Investigation of the hydrolysis of ginsenosides by high performance liquid chromatography-electrospray ionization mass spectrometry

The hydrolysis of ginsenoside standards and the crude extracts of ginseng has been investigated at different pH values (2.4 - 11.2) using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). The experimental results indicated that the pH value of aqueous solutions is an important factor in changing the composition of ginsenosides. For (20S)-protopanaxadiol ginsenosides, ginsenosides with a large mass hydrolyzed to form hydrolysates (20S)-Rg(3) and (20R)-Rg(3) at pH 4.3. There were more hydrolyzed products observed at pH 3.3: (20S)-F-2, C-25,26 hydrated ginsenoside "C-Y-1" and "C-Y-2" (MW = 802 Da) accompanied with (20S)-Rg(3), (20R)-Rg(3). At pH 2.4, only (20R)-Rg(3), (20S)-F-2, a small quantity of (20S)-Rg(3) and three C-25,26 hydrated ginsenosides were obtained. For (20S)protopanaxatriol Re, no hydrolysates were observed at pH 4.3; it was hydrolyzed at pH 3.3 to form hydrolysates (20S)-Rg, (20R)Rg(2) and hydrated C-25,26 (MW = 802 Da) and at pH 2.4 only C-25,26 hydrated ginsenosides "C-Y-1" and "C-Y-2" (MW = 802 Da) were left in the solution. Similar hydrolysis reactions could be also observed for the crude extracts of ginseng. It showed that HPLC/ESI-MS is a fast and convenient method to study the hydrolysis of ginseng.

Genetic characterization of asymmetric reciprocal hybridization between the flatfishes Paralichthys olivaceus and Paralichthys dentatus

Interspecific reciprocal crosses between the two flatfishes Paralichthys olivaceus and P. dentatus yielded hybrids with different viabilities. Specifically, the hybrids of P. olivaceus female and P. dentatus male (HI) were found to be viable, while the reciprocal hybrids from P. dentatus female and P. olivaceus male (HII) were completely inviable. All the HII individuals showed morphological deformities and died before first feeding. The chromosome analysis showed that HI individuals had the same chromosome number as parents. However, two chromosomes were missing in HII offspring indicating that the latter were aneuploids. Genomic inheritance from the parents to F-1 progeny was also examined by amplified fragment length polymorphism (AFLP) analyses, and the results showed differences between reciprocal hybrids. Almost all AFLP bands (97.71%) observed in parents were passed on to HI individuals. In contrast, only 86.64% of the AFLP bands from parents were scored in HII individuals. Frequency of lost parental bands was thus significantly higher in HII than that in HI and intraspecific crosses, which was probably associated with chromosomal elimination. In addition, higher segregation distortions were found in hybrids than in controls, although these differences were not significant. The present study indicates that chromosomal elimination and loss of AFLP loci occurred in inviable HII individuals, while such genomic changes were not found in viable HI individuals. Possible implications of such difference on genomic changes for asymmetric viability in reciprocal hybrids are discussed.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

Comparative molecular population genetics of phycoerythrin locus in Prochlorococcus

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.

Genetic variation in the endangered Anisodus tanguticus (Solanaceae), an alpine perennial endemic to the Qinghai-Tibetan Plateau

We used random amplified polymorphic DNA markers (RAPDs) to assess genetic variation between- and within-populations of Anisodus tanguticus (Solanaceae), an endangered perennial endemic to the Qinghai-Tibetan Plateau with important medicinal value. We recorded a total of 92 amplified bands, using 12 RAPD primers, 76 of which (P = 82.61%) were polymorphic, and calculated values of H-t and H-sp of 0.3015 and 0.4459, respectively, suggesting a remarkably high rate of genetic variation at the species level. The average within-population diversity also appeared to be high, with P, H-e and H-pop values of 55.11%, 0.1948 and 0.2918, respectively. Analyses of molecular variance (AMOVA) showed that among- and between-population genetic variation accounted for 67.02% and 32.98% of the total genetic variation, respectively. In addition, Nei's coefficient of differentiation (G(ST)) was found to be high (0.35), confirming the relatively high level of genetic differentiation among the populations. These differentiation coefficients are higher than mean corresponding coefficients for outbreeding species, but lower than reported coefficients for some rare species from this region. The genetic structure of A. tanguticus has probably been shaped by its breeding attributes, biogeographic history and human impact due to collection for medicinal purposes. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this species.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA rpl16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA rpl16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.