| Rhodiola fastigiata, one of the important traditional Chinese herbal medicines, is a perennial herbaceous plantof the family Crassulaceae. Salidroside and tyrosol have been shown to possess the medical functions. In the different environment of growth of Rhodiola fastigiata, the level of genetic diversity is high. Intra- and extra-species genetic diversity of Rhodiola fastigiata reflects evolution and development to the special entironment。In this paper, the alpine plant Rhodiola fastigiata of Sejila is used as the material, selecting three populations of Rhodiola fastigiata distributing the elevation of 4300-4700m for research. The technology of AFLP is used to determine and code the bands of Rhodiola fastigiata , which are from the 30 individual functional leaves of three populations of Rhodiola fastigiata. and then according to the results of the genet bands, we do cluster analysis. At the same time, with three genetic indicators of Rhodiola fastigiata correlation analysis is done,Our aim was to provide the theory of the artificial cultivation, the relationship within the active contents, zone and species. It is available for the conservation and sustainable development. The results show:1,The improvement methods of extract high-quality genome DNAThrough the modified CTAB method, the success of the genus Rhodiola fastigiata extract genomic DNA, by agarose gel electrophoresis, DNA gel electrophoresis made clear after the order was a band-like hole near the point of no residual material, and no degradation of tailing phenomenon, and the oth0er by UV detection, OD260/OD280 value of around 1.8. The high-quality genome DNA method of abstraction that is another set up in this research has already been applied to other high and cold plants(Paeonia ludlowii,Arenaria pulvinata Edgew.)。2,Primer pairs and multi-stateMorphology in the 2 different species of Rhodiola (Rhodiola fastigiata, Rhodiola yunnanensis) for the test material, from the 64 pairs of primers, the selected four pairs of high polymorphism AFLP primer combinations (E-ACA: M -CAC; E-ACA: M-CAT; E-ACA: M-CAG; E-ACT: M-CTA;). Four pairs of primers that Tibet Hill Sejila Rhodiola alpine plants of the 30 test samples for analysis of the AFLP, amplified 510 clear bands, of which 345 polymorphic band.3,Simple silver staining methoda,liquid (100ml ethanol +5～10ml glacial acetic acid +900 ml deionized water) (12 minutes);b, silver dye (1.5～2ml formaldehyde +1.5～2g silver nitrate +1000 ml deionized water) (10-14 minutes) deionized water;c,1000ml washed 15 seconds ;d, developer (2～3ml formaldehyde +15～25gNaOH +1000 ml deionized water) (until clear bands);e ,with the above-mentioned "1" of liquid with a fixed 2～3 minute;f,deionized water 1000ml elution 2～3 minutes later, the room temperature plastic.4,AFLP analysis systemGenomic DNA template 300ng, 20ul digestion and connection system, digestion and connected in one step (37℃, 5h; 8℃, 4h; 4℃, overnight); 25ul of pre-amplification system, digestion and connected after the template DNA 2ul, Taq enzyme 0.5ul, Pre-ampmix 1ul, 10×PCR buffer 2.5ul, sterile double-distilled water 18.5ul; 25ul selective amplification system, the sample diluted preamplification 2ul, 10×PCR buffer 2.5ul, dNTPs 0.5ul, EcoRⅠprimer 2ul, MseⅠprimer 2ul, Taq enzyme 0.5ul, sterile double-distilled water 17.5ul;5,Analysis of genetic diversity indicators The genetic diversity of Rhodiola fastigiata. at the species level is higher than that at the population level.As for wild population at species level, the percentage of polymorphic loci(PPL) was 96.61%, the Nei's gene diversity(H) was 0.3329, and the Shannon's information index(I) was 0.4893.At the population level, the estimates for the wild population PPL=64.97%, H=0.2404, I=0.3545,Na=1.4252 .Genetic differentiation was high among populations, Gst was0.2779.This means 27.79% genetic variation that occurred between populations. The gene flow (Nm) was 1.2995.6,Results show that the AFLP silver-staining technique could be used to study the genetic diversity of Rhodiola fastigiata. |
PDF Full Text Request