Study On Dandelion With ISSR Molecular Markers

Objective: To study the genetic diversity and phylogenetic relationships of germplasmresources for dandelion by ISSR and sequencing ITS (internal trancried space) sequences.Methods: Based on the analysis of orthogonal design test, an orthogonal design was usedto optimize the ISSR-PCR amplification system on dandelion by three factors (Taqpolymerase Mix, DNA template and primer) at two concentration levels, respectively. PCRprogram was optimized by single factor experiments. On this basis, screen primers withstable amplification and rich polymorphism from50ISSR primers. The optimal annealingtemperature for ISSR-PCR reaction was proposed by gradient PCR. The21populations ofgermplasm resources for dandelion were analyzed by ISSR molecular markers．Nei’s (He)and Shannon (I) were calculated by POPGEN32. Make up the systematic diagram of geneticrelationship by NTSYS2.1e and Cluster by UPGMA method. The ITS sequence wasamplified by PCR with universal primer of ITS and sequencing ITS on PCR product wasdirectly sequenced after purification. The results of ITS sequence were analyzed byDNAstar.Results:1The suitable PCR reaction system contained15μLTaq polymerase Mix(1.25UTaqDNA polymerase,1.8mmol/LMg2+, dNTPs0.24mmol/L),0.3μmol/L primer and5ng template DNA in total25μl reaction solution. The optimal amplified procedure were asfollows: after apredenaturing of5min at94℃,40cycles were performed with denaturing of30s at94℃, annealing of45s due to denaturing temperature of different primer, extension of2min at72℃, a final extension step of7min at72℃and hold at4℃.2On the basis of TEXT1,16primers were screened with stable amplification andrich polymorphism from50ISSR primers. The optimal annealing temperature for ISSR-PCRreaction was proposed by gradient PCR. 3A total of166ISSR bands were scored for16primers, among which150werepolymorphic bands. The average percentage of polymorphic bands was90.4%. Ner’s genediversity index (H) was0.2865, Shannon information index (I) was0.4370. The geneticdistance coefficient and genetic similarity were0.1562~0.5902and0.5542~0.8554respectively.4By cluster analysis, the geographical distribution is mutually related to therelationship and it was also showed some of dandelion from the same region were in thesame group which presented the rule of geographical distribution in the tested materials.5The total length of ITS sequence is711bp in the different samples. The ITSsequence can be divided into three fragments: ITS1is225bp,5.8S is262bp and ITS2is226bp. In all samples5.8S is comparatively conserved, ITS1and ITS2have bigger diversityin base sequence among the various specimens. ITS sequence variation is correlated to thegeographic distribution.Conclusion: It is a way to establish the ISSR-PCR system for orthogonal designcombining with single factor test. This optimized ISSR reaction system would provide thebasis for the genetic analysis of dandelion. The diversity level of the different germplasmresources for dandelion higher The ISSR marker could be used for the analysis of the geneticdiversity and genetic variation of dandelion. ITS sequence can be used to analysisphylogenetic relationships of different variety dandelion.