فصلنامه ژنتیک نوین
سال دوازدهم شماره 2 (پیاپی 49، تابستان 1396)

Mutation breeding in order to create mutations and diversity in crop genetics structure have been used in breeding for many years. In this study, to determine the induced genetic variation and select lines with desirable agronomic traits, 16 rapeseed mutant lines from three rapeseed cultivars Talayeh, Zarfam and Express obtained through gamma radiation with 800, 900 and 1200 gray dose rates followed by 7 selfing generation. These cultivars were compared in a randomized complete block design with three replications. Traits such as plant height, number of pod in main branch, secondary branches and the whole plant, number of branches, the length of main branch, number of seeds per major and minor pod, first pod height from the ground, stem diameter, length of main and secondary pods, days to flowering, end of flowering, days to maturity, grain weight, yield, oil content seeds and oil yield was evaluated. The highest variation coefficient of the number of pods per branch (19/23 percent) was in the Zarfam mutant lines. The mutant line, Z-800-6 had highest grain and oil yield compared to control Zarfam. The mutant line T-800-6 showed the largest grain and oil yield compared to control Talayeh and mutant E-900-1 had the highest grain and oil yield compared to control Express. Among all the lines and cultivars Z-800-6 with best kernel yield (5277kg ha-1) were considered as superior mutant lines.

The purpose of this study was to compare the accuracy of three genomic prediction methods based on traditional BLUP, G-BLUP and single step method ( or SS-BLUP) for milk (MY), fat (FY), protein yield (PY) and somatic cell count (SCC) in Iranian Holstein dairy cattle. In this study the genomic data records of 345 Iranian Holstein dairy cattle, genotyped using the Illumina Bovine SNP50 BeadChip, were used. Genomic breeding values for the studied traits was estimated by MIXBLUP software. Results showed that the mean of genomic prediction accuracy of traditional BLUP, G-BLUP and SS-BLUP was 0.39, 0.47 and 0.54 respectively. Accuracy estimation ranged from 0.32 for somatic cell count trait in traditional BLUP method to 0.59 for milk production trait in SS-BLUP method. Genomic prediction accuracies under SS_BLUP for the considered traits including MY, FY, PY and SCC were 0.1, 0.07, 0.09 and 0.07 higher than the corresponding values obtained under G-BLUP, respectively. The corresponding values under SS-BLUP were 0.16, 0.14, 0.17 and 0.14 higher than the values obtained under traditional BLUP. Mean squared error for SS-BLUP compared to other methods was lower for all traits. Genomic prediction regression coefficients under tradition BLUP, G-BLUP and SS-BLUP methods were 0.77, 0.88 and 0.94, respectively. Therefore, compared to traditional BLUP and G-BLUP, the SS-BLUP approach can significantly improve the accuracy of genomic prediction for milk production traits in Iranian Holstein cattle.

The health aspects of vegetables containing high anthocyanin has increased the consumption of colored potatoes rich in anthocyanin in recent years. Anthocyanin’s in blue and purple potatoes are derived from delphinidin, while in the red ones they are resulted from pelargonidin. Three genes, D, P and R interact for production of delphinidin and pelargonidin within the anthocyanin biosynthesis pathway that eventually results in colored potato flesh. Studying genetic and allelic relations among these three major genes provides a better understanding of anthocyanin biosynthesis pathway in potato. In this research, 17 potato genotypes of varying tuber color were used to detect the three key genes of D, P and R using specific primers and to compare their restriction fragment patterns. Results showed that purple genotypes contained the dominant allele for the P locus and the white and the red genotypes had the recessive allele. The white colored potatoes contained only the r allele in a homozygous state while the red and purple colored potatoes had both R and r alleles in a heterozygous condition masking the phenotypical presence of r. Restriction fragment patterns of the PCR products clearly revealed that none of the white potato genotypes possessed the D allele but the colored genotypes contained both D and d alleles in a heterozygous condition with an unknown allelic arrangements. D is an important regulatory gene for both P and R genes and the dominant allele D is required for their expression and anthocyanin production in the potato tuber. In other words, having dominant alleles of P and R in a potato plant does not necessarily give colored potato if the dominant allele D is not present. Albeit, restriction fragment pattern procedure discriminated the colored genotypes from the white tuber ones in P and R genes but primarily determination of D locus dominant status as a key regulatory factor in anthocyanin biosynthesis pathway is more important.

Citrus canker is a devastating disease that affects most commercial citrus varieties. In the present study changes of apoplastic proteome upon infection were put under scrutiny to define Citrus aurantifolia molecular response. In order to evaluate the changes of extracellular proteome, we employed proteomics to identify apoplastic proteins changed during infection. Citrus plants inoculated with X.citri subsp.citri and inoculated leaves and control leaves (mock inoculated) sampled at 1, 4, 7 days after inoculation in three biological replicates. Apoplastic proteins resolved by 2-DE and differentially expressed spots were identified by MS analysis. In two-dimensional electrophoresis analyses, 63 spots on the gels showed significant changes in abundance at 1,4 and 7 day after inoculation, as compared to non-inoculated plants. Of these 63 proteins, 35 were identified by mass spectrometry analysis using the NCBInr. These proteins were associated with the defense response, photosynthesis, metabolism, and other cellular processes in citrus aurantifolia. It seems that pathogen, by manipulation of host homoeostasis, proliferate and develop disease.

In this research population structure of Botrytis cinerea (tel: Botryotinia fuckeliana) determined by vegetative compatibility groups (VCGs) using sulfate non utilizing mutants (sul). For this purpose 26 isolates of B. cinerea were collected from different hosts in Varamin county greenhouses. All isolates identified and purified by single spore method and cultured on BSM medium. In order to determine the vegetative compatibility groups, all isolates were cultured on Vogel's basal medium amended with sodium seleanate (1-3 gr/lit). Selenate is a toxic substance for fungi and it is an analogue for sulfate in fungal metabolic pathway. Selenate-resistant mutants were isolated from all strains as spontaneous, fast- growing sectors arising from restricted colonies on medium amended with sodium selenate. Sectors were sulphate non-utilizing (sul) mutants if grew as thin expansive colonies with little or no aerial mycelium on MM (minimal media), but similarly to the wild-type on L-methionine- amended MM medium. MM amended with potassium chromate also used for partial phenotyping of sul mutants. Mutants that grew on MM amended with potassium chromate were chromate-resistant. Also mutants with inability for growing on this medium were indicated as chromat sensitive. In order to evaluation of vegetative compatibility groups (VCGs), chromate resistance and chromate sensitive mutants were paired in all possible combinations. All plates were monitored for formation of dense aerial mycelia (prototrophic growth pattern) at the contact zone of each two isolates up to 30 days. Isolates were assigned in the same vegetative compatibility group if represented prototrophic growth pattern along their contact zone. Among 26 isolates of B. cinerea, 15 VCGs were determined. No correlations were observed between VCGs with hosts and geographic origins of isolates.

Metallothioneins (MTs) are low molecular weight, Cys-rich proteins that are able to bind metals through the thiol groups of their Cys residues. Plants have multiple MT isoforms which are divided in 4 types based on distribution of Cys residues. In the present work, the gene encoding OsMTI-2b, a rice MT type 2 (Oryza sativa), was cloned into pET41a vector. The resulting construct was transformed into Escherichia coli strain Rosetta (DE3). Following the induction with Isopropyl β-D-1-thiogalactopyranoside the OsMTI-2b was expressed as carboxyl-terminal extensions of glutathione-S-transferase (GST). As compared with control, the E. coli cells expressing GST-OsMTI-2b showed increased tolerance against metals Ni, Cd, Pb and Zn. Furthermore in the medium of cells expressing GST-OsMTI-2b the concentration of Pb2, Cd2, Ni2, Zn2 and Cu2痧⭹꬞ 15.85±0.7, 13.07±0.8, 11.8±0.8, 14.51±0.75 and 7.47±0.6 ppm, respectively. In comparison, the concentration of these metals did not change in the medium of control strain.

Local species are considered as a part of national capital and strategic recourses. Due to significant decline of their population, identification, preservation and reproduction of local breeds is very important. In this study D-loop region of mtDNA was used for analyzing genetic diversity, phylogenetic and genetics structure of Khalkhali goat population. Blood samples were collected from 100 Khalkhali goats and, genomic DNA was extracted using DNA extracting kit and amplified with specific primers. Then, genotyping were performed using SSCP method and were sequenced through detection of different pattern. mtDNA sequences of other species were collected from NCBI (as outgroups sequences) and analyzed by using bioinformatics software’s. In total 62 mutations were identified in 126 sequences that cause 37 haplotype. Haplotype diversity, nucleotide diversity between species were 0.915 and 0.069 respectively. Genetic diversity and haplotype diversity of Khalkhali goat was 0.008 and 0.834 respectively, which indicated low genetic diversity in this breed. The comparison between khalkhali goat and others haplotypes showed that khalkhali goat were located on Haplogoup A.

Since the economic value of cultivars depends on different characteristics, thus procedure of selection for several traits to maximize the economic value has been always considered by plant breeders. Detailed information on genetic behavior and association between economic traits will help breeders to improve plant populations. In order to identify retrotransposon-based-molecular markers associated with agro-morphological traits in confectionery sunflower, a collection of 50 accessions were studied for 20 traits according to IBPGR sunflower descriptor in a completely randomized design with 10 replications. Genetic diversity of populations was assessed using 20 retrotransposon primers (7 IRAP markers and 5 REMAP primers). Genetic structure analysis of germplasm using Bayesian method revealed three sub-populations. Association analysis using mixed linear model (MLM) identified 37 loci significantly (P

In order to study the effects of Bacillus amyloliquefaciens1732 and salicylic acid on defense enzymatic and non-enzymatic mechanisms and gene expression of limonene hydroxylase using Real time PCR in green mint under drought stress, a factorial experiment based on completely randomized design wih three replications was conducted There levels of drought stress including 80% field capacity as a control, 60 and 40% of field capacity and , factor B including biological fertilizer (inoculated and non-inoculated), and factor C as salicylic acid (zero as control and 1 milli molar). The results of experiment showed that Bacillus amyloliquefaciens1732 and salicylic acid increased the non-enzymatic antioxidant traits of phenol and flavonoid. Also expression of limonene hydroxylase gene as an involving gene in monoterpenes synthesis under stress and treatment of bacteria and salicylic acid was increased. Application of bacteria and salicylic acid treatment did not have significant effect on antioxidant enzymes whereas drought stress significantly increased the antioxidant activities. The results showed that Bacillus amyloliquefaciens1732 and salicylic acid through non-enzymatic traits of total phenols and flavonoid and enhancement of second metabolites as the first defense reduced the destructive impacts of drought stress. In conclusion the plant showed better resistance and used lower energy for production of antioxidant enzymes.

As the most easterly species of the subgenus Naja, the Caspian Cobra (Naja oxiana) has long remained a case of major controversy with regard to phylogenetic relationships. The aim of the present study is to analyze the evolutionary relationships, genetic diversity, structure and variations of the Caspian Cobra in north and northeastern populations of Iran. To this end, 5 to 10 abdominal scales were obtained from 19 collected individuals from natural habitats in the north (Golestan province) and northeast (Khorasan Razavi and South Khorasan provinces) of Iran. DNA extraction was done using Ammonium acetate. Forward and reverse primers were used to amplify a segment of the D-Loop gene with 589 base pairs in length. The genealogy analysis was conducted to determine the best evolutionary model using Bayesian and maximum likelihood analyses. Analysis of haplotype relationships was performed using maximum parsimony. The results identified seven unique haplotypes and one common haplotype among populations of the north (Golestan province) and northeast (Khorasan Razavi and South Khorasan province) of Iran. Genealogical analyses revealed that populations of the Caspian Cobra have bifurcated from the Chinese Cobra, supported with rather inadequate bootstrap and posterior probability values of 54.31 and 0.61, respectively. Eventually, Analysis of Molecular Variance (AMOVA) found no significant genetic variation (Fsc 0.05, p 0.05) between populations of the Caspian Cobra in the north and northeast of Iran, whereas significantly large genetic variations (Fsc 0.25, p 0.01) were detected between Caspian and Chinese Cobra populations. Our data set demonstrated that the Caspian Cobra lineage is genetically relatively homogenous, as we found no significant genetic variation between populations of the species in Iran, possibly suggesting their historically recent radiation in the most western part of the species range in Asia. It is demonstrated that an integrated conservation plan is needed to protect the Caspian Cobra in populations of the north and northeast of Iran.

Bread wheat is one of the most important plant and hybrid varieties plays an important role with regard to the food supplement in the world. Some of the traits of wheat that show significant differences in hybrid more than parents should be responsible for the observed heterosis. This phenomenon is controlled by genes/proteins that identification of these genes in molecular approach is important issue for plant breeding projects. The purpose of this research is identification of heterosis related proteins in bread wheat via proteomics. Three varieties, zagros, N8118 and their hybrid were cultured in randomized complete block design in 3 replications. Morphological analysis with SPSS shows significant difference between hybrid and parents in stem length and spike length. Also the results of proteomics show that the expression of one protein spot has changed in type of position shift in zagros variety and this spot is related to cytochrome b6/f complex. This protein plays an important role in production of ATP and NADPH in photosynthesis pathway.

Salt stress negatively affects many physiological processes in plants. Some of these effects may involve the oxidative damages of cellular components, which can be created by reactive oxygen species (ROS) and prevented by antioxidant enzymes. In this study, FeSOD and Ferritin-3 genes expression under salt stress (0, 25, 50, 75, 100 and 150 mM Nacl) in peppermint using quantitative real time PCR method were investigated. Samples of the leaf and root were collected 24, 48 hour, one week and one month after exposure to stress. Results showed that expression of FeSOD gene up-regulated by increasing salt stress in both leaf and root, but it was higher and more stable in leaves. Expression of this gene was down-regulated after 24 hour, but up-regulated after 48 hour and one week in the leaves, and down-regulated significantly after one month again. In the roots, the FeSOD gene expression was down-regulated after 24 hour, alike leaves, but up-regulated after 48 hour, one week and one month. The expression pattern of Ferritin gene was various in the leaf and root. In the leaves, expression of Ferritin gene was up-regulated in low concentration of salt stress (25 & 50 mM NaCl), then down-regulated by increasing of salinity levels but in the roots, the gene expression down-regulated in all of the salinity levels. The Ferritin gene expression was up-regulated significantly in the early stages of exposure to the salt stress (after 48 hour), and then down-regulated in late time course of exposure to the stress in the leaves. In the roots, the expression of this gene was down-regulated after 24 and 48 hours and one week exposure to the stress, but up-regulated significantly after one month.

Cumin (Cuminum cyminum) an annual herbaceous plant from Apiaceae is the most important medicinal plant for export in arid and semi-arid of Iran. Seeds contain phytochemicals with antioxidant and anti-flatulent properties. Till now, no cultivars have been introduced in the country. Getting an information about genetic diversity extent is a perquisite of any breeding program. In this study, ten SCoT primers were used to assess genetic diversity of 49 endemic varieties from different areas of Iran including nine provinces. Totally, 48 bands were amplified of which 41 were polymorphic resulting in a polymorphism frequency of 62.03% and an average of 5.1 polymorphic bands per primer. Out of nine primers, one primer (SCoT26) had no polymorphic band and one primer was not amplified. The size of bands varied between 250-3000 bp. Dendrogram was gained using Centroid Linkage and revealed five main clusters. Each cluster was subdivided into subgroups. The farthest genetic distance demonstrated among Esfahan-Feridoon and Northern Khorasan-Shirvan ecotypes and shortest revealed among Northern Khorasan-Esfarayen and Northern Khorasan-Maneh. According to Shannon-Weaver and Nie diversity indices, Kerman population have the maximum within population diversity and Semnan population have the minimum within population diversity. Fst and Nm amounts indicating high level of gene flow among nine Iranian cumin populations. Due to values of heterozygosity of subpopulations (Hs), total heterozygosity (Ht), there is more diversity among populations than diversity of within populations. Analysis of molecular variance within and among populations (AMOVA) evaluated. The present investigation revealed that significant genetic variations present among different ecotypes that could be successfully utilized in cumin breeding programs.

For investigating mechanisms of tolerant to slat and drought stress in canola (Brassica napus L.), two spring canola cultivars such as SW5001 as tolerant cultivar and Sarigol as sensitive cultivar were evaluated at two levels of salinity and drought in factorial based on randomized complete block design with four replications using a hydroponic culture system. Sampling for proteomic analysis was performed in the rosette stage and before the stem elongation stage (two weeks after salt stress treatment ) in greenhouse. To investigate the proteome response of rapeseed to salt stress and drought stress, protein extraction from leaf was performed and subjected to first dimensional electrophoresis using IPG strips for both control and stressed plants. In the second dimension, proteins were separated by SDS-PAGE and then, staining with commassie brilliant blue, gel acquisition and quantitative analysis of protein spot by PDQuest software. Finally, 17 and 25 protein spots between control and treatment plants for salinity and drought, were detected, respectively. 10 and 15 protein spots were common between two cultivars under salt and drought stress separately. Also, four and six digit unique protein spots for tolerant cultivar under salt and drought stress respectively while for susceptible cultivars were three and four unique protein spots, respectively. The total of all proteins in response to salt stress and drought in the SW5001 and Sarigol, three proteins common in both cultivars and both the stresses that such as Oxygen-evolving enhancer protein 2-chloroplastic, Ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit of ATP synthase CF1 beta subunit of the protein were identified responding to drought and salinity. This proteins are involved in cell three members of the complex in photosystem II, ATP synthase and Rubisco that seems to be the most important cell components under salinity and drought. Thus, increasing the efficiency of the three complex cause to increase the efficiency of the cells and decrease the harmful effects of salinity and drought.

This study was conducted in order to identity mutation in GDF9 gene and possible association with the sperm quality and testicular biometric traits using PCR-RFLP method. For this purpose, the blood and sperm samples were collected from 96 rams belonged to Sanjabi breed. After the extraction of DNA, amplification of 462bp fragment including GDF9 gene exon 1 region was performed using a pair of specific primers. Then, enzyme digestion of products was performed using Hhal restriction enzyme. The results of digestion process approved absence of G to A mutation in the studied population and all samples showed to be wild-type genotype (GG) in this position. In regard to significant differences in sperm quality traits in sampled rams and as well as the incidence of twining in Sanjabi breed ewes, the results of this study showed that other studies must be performed in order to identify mutations in other regions of this gene and other genes to know responsible genomic regions for the sperm quality in rams and also twining in Sanjabi breed ewes in order to improve fertility in flock.

The use of molecular markers clearly reveals the diversity of pest populations, to take a convenient approaches of controlling them. In this study, the genetic diversity of Leopard moth (a borer pest of walnut trees) from different parts of Iran was studied by using primers of the COI and ITS regions. The sequences were aligned by the BioEdit program and analyzed by the PAUP program. For this propose, specimens of the pest from different geographical regions were collected and the COI and the ITS region were amplified by appropriate primers (four pairs) using PCR reaction and were subsequently sequenced. By following alignment of the COI sequences, 654 nucleotides positions were distinguished that 459 of them were constant, 149 positions were uninformative and 49 positions were informative. While for the ITS1 region, 504 positions were distinguished that 108 positions of them were constant, 378 positions were uninformative and 18 positions were informative. The constructed phylogenetic tree for the COI sequences showed that the population from Gonabad, Shahrood, Tabriz, Kerman and Karaj were classified in one group, representing minor differences among them. These minor differences among populations were also seen for the ITS1 sequences more clearly. The result showed, the population of different regions like Karaj, Shahriar, shahrood and Khorasan were very close in the constructed phylogenetic tree, while the population of Sarayan (South Khorasan province) and Gonabad (Razavi Khorasan) that are geographically contiguous were much distant from each other. Accordingly, it can be said that the differences among the leopard moth populations in Iran, are not affected by genetic divergence and the other factors, such as host diversity, have the major role.