Identification And Genetic Diversity Analysis Of Dendrobium Officinale Germplasm Resources

Dendrobium officinale is a valuable herb,which has important theoretical and practical significance for its genetic diversity and DNA barcode identification because of its medicinal,edible and ornamental value.Dendrobium officinale was collected in eight regions of Hunan,Anhui,Yunnan,Zhejiang,Fujian and Jiangxi,and they were subjected to ISSR analysis and DNA barcode identification of germplasm resources.This study provides a reliable theoretical support for comprehensively mastering the genetic relationship of different populations of Dendrobium officinale,selecting,cultivating and promoting excellent Dendrobium officinale varieties.The main results in this study are as follows:(1)Dendrobium officinale plants contain polysaccharides,phenolic acids,flavonoids and other raw metabolites,which will irreversibly bind to DNA fragments during DNA extraction,increasing the difficulty of DNA extraction.The DNA extracted by the conventional CTAB method contains more impurities and needs further purification to be used in subsequent experiments.In this paper,the DNA in the leaves was extracted by the CTAB-based kit.The samples were pure and can be directly used in subsequent ISSR labeling experiments and DNA barcode studies.(2)Genetic diversity analysis of Dendrobium officinale in 24 different regions was performed using ISSR markers.A total of 92 DNA bands were amplified from Dendrobium officinale using 7 ISSR primers,of which 84 were polymorphic bands with a polymorphic ratio of 91.3%.The polymorphism of Dendrobium officinale at the species level was calculated using POPGENE 32 software.The average observed allele was 1.9130,the average effective allele number was 1.5283,the average Nei's gene diversity index was 0.3100,and the average Shannon's information index was0.4650.The band ratio was 91.3% and the average on the population was 36.41%.It can be seen from NTSYS-pc software that the genetic similarity coefficient of 24 Dendrobium officinale was between 0.4783 and 0.9130,and the range of variation was relatively large.It indicates that there are abundant genetic diversity among 24 different populations of Dendrobium officinale.(3)Using the NTSYS-pc software to analyze the results of PCR,the molecular clustering map was obtained.With the genetic similarity coefficient of 0.64 as the threshold,24 Dendrobium officinale could be divided into three groups,and the Dendrobium officinale in different regions showed the general trend in the molecularclustering diagram.It is consistent with the source of the material,but there are still differences among individuals.This difference indicates that there is a rich genetic diversity between Dendrobium officinale from different populations,which is conducive to species adaptation to the environment.(4)In this experiment,DNA barcode identification was performed on 22 Dendrobium officinale collected in 8 regions.The matK and rbcL genes of the chloroplast coding region of Dendrobium officinale were amplified by PCR,and the amplified products were sequenced and analyzed.The genetic distance analysis of the two sequences showed that the variability of the matK sequence fragment is higher than that of the rbcL sequence fragment,and has certain conservation.The matK sequence may be a more ideal Dendrobium officinale DNA barcode.Single matK sequence or rbcL sequence could not identify all germplasm resources well.The combined sequence matK+rbcL is an ideal way to identify the germplasm resources of Dendrobium officinale.Most of the Dendrobium officinale from different regions can be successfully clustered into single branches.