مجله بیوتکنولوژی کشاورزی
سال پانزدهم شماره 2 (پیاپی 51، تابستان 1402)

Due to adaptation to environmental conditions, different ecotypes of native goat can be seen in different geographical regions of the country, which are very diverse in terms of appearance and production characteristics. So far, no comprehensive study has been done at the whole genome level to identify the genetic diversity of native goats. Therefore, the aim of this study is to identify the genomic characteristics of these native reserves in order to organize appropriate programs for their exploitation and protection.

In this study, the whole genome sequence of 36 Iranian native goats were downloaded from the NCBI database and analyzed. Whole genome sequencing of the studied data was done by Illumina company and Hiseq2500 and Hiseq2000 sequencing machines. Quality control of raw sequence data was done by FastQC program. The BWA-MEM algorithm applied in the BWA software package was used to align the data sequence with the goat reference genome (ARS1, GCF_001704415.1). The alignment outputs with the reference genome were processed in two steps, including realignment around small deletions and insertions and base quality score recalibration using GATK program.The mean depth for the alignment output with the reference genome was calculated using the depth command applied in the samtools software. Single nucleotide polymorphisms (SNPs) were identified by the UnifiedGenotyper tool used in the GATK program. The values of nucleotide diversity and genomic inbreeding coefficient based on homozygous SNPs for each individual were calculated using the het command used in the VCFtools program.

The mean depth of the used data in this study was 11.25 X. The average number of single nucleotide polymorphisms in the 36 Iranian native goat genomes was 7495554. The genomic inbreeding coefficient values in different ecotypes of Iranian native goat varied from -0.01 to 0.4. The lowest genomic inbreeding coefficient value was observed in the Hamedan native goat genome (-0.01) and the highest genomic inbreeding coefficient value was observed in the Balochi goat ecotype genome of Sistan and Baluchistan. Also, the average of percentage observed and expected of heterozygosity values was calculated for single nucleotide polymorphisms in the o Iranian native goat ecotype genomes varied from 10.27 to 17.75 and from 17.33 to 17.57.

This research shows an important insight about the structural diversity of the Iranian native goat genomes, which have not been evaluated genetically so far. The results obtained from this research can be used in the design of conservation and breeding programs for Iranian native goats.

Natural and artificial selections change the allele frequencies among populations and consequently leave detectable patterns on the genome during domestication. A little information about the differences of genetic structures of Iranian native breeds that result in these selective pressures are available. Therefore, in this study, we examined genetic diversity using four native horse breeds including Turkmen (29 samples), Caspian (21 samples), Kurdish (67 samples) and Persian Arabian (52 samples).

For this purpose, three methods including Principal Component Analysis (PCA), neighbor joining (NJ) and admixture were investigated. In addition, with the integrating of three different selection of signature methods, including TajimaD, nucleotide divergency (pi) and integrated haplotype homozygosity score could identify the selection traces in these breeds. To integrate the different results of these three methods, De-correlated composite of multiple signals framework was used.

All the methods used to examine the genetic structure well separated these four breeds and showed a pattern related to geographical origin for their genetic diversity. By combining different methods of identifying the selection signatures, many genes affected by the selection pressure were identified in all four breeds. These genes were associated with specific GO terms and QTL, in which 16 shared candidate genes among all four breeds were identified as suggested candidates for selection. Additionally, 11 different QTL types was identified in all four studied breeds, which was divided into the category of traits related to adaptation, fitness through genetic disorders or morphological and behavioral traits.

Overall, the results of this study can help better understand the process of natural and artificial selections in Iranian horses. In addition, this research has helped to improve our understanding about the genetic differences between studied Iranian horse breeds, which can help in finding the best solution to preserve and improve genetic diversity.

Bovine ephemeral fever (BEF) is an arthropod-borne viral disease of cattle and water buffalos which causes considerable economic and commercial losses in the cattle industry. The G glycoprotein of bovine ephemeral fever virus (BEFV) has been identified as a plausible candidate to product recombinant vaccines against this disease. The aim of this study was to investigate the immunogenicity of a eukaryotic plasmid expressing the G1 epitope of bovine ephemeral fever virus G glycoprotein as a possible DNA vaccine in mice.

At first, a eukaryotic plasmid expressing the G1 epitope of bovine ephemeral fever virus G glycoprotein was constructed and designated as pEGFPN1-G1. After transfection of the pEGFPN1-G1 recombinant construct to human embryonic kidney 293 (HEK 293) cells, the expression of G1 protein was confirmed by indirect immunofluorescent staining (IFA). Immunization experiments were intramuscularly carried out by inoculating 6-8-week-old female mice in four groups with five repeats, including the pEGFPN1-G1 recombinant construct, bovine ephemeral fever-inactivated vaccine, pEGFP-N1 plasmid alone, and phosphate-buffered saline (PBS) (1X) three times with 2-week intervals. Fourteen days after the last immunization, the animals were bled and the resulting sera were tested for anti-G1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralisation (VN) test. The optical density values of all the serum samples obtained from the ELISA assay were statistically analyzed in the form of a completely randomized design with four treatments and five replications using SAS software.

Immunoblotting analysis and statistical analysis of serum samples obtained from the ELISA assay showed that the pEGFPN1-G1 recombinant construct was able to elicit specific antibodies against G1 antigen of bovine ephemeral fever virus in mice. However, the serum of mice inoculated with this plasmid could not neutralize the replicating bovine ephemeral fever virus in virus neutralization assay.

Since the pEGFPN1-G1 recombinant construct had the ability to produce specific antibodies against the G1 antigen of bovine ephemeral fever virus, the results of this study can be a step towards the development of new vaccines for bovine ephemeral fever in the future.

This study was conducted to evaluate the genetic diversity of tomato accessions selected from the core collection of National Plant Gene Bank of Iran (NPGBI) based on a comparative assessment of SSR and ISSR markers. Genetic diversity of species and cultivars is necessary to increase productivity and production, and if diversity is reduced, species and cultivars are in danger of extinction. Due to the importance of tomato as the second most consumed crop among vegetables, it is necessary to study it. Other objectives of this study are to compare the performance of SSR and ISSR markers in differentiating tomato genotypes and to find primers with the highest polymorphism.

In this study, genetic diversity among and within tomato accessions collected from 8 geographic regions of Iran and 22 countries from the world were evaluated using 13 ISSR and 5 SSR primers.

The number of polymorphic alleles and the average of resolving power and marker index were related to the ISSR marker. However, the highest values of PIC for studied markers were obtained in the SSR primers. The molecular analysis of variance showed that both markers were suitable for the evaluation of diversity among species. Also, genetic diversity among the species was higher than within the species. The highest values of genetic diversity features were obtained in SSR primers. The lowest parameters were observed in the S. lycopersicum cherry in comparison to S. lycopersicum esculentum. In the cluster analysis, tomato accessions were distributed into four groups. PCoA was obtained for a more accurate explanation of the grouped accessions. These results indicated that SSR primers have had more tangible efficiency in showing the genetic diversity between Iranian and S. lycopersicum Cherry populations, while ISSR primers distinguished the S. lycopersicum esculentum accessions better than others.

The results indicated high genetic diversity among and within tomato species. According to genetic diversity features in both markers, it can be expected that the use of ISSR and SSR primers will be more effective for preparing genetic maps and studying population structure and/or accession grouping, respectively.

Medicinal plant periwinkle with the scientific name Catharanthus roseus is an important source of anticancer and antihypertensive alkaloids. Due to the high price of these metabolites and their small content in periwinkle plant, tissue culture techniques have been suggested to increase their production. Therefore, this study was carried out with the aim of evaluating the effect of titanium oxide nanoparticles (TiO2) on the expression of key genes of the biosynthetic pathway of important medicinal constituents in periwinkle.
 
Murashige and Skoog (MS) medium was used as the basic medium along with 2,4-D (1 mg/L) and BAP (0.5 mg/L) plant growth regulators for leaf cultivation, callus induction, and cell suspension production. Periwinkle suspension culture was treated with concentrations of 0, 50, and 100 mg/l TiO2 nanoparticles at the peak of cell growth. Then, the expression of STR, SGD, DAT, and PRX genes was measured 24, 48, and 72 h after the treatment by Real-Time PCR. Tetrazolium test was also used to measure cell viability.
 
Although there was no significant difference between the percentage of cell viability after 50 and 100 mg/L NP-TiO2 treatment; however, the passage of time (from 24/48 h to 72 h) caused a decrease in cell viability. The application of a higher concentration of nanoparticles increased the expression of the key genes involved in the biosynthetic pathway of indole alkaloids. This increase continued up to 48 h after treatment, but then reduced. The highest expressions of STR, SGD, DAT, and PRX genes were obtained by 290, 186, 193, and 287% increase, respectively, in 48 h after the treatment of 100 mg/l TiO2 nanoparticles (as the most effective treatment with the highest percentage of cell viability).
 
In the face of TiO2 nanoparticles, as an elicitor and stress-causing agent, the expression of the genes involved in the biosynthesis of indole alkaloids is induced to achieve defense against the stressful agent and then the expression of the genes decreases after the passage of time and reduction of stressful symptoms. In general, the concentration of 100 mg/l TiO2 and the extraction of metabolites in 48 h after the treatment can be introduced as a promising stimulus to increase the content of indole alkaloids. According to the results, it is suggested that the exact mechanism involved in this metabolite increase be further investigated.

Bacterial leaf streak (BLS) caused by of Xanthomonas translucens, is a serious bacterial seed-borne disease of wheat (Triticum aestivum L.) worldwide. This research was planned to study pathogenicity of the isolates obtained from bacterial leaf streak on wheat plants. Resistance of different wheat cultivars against the isolate with the highest pathogenicity was investigated. Molecular tools were used to identify bacterial isolates and their genetic diversity.
Bacterial strains with yellow and soft colonies were isolated and their pathogenicity indicated that all selected isolates are pathogen. Aggressiveness of the isolates was differing among isolates. Wheats cultivars showed different responses to selected isolate, but all cultivars were susceptible to the pathogen. Result of molecular analysis with DNA fingerprinting techniques showed genetic diversity among the bacterial isolates obtained from different wheat growing regions. In the cluster analysis of the banding pattern obtained with ERIC and BOX primers, the isolates were divided into 5 main groups at 33% similarity. The results showed significant genetic diversity among isolates causing bacterial leaf streak disease in these provinces. Sequences comparison of gyrB and dnaK genes with similar sequences in the gene bank showed high homology with sequences from Xanthomonas translucens pv. undulosa.
Bacterial strains with yellow and soft colonies were isolated and their pathogenicity indicated that all selected isolates are pathogen. Aggressiveness of the isolates was differing among isolates. Wheats cultivars showed different responses to selected isolate, but all cultivars were susceptible to the pathogen. Result of molecular analysis with DNA fingerprinting techniques showed genetic diversity among the bacterial isolates obtained from different wheat growing regions. In the cluster analysis of the banding pattern obtained with ERIC and BOX primers, the isolates were divided into 5 main groups at 33% similarity. The results showed significant genetic diversity among isolates causing bacterial leaf streak disease in these provinces. Sequences comparison of gyrB and dnaK genes with similar sequences in the gene bank showed high homology with sequences from Xanthomonas translucens pv. undulosa.
The rep-PCR techniques are suitable for studying genetic diversity of the obtained isolates. In this study, geographical location had no effect on the grouping of the isolates. Bacterial leaf streak disease is found in almost every wheat growing area and better understanding of the Xanthomonas translucens-wheat interactions are necessary to find and develop wheat cultivars with resistance to the disease.

Due to its sterility, saffron reproduces only asexually and for this reason, except for some spontaneous mutations, other genetic changes do not occur naturally in it. Therefore, induced mutations can act as a suitable method to create diversity in the genetic structure of the plant and improve its growth, performance and quality characteristics. The present study was conducted to investigate the genetic diversity of irradiated saffron plants compared to control samples (without irradiation) using the SCoT markers.
 
Healthy saffron corms were grown in the greenhouse along with control corms at two levels of 15 and 18 Gy gamma rays’ radiation and immediately after irradiation. In order to investigate the created genetic diversity, after extracting DNA from the leaf sample and determining the quality and quantity of the extracted DNA using the nanodrop device, out of the 30 SCoT primers used, 9 primers were finally analyzed for this study.
 
A total of 46 strips were scored, 31 strips had polymorphism, so that the highest number of polymorphic strips was related to SCoT05 (6 strips) and the lowest number was related to SCoT04, SCoT11 and SCoT12 (2 strips). The average percentage and Polymorphic Information Content (PIC) were 67.13 and 0.35, respectively, and the highest polymorphic content was related to SCoT11 and SCoT17 (0.45) and the lowest was related to SCoT13 (0.13). SCoT17 (0.33) had the highest Marker Index (MI). Based on the results of the Jaccard’s similarity coefficient matrix, the range of changes varied from 0.45 to 0.88 and its average was reported as 0.70. The results of cluster analysis also showed that two control samples in the first cluster, 3 out of 4 irradiated with 15 Gy dose in the second cluster, and except for 18G105 irradiated which was placed in a separate cluster, the other 18 Gy radiation along with 15G132 irradiated from the 15 Gy level were grouped in a cluster. The most observed alleles (Na) were estimated in the 15 Gy treatment (1.55) and the lowest in the control (1.2767). The amount of diversity in the population of 15 Gy based on Shannon and Ni indices (I=0.2061 and He=0.3064) was more than control and 18 Gy.
 
The results showed the high efficiency of SCoT17 in the incidence of polymorphism among irradiated and control samples of saffron. Molecular variance analysis (AMOVA) evaluated the diversity within groups more than between groups. In addition, the difference in the band pattern of SCoT markers, the clustering pattern and the genetic distance showed the usefulness of gamma irradiation and the efficiency of mutagenesis to create diversity in the saffron plant.

Bee venom contains various enzymes such as hyaluronidase and phospholipase A2, which have medical and pharmaceutical applications. In several studies, the activity of phospholipase A2 and hyaluronidase has been reported in different bee species, but there is no report about these enzymes and their activities in Iranian honey bee venom. Therefore, the purpose of this research was to isolate, identify phospholipase A2 and hyaluronidase enzymes and measure their activity in crude venom and its fractions of Iranian honey bee (Apis mellifera meda).

One hundred mg of the crude venom was purified using gel filtration chromatography on sephadex G-50, equilibrated with 0.05mM ammonium acetate buffer. SDS-PAGE was used to determine the protein profile of BV and its fractions. Protein concentration, phospholipase A2 and hyaluronidase enzyme activity of Apis mellifera crude venom and its fractions were measured. Protein concentration of Apis mellifera crude venom and its fractions was determined using Bradford method. Phospholipase A2 (PLA2) activity was determined using suspension of egg yolk as substrate. The activity of hyaluronidase was determined turbidimetrically.

The preliminary results showed that the 56% of crude venom was protein. Crude venom produced three fractions. Phospholipase A2 activity was observed in the crude venom as well as in the first and second fractions. The second fraction showed the highest phospholipase activity. Hyaluronidase activity was observed in crude venom and only in the first fraction. The optimal activity of bee venome phospholipase A2 enzyme was obtained at pH 7 and 37 °C. In this study, it was found that the activity of hyaluronidase in Iranian honey bee venom has the highest activity at 37 to 39 °C and gradually loses its activity as the temperature increases. Also, the results of this study indicate that the optimum pH for hyaluronidase enzyme activity is 5.5.

Iranian honey bee venom has both phospholipase and hyaluronidase activities, which can be separated using gel filtration chromatography. This study can be introduced as a simple method to isolate, purify and measure the activity of hyaluronidase and phospholipase A2 enzymes of Iranian honey bee venom. Purified hyaluronidase and phospholipase A2 enzymes can be used in industry and research.

The existence of molecular relationship between different organisms through diet-derived plant miRNAs is one of the most challenging debates of the last decade. Considering the mutual relationship between plants and pollinating insects, including honey bee as the most important pollinating insect, the aim of the present study is to investigate the existence of molecular relationship in the interaction between plants and honey bee through plant-derived xenomiRs can be very important and create a new perspective of the beneficial effects of this relationship.
 
Pollen was collected by pollen trap device from honey bee colonies that were located near Sidr trees. Honey bees were fed under controlled conditions in two groups (fed by Sidr pollen as treatment and fed by sugar syrup as control). Following the feeding experiments, the bees were anesthetized using cold and their midgut tissue was collected and used for RNA extraction. After the Small RNA sequencing of the samples, the identification of pollen miRNAs and their tracking in the bee body was done using different bioinformatics analysis. Finally, the target genes of the detected plant miRNAs in the bee body and the molecular pathway involved by them were determined.
 
The results of bioinformatics analysis indicate the detection of eleven plant miRNAs including miR-148a, miR-26a, miR-21-5p, miR-143, miR-27a,miR-203, let-7g, miR-126, miR-206, miR-30d and miR-199b into the tissue of honey bees fed by Sidr pollen. miRNAs participate in various biological processes through their target genes. In the present study, 99 target genes for the detected plant miRNAs were predicted in honey bee genome. The result of KEGG pathway analysis showed that target genes are significantly involved in 23 different biological pathways.
 
The result of the current study clearly present the role of plant-derived xenomiRs in the regulation of honey bee gene expression. Therefore on one hand, these findings extend our understanding of the molecular interaction between honey bees and flowering plants, and on the other hand, it can be used as a practical road map for breeding studies in order to improve honey production and deal with diseases related to bees, as the most important pollinating insects in the 21st century’s agriculture.

Wheat is the most important cereal crops; it is a stable diet for more than one third of the world population. DNA markers play the most important role in diversity due to the relative ease in their generation and elimination of the influence of environment. The objective of this research were study of genetic diversity of bread wheat cultivars using RAPD markers and efficiency of these markers in grouping and distinguishing wheat cultivars based DNA fingerprint. 

In this study 42 wheat cultivar was evaluated with using 20 RAPD markers. The amplified fragment profiles were visually scored for presence (1) and absence (0) of bands and entered in a binary matrix. 

The results showed that, RAPD primers detected 132 fragments and 88 of them (66/67%) were polymorphic. The amplified DNA fragments varied in size from <100bp to 3000bp. The number of polymorphic fragments primer ranged from 2-7 with an average of 4/4. RAPD68 and RAPD28 each whit 7 polymorphic bands showed the highest amount of polymorphism. Primer RAPD68 were able to distinguish the cultivars omid and azadi, primer OPB08 cultivars Chenab. Sepahan, Salyasonez and primers TIBMBD17 and TIBMBB09 cultivar Atrac from other cultivars. Cluster analysis using Jaccard matrix and UPGMA method was performed, wheat genotype clustered in seven distinct groups, and the genetic similarity values ranged from 0.74 to 0.94. sistan and gasparo showed the most genetic similarity (94%). Atrac, Azadi and vrinac were clustered as outliers. Analysis of molecular variance (AMOVA) determined 1% inter group diversity and 99% intra group diversity for studied genotypes. 

The results of this research showed that there was not genetic variation among bread wheat cultivars, that can be due to the low number of primers and cultivars under investigation. Suggested to use other primers such as SSR, which shows more diversity.

Compared to other primary photosynthetic products (such as sucrose and starch), little is known about sugar metabolism and how it is integrated with others. Mannose-6-phosphate reductase (M6PR) is a key enzyme involved in mannitol biosynthesis in celery. This study aimed to clone the gene, express and purify the M6PR enzyme and investigate its function on mutated genes in a laboratory environment.
 
First, the mRNA was extracted from the celery plant, then the cDNA was synthesized, and the product was used as a template to amplify the M6PR gene. The PCR product was purified in a DNA gel extraction kit. The purified PCR product was cloned into the pTZ57R vector according to the T/A Cloning recipe (Fermentase Company). Susceptible cells of E.coli strain Top10 were prepared using the biochemical method of calcium chloride, and the recombinant vector inside it was transformed and cultured on a plate containing ampicillin. The cloning accuracy was done using M6PR gene PCR and enzymatic digestion of the recombinant plasmid by BamHI and SacI enzymes (Fermentase Company). The M6PR gene was homogenized in the expression plasmid pET32a and transferred into E.coli strain BL2I. The promoter was induced with IPTG and analyzed by western blotting. The protein was purified by affinity chromatography column (His. Tag/S.Tag).
The results showed that the enzyme could identify the heteroduplex regions of the gene. The recombinant M6PR purified from Escherichia coli had specific molecular activity. The results of double digestion of the plasmid with SacI and BamHI enzymes were 2870 bp and 186 bp fragments. According to the blast test result, the current fragment had 100% similarity with the M6PR gene of the celery plant. M6PR recombinant gene transcription results showed that the M6PR recombinant gene transcription rate was 2.3 in the transgenic strains and 0.32 in control, which showed a statistically significant difference at the P<0.01 level. After induction of the promoter and sampling at different times, the samples on the SDS-PAGE gel showed a protein band in the region of 42 kDa, indicating the protein's successful expression.
 
Homology of M6PR enzyme gene obtained from celery plant and then recombinant production of this enzyme in the laboratory can lead to its high expression in the prokaryotic system so that the enzyme has activity. Also, the present study showed that plant enzymes are active when expressed in bacteria and can be used as a suitable source to accelerate catabolic activity.

Skeletal muscles make up about 40% of body weight and are responsible for many functions, such as maintaining required energy, maintaining body condition, and protecting tissues. Normal growth of skeletal muscles is a prerequisite for animals to maintain normal functions and metabolism, and any abnormal growth of muscles leads to disease. Based on the analysis of gene expression profiles in skeletal muscle, MYH7 gene has been identified as a candidate gene related to muscle contraction. The aim of this study was to investigate the expression of MYH7 gene in femur, humeral muscle and back muscle tissues of fattening lambs of Kermani breed.

In this research, six Kermani male lambs were sampled from femur, humeral muscle and back muscle tissues at the time of slaughter. The total number of samples was equal to 54 samples (6 animals, 3 tissues and 3 repetitions of each tissue). Total RNA was extracted using a standard extraction kit. The quality and quantity of extracted RNA was evaluated using electrophoresis on 2% agarose gel and using nanodrop device. Parstous cDNA synthesis kit was used for the cDNA synthesis from the extracted RNA. Syber Green real time PCR reaction was used to check the relative level of gene expression. The method of Pfaffl et al. (2002) was used to analyze the data obtained from real time PCR.

The results of RNAs extracted at A260/A280 wavelength showed that the quality is suitable and desirable (1.9-1.8). In addition, the observation of two bands of 18S and 28S for RNA on agarose gel indicated that the RNA was intact and the absence of additional bands on the gel indicated that the extracted RNA was pure. The results of real time PCR curves and electrophoresis of PCR products on agarose gel (2%) showed that the MYH7 gene is amplified and expressed in the femur, humeral muscle and back muscle tissues of Kermani male lambs. For the studied tissues (femur, humeral muscle and back muscle), a band of 283 bp was observed for the MYH7 gene and a band of 112 bp was observed for the beta-actin gene in all samples. These observations indicated that the correct experiment was performed and the desired fragments were amplified correctly. The results of this research showed that this gene is expressed in the femur, humeral muscle and back muscle tissues.

In the present study, it was found that the MYH7 gene is expressed in the femur, humeral muscle and back muscle. Therefore, it can be concluded that MYH7 gene plays a role in different muscles, which are very necessary for more meat production in the sheep breeds