[Herbalogical study of Colla Corii Asini(Ejiao)].

Colla Corii Asini(Ejiao)is an important Chinese medicine used in China for thousands of years, and is well known for its famous tonic properties. The herbalogical study was detailed carried out based on the naming, habitat, harvesting, processing, medicinal properties and clinical efficacy. The results showed that the name of Ejiao could be traced back to Shennong's Materia Medica, and various names of Lvpi Jiao, Penfu Jiao and Fuzhi Jiao were recorded in other ancient books. In the many intervening centuries, the main materials of Ejiao had been replaced from cow leather before Tang Dynasty to donkey skin in the middle to late Tang Dynasty. This phenomenon could be probably caused by complicated social factors of various periods and different efficacy of Ejiao made by all kinds of raw materials. Ejiao was merely processed with the simple methods before Tang Dynasty, which subsequently improved avariety of methods to enhance the supplementation action. Most importantly, Ejiao has a wide clinic application along with the development of processing theories and methods, which can be found in various Classics, especially in imperial medical case record in Qing Dynasty.

[Recent advances on genuineness evaluation of Lingnan herbal drugs].

Chinese medicine is the traditional treasure of China nation. As the basis of Chinese medicine, traditional Chinese medicine(TCM) plays an important part for the development of Chinese medicine. Genuine medicinal materials with special characteristics of TCM growing in special ecological environment, is recognized as the high quality medicine. Research on genuineness evaluation of TCM is the key to ensure its clinical applications, efficacy and the process of modernization and internationalization for Chinese medicine. Lingnan region of China is situated in the tropical and subtropical zones, where there are rich geothermal and hydrothermal resources. The superior natural and geographic environment of Lingnan has given birth to a variety of native herbal drugs. And treating and preventing diseases with Lingnan herbal drugs has a long story. This study mainly evaluated the genuineness of Lingnan herbal drugs from the aspects of ecological factor, thegenetic information, the history, the culture, the clinical efficacy and the processing, and proposed a new idea to investigate the genuineness of TCM, aiming to provide a scientific basis for genuineness evaluation.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.

Characterization of 39 novel EST-SSR markers for Liriodendron tulipifera and cross-species amplification in L. chinense (Magnoliaceae).

PREMISE OF THE STUDY: A novel set of simple sequence repeat (SSR) markers were developed and characterized from the expressed sequence tag (EST) database of Liriodendron tulipifera for application in population genetic studies of Liriodendron. METHODS AND RESULTS: Thirty-nine polymorphic EST-SSR loci were identified among 27 individuals sampled from a cultivated population of L. tulipifera. The number of alleles per locus ranged from three to 18. The average observed heterozygosity and expected heterozygosity were 0.684 and 0.778, respectively. Of the 39 loci, 32 showed interspecific transferability and polymorphism in a related species, L. chinense. The number of alleles per locus ranged from two to 11, and the average observed heterozygosity and expected heterozygosity were 0.475 and 0.736, respectively. CONCLUSIONS: The developed EST-SSR markers will be useful for investigating adaptive genetic differentiation in Liriodendron.

Chloroplast microsatellite markers in Liriodendron tulipifera (Magnoliaceae) and cross-species amplification in L. chinense.

PREMISE OF THE STUDY: A set of cpSSR markers were developed for the tree genus Liriodendron L. to investigate population genetic structure and phylogeographic history. METHODS AND RESULTS: Primers were designed directly from the chloroplast genome sequences of Liriodendron tulipifera. Among the 55 cpSSR markers tested, 11 polymorphic markers were identified in L. tulipifera. The number of alleles in the population tested ranged from two to five, and the unbiased haploid diversity per locus ranged from 0.074 to 0.644. Eighteen primer pairs generated polymorphic amplification in L. chinense. The number of alleles per locus ranged from two to seven, and the unbiased haploid diversity per locus was from 0.250 to 0.964. CONCLUSIONS: cpSSR markers developed here will be useful for phylogeography and population genetics studies of Liriodendron.

Spontaneous interspecific hybridization and patterns of pollen dispersal in ex situ populations of a tree species (Sinojackia xylocarpa) that is extinct in the wild.

For endangered plants interspecific hybridization occurring in ex situ collections may lead to failure of reintroduction actions. We used Sinojackia xylocarpa, a well documented Chinese endemic species that is extinct in the wild, as a model case to address this concern. We used paternity analyses to assess the spontaneous hybridization and patterns of pollen flow between S. xylocarpa and its congener species, S. rehderiana, in conserved populations in Wuhan Botanic Garden. Interspecific hybridization events were detected in seven out of eight maternal trees of S. xylocarpa, and an average of 32.7% seeds collected from maternal trees of S. xylocarpa were hybrids. The paternity of 93 out of 249 seedlings from S. xylocarpa assigned to S. rehderiana provided convincing evidence that spontaneous interspecific hybridization occurred extensively in the living garden collection we studied. Different patterns of pollen dispersal (predominantly short-distance vs. long-distance pollination) were observed between intra- and interspecific hybridization events in the garden. Pollen dispersal within the ex situ populations was not restricted by distance, as evidenced by a lack of significant correlations between the average effective pollen dispersal distance (delta) and the geographic distances (d1 and d2) between maternal and paternal trees. The interspecific pollen-dispersal distance ranged from 10 to 620.1 m (mean 294.4 m). Such extensive hybridization in ex situ collections could jeopardize the genetic integrity of endangered species and irrevocably contaminate the gene pool if such hybrids are used for reintroduction and restoration. We recommend strongly that measures be taken to minimize the genetic risks of this kind of hybridization, including establishing buffer zones in ex situ collections, manipulating flowering phenology, testing seed lots before use in reintroduction programs, and controlling pollination for seed purity.

Characterization and development of EST-derived SSR markers in Sinowilsonia henryi (Hamamelidaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to reveal the genetic diversity of extant populations and the mating system of Sinowilsonia henryi (Hamamelidaceae). METHODS AND RESULTS: In this study, nuclear simple sequence repeat (SSR) markers were developed using the Illumina high-throughput sequencing technique (RNA-Seq). The de novo-assembled transcriptome generated a total of 64,694 unique sequences with an average length of 601 bp. A total of 2941 microsatellite loci were detected. Of the 121 tested loci, 13 loci were polymorphic and eight were monomorphic among 72 individuals representing three natural populations of the species. The number of alleles per locus ranged from one to four, and the observed and expected heterozygosity at population level were 0.00-1.00 and 0.10-0.66, respectively. CONCLUSIONS: The developed expressed sequence tag (EST)-SSRs will be useful for studying genetic diversity of S. henryi as well as assessing the mating system among Sinowilsonia species.

Evidence for asymmetrical hybridization despite pre- and post-pollination reproductive barriers between two Silene species.

Interspecific hybridization is widespread among plants; nevertheless, pre- and post-zygotic isolating mechanisms may maintain species integrity for interfertile species in sympatry despite some gene flow. Interspecific hybridization and potential isolating barriers were evaluated between co-flowering Silene asclepiadea and Silene yunnanensis in an alpine community in southwest China. We investigated morphological and molecular (nuclear microsatellites and chloroplast gene sequence) variation in sympatric populations of S. asclepiadea and S. yunnanensis. Additionally, we analyzed pollinator behaviour and compared reproductive success between the putative hybrids and their parental species. Both the molecular and morphological data indicate that there were putative natural hybrids in the field, with S. asclepiadae the ovule parent and S. yunnanensis the pollen parent. Bumblebees were the primary visitors to S. asclepiadae and putative hybrids, while butterflies were the primary visitors to S. yunnanensis Pollen production and viability were significantly lower in putative hybrids than the parental species. The direction of hybridization is quite asymmetric from S. yunnanensis to S. asclepiadea Protandry combined with later peak flowering of S. yunnanensis, and pollinator preference may have contributed to the asymmetric pattern of hybridization, but putative hybrids were rare. Our results thus suggest that despite gene flow, S. asclepiadea and S. yunnanensis can maintain species boundaries, perhaps as a result of floral isolation and low fecundity of the hybrids.

An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.

Real-time polymerase chain reaction (RT-PCR) enables effective and sensitive screening for infectious risk in the field of blood safety. However, when using RT-PCR to detect bacterial contamination, several intractable points must be considered, one of which is the lack of appropriate quality control. In this study, we developed a simplified RT-PCR assay in which the same primer set and two distinct probes were used to detect both, an internal reference control and the target in a reaction. The copy number of the internal reference control represents the positive detection limit of the assay; therefore, when the threshold-cycle value of the target is less than or equal to that of the internal reference control, the result obtained for the target can be considered to be a true positive. When human gDNA was spiked with Escherichia coli gDNA and the detection limit for the internal reference control was set to five copies, the measured detection limit for E. coli gDNA was two copies. The internal reference control duplex RT-PCR assay showed high efficiency (0.91-1.02), high linearity (R2 > 0.99), and good reproducibility in intra- and inter-assay comparisons. Lastly, when human platelet-rich plasma samples were spiked with E. coli or other bacterial species, all species were detected efficiently, and the results of a two-sample pooled t test showed that the limit of detection for E. coli was 1 cfu/mL. Here, we present a synthetic internal reference control molecule and a new statistical method for improving the reliability of RT-PCR assays when screening for bacterial contamination in blood products.