Quality evaluation based on color grading: quality discrimination of the Chinese medicine Corni Fructus by an E-eye.

'Quality evaluation based on color grading' is one of the features used in Chinese medicine discrimination. In order to assess the feasibility of electronic eye (E-eye) in implementing 'quality evaluation based on color grading', the present study applied an IRIS VA400 E-eye to test 58 batches of Corni Fructus samples. Their optical data were acquired and combined with their corresponding classes. A total of four quality discrimination models were produced according to discrimination analysis (DA), least squares support vector machine (LS-SVM), partial least squares-discrimination analysis (PLS-DA), and principal component analysis-discrimination analysis (PCA-DA). The accuracy rate of the aforementioned 4 cross evaluation models were 86.21%, 89.66%, 81.03% and 91.38%, respectively. Therefore, the PCA-DA method was used to build the final discrimination model for classifying Corni Fructus or discriminating its quality.

Confirmation of independent introductions of an exotic plant pathogen of Cornus species, Discula destructiva, on the east and west coasts of North America.

Cornus florida (flowering dogwood) and C. nuttallii (Pacific dogwood) are North American native tree species that belong to the big-bracted group of dogwoods. Cornus species are highly valued for their ornamental characteristics, and have fruits that contain high fat content for animals. Also, they are an important understory tree in natural forests. Dogwood anthracnose, caused by Discula destructiva, was observed in the late 1970s on the east and west coasts of the United States and by 1991 had quickly spread throughout most of the native ranges of C. florida and C. nuttalli. We investigated the genetic diversity and population structure of 93 D. destructiva isolates using 47 microsatellite loci developed from the sequenced genome of the type strain of D. destructiva. Clone-corrected data indicated low genetic diversity and the presence of four genetic clusters that corresponded to two major geographic areas, the eastern United States and the Pacific Northwest, and to the two collection time periods when the isolates were collected (pre- and post-1993). Linkage disequilibrium was present in five out of six subpopulations, suggesting that the fungus only reproduced asexually. Evidence of population bottlenecks was indicated across four identified genetic clusters, and was probably the result of the limited number of founding individuals on both coasts. These results support the hypothesis that D. destructiva is an exotic pathogen with independent introductions on the east and west coasts of North America. We also tested the cross-amplification of these microsatellite primers to other Discula species. Genomic DNA from 17 isolates of four other Discula species and two isolates of Juglanconis species (formerly Melanconis species) were amplified by 17 of 47 primer pairs. These primers may be useful for investigating the genetic diversity and population structure of these Discula species.

A SCAR marker for the analysis of chloroplast DNA from different cultivars of Cornus officinalis.

The aims of this study were to establish a random amplified polymorphic DNA (RAPD) fingerprint database of chloroplast DNA (cpDNA) from different cultivars of Cornus officinalis and to convert RAPD markers to sequence characterized amplified regions (SCAR) markers. A method of extraction was established that was suitable for obtaining cpDNA from samples rapidly dried in silicone; an RAPD fingerprint database was built; and the genetic distance between samples was used as statistical clustering variables for calculating DICE genetic similarity coefficients and for building a kinship tree chart. RAPD markers were converted to SCAR markers to design specific primers, and samples from C. officinalis cultivars, plants of the same family, and its adulterants, were used for amplification and identification. Fifteen amplified primers with stable polymorphisms were screened for amplification of 130 copies of materials. In total, 57 sites were achieved, 40 of which were polymorphic, and the polymorphic rate was up to 70.18%. A genetic tree was built based on seven cultivars. SCAR markers of C. officinalis cpDNA were successfully converted into RAPD markers. cpDNA samples from hawthorn, C. officinalis, Cornus wood, and grape were used for SCAR amplification, and their bands were distinctly different. In conclusion, SCAR markers and cpDNA may be used for research on C. officinalis and its adulterants, and the results may provide a basis for identifying germplasm and screening fine varieties at a molecular level.

Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences.

UNLABELLED: The DNA barcoding method was used to accurately and rapidly identify Corni Fructus and its adulterants. METHODS: Genomic DNA extracted from Corni Fructus and its adulterants were used as templates. The ITS (internal trascribed spacer) regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining (NJ) trees. RESULTS: The ITS sequence length of Corni Fructus was 659 bp. The average intra-specific genetic distance of Corni Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants (0.357). The ITS2 sequence length of Corni Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. CONCLUSION: ITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Corni Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding.

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.

[Study on sequence characterized amplified region (SCAR) markers of Cornus officinalis].

OBJECTIVE: To establish sequence characterized amplified region markers of Cornus officinalis and provide a scientific basis for molecular identification of C. officinalis. METHOD: The random primer was screened through RAPD to obtain specific RAPD marker bands. The RAPD marker bands were separated, extracted, cloned and sequenced. Both ends of the sequence of RAPD marker bands were determined. A pair of specific primers was designed for conventional PCR reaction, and SCAR marker was acquired. RESULT: Four pairs of primers were designed based on the sequence of RAPD marker bands. The DNA of the seven varieties of C. officinalis was amplified by using YST38 and YST43 primer. The results showed that seven varieties of C. officinalis were able to produce a single PCR product. It was an effective way to identify C. officinalis. The varieties with cylindrical and long-pear shape fruits amplified by YST38 showed a specific band, which could be used as the evidence of variety identification. Seven varieties of C. oficinalis were amplified by using primer YST39. But the size of band of the variety with spindly shape fruit (35,0400 bp) was about 300 bp, which was shorter than those of the variety with the other shape fruits of C. officinalis (650-700 bp). The variety with the spindly shape fruit could be identified through this difference. The primer YST92 could produce a fragment from 600-700 bp in the varieties with cylindrical and long-pear shape fruits, a fragment from 200-300 bp in the varieties with oval and short-cylindrical shape fruits and had no fragment in the varieties with long cylindrical, elliptic and short-pear shape fruits, which could be used to select the different shapes of C. officinalis. CONCLUSION: SCAR mark is established and can be used as the basis for breeding and distinguishing the verieties of C. officinalis.

Rates of nucleotide substitution in Cornaceae (Cornales)-Pattern of variation and underlying causal factors.

Identifying causes of genetic divergence is a central goal in evolutionary biology. Although rates of nucleotide substitution vary among taxa and among genes, the causes of this variation tend to be poorly understood. In the present study, we examined the rate and pattern of molecular evolution for five DNA regions over a phylogeny of Cornus, the single genus of Cornaceae. To identify evolutionary mechanisms underlying the molecular variation, we employed Bayesian methods to estimate divergence times and to infer how absolute rates of synonymous and nonsynonymous substitutions and their ratios change over time. We found that the rates vary among genes, lineages, and through time, and differences in mutation rates, selection type and intensity, and possibly genetic drift all contributed to the variation of substitution rates observed among the major lineages of Cornus. We applied independent contrast analysis to explore whether speciation rates are linked to rates of molecular evolution. The results showed no relationships for individual genes, but suggested a possible localized link between species richness and rate of nonsynonymous nucleotide substitution for the combined cpDNA regions. Furthermore, we detected a positive correlation between rates of molecular evolution and morphological change in Cornus. This was particularly pronounced in the dwarf dogwood lineage, in which genome-wide acceleration in both molecular and morphological evolution has likely occurred.

Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins.

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.