Potential DNA Barcoding of Local Stingless Honeybee (Tantadan) From Kiulu, Sabah using 28S Ribosomal DNA

Abstract

Abstract This study aims to use DNA sequence and perform classification of eight Tantadan honey bees species from Sabah that were morphologically identified through Pictorial Identification Guide and Composite Algorithm. The species involved were Tetragonula fuscobalteata, Tetragonula laeviceps, Tetragonula drescheri, Lepidotrigona cf. ventralis, Lepidotrigona terminata, Heterotrigona itama and Tetragonula melanocephala. Normally, coI gene is used as it has elevated evolutionary rates higher than nuclear genes and is more specific in discriminating between closely related taxa. However, in this study, the nuclear gene was used as only this region could be amplified from this species. DNA barcoding is a method used for identifying unknown species and taxonomic classification at the molecular level using DNA fragments found in the genome of a species that is unique for the purpose of identification. However, morphological identification is also crucial as it provides the means of observing the differences between species based on the naked eyes. The methods used were DNA extraction, amplification of 28S nuclear DNA by PCR method, DNA sequencing and bioinformatics analysis of DNA to complete the identification process of these species based on their genetic material. Homology search of the 28S partial gene sequences revealed only three out of eight species were in agreement with taxonomic classification but five out of eight were not. Those five species are as follows; Tetragonula laeviceps identified as Tetragonula carbonaria, Tetragonula drescheri identified as Lepidotrigona terminata, Lepidotrigona cf. ventralis identified as Tetragonula carbonaria, Heterotrigona itama identified as Heterotrigona bakeri and Tetragonula melanocephala identified as Tetragonula carbonaria. The study revealed that 28S fragment of nuclear DNA is a suitable candidate to identify Tantadan honey bees species but it was not distinct enough for specific species identification, thus the result obtained here can be used for further study with primers targeting amplification of coI gene. In a nutshell, this study successfully demonstrated the use of DNA barcode using 28S rDNA in differentiating closely related species.