ISSR Analysis For Genetic Diversity And Rapid Propagation System Of Goodyera Foliosa

Goodyera foliosa is a perennial belonging to the Goodyera(Orchidaceae),its plant morphology is easily confused with that of G.henryi,and in the absence of flowers,it is difficult to identify the species only by the leaves.Since G.foliosa is clustered in the wild,it is very easy to be extensively damaged and excavated.Therefore,understanding the genetic diversity characteristics of its wild population can provide research information for formulating corresponding conservation measures.In addition,G.foliosa has high ornamental value due to its colorful flowers and long flowering period,as well as its potential medicinal value,and has a broad prospect of development and application.On the basis of a comprehensive survey of the wild resources of G.foliosa in Fujian Province,ITS,mat K and chloroplast genome are used to identify the wild samples respectively.And the genetic diversity and genetic structure of 190 individual materials from 19 wild populations are analyzed by ISSR molecular marker technology,designed to know the genetic diversity,degree of genetic differentiation,gene exchange and genetic structure of wild populations and to propose corresponding conservation measures;finally,the establishment of asymbiotic germination and artificial fast propagation system was investigated,which provided scientific basis for the subsequent germplasm reserve and rational exploitation of resources.The main results of the study are summarized bellow:(1)ITS,mat K and chloroplast genome were used as DNA barcodes to assist in the identification of the field collection G.foliosa.In the three phylogenetic topology trees showed that the collected samples of G.foliosa clustered with the data on NCBI as the same species,all of which are strongly supported;the interspecific discrimination is credible.It indicates that both of the above two gene fragments as well as the chloroplast genome can be used as DNA barcodes for molecularly assisted identification of G.foliosa.Combining the phylogenetic tree with the key to species in the genus Goodyera,the phylogenetic position and the close relatives of G.foliosa are determined,and G.foliosa belonged to the subsection.Fliosum of section.Reticulum,and G.henryi is a close relative of G.foliosa.(2)7 primers for ISSR-PCR amplification of G.foliosa are obtained by screening100 primers.56 bands are amplified,including 54 polymorphic bands,with a polymorphism ratio of 96.43%.In terms of genetic diversity,the genetic parameters at the total level of the 19 populations are PPF=96.43%,Na=1.9643,Ne=1.5373,He=0.3189,I=0.4821.There is rich genetic diversity at the total level of the populations,but the level of genetic diversity is low within each population,with the highest in the XDJ population:PPF=26.79%,Na=1.2679,Ne=1.2296,He=0.1215,I=0.1723;the lowest in the JS population: PPF=5.36%,Na=1.0536,Ne=1.0358,He=0.0209,I=0.0308.In terms of genetic structure,the level of genetic differentiation between populations is high(Gst=0.7789>0.25),and the genetic differentiation between populations(77.89%)exceed that within populations(22.11%);gene exchange level between populations(Nm=0.1419<l)is low.Therefore,the level of genetic diversity within populations is low,genetic differentiation between populations is high,and gene exchange is hindered,which may be caused by the vegetative reproduction of individuals in the population.The UPGMA cluster analyses illustrates that 19 populations could be divided into 7branches when the threshold value is 0.80,Among them,the first branch consists of LT1,LT2 and DF populations and belongs to Sanming City,Fujian Province;the second branch consists of the GG,ZL,LJ,YS and TT populations and belong to the Fuzhou City,Fujian Province;the third branch consists of GYS1 and GYS2 populations and belongs to Huaan County,Fujian Province;the fourth branch consists of NTC1 and NTC2 populations and belongs to Longyan City,Fujian Province;the fifth branch consists of DG and XDJ populations and belongs to Longyan City,Fujian Province;the sixth branch consists of CD and ZX populations and belongs to Wenzhou City,Zhejiang Province and Wuyishan City,Fujian Province;the seventh branch consists of JZ,JS and CB populations and belongs to Zhangzhou City,Fujian Province.The clustering results shows that most wild populations clustered together in close geographical proximity and their genetic distances are related to geographical distance.(3)The results of orthogonal experiments shows that the effects of different factors on the asymbiotic germination of seeds of G.foliosa varied from high to low:sucrose>NAA>medium>potato juice,and the optimal medium for asymbiotic germination of seeds is: 1/2MS+20 g/L sucrose+5% potato juice+0.2 mg/L NAA,and the germination rate is up to 92.96±2.28%.The effects of different factors on the proliferation and differentiation of protocorms of G.foliosa varied from high to low:NAA>KT>activated charcoal,and the optimal medium for proliferative differentiation of protocorms is: 1/2MS+1.0 g/L activated charcoal+1.0 mg/L KT+0.5 mg/L NAA,with a proliferation coefficient is up to 4.67±0.12,and a differentiation rate is up to99.30±0.99%.Some protection strategies for G.foliosa are suggested according to the its reproductive pattern and genetic diversity characteristics,such as protecting G.foliosa in its native environment,transplanting G.foliosa among populations.A large number of seedlings can be obtained through the artificial fast breeding route,which can lay the foundation for the development and utilization of ornamental flowers as well as medicinal resources.