Study and assessment of the applicability of the mitochondrial COI and COII gene regions for molecular genetic diagnostics of the Rhagoletis indifferens Curran species
The Western cherry fruit fly Rhagoletis indifferens Curran, 1932 belongs to the Tephritidae family. This North American species is a pest of sweet cherries and cherries, as well as some other members of the genus Prunus. The object of this study, Rhagoletis indifferens, belongs to the “cingulata” group of species, which, in addition to it, includes the species Rhagoletis cingulata (Loew, 1862), Rhagoletis osmanthi Bush, 1966, and Rhagoletis chionanthi Bush, 1966. Since the species Rh. indifferens and Rh. cingulata are not only morphologically very similar, but also affect similar production, there is a need for molecular genetic diagnostic methods that will reliably distinguish these two species from other species and between 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 Rh. cingulata and Rh. indifferens on the dendrogram form a clade corresponding to one species, therefore, it is impossible to distinguish them using the sequences of the COI gene region. However, based on this sequences, it is possible to narrow down the identification by molecular genetic methods to these two species, since they are well separated from other studied species. According to the graph of the distribution of pairwise genetic distances and the dendrogram for the COII gene, there is a lower variability than for the COI gene, and hiatus is not observed not only between the species Rh. cingulata and Rh. indifferens, but also in the whole sample. Thus, based on the COI gene sequence, it is possible to separate the species Rh. indifferens and Rh. cingulata from other species of the genus Rhagoletis studied by us. That is, the COI gene sequence can potentially serve as the basis for further development of molecular genetic identification methods. To study the relationships within the group of “cingulata” species and search for the gene sequence by which it will be possible to reliably distinguish between the species Rh. indifferens and Rh. cingulata, 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 Rhagoletis. The dendrogram was constructed using the MEGA6 program using the Neighbor-Joining (NJ) method. Bootstrap support (1000 replications) for each branch are indicated on the tree. 86 sequences of the COI gene region of representatives of the genus Rhagoletis were taken from the GenBank NCBI database with a view to construct trees. Seven sequences of the species Bactrocera dorsalis (Hendel, 1912) were taken as the data of the outgroup. 76 sequences of the COII gene region of representatives of the genus Rhagoletis and 11 sequences of the species Bactrocera dorsalis as an outgroup were used for analysis.