ISSR Analysis Of Genetic Diversity Among The Part Of Bamboos In Phyllostachys

Bamboos are the Earth's terrestrial ecosystems an important component of the fast-growing and widely used.Not only have important economic value,but also have a good water conservation and soil conservation function.Phyllostachys is the largest type in the subfamily of bamboo,up to 50 species.This is all the origin of almost all types of bamboo in China,some of which enjoy higher-value species of bamboo in the garden has been widely applied.Therefore,the future research in Phyllostachys have been emphasized.DNA molecular marker is a new genetic marker,it is expressed form of specifically genotype and have some merits of easily identifcation,so it was considered as one important technical index in study of biological taxology,breeding,genetics and species origination and evolution.Molecular markers directly reveal differences in the levels of DNA,using it to describe the genetic diversity of plants is more intuitive and accurate.ISSR(Inter-Simple Sequence Repeat) is an ideal molecular marker based on PCR technology.In germplasm identification,construction of genetic map,gene mapping,genetic diversity and molecular ecology are widely used in the study.Both intraspecific and interspecific relationships of the 14 ornamental bamboos of Phyllostachys in the section were analyzed using ISSR.The species as follows:Three cultivars species of P.sulphurea:P.sulphurea cv.houzeau,P.sulphurea cv.robert and P. sulphurea cv.viridis;P.heterocy and three cultivars species of:P.heterocycla cv.pubescens, P.heterocycla cv.tao and P.heterocycla cv.luteosulcata;P.nigra and varieties specie of P. nigra var.henonis;P.bambusoides f.lacrimadeae;P.praecox;P.aureosulcata and cultivars specie of P.aureosulcata f..spectabilis;P.vivax f.aureocaulis.1,Based on the analysis of SDS and CTAB methods about extraction and purification of P.sulphurea cv.robert genetic DNA,it was found that the improved CTAB method was optimal for Phyllostachys.2,The orthogonal design L₁₆(4⁵) was used to optimize amplification system on Phyllostachys in four levels of five facrors(Taq DNA polymerase,Mg²⁺,template DNA, dNTP and primer) respectively.As a result,a satisfactory ISSR reaction system for Phyllostachys was established.In a total volume of 20μL system which contained 1×buffer, 1.5 U Taq DNA polymerase,2.5 mmol/L Mg²⁺,80 ng template DNA,0.15 mmol/L each of dNTP and 0.4μmol/L Primer.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. 3,The genetic polymorphism of the 14 ornamental bamboos of Phyllostachys was investigated using ISSR.10primers,which could amplified clearly bands,were selected out from 40 ISSR primers.As a result 75 bands were generated,of which polymorphic bands number was 60,the percentage of polymorphism was 80%.The length of most amplified fragments ranged from 0.3 to 2.0 kb.4,Amplified fragments were used in the analysis of the relationship of the 14 species. The dendrogram was constructed by using UPGMA methods:(1) The genetic distance between P sulphurea cv.viridis and P sulphurea cv.houzeau,P sulphurea cv.robert were the further,it proved that they are larger in the extent of genetic variation;(2) P vivax f.aureocaulis and P bambusoides f.lacrimadeae clustering for a class,their genetic distance is close;(3) Others species clustering accorded with morphological classification system.P.praecox;P.aureosulcata f.spectabilis and P.aureosulcata;P.nigra and P.nigra var.henonis;P.heterocycla cv.pubescens,P.heterocycla cv.tao,P.heteroey and P. heterocycla cv.luteosulcata;(4) The genetic distance was 0.0134 between P.heterocycla cv.tao and P.heterocycla cv.luteosulcata were the closest,P.praecox and P.sulphurea cv.pubescens were the furthest for 0.7621.It proved that using ISSR is very effective on classification and relationship research on Phyllostachys.In sum,establishment on extraction method of Phyllostachys genome DNA samples, optimize the ISSR-PCR reaction system,studies on ornamental bamboos of Phyllostachys in the section using ISSR.Test results not only can improve varieties and genetic breeding,and also provide theory and the technology basis about classification system of bamboos.