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.

Cytomolecular analysis of mutants, breeding lines, and varieties of camelina (Camelina sativa L. Crantz).

Camelina sativa L. Crantz (Brassicaceae family), known as camelina, has gained new attention as a re-emerging oil seed crop. With a unique seed oil profile, with the majority of the fatty acids consisting of linolenic (C18:3), oleic (C18:1), linoleic (C18:2), and eicosenoic (C20:1), camelina oil is reported to be useful as a food oil and biofuel. However, there are still many unknown factors about the structure and genetic variability of this crop. Chromosomal localization of ribosomal DNA was performed using fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as molecular probes on mitotic chromosomes of enzymatically digested root-tip meristematic cells. Here, we present for the first time a comparative analysis of selected genotypes (cultivars, breeding lines and mutants) of C. sativa with the use of cytogenetic techniques. The main aim of the study was to determine the intraspecific and interspecific polymorphisms in the structure of chromosomes of selected accessions using conserved 5S and 25S rDNA repetitive sequences as molecular probes. The results were compared with C. microcarpa (closely related to C. sativa) rDNA gene loci distribution. The presence of minor rDNA sites was discussed and compared with other Brassicaceae species. In addition, demonstration karyograms of C. sativa and C. microcarpa mapped with rDNA probes were prepared based on the cv. "Przybrodzka" and GE2011-02 genotype, respectively. The use of 5S and 25S rDNA probes provided an insight on the genome structure of C. sativa at the cytogenetic level and can help to understand the genome organization of this crop. The putative role of cytogenetic markers in phylogenetic analyses of camelina was discussed, as well.

Linkage of 35S and 5S rRNA genes in Artemisia (family Asteraceae): first evidence from angiosperms.

Typically in plants, the 5S and 35S ribosomal DNA (rDNA) encoding two major ribosomal RNA species occur at separate loci. However, in some algae, bryophytes and ferns, they are at the same locus (linked arranged). Southern blot hybridisation, polymerase chain reactions (PCR), fluorescent in situ hybridisation, cloning and sequencing were used to reveal 5S and 35S rDNA genomic organisation in Artemisia. We observed thousands of rDNA units at two-three loci containing 5S rDNA in an inverted orientation within the inter-genic spacer (IGS) of 35S rDNA. The sequenced clones of 26-18S IGS from Artemisia absinthium appeared to contain a conserved 5S gene insertion proximal to the 26S gene terminus (5S rDNA-1) and a second less conserved 5S insertion (5S rDNA-2) further downstream. Whilst the 5S rDNA-1 showed all the structural features of a functional gene, the 5S-rDNA-2 had a deletion in the internal promoter and probably represents a pseudogene. The linked arrangement probably evolved before the divergence of Artemisia from the rest of Asteraceae (>10 Myrs). This arrangement may have involved retrotransposons and once formed spread via mechanisms of concerted evolution. Heterogeneity in unit structure may reflect ongoing homogenisation of variant unit types without fixation for any particular variant.

Identification of Swertia mussotii and its adulterant Swertia species by 5S rRNA gene spacer.

OBJECTIVE: This research focused on analyzing the differences of 5S rRNA gene spacer sequences on Swertia mussotii and its commonly used adulterants, including S. franchetiana, S. wolfangiana and S. chirayita. METHOD: DNA was extracted from the collected Swertia samples. 5S rRNA intergenic spacers were amplified by PCR, sequenced and analyzed. RESULT: 5S rRNA gene spacer sequences were different between S. mussotii and its other three adulterants. Sequence divergence among species ranged from 30.6% to 65.0%. CONCLUSION: 5S rRNA spacers may be used as molecular authentication markers to differentiate S. mussotii and other commonly used Swertia adulterants. This result provides reliable and simple reference for the authentication of Swertia genus species.

Authentication of Saussurea lappa, an endangered medicinal material, by ITS DNA and 5S rRNA sequencing.

Wild SAUSSUREA LAPPA in the family Asteraceae is a highly endangered plant. On the other hand, the dried root of cultivated S. LAPPA (Radix Aucklandia, Muxiang) is a popular medicinal material for treating various gastrointestinal diseases. In the market, several medicinal plants including VLADIMIRIA BERARDIOIDEA, V. SOULIEI, V. SOULIEI var. MIRABILIS, INULA HELENIUM and I. RACEMOSA in the family Asteraceae and ARISTOLOCHIA DEBILIS in the family Aristolochiaceae have the trade name of Muxiang. To manage the concerned medicinal material, we investigated if the ITS and 5S rRNA intergenic spacers are effective for discriminating S. LAPPA from its substitutes and adulterants. Sequencing results showed that the similarities of ITS-1, ITS-2 and 5S rRNA intergenic spacers among S. LAPPA and related species were 56.3 - 97.8 %, 58.5 - 97.0 %, and 26.4 - 77.9 %, respectively. The intraspecific variation was much lower. There are also several unique changes in the S. LAPPA sequences that may be used as differentiation markers.

Molecular authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii by ITS and 5S rRNA spacer sequencing.

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (Willd). Ohwi, P. thomsonii Benth., and P. montana (Lour.) Merr. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.

5S rDNA gene diversity in tea (Camellia sinensis (L.) O. Kuntze) and its use for variety identification.

Variability in the organization of repeats of 5S rDNA is useful for phylogenetic studies in various crops. We found variable repeats of 5S rDNA gene in the genome of tea (Camellia sinensis (L.) O. Kuntze) during Southern hybridization. Variability in the repeats of 5S rDNA with specific restriction endonucleases (Sau3AI, BamHI, and ApoI) was analyzed in 28 different tea clones representing 3 types of tea. Our results clearly show that the 5S rDNA gene in tea could be used as a molecular marker to distinguish C. sinensis Chinary tea from the other important types of tea, namely Assamica and Cambod. Upon analysis with restriction endonucleases, the 5S rDNA gene in the tea genome was found to be heavily methylated.

Induced fit and "lock and key" recognition of 5S RNA by zinc fingers of transcription factor IIIA.

Transcription factor IIIA (TFIIIA) is a Cys2His2 zinc finger protein that regulates expression of the 5 S ribosomal RNA gene by binding specifically to the internal control element. TFIIIA also functions in transport and storage of 5 S RNA by binding directly to the RNA transcript. To obtain insights into the mechanism by which TFIIIA recognizes 5 S RNA, we determined the solution structure of the middle three zinc fingers bound to the central core of 5 S RNA. Finger 4 utilizes "lock and key" recognition to bind in the widened major groove of the pre-structured RNA loop E motif. This interaction is mediated by direct hydrogen bonding interactions with bases. In contrast, recognition of loop A, a flexible junction of three helices, occurs by an induced fit mechanism that involves reorganization of the conserved CAUA motif and structuring of the finger 5-finger 6 interface to form a complementary RNA binding surface.

Molecular genetic and chemical assessment of Rhizoma Curcumae in China.

Rhizoma Curcumae (Ezhu) is a traditional Chinese medicine that has been used in removing blood stasis and alleviating pain for over a thousand years. Three species of Curcuma rhizomes are being used, which include Curcuma wenyujin, Curcuma phaeocaulis, and Curcuma kwangsiensis. In China, the production of Rhizoma Curcumae largely depends on agricultural farming. The essential oils are considered as active constituents in Rhizoma Curcumae, which include curdione, curcumol, and germacrone. On the basis of the yield of curdione, curcumol, and germacrone in an orthogonal array design, the optimized extraction condition was developed. The amounts of these compounds within essential oils in Rhizoma Curcumae varied according to different species and their regions of cultivation. Chemical fingerprints were generated from different species of Curcuma, which therefore could serve as identification markers. In molecular genetic identification of Rhizoma Curcumae, the 5S-rRNA spacer domains of 5 Curcuma species, including the common adulterants of this herb, were amplified, and their nucleotide sequences were determined. Diversity in DNA sequences among various species was found in their 5S-rRNA spacer domains. Thus, the chemical fingerprint together with the genetic distinction could serve as markers for quality control of Curcuma species.

Abridged 5S rDNA units in sea beet (Beta vulgaris subsp. maritima).

Amplification by polymerase chain reaction of the 5S rDNA repeat units of Beta vulgaris subsp. maritima resulted in a 350-bp product corresponding to the full-length 5S unit, but also revealed 4 abridged unit classes, each with a deletion that removed most of the spacer and 12-76 bp of the coding sequence. Each abridged type lacks at least 1 of the conserved elements involved in transcription of the 5S gene, and so appear to be nonfunctional. Network analysis revealed that the abridged units are evolving in the same manner as the full-length versions.

Characterization of medicinal Epimedium species by 5S rRNA gene spacer sequencing.

Sequences of 5S rRNA gene spacer were used to identify Epimedium brevicornu Maxim., E. sagittatum (Sieb. et Zucc.) Maxim., E. wushanense T. S. Ying, E. pubescens Maxim., and E. koreanum Nakai. These species are listed as source plants of Chinese medicine 'Ying Yang Huo' in the Chinese Pharmacopoeia. The neighbor-joining method was used in a sequence analysis of Epimedium species. A position-specific nucleotide was found in the 5S rRNA gene spacer for E. pubescens, E. wushanense, and E. brevicornu. A 19-bp deletion was found for E. koreanum in the 5S rRNA gene spacer. E. koreanum was most divergent from the other four endemic Chinese species of Epimedium.

Molecular detection of cashew husk (Anacardium occidentale) adulteration in market samples of dry tea (Camellia sinensis).

Species-specific PCR primers were developed from intergenic spacer regions of 5S ribosomal RNA genes and used successfully in the detection of adulteration of cashew husk (Anacardium occidentale L.) in tea [Camellia sinensis (L.) O. Kuntze] samples. This is the first report of detecting adulteration in tea using molecular tools. Application of this approach in detecting adulteration of other biological materials in tea, medicinal herbs and the composition of admixtures of ayurvedic herbs has been discussed.

Phylogeny of Astragalus in China: molecular evidence from the DNA sequences of 5S rRNA spacer, ITS, and 18S rRNA.

Radix Astragali (root of Astragalus; Huangqi) is a traditional Chinese medicine commonly used as an immunostimulant, hepatoprotective, diuretic, antidiabetic, analgesic, expectorant, and sedative drug. Although the species of Radix Astragali have been defined as Astragalus membranaceus and A. membranaceus var. mongholicus in Pharmacopoeia of China, their taxonomy remains controversial. The phylogenetic relationships among 10 Astragalus taxa, which are commonly found in China including A. membranaceus, A. membranaceus var. mongholicus, Astragalus propinquus, Astragalus lepsensis, Astragalus aksuensis, Astragalus hoantchy, Astragalus hoantchy subsp. dshimensis,Astragalus lehmannianus, Astragalus sieversianus, and Astragalus austrosibiricus, were determined using the DNA sequences of the 5S ribosomal RNA (5S rRNA) spacer, internal transcribed spacer region (ITS), and 18S rRNA coding region. The 5S rRNA spacer, ITS, and 18S rRNA, amplified by polymerase chain reaction from the isolated genomic DNAs, were sequenced. By using neighbor-joining and maximum parsimony analyses, phylogenetic trees were mapped by their sequence diversity. A. membranaceus and A. membranaceus var. mongholicus shared the greatest sequence homology. In addition, A. propinquus shared a closer relationship with A. membranaceus and A. membranaceus var. mongholicus, while other Astragalus species were less closely related. This is the first paper to show the phylogenetic relationship of Astragalus species related to Radix Astragali in China by the molecular genetic approach.

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis.

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.

Molecular genetic and chemical assessment of radix Angelica (Danggui) in China.

The roots of Angelica sinensis (Danggui), a traditional Chinese medicine, have been used for invigorating blood circulation for over 2000 years in China. Three common species of Angelica roots are found in Asia: A. sinensis from China, A. acutiloba from Japan, and A. gigas from Korea. By using a molecular genetic approach, the 5S-rRNA spacer domains of the three species of Angelica were amplified, and their nucleotide sequences were determined. Diversity in DNA sequences among various species was found in their 5S-rRNA spacer domains, which could serve as markers for authentic identification of Angelica roots. In chemical analyses, the main constituents of Angelica roots including ferulic acid and Z-ligustilide were determined by HPLC; roots of A. sinensis were clearly distinct in that they contained approximately 10-fold higher levels of ferulic acid and Z-ligustilide as compared to roots of A. acutiloba and A. gigas. In addition, the amounts of main constituents in roots of A. sinensis varied according to different regions of cultivation and different methods of preservation. The chemical profile determined by HPLC therefore could serve as a fingerprint for quality control of Angelica roots.

Identification of bulb from Fritillaria cirrhosa by PCR with specific primers.

Bulb of Fritillaria cirrhosa is an important traditional Chinese herbal medicine. According to the Chinese Pharmacopoeia (1995), it is commonly used as an antitussive and expectorant. Many young bulbs from species of Fritillaria are similar to those of F. cirrhosa, but they are different in price and quality. Therefore, there are many young bulbs from species of Fritillaria that could fake those of F. cirrhosa on the commercial market. The coding region of 5S-rRNA is highly conserved in higher eukaryotes. The 5S-rRNA spacer region sequences of F. thunbergii, F. pallidiflora, F. ussuriensi, F. delavayi, F.cirrhosa, F. anhuiensis, F. puqiensis were cloned by PCR with a pair of primers located within the conserved coding region. Based on sequences analyses of the 5S-rRNA spacer region from the 7 species, a specific sequence was found in F. cirrhosa. A pair of specific primers was designed for differentiating the bulbs of F. cirrhosa from each other by PCR. This result indicated that the method is rapid, more accurate and applicable in identification of the bulbs of F. cirrhosa at the DNA level.

Genetic distinction of radix adenophorae from its adulterants by the DNA sequence of 5S-rRNA spacer domains.

Radix Adenophorae (Shashen), a traditional Chinese medicine commonly used as an antitussive and expectorant, is derived from roots of Adenophora stricta Miq. and Adenophora tetraphylla (Thunb.) Fisch. Twelve species and varieties of Adenophora and Glehnia, however, could act as substitutes or adulterants of Radix Adenophorae on the commercial markets in South East Asia, and roots of Adenophora hunanensis Nannf. and Glihnia littoralis F. Schmidt ex Miq. are the most common examples. The authentic identification of dried roots of A. stricta and A. tetraphylla, however, is difficult on the basis of appearance and morphology. A molecular genetic approach was developed here to identify the species of Radix Adenophorae. The 5S-rRNA spacer domains (approximately 250 bp) were amplified by the polymerase chain reaction (PCR) from genomic DNAs isolated from A. stricta, A. tetraphylla, A. hunanensis and G. littoralis, and subsequently, the nucleotide sequences were determined. Diversity in DNA sequence and restriction enzyme mapping among various species were found in their 5S-rRNA spacer domains, which could serve as markers for authentic identification of Radix Adenophorae.