DNA Barcoding Research And Medicinal Compositions Analysis Of Gastrodia Elata (Orchidaceae)

Gastrodia elata Bl. is a rootless and leafless saprophytic perennial herb of theOrchidaceae, Subfam. Orchidoideae, Trib.Epidendreae, Subtrib. Gastrodiinae,Gastrodia, symbiosed with Armillaria. It is a kind of precious traditional Chinesemedicine with rhizome as its medicinal part, which has a history of over two thousandyears. There are thirteen species of Gastrodia distributed in China as recorded in Floraof China. Zhaotong, Yunnan province mainly produces four forms of G. elata. Theyare G. elata Bl. f. elata, G. elata Bl. f. viridis Makino, G. elata Bl. f. glauca S.Chowand G. elata Bl. f. flavida S. Chow. In this thesis, morphology, DNA barcoding andchemical compositions analysis were exploited to study on the classification of threeforms of G. elata grown in Zhaotong. In addition, we also discuss on varioussymbiotic fungi that symbiosed with different forms of G. elata produced in Zhaotong.The results are as below.1．Study on morphology classification of Gastrodia elataThere are significant differences in morphological characteristics among differentforms of G. elata produced in Zhaotong. G. elata Bl. f. glauca, G. elata Bl. f. flavida,and G. elata Bl. f. viridis distributed in Zhaotong can be easily classified according totheir differences in height, color of flowers and stems as well as the phenotypiccharacteristics. In comparison with G. elata Bl. f. flavida and G. elata Bl. f. viridis, G.elata Bl. f. glauca possesses larger rhizome and more stable genetic variation. So itcan stand as qualified seed source for cutivation.The morphological variations amongrhizome of each form of G. elata are greater than that within each form. In this sense,variation within each form is the main source of variations. To distinguish processeddrugs derived from three forms of G. elata by morphological characteristics isdifficult for the morphological differences among them are extremely minor. 2．DNA barcoding research on Gastrodia elata of ZhaotongIdentification efficacy of five candidate DNA barcode sequences (ITS, matK,rbcL, trnH-psbA, trnL-F) within forms and among forms of G. elata were analyzedand compared. The identification efficacy of combined fragment ITS+rbcL was alsoevaluated. Only ITS and rbcL fragments were amplified preferably but no obviousbarcoding gap existed within and among each form of G. elata. Consequently, none ofthe above universal barcode sequence (ITS, matK, rbcL, trnH-psbA, trnL-F) weresuitable for the identification of different forms of G. elata. But ITS sequence can beused for the identification of different populations G. elata. In addition, successfulamplification of rbcL and trnH-psbA fragments of chloroplast of G. elata，So weconjectured that chloroplast of G. elata didn’t degenerate completely.According to ITS sequence analysis, the genetic relationship between G. elata Bl.f. flavida and G. elata Bl. f. glauca were proved to be the closest among three forms,followed by relationship between G. elata Bl. f. viridis and G. elata Bl. f. flavida.Relationship between G. elata Bl. f. glauca and G. elata Bl. f. viridis were proved tobe the farthest. The variation rate of ITS sequence in most individuals within oramong each form is low and only that of few individuals is higher. The variationsmainly occur in the ITS1region.Within each form of G. elata, G. elata Bl. f. viridis showed the most geneticvariation, while G. elata Bl. f. glauca and G. elata Bl. f. flavida displayed smallgenetic variations and were genetically stable, which was beneficial to geneticbreeding. The genetic relationships between Zhaotong population and Guizhoupopulation is closer than that between Zhaotong population and Hanzhong of Shanxipopulation. ITS sequence of certain form from different population showed significantdifference while that of different forms from the same population showed highhomology.Armillaria mellea is not the only symbiotic fungi of G. elata, and most of themare other species of Armillaria. The symbiosis is not one-to-one correspondencebetween Armillaria spp. and forms of G. elata. The fungi symbiosed with G. elatamay also contain Mycena. However, we didn’t find Myeena osmundieola reported tosymbiose with the rhizome of G. elata.3．Medicinal compositions study for Gastrodia elata of ZhaotongAfter HPLC analysis, this study found that the content of gastrodin in different forms of G. elata from highest to lowest are dry G. elata Bl. f. glauca, fresh G. elataBl. f. elata, fresh G. elata Bl. f. flavida, fresh G. elata Bl. f. viridis, and fresh G. elataBl. f. glauca., the content of p-hydroxybenzyl alcohol in different forms of G. elatafrom high to low are fresh G. elata Bl. f. viridis, fresh G. elata Bl. f. elata, fresh G.elata Bl. f. flavida, fresh G. elata Bl. f. glauca, and dry G. elata Bl. f.glauca., thecontent of adenosine in different forms of G. elata from highest to lowest are fresh G.elata Bl. f. elata, dry G. elata Bl. f. glauca, fresh G. elata Bl. f. flavida, fresh G. elataBl. f. glauca, and fresh G. elata Bl. f. viridis., and the content ofp-hydroxybenzaldehyde in different forms of G. elata from highest to lowest are dryG. elata Bl. f. glauca, fresh G. elata Bl. f. glauca, fresh G. elata Bl. f. viridis, fresh G.elata Bl. f. flavida, and fresh G. elata Bl. f. elata. Only dry G. elata Bl. f. glauca’scontent of gastrodin reached0.2%in all the tested samples. Maybe it’s just becausethat gastrodin in fresh G. elata were hydrolysed after long-term preservation or thatcontent of gastrodin increased by processing. Therefore, with regard to G. elata Bl.used as drug, it is better to use the processed ones. Differences were tested in contentsof gastrodin,p-hydroxybenzyl alcohol, adenosine and p-hydroxybenzaldehyde amongforms of G. elata while no regulation were obtained.