مجله بیوتکنولوژی کشاورزی
سال دوازدهم شماره 3 (پیاپی 40، پاییز 1399)

Metabolic adaptation to cold stress plays an important role in the growth, survival and yield of crops. Gamma aminobutyric acid (GABA) as an osmolyte may take part in counteracting the oxidative stress induced by cold stress in chickpea.

In this experiment, content of Putrescine (Put),GABA, Hydrogen Peroxide (H2O2), activity of Diamine Oxidase (DAO) andrelative expression ofGlutamate Gecarboxylase1 (GAD1)gene in cold-tolerant (Sel96th11439) and cold-sensitive (ILC533) chickpea (Cicer arietinum L.) genotypes under cold stress (4°C)as a factorial experiment in a Completely Randomized Designwere studied.  

In tolerant genotype H2O2 content after a significant increase on the first day of cold stress decreased significantly on the sixth day of cold stress compared to control conditions (up to 4.7%) while its accumulation was observed in sensitive genotype (up to 50%). These results indicated a relative acclimation to cold stress in tolerant genotype. Under cold stress, GABA and Put contents in tolerant genotype was higher compared to sensitive genotype (up to 14% and 35%, respectively). Under cold stress, in tolerant genotype increasing GABA content was accompanied with an increase in DAO activity and relative expression of GAD1 gene as biosynthetic pathways of this metabolite (up to 3- and 17-fold, respectively). The maximum activity of these two pathways was observed in tolerant genotype on the sixth day of cold stress. 

 Under cold stress, the accumulation of GABA in tolerant genotype led to reduced cell damage (H2O2 results) and improved cold tolerance. These indices were useful in assessment of chickpea genotypes under cold stress and breeding programs.

 Photoperiod controlling genes (Ppd) are the most important genes groups that modify early heading in bread wheat. The aim of this research was to evaluate Ppd genes in Iranian cultivars, and also assessing the effect of Ppd-D1a on earliness trait.

Material and methods

 Earliness was transferred from Excalibur to Kalhaydari, Roshan and Mahdavi cultivars using backcross method to generate BC1F3 generation. Three photoperiod controlling genes including Ppd-A1, Ppd-B1 and Ppd-D1 were assessed in Iranian cultivars using PCR by specific primers. Bulk segregation analysis of Ppd-D1 loci was done for early heading genotypes in three populations. Two years field evaluation were conducted to show the effect of selection for earliness on important agronomic traits of bread wheat.

 Mahdavi, Ghods, Kalhaydari, Sardari, Roshan, Kavir, Excalibur and Shahpasand were containing Ppd-A1b and Ppd-B1b. Kalhaydari, Sardari, Roshan and Kavir were containing Ppd-D1b, while Mahdavi, Ghods, Excalibur and Shahpasand were possessed Ppd-D1a. Bulk segregation analysis of 10% of the earliest heading BC1F3 populations showed that early heading progenies were possessing Ppd-D1a. Positive significant correlation was observed between early heading and earliness. Meanwhile, days to heading had a negative correlation with grain filling period. Longer grain filling period improved 1000-grain weight, grain number per plant and grain yield. Selection for early heading improved plant height, leaf size, spike number per plant, seed number per plant, 1000-grain weight and grain yield, while decreased spike length, awn length and infertile tiller number.

 Marker assisted selection or marker assisted backcross of Ppd-D1a gene could improve earliness in bread wheat. Early heading was improved in Roshan, Kalhaydari and Mahdavi background for 13, 14 and 1o days, respectively. Selection for early heading improved the most important grain yield related agronomic traits in Kerman condition.

The analysis of mitochondrial DNA has been a potent tool in our understanding of evolution, owing to characteristics such as high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. This study aims to carry outgenetic and phylogenetic analysis on the D-Loop region of the mitochondrial genome in Najdi goats and to evaluate the relations between Najdi goats and other goat breeds available in Iran and around the world.

12 blood samples from unrelated Najdi goats from various areas of Khuzestan Province were collected. After extraction of DNA, a 968 nucleotide fragment of the D-loop region of the mitochondrial genome was amplified and sequenced. Then, the fragments were compared to the other breeds submitted in NCBI database. Finally, a phylogenetic tree was constructed using MEGA6 software.  

The results showed that the Najdi goat breed classified into A Haplogroup and has the lowest genetic distance with the breeds of Chinese and Pakistani goats. The results of genetic variation led to the identification of 20 mutations with 5 Haplotypes in Najdi goat population. Moreover, the haplotype, nucleotide diversities and the average number of pairwise differences were estimated as 0.727, 0.009 and 0.540, respectively; which indicates a high diversity in the Nadji goat. The results obtained from Tajima's D show that in the population of the Najdi goat, a balanced natural selection is going on and the frequency of rare alleles in this population is very low.

The genetic diversity of Najdi goats has increased over the years, while the number of livestock in Khuzestan province has decreased. The increase in genetic diversity could be due to crossbreed of this breed with Pakistani breeds and so on. The result of these crossbreeding in the near future will be the extinction of Najdi goats, and this issue requires more attention from the authorities.

Chicory (Cichorium intybus L.) is an important medicinal plant from Asteraceae family; contain a number of valuable medicinal compounds. Hairy root induction by Agrobacterium rhizogenes are an effective method for production of secondary metabolites.

In this study, through first experiment, different concentrations of macro elements (0.5x, 1x, 1.5x, 2x, 2.5x and 3x concentrations of MS base medium) were investigated. In second experiment inoculation time was examined to establish an efficient transformation system for chicory. Molecular confirmation of transgenic hairy roots was done with PCR using gene-specific primers for rolB gene and also growth rate of hairy root lines obtained from different explants were investigated. Also the effect of hormonal elicitor and carbon source were studied in hairy roots growth.

The results show that maximum chicory hairy roots induction was observed by using 1x KNO3, 1.5x NH4NO3, 1x and 1.5x MgSO4, 1.5x CaCl2 and 0.5x KH2PO4 (45.66, 53.33, 46.66, 53.33 and 66.66). The highest phenolic content was obtained by using 1x KNO3, 2x NH4NO3, 1.5x MgSO4, 1.5x CaCl2 and 0.5x KH2PO4 (4.21, 4.33, 4.6, 4.58 and 4.84). Also 1.5x KNO3, 2x NH4NO3, 1x MgSO4, 1.5 CaCl2 and 0.5x KH2PO4showed maximum flavonoid content (17.25, 18.52, 17.22, 18.3, 17.96 mg g DW respectively).The observation confirmed that grow of hairy root lines were significantly different and Line C showed higher biomass (0.36 g). Also the results of experiments revealed that 1.5 mg l-1 NAA in combination with 3 and 4% sucrose were superior for highest fresh (1.96 and 1.72 g) weight.
 Conclusions

Hairy roots culture was developed as the innovative path for bulky production of secondary metabolites which find relevance in the pharmaceutical, food and flavor industries. The results indicated significant increases in hairy root induction and total metabolites content by ATCC15834 strain and 0.5x KH2PO4 co-culture MS medium.

Identifying genes with large effects on economically important traits, has been one of the important goals in chicken breeding. The present study aimed to conduct a genome wide association studies (GWAS) based on Gene-set enrichment analysis for identifying the loci associated with egg weight in Rhode Island Red chicken using the high-density SNPs.

Phenotypes records and genotypic data were obtained from the Figshare online public repository. The gene set analysis consists basically in three different steps: the assignment of SNPs to genes, the assignment of genes to functional categories, and finally the association analysis between each functional category and the phenotype of interest. Genome wide association study for 1,078 hens was performed with egg weight including first egg weight, eggs weight at 28, 36, 56, 66, 72, and 80 weeks of age using GenABEL software. Using the biomaRt2 R package the SNP were assigned to genes if they were within the genomic sequence of the gene or within a flanking region of 20 kb up- and downstream of the gene. Subsequently, gene enrichment analysis was performed with the goseq R package and bioinformatics analysis was implemented to identify the biological pathways performed in GO, KEEG, DAVID and PANTHER databases.

In this research, 9 SNP markers on chromosomes 3, 4, 6, 7, 8, 9, 19, 20 and 22 located in MC3R, LEPR, ECT2, SH3GL2, KCNMA1, SPP1, PCK1, MMP9, PPP1CB, ACOX1, and IGFBP2 genes were identified. Some of the genes that were found are consistent with some previous studies related to egg weights. According to pathway analysis, 28 pathways from gene ontology and biological pathways were associated with the egg weight (P˂0.01). Among these pathways, the regulation of feeding behavior, positive regulation of the apoptotic process, positive regulation of protein phosphorylation, osteoblast differentiation, positive regulation of gluconeogenesis, cell-cell junction, and focal adhesion have important functions in creating the egg weight and process production through the development and ovulation of the oocytes, the formation of albumen, and the formation of eggshell.

 In total, this study supported previous results from GWAS of egg weights, also revealed additional regions in the chicken genome associated with these economically important traits, using these findings could potentially be useful for genetic selection in the breeding programs.

This study aimed to investigate the genetic diversity of different genotypes of this valuable medicinal plant under the same culture condition which can be used as an introduction to domestication, germplasm conservation, and possible cross in the future.

In this study, after preparing 20 different genotypes from all over Iran, they were cultivated in complete randomized blocks design with three replications under the same condition. After extraction of DNA, the survey of genetic diversity using 12 ISSR markers from 15 markers was conducted by polymerase chain reaction (PCR). The essential oil for each genotype was extracted by water distillation. The dry yield based on gr/m2 and essential oil content (w/w, based on dry weight) were measured. Furthermore, the correlation of molecular markers with dry yield and essential oil content from each genotype was determined using stepwise regression. 

The mean percentage of polymorphism determined in all the genotypes was 91.97. The number of polymorphic bands for each primer varied from 5 to 9 and a total of 89 replicate bands were scored, of which 82 bands showed polymorphism. The average content of primer information polymorphism (PIC) was estimated to be 0.31 and the IS1 primer showed the highest PIC (0.46). The Ni and Shannon indices for IS1 primers were 0.43 and 0.61, respectively. Cluster analysis using the Jaccard similarity coefficient and UPGMA algorithm divided the studied genotypes into four groups. The highest genetic distance was observed between Khuzestan and Qazvin genotypes with a coefficient of 0.39 and the lowest between Kerman-2 and Kerman-4 genotypes with a similarity coefficient of 0.77. Stepwise regression showed that the IS10 primer has a coefficient of 0.70 with the essential oil percentage and dry matter yield.

The results of this study showed that ISSR markers can be effectively used to study the genetic diversity of wild mint genotypes and will provide the possibility of breeding. So that, IS1 and IS10 primers were introduced as the best markers. Also, Khuzestan and Qazvin genotypes had the highest genetic distance.

Enzymes are noble metabolites which extensively utilize in different industries. Currently, the production of enzymes by microorganisms is one of the best and most effective methods for the production of these enzymes. Hydrolytic enzymes are one of the most important enzymes that produced by various microorganisms, including endophytes.

In order to investigate the presence of endophytic fungi from two plants Euphorbia esula L. and Chenopodium album L, in summer of 1395, six different plants parts including flowers, leaves, seeds, roots, crowns and stems were sampled from healthy plants in Hamadan province. After fungal purification, the presence of extracellular hydrolytic enzymes such as amylase, catalase, protease, cellulase, pectinase, and xylanase were investigated by adding the corresponding substrate in the endophytic fungal culture medium.

After sterilizing the surface of collected plants and transferring samples to Potato Dextrose Agar culture media, a total of 31 endophytic fungi were isolated. The results showed that 84% of fungal isolates had amylase, 81% catalase, 71% protease, 61% cellulase, 39% pectinase and 26% xylanase activity. Morphological studies of the isolated fungi showed that some them belongs to the Fusarium, Alternaria and Trichoderma spp.

The results obtained in this study showed that endophytic fungi isolated from Euphorbia esula L. and Chenopodium album L, plants are capable of in vitro production of various enzymes. Among them, the presence of important enzymes like cellulase, pectinase and protease in some isolates, emphasis the significance of results. Based on the vast application and magnitudes of these enzymes in different industries, the importance of these fungi and demands for more experiments on enzyme activity arespecified.

The wheat is one of the most important sources of human food. In production of this product abiotic stresses such as drought, salinity, temperature, etc., involved in cultivation. Therefore, identification and evaluation of genes involved in stress resistance in this plant is very importance. Aim of this study was identifying responsive genes to drought and their expression variation in, resistant and susceptible wheat.

 The data available on the NCBI GEO microarray data were collected in 2018. The libraries were belonging to drought resistant and drought susceptible wheat under stress and control conditions. Using meta-analysis, altered genes expressed under stress conditions were identified for each group of cultivars. Significantly expressed altered genes were identified, the aci-reductone-dioxygenase-like protein (ARD) gene was evaluated for laboratory confirmation. Leaf sampling was performed in 4-leaf stage at three times 2, 4 and 7 days after stress with a control sample with three biological replications. RNA extraction from leaf samples was performed using RNx ™ -plus extraction solution and Real time PCR reaction using specific primers of d ARD gene.

Among differential expressed genes of resistant cultivars, transcription factors genes were Myb3, ethylene responsive 5a, MIKC-type MADS-box WM24B and salinity inducible ERF4 and in sensitive cultivars, transcription factors such as WRKY15, MADS-box TaAGL8, WRKY39 and Myb have increased expression. By identifying the genes and transcription factors mentioned and changing their expression, wheat cultivars can tolerate drought stress. Real time PCR results of ARD Gene for 6o4 and Sundor, drought resistant and drought susceptible wheat varieties was consistent with the results of the meta-analysis and it confirmed the result.

 According to gene expression results and other outcomes of metanalysis in this study, an effective step can be taken in predicting drought resistance and susceptibility in different wheat cultivars.

Salmonella is a Gram-negative, anaerobic, flagellated bacterium of Salmonella type in Enterobacteriaceae family, which is known as a zoonosis infectious agent. In recent years, different types of antibiotics have been used to overcome the Salmonella infection; however, the increasing number of antibiotic-resistant bacteria, the provision of alternative methods seems be necessary. Specific immunoglobulin Y (IgY) originated from egg yolk causes passive immunity in the fetus, and extracting these antibodies from egg yolk is cheaper and more feasible than other antibodies. Numerous studies have shown that the egg yolk IgY has immunogenic function against a wide range of bacterial and viral infections in mammals. The purpose of this study was to produce specific immunoglobulin Y against Salmonella typhimurium and Salmonella enteritidis bacteria in laying hens.

 In the current study, six pullets were inoculated and boosted with one milliliter of killed Salmonella typhimurium and Salmonella enteritidis. Injections were done fortnightly until one week before egg production. After breast muscle injection of chickens, the eggs were collected, and the IgY was extracted from yolk by the Polson method with polyethylene glycol 6000. The produced IgY was approved by SDS-PAGE method. The specific indirect ELISA done by specificity test for purified IgY also approved the specific IgY against both Salmonella typhimurium and Salmonella enteritidis.

The results of SDS - PAGE demonstrated the presence of two protein bands of 27 and 67 kDa representing light and heavy chains of IgY, respectively. The specific indirect ELISA method proved that the specific IgY against Salmonella typhimurium and Salmonella enteritidis was produced in the egg yolk.    

 It might be possible to administer the egg yolk containing specific IgY against both Salmonella typhimurium and Salmonella enteritidis, to induce passive immunity for preventing some pathogenic diseases in calves such as diarrhea.

Three fecundity genes have been identified, namely bone morphogenetic protein receptor type 1B (BMPR1B; or activin-like kinase 6, ALK6) known as FecB on chromosome 6, growth differentiation factor 9 (GDF9) known as FecG on chromosome 5, and bone morphogenetic protein 15 (BMP15) known as FecX on chromosome X. Bone morphogenetic protein (BMP) genes are members of the transforming growth factor-beta (TGF-β) super-family which are multifunctional cytokines with a 2-fold function and are expressed in a variety of cells. BMPs were originally identified based on their ability to produce ectopic bone formation when implanted within the soft tissue in vivo. They also play roles in embryonic development, homeostasis, repairing of various tissue patterning, cell differentiation, and apoptosis. The BMP15 gene regulates granulose cell proliferation and differentiation by promoting granulosa cell mitosis, suppressing follicle-stimulating hormone receptor expression, and stimulating the kit ligand expression; hence, this gene plays a pivotal role in female fertility in mammals. The aim of this study was to investigate tissue-specific mRNA expression profile of BMP15 gene in Raini cashmere goat using Real Time PCR.

Tissue samples (3 repetitions of each tissue) were taken from ovary, uterus, uterine horn, oviduct, skeletal muscle, subcutaneous fat, heart, liver, lung, kidney, spleen and adrenal   during slaughter at the slaughterhouse from two goats. RNA extraction from tissues and cDNA synthesis were performed. Relative gene expression of BMP15 was done applying Real Time PCR using SYBR Green method. GAPDH gene was used as housekeeping gene. Pfaffl method was used to analyze achieved data.

BMP15 gene expression in ovary, uterus, uterine horn, oviduct, skeletal muscle, subcutaneous fat, heart, liver, lung, kidney, spleen and adrenal tissues using Real Time PCR in studied goats demonstrated that BMP15 gene is expressed only in the ovary tissue and is not expressed in other studied tissues.

Based on the results of the present study and the results of other researchers, it can be concluded that BMP15 gene plays an important role in ovary and as a result in fertility, because in the present study it was found that BMP15 is expressed only in ovary tissue. Therefore, BMP15 is likely to be essential for female fertility, and the results of this study provide a basis for future research to describe the role of BMP15 gene as a candidate gene for better fertility and normal physiology in domestic animals, especially goats.