| Paeonia rockii belongs to ranunculaceae paeoniaceae paeonia group(Section Moutan DC.).It is a special plant in China whose flowers are big and plaque are on the bottom of them.With the efforts of many scholars,peony and dwarf peony in the central plains were introduced into non-directional hybridization with paeonia rockii.At present,the variety group of paeonia rockii is growing with thousands of species,and the genetic background is very complex.And different regions have their own names,so homonyms is very common,which brings a lot of difficulties to the identification and breeding of varieties of peony purple-spotted.The development of simple,fast,informative and discriminative molecular marker technology is of great significance for the identification and breeding of plant varieties.EST-SSR and CDDP are novel molecular markers,both of which are dominant markers with good polymorphism,simple operation and relatively low cost.First of all,this study compared the two different molecular marker methods-EST-SSR and CDDP in 12 paeonia rockii varieties of the same identification,a short-list identification ability of the method used in 47 paeonia rockii species identification,discusses different colors,designs the paeonia rockii germplasm genetic diversity and molecular marker method in the role of paeonia rockii species identification,paeonia rockii varieties provide important basis for classification and identification.The main conclusions are as follows:1.Orthogonal test design was used to optimize the primer concentration(5pmol,10 pmol,15pmol,20pmol)and template DNA concentration(15ng,30 ng,45ng,60ng)at two factors and four levels,and a CDDP-PCR reaction system suitable for paeonia rockii was establishedThe total reaction system was 20?l,including 2× Es Taq MasterMix enzyme(containing dye)10?l,1.0pmol/?l primer 1.5?l,15ng/?l DNA template 4?l,ddH2 O 4.5?l.2.Orthogonal test design was used to optimize the primer concentration(5pmol,10 pmol,15pmol,20pmol)and template DNA concentration(15ng,30 ng,45ng,60ng)at two factors and four levels,and a EST-SSR-PCR reaction system suitable for paeonia rockii was established.The total reaction system was 15?l,including 2× Taq PCR MasterMix enzyme(containing dye)7.5?l,10pmol/?l primers 0.5?l each,15ng/?l DNA template 3?l,ddH2O3.5?l.3.Compared with EST-SSR,CDDP is more suitable for the identification of paeonia rockii varieties with strong identification ability.CDDP markers had more amplification sites,higher PIC values and higher resolution for identification of paeonia rockii cultivars.4.19 CDDP primers were selected for amplification of 47 materials of paeonia rockii.A total of 112 bands were amplified,97 of which were polymorphic,and the polymorphism rate was as high as 86.61%.A total of 30 specific bands were obtained from 19 CDDP primers,with a specificity ratio of 26.79%.The specific bands played an important role in variety identification.The results showed that the CDDP marker technology was rich in information and could better reveal the genetic diversity among the varieties of paeonia rockii.5.CDDP primer Pr11(ERF3)has the most distinguishable samples,including 12 samples,which are ‘Lixiang,Yuguancaidai,Jinhuanyinxian,Zihai,Beijixiong,Fenloupiaocai,Lantadianjin,Mohaiyinbo,Heifanvlang,Hepingerqiao,Heitiane,Moguanyuzhu and Mohai'.6.In Ntsys 2.10 e,UPGMA method was used for cluster analysis of CDDP amplification results.When the genetic similarity coefficient was 0.72,all tested materials were clustered into two categories.The second category consists of two yellow varieties:‘ Haihuang' and‘Huaxiajinlong'.From the analysis of flower type,there was no clustering of the same flower type,which indicated that there was no obvious correlation between the division of genetic clustering group and flower type.In terms of design and color,the varieties of the same design and color are generally clustered together,but they are not completely. |
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