Nucleotide Sequence Analysis of ITS2 Gene in Cassava for DNA barcode

Abstract

This research aimed to study of construct DNA barcode based on nucleotide sequence of ITS2 gene. In this study, 17 cassava cultivars were recorded the characteristics that their decided the correct naming of each cultivar. These cultivars were made herbarium specimen and remained in Bangkok Herbarium (BK), Department of Agriculture. DNA extracts from each cultivar used as a template for amplified and universal primer, matching ITS2 region using polymerase chain reaction technique. The sequences length of DNA fragment were 317 base pairs in all samples. Sequencing analysis was compared with GenBank. The nucleotide sequences showed a value of 99.05 to 100 percent similarity of gene among M. esculenta Crantz. The ITS2 sequences found the variation among cassava cultivars contain unique signatures at 5 nucleotide positions. All sequences were validated and deposited in GenBank. When analysis the phylogenetic tree using rubber as an outgroup, results presented that could effective divided of 3 groups and distinguished Hanatee, Rayong 3 and Rayong 72 from cassava cultivars. 50 samples from several crops have accurate characteristics as Rayong 72 and Kasetsart 50. All samples were collected and were successfully DNA extracted and nucleotides sequenced. The ITS2 nucleotide sequences were determined alignment search tool of GenBank. The result found that nucleotide sequence data were highly similar (100 percent) to deposit nucleotide sequences of Rayong 72 and Kasetsart 50 into GenBank. Results suggested that ITS2 sequences facilitates independent assessment one of DNA barcode as an effective tool for cassava cultivar identification. However, a combination with other DNA barcode could increase effective identification. This research information can be applied to research, especially plant genetic conservation and breeding