Analysis And Identification Of AFLP Genetic Diversity Of White And Genus Plants

Bletilla H.G.Reichenbach,a kind of precious medicinal plants,belongs to Orchidaceae.Its plants are widely distributed and there are four species of in China,which are Bletilla Striata,Bletilla Ochracea,Bletilla Formosana,and Bletilla sinensis.Four species of Bletilla plant are high ornamental value and medicinal value.Due to excessive excavation of human and the destruction of habitat,wild resources of Bletilla are in danger of extinction,needed to take effective protection immediately.Therefore,this research intended to provide theoretical basis of plant resources protection and mixed falsify for the appraisal from the following three aspects.In order to research the genetic diversity of Bletilla accurately using AFLP molecular marker technology,this thesis built the best reaction system of Bletilla from the following gradient analysis,such as the template DNA dosage,enzyme digestion and connection time,the results of DNA amplification products diluted multiples and amplification electrophoresis factors.The results showed the best reaction system of 500ng DNA of enzyme-connection reaction in 4 hours,pre-amplification product after diluted 10 times to the selective amplification.Finally,the selective amplification products were electrophored and detected using the 3500 x1 based Analyzer,then recorded and analyzed the strips Using GeneMapper v.4.1.this thesis research genetic diversity and genetic structure analysis of 10 populations of Bletilla using AFLP molecular markers,which lay the foundation of protection and usage of the genetic resources.This study used 4 most suitable primers pairs from 64 pairs to maker AFLP analysis from 78 samples of Bletilla.The results showed that the 4 primer combinations amplified out of a total of 640 rules,the polymorphism proportion was as high as 72.5%.The genetic diversity index H was 0.1974.The variation among populations of Bletilla Striate was 9.88%,and within populations was 90.12%.Therefore,the with-in population variation is greater than the among-population variation.To identify adulterants from medicinal plants of Bletilla H.G.Reichenbach,the suitable candidate DNA barcoding of Bletilla were evaluated In this study,the internal transcribed spacer(ITS)of nuclear ribosomal DNA,the LFY homologous gene intron 2 and chloroplasty cffl gene were amplified and sequenced from forty-one samples.The result showed the intra-specific divergence of nrDNA ITS and ycf1(0.022-0.106 and 0.017-0.106)were obviously higher than the inter-specific divergence(0-0.012 and 0-0.015),and four species of Bletilla were also accurately distinguished in NJ trees.Whereas,there was no Barcoding Gap on LFY homologous gene intron 2,thus it cann't effectively identify species of Bletilla.Using NJ tree of nrDNA ITS and ycfl gene were also successfully unidentified powdery medicine and the adulterants of Bletilla.In conclusion,nrDNA ITS and ycfl can be used as a potential DNA barcoding to identify the medicinal plants in Bletilla and its adulterants.There were only three base differences on nrDNA ITS between "Jujing baiji" and Bletilla striata of Lu'an in Anhui province,and two base differences in ycfl.Based on morphological and molecular data,"Jujing baiji" could be recognized as the species of Bletilla striata.