بررسی کارایی نرم افزارهای متاژنومیک در شناسایی تنوع گونه‏ های میکروبی اندوفیت در گیاه سنجد

Introduction
One of the most important factors for the sustainability of any type of ecosystem is the biodiversity and natural diversity of the organisms of that ecosystem. Therefore, recognizing the species’ diversity within each ecosystem helps preserve that environment. Endophytic microorganisms are the most unknown species in any ecosystem because these microorganisms are inside other living species, so they are usually overlooked by biologists unless they cause a specific disease or disorder in their host. On the other hand, because most of these microorganisms are uncultivable, it is impossible to identify them through conventional methods.
With the development of the next generation of sequencing (NGS), extracting total DNA from the host is only done once without further repetition of the process in order to sequence all its genomic content, so the sequence of associated species or its endophytes are also found. Metagenomics has been used for microbiota analysis within different ecosystems. However, few reports for its use to identify plant endophytic bacteria are available. Recently, in most Russian Olive trees (Elaeganus angustifolia L.) in and around the city of Tabriz, there have been very clear signs of the leaflet, dwarfing, and rosseting. The cause of this infection has been associated with phytoplasma disease. However, the type and severity of the symptoms indicated the possibility of co-infection with other phytoplasma or other plant pathogens, necessitating a careful study the pathogens in the plant. Therefore, the present study was conducted to identify the genomic content of Russian olive trees infected with phytoplasma disease in the Tabriz region.

Methodology
To evaluate the efficiency of NGS (Next Generation Sequence) for species diversity, the total-DNA of selected Russian olive trees in Tabriz city was extracted and extracted DNAs were stored at -20°C for further use after dissolving in distilled sterile water. Three different DNA samples were prepared for NGS analysis. Each sample consisted of DNA of about 15 different trees. To ensure the removal of accidental microorganisms or surface contaminants, all samples were thoroughly rinsed with sterile distilled water and surface disinfected with alcohol before DNA extraction. The quantity and quality of obtained DNA, with spectrophotometer (Genway-6850 model) and electrophoresis in 1% agarose gel were evaluated. Samples were then sent to Novagen Co. for NGS sequencing. Each sample sent was about 100μL with a concentration of 80 ng/μL. The samples were used by the Illumina 1.9 Novaseq 6000 platform to produce paired-end reads. The requested data volume was set to 12 GB. After receiving NGS data, CLC workbench and Fastqc software were used for the initial evaluation of readings. In the next step, using Metaphylan2 and Kraken software, the genome abundance of each microbial phylum (fungal, bacterial and protozoan) in the data was evaluated. Then, using Bowti2 v2.4.2 software, all readings obtained were aligned with the reference sequences in the NCBI genebank.

Results
Based on the output of CLC workbench and Fastqc software, the quality of data received from Novagen Company was appropriate and acceptable. Data analysis showed that there were a total of 45 million READs inside the submitted file, the reading fragment length was 150bp and the GC% of the total readings in the three submitted samples was between 31-39%. The Phred Score for the readings was about 36 for the whole data, which indicates the good quality of the reads (above 20 is considered acceptable). Sequence Duplication Levels were also obtained very low, which indicates that the reads are appropriate and the data is reliable.
The results obtained from Metaphylan2 and Kraken software showed that the inside of the Russian Olive plant is rich in the genomic content of various bacterial, fungal, and protozoan species and so on. Out of 45 million readings, about half a million reads (1.12% of the total) were classified into different microbial phyla and the remaining 98.88% belonged to the host genome (Russian Olive). Only the species that were found in all three submitted samples are listed. The genomes of more than nine prokaryotes within the Russian Olive trees have been identified. With the exception of one species of archaea, the rest belonged mainly to gram-negative bacteria of the Proteobacteria group. Most of the bacterial genomes identified inside Russian Olive belonged to Mollicutes and Phytoplasma species, which, contrary to expectations, did not include only one species, and the genomes of four different species of Phytoplasma were observed inside the samples.
In the analysis of viral genomes, the presence of the genomes of three types of viruses and one type of satellite virus inside the examined samples was confirmed. With the exception of one virus that was identified as an entomopathogen, the rest were plant pathogens. Bombyx mori nucleopolyhedrovirus, which consisted of a large part of the viral genome, is a major pathogen of insects and has been used in the biological control of several species of insects to date.
In the study of eukaryotic genomes, the presence of the genomes of yeast fungi of the saccharomycete class and protozoa from the group of Apicomplexa and Amoebae in the samples was confirmed. One of the identified fungi (Eremothecium sp.) belonged to a genus that mainly plant pathogenic and some of its species cause economic damage to plants such as beans and cotton, and other species (Schizosaccharomyces sp.) was a Yeast which has been found in various environments so far. One of the identified protozoa (Eimeria tenella) has been reported to be a pathogen of birds and the other species belonged to the genus (Dictyostelium sp.) which is a type of amoeba whose species have already been identified in plant debris in different countries.
According to these reports, there was no doubt that Iranian Russian Olive trees are one of the main but overlooked hosts for phytoplasma diseases and some other diseases. Therefore, identifying the internal microbiome of Russian olive trees in the region seemed interesting and necessary. Identifying every single infection inside a tree or any other environment is very time-consuming and sometimes impossible because there is no mindset about the number and type of microorganisms inside it. Recently, with the development and completion of NGS-based metagenomic experiments, it has become possible to obtain an overview of potential organisms within an ecosystem without spending much time and money.

Conclusion
Identifying each species in an ecosystem and obtaining genomic information from each is a very long and costly task that was impossible to imagine for several years. However, the present study, as an example, shows that the NGS technique can easily and with the least cost and time can obtain valuable information from unknown ecosystems that were previously impossible or very difficult to obtain. This study not only proved that Russian Olive trees in northwestern Iran are infected with more than one phytoplasma, but also the existence of various types of other beneficial or harmful microbes in them was proved for the first time. Knowing about the infection of Russian Olive trees to several pathogens can help plant protection officials and other experts to make better decisions about future development or removal of these trees in the area in future management plants.

Keywords: Biodiversity, Metagenomics, NGS, Metaphylan2