FISH-Based Karyotype Analyses of Four Dracaena Species.

The genus Dracaena is the main source of dragon's blood, which is a plant resin and has been used as traditional medicine since ancient times in different civilizations. However, the chromosome numbers and karyotypes present in this genus remain poorly understood. In this study, fluorescence in situ hybridization (FISH) using oligonucleotide probes for ribosomal DNAs (5S and 45S rDNA) and telomeric repeats (TTTAGGG)3 was applied to analyze 4 related species: Dracaena terniflora Roxb., Dracaena cambodiana Pierre ex Gagnep., Aizong (Dracaena sp.), and Dracaena cochinchinensis (Lour.) S.C. Chen. In all 4 species, both 5S and 45S rDNA showed hybridization signals in the paracentromeric region of a pair of chromosomes; the sizes of the 45S rDNA signals were larger than those of the 5S rDNA. Importantly, the telomeric repeat signals were located in the telomeric regions of almost all chromosomes. The results indicated that the chromosome number of all 4 Dracaena species is 2n = 40, and the lengths of the mitotic metaphase chromosomes range from 0.99 to 2.98 µm. Our results provide useful cytogenetic information, which will be beneficial to future studies in genome structure of the genus Dracaena.

Tertiary origin and pleistocene diversification of dragon blood tree (Dracaena cambodiana-Asparagaceae) populations in the Asian tropical forests.

BACKGROUND: The origin of extraordinarily rich biodiversity in tropical forests is often attributed to evolution under stable climatic conditions over a long period or to climatic fluctuations during the recent Quaternary period. Here, we test these two hypotheses using Dracaena cambodiana, a plant species distributed in paleotropical forests. METHODS: WE ANALYZED NUCLEOTIDE SEQUENCE DATA OF TWO CHLOROPLAST DNA (CPDNA: atpB-rbcL and trnD-trnT) regions and genotype data of six nuclear microsatellites from 15 populations (140 and 363 individuals, respectively) distributed in Indochina Peninsular and Hainan Island to infer the patterns of genetic diversity and phylogeographic structure. The population bottleneck and genetic drift were estimated based upon nuclear microsatellites data using the software programs BOTTLENECK and 2MOD. The lineage divergence times and past population dynamics based on cpDNA data were estimated using coalescent-based isolation-with-migration (IMa) and BEAST software programs. RESULTS: A significant phylogeographic structure (N ST = 0.876, G ST = 0.796, F ST-SSR = 0.329, R ST = 0.449; N ST>G ST, R ST>F ST-SSR, P<0.05) and genetic differentiation among populations were detected. Bottleneck analyses and Bayesian skyline plot suggested recent population reduction. The cpDNA haplotype network revealed the ancestral populations from the southern Indochina region expanded to northward. The most recent ancestor divergence time of D. cambodiana dated back to the Tertiary era and rapid diversification of terminal lineages corresponded to the Quaternary period. CONCLUSIONS: The results indicated that the present distribution of genetic diversity in D. cambodiana was an outcome of Tertiary dispersal and rapid divergence during the Quaternary period under limited gene flow influenced by the uplift of Himalayan-Tibetan Plateau and Quaternary climatic fluctuations respectively. Evolutionary processes, such as extinction-recolonization during the Pleistocene may have contributed to the fast diversification in D. cambodiana.

Cloning, bioinformatics and the enzyme activity analyses of a phenylalanine ammonia-lyase gene involved in dragon's blood biosynthesis in Dracaena cambodiana.

Phenylalanine ammonia-lyase (PAL) is the key enzyme of the phenylpropanoid pathway, playing an important role in plant development and defence. We cloned a partial cDNA of PAL gene, DcPAL1, from Dracaena cambodiana seedlings using RT-PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. DcPAL1 shows highly homologous to other known PAL genes registered in GenBank, being closest to that of Musa acuminata. DcPAL1 has a relatively high GC content and most of the GC is in the third codon position. It has 768 bp in size with a maximum open reading frame (ORF) of 765 bp, encoding a 255 amino acid-polypeptide. The deduced PAL protein is a stable protein, having classical PAL domains and consisting of three major hydrophobic domains. Analysis of effective number of codons (ENC) shows that DcPAL1 codons are used at equal frequency. Relatively higher usage frequency appears randomly in codons ended with any of the four bases; six codons have no usage bias. There are 45 codons showing distinct usage preference between DcPAL1 and E. coli, 20 between DcPAL1 and yeast. Therefore, the yeast system may be more suitable for the expression of DcPAL1. Upon the elicitation of Fusarium proliferatum, a potent elicitor of dragon's blood, the PAL enzyme activity in the leaves and stems of D. cambodiana and other two Dracaena spp. significantly increased, accompanying with the formation of dragon's blood, indicating the involvement of PAL in the biosynthesis of dragon's blood, a precious traditional medicine.