| Endogenous reference gene is a specific marker of a particular species at the molecular level,and it is widely used.At present,a number of endogenous reference genes have been validated for many species,yet no endogenous reference gene has been developed forsafflower.The transcription spacer sequence of nuclear rDNA,which called ITS,has great significance for the research about plant identification and phylogenetic analysis.In this study,we are working from two aspect.On the one hand,developing the endogenous reference gene of safflower,on the other hand,studying the speciesidentification and phylogenetic analysis of Cynareae Less.by ITS.1.Based on genetic information of safflower in the Genebank,the CTOS gene were selected as candidate gene of endogenous reference gene.And according to the information of CTOS gene,pairs of primers were designed.Corresponding qualitative PCR detection system was developed,two pairs of primers were selected out,both of them had a amplification in 23 kinds of safflower,amplifying a 171 bp anda 280 bp fragment.The PCR system had high detection sensitivity.The qualitative PCR assay can detect as low as 0.05 ng DNA of safflower.Corresponding quantitative PCR detection system was also developed.A standard curve was generated by plotting the Ct values obtained in each real-time PCR reaction against the concentration of safflower DNA.The square regression coefficient(R2)value was 0.996,and the PCR efficiency was more then 90%.And by using the developed PCR assays,the safflower component in different organizations was detected successfully.2.According to the analysis results of ITS sequences about 10 different kinds of lappa.We found the ITS sequences were highly conserved,only 7 base mutation,the difference value of ITS sequences in 10 different kinds of lappa were less than 1%.Therefore,ITS sequences could be used as a maker for the identification of lappa at the molecular level.The ITS sequences of the five wild lappa were all the same,and the ITS sequences of the five cultivated lappa were having mutations.Therefore,according to the sequence variation of ITS,could distinguish between the wild and cultivated lappa effectively.Based on the results of clustering analysis by ITS sequences,10 different kinds of lappa had been clustered into two groups.One group had the close relationship with the wild lappa,the other one were having a base transversion in ITS1 area at 229 bp.3.The analysis of ITS sequences were used in 36 different kinds of Cynareae Less.samples and 3 different kinds of Astereae Cass.samples.And based on the analysis results,the ITS sequence were obvious different between the different groups,the groups with closer relationship,the ITS sequences were more similar.The results of clustering analysis by ITS sequences showed,the samples with the same genus can be gathered together,results are basically consistent with the traditional phylogenetic analysis.The samples of Centaureinae O.can gather together,but the samples of Carduinae O.were clustered into different kind of groups,which showed that the different ways of the evolution in different samples.And we had predicted the second structure of ITS2 sequences,the basic structure of ITS2 sequences have 4 helix arms,which called A,B,C and D.Analysis the difference among 36 samples,we found the helix D arm is different in different tribal groups,and helix A arm is different in different genus groups,the helix B and C were polytropic,especially C arm,concluded they will play an important role in variety identification and phylogenetic analysis. |
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