Analysis Of Main Components And Genetic Diversity Of Dendrobium Officinale Germplasm Resources

Dendrobium Officinale Kimura et Migo,a rare and endangered species of Dendrobium in Orchidaceae,is a traditional precious and rare traditional Chinese medicine with unique functions such as benefiting stomach,nourishing yin and clearing heat,anti-cancer and anti-aging.The sorely lacking of D.Officinale natural resources due to its harsh growth environment and low seed reproduction rate.However,as self-sterility,most of the lines used in D.Officinale production are randomly hybridized based on existing resources.As a result,the genetic characters of germplasms used in large-scale production are discrepant,with unstable quality,low content of effective components and soluble polysaccharides,as well as the vulnerable vitality.Our research team has been devoted to the investigation,collection and evaluation of D.Officinale resources.On this basis,more than 200 resources were collected from Zhejiang,Jiangxi,Yunnan Province and so on.According to morphology,geographical origin,and the condition of variety planting of Jiangxi enterprises,four samples from Wenzhou,Zhejiang,Yingtan,Jiangxi,Kunming,Yunnan and Zhangzhou,Fujian were selected,which were used to determine the content of polysaccharides and volatile components.Given the variance on morphology and geographical origins,81 germplasms were selected for genetic diversity analysis with EST-SSR molecular markers.And several SSAP primers were designed,which will be helpful to lay foundation for subsequent genetic diversity research,germplasm resources protection and variety breeding.The main results are as follows:(1)The polysaccharides capacity of four D.Officinale germplasms collected in February from Wenzhou,Zhejiang(ZJ-6),Yingtan,Jiangxi(JX-1),Kunming,Yunnan(YN-1)and Zhangzhou,Fujian(FJ-2)were measured by colorimetry.And results showed that the method of colorimetry with good reproducibility could be used to determine the capacity of polysaccharides.And the polysaccharides content was followed by ZJ-6(36.4%),JX-1(38.43%),YN-1(33.13%)and FJ-2(28.4%).The polysaccharides content between four varieties were the pattern of JX-1 > ZJ-6 > YN-1 > FJ-2,which indicated that there were significant differences in polysaccharides content of four D.Officinale germplasms.(2)The volatile components of ZJ-6,JX-1,YN-1 and FJ-2 were determined and analyzed by GC-MS technology,83 volatile compounds were obtained,including esters,alcohols,organic acids,terpenes and alkanes.51 substances were detected in ZJ-6(7 esters,9 alcohols,8 organic acids,5 terpenes and 11 alkanes),44 substances in JX-1(6 esters,10 alcohols,8 organic acids,4 terpenes and 10 alkanes)and 48 substances in YN-1(10 esters,6 alcohols,8 organic acids,3 terpenes and 8 alkanes),and 39 substances in FJ-2(3 esters,8 alcohols,8 organic acids,5 terpenes and 9 alkanes).The analysis of pharmacological and toxic components in volatile components showed that a total of 18 pharmacological components and 4 toxic components were found,with 2 toxic components found in ZJ-6,no toxic components were detected in JX-1,2 and 3 toxic components were find in YN-1 and FJ-2 respectively.And butyl tetradecyl phthalate had the highest content of toxic components in ZJ-6 and FJ-2.The contents are 0.26% and 0.22% respectively.While in YN-1 were butyl tetradecyl phthalate and ethyl hexadecanoate both contents were 0.26%.The results revealed that there were great differences in the types of volatile components and extremely significant differences in the contents of pharmacological components among the four D.Officinale germplasms,which can provide a certain reference value for genetics,breeding and planting of D.Officinale.(3)81 germplasms with different morphology from Jiangxi,Zhejiang,Yunnan,Fujian,Anhui and Guangxi Province were collected for genetic diversity analysis.75 pairs of EST-SSR primers were used for PCR amplification,a total of 480 bands were obtained,and the average number of alleles ranged from 1.96 to5.16,with a average of 3.16,the average expected heterozygosity(He)was 0.49 and the Shannon index(I)was 0.86,indicating that D.Officinale with a rich genetic diversity.Principal component analysis and cluster analysis showed that most of the D.Officinale collected from Zhejiang and Yunnan provincial were clustered with D.Officinale from other province.And the D.Officinale from Jiangxi Province clustered together alone.It is revealed that most of the germplasm resources may come from the varieties of D.Officinale from Zhejiang and Yunnan Province,while the germplasm of D.Officinale in Jiangxi Province remains to be reasonably developed and effectively utilized to provide high quality germplasm resources for the D.Officinale industrialization.(4)In order to develop SSAP molecular markers to analyze the genetic diversity of the whole genome of D.Officinale,we cloned the retrotransposon reverse transcriptase Ty1-copia and Ty3-gypsy of D.Officinale.The fragment size was 258 Mel 268 bp and 307 Mel 406 bp respectively.Multiple alignment of Ty1-copia reverse transcriptase sequences with transcriptional activity showed conserved amino acid fragments of ‘TAFLHG’,‘YGLK’ and ‘YVDDM’,as well as highly conserved ‘RMCVD’ motifs of Ty3-gypsy.Based on the sequence analysis of the clones,five pairs of SSAP primers were designed.