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2019, № 80

UDC: 632.7:575.116.4
GSNTI: 68.37.29, 34.15.23

Design and testing of the molecular markers in silico to identify 16 species of insect pests in the Krasnodar Territory

The preparatory stage of the PCR of the agricultural pests DNA from the gut contents of the predatory entomophages in conditions of the Krasnodar Territory is characterized. Primer pairs are developed and analyzed in silico using international databases Ensembl Genomes and NCBI Nucleotide, available on the Internet. Sixteen species of pests, widespread in the Krasnodar Territory and in the south of the Russian Federation, were selected for the study. Cytochrome b (Cytb) and cytochrome oxidase subunit 1 (COI) were selected as target genes to identify their genotypes. The choice of these genes is due to the fact that they are a priority in the development of the primer pairs not only for species identification, but also for sequencing. The primer pairs were tested for the problems such as "Conformation hairpins" and self-complimentariness. According to analysis in the Oligo Calc program, unsuccessful oligonucleotide variants were rejected. As a result, successful primer pairs were identified and tested again using NCBI Primer-BLAST algorithms to find possible complimentariness with other species. Despite the fact that such complimentariness has been revealed, the created primer pairs are relevant since the coincidence with other species included non-complimentary nucleotides and the recommended temperature of the primers is high to provide the required specificity. Thus, the developed primer pairs are recommended to be used for both species identification of pests and sequencing for the purpose of phylogenetic analysis of invertebrates.
Keywords: In silico, DNA, PCR, primers, agricultural pests, predatory entomophages, gut content, Krasnodar Territory
DOI: 10.21515/1999-1703-80-200-208

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Authors:

1. Khomitskiy Evgeniy Evgenievich, post-graduate student, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
2. Zamotajlov Alexandr Sergeevich, Dr. Sc., Professor, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
3. Savenkova Dar'ya Sergeevna, student, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
4. Milovanov Aleksandr Valerievich, PhD in Biology, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
5. Belyi Alexandr Ivanovich, PhD in Agriculture, Assistant Professor, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.