Molecular identification of fruit flies, Bactrocera spp. (Diptera: Tephritidae) infesting guava fruits by using mitochondrial COI gene
Nishant Ukey, Ajit Chandele, Shubhangi Khandare and Vivek Chimote
Molecular identification of fruit flies, Bactrocera spp. (Diptera: Tephritidae) using mitochondrial COI gene using primer COI F, COI R, UEA7 and UEA10 and restriction enzymes (Msel and Alul) infesting guava fruits was carried out. The banding profiles in the electrophoresis gel were analysed. For the molecular studies, two set of mtDNA gene specific primer pairs i.e. COI F and COI R and UEA 7 F and UEA 10 R were used for polymerase chain reaction amplification of cytochrome oxidase I gene. In COI primer, samples yielded bands from all five Bactrocera spp. except B. zonata within the range of 1000-1500 bp, while UEA primer pair fragments were amplified PCR fragments from all five species within the range of 500-750 bp. The sequencing results showed total nucleotide length was 450-680 bases. Alignment of above sequences with NCBI database revealed that B. dorsalis and B. cucurbitae showed 97-99% similarity while, B. zonata, B. verbascifoliae and B. correcta showed 85-94% similarity. In B. correcta where there was deletion of 28 bp nucleotides as compared to sequence reported earlier. The restriction sites of DNA sequences were examined by Insilico approach in Bioedit program by selecting two restriction enzymes i.e. Alul and Msel which cut the sequence according to their sites and form a specific restriction pattern within five Bactrocera spp.