فصلنامه ژنتیک نوین
سال هجدهم شماره 1 (پیاپی 72، بهار 1402)

Mitogen activated protein kinase (MAPK) cascades are key players in plant immune signalling pathways. MAP kinase singling consists of a number of associated proteins where MAPKKKs are located upstream of MAPK cascade activating multiple MAPKs. MAPKKKε, a positive regulator of cell death, is associated with plant immunity. Phytophthora infestans is a pathogenic oomycete that inflicts potato late blight disease, delivering RXLR effector proteins to plant cells to modulate host immune signalling and colonization incident. The RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε. Using a CRISPR/Cas9 system, a construct was made to introduce four guide RNAs (gRNAs) in potato cultivar, Agria. Thirty-five mutant lines were regenerated, in which three and two transgenic lines with deletions and insertions were analysed, respectively. A mutant line with a three-allelic knock down of MAPKKKε was obtained, showing ~70% reduction in MAPKKKε expression level resulting in enhanced susceptibility to P. infestans. The results of this study provide evidences that editing MAPKKKε to modulate PexRD2 interaction, enhances susceptibility to P. infestans, suggesting that MAPKKKε plays a key role in resistance to P. infestans in potato. Therefore, it can be anticipated that MAPKKKε gene can be used in future potato breeding programs to generate biotic stress resistance plants.

Vernalization genes (Vrn) control wheat growth habit (spring and winter habit). These genes play a key role in flowering time and earliness of bread wheat and are of great interest in wheat drought tolerance studies. In a research that was designed to develop isogenic lines for Vrn-B1 and Vrn-D1 genes, in the BC5F5 generation of Roshan (recipient parent) and Excalibur (donor parent), some isogenic lines were awned. Since the recurrent parent was awnless and the genetic background of the isogenic lines were similar with a probability of 99.99%, the possibility that the awn controlling gene is independent of the Vrn-B1 and Vrn-D1genes was rejected. Results showed that the awned and awnless isogenic lines carry Vrn-D1 and Vrn-D1a alleles, respectively. Therefore, the evidence can confirm the association of a gene controlling awn and Vrn-D1. The hypotheses of genetic linkage and pleiotropic effect were proposed to explain this association. To find the genetic reason for this relationship, in addition to Roshan and Excalibur, Rakhshan, Shahpasand, Mahdavi and Kalhaydari cultivars were also genetically evaluated. The results showed that Shahpasand, Mahdavi and Kalhaydari cultivars had Vrn-D1/Vrn-D1 genotype while Rakhshan had Vrn-D1a/Vrn-D1a genotype. Therefore, the hypothesis of pleiotropic effect was rejected and it was determined that the gene which is located at a close distance from Vrn-D1 is controlling the awns. The distance between these two genes is so tight that no crossing over has been occurred between them in 14 generations of sexual reproduction. Three known genes on chromosomes 4A, 5A and 6B are controlling awns. Yet, there is not known gene on chromosome 5D controlling awns. Therefore, the gene that is located at a short distance of Vrn-D1 and controls the awns has remained unknown till now. In this research, it was found that the awned phenotype is linked with the Vrn-D1a allele. Since awnless phenotype is not a desirable trait, the genetic linkage of these two genes reduces the agronomic value of cultivar.

Flax (Linum usitatissimum L.) is cultivated all over the world on a large scale due to its wide use in the food, textile, and pharmaceutical industries. One of the factors that affect the cultivation of this plant is abiotic stresses, such as inappropriate pH and soil with a lack or toxicity of nutrients, which leads to a decrease in its quantity and quality. Therefore, it is necessary to identify genes responsive to different stresses. In the present study, using RNA-Seq data obtained from the roots of flax plants under the stress of aluminum toxicity and zinc deficiency, DEGs were investigated to identify stress-responsive changes in two resistant and sensitive cultivars. In total, 30 common DEGs were identified between resistant cultivars and 97 common DEGs between sensitive cultivars in both stresses in which, the number of DEGs was more in the sensitive cultivar and four genes were common between sensitive and resistant cultivars. The most general changes of common DEGs were related to flax-specific biological processes, metabolic processes, and cellular processes, which indicate the metabolically active state of the plant cell. Resistant cultivars responded more specifically and most of the genes were grouped in GO related to response to stimuli. In the protein-protein interaction network of susceptible cultivars, chitinases and the PR4 gene had the most related to other proteins, while in the resistant cultivars, the resistance strategy was different, and the expression of cell wall modifying genes showed the highest relation with other proteins. In general, the expression profile of stress-responsive genes showed that the PR1 gene had the most changes in both cultivars and can be used as a resistance marker for plant breeding purposes. Also, the biosynthesis of secondary metabolites, which is a response to stress, increased significantly. The obtained results provide new insights into the mechanism of tolerance to abiotic stress in flax, which can be used to improve the quantitative and qualitative yields.

European black cumin (Carum carvi L.) is an important species of the Apiacea family that has valuable terpene compounds. The aims of this study were, to determine the effect of different hormonal treatments on the production of callus from leaf and root explants and also to select the best explant and hormonal treatment for cell culture, and then to investigate the effect of silver nanoparticles on the stimulation of the production of secondary metabolites and the expression of the Limonene synthase gene in callus was obtained from cell culture. First, the effect of 6 hormone treatments in MS culture medium on the callus produced from two explants (leaf and root) were investigated by measuring three quantitative and qualitative traits in each of the treatments. Considering the above traits, leaf explant in MS2 hormone treatment, which included IBA (0.1 mg/l), 2IP (0.5 mg/l) and BA (0.5 mg/l), were identified as superior explant and hormone treatment. To continue the studies, the calluses obtained from the superior explant on the superior hormone treatment were transferred to the cell culture medium containing MS culture medium without agar. Then the samples were placed in a shaker at 120 rpm under dark conditions at a temperature of 24°C. After multiplying the callus and reaching the desired growth, the samples were treated with silver nanoparticles in two concentration of 50 and 100 mg/l, and samples were taken from the calluses at zero, 24 and 48 hours after the treatment. The data obtained from GC and GC/MS and the results of limonene synthase gene expression showed that silver nanoparticles as a non-living stimulus by intensifying defense responses in black cumin, caused a change in the synthesis rate of secondary  metabolites such as Limonene, Carvone, Carvacrol, Thymol, p-Cymene and γ-Terpinene and also increased the expression of Limonene synthase gene.

For many years, different species of the genus Hypericum spp. used to treat neurological disorders and depression. The high susceptibility and fluctuation of the production of secondary metabolites caused by climate changes in this plant can be controlled by in vitro culture systems. Hairy roots (transgenic) has been used as an alternative for production of medicinal plants to produce secondary plant metabolites in many species, but the production of these roots have always faced challenges, one of them is the difficulty of induction. Therefore, the present study aimed to introduce practical guidelines for induction of adventitious roots from Hypericum perforatum leaf explants treated with different concentrations of indole-3-butyric acid (IBA) and different light sources including red, far red, full light, blue and flourcent , as well as absolute darkness as control treatment. The results of this study showed that the concentration of 2 mg/l IBA under dark treatment had the best inductive effect on adventitious root production from leaf explants. Higher concentrations of IBA caused callus formation in both light and dark conditions. In investigating the effect of light source on the frequency of adventitious root induction, the highest number of roots was recorded in far red and red, while the lowest number of adventitious roots was recorded under full light treatment. The highest level of hypercin was in the dark followed by fluorescent light and the lowest in blue light, but different light sources did not show a significant effect on hyperforin content.

Bread wheat (Triticum aestivum L.) is an allohexaploid plant consisting of three genomes (A, B and D) that originated from three ancestral diploid species via multiple hybridizations. Despite the availability of the wheat genome sequence and the related studies conducted, more information about its genome and transcriptome can still be obtained. The aim of this study was to extract descriptive information about wheat genes and transcripts and investigate the similarities and differences of wheat genomes from different points of view such as number of genes and transcripts, gene density, number of introns, alternative splicing, CDS and UTR length, and GC content. Results showed that the gene density in wheat is about 7.5 and the D genome has a higher density than the other two genomes. In addition, despite the difference in the average of genes and introns lengths, high similarities were found in the average of exon, transcripts, and coding region lengths of A, B, and D genomes. Comparison of transcripts and genes represented approximately 80% of the genes contained introns. This comparison also revealed alternative splicing occurred in approximately 16% of the genes. Our study showed most of the splicing sites occurs in the CDS and a small part of the UTR region. By focusing on CDS regions, more abundance of splicing was observed between codons than within codons. It was also shown most of wheat transcripts contain UTR and almost 10% of UTRs contain uORF. Although the amount of GC content of genes, transcripts, introns and UTRs were between 30-80 percent, the relationship between GC content and the length of mentioned cases showed a different pattern. Finally, it could be concluded, despite differences in the origin of bread wheat genomes, many similarities in terms of chromosome length, gene density, alternative splicing and various parameters such as CDS length, UTR length and GC percentage would be seen.

In this study, ISSR marker was employed to measure the genetic diversity within and among 29 accessions of Ziziphora specie from different regions of Iran including Z. tenuior, Z. persica and Z. capitata from 19, 5 and 5 geographical region, respectively. A total of 154 bands were amplified, and 86.67%, 81.13% and 84% were polymorphic in Z. tenuior, Z. persica and Z. capitata, correspondingly. Average number of amplified bands for each primer in 29 accessions was 10.26, while average number of amplified bands for each primer in Z. tenuior, Z. persica and Z.capitata was 3.8, 7.6 and 6.8, respectively. The most Polymorphic information content among tenuior, persica and capitata species belonged to UBC-805 (0.46), UBC-811 (0.35) and UBC-824 (0.37) primers respectively. The results of cluster analysis indicated that the ISSR marker could easily separate the tenuior species from the other two species, persica and capitata, although it was unable to separate the latter two species. Analysis of molecular variance (AMOVA) showed that there is a significant differences within the samples. Indeed, 38% of the observed genetic diversity is related to genetic diversity between populations and 62% is related to genetic diversity within populations. The results of principal coordinate analysis (PCoA) demonstrated that the first two vectors explained an 80.3% of the total variance, which shows the high correlation of the selected primers. Therefore, with a smaller number of primers and, as a result, the cost, it is possible to separate the tenuior species from the other two species. Considering that the primers UBC-805, UBC-811 and UBC-812 in the tenuior species contained the most polymorphic information and marker index and also showed 100% polymorphism, they can be used as efficient primers to investigate genetic diversity in this species. Overall, high genetic diversity in the Ziziphora sp. plants were identified in this study, which can be useful in the management and maintenance of the germplasm of this plant.

Proteases are the most important industrial enzymes. Microbial proteases, especially from Bacillus sp. are most widely exploited industrially. The aim of this study was isolation, cloning, sequencing and bioinformatics study of htrB protease serine gene extracted from Bacillus licheniformis. In this study, after extraction of bacterial DNA, one of the serine protease genes, htrB, was isolated from Bacillus licheniformis using the polymerase chain reaction technique. cloned into pTG19-T vector. DNA sequencing results confirmed the cloned segment. The cloned gene in the recombinant plasmid pTG19-htrB was cloned in pET41a (+). Based on nucleotide sequencing, the target gene has 1371 base pairs and encodes a protein with 456 amino acids. The calculated molecular weight and its predicted isoelectric point were 48.66 kDa and 4.85, respectively. Studies have shown that the enzyme is in the category of stable enzymes and will be expressed as a solution in Escherichia coli bacteria. The molecular structure, its biochemical and phylogenetic properties were investigated. Based on the results of phylogenetic studies, the obtained protein sequence showed high similarity to the sequences of other Bacillus species, such as B. subtilis, B. gobiensis and B. pumilus. The three-dimensional structure of the cloned enzyme was predicted using the PyMOL, I-TASSER, PHYRE2, RAPTORX and Modeller tools. After evaluating the drawn models, it was determined that the models provided by PHYRE2 and RAPTORX software are desirable models for predicting the three-dimensional structure of this protease.

Grape is one of the most important horticultural products in the world and in Iran and has a high genetic diversity. Determining the level of genetic diversity is the first step in plant breeding programs. Knowledge of genetic diversity, in addition to providing information on evolutionary and phylogenetic relationships, genetic mapping, ecological and environmental adaptations, and ultimately selecting the right parents for breeding programs, will reduce the time and cost of breeding projects. This study was performed to investigate the genetic diversity of 69 cultivars and promising genotypes of Russian grapevines with three Iranian commercial grapevine cultivars using 15 microsatellite markers (ISSR). Polymerase chain reaction (PCR) samples were electrophoresed in 1.3% agarose gel. Cluster analysis with the UPGMA method was used based on the Jaccard coefficient after assigning one and zero to the presence or absence of a band in each primer for each genotype. The highest percentage of polymorphisms (100%) was related to UBC-823, UBC-825, UBC-841, UBC-846, UBC-855, and UBC-873 primers, and the lowest (66.7%) was related to UBC-817 primer. The highest and lowest levels of the polymorphic content index were observed in UBC-825 (0.487) and UBC-856 (0.216) primers, respectively. The highest and lowest levels of marker index were 2.718 and 0.904 related to UBC-889 and UBC-817 primers, respectively. The highest and lowest values of the effective multiplex ratio index were 7 and 1.33 for UBC-841 and UBC-817 primers, respectively. The highest and lowest resolving power were 8.11 and 2 for UBC-877 and UBC-841 primers, respectively. The UBC-815 and UBC-834 primers had no clear and concise bands. Overall, 73 bands were obtained from the 13 primers of the ISSR DNA marker which 64 bands were polymorphic and the total polymorphism was 88.1%. The highest and lowest numbers of bands were eight and two bands for UBC-826 and UBC-817 primers, respectively. The cluster analysis was performed by the UPGMA method based on Jaccard's coefficient of similarity, which led to the classification of 72 grape varieties with a similarity value of 0.12 to 0.89 in seven different groups. In the present study, high diversity was observed among the studied grapevine cultivars. This indicates the different geographical origins of the cultivars and genotypes under study.