Modern methods of molecular diagnostics and study the genetic diversity of fruit and small fruit crops viruses based on sequencing

Abstract

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.