فصلنامه ژنتیک نوین
سال شانزدهم شماره 3 (پیاپی 66، پاییز 1400)

In this experiment, the effect of cold stress on cell damage index (H2O2) and photosynthetic enzyme activity of phosphoenolpyruvate carboxylase (PEPC) and relative expression of small Rubisco and large Rubisco subunit genes and the chlorophyll a/b binding protein gene were studied in two chickpea (Cicer arietimun L.) genotypes differing in cold sensitivity (tolerant Sel96th11439 and sensitive ILC533) during cold stress (4˚C). Under cold stress, H2O2 was significantly lower in the tolerant genotype compared to the sensitive one (by 40.8%). Results showed that PEPC activity increased in both genotypes particularly in tolerant genotype (by 69%). However, on the sixth day of cold stress, PEPC activity in tolerant genotype was 16.9% more than sensitive genotype. In tolerant genotype, the expression of small and large Rubisco subunit genes on the first day of cold stress showed significant increase (by 80.3% and 70.6%, respectively) compared to control conditions, while on the sixth day of cold stress, the expression of these genes significantly declined (93.5% and 38.2%, respectively) compared to control conditions. In sensitive genotype, the expression of studied genes significantly declined compared to control conditions. The relative expression of chlorophyll a/b binding protein gene has shown almost the same trend compared to small and large subunit, with the difference that in tolerant genotype, on the sixth day of cold stress was equaled to control plants. These results are probably referred further homeostasis of cell through metabolic activities and photosynthesis for acclimatization the plant to cold stress. The reduction of cell damage index (H2O2) in tolerant genotypes on the sixth day of cold stress compared to sensitive genotype confirms this issue. Therefore, changes in metabolism involved in photosynthesis can be an indicator for assessing the degree of cold tolerance in chickpea.

In order to identify the QTLs controlling the morphologic traits of bread wheat, 148 recombinant inbred lines of spring wheat derived from American Yecora Rojo (early maturity and dwarf as the male parent) and No.49 genotype from Sistan Baluchestan (late maturity and tall as the female parent) were studied with the parents. The studied traits included peduncle length, spike length, awn length, flag leaf length, the number of spikes, spike weight, and the number of grains in spike. In this study, linkage map was used based on 202 markers (177 SSR markers and 51 Retro transposon Markers). The map length was about 691.36 cM with the average distance of 3.42 cM in every marker pair and covered 21 chromosomes in bread wheat. QTL analysis was done based on composite interval mapping (CIM) method, and additive effects were estimated for the identified QTLs. The results of QTL analysis showed that one QTL was detected on chromosomes 7B, 5A, 3A, 4A, 3A and 3A respectively for plant height, peduncle length, spike length, awn length, flag leaf length, the number of seed per spikes; two QTL was detected on chromosomes 3A and 2D for the number of spike and seed wieght per spike; and three QTLs were detected and mapped on chromosomes 3A, 4A, and 3A for spike weight. Among the detected QTLs, QSNS3A had the highest additive effect. For spike length, spike weight, the number of grains in spike, and seed weight per spike. The negative additive effect of the mapped QTLs indicate the optimal allele inheritance to crosses in this position of No.49 parent. The phenotypic variance explained by these QTLs varied from 4.93 to 13.63. In this study, the number of QTLs found for spike traits was so limited; it can be due to the large number of small-effect QTLs, mutual effects, and environmental effects.

Hot spring extreme ecosystems are important biological resources for identifying the genetic stocks of thermophilic microorganisms and their biological characteristics in terms of tolerance and survival in high temperatures in order to use of their potential in various industries and biotechnology. Although there have been many studies on the microbiome of these biomass in recent years in the world, so far few studies have been done on such indigenous extreme environments of Iran. Iran has a variety of geothermal and volcanic areas which contained numerous unique springs with antimicrobial and antifungal, anti-rheumatism and other muscle aches properties. In this study, the identification of composition of different taxa of the bacterial and archaeal community of Ghotoursooyi hot spring as one of the most acidic chloride springs in the world with the pH of 2.87 was performed by independent cultivation methods using next generation sequencing techniques. For quantitative profiling of the relevant microbial community or so-called metagenomics analysis, 16s rRNA gene libraries were constructed from two functional regions of taxonomic classification V4 to V5 and V3 to V4 with barcoded primers for bacteria and archaea, respectivly. Using bioinformatics analysis, 26 and 11 different taxa belonging to different bacterial and archaeal families and genera were identified with MG-RAST systems, respectivly.

Basil (occimum basilicum L.) essential oil is a rich source of phenylpropaniod compounds such as phenyl cinnamate, methyl chavicol, methyl eugenol and eugenol. Cinnamate-4-hydroxylase (C4H) is one of the important key genes involved in phenylpropanoid biosynthesis. Nowadays considering the importance of the promoter in genetic manipulation, gene transfer and the production of secondary metabolites in plants, identification of the promoter sequence and its constituent elements is necessary. Hence, in the current investigation, thermal asymmetric interlaced PCR (TAIL-PCR) was used to identify the upstream sequences of the C4H gene in O. basilicum L. In this method two series of primers differing in length and annealing temperature were applied to amplify the target sequence: three specific primers with high annealing temperature and different sequences and six random primers with a short length and low annealing temperature. TAIL-PCR was performed in three stages which in the second and third stages, diluted PCR product of the first and second stages were used respectively as the template. After electrophoresis of the PCR products and identification of the interested band, the fragments were extracted and purified from the agarose gel, and sequenced in both directions. Subsequent bioinformatic analysis of the sequenced fragment using PLACE, PLANT CARE, Softberry and Neural Network Promoter Prediction softwares revealed a sequence with a length of 794 bp. The TATA Box of the promoter was recognized around nucleotide -35 and the transcription initiation site was 170 nucleotides upstream of the initiation codon. A group of various putative functional regulatory elements such as CAAT box, W box, Gbox and I box were also identified in the promoter. Therefore, the promoter detected (after validation), may be used in the gene transfer and manipulation programs in basil for enhancement of the valuable metabolites.

Different techniques are used nowadays to assess genetic diversity in insects. Due to the importance of studying the genetic diversity and populationchr(chr('39')39chr('39'))s genetic structure in the conservation of species, the genetic diversity of Iranian honey bees collected from 20 provinces of Iran was investigated using PCR-RFLP markers. The 300 samples of young honey bee workers were collected from different parts of the country, and their DNA was extracted from the head and thorax according to the salting-out method with minor modifications. In this study, PCR-RFLP analyses of mitochondrial DNA regions (COI and 16S rDNA genes) were used, and each of these genes was digested by four restriction enzymes. The results showed very low genetic diversity in the studied honey bee populations. The observed and expected heterozygosity, Shannon index and the number of observed and effective alleles were calculated and found to be zero in most of the populations. The results of cluster analysis showed that the studied honey bee samples were scattered and integrated throughout the branches, none of them formed a separate population, and all of them formed a single group which can be due to the nature of the PCR-RFLP markers, low enzymatic polymorphism, haplodiploidy reproductive system of the honey bee, human activities, queen and hive trade among beekeepers, winter-summer displacements and migration to similar areas. Therefore, according to the results of the present study and its inconsistency with previous research, using PCR-RFLP marker is not recommended for future attempts to investigate the genetic diversity of honey bee populations.

Knowledge of genetic diversity along with the management of germplasm can be used to select genotypes in breeding programs and to protect plant genetic resources. Molecular methods, along with multivariate statistical methods, have a high potential for studying relative relationships, evolution and genetic variation of plants. In this research, 20 pistachio cultivars and genotypes were evaluated in the greenhouse for root and crown rot (gummosis). The traits; disease score, mortality and dry weight were measured. Twelve SCoT primers were used to evaluate the genetic diversity of these cultivars in response to gummosis. The results of greenhouse experiment, divided the cultivars into four groups as: resistant, moderately-resistant, moderately-sensitive and sensitive. The results showed that the SCoT primers, produced 122 loci, of which 105 loci were polymorphic. Cluster analysis of genotypes based on obtained bands did not fit well with phenotypic grouping. In order to identify the bands that have the most relationship with phenotypic attributes related to gummosis, PLS and stepwise regression methods were used. Through stepwise regression, 7 loci were entered into the model, which explained the resistance to gummosis by77 to 86 percent. By cluster analysis based on the reduced variables (7 markers), two groups were obtained that association analysis (contingency table) showed association between this grouping with the phenotypic grouping. These 7 loci greatly explained the genetic variation of pistachio genotypes in response to gummosis disease. In general, according to the results, SCOT12, SCoT16, SCoT17 and SCoT20 primers could be used to study the genetic diversity of pistachio cultivars against gummosis.

During domestication and artificial selection, horse breeds have experienced intense selection pressures for the development of desirable traits. This has resulted in a large diversity of breeds that display variation in many phenotypic traits, such as body size, muscle composition, growth rate, reproduction and behavior. The main goal of this study was detection of signatures positive selection associated with body size and athletic performance traits in horse breeds. Herein, we used whole genome sequencing data from 86 horses. To detect genomic signatures of positive selection, we applied fixation index (FST) and cross-population composite likelihood ratio (XP-CLR) methods. For body size trait, genetic study was performed based on the small body size of Debao pony. The most significant signal of positive selection was mapped to the FGFR1 gene, which may have independently been selected for short stature in the Chinese pony population. In addition, four other loci on chromosomes 2, 3 and 4 were identified to be potentially involved in the development of body size. The screen of the potential candidate genes involved in athletic performance was carried out based on Hanoverian horse breed.  Genetic analyses showed that the ACTA1 gene is potentially involved in athletic performance in this breed, consistent with its role observed in human population. In addition, three other loci on chromosomes 1 and 20 were identified to be potentially involved in the equine physical performance. These novel candidate genes identified in this study may be useful targets for clarifying our understanding of the molecular basis of body size and athletic performance in horses. These results can provide new insight for future research into the genetic architecture of relevant traits in horse breeding program.

Energy consumption and its impact on the environment is one of the greatest human challenges and the production of bioethanol or other chemicals by cellulose enzyme hydrolysis can have an important effect in replacing fossil fuels and reducing of CO2 production in the atmosphere. The Cel6B enzyme from Thermobifidia fusca, a thermal actinomist, a CBHII, belongs to the family of cellulases B, which is highly resistant to heat. Due to the low level of production of this enzyme in the primary host for industrial use, it must be optimized in systems such as bacteria. In this research, specific primers were used to confirm the presence of Cel6B gene in PSZ143 vector. Then, the PSZ143 vector containing the Cel6B gene was transferred by heat shock to E. coli BL21 (DE3) competent cells. After protein extraction, expression of a protein with a molecular weight of 59.6 KDa was confirmed using SDS-PAGE. Optimization results of protein production showed that the best time and best concentration of IPTG for induction and expression of the protein in E. coli was obtained 6 hours after induction and 0.6 mM, respectively. Also, cellobiohydrolase (CBH) activity on Phosphoric acid pretreated cotton (PC) substrate in lysed bacteria, was investigated by DNS method. The enzyme activity of cel6B in cellulose hydrolysis, in bacterial lysis and its culture media was obtained after IPTG induction of 0.8 mM, 1.21 and 0.07 U(mmol/min)/ml, respectively. Finally It has been shown that the expression of the cel6B protein is low due to the low CAI (Codon Adaptation Index) and the difference in codons preferred by E. coli. Also, by checking the native signal sequence of the cel6B gene in the Signal P4.1 server, the inability to secrete this protein in E. coli was proven.

Thermophilic fungi produce a variety of enzymes that in addition to biocatalytic activity, are widely used in biological processes in many industries, including food, paper, detergents, pharmaceuticals, toxic waste removal, and oil drilling. Extraction of enzymes, especially amylase, from thermophilic fungi has greatly contributed to industry and the global economy, so that one of the indicators of economic prosperity in developed countries. The aim of this study was to isolate and identify thermophilic fungi from soil, municipal waste compost and mushroom compost and to investigate the ability of amylase production of isolates. Isolation was performed by serial dilution method on potato-dextrose-agar extract at 45 and 50 °C. Molecular identification of isolates was performed by amplification of the ITS rDNA region (ITS1–5.8S – ITS2) using the general primers ITS1 and ITS4 during the polymerase chain reaction. The amylase activity of the isolates was studied in agar-bearing culture medium containing starch. Due to the bright halo created around the colonies after 48 hours at 37 °C, most of the isolates were able to decompose and enzymatic activity. The highest bright halo and amylase activity belong to a species of Thermomyces sp. and followed by Malbranchea cinnamomea, Thermomyces dopuntii, Thermomyces lanuginosus, Absidia sp., Aspergillus nidulance, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus had the highest amylase activity, respectively. Thielavia arenaria and Melanocarpus albomyces showed no activity of producing amylase. This is the first comprehensive study on thermophilic fungi with ability of producing amylase in Iran. All isolates of the present study were deposited to the Iranian Plant Protection Research Institute.

Sheep are one of the most important farming animals in the world, which has a special role in providing protein materials, raw materials needed by related industries, and creating employment for a group of the population. The importance of fleece in textile production has led to extensive research into its genetic structure and basis since 1960s. Milk is a complete diet and contains nutrients necessary for the vital functions of the body and the main ingredient in dairy products. Sheepchr('39')s milk is no exception to this rule, especially in countries that lack fertile land. Sheep are the main source of milk production. To achieve high-quality products, we need to know the genes involved in the production of these products and their relationship with each other. Today, with access to genomic data, development and advancement of molecular techniques in addition to understanding the genetic context of economic traits, several functional genes related to these traits have been identified and comprehensive information about them has been providedthat by using the functional properties of these genes, the most use can be made of the genetic potential of the animal to obtain their products with the best qualitative and quantitative parameters. In this study, a comprehensive search of genes effective in milk and wool production in sheep around the world was conducted and then with STRING software, the relationship between genes involved in milk production, correlation between genes effective in wool production and the relationship between these two groups of genes was investigated. The results showed that the genes effective in milk production CSN1S1, CSN2, CSN3, LALBA, BTN1A1 and PRLR are in one cluster and the genes LEP, FASN, SREBF1, ACACA, LPL, SREBF2 and VLDLR are in the second cluster. The TLR4 gene links the two clusters. These results confirm the reported functions for the TLR4 gene. Genes involved in wool production including GPRC5A, KRTCAP3, NBEA, EPHA5, GCFC2, K1F16B, DDX47, TSPEAR, NLGN1, RHPN2, USP13, UBE2E3, CALN1, PAPOLA, PGM2L1, SRC and YWHAZ are in cluster 1 and genes VAV3, NRG3, KAP6-L, PITPNC1, BCO2, FAT1, NRXN1, ASIP, TYRP1, MITF, MC1R, KIT, DCT, TYR, FAM204A, EWSR1 and KRTAP11-1 are in the second cluster and are related. The identification of these genes is an important step toward the goal of increasing milk and wool yields and improves their quality.