vstispСадоводство и виноградарствоHorticulture and viticulture0235-25912618-9003Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»10.31676/0235-2591-2020-3-44-50vstisp-532Research ArticleМЕТОДЫ И СПОСОБЫ ЗАЩИТЫ РАСТЕНИЙ ОТ ВРЕДИТЕЛЕЙ И БОЛЕЗНЕЙMETHODS AND WAYS OF PROTECTING PLANTS FROM DISEAS AND PESTSСовременные методы молекулярной диагностики и изучения генетического разнообразия вирусов плодовых и ягодных культур на основе секвенированияModern methods of molecular diagnostics and study the genetic diversity of fruit and small fruit crops viruses based on sequencinghttps://orcid.org/0000-0002-5820-102XРадзениецеС. Б.RadzenieceS. B.

Радзениеце Светлана Борисовна - младший научный сотрудник, лаборатория репродуктивной биотехнологии

Загорьевская ул., д.4, Москва, 115598

Svetlana B. Radzeniece - Junior Researcher, Laboratory of Reproductive Biotechnology

4, Zagorevskaya str., Moscow, 115598

vstisp@vstisp.orghttps://orcid.org/0000-0003-1069-3771УпадышевМ. Т.UpadyshevM. T.

Упадышев М. Т. – доктор сельскохозяйственных наук, членкорреспондент РАН, главный научный сотрудник, заведующий лабораторией вирусологии

Загорьевская ул., д.4, Москва, 115598

Upadyshev M. T., Dr. Sci. (Agric.), Corresponding Member of the Russian Academy of Sciences, Senior Researcher, Head of Laboratory of Virology

4, Zagorevskaya str., Moscow, 115598

https://orcid.org/0000-0003-1182-0867ЧердаклиА. А.CherdakliA. A.

Чердакли А. А. – младший научный сотрудник, лаборатория репродуктивной биотехнологии

Загорьевская ул., д.4, Москва, 115598

Cherdakli A. A., Junior Researcher, Laboratory for Reproductive Biotechnology

4, Zagorevskaya str., Moscow, 115598

ФГБНУ «Всероссийский селекционно-технологический институт садоводства и питомниководства»РоссияAll-Russian Horticultural Institute for Breeding, Agrotechnology and NurseryRussian Federation202002082020034450Copyright © Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture», 20202020Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»Autonomous non-profit organization Editorial Board of journal «Horticulture and viticulture»https://www.sadivin.com/jour/about/submissions#copyrightNoticehttps://www.sadivin.com/jour/article/view/532

Плодовые и ягодные культуры поражаются различными вирусными болезнями, приводящими к снижению урожайности и качества продукции, в связи с чем актуальной задачей является повышение достоверности, чувствительности и производительности диагностики вирусов и других опасных патогенов. Решение этой задачи осуществляется путем внедрения новых методов и технологий молекулярной диагностики, причем основное внимание уделяется расшифровке нуклеотидных последовательностей путем секвенирования. Секвенирование дает детальную характеристику генома вируса и позволяет получить полноценную эпигеномную информацию. Методы генерационного секвенирования (NGS – next generation sequencing) обеспечивают параллельное тестирование на наличие всех вредоносных вирусов в одном образце, включая идентификацию с высокой степенью достоверности неспецифичных и новых вирусов при возможности использования различных видов образцов, например, пыльцы. Примером NGS является метод Illumina, основанный на секвенировании и биоинформатическом анализе коротких РНК. Современные секвенаторы могут генерировать от 4 миллионов до 20 миллиардов операций чтения за цикл с длиной считывания от 50 до 300 нуклеотидов. Применение высокопроизводительного секвенирования (HTS – High Throughput Sequencing) совместно с баркодированием позволяет проводить массовое генотипирование и давать характеристику вирусов, анализировать и устранять ошибки ПЦР при сохранении реального разнообразия библиотек генов, а также распознавать мутации в образцах. Новые методы секвенирования позволяют более глубоко изучить генетическое разнообразие штаммового состава вирусов плодовых и ягодных культур. Некоторые из недавно идентифицированных и инфицирующих плодовые культуры вирусов относятся к родам вирусов, ранее неизвестных для данных видов растений (например, Fabavirus, Luteovirus). Полное РНК-секвенирование использовалось для идентификации и характеристики вирусов винограда, яблони, груши, вишни. У возделываемых видов Prunus были идентифицированы 44 вируса. На яблоне обнаружен новый иларвирус – мозаика некроза яблони. Применение HTS для анализа вирусов плодовых и ягодных культур становится все более масштабным. При снижении стоимости секвенирования внедрение и валидация новых молекулярных методов позволит в ближайшее время применять их в диагностике вирусов органами службы государственного надзора.

Fruit and small fruit crops are aff ected by various viral diseases, leading to a decrease in yield and product quality, in connection with which the urgent task is to increase the reliability, sensitivity and productivity of diagnosing viruses and other dangerous pathogens. The solution to this problem is carried out by introducing new methods and technologies of molecular diagnostics, with the main attention being paid to decoding nucleotide sequences by sequencing. Sequencing provides a detailed description of the genome of the virus and allows one to get complete epigenomic information. Next generation sequencing (NGS) methods provide parallel testing for the presence of all malicious viruses in a single sample, including identifi cation with a high degree of certainty of non-specifi c and new viruses with the possibility of using diff erent types of samples, for example, pollen. An example of NGS is the Illumina method, based on sequencing and bioinformatics analysis of short RNAs. Modern sequencers can generate from 4 million to 20 billion reads per cycle with read lengths from 50 to 300 nucleotides. The use of high-throughput sequencing (HTS) in conjunction with barcoding allows mass genotyping and characterization of viruses, analysis and elimination of PCR errors while maintaining the real diversity of gene libraries, as well as recognition of mutations in samples. New sequencing methods allow a deeper study of the genetic diversity of the strain composition of the viruses of fruit and small fruit crops. Some of the recently identifi ed and infecting fruit crops belong to the genera of viruses previously unknown for these plant species (for example, Fabavirus, Luteovirus). Complete RNA sequencing was used to identify and characterize the viruses of grape, apple, pear, and cherry. In cultivated Prunus species, 44 viruses have been identifi ed. A new ilarvirus was discovered on the apple tree – the Apple necrotic mosaic virus. The use of HTS for the analysis of fruit and small fruit viruses is becoming increasingly widespread. With a decrease in the cost of sequencing, the introduction and validation of new molecular methods will make it possible in the near future to use them in the diagnosis of viruses by State surveillance authorities.

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The authors declare that there are no conflicts of interest present.