شناسایی متاژنومیکس چند گونه باکتری همزیست حشرات با استفاده از روش توالی یابی نسل جدید از استان آذربایجان شرقی

Metagenomics studies have made it possible to achieve the population composition of living organisms of any ecosystem with high accuracy at a lower cost and at high speed. Russian olive trees are found as a native tree of Azerbaijan in and around most of the orchards of this region and it is possible to act as a reservoir for beneficial and harmful microorganisms for nearby fruit trees. In order to study the microbial population on the foliage of Russian olive trees, native to East Azerbaijan, the whole genome sequence of Russian olive trees collected from Tabriz city green space was obtained by next-generation sequencing (NGS) using llumina 1.9 Novaseq 6000 platform. To extract total DNA, the leaves and soft branches were separated and completely powdered using liquid nitrogen without surface disinfection. For Next-generation sequencing, 100 μl of DNA solution at a concentration of 5 μg/μl, was sent in completely closed vials to Novogen company, China. Finally, the paired end sequences were received as 12 GB of raw data. In the first step, the quality of the sequences was evaluated using FastQC software. Then, to determine the composition of the microbial population on the Russian olive trees branches and leaves, the sequences were analyzed using MetaPhlan2 software. The results indicated that the complete genome of Buchnera aphidicola, which is an endosymbiotic bacteria associated with aphids, Candidatus Carsonella ruddii, which is an endosymbiotic bacteria associated with psyllids and Candidatus Sulcia muelleri which is an endosymbiotic bacteria associated with leafhoppers were respectively comprised 4.2%, 4% and 0.1% of the total microbial population on Russian olive trees. Therefore, the reference genome of these endosymbiotic bacteria was obtained from NCBI and was mapped on the primary raw sequence using Bowtie2 software. Sequences were then assembled and bacterial genomes were extracted as contigs. Finally, the complete genome of Buchnera aphidicola in 657623 bp size, Ca. Sulcia muelleri in 270516 bp size and Ca. Carsonella ruddii in 175756 bp size were derived from the raw sequences and recorded in the NCBI database for first time from Iran. In addition to the complete genome of these three endosymbiotic bacteria, partial genomes of other endosymbiotic bacteria including Regiella insecticola, Ca. Zinderia insecticola, Can. Portiera aleyrodidarum and Serratia symbiotica were also predicted within the raw sequences. Genomic characterization of insect endosymbiotic bacteria associated with Russian olive trees showed that insects or their larvae and eggs, especially aphids, were present on Russian olive foliage at the time of sampling and DNA of endosymbiotic bacteria has been extracted and sequenced along with Russian olive DNA. The results of this study indicated that the next-generation sequencing is a very useful method to determine the microbial population on tree foliage and other living environments.