فصلنامه ژنتیک نوین
سال دوازدهم شماره 3 (پیاپی 50، پاییز 1396)

Plant diseases are the most critical limitation factors in agricultural products. Citrus bacterial blast disease caused by Pseudomonas syringae pv. syringae is one of the most common diseases in in citrus gardens except in tropical region of the world. Host plants interact to bacterial pathogenes by inducing of resistance genes. Accumulation of pathogenesis related proteins such as, chitinase, ß-1, 3 and some of the other genes like phenyl alanine ammonia lyase and transcriptional factor WRKY40 are among the defens mechanisms in challengins to pathogens. In this research, the transcript levels of PR2, PR3, PAL and WRKY40 genes were evaluated in sour orange, limequat and okitsu lines in stimulating with Pss using quantitative real time PCR technique at different time courses. After inoculation of citrus lines to Pss, total RNA were extracted from samples at different time courses. Complementary DNA were synthesized and the expression levels of mentioned genes evaluated. Result of this study showed that transcripts levels of PR2, PR3, PAL and WRKY40 genes increased faster and significantly higher in okitsu and sour orange than limequat.

The purpose of this study was applying artificial intelligence for estimating breeding values of growth traits in Kermani sheep. The records of 867 lambs including animal sex, maternal age, birth weight and weaning weight (3 months old) were used for this purpose. These data were firstly analyzed using ASReml software and then was pre-processed for applying by the MATLAB software. After initial experiments on appropriate architecture for neural networks, it was found that numbers of neurons for input layer were 3 neurons for birth weight and 6 neurons for 3 months weight. The used model in this study was multilayer perceptron (MLP) and applied algorithm was back-propagation to minimize the mean square error. From total data, 70% used as training, 15% for testing and 15% were considered as validation. During the training process, network learning rate was measured regularly by the objective functions and was used network having the minimum error and maximum accuracy. The MLP neural network with 3 input variables, 8 neurons in hidden layer and with correlation coefficient 0.73 has ability to predict breeding values of birth weight and also the MLP neural network with 6 input variables, 3 neurons in hidden layer and with correlation coefficient 0.74 has ability to predict breeding values of 3 months weight. It can be concluded that artificial neural network has a good ability to predict breeding values of growth traits in Kermani sheep with acceptable speed and accuracy and can be used to estimate breeding values for all traits in livestock.

Fire blight is the most destructive vascular necrosis disease of pome fruits. Furthermore, secretion of bacterial effector proteins including HrpN, HrpW and DspA/E to host cells is the most important virulence factor of the bacterium. In order to study the role of these effector proteins on hosts, three mutant strains of pathogen including hrpN-, hrpW-,dspA/E- and wild type strain Ea 273 were used for inoculation of various pear cultivars including, Bartlett, Harrow Sweet and Dargazi. Due to the chloroplasts role in this interaction, all in vitro tests performed in two active and inactive chloroplast electron transport chain conditions. Evaluation of necrosis development index in post inoculation periods showed that the lack of the HrpN and DspA/E effector proteins, in hrpN- and dspA/E- mutant strains respectively, postponed the first necrosis triggering and decreased necrosis progress rate in comparison with the control strain. However the effects of dspA/E- mutant were more considerable. Moreover mutation in hrpW gene had the least decline in bacterial pathogenicity in comparison with the control strain. The effects of mutant strains on severity of pathogenicity and its comparison in two active and inactive chloroplastic conditions revealed that DspA/E, HrpN and HrpW had the most impact on pathogenicity of the bacteria respectively; likewise the effect of HrpW is inconsiderable. The results also revealed that active chloroplasts are necessary for HrpN impact on hosts. It demonstrates that HrpN can be the main factor for interaction of pathogen with host cell chloroplasts which is in accordance with its role on activation of systemic acquired resistance.

Melon is an important crop cultivated in moderate climate regions of the world. One of the most important diseases of this plant is vascular wilt caused by Fusarium oxysporum f.sp. melonis (Fom). The infection of farm with this pathogen can result in huge loss of yield and fruit quality of melon around the world. Genetic improvement for resistance is the best way of control of this disease. Up to now four races of 0, 1, 2 and 1,2 have been reported among which race 1 is more versatile and dangerous than other races especially at the north part of the country. Land races such as Saveh, Rish-baba and Tile-torogh are susceptible to this race. In this research the resistance gene was transferred from Isabelle, a resistant cultivar to susceptible varieties via marker assisted backcrossing. The screening of plants in BC1F1 and BC2F1 generations was carried out using artificial inoculation. Single dominant gene of Fom-2 induces resistance toward race 0 and 1. This gene has been sequenced and there are differences between susceptible and resistant allele. In order to verify the resistant plants survived after artificial inoculation in BC2F1, the plants were genotyped by SCAR marker. The resistance of most plants was verified however there were some susceptible plants among them implying the shortcomings of artificial inoculation method and more strength of molecular method. The verified plants were crossed to recurrent parents to produce BC3F1 generation. Considering acceptable yield and fruit quality of donor cultivar and evaluation results of BC2F1 it seems that no more back crossing is needed and with the selfing of BC3F1 plants new homozygote resistant varieties will be produced.
The screening of plants in BC1F1 and BC2F1 generations was carried out using artificial inoculation. Single dominant gene of Fom-2 induces resistance toward race 0 and 1. This gene has been sequenced and there are differences between susceptible and resistant allele. In order to verify the resistant plants survived after artificial inoculation in BC2F1, the plants were genotyped by SCAR marker. The resistance of most plants was verified however there were some susceptible plants among them implying the shortcomings of artificial inoculation method and more strength of molecular method. The verified plants were crossed to recurrent parents to produce BC3F1 generation. Considering acceptable yield and fruit quality of donor cultivar and evaluation results of BC2F1 it seems that no more back crossing is needed and with the selfing of BC3F1 plants new homozygote resistant varieties will be produced.

For economic reasons as well as animal breeding of farm animals, fertility rate is of particular importance. Bone morphogenetic protein15 (BMP15) is one of the genes that associated with fertility in sheep. BMP15 is located on the X chromosome and one of the X chromosomes is inactivated in female mammals because of dosage compensation comparing to males. This study aimed to evaluate the methylation status of BMP15 promoter on the inactive X chromosome. To do this, samples were taken from ovaries of 22 nonpregnant, and 8 pregnant ewes. DNA and RNA was extracted from the follicles. Using specific primers for the bisulfite converted DNA and wild type, DNA, the methylation status of the cytosine was investigated. Expression of this gene in pregnant and nonpregnant group was also studied. Results showed that promoter of BMP15 is not methylated even in inactive chromosome and unlike most genes in this chromosome escapes from methylation. This shows that despite the inactivation of one of the X chromosomes, both alleles of this gene in mutant homozygous individuals are active and this resemble the functional pattern of the genes that are located on autosomal chromosomes. Expression of the gene in different physiological steps supports this. Results showed that mRNA expression of the gene among pregnant and nonpregnant groups was not significantly different. It seems that, inactivating an allele of BMP15 in early life of animal permanently will make the mutant homozygotes to be fertile.

Due to high adaptability and tolerance to stressful condition, genus hawthorn (Crataegus) is considered as useful rootstocks for several species of pome fruits. On the contrary, due to high genetic diversity of this genus little information is available on its genetic relatedness and accurate use of each species. This research was considered in order to evaluate genetic diversity of several species and sub-species of hawthorn species that have been collected from several habitats of this species in Iran. 32 hawthorn genotypes including 14 species and 10 sub-species were evaluated by 32 pairs of specific SSR primers of apples and pears. The results presented totally 422 amplified bands and mean PIC parameter was 77 percent for each individual primer pair. Evaluation of genetic distance between hawthorn habitats in Iran by matrix of Nei similarity showed the highest distance between Kermanshah and Mazandaran, while the least distance was observed between West Azarbaijan and Guilan habitats. Cluster analysis by using Jaccard`s coefficient and UPGMA method demonstrated low similarity of accumulated hawthorn genotypes and therefore high distance of clustering in hawthorn genotypes, that could be a consequence of numerous species and poloidy levels of evaluated genotypes. Finally this research demonstrated high dispersal and complexity of genetic diversity of this genus in Iran that presents necessity of a comprehensive program for collecting and evolution of hawthorn species in their habitats.

A gram-positive bacterium Bacillus subtilis MJ01 strain was isolated from crude oil contaminated soil, Pazanan oil field in Khuzestan province, in order to produced surfactin and applied as microorganism enhanced oil recovery(MEOR). The primary research on this strain revealed that chemical and characteristics its surfactin was different among other surfactin variants. In the next step, whole genome sequencing of MJ01 strain was designed due to better understanding about variation of this strain and other close relative bacteria. The 16SrRNA genes analysis categorized MJ01 strain in Bacillus subtilis cluster close to spizizenii TU-B-10 strain in the same group. Multi-locus sequence typing(MLST) results was confirmed four of seven housekeeping genes in B. subtilis family were new mutant loci and one of them was inverted-competently in MJ01 genome. These results were shown that MJ01 MLST profile template have high similarity with spizizenii BGSC3A17 strain. ANIb (Average Nucleotide Identity based on BLAST) analysis with 12 close related genomes demonstrated that this strain has high identity to three spizizenii subspecies strains include TU-B-10 (99.13), NRS 231(96.52) and W23(96.52). Also based on ANI analysis revealed that it was closely related with Jeotglibacillus marinus DSM 1297. Non-ribosomal lipoprotein prediction online tools were used for located surfactin operon and flanking regions in MJ01 genome. Comparison of this operon and flanking region with other close related B. subtilis complete genome indicated that MJ01 placed in the spizizenii subspecies group. In summary, integrative phylogeny analysis and DNA homology using comparative genomics approaches supported that MJ01 to be included as a member of spizizenii subspecies group, although MJ01 have some specific strain differences.

Root-knot nematode (RKN) [Meloidogyne incognita] is one of the main pests of tobacco. Determination of genetic behavior for RKN resistance can be useful to improve the resistance of tobacco against RKN. This study was conducted to determine the combining abilities and genetic parameters of RKN resistance in tobacco using a five-parent diallel analysis by Griffing’s method. Three resistant parents (Urmia3, KY9, K17) and two susceptible parents (Ergo, Burley TMV4) were crossed based on half diallel. Fifteen genotypes (Parents and F1s) were planted in the greenhouse based on completely randomized design with five replications and inoculated with 2500 J2 M. incognita race 2 one week after transplanting. Investigation of Gall Index (GI), Number of Egg Masses (NEM) and Average of Eggs per Mass (AEM) showed significant differences among genotypes. The general combining ability was more important than the specific combining ability in GI and NEM. Non-additive effects in AEM and additive effects in GI and NEM had more important role. Narrow-sense heritability was 0.73, 0.54 and 0.07 for GI, NEM and AEM, respectively, which revealed that GI and NEM are suitable traits for RKN resistance identification. KY9 had the highest negative GSA in all traits. So this genotype is appropriate donor parent for RKN resistance.

The relationships between D, S, U and A genomes were investigated using 30 populations from 6 diploid species of Triticum and Aegilops based on karyological characters. Karyotype was prepared for 5 metaphases cells and the karyotypic traits were calculated in each population. The studying genotypes had a symmetric karyotype. The karyotypic traits showed variation between species clearly. Intraspecific diversity of each species were observed between populations. The species were separated based on cluster analysis, but Triticum species dont completely separation from Aegilops species and Ae. umbellulata had the more distance with other Aegilops species, also, Triticum species were separated in closer distance with Ae. Speltoides based on cluster analysis graph. Principal components analysis showed that interspesific variation was further based on intrachromosomal asymmetry and chromatin value which Ae. umbellulata had the most intrachromosomal asymmetry and the lowest chromatin value. On the other hand, Ae. speltoides, T. urartu and T. boeticum had the lowest intrachromosomal asymmetry and the most chromatin value. The total length of chromosomes and interchromosomal asymmetry was causes of intraspecific diversity. In addition, the U genome had the most distance with A genome, the S genome had the most similarity with A genome and the D genome had a moderate similarity with A genome.

Resistant cultivars are the best and the most effective solution for plants disease management Host and non- host, are to kinds of plant innate resistance. Functional analysis of mapped QTLs for resistance can be served as an important method to study the genetics of resistance for host and non-host plant pathogens. For this purpose, QTLs which were related to the rust resistance in the previous studies were collected from two mapping populations named Suspttit×Vada and Susptrit× cebadacab,. For both mapping population, a set of sequence- based SNP markers were developed in Wageningen University, The Netherlands,. The peak AFLP markers and Sequence based snp markers were used for estimation of correlation the peak markers related to each QTL with SNP markers. SNP markers with a high association were selected as candidate gene then used bioinformatics tools for investigation on their function. Functional analysis of genes in ontology terms showed that the most of host and non-host resistance genes involved in transferase activity, signal transduction and DNA transcription. Study on resistance gene ontology showed that in the molecular functionality, the most of the host and non- host resistance genes have similar behavior. The results of analysis for cellular components showed that plasma membrane, nuclear and chloroplast, are the three most common cell parts in which host and nonhost resistance genes were located. The activity of both genes in biological process, metabolic response to stress, RNA processing, were almost identical.

To evaluate the karyotype and genome size of Ae. triuncialis of Iran, 209 accessions from the collection of National Plant Gene Bank of Iran were studied using flow cytometry and cytogenetic traits. Accessions were analyzed by flow cytometry using DAPI method. The flow cytometry peak for Aegilops samples were in the range of 42 to 95 with average of 63.74 and standard deviation was 11.83.The average Peaks for control sample of hexaploid wheat was 120.67 with standard deviation 5.40. The mean ratio of sample peaks to check peaks for all samples was 0.44 -0.57 with average of 0.53. Karyotype studies showed that all Aegilops accessions were tertaploied and number of their genome was x=7. Karyotype of Ae. triuncialis was include 5-10 pairs of sub metacentric and 4-9 metacentric chromosomes, classified according to Levan et al. (1964). Two Pairs of satellite observed on short arms of chromosome number 8 and 12. The average of tallest chromosomes in accessions was 14.87 and average of shortest was 12.12 micrometer. Ratio of long to short arms observed from 1.66-2.03 and centromer indices from 0.35 to 0.39. So, Ae. triunciallis is in A2 according to Stebins table. Total form percentage (TF %) observed from 34.63 to 38.76. So, these characteristics show a relative symmetry for chromosomes of Ae. triuncialis and suggested that this species is in early steps of its evolutions.

Diabetes is a disease in which the body does not produce or properly utilize insulin. Although insulin is predominantly made in bacteria or yeast for commercial production, there is a burgeoning need for alternative systems in the production of insulin. In this regard, edible oyster mushroom was studied as host for the production of insulin. Therefore, a binary vector pCAMBIACTB-INS was constructed containing the cholera toxin B subunit fused with human proinsulin gene under the control of glyceraldehyde-3-phosphate dehydrogenase as homologous promoter. Then it introduced into gill tissue of edible oyster mushroom, Pleurotus ostreatus isolate Iran1649c, via Agrobacterium Tumefaciens strain GV3101 (OD600nm) and co-cultivated with P. ostreatus at 25 °C on selective medium containing hygromycin and cefotaxime. After confirming Putative transformants by PCR using gene specific primers, stable integration of the CTB-proinsulin fusion gene into P. ostreatus genome was confirmed by Southern blotting analysis. Also, the expression of transgene was confirmed by RT-PCR, ELISA and immunoblotting methods and in transgenic mycelia the recombinant CTB-proinsulin showed an expression level by 0.04% of total soluble protein. In conclusion successful expression of foreign genes in transgenic mushrooms is promising for development of biopharmaceutical in edible mushrooms.
medium containing hygromycin and cefotaxime. After confirming Putative transformants using PCR with specific primers, stable integration of the CTB-proinsulin fusion gene into P. ostreatus genome was confirmed by Southern blotting analysis. Also, the expression of transgene was confirmed by RT-PCR, ELISA and immunoblotting methods and in transgenic mycelia the recombinant CTB-proinsulin showed an expression level by 0.04% of total soluble protein. In conclusion successful expression of foreign genes in transgenic mushrooms is promising for development of biopharmaceutical in edible mushrooms.

Drought stress is an important yield-restricting factor in arid and semi-arid areas. Drought tolerance is a multigenic character and it occurs in various stages of growth. Since the mutagenesis is a fast and safe method, it can be used to diversify genetic contents of plant with the purpose of enhancing quantitative and qualitative traits. In this research, expression pattern of some genes involved in ABA-independent pathway in Tabasi Cultivar (drought sensitive) and mutant line T65-58-8 (drought tolerant, treated with 250 gray gamma radiation) were evaluated under drought stress conditions at maturity stage. In order to study the expression of genes involved in production of proline (P5CS, P5CR and P5CDH) and measuring its amount under drought stress and re-irrigation condition, seeds of two genotypes mentioned above were cultured in 24 cm pots. At the anthesis stage, irrigation was stopped until wilting symptoms appeared, then sampling from flag leaf was done at appropriate intervals (two, three, four, five and six days after irrigation stoppage) and also 12 hours, three, four and five days after re-watering. The control treatment underwent sampling simultaneously. The study was conducted as a factorial experiment based on completely randomized design with two biological replicates. The results demonstrated that the increment of drought stress led to higher expression of P5CS gene in Tabasi cultivar compared to the mutant line, while P5CR gene was more expressed in the latter. The expression of P5CDH in mutant line decreased, while Tabasi cultivar showed an increase in this regard. After re-irrigation, the expression of P5CS and P5CR in both genotypes decreased compared to the control, whereas P5CDH gene was expressed less and more in mutant line and Tabasi cultivar, respectively. Also, the results indicated that proline concentration in the mutant line increases more than Tabasi cultivar under drought stress, while it decreases in both genotypes as a result of re-irrigation. In general, mutant line T-65-58-8 adopts better remedial responses for expression of genes involved in proline production compared to Tabasi cultivar so as to remain undamaged, and it is probable that the osmolyte pathway underwent some changes as result of exposure to gamma rays.

In the present study in order to investigate the photosynthetic mechanism of this plant under salinity stress , and 600 mM NaCl were applied for 10 days then plant responses in physiological and molecular levels were assessed and compared to the control plants ( 0 mM NaCl). For this end, the coding sequence of this gene was isolated from Aeluropus littoralis for first time and the bioinformatic investigation were done. Results showed that the chlorophyll amount, the starch content and Sodium excretion of leaves in 600 mM NaCl were significantly (P

Cucumber (Cucumis sativus) is the most important greenhouse product in Iran,Yazd province. Fusarium crown and stem rot have been the major disease of greenhouse cucumber in Yazd Province. Recently various methods have been proposed to identify isolates of F. oxysporum. Due to defects of morphological characteristics to identify species and sub groups of Fusarium genus, research on molecular tools to determine evolutionary relationships of species have been studied. ForcF1 and ForcR2 specific primers to identify isolates of F. oxysporum f. sp. radicis-cucumerinum were used to amplify genomic regions OPB-07. Bands with molecular weights of bp277 were amplified using gene specific primers for all isolates. This is the first report of using SCAR primers for molecular identification of this form of F. oxysporum f. sp. radicis-cucumerinum.

The genus Potyvirus is one of the largest and economically important groups of plant viruses, due to their effects on crops worldwide. Potyviruses are accounted for 40% of viral diseases of various plants. Iranian johnsongrass mosaic virus (IJMV) is one of the important potyviruses infecting cereal crops and one of the main causal agents of maize mosaic in Iran. In order to study the this virus, 90 samples showing virus-like symptoms were collected from different maize fields in Mazandaran province during 2013-2014 and evaluated with DAS-ELISA test using polyclonal specific antibody. RT-PCR assay with degenerate primers corresponding to the virus CI gene led to the amplification of the expected DNA fragment with a length of 680 bp in all ELISA-positive samples. Two fragments were cloned in a pTG19-T vector and sequenced. BLAST analysis of the sequenced data confirmed that the PCR-amplified fragments belonged to IJMV. Phylogenetic analysis based on partial sequences of CI gene showed that these two isolates of IJMV had the highest nucleotide (84.2%) and amino acid (97.3%, 97.8%) identities and a close phylogenetic relationship with another Iranian isolate of IJMV namely Shz (JQ692088) isolated from Johnsongrass and previously reported from Shiraz. High-nucleotide differences between the IJMV isolates obtained from maize and Shz isolate from johnsongrass indicating high nucleotide variability of this potyvirus. These results demonstrated the occurrence and spread of IJMV in the maize fields of Mazandaran province based on serological test, RT-PCR, and CI gene analyses.