Open AccessDNA barcoding of Thrixspermum longipilosum based on Internal Transcribed Spacer 2 (ITS2) region
Author(s) -
Siti Rohimah,
Tri Ratnasari,
Mukhamad Su’udi
Publication year - 2021
Publication title -
iop conference series. earth and environmental science
Language(s) - English
Resource type - Journals
eISSN - 1755-1307
pISSN - 1755-1315
DOI - 10.1088/1755-1315/743/1/012092
Subject(s) - amplicon , internal transcribed spacer , dna barcoding , biology , dna , genetics , phylogenetic tree , polymerase chain reaction , genomic dna , dna sequencing , homology (biology) , nucleotide , computational biology , microbiology and biotechnology , evolutionary biology , gene
Thrixspermum is an orchid that has the characteristics of a very short flowering period. This is one of the obstacles in the conventional identification process that uses morphology, both vegetative and generative. For this reason, a molecular approach is needed as an alternative identification tool for the Thrixspermum orchid. For molecular identification, CBOL recommends three markers, namely matK, rbcL , and ITS . In this study, only ITS2 was used since ITS have been reported for its ability to discriminate plant sample up to species levels. The genomic DNA of T. longipilosum was isolated using the CTAB method with minor modification, then used as a template in PCR amplification. The amplicon resulted from the PCR reaction (± 450 bp in size) was purified and further determined using DNA sequencer. The results showed that T. longipilosum has a high homology level with its close relatives (per ident 94.07%-96.91%). From alignment analysis, the ITS2 sequence of T. longipilosum showing differences nucleotides located in sequences 3 (A→G), 55 (G/A→T), and 64 (A→G). Meanwhile, phylogenetic analysis showed that T. longipilosum formed one cluster with T. linusii . This shows that ITS2 are highly recommended as a molecular marker to determine the barcode of the T. longipilosum .