مجله بیوتکنولوژی کشاورزی
سال هفتم شماره 3 (پیاپی 19، پاییز 1394)

In this study 17 ISSR molecular markers were employed to investigate the genetic diversity among 25 female pistachio genotypes. The markers produced 148 bands in total, of which 123 (83%) was polymorphic and the average of polymorphism for each primer was 7.23 band per primer. Cluster analyses of genotypes were estimated based on Jaccard's similarity algorithm and UPGMA clustering method and placed the genotypes in seven groups. The results obtained by similarity matrix revealed the highest similarity between cultivars "Khanjari Damghan" and "Fandoghi 48" (63%) and the lowest similarity between cultivars "Ebrahimi" and "Shasti" (2%) which is due to high amount of polymorphism among the genotypes. This experiment shows that ISSR markers are reliable for assessment of pistachio female genotypes and genetic diversity study. Moreover, these markers can be used to provide molecular genetics identity for Iranian pistachio populations.

Although wolves have the ability to live in different habitat types, in recent years their population has been drastically reduced in most parts of the country and in some areas has become extinct. Conservation programs are essential to ensure the viability of the remaining wolf populations. Wildlife management programs are depended on estimation of population size. With the development of molecular ecology in recent years and considering the ineffectiveness of traditional methods to estimate population size, population genetics methods have been developed to estimate population size more accurately than traditional methods. The mitochondrial genome is a strong indicator in predicting changes of population size of wild animals and calculating the effective population size (Ne) of female animals. Using mtDNA markers, we calculated the female effective population size in Hamedan, Qazvin, and Zanjan provinces. According to sex ratio (1:1) and ratio of effective population size to the census population size (Ne/N), the total population size was calculated between 90 to 360 wolves in these provinces. Our results suggest that estimating the effective population size by molecular markers is a useful tool for management of small and isolated populations.

To meet the growing demand of world’s current population for food, devising specific strategies are required. Production of genetically modified crops has been considered as a suitable strategy so that the number of countries producing GM crops rose from 29 countries in 2012 to 29 in 2013 with 175.2 million hectare cultivation area. Among the developed GM crops, Bt crops with more than 24 million ha cultivation area have been accepted greatly. Although they offer too many advantages, concerns regarding biosafety issues exist. However, no report against the of undesirable effects of GM crops exists, risk assessment of certain concerns relating to biosafety of Bt crops is necessary. Some of these concerns include risk to non-target organisms, persistence of Bt residue in Soil, acquisition of weediness potential, monoculture and eventual loss of biodiversity, unintended effects of gene transfer on plant metabolism and gene flow from Bt transgenic crops. The present reviews emphasis the considered concerns in relation to Bt transgenic crops, their assessment and management that can be used by policy maker or experts.

1-aminocyclopropane-1-carboxylic acid oxidase (ACO) enzyme is a member of the Fe (II-dependent family of oxidases/ oxygenases) and for activity requires Fe2+ as cofactors and ascorbate as a co-substrate. This enzyme is the only oxidoreductase in the ethylene biosynthesis pathway and catalysis the biosynthesis terminal step. In the present study, total RNA was extracted from tomato fruit (cv, Memory I), and then, coding cDNA of the LeACO1 gene was synthesized by reverse transcription polymerase chain reaction (RT-PCR). The PCR product was cloned into a pUC19 plasmid, and then, their biochemical, structural and phylogenetic characteristics were analyzed. LeACO1 amino acid sequence studies in tomato and other plant of such family were indicated that His177, His234 and Asp179 amino acids are formed a triangle catalytically site to play a role in binding Fe2+ cofactor. Phylogenetic and multiple alignment analysis were illustrated a high degree of identity to those of the ACO1 gene from other plants.

Salmonella is one of the causes of infectious diseases as a common disease in human and animals, has important health and economic terms, which has so far of a vaccine for it not been established. The InvG gene due to structural and Type three secretion systems as one of the primary genes located in the membrane of Bacteria also play a major role in the initial binding of the Bacteria to the host cell. The main aim of the present study was to introduce recombinant protein expression InvG as an effective adjuvant and a DNA vaccine. The design makes use of specific primers and reaction PCR, InvG gene was amplified Salmonella. After purification, the gene in the plasmid vector pET32a(+) cloned for expression. The recombinant plasmid pET32a-InvG into Escherichia coli strain became BL21-DE3. To produce recombinant protein expression system, the Bacterial expression vector containing the target gene (InvG) induced using IPTG. The results of sequencing showed that the sequence of the amplified gene cloned into pET32a expression vector for gene InvG with sequences recorded in the same gene bank. Recombinant protein production with IPTG induction to vector containing the plasmid pET32a-InvG performed successfully. Confirmation of InvG gene expression in the expression system performed by SDS-PAGE and Dot blotting analysis. The present study showed that the production of InvG recombinant in E. coli hosts is possible and protein with a weight of about 81 kDa was produced.

Wild sheep is belonging to Bovidae family and genus ovis which living in the most regions of Iran. There are many challenges between researchers about Wild Sheep species taxonomy. This study aims to investigate genetic and phylogenetic analysis of cytochrome b region of ovis orientalis's mtDNA in the Buruieh wildlife refuge in order to determine the correct taxonomy. With this regard, 17 samples of ovis orientalis stool and tissue were collected from Buruieh Wildlife Refuge in Khatam Township. After DNA extraction 1140, 741, 765 bp fragment of the mitochondrial cytochrome b was amplified with primers Cytb F-Cytb R, Cytb F- Cytbin R and Cytb R-Cytbin F under polymerase chain reaction. Amplified fragments were sequenced by ABI 3130 automated device with Sanger method. The results were compared with sequences from other studies to determine the genetic distances. Hoplotype variations were not observed in the nucleotide sequences obtained in this study. Using the Neighbor-Joining phylogenetic test results showed that the Buruieh Wildlife Refuge wild sheep is placed in the group of ovis orientalis and is different from other ovis orientalis populations all around Iran in the terms of Hoplotype. Our results demonstrated that the Buruieh Refuge Wildlife Sheeps have low genetic diversity which should be considered at the long-time managment activities in the future.

The aim of this study was to investigate polymorphism in the exon 3 of myostatin gene and its relationship with twining rate in Markhoz Goat. In this study, blood samples of 150 goats were collected from Animal Science Research Station of Sanandaj province. After DNA extraction, one set of specific primers was designed and used to amplify a 475bp fragment of the myostatin gene exon 3. The enzyme digestion of the PCR products confirmed presence of a mutation from T to G nucleotide in position 169 of the amplified fragment. The genotype frequencies of TT, TG, and GG were estimated 81.3%, 12.7% and 6%, and allele frequencies estimated were 88% and 12% for T and G alleles, respectively. The Chi-Square test showed Hardy-Weinberg equilibrium in myostatin gene (P<0.01). The amounts of Odd Ratio estimated for twining rate were 1.1032 for first parity on second parity (P>0.05) and 4.9497 on third parity (P<0.1), and 5.4605 for second parity on third parity (P<0.1). Also, the Chi-Square statistic (5.68) showed that twining rate in Markhoz goat breed is statistically affected by myostatin gene genotypes (P<0.05). The Odds Ratios obtained to compare the different genotypes of the myostatin gene showed superiority for twining rate as TT>TG>GG genotypes. The Chi-Square statistics equal to 2.65 demonstrated significant differences between two TT and GG genotype groups for twining rate in Markhoz goat. The results of this research demonstrate that the myostatin gene can be considered as a suitable marker to improve twining rate in breeding programs of Markhoz goat.

Filia is a maternal effect gene, disorder in which causes decreased fertility in mouse and abortion in human. Bovine Filia transcript is predicted in NCBI database. Nucleotide analysis shows that it has a higher GC content in comparison with its mouse or human orthologs, and this can make problems in PCR amplification. This study was performed to investigate the possibility of amplification of bovine Filia transcript with high GC content by optimization of primer design. Two pairs of primers were designed to amplify 443 and 230 bp fragments. High Tm of primers and low ΔTm conditions were considered to design only the first pair of primers. Bioinformatic analysis was performed on the two fragments to analyze the GC contents and possible secondary structures. RNA was extracted from bovine oocytes. After reverse transcription, PCR amplification was performed with different annealing temperatures in two or three steps. Results showed that the 443 bp fragment has a CpG island and a higher GC content and more possibility of secondary structure formation compared with the 230 bp fragment. Nevertheless only the 443 bp fragment with the primers of interest was amplified with a sharp band. In conclusion we showed that low ΔTm, high Tm of primers and annealing temperatures and also a two-step PCR help specific amplification of GC-rich sequences.

Plasma membrane proton pump (H+-ATPase) has important roles in various physiological processes. Hence, the expression pattern of the gene which encodes a plasma membrane proton pump was surveyed in the shoots of resistant and sensitive wheat cultivars and its wild relative, Aegilops crassa under salinity stress. Salinity stress induced by adding 150 mM NaCl to the basic solution and sampling was performed in three intervals: 0, 12 h and 3 weeks after applying salinity treatment. A quantitative PCR technique was used to study the relative expression of the gene of interest and a gene encoding actin was used as the reference gene to normalize the samples. The results showed that the expression level of the gene encoding plasma membrane proton pump significantly increased in response to salt stress after 3 weeks in comparison to 12 h after applying salinity treatment. It also revealed that the expression level of the gene was significantly higher in the resistant cultivar, Arg, than sensitive cultivar and Aegilops crassa. It revealed that the expression level of the gene was not significantly different in sensitive cultivar under salinity treatment in different times after salinity treatment. The bioinformatics analysis of promoter region of the gene encoding plasma membrane proton pump showed that various motifs within this region involved in response to abiotic stress and light. Overall, bioinformatics and laboratory studies revealed that gene encoding a plasma membrane proton pump plays a role in induction of salinity tolerance.

In this study genetic variation of Artemia urmiana and Artemia franciscana populations were assessed using five microsatellite markers including Af-B105TAIL, Af-A136, Apdq03TAIL, Apdq04TAIL and Apdq05TAIL from Artemia franciscana and Artemia parthenogenetica. DNA was extracted from 50 cysts of Artemia urmiana and Artemia franciscana populations individually by Hot Shot method. Polymerase chain reactions (PCR) were successfully conducted with all primers and then the PCR products were electrophoresed using 6% none denaturing gel and stained using silver nitrate method. Hence, all alleles were polymorphic. Average number of alleles and polymorphic information content (PIC) for Artemia urmiana and Artemia franciscana populations were 3.0, 0.54 and 2.5, 0.38, respectively. All loci in Artemia urmiana were in HWE but only the Af-A136 locus in Artemia franciscana was in HWE. The average expected heterozygosity for Artemia franciscana and Artemia urmiana were estimated as 0.6209 and 0.4531, respectively. The phylogeny dendrogram based on the Distance Matrix was drawn using UPGMA for within populations. Our findings demonstrated that microsatellite markers could be an appropriate tool for screening biodiversity in animals. Therefore, extinction of these invaluable genetic resources can be preserved using accurate breeding and management programs.

Keeping genetic diversity among native Iranian breeds is very important as a national resource. Studying mitochondrial genome between or within breeds can be a useful indicator of genetic diversity of the population to be studied. The aim of current study was bioinformatic and phylogenetic investigation of NADH3 and NADH4L genes of mitochondrial genome in Camelus bactrianus in Iran. For this purpose, blood samples were collected from 10 Camelus bactrianus in Iran. After extracting DNA, fragment 971 bp of genome mitochondrial of Camelus bactrianus amplified by primers. The amplified fragments were sequenced after purification. Results indicated two different haplotypes based on one single nucleotide polymorphism sequence. The final sequences of each haplotype had a length of approximately 715 bp which included 27/70 % adenine, 13/80 % guanine, 25/60 % cytosine and 32/80 % thymine. Comparison of nucleotide and amino acid sequences of NADH3 and NADH4L genes among Iranian Camelus bactrianus demonstrated that this specie had close genetic distance with domestic Camelus bactrianus. Phylogenetic analysis using Neighbor-Joining method showed that this specie has the lowest similarity with Lama among the Camelidae family.

Today, Karyotying studies are important to understand the genetic structure of animals. In the current research to evaluate the chromosomal structure of some Iranian native dogs, blood samples were collected from the Kurdi, Sarabi and samples of Alborz province. Blood samples were cultured in defined medium for 72 h at 37 ˚С. Then the cell divisions were stopped at metaphase stage and chromosomal specimens were prepared for karyotype analysis. Next, centromeric index, total length of chromosome, chromosomal arm ratios and relative length of chromosomes were analyzed and the ideograms were created. The results indicated that the dog genomes contain 78 chromosomes including 38 acrocentric pairs in each sex, a pair of metacentric X in males and females and one metacentric Y chromosome in males. The average relative length of the autosomal chromosomes ranges from 1.13 to 4.89 μm in males and 1.10 to 5.21 μm in females. The ANOVA of the chromosomal data indicated that significant (P≤0.05) differences in chromosomal parameters were observed in animals. The results indicate a bias of chromosome asymmetry in animals which could be related to evolutions of the canine chromosomal structures.