Study On Genetic Diversity Of Nervilia Fordii

The wild Nervilia fordii used to be grow in the lacune between the stones,next to the stone,brushwood or hurst moist humus.Nervilia fordii only growth one leaf,singularly 2-3 leaves every year.It have a very short growing season,only 180 days.Nervilia fordii use the tuber as a way of breeding,bornth 1-2 new tubers annually.Because of higher prices in recent years,the herba are disroot,the resource was profoundly damaged. However,the demand for domestic and foreign markets has increased year by year, resulting in species Nervilia fordii market are confused,some substitute and counterfeit are used.The objetive:Collect Nervilia fordii wild species,cultivars,relatives species from Guangdong,Guangxi and Hainan main producing district.Identification the Nervilia fordii through the triditional morphological and molecular way.Analyse the genetic diversity on morphologic and molecular of Nervilia fordii from different district and resourse.Compare the genetic relationship and similary of Nervilia fordii.This meathod are used:Analyze leaf type,vein,size of leaves,weight of leaves and other 10 modality ingredient,and classify by SPSS.Ameliorate Low pH extraction medium with high salt method to extract genomic DNA,use Randomly Amplified PolymorpHic DNAs(RAPD),select 19 polymorphism primer from 49 random primer,22 samples were analysis by RAPD,and classify by SPSS.The result shows that:1.The Nervilia fordii with small leaves length is about 3cm,width is about 4cm;petiole is between 4 to 6 cm long;the Nervilia fordii with middle size leaves length is about 4cm, width is 5-6cm;petiole is between 6 to 10cm long;the Nervilia fordii with big leaves length is more than 6cm,width is more than 7cm;petiole is more than 10cm long.2.Different Nervilia Fordii breed are different in leaf type,vein,size of leaves,weight of leaves and other 10 modality ingredient;From the result of classify,it can be divided into two kinds by Euclidean distance 15 in general,Nervilia fordii as a genus and Nervilia plicata as another genus,it is the same with the botanic taxonomy.In the genus of Nervilia fordii,it is can divide into 3 kinds,the breed with small leaves,the breed with middle size leaves,and the breed with big leaves.The diversity of breed with big leaves is more likely have relationship with the clime.3.Low pH extraction medium with high salt is better to extract fresh leaves DNA than medical materials leaves,but both of which can run RAPD.The optimize of RAPD condition is:20μL solution contains 2μL 10×Buffer solution,0.5μL dNTP(2.5mM each),0.8μL Mg²⁺(25mM),0.5μL Primer(20 pinol/μL),0.2μL Taq DNA polymerase(5 U/μL),1μL DNA(50ng),1μl BSA(20mg/mL).RAPD program is 3 min at 94℃for predenaturation,then 40 cycles of 30 sec at 94℃for denaturation,30 sec at 40℃for annealing,1 min at 72℃for extension,finally extension at 72℃for 5 min.4.Select out 19 polymorphism primer from 49 primer.Divide 22 sample into fresh and medicinal materials to classify.In the medicinal materials,breed with small leaves and breed with middle size leaves are closer,breed with big leaves and Nervilia plicata are far from others sample.In the fresh materials,there breeds of Nervilia fordii are in one kind, closer with Nervilia plicata,Pachyrhizus erosus and culture breed than Plantago asiatica and Centella asiatica.5.Use Shannon's and Nei's index estimated the genetic variation of the number of different species Nervilia fordii.Shannon's index showed that the total genetic variation is 0.463,and Nei's index shows that the total genetic variation is 0.276;in the Shannon's index of within populations genetic variation is 49.5 percent of total variation;and Nei's index of genetic variation within populations accountedis 65.1 percent of the total variation, Nervilia fordii gene flow is 0.949.The genetic variation within the small leaves,middle size leaves and big leaves breeds are low,the genetic variation from different geographic location are high.6.The Concultion is:Different breed Nervilia Fordii and its substitute have genetic diversity on morphologic and molecule.RAPD can be used to identify the Nervilia Fordii. The genetic diversity of Nervilia fordii are came from grographic reigion and ecological environment.