phylogenetic analysis

The chickpea (Cicer arietinum L.) is grown in more than 50 countries and is the third most important food legume in the world after beans and peas in terms of both cultivated area and total production. Chickpea is affected by a wide range of fungal and viral diseases that can cause economic losses. Among these diseases, fusarium wilts and root rot, caused by Fusarium oxysporum f.sp ciceris and F. solani, respectively Are the most important soil-brone pathogens affecting chickpea. These assays will provide plant pathologists a valuable tool to refine the control and management of diseases caused by Fusarium spesies in chickpea. The present study aimed to identify the Fusarium species associated with root rots and wilting of chickpea, as well as the pathogenic strains affecting the crop.
 

Fusarium species were isolated from roots of chickpea plants showing wilt and yellowing symptoms in different chickpea-growing regions of Kermanshah, Illam and Golstan provinces. Plant samples were cut into small pieces, washed under running water for 20 mins, sterilized with 5% sodium hypochlorite for 3 minutes, rinsed in sterile distilled water, and then placed on filter papers. Afterward, the pieces were transferred to petri dish containing potato dextrose agar media (PDA). All the plates were incubated at 25° C for 5-10 days. To determine pathogenicity, purified isolates of Fusarium species were used. Total DNA was extracted using a modified protocol previously described by Talbot et al.  Each fungal species was characterized morphologically and molecularly based on DNA sequence data for ITS-rDNA. The PCR sequencing products of the tested isolates were aligned with Clustal (ver. 2). The MEGA6 program was used for phylogenetic analysis. Aligned sequences were checked for quality and compared with deposited sequences in Gene-Bank, NCBI, using BLAST. 

Based on morphological characteristics and molecular data obtained from ITS-rDNA amplification, F. solani, F. acuminatum, F. equiseti, F. oxysporum f. sp. ciceris and Fusarium spp. were identified in the samples, among which F. oxysporum was the most dominant and common species and had the highest abundance. F. acuminatum and F. equiseti were mostly isolated from adult plants, while F. oxysporum and F. solani were isolated at all stages of growth, from seedling to podding. The phylogenetic tree, based on the comparison of ITS-rDNA sequences, classified the identified Fusarium species into four phylogenetic groups. The results showed that the use of ITS-rDNA sequencing can be used as a suitable complementary method for the identification of different Fusarium species.

Grapevines, as a vital agricultural product, face significant challenges from pests and diseases that adversely affect both the quality and quantity of the crop. Among these, viruses are particularly detrimental, with 86 different species identified in grapevines. The viruses associated with grapevine leafroll disease (GLD) are considered the most prevalent in vineyards. Specifically, six species of grapevine leafroll-associated viruses (GLRaVs), belonging to the family Closteroviridae, are linked to this disease. These viruses typically enter vineyards through infected cuttings and are disseminated by vectors, notably mealybugs. The symptoms of GLD manifest as downward rolling of leaf margins, leaf interveinal reddening, and leaf interveinal chlorosis across various grapevine varieties. These symptoms negatively impact the quality of the berries, ultimately affecting yield and marketability. The GLRaV-1 genome is composed of positive-strand RNA and contains ten open reading frames (ORFs), which play crucial roles in its biology and pathogenicity. In Iran, GLRaV-1 has been identified as one of the most widespread grapevine viruses, exhibiting high genetic diversity that complicates management and control efforts. This study focuses on the identification of GLRaV-1 and the phylogenetic analysis of its isolates in the vineyards of Razavi Khorasan province. By understanding the genetic relationships and variability of GLRaV-1 isolates, this research aims to provide insights which could be useful for designing effective management strategies and improving the overall health of grapevine in the region.

In the spring of 2020, a study was conducted in the Kashmar region of Khorasan Razavi province, focusing on grapevine plants exhibiting distinct symptoms of viral infection. A total of 70 samples were collected from affected plants. The RNA was extracted from these samples to facilitate the detection of grapevine leafroll-associated virus 1 (GLRaV-1). The initial identification process involved reverse transcription-polymerase chain reaction (RT-PCR). This method confirmed the presence of GLRaV-1 in 22 samples, marking a crucial step in understanding the viral infection's prevalence. To ensure the accuracy of the RT-PCR results, Sanger sequencing was used on the amplified fragments from three selected samples. This technique provided definitive confirmation of the viral identity by analyzing the nucleotide sequences, thereby validating the initial findings from the RT-PCR. Data analysis included several critical components. Nucleotide sequence similarity analysis was performed to compare the identified GLRaV-1 isolates against known sequences in databases, assessing their similarity and identifying potential genetic variants. Following this, multiple sequence alignments were conducted using MAFFT software, arranging the sequences to highlight similarities and differences among the isolates. The phylogenetic tree was constructed using the Maximum-Likelihood method with MEGA7 software.

The samples collected from black grape vineyards in Kashmar exhibited mild yellowing and downward rolling of leaf margins, symptoms commonly associated with increased anthocyanin levels in red grape varieties. In samples infected with GLRaV-1, a fragment of the minor capsid protein 2 (Cpm2) gene was successfully amplified, while no such fragments were detected in healthy plants. This indicates the presence of GLRaV-1, a virus recognized as one of the common pathogens affecting grapes across various regions of Iran. The GLRaV-1 is primarily transmitted through vectors or contaminated cuttings, which emphasizes the importance of monitoring and controlling these pathways to prevent outbreaks. Sequence comparisons revealed that nucleotide identity among Iranian isolates ranged from 84.2% to 100%, while identity with GenBank isolates varied between 77.6% and 87.7%. This high level of genetic diversity among the isolates may enhance the virus's adaptability to different grape varieties, posing challenges for management and control. The observed diversity at the genomic level was consistent with previously reported data regarding the sequence divergence between the two GLRaV-1 groups (Alabi et al., 2011; Sabella et al., 2018; Fan et al., 2015). Phylogenetic analysis further demonstrated that Iranian isolates of GLRaV-1 clustered into two main groups, indicating that the phylogenetic relationships are not strictly related to geographical origin. In previous studies, global isolates of GLRaV-1 have also been distinguished into two different groups based on the gene encoding the minor coat protein 2 (Alabi et al., 2011; Elci, 2019). This finding suggests that the spread of the virus may occur independently of the location, highlighting the need for comprehensive management strategies. To mitigate the spread of GLRaV-1, the use of virus-free plant materials is essential (Alabi et al., 2011; Bruisson et al., 2017). The genetic diversity of GLRaV-1 significantly influences the epidemiology of grapevine leafroll disease, underscoring the importance of producing virus-free plants. Continued research is crucial to understand the underlying causes and effects of this virus. Additionally, developing more sensitive and specific diagnostic tests, along with new control strategies, is vital for effectively managing GLRaV-1 and safeguarding grapevine health.

In spring and summer 2023, a survey of plant samples from green spaces and ornamental greenhouses, along with adjacent weeds in Yazd City, showed symptoms likely linked to phytoplasma disease. Notable symptoms included stem flattening, phyllody, and yellowing observed in Angelica archangelica, Ligustrum sp., Mirabilis jalapa, and Hibiscus cannabinus. Five samples from each plant were sent to the Plant Virology Research Center at Shiraz University for DNA extraction via the CTAB method. Polymerase chain reaction (PCR) was conducted using general primers (P1/P7) and nested primers (R16MF2/R16MR2) specific for phytoplasma. As expected, fragments of 1800 bp and 1250 bp were amplified with the general and nested primers, respectively. These fragments were excised from the gel, purified, and sent to Sinohe Company for sequencing. Comparison of the sequences with those in the NCBI database using BLAST showed that the phytoplasmas from Angelica archangelica, Ligustrum sp., Mirabilis jalapa, and Hibiscus cannabinus were closely related to Indian (MH393563.1; host: Datura stramonium; identity: 98.94%), Turkish (HE649495.1; host: Ligustrum sp.; identity: 99.24%), Ugandan (HE649495.1; host: Hibiscus rosae; identity: 99.14%), and Iranian (KX685880.1; host: Eucalyptus sp.; identity: 98.64%) isolates. A phylogenetic tree was created to compare Iranian isolates with similar global isolates using MEGA 8.0 software.

 Vinca minor or lesser periwinkle is a perennial, herbaceous and creeping plant belonging to the genus Vinca and the family Apocynaceae. In addition to being an ornamental and cover plant, V. minor is a valuable herbal plant in traditional medicine, and it also acts as a natural source for the industrial manufacture of brain blood flow stimulants. Periwinkle contains more than 150  alkaloids, which have been isolated from the aerial parts and roots of the plant, so far (Proksa et al.,1988), consisting of vincaminorine, vincaminoreine, minovine, minovincine, vincamine, which has antihypoxic and neuroprotective properties as well as modulatory effects on brain circulation and neuronal homeostasis (Farahanikia et al., 2011). Vinca mosaic virus (VMV), belonging to the genus Potyvirus and the family Potyviridae, causes severe to mild mosaic symptoms, yellowing, blistering and leaf curl in the leaves of the periwinkle plant. In this study, the molecular and phylogenetic characteristics of two isolates of VMV were investigated via analysis of p1 and cp genes.

 Virus-like symptoms, such as mosaic and mottling, yellowing, blistering and leaf curling were found on the leaves of V. minor plants in the pardis campus of Ferdowsi University of Mashhad, Razavi Khorasan, Iran, during a survey in September 2019. Total RNA was extracted from the leaves with the SV Total RNA Isolation Kit to determine the causal agent(s) (Promega, USA). Deep sequencing was carried out on the samples by Macrogen Company in South Korea. Analysis of the results with CLC Genomics Workbench v.12.0.3 software showed that these plants were infected with two isolates of VMV. The cp and p1 gene sequences of VMV isolates were determined. Clustal Omega software was used to compare multiple sequence alignments among sequences and determine the percentage of identities at the nucleotide and amino acid levels. To determine phylogenetic relationships and evolutionary origin of Iranian VMV isolates, phylogenetic tree was drawn based on nucleotide and amino acid sequences of cp and p1 genes using MEGA 7 software and Maximum-Likelihood (ML) method with 1000 replications (Bootstrap). The SDT software (v.1.2) and Muscle sequencing were used to plot the similarity matrix among the isolates at the nucleotide and amino acid levels.

 The identities between the cp gene of two Iranian VMV isolates (VMV-IR-1 and VMV-IR-2) at the nucleotide (nt) and amino acid (aa) levels were 93.11% and 97.00% respectively. For the p1 gene, the obtained results showed 68.35% identity at the nt level and 97.00% at the aa level. The highest percent of nt identity of the VMV cp with other potyviruses in the GenBank was with ASV1 (asparagus virus 1) (75.00% with VMV-IR-1 and 73.82% with VMV-IR-2) VMV-IR-1 and VMV-IR-2 showed the highest aa identity with VDMV (vanilla distortion mosaic virus) (70.39%). The highest nt and aa identity of p1 gene of VMV isolates was with CatMV (catharanthus mosaic virus) (52.29% VMV-IR-1 and 51.31% with VMV-IR-2) and ZTMV (zucchini tigre mosaic virus) (28.70%), respectively. Protected motifs were also identified in the cp and p1 gene sequences. Dendrograms obtained from phylogenetic analysis based on nt and aa acid sequences of these genes placed two Iranian VMV isolates, along with other species of potyviruses in the GenBank, viruses in two and three separate groups respectively. Based on cp gene sequences, at the aa level Iranian VMV isolates is most closely related to yam mosaic virus (YMV) virus, and at the nt level with the closest viruses are include sweet potato virus 2 (SPV2), sugarcane mosaic virus (SCMV), onion yellow dwarf virus (OYDV) and johnsongrass mosaic virus (JGMV) in the same group. Based on the p1 gene sequences, VMV isolates at the nt level were most closely related to the JGMV virus and at the aa level were most closely related to the YMV and celery mosaic virus (CeMV) viruses.

 In this study, for the first time, the molecular properties of VMV isolates were determined based on the cp and p1 genes, and the phylogenetic position of two Iranian VMV isolates was drown. The results showed that the cp gene can be used for taxonomic purposes. Considering the medicinal and ornamental properties of periwinkle and effect of the viruses on its properties, special attention should be paid to viral diseases of this plant.

Heavy metals are a major environmental issue, severely affecting plant growth. Phytoremediation, which involves using plants to extract, separate, or eliminate pollutants, offers a practical, cost-efficient, and environmentally friendly solution. Several protein families, such as the Metal Tolerant Protein (MTP) family, play a key role in the phytoremediation of heavy metals in plants like Euphrates poplar. With advances in genomics, especially the sequencing of various organisms' genomes and data availability from public databases, researchers can now identify relevant genes and their functions. This study aims to explore the genetic diversity and relationships among the members of the MTP family in Euphrates poplar. 

Members of the MTP family in the Euphrates poplar genome were identified using homologous protein sequences of the MTP family from two model plants of Arabidopsis and rice using BlastP in the NCBI database. Sequence alignment was performed, followed by phylogenetic tree construction using the Neighbor-Joining method. Various software tools were used to predict the physicochemical properties and protein localization of MTP members and to assess the selection pressure, exon-intron distribution patterns, and specific motifs within gene family members. Gene ontology was performed at three levels: biological processes, molecular function, and cellular components, including identifying unique simple sequence repeat (SSR) markers. The microRNA network regulating gene expression for each member was also identified and, the communication network between genes and MicroRNA was drawn. Gene expression was evaluated in various Euphrates poplar tissues, and RNA-seq data were normalized and analyzed for expression under 200 mM salt stress. Heat map visualizations were generated using Mev4.0 software. 

A total of 26 MTP gene family members were identified in the P. euphratica genome through bioinformatics analysis. Phylogenetic analysis classified these into three major groups (Mn-MTP, Zn/Fe-MTP, and Zn-MTP) and seven subgroups. The encoded proteins were found to be associated with the plasma membrane and the transfer of metals to the vacuole, supporting their role in detoxifying heavy metals. Negative selection pressure was observed across all members, indicating the effect of gene divergence. Gene structure analysis showed the highest integration within groups and the lowest integration between groups. Out of the 15 gene sequences analyzed, 52 SSR markers were identified for marker-assisted selection. MTP11.2, MTP3.2, MTP12, and MTP7 genes exhibited the highest expression levels across various tissues. The MTP1.1 gene showed the most significant increase in gene expression 48 hours after salt application, indicating a potential role in plant responses to salinity and heavy metal stress. 

The presence of repeated gene sequences reflects the plant's high demand for the specific function of these genes and their differential expression during various growth stages and stress conditions. Euphrates poplar possesses numerous MTP family members, which contribute to removing metals from the soil and are linked to increased growth and wood production. This research demonstrates the utility of bioinformatics data in facilitating the identification of metal-resistant populations more efficiently and cost-effectively.

The aim of this investigation was to examine the alterations in the nucleotide sequence of CAPN2 and CAPN3 genes among a population consisting of nine Japanese quails. The study also aimed at determining the genetic diversity within these populations, as well as establishing their phylogeny relationship with other species. To achieve these goals, data on nucleotide sequences for both CAPN2 and CAPN3 were extracted from 62 different species available in World Gene Bank (NCBI), alongside that found for Japanese quail samples and analyzed using DnaSP software coupled with MEGA program techniques. The results showed that CAPN2 gene polymorphism was more than the CAPN3 gene in Japanese quail and as a result, the conserved regions in CAPN3 gene were more compared to the CAPN2 gene. The numerical value of dN/dS ratio for Japanese quail was estimated in the CAPN3 gene (0.94) and in the CAPN2 gene 0.68, which respectively indicate neutral selection and purifying selection on these two genes during evolution. The results of the neutral evolution test (Tajima's D) for CAPN2 and CAPN3 genes in all the discussed species were positive and more than one, which indicates positive selection for these genes in all the studied species. The findings of this investigation can aid in identifying the areas within CAPN2 and CAPN3 genes linked to distinct phenotypic traits seen in the examined species. Analysis of phylogeny revealed that these two genes have displayed varying evolutionary responses along different studied species,

Grapevine virus A (GVA) belongs to Vitivirus genus and is one of the most important causes of wood rugose in grapes. During the years 2019 to 2021, a total of 79 leaf samples showing symptoms of leaf yellowing and reddening from vineyards of Khorasan Razavi Province (including Bardaskan, Kashmar, Khalilabad, and Mohammadiyeh) were collected. The initial infection of the samples to GVA was directly checked using an ELISA test, and the final confirmation of results was performed by RT-PCR with specific primers. Total RNA extraction was performed from scraped bark of young stems using CTAB method. GVA was detected in 14 samples through the ELISA test, and a fragment of 238 base pairs from the p10 protein was amplified in these 14 samples using RT-PCR. The amplified products from four samples were ligated into the pTG19-T vector, and the recombinant plasmids were sequenced bidirectionally. The obtained sequences were compared with the other GVA sequences in GenBank using BlastN tool and the nucleotide similarity was 85.7-95%. The phylogenetic tree based on p10 protein showed there are four main groups, and Khorasan Razavi isolates classified within the fourth group. Comparison of p10-protein sequence of Iranian isolates with other isolates showed there is a conserved region in N-terminal region, which includes an arginine-rich motif followed by a zinc-finger motif. There have been many studies on other viruses of grape in this province, but this is the first study on ithe phylogenyof GVA isolates in the vineyards of Khorasan Razavi province.

The Saffron Latent Virus (SaLV) is new species in the Potyvirus genus, which has been recently characterized from Iran. Even though most of the symptoms on infected plants remain hidden, it causes considerable damage to saffron fields. There is no data available on the phylogenetic relationship among different strains of this virus based on its coat protein, despite its high incidence and economic significance. To address this, we collected 152 samples of saffron leaves, flowers, and corms from fields in South Khorasan and Razavi Khorasan provinces, both with and without symptoms like stunting, yellowing, and twisting. After extracting RNA, the presence of SaLV was examined using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers targeting the coat protein. The findings revealed that 92 out of 152 samples (60%) were infected with the virus. Subsequently, seven representative samples, each from a selected region were sequenced. Phylogenetic analysis using MEGAX software showed that the virus strains formed two distinct groups, and the strains studied were placed in the second group. Interestingly, strains from the same geographical region and product were distributed among different groups, suggesting that geographical separation and host type did not significantly impact the viral diversity. This study is the first in detecting of SaLV in saffron flowers and corms and pinpointing the diversity of its new strains based on the coat protein in Iran.

AT-hook motif nuclear localization (AHL) proteins play a critical role in plant biological processes, and it is highly conserved in terrestrial plants. In Arabidopsis thaliana this gene family data is available. We used this data to detect and interpret the AHL gene family in the Cardamine hirsuta. In Silico analysis was launched and initially 29 AHL genes were detected. Phylogenetic analysis afterward exhibits that AHL in C. hirsuta has been grouped into two clades, clade B with 3-5 introns, and clade A intron-free similar to A. thaliana. According to the combination of AT-hook motif(s) and PPC domain, AHL proteins are divided into three subclades (sub-clades-I/-II/-III). Subclades-I are related to Clade-A, while subclades-II and subclades-III are related to Clade-B. The motif analysis exhibits that the three genes including AHL1, AHL10, and AHL11, although had the AT-hook motifs, lacked the PCC domain, and therefore the actual number of AHL family genes decreased to 26. Looking at duplication events in the AHL gene family in C. hirsuta, 16 homologous genes were found due to segmental duplication, and no tandem sequences were observed in these genes. In-depth inspection showed that segment genomic duplication events could cause the expansion of these genes. Examining ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates, i.e. Ka/Ks, in 5 pairs of homologous genes showed those with Ka/Ks < 1 were exposed to the adverse selection. These findings reveal a possible evolutionary relationship for the AHL gene family in C. hirsuta, providing helpful information for future analysis to investigate their biological functions.

In the visits of the spring, summer, and autumn of 2021 and 2022 to the orchards of stone fruit trees in Golestan province, suspected symptoms of phytoplasma disease such as yellowness, deterioration, reddening, and small leaves were observed, and 28 samples of leaves from different hosts of stone fruit trees were collected. P1/P7 general primers were used for general PCR, and R16F2n/R16R2 primers were used for nested PCR. Infected samples amplified 1800 bp fragments in general PCR and 1250 bp fragment in nested PCR. Out of the 28 samples collected, 3 samples of peach trees had a band in the target area. The nested PCR product after sequencing was registered in the World Gene Bank with accession numbers OQ945143 and OP055811. At the same time, transplanting was done on the benchmark plant, Periwinkle, with the aim of gene storage and observation of phytoplasma symptoms. The comparison of the obtained sequences with the sequences available on the NCBI by the BLAST showed the most similarity to Aster yellows phytoplasmas, and the phylogenetic analysis of the target sequence with the maximum likelihood estimation algorithm in comparison with the different phytoplasma subgroups reported in Iran also showed the results confirmed the blast. Also, the comparison of patterns obtained from virtual RFLP identified the most similar reference pattern of the 16SrI group, subtype AD (GenBank accession: DQ286577), with a similarity coefficient of 0.77, which indicates that this strain may represent a new group. The results of this study confirm the presence of phytoplasma in peach trees in Golestan province for the first time and indicate the presence of a phytoplasma species close to Candidatus phytoplasma asteris in Golestan province.

Saffron is the most expensive spice in the world. The economic value of saffron is due to the existence of apocarotenoids in its stigma. Therefore, the isolation and characterization of genes involving in apocarotenoids metabolism is on particular importance. In this research, beacause the importance of CsUGT gene in crocin biosynthesis, it was isolated, cloned the E.coli strain DH5α. The full length gene was sequenced and registered in the NCBI. In order to characterize CsUGT gene, first the protein sequence was obtained, and then the physical and chemical characteristics and physiology of the CsUGT protein were analyzed by Protparam, SOPMA, ProtScale, Pfam, ProtComp, SignalP, TMHMM and ChloroP servers and tools. Also, using the Swiss-Model server, the 3D structure of this protein was investigated and Ramachandran diagram was drawn to validate the 3D drawn model structure. According to the results, CsUGT protein with 462 amino acids has the conserved sequence of glycosyltransferase family proteins and was identified as a polar protein, stable at high temperatures and without hydrophobic domain. CsUGT protein has no peptide signal or binding signals and has a cytoplasmic location. This research made it possible to isolate the CsGTS gene of saffron and optimized the conditions of its transfer into a vector. In addition, the results of CsUGT protein structure analysis provide the basis for future functional studies and can also provide valuable information regarding the behavior and reaction of this enzyme in the synthesis of saffron apocarotenoids. In addition, these results can be useful in the future programs of Iranian saffron genetic modification.

DNA Mitochondria is one of the most commonly molecular markers for phylogenetic studies in animals due to its simple genome structure, which presents an ideal model to study evolution and genetic similarity. The aim of the present study was to investigate the divergence and percentage of genetic similarity along with the phylogenetic analysis of the eight species of wild and domestic goat based on the complete sequence of the mitochondrial genome and the separate sequences of 13 protein-coding genes for each genome.

In the present study, complete mitochondrial genome sequences along with separate sequences of 13 protein-coding genes per each genome from seven wild species of goat including Markhor (C. falcoeri), Capra ibex, Capra nubiana, Capra pyrenaica, Capra sibirica, Capra caucasica, Capra aegagrus, and domesticated species of goat (Capra hircus) were retrieved from NCBI database and compared to each other. Mitochondrial genomes and genes alignment were accomplished by the MegAlign module of DNASTAR software and compared by the Clustal W method. The Sequence Distances sub-section of the MegAlign module of DNASTAR also was used for the analysis of complete genome and gene sequences divergence and similarity percentage. For phylogenetic analysis, complete mitochondrial genomes and protein-coding genes’ sequences were aligned using MEGA7 software.

Based on the analysis, it was found that the highest similarity between the nucleotide sequences of the complete genome (99.50) of domestic goat (Capra hircus) and wild Capra aegagrus goat and the lowest percentage of similarity (93.79) between the nucleotide sequences of the complete genome of the breed Capra nubiana) with wild goat Capra sibirica goat. Also, based on the results of the phylogenetic tree, it was found that domestic goat breeds (Capra hircus) and wild Capra aegagrus goat are divided into one group and wild Capra ibex and Capra pyrenaica breeds are divided into another distinct cluster.

In this study, all the results obtained from complete sequences of the mitochondrial genome analysis are consistent with the results obtained from the sequence of 13 protein coding genes per each genome, which indicates the correct clustering of wild and domestic goat breeds. Therefore, mitochondrial genome sequences could be used as a suitable genetic marker for accurate phylogenetic analysis and clustering of different species of sheep.

Squalane is an unsaturated triterpene that has wide applications in pharmaceuticals. In this research, the production of squalene and its bioinformatic analysis in four species of unicellular and multicellular eukaryotes and prokaryotes were investigated in order to determine the difference of this gene in eukaryotes and prokaryotes. The results of phylogenetic analysis showed that algae as multicellular eukaryotes, yeast as single-celled eukaryote and bacteria as single-celled prokaryote were placed in one group and plants were placed in a separate group. GC percentage of SQS protein was evaluated by GC Content Calculator, as well as aliphatic index and instability index by protparam. The results showed that the SQS gene is in a range from unstable to stable. The analysis of the presence of signal sequences and the analysis of the detection of the final location of the protein showed that the possibility of transferring the SQS protein to the mitochondria, chloroplast and secretory pathway is very low and it is not among the signal proteins. It was also found in Gymnema sylvestre that this protein has three protected domains. The comparison of the secondary structure of the protein confirmed the existence of alpha sheets. 3D modeling of this protein in plant was done by homology modeling method and using Swiss Model database after selecting a suitable model with high similarity which was extracted from PDB database. In order to validate the structure of the drawn three-dimensional model and stereochemical analysis, the Ramachandran diagram was drawn and the dihedral angles were calculated. The results of structural quality evaluation showed that the proposed models have good quality and stability. The study of the protein structure can help to understand the function of the protein, and studying the details of its structure can be useful in the studies of the active site of the protein and docking.

Iris spp. is reported to be affected by several viruses in the family Potyviridae including iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV), iris fulva mosaic virus,  bean yellow mosaic virus (BYMV), turnip mosaic virus (TuMV), ornithogalum mosaic virus (OrMV), narcissus latent virus (NLV), butterfly flower mosaic virus (BFMV), and gladiolus mosaic virus (tentative name). Narcissus latent virus (NLV) is a member of the genus Macluravirus in the family Potyviridae. It has non-enveloped flexuous filamentous virions of 657 nm long and 13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 8,000 nt long. NLV is distributed widely throughout the major planting areas of Japan, New Zealand, and European countries. It is one of the most common viruses infecting narcissus, iris, gladiolus, and nerine, causing significant yield losses and quality deterioration in their bulbs and flowers. Due to the presence of asymptomatic infection of NLV in iris and narcissus, the relevance of its infection in host plants may be severely underrated. As Khorasan Razavi province is one of the major producing areas of ornamental plants in Iran, identification of this virus is a concern. In this study, we attempted to identify NLV infecting iris plants and compare Iranian NLV isolates with other sequences from different geographical regions to provide the first detailed information of phylogenetic characterization of this virus in Iran.

Iris leaf samples showing virus-like symptoms of leaf chlorosis and mosaic were collected from field-grown plants in Khorasan Razavi province. Total RNA was extracted from the field samples using Promega SV Total RNA Isolation Kit (USA). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using specific primer pair CPU-F (5΄-CATTACACCCGACCTGGAACT-3΄) and CPU-R (5΄-CCATTTCAGGGCATTGGAGGA-3΄), which were designed to amplify a 1066 bp fragment of the 3΄-region of NLV genome (encompassing partial NIb (25 nt), complete CP (894 nt), and partial 3'UTR (147 nt)). PCR products and DNA ladder were separated by agarose gel electrophoresis, visualized using DNA Green viewer staining, and photographed with ultraviolet-illumination. Amplified fragments of the expected size were purified, cloned into pTG19-T vector and bi-directionally sequenced. Obtained sequences were phylogenetically compared with the corresponding isolates available in the GenBank after multiple alignments. The phylogenetic tree was constructed based on the nucleotide sequences of the CP-UTR using the neighbor-joining method by MEGA11.

Amplification product (1066 bp) was obtained from five infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, and interveinal chlorosis. Three selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to NLV. Three selected isolates which are referred to as IR, IR2, and IR3 were deposited in GenBank. The previously identified and conserved amino acid sequence motifs described in CP of macluraviruses were present in Iranian CP sequences. The phylogenetic tree placed the NLV sequences into two distinct phylogroups I and II; the Iranian isolates clustered together with isolates from Poland, New Zealand, and United Kingdom into group II. Phylogenetic analysis showed that Iranian isolates shared 77.47 to 98.12% nucleotide sequence identity and 77.70-99.34% amino acid sequence identity with other isolates of NLV. Also, identity of these three isolates in the nucleotide and amino acid levels ranged between 97 to 97.84% and 97.38 to 99.02%, with each other, respectively. Iranian isolates showed the highest nucleotide sequence identity with NLV5_1 isolate (JX270766) from Poland (between 97.65 to 98.12 %) and the lowest with NLV3 isolate (JX270762) from Poland (between 77.47 to 77.95 %).

NLV is a major constraint to iris and narcissus production worldwide. The phylogenetic analysis showed a low correlation between genetic and geographic distances which further emphasizing the importance of the exchange and use of virus-free propagating organs in preventing the dissemination of this virus. It seems that contaminated vegetative organs from some European countries (e.g. Netherlands), which are the major producer and the largest exporters of flowers and ornamentals in the world, can play a significant role in the worldwide distribution of the virus. Identification and the use of more isolates are recommended for a better understanding of the genetic structure and variation of NLV populations on a large geographical scale. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran.