مجله بیوتکنولوژی کشاورزی
سال یازدهم شماره 3 (پیاپی 36، پاییز 1398)

 Dracocephalum kotschyi is an endemic medicinal plant in Iran belonging to the Lamiaceae family. One of the most important monoterpenes identified in this plant is limonene. Drought stress is one of the factors affecting the content of monoterpene in medicinal plants.  In this study, the effect of different levels of drought stress was investigated on the expression of some genes involved in limonene biosynthetic pathway in D. kotschyi at two growth stages.
 

 This experiment was conducted as a factorial experiment with two factor included drought stress and growth stages in a completely randomized design with three replications. Drought stress consisted of severe (25% FC), moderate (50% FC), low (75% FC) stress and non-stress or control (100% FC) and growth stages consisted of vegetative and flowering. Secondary metabolites were analyzed and the amount of limonene was measured by GC / MS using Head-space method and the relative expression of key genes involved in limonene biosynthesis was measured using Real Time PCR.
 

 Analysis of secondary metabolites showed, one of the main monoterpenes was limonene whose amounts was 20.57 % and 13.41% during vegetative and flowering stages, respectively. Drought stress significantly increased HMGR and DXS gene expression during flowering stage, while during vegetative stage no significant change was observed on expression of both genes. In both growth stages, low drought stress increased HDR gene expression, although at flowering stage this increase was not significant but severe drought decreased HDR gene expression, although this decrease was not significant at vegetative stage. Drought stress decreased LS gene expression during flowering stage and increased it during vegetative stage.
 

 The results indicated that limonene content and expression of limonene biosynthesis key genes in D. kotschyi under drought stress are growth stage-specific.

Gene expression studies by Real-Time PCR constitute a powerful tool to analyze the mechanisms underlying plant biotic-stress tolerance. One of the crucial steps of this technique is the selection and validation of reference genes to normalize target gene expression under different stress conditions. In this study, the expresion of candidate gene in oryza sativa was investigated under biotic stress at different developmental stages.
 

Eight internal control genes consists of eIF-4A, UBQ5, UBC, Actin1, Actin11, GAPDH, Cyclophilin and 18SrRNA which are commonly used as housekeeping genes in plants, were selected and their expression stability were examined in present of Rhizoctonia solani RBL1 strain, potasium silicat as tolerance inducer and different growth stages in three time periods (6 h, 24 h and 72 h) using BestKeeper and NormFinder softwares.
 

Based on the results gained through Best Keeper, the UBC has the higher expression than the other genes under biotic stress in rice leaf and also the UBC and Actin11 genes poses the highest correlations with the BestKeeper index (0.97). Additionally, it was shown that the UBC and Actin has the lowest coefficient variation. Also, the evaluation of the reference genes expression using geometric mean of the UBC and Actin11 compared to the Actin1 gene indicated the necessity of appropriate selection of the reference gene. Taken together, it was evidently demonstrated that the UBC and Actin11 genes are the proper reference gene to be employed for the normalization of expression data in the Oryza sativa L. using by Real-Time PCR.

 Sulforaphane with several pharmaceutical effects is an isothiocyanate which derived of glucosinolate glucoraphanin under myrosinase hydrolysis activity. This glucosinolate is found in huge scale in Lepidium draba plant from the brassicaceae family.
 
In this research, sulforaphane production and the gene expression level of CYP79F1 (one of the critical enzyme in glucoraphanin biosynthesis pathway) and myrosinase were investigated in Lepidium draba seedlings under treatment with different concentrations of ZnO (0, as a control, 25, 100, 500 and 1000 mg/L).
 
 The results showed that sulforaphane content in treated seedling significantly increased in comparison with the control in the presence doses more than 100 mg/L ZnO. Based on the results, gene expression level of CYP79F1 decreased by the increase of ZnO concentration in media, and the increment was significant at the 5% level in the presence of the highest dose in compared to the control. In contrast, the myrosinase   gene expression level evaluated by increasing ZnO concentrations in media and the increment was significantly at the doses more than 500 mg/L ZnO concentration.
 
According to the results, it seems that evaluating of the sulforaphane production level in the treated seedlings is more attributed to the increased gene expression level of myrosinase which resulted in the enhanced enzyme production.

Genetic diversity in field crops has important role in breeding programs. The object of this study were to evaluate the genetic diversity and association analysis of morphological traits and mildew disease of wheat germplasm.

In the research 116 wheat genotypes were assessed for morphologic traits using a lattice design with 3 replications in the research field of Gonbad Kavous University. Plants response to powdery mildew was also evaluated. 10 ISSR and IRAP markers were applied for molecular analysis.
 

There was a significant difference between genotypes for all measured traits except 1000 grain weight. In molecular evaluation using ISSR and IPBS markers, 57 bands were amplified where 47 bands were polymorphic. The number of polymorphic bands was ranged from 3 to 6 bands for each primer. The maximum polymorphic bands belonged to PRI-14, PRI 43 and PRI 50 primer. The PIC and MIC values ranged from 0.35-0.88 and 0.62-3.14 respectively. The maximum percent of polymorphism belonged to PRI-13, PRI-14, PRI-43, PRI-20 and PRI-10. The maximum Molecular Index was 3.14 in PRI-7. Cluster analysis was grouped lines into three groups, which genotypes 60 and 786 were the most resistance to Powdery mildew. Association analysis between markers and resistance to powdery mildew revealed that total of 9 primers showed a connection to the character.
 

Based on the results of this research, genotypes with acceptable morphological traits and resistance to powdery mildew could be screened for advanced wheat breeding programs.

This study was conducted to investigate the effects of green tea and rosemary extracts and their mixtures on growth performance, carcass characteristics, blood biochemical parameters, oxidative stability of meat and expression of interleukin-6 genes and interferon gamma in broiler chickens.
 
 

Birds fed on a Corn-Soybean. The study was conducted in a completely randomized design with a 3 × 3 factorial arrangement with 9 treatments and 4 replications, each replication containing 15 birds. (540 Ross 308 broiler chickens). The experimental diets included the control diet (without extracts) and the levels of 0.5 and 1 g / kg extracts of green tea and rosemary.
 

The results showed that the use of mixture of rosemary extracts increased feed intake, increased weight and improved feed conversion ratio at the end of broiler chickens breeding (P<0.05). Among the experimental groups, 0.5 rosemaryextract (g/kg of feed) had the best production index (P<0.05). The use of these extracts in the diet of broiler chickens has significant effect on liver enzyme (P<0.05).These exteracts reduce the amount of malondialdehyide produced in the drumsticks of broiler chickens (P<0.05). The different levels of these plant compounds increased the expression ofinterleukin-6 and gamma interferon liver genes.
 

In general, it can be stated that the mixture containing 0.5 rosemary extract (g/kg of feed) has the most effect on performance improvement, oxidative stability of meat and expression of liver genes in broiler chickens.

Lettuce is among one of the oldest vegetables in the world originally lodge in India and Central Asia. This study aims to evaluate the genetic diversity of fifteen lettuce genotypes by using the rbcl gene and protein sequences.
 

Using sequencing method, the rbcl gene sequence of of fifteen lettuce were sequenced. Chromas 2.1.1 software, MegAlign ver5 software (using ClustalW approach) were applied for generating the similarity matrix, genetic distances as the same as building phylogram of the studied sequences. The modeling and validation of rbcl protein structure were performed through SWISS-MODEL and QMEAN, respectively. Further steps were progressed using Ramadan and Pro-SAP servers.
 

Investigating protein-protein cross-links using STRING showed most of proteins encoded by matK, psb (A, D and C)-tranH have the interaction with rbcl protein. The results of cluster analysis, similarity matrix, molecular-variance and dn/ds was indicated that the sequence of rbcl gene and protein is very similar and conserve among all genotypes.
 

The results were indicated that the rbcl sequence is not useful to study the phylogenetic relationships inter-species. Therefore, this point is trustworthy to say that one of the reasons for failing the segregation all lettuce genotypes is the tiny diversity of rbcl gene.

Ajowan (Trachyspermum ammi L.) is an annual plant in Apiaceae family which is used in traditional medicine and medical science. This study was carried out to determine the quantitative expression pattern of some genes on monoterpenoid biosynthesis pathway and essential quality in Ajowan treated by methyl jasmonate. Ajowan essential oil compounds and the amount of MECPS, DXR,  and GDs genes expression as main genes related to Thymol biosynthesis pathway in response to methyl jasmonate in 4 different times were analyzed.  

After flowering, the 0.1 mM methyl jasmonate hormone was sprayed onto the plants. Samples were taken in 4 times (after 8, 24, and 48 treatment and control treatment). RNA extraction, cDNA synthesis, and Real-Time PCR have done from flowers and the date from these gene expressions were analyzed. Essential chemical compounds were studied by GC-GC/MS. Generally, in 4 samples (samples were taken after 8, 24, and 48 treatment and control treatment), 10 different compounds were distinguished.  

Based on results, the study of the essential oils of Ajowan showed that the changes of the amount of secondary compounds in the essential oil, the amount of γ-Terpinene (precursor of monoterpenoids like Thymol) went up after 24 hours methyl jasmonate treatment. Due to Real-Time PCR dates, the quantity of all 3 studied genes increased after 24 hours methyl jasmonate treatment. The increasing of these genes expression can be causes the incline level of γ-Terpinene in essential oil.  

Increasing the amount of 3 mentioned genes expression and also a high amount of γ-Terpinene in essential oils after 24 hours in flowers show that this hormone can affect the gene expression in MEP pathway which causes monoterpenoids production increase after one day of using the hormone.

The aim of this research was to identify marker loci associated with some agronomic traits under drought stress conditions in Common millet.
          

In this study, 30 genotypes of Common millet were planted as augmented design at research farm of Shahid Bahonar University of Kerman in April 2016. In order to induce stress irrigation was stopped at 50% panicling stage. Traits including grain yield, biological yield, 1000- grain weight, number of grain in tiller, number of tiller in plant, panicle length, number of panicle branches, plant height, number of leaf in plant, leaf length, leaf width and harvest index were measured. The AFLP Technique was performed by the six EcoR1/Mse1 primer combinations.
         

In total, 246 polymorphic bands were generated and the average percentage polymorphic was 89.13%. Association analysis based on GLM and MLM model indicated that 52 and 64 markers associated to traits under drought stress condition, respectively. Among them, four markers (M4/E10-65, M4/E10-74, M4/E10-67 and M59/E36-105) with grain yield, three markers (M59/E36-84, M10/E1-244 and M10/E1-245) with 1000- grain weight, two markers (M59/E36-106 and M10/E1-224) with number of tiller in plant and five markers (M3/E2-31, M3/E2-35, M3/E2-38, M59/E36-114 and M4/E8-128) with number of grain in tiller showed highly significant and strong relationship under drought stress condition in both models.
         

In this study, several common genetic locations for the studied traits were identified. The existence of common markers between different traits may be due to pleiotropic effects or to linked genomic regions affecting several traits.

A molecular study of the buffalo genetic structure can be effective for better understanding the origin of this animal. Among the molecular markers, mitochondrial genomic sequencing is one of the best and most commonly used methods for genetic classification of populations and species close together, studying the possibility of deriving different species from a common ancestor, studying the phylogenic relationship of each species with other species and races, and obtaining solutions for conservation genetic resources. The purpose of this research was to determine the sequence of 12s rRNA and 16s rRNA regions of Khouzestan buffalo.

For this study, 30 blood samples collected from both sexes of unrelated Khuzestan buffalo. After DNA extraction, the target regions amplified by corresponding specific primers by PCR technique and sequenced after purification.
 

The results of matching the sequences of 12srRNA and 16srRNA regions of Khuzestan buffalo showed that there was no mutation in this population, which indicates low diversity in Khuzestan buffalo population. According to this study, the 12srRNA and 16srRNA regions are encoding regions and the mutation and diversity are low. The results of the phylogenetic test using UPGMA for both sites showed that the buffaloes of Iran with Indian and Italian buffaloes are in a closely spaced cluster.
 

Finally, the sequences generated from these regions were the first recorded in the gene bank with MG650115 access code and the name of the Khuzestan buffalo brought to the World Bank Gene, and this race introduced to international associations.

Dlk1 plays an important role in regulating skeletal muscle development and regeneration, differentiation of adipocytes, regulation of muscle cell growth, hepatoblast proliferation and blood cells formation. The aim of this study was to investigate Dlk1 gene expression in different tissues of Raini cashmere goat using Real Time PCR.

Tissue samples (18) including muscle, kidney, spleen, brain, liver and back fat tissues were taken from 3 Raini cashmere goats. RNA was extracted and cDNA was synthesized. Real Time PCR was performed using SYBR Green method to study relative gene expression. Beta actin gene was used as housekeeping gene. Pfaffl method was used to analyze achieved data.  

Results of this study showed that Dlk1 gene is expressed in all studied (muscle, kidney, spleen, brain, liver and back fat) tissues and the highest level of expression was observed in muscle and kidney tissues and the lowest level was seen in spleen and brain tissues.

This study would lay a foundation for further Dlk1 research in Raini cashmere goat and other goat breeds and also native animal and poultry. It is suggested that this study be conducted with greater number of livestock, different sexes, different ages and different physiological stages in different breeds of goats in order to reach a comprehensive conclusion.