Molecular genetic analysis of promising potato hybrids of the Komi Republic (Russia)

. Potato varieties with a complex of economically valuable traits, such as high yield, quality, disease resistance, are the key to successful competitive production in the Komi Republic (Russia). Agribusiness is increasingly demanding high field resistance of varieties to the main diseases and pests common in the region - viruses (X, Y), nematodes, etc. The


Introduction
Potato is the most common tilled crop cultivated by agricultural producers in the Komi Republic [1][2].However, the instability of growing conditions in this region requires the continuity of the breeding process and its improvement.Global climate change is taking place, various diseases and pests of agricultural crops are more and more successfully resisting the used plant protection preparations, infecting varieties that were previously considered resistant to them [3][4][5][6].At the present stage, science makes it possible to select varieties and hybrids for each region of Russia in accordance with its agro-climatic and phytosanitary characteristics.In recent years, the method of genetic screening for the presence of markers of genes responsible for resistance to cancer, golden and pale potato nematodes (GPN and PPN), potato viruses X, Y (XVP and YVP) and other diseases, including quarantine ones, has become widespread, which makes it possible to select the most preferred hybrids at early stages [7][8][9].

Materials and methods
For a comprehensive assessment of the five potato hybrids presented in table 1, scientific studies were carried out, which included genetic certification for the presence of 10 main markers of genetic resistance to pathogens.Genetic screening using 10 markers of resistance genes to cancer, golden potato nematode, pale potato nematode, X-virus and Y-virus, presented in table 2, was carried out in the laboratory of PCR diagnostics of the Institute of agrobiotechnology of the Federal Research Center of the Komi Research Center of the Ural Branch of the Russian Academy of Sciences and on the basis of the scientific and production company "Syntol" (Russia, Moscow) using a set of reagents "GenExpert "Markers of potato resistance genes" (Syntol, Russia) [10][11].The leaves of test-tube plants of the corresponding potato hybrids were chosen as the object of research (DNA analysis preparation).According to the analysis protocol [21], water was used as a negative control, and a mixture of 10 plasmids containing the desired potato DNA fragments was used as a positive control.Amplification of the samples was carried out by PCR analysis using an Applied Biosystems 2720 Thermal Cycler (USA).The sample preparation consisted in mixing the reaction mixture and the primer mixture in one tube, bringing it to a volume of 23 μl with water, adding control samples in a volume of 2 μl and analyzed samples at a concentration of 5-15 ng.The reaction of the conducted studies on the amplification mode is presented in table 3.
After completion of the amplification process, the samples were denatured for 5 minutes at a temperature of 95°C.To do this, 1 µl of the PCR mixture, 9 µl of formamide were poured into a 0.2 ml tube, and 1 µl of the CD-600 molecular weight marker (Syntol, Russia) was added.In order to determine the specificity of the amplification reaction, positive control (PCS) and negative control (NCS) samples were used.Deionized water was chosen as the latter.The further process of electrophoresis of the studied samples was carried out on an 8capillary sequencer (genetic analyzer) Nanofor 05 (Institute of Analytical Instrumentation, Russian Academy of Sciences, Russia) in accordance with the manual and instructions for use [22].During electrophoresis, the obtained information about fluorescence detection in automatic mode without the participation of an operator was transferred to a control personal computer and processed by the Nanofor 05 program.Analysis results were processed and alleles were identified automatically using the GenExpert program version 5.0.1.6(Institute of Analytical Instrumentation RAS, Russia).
In a positive control DNA sample, all 10 markers of resistance genes were detected, corresponding to the standard ones from the manual and instructions for the set "GenExpert "Potato resistance gene markers" (Syntol, Russia), which allows us to conclude that there are no false negative results in the obtained data.No peaks of amplification products were found in the negative control sample, which confirms the absence of a false positive test result.The presence of peaks of amplification products of known length indicates the presence of markers of resistance genes, and their absence indicates the absence of the corresponding markers [21].
The studied samples of potato hybrids were tested for resistance to certain types of pathogens (Y-virus, X-virus, S-virus, A-virus, M-virus, potato cancer (Synchytrium endobioticum), ring rot (Clavibacter michiganensis subsp.Sepedonicus), brown bacterial rot (Ralstonia solanacearum), leaf curl virus) in the field on experimental plots of the Institute of agrobiotechnology of the Federal Research Center of the Komi Research Center of the Ural Branch of the Russian Academy of Sciences.To do this, breeding nurseries for the reproduction of hybrids, 400 tubers each, were planted in four-row plots (the area of one nursery is 84 m 2 ), and a visual record was made of the defeat of the tops and tubers of potatoes during the growing season [23].The leaves of the studied five samples of potato hybrids were taken in the budding-flowering phase from the middle tier of the bush (one leaf per plant) and the material was taken directly from the tuber in the areas of the buds during the harvesting period.
After a visual assessment of the objects in the field, laboratory methods were used to study the infestation with phytopathogens in the genomic and analytical laboratories of the Institute of agrobiotechnology of the Federal Research Center of the Komi Scientific Center of the Ural Branch of the Russian Academy of Sciences using DNA/RNA assessment methods for pathogens of potato diseases.For the isolation of plant nucleic acids (DNA/RNA), a set of reagents "Proba-GS" [24] was used.After preparing for the PCR reaction, the analyzed samples were placed in the amplifier and the PCR amplification process was carried out in the appropriate mode according to the instructions [26][27][28] and the modes presented in table 4. The length of the amplification products and the programs used during detection and taken into account as a result of PCR amplification are shown in table 5.

Results and discussion
The results of molecular genetic analysis of potato gene markers for resistance to pathogens are shown in table 6. Hybrid 2341-265 contains only one marker of the potato cancer resistance gene NL25 (Sen1).This makes this hybrid the least promising for its further use in breeding new potato varieties.
Hybrids 2339-9 and 2000-60 contain three markers of the resistance gene to the golden potato nematode H1 (TG-689, 57R, N195), a marker of the potato cancer resistance gene NL25 (Sen1).This set of genes is the minimum necessary for supplying hybrids to a variety, since these diseases and pests are quarantine objects [23].
The hybrid 1992-14 contains three markers of the golden potato nematode resistance gene H1 (TG-689, 57R, N195), the pale potato nematode resistance gene marker Gpa2 (Gpa2-2), and the potato cancer resistance marker NL25 (Sen1).The presence of the pale potato nematode resistance gene makes this hybrid promising for further breeding of new varieties for areas where the pale potato nematode is common.
According to the results of field studies during the growing season, wrinkling and twisting of the leaves were visually noted in the studied potato hybrids, which may indicate the defeat of plants by viral diseases.The leaves were selected for PCR analysis, according to the results of which phytopathogens were not confirmed.Thus, it can be assumed that these changes are either a physiological feature of hybrids or are explained by the reaction of plants to a lack of moisture and micronutrients directly in the soil [2], which requires further study.

Conclusion
According to the research results, in the course of a comprehensive assessment of potatoes by the methods of molecular genetic analysis of gene markers, a promising potato hybrid 2000-60 was identified.This hybrid is characterized by a complex of genes for resistance to the golden potato nematode H1 (TG-689, 57R, N195) and potato cancer NL25 (Sen1).Based on the molecular genetic analysis of potato genes, hybrids 2339-8 and 1992-14 are recommended for crossing and obtaining new breeding lines in order to develop new potato varieties resistant to various types of nematodes.

Table 2 .
Markers and genes of potato disease resistance.

Table 5 .
Lengths of DNA PCR amplification products and amplification program.

Table 6 .
The results of genotyping of the studied samples using the set of reagents "GenExpert "Potato resistance gene markers".