فصلنامه ژنتیک نوین
سال هجدهم شماره 3 (پیاپی 74، پاییز 1402)

Almonds (Prunus dulcis Mill.) are a crucial deciduous nut crop on a global scale. The development of fruiting buds necessitates a period of chilling during the winter. However, the presence of early or late spring frost may cause harm to reproductive tissues, particularly anthers, resulting in a decrease in productivity. The critical roles of carbohydrates and the key enzymes involved in carbohydrate metabolism in plants are well-known for achieving maximum frost tolerance. Despite the significance of genes associated with carbohydrate metabolism, little is known about the expression patterns of the key genes in almond anthers. In this study, the expression profiles of genes involved in carbohydrate metabolism were compared between cold-sensitive and cold-tolerant almond genotypes  and cultivar. Cold-tolerant genotypes demonstrated higher expression levels of PdHEX1, PdINV, PdPEPcK, PdPGK, PdSUS, and PdUGPase genes in comparison to sensitive genotypes. This could imply a positive role of these genes in the cold tolerance of almonds. To conclude, examining the expression profile of genes involved in carbohydrate metabolic processes can provide valuable insights into the response of almonds and other Prunus species to cold stresses. Additionally, the H genotype, which blooms late and is frost-tolerant, has the potential to be introduced as a cold-tolerant genotype.

The understanding of fundamental immune indicators, especially T-lymphocyte parameters, is crucial for research into sheep diseases and vaccines, as well as to reduce antibiotic usage and improve sheep welfare. CD8 (gene encoding beta chain of T-cell surface glycoprotein) is a co-receptor molecule expressed on the surface of Cytotoxic T cells. Due to the role and importance of this gene in the immune system, this gene's expression was examined in different Kermani sheep tissues. Samples were taken from the spleen, liver, and heart of three Kermani lambs with almost the same weight at slaughter. Total RNA was extracted and cDNA was synthesized. Real-Time PCR using Syber Green was applied for estimating gene expression levels. Beta-actin gene was used as an internal control. For analyzing Real-Time PCR data, Prism software, ΔΔCT method was used. The results showed that CD8B was expressed in all three tissues, with more expression in the liver (18.06) and spleen (16.28) and less expression in the heart (1.94). There was a significant difference (P<0.05) between all tissues. Expression in the liver and spleen tissue was significantly higher than in heart tissue (P<0.001). While CD8B expression was higher in immune-related tissues, there were significant differences in all tissues. Thus, by conducting supplementary experiments and understanding its mechanisms, we can find out why animals exhibit different levels of expression and use this variation to enhance animal performance.

In plants, putrescine (Put), spermidine (Spd), and spermine (Spm), all belong to the polyamine family, and their roles in several biological processes such as growth, development, and various responses to stimuli have been little studied. This report deals with the in-silico genome-wide evolutionary characterization of polyamine biosynthetic genes family members (including arginine/ornithine decarboxylase (ADC/ODC), Spd synthase (SPDS), and Spm synthase (SPMS), respectively) and their digital gene expression profiling analysis in different tissues along with gene ontology (GO) and gene-gene based network analysis in chickpea (Cicer arietinum L.). Based on bioinformatics analysis, identified five different putative polyamine biosynthetic gene pathways located on chromosomes 2, 4, 5, 6 and 7, respectively. Their open reading frames ranged in size from 990 to 2199 bp, encoding proteins with lengths of 329 to 732 aa. The MW values for these 5 sequences ranged from 36.59 to 78.74 kDa while pI values were 5.08 to 6.67. CaADC, CaSPDSs and CaSPMSs were predicted to localize in apoplast (extracellular). CaODC were found primarily in mitochondria. The prediction of the three-dimensional shape (3-D) of these proteins shows their structural diversity. The analysis of the second structure showed that these proteins have different percentages of Alpha-helices, Beta sheet, Random coil and Turn. Analysis of gene structure showed that the sequence of ADC and ODC were singleton while the SPDS and SPMS sequence showed dispersed duplication. The phylogenetic analysis provides further insights into the evolutionary relations between these proteins, as well as their putative functions. Multiple alignments revealed high similarity in their conserved domain but divergence in the N- and C-terminals. The phylogenetic tree showed that the ADC and ODC sequences in chickpea have very conserved protein motifs and there are no introns in the sequence of these two genes. SPDSs and SPMSs are divided into two distinct groups based on the presence of similar protein motifs. The results indicate that SPDSs and SPMSs contain seven to ten introns and most of these isoforms have the same number of exons but different lengths of exons and introns. Analyzing digital gene expression during developmental processes revealed that polyamine biosynthetic genes play a more significant role in fine-tuning plant life cycle. According to the results of data mining in the EST database, high expression levels of PAs biosynthesis genes, especially ADC, were identified in leaf and root, while, ODC had low or no expression level in all of this tissues. When considered together, these results provide valuable information for future research with regard to polyamine biosynthetic gene family members' function and structure in plants and also provide applying and manipulation of PA biosynthetic gene pathways in chickpea breeding programs.

In this study, the effect of dietary corn replacement with oak acorn on the expression of pancreatic chymotrypsin gene in broilers was investigated. The use of oak acorn in poultry feed can reduce feed intake, nutrient digestion and poultry performance by binding to dietary proteins and digestive enzymes. Therefore, the aim of this study was to evaluate the expression of pancreatic chymotrypsin gene in broilers fed with different levels of oak acorn. For this purpose, broilers were fed with three treatments (control, 15% and 20% oak flour) from 1 to 42 days of age. At 21 and 42 days of age, pancreatic tissue was isolated and weighed from 36 chickens (6 from each treatment at each age) after slaughter and then total RNA was extracted. To evaluate gene expression, chymotrypsin gene expression was compared with beta-actin gene as reference gene. To analysis of gene expression data, REST V2.0.13 software and SAS 9.1 software were used. At 21 days of age, no significant difference was observed in relative weight of pancreas between different treatments (p> 0.05), while at 42 days of age, relative weight of pancreas in 20% oak acorn treatment was significantly higher in compared to 15% oak acorn and control treatments (p <0.05). At 21 days of age, expression of chymotrypsin gene in broiler pancreas was not significantly different between treatments containing oak acorn compared to control, while on day 42, expression of chymotrypsin gene was significantly higher in treatments containing 15% and 20% oak acorn in relation to the control group (P <0.05). In general, increasing the level of oak acorn in the broilers diet due to the increase of phenolic compounds (tannins) and their binding to dietary proteins and digestive enzymes can reduce the digestion and absorption of proteins and amino acids and increased expression of protein digestive enzymes in the gastrointestinal tract.

In this study, the effect of dietary corn replacement with oak acorn on the expression of pancreatic chymotrypsin gene in broilers was investigated. The use of oak acorn in poultry feed can reduce feed intake, nutrient digestion and poultry performance by binding to dietary proteins and digestive enzymes. Therefore, the aim of this study was to evaluate the expression of pancreatic chymotrypsin gene in broilers fed with different levels of oak acorn. For this purpose, broilers were fed with three treatments (control, 15% and 20% oak flour) from 1 to 42 days of age. At 21 and 42 days of age, pancreatic tissue was isolated and weighed from 36 chickens (6 from each treatment at each age) after slaughter and then total RNA was extracted. To evaluate gene expression, chymotrypsin gene expression was compared with beta-actin gene as reference gene. To analysis of gene expression data, REST V2.0.13 software and SAS 9.1 software were used. At 21 days of age, no significant difference was observed in relative weight of pancreas between different treatments (p> 0.05), while at 42 days of age, relative weight of pancreas in 20% oak acorn treatment was significantly higher in compared to 15% oak acorn and control treatments (p <0.05). At 21 days of age, expression of chymotrypsin gene in broiler pancreas was not significantly different between treatments containing oak acorn compared to control, while on day 42, expression of chymotrypsin gene was significantly higher in treatments containing 15% and 20% oak acorn in relation to the control group (P <0.05). In general, increasing the level of oak acorn in the broilers diet due to the increase of phenolic compounds (tannins) and their binding to dietary proteins and digestive enzymes can reduce the digestion and absorption of proteins and amino acids and increased expression of protein digestive enzymes in the gastrointestinal tract.

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.

Fenugreek (Trigonella foenum-graecum L.) as an important aromatic and medicinal plant is a rich source of the Diosgenin and Trigonelline. Genetic diversity analysis using reliable methods provides useful information for the crop breeding programs and conservation of genetic resource. In the present study, 40 Fenugreek genotypes were evaluated for genotypic diversity using two gene-targeted markers. Seven SCoT and ten CBDP primers amplified 78 and 93 fragments in which 65 and 78 were polymorphic, respectively. The average of polymorphism information content (PIC) for SCoT and CBDP Primers were 0.37 and 0.29 respectively, that showed a higher resolving power of SCoTs than CBDPs. The dendrogram generated using the UPGMA algorithm based on Dice distance coefficient, classified all 40 investigated samples into four main groups. Furthermore, these results were confirmed by principal coordinate analysis (PCoA). Analysis of molecular variance (AMOVA) showed a higher distribution of genetic diversity within populations (66%), than between them (34%). Among all five investigated populations, the highest values of diversity indexes such as number of effective alleles (1.54), Shannon’s index (0.46) and Nei’s gene diversity (0.31) were estimated for population IV indicating the higher variation within this population than others. These results showed a high amounts of genetic variation among tested genotypes, which can be used in breeding programs. Moreover, the results revealed that these two gene-targeted markers are suitable and reliable techniques for DNA fingerprinting and genetic diversity investigations.

Soybean plant has a lot of value in human nutrition, so the processed soy protein is a substitute for animal proteins, which is increasingly consumed. The main environmental stress in most regions of the world is salt stress, which has limited the growth and performance of crops. Therefore, the purpose of this research is to investigate the changes in the protein pattern of two soybean cultivars under salt stress using two-dimensional electrophoresis technique. An experiment was carried out in the crop year of 2018-2019 as a factorial based on a completely randomized design with three replications in University of Mohaghegh Ardabil. The test factors included the first factor of salinity stress (0, 3, 6 and 9 dS/m) and the second factor of soybean genotypes (DPX and Arian). To study and identify the proteins related to leaf development stages, total protein was extracted from soybean leaves based on TCA-acetone method and separated by two-dimensional electrophoresis method. In order to perform electrophoresis in the first dimension, an 18 cm strip gel with pH = 4-7 was used. The second dimension was put on 11 Percent acrylamide gel by SDS-PAGE method, and the electrophoresis device was first performed for 30 minutes with 50 V and then for 3 hours with 165 V. The findings showed that by performing two-dimensional electrophoresis, a number of reproducible protein spots were identified, of 14 protein spots related to DPX cultivar were significant at the five percent probability level, and 11 protein spots were in Arian cultivar. They showed significance in different levels of salinity stress. Also, 55 Percent of the detected proteins showed decreased expression and 19 Percent increased expression. 26 Percent of the identified proteins did not have regular and significant changes. The identified proteins are involved in various metabolic pathways such as energy metabolism, photosynthesis, respiration, antioxidant activities, message transmission, translation, transcription and cell wall. Most of the common proteins in DPX and Arian cultivars were chloroplastic enzymes and some of them were mitochondrial enzymes. The most changes were observed in the DPX variety (tolerant to salinity) and finally the least changes were observed in the sensitive Arian variety, which indicates the greater effort of the DPX variety to reduce the damage caused by salinity stress.

Myostatin, also called growth and differentiation factor 8 (GDF8), acts as a negative regulator of muscle growth and development; Therefore, its role in animal growth and meat production is very important. Functional studies showed that a single nucleotide polymorphism in the 3'UTR region of this gene causes the elimination of its growth inhibitory role and finally the occurrence of double - muscled sheep. The tetra primer ARMS PCR method is an easy, fast, reliable and cost-effective method for the detection of nucleotide polymorphism. The present study was conducted with the aim of identifying single nucleotide polymorphism of myostatin gene (g+6723G>A) using tetra primer ARMS PCR technique in 86 Karakul sheep. Polymerase chain reaction was optimized using 3 control samples including 1 homozygous wild-type, 1 heterozygous and 1 homozygous mutant genotype of sharole breed sheep. All the studied Karakul sheep had the G allele, and the mutant type allele (A allele) was not observed, so the frequencies of the mutant type (A) and the wild type (G) alleles were 0 and 100%, respectively. To evaluate the accuracy of tetra primer ARMS PCR technique in this locus, all tested samples were also genotyped by PCR-RFLP method. The results indicated that two methods are the same as therefore using Tetra Primer ARMS PCR method been as economical to genotyping

at the seedling and adult plant stagesAegilops species are abundantly found in Iran, of which many could be resistant to wheat rusts .However, the researches that have been conducted to study their resistance and exploit them, are insufficient. Despite having different ploidy levels, some Aegilops species are similar in appearance and hence, flow cytometry was used to determine their ploidy level.  Three species i.e. Ae. crassa, Ae. neglecta and Ae. vavilovii were studied where Ae. tauschii (2n=2x=14) was used as a reference genome. The DNA content of the species Ae. crassa and Ae. neglecta was twice that of Ae. tauschii, and hence they were tetraploid (2n=4x) while DNA content in Ae. vavilovii was three times of the reference genome, and therefore it was hexaploid (2n=6x). To identify susceptible and resistant genotypes, a greenhouse study was conducted of 49 genotypes of Ae. crassa, Ae. neglecta, Ae. geniculata, Ae. kotschyi, Ae. biuncialis and Ae. vavilovii to wheat leaf rust at the seedling and adult plant stages. First, single spores of four pathotypes of Puccinia triticina were multiplied on the wheat susceptible cultivar “Roshan” to obtain pure and fresh urediniospores. Then, at the two-leaf stage, the plants were inoculated with these pathotypes and were kept at 20 ℃ under 100 % humidity for 24 hours in dark. Two weeks later, the seedlings were scored using the 0-4 scale where different infection types from completely resistant (0;=) to highly susceptible (3+) were detected. Additionally, at the adult plant stage, different levels of resistance (R to MR) were observed. This study lays the foundation for identifying the number of resistance genes in these genotypes and their modes of action in future studies.