| The protection of non major crop varieties has changed from examination and approval to registration system in China,which caused the demand for the identification and protection of sugarbeet varieties urgent.How to accurately identify applied sugar beet varieties is of great significant value for the protection to breeders’rights and interests and the promotion of new varieties.The DNA barcoding technology are easy to be amplified sleceted DNA fragments which have enough intraspecific and interspecific differences to identify species or subspecies units.At present,the DNA barcoding technology t has not been reported the application of sugar beet identification,so screening the core library of DNA barcoding fragments was selected from the nuclear genome and chloroplast genome,and the phylogenetic tree is constructed by analyzing the genetic difference and genetic relationship between different sugar beet varieties using single and multi fragments combination.The reliability of DNA barcoding technology for the classification and identification to sugar beet varieties was evaluated,finaly,the identification system of sugar beet varieties based on the DNA barcoding technology is established in this study.In this study,7 fragments,including 2 nuclear fragments(ITS,ITS2)and 5 chloroplast fragments(two coding region fragments rbcL and matK,three non coding region fragments trnL-F,psbA-trnH,trnT-trnL)were selected to amplify the corresponding fragments of 84 sugar beet varieties.The success rate of amplification and sequencing of each fragment was 100%.After sequencing,the length of the fragments was compared as follows:trnL-F>matK>trnT-trnL>rbcL>ITS>ITS2>psbA-trnH,in which the longest trnL-F sequence is about 896bp,the shortest psbA-trnH sequence is 232bp,and the length of fragments meets the requirements of DNA barcoding.Based on the molecular evolution theory,84 sugar beet varieties were identified by using single fragment and combination respectively,and the identification rate of each single sequence and combination was compared.The results showed that the number of haplotypes and the diversity of haplotypes of trnT-trnL sequence were the highest,there were 17 haplotypes,and the diversity of haplotypes was 0.583,which indicated that the evolution speed of trnT-trnL sequence in sugar beet was faster and the genetic difference was larger.The number and diversity of haplotypes in trnL-F,matK,rbcL and ITS sequences were 6 and 0.115 on average,indicting the moderate evolution rate.The number of haplotypes and the diversity of haplotypes in psbA-trnH sequence were the lowest,which were 4 and 0.024,respectively,show a low evolution rate.The trnT-trnL has the highest success rate in single segment,94.0%.It could be used as a candidate sequence of DNA barcoding,suitable for DNA barcoding research for sugar beet.The successful identifications rate of matK was the lowest(8.3%),as for trnL-F,psbA-trnH,rbcL and ITS were 17.9%,15.5%,14.3%and 10.7%,respectively.Based on the combination of five chloroplast DNA fragments,84 sugarbeet varieties amplified 24 haplotypes in total,and the phylogenetic analysis divides all sugar beet varieties into three groups.Based on the combination of ITS+ITS2 fragments,84 sugarbeet varieties have 10 haplotypes in total,and the phylogenetic analysis divides all varieties into two groups,both combinations phylogenetic analysis revealed that the varieties within the same group had relatively close phylogenetic relationship,that is,the possibility in breeding They are closely related.Compared with the single segment,the successful identification rate of the combination segment was significantly improved.The success rate of identification using the fragments combination as the DNA barcoding of sugar beet was better than that of the single fragment.The combination of trnT-trnL+trnL-F+rbcL+matK+ITS+ITS2 fragments and the combination of chloroplast trnT-trnL+trnL-F+psbA-trnH+rbcL+matK identified all sugar beet varieties in this experiment.Considering the cost and complexity of the experiment,the combination of chloroplast trnT-trnL+trnL-F+psbA-trnH+rbcL+matK is recommended as the DNA barcoding for the identification of sugar beet varieties. |
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