found in comparisons among the four geographic regions. Moreover, essentially no genetic structuring was found for the three regions west of the CMR. A fit of spatial range expansion was found for pooled and regional samples according to the non-significant values of the sum of squared deviations. Using the Bayesian skyline plot (BSP) method, a recent bottleneck after the LGM expansion was detected in both regional and pooled samples. CONCLUSIONS: Common haplotype distributions among geographic regions and the relatively shallow genetic structuring displayed are the result of historical gene flows. Southward dispersals in an earlier time frame from the NW region and in a later time frame from the SE region were inferred. The BSP analysis suggested a postglacial expansion event. Recent trends, however, refer to a bottleneck due to human interventions observed for both pooled and regional C. kanehirae samples.

A geraniol-synthase gene from Cinnamomum tenuipilum.

Geraniol may accumulate up to 86-98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC-MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 microM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.

Temporal aspects of the fine-scale genetic structure in a population of Cinnamomum insularimontanum (Lauraceae).

Cinnamomum insularimontanum Hayata (Lauraceae) is an insect-pollinated, broad-leaved evergreen tree with bird-dispersed seeds. We used allozyme loci, Wright's fixation index, spatial autocorrelation statistics (Moran's I), and coancestry measures to examine changes in genetic structure among four age-classes within a recently founded study population (60 x 100 m area) in southern Korea. There were no significant differences in expected heterozygosity among age classes. However, significant genetic differentiation among age classes was detected (P<0.0001). Fixation indices within age classes showed significant deficits of observed heterozygosity, which may be caused by partial selfing. The homogeneity of genetic structure among four age-classes may reflect similar spatial patterns of seed immigration from surrounding populations occurring year after year. Finally, the average Moran's I and coancestry estimates indicated essentially random spatial distributions of alleles for each of the four age-classes and between seedlings and 2-4 year juveniles vs adult trees. These findings are very similar to those observed in the same study area for another member of the Lauraceae, Neolitsea sericea, which has a very similar life history and ecological characteristics (ie, bird-dispersed fruits, insect pollination, and a similar age structure). Together, these results suggest that the fleshy drupes of lauraceous species represent an adaptation to aid in the independent dispersal of seed by birds, which in turn may increase the genetic diversity of founders colonizing new habitats.