A Study On The Genetic Diversity And The Identification Of DNA Barcoding Of Medicinal Plants Rosa L.

Rosa L.belongs to Rosaceae and there are about 200 species and variant in the world.In the Rosa.L,R.rugosa and R.chinensis are very famous ornamental plants and also important traditional medicinal plants,but due to the recent years on the market demand for viewing the quantities of R.rugosa and R.chinensis,cross-fertilizing in a large number of different species of Rosa L.makes new varieties emerging endlessly,which leads to confusion of medicinal plants R.rugosa and R.chinensis in traditional chinese medicine market.The experiment analyzes 15 kinds of 33 samples of medicinal plants R.rugosa,R.chinensis and its related species by using ISSR molecular marker technology.Combining the experiment and the Gen Bank database,the study analysises the ability of identity about ITS,trn H-psb A,mat K,rbc L,rpo C1 five frequently-used barcodes in intra-specific discrimination and inter-specific variation,barcoding gap,phylogenetic tree and so on on the plant of Rosa.L by DNA barcoding technology,which is aimed at evaluating the identification of ISSR molecular marker technique and DNA barcoding on medicinal plants of the Rosa.L,discussing the new identification methods of medicinal plants of Rosa L..The results of the study are as follows:1)Six ISSR primers generated 110 loci of which 109 loci were polymorphic.The average percentage of polymorphie bands(PPB)was 99.09%.Nei's genetic diversity index(H)and Shannon's information index(I)were 0.3705 and 0.5464,the coefficient of genetic differentiation(Gst)and gene flow(Nm)were 0.8868 and 0.0638 between the species levels.The genetic distance(D)varied from 0.1691 to 0.7302.The results of ISSR analysis reveal that medicinal plants R.chinensis,R.rugosa and their relative species had the plentiful genetic diversity.2)The results of cluster analysis show that 15 species of Rosa L.are clustered into I,II,III,IV,V and VI 6 groups at the level of genetic similarity coefficient(GS)0.60.Group I includes three species of Sect.Synstglae DC.in Subgen.Rosa of Rosa L.,which is R.multiflora,R.maximowicziana(J)and R.wichuraiana(I),wherein A1,A2,A3,A4,A5,A6 and A7 seven samples of R.multiflora get together;Group II includes only a specie R.chinensis,and C1,C2,C3,C4,C5,C6,C7 and C8 eight different cultivars of R.chinensis get together,which belongs to Sect.Chinenses DC.in Subgen.Rosa of Rosa L.;Group III is R.laevigata(O)of Sect.Laevigatae Thory;The IV group includes six species of Sect.Cinnamomeae DC.in Subgen.Rosa of Rosa L.,which is R.rugosa,R.Sertata(D),R.setiipoda(E),R.persetosa(F),R.chengkouensis(L),and R.davurica(G),wherein B1,B2,B3,B4,B5 and B6 six cultivars of R.rugosa get together;The V group includes three species of Sect.Rosa in Subgen.Rosa of Rosa L.,which is R.gallic(H),R.damascena(N)and R.xalba(M);VI group is R.banksiae(K)of Sect.Banksianae Lindl.in Subgen.Rosa of Rosa L..The results of cluster analysis are consistent with the taxonomy of morphological characters.ISSR molecular marker technology is an efficient method for identification of R.chinensis,R.rugosa and their related species.3)As the two fragments of chloroplast genome,the evolution rate of rbc L and rpo C1 gene is slower,and the variable loci and informative sites are less(22 and 5,respectively;11 and 1)in inter-specific of Rosa L.;as well as it is the presence of overlapping in inter-specific and intra-specific genetic distance;barcoding gap test results show that although the crossover and mutation of rbc L and rpo C1 in intra-specific and inter-specific is small relatively,but distinction is not obvious.In addition,in the NJ phylogenetic tree of rbc L and rpo C1,the different populations of R.rugosa,R.chinensis and R.laevigata three species which are medicinal plants of Rosa L.,are not formed their own monophyletic group,and the resolution of the species is extremely low.Therefore,this study suggests that rbc L and rpo C1 genes are not suitable as a sequence of DNA barcoding to identify the medicinal plants of Rosa L..4)The analysis of sequences information and intra-specific or inter-specific variation about nr DNA ITS sequences,chloroplast gene trn H-psb A and mat K sequences showed that the variation sites and information site of chloroplast gene trn H-psb A fragment sequence is the most(80 and 62),followed by ITS fragments(59 and 39),mat K fragments(45 and 17),which indicating that ITS,trn H-psb A and mat K sequences can provide enough variation sites.Derived from the analysis of intra-specific and inter-specific genetic variation it is concluded that: the intra-specific maximum variation and inter-specific minimum variation of five sequences are relatively small,the inter-specific minimum variation of trn H-psb A sequences is greatest(0.00640),followed by ITS sequence(0.0026),mat K sequence(0.00095);the intra-specific maximum variation of trn H-psb A sequences is largest(0.00060),followed by ITS sequence(0.00046),mat K sequence(0.00024),and the average inter-specific variation of ITS,mat K and trn H-psb A three sequences are greater than the average intra-specific variation.The results of different sequence barcoding gap test and analysis show that the five sequences have no obvious interval area.Relative to other sequences,trn H-psb A although has no obvious interval area,but the intra-specific and inter-specific variations obviously divided into two parts,higher genetic variation distribution can be clearly divided.In NJ tree,ITS sequence can distinguish the medicinal plants of R.rugosa and R.laevigata from related species of Rosa.L;trn H-psb A sequence can distinguish the medicinal plants of R.laevigata from related species of Rosa L.;ITS + trn H-psb A + mat K sequences can distinguish the medicinal plants of R.chinensis,R.rugosa and R.laevigata very well from related species of Rosa L..5)The analysis of phylogenetic relationship in Rosa L.show that: medicinal plants R.rugosa,R.persetosa,R.sertata,R.davurica,R.macrophylla and R.willmottiae cluster a monophyletic clade,the branch agrees well with the flora of China,which based on the results of morphological study,belonging to Sect.Cinnamomeae DC.,thereinto the genetic relationship of R.rugosa,R.persetosa and R.Sertata is nearest and getting 100% support.The R.laevigata population of Sect.Laevigatae Thory cluster into a single line,and also reach the highest support 100%.Several sampling varieties of R.chinensis constitute a single branch,and also constitute a single with R.multiflora and R.wichuraiana together,which getting a 75% support rate.Building NJ phylogenetic tree based on DNA barcoding sequences is in conformity with the traditional morphological classification system research.