Evaluation And Identification On The Genetic Diversity Of Different Germplasm Resources Of Anoectochilus Roxburghii(Wall.)Lind

Anoectochilus roxburghii(Wall.)Lindl,was a precious perennial herb of Orchidaceae,cultivated widely in Fujian province and other southwest regions of China.Whole plants of A.roxburghii were used for raw marterials of Chinses medicine.Furthermore,A.roxburghii have typical medecial properties with sweet in flavor and cold in property.However,some common problems were encountered during the process of cultivation and production of A.roxburghii plants,such as there were wide planting area for A.roxburghii but messy,the numberous cultivars but less excelent,and the multiple cultivation techniques but inconsistent standarts.Many cultivars of A.roxburghii used by different A.roxburghii manufacturers were breeded by theirself through self screeing and identifying.Therefore,there were confusions phonomens among different cultivars from different planting areas ofA.roxburghii,the same eultivars of A.roxburghii have different names in different areas,and simtanously the cultivar have indentical calls in same areas.In order to explore genetic and evolutionary relationship among different A.roxburghii gernplasms,and construct a standard cultivar in breeding system for A.roxburghii,this study selected the representive A.roxburghii cultivars from Fujian province and identify the difference presented in A.roxburghii germplasms based on the phenotype,physiology,and genetic level.The research content and results are as follows:1.The differences among A.roxburghii germplasm were revealed on phenotype and phisilogicallevels.In this study,the select of 9 A.roxburghii,which named FJQG,FJCY,FJSM,FJTN/1,FJTN/2,GXJX,GXDY,GXHZ,TW,FJCY repectively,were collected from Fujian province.These germplasms were in detail evalued by using morjphological analysisand physiological index detection.As a result,there were obvious differences for the roots,stems and leaves among 9 A.roxburghii.The different A.roxburghii ranged from 2.63 to 4.5 cm for leaf length,from 11.74 to 15.96 cm for stem length.At the same time,dry weight ratio of leaves and stems located from 0.085 to 0.252 and from 0.046 to 0.076 respectively.Additionaly,the antioxidant activity including CAT,POD,SOD and the medical acitive components including polysaccharides and total flavonoids,soluble sugars,and protein content were in detail studed among different A.roxburghii germplasms.Resulting for SOD and POD activities,"GXDY" was the highest level activty,"TW" and "FJTN/2" were the lowest repectively among 9 species of A.roxburghii.The highest CAT activity was found in "FJTN/1",the lowest in "FJTN/2".For active ingredient contents,the soluble protein,polysaccharide,and flavonoids have the highest contents for "GXDY"obviously,the lowerst for“FJSM","FJQG" and "FJJY".In the soluble sugar content,the highest and the lowest were "FJTN/1" and "FJTN/2"repectively.The results indicated that there were significant differences among different A.roxburghii germplasms in phenotypic,physiological level and medicinal active ingredient content.These differences supplied an important candidate resource on selecting diversification and wide adaptation of A.roxburghii.2.The genetic background differences of different A.roxburghii resources were analyzied at the cytological levels.In order to reveal the difference genetic background of different A.roxburghii resources,the karyotype analysis of A.roxburghii chromosomes were performed.The results showed that the karyotypes of A.roxburghii belong to "2B" type.Of which,the karyotype formulas of "GXHZ" belong to 2n=64m+16sm,"GXJX" to 2n=18m+22sm,"GXDY" to 2n=1st+20m+1gsm,"FJQG" to 2n=18m+22sm,"FJTN/1" to 2n=68m+12sm,"FJTN/2" to 2n=29m+11sm,"FJCY" to 2n=38m+2sm,"FJSM" to 2n?14m+26sm,"FJJY" to 2n=74m+6sm.The result indicated that all kind of A.roxburghii had a primitive level in evolution."GXDY" and "FJSM,were the higher evolution levels among different A.roxburghii germplasms.The karyotype clustering of A.roxburghii germplasms indicated that there was the closest genetic relationship between "GXDY" and "FJCY".In addition,flow cytometry used to undertake ploidy characteristics of different A.roxburghii resources.Resulting all of 9 A.roxburghii resource were identified as tetraploid using the standard diploid of Dendrobium nobile Lindl as controls.3.Identification of relationship of different A.roxburghii resources at the molecular level.This study installed the published transcriptome data of A.roxburghii seeds and tried to find the available EST?SSR markers.The results showed that 82723 unigenes were got from the transcriptome data of A.roxburghii,and 9401 simple sequence repeat(S SR)were got when using MIS A software,and 200 from them were randomly selected to validate their effectiveness.Consequently,144 primer pairs could amplify target products,and the amplification efficiency was up to 72%,among which 60%primer pairs showed polymorphism.The results showed that when using polymorphic primers to analysis clustering on A.roxburghii,in which devided into three groups.Group 1 included 7 different resources from 9 different A.roxburghii resources,group 2 included "GXDY" and group 3 included"FJCY",which was the cultivation species and wild species respectively.The results demonstrated that the EST-SSR loci using transcriptome data,with the advantages of steady amplification,high polymorphism and high transferability.It can be deduced that the genetic relationship of A.roxburghii.This study identified in detail the differences for A.roxburghii from description of phenotypic character to identification of inside molecular levels through different perspective from phenotypic,physiological,and genetic background levels.Based on above study,the evolutionary relationship among A.roxburghii was in detail evaluated at the molecular and cytological level.This study provided important data support for further screeing and identifying A.roxburghii germplasm.At the same time,this study provided a technical basis and a good theoretical basis for the further identifying genetic resources of A.roxburghii on wide scales and the good combination of different A.roxburghii germplasms that used to breed A.roxburghii cultivars.