METHODS AND RESULTS: The amplification of genomic DNA with 32 ISSR markers detected an average of 97.64% polymorphism while 35.15% and 51.08% polymorphism per population and geographical zone, respectively. Analysis of molecular variance revealed significant variation within population 75% and between population 25% whereas within region 84% and between region 16%. The Bidillali exposed greater number of locally common band i.e., NLCB (≤ 25%) = 25 and NLCB (≤ 50%) = 115 were shown by Cancaraki while the lowest was recorded as NLCB (≤ 25%) = 6 and NLCB (≤ 50%) = 72 for Roko and Maibergo, accordingly. The highest PhiPT value was noted between Roko and Katawa (0.405*) whereas Nei's genetic distance was maximum between Roko and Karu (0.124). Based on Nei's genetic distance, a radial phylogenetic tree was constructed that assembled the entire accessions into 3 major clusters for further confirmation unrooted NJ vs NNet split tree analysis based on uncorrected P distance exposed the similar result. Principal coordinate analysis showed variation as PC1 (15.04%) > PC2 (5.81%).
CONCLUSIONS: The current study leads to prompting the genetic improvement and future breeding program by maximum utilization and better conservation of existing accessions. The accessions under Cancaraki and Jatau are population documented for future breeding program due to their higher genetic divergence and homozygosity.
MATERIALS AND METHODS: The object of the study were samples of biological substrates (leukocyte mass, saliva, urine) taken from patients who underwent liver and kidney transplantation. Detection of CMV DNA was carried out by a real-time PCR using commercial diagnostic AmpliSense CMV-FL test systems (Central Research Institute for Epidemiology, Moscow, Russia). DNA extraction was performed using DNA-sorb AM and DNA-sorb V kits (Central Research Institute for Epidemiology) in accordance with manufacturer's manual. The quality of the prepared DNA library for sequencing was assessed by means of the QIAxcel Advanced System capillary gel electrophoresis system (QIAGEN, Germany). Alignment and assembly of nucleotide sequences were carried out using CLC Genomics Workbench 5.5 software (CLC bio, USA). The sequencing results were analyzed using BLAST of NCBI server.
RESULTS: CMV DNA samples were selected for genotyping. The two variable genes, UL55(gB) and UL73(gN), were used for CMV genotype determination, which was performed using NGS technology MiSeq sequencer (Illumina, USA). Based on the exploratory studies and analysis of literature sources, primers for genotyping on the UL55(gB) and UL73(gN) genes have been selected and the optimal conditions for the PCR reaction have been defined. The results of sequencing the UL55(gB) and UL73(gN) gene fragments of CMV clinical isolates from recipients of solid organs made it possible to determine the virus genotypes, among which gB2, gN4c, and gN4b were dominant. In some cases, association of two and three CMV genotypes has been revealed.
CONCLUSION: The application of the NGS technology for genotyping cytomegalovirus strains can become one of the main methods of CMV infection molecular epidemiology, as it allows for obtaining reliable results with a significant reduction in research time.