Assessment of the applicability of COI and 16S gene regions for molecular genetic diagnostics of the species Bactrocera tryoni (Froggatt)
The Queensland fruit fly Bactrocera (Bactrocera) tryoni (Froggatt, 1897) is a member of the Tephritidae family and is a serious pest of a wide range of fruit and vegetable crops in Australia. The object of this study, Bactrocera tryoni, belongs to the complex of B. tryoni species, which, in addition to it, includes the species Bactrocera aquilonis (May, 1965), Bactrocera neohumeralis (Hardy, 1951), and Bactrocera melas (Perkins & May, 1949). Since these species are morphologically very similar and some may infect similar products, there is a need for molecular genetic diagnostic methods that will reliably distinguish these species from other species and among themselves. According to the obtained data on the COI gene generally in the sample matrix, there is a hiatus - a gap between intra- and interspecific distances. However, the species B. tryoni, B. aquilonis, and B. neohumeralis on the dendrogram form clades corresponding to the same species, therefore, it is impossible to distinguish them using the sequences of the COI gene region. However, based on this sequence, it is possible to narrow down the identification by molecular genetic methods to these three species, since they are well separated from other studied species. According to the graph of the distribution of pairwise genetic distances for the 16S gene, there is a lower variability than for the COI gene. On the dendrogram, the species B. tryoni, B. aquilonis, and B. neohumeralis form the one clade, but other species formed separate clades. It is necessary to increase the number of 16S gene sequences in the sample for each species. Thus, based on the COI gene sequence, it is possible to separate the species B. tryoni, B. aquilonis, and B. neohumeralis from other species of the genus Bactrocera studied by us. To study the relationships within the complex of B. tryoni species and search for a gene sequence by which it will be possible to reliably differentiate the B. tryoni species, another, possibly more rapidly evolving gene is required.
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To construct trees, we used sequences taken from the GenBank NCBI database. The matrix of pairwise genetic distances of the studied species, the frequency of pairwise genetic distances, which were calculated in the MEGA6 program. We used the method of detecting hiatus between intra- and interspecific genetic distances (“barcoding gap”) implemented in the ABGD program with a view to analyze the taxonomic status of the studied representatives of the genus Bactrocera. The dendrogram was constructed using the MEGA6 program using the Neighbor-Joining (NJ) method. Bootstrap supports (1000 replications) for each branch are indicated on the tree. 80 sequences of the COI gene region of representatives of the genus Bactrocera were taken from the GenBank NCBI database with a view to construct trees. Seven sequences of the species Dacus ciliatus Loew, 1862 were taken as the data of the outgroup. 11 sequences of the 16S gene region of representatives of the genus Bactrocera and 9 sequences of the species Dacus ciliatus as an outgroup were used for analysis.