JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2014 | Volume:6 | Issue:2|Papers Count:16

Carnation etched ring virus (CERV) is a member of family caulimoviridae, caulimovirus genus. The virus is the second important virus after Carnation mottle virus to infect carnation. In order to investigate the presence of this virus in greenhouses carnation plats of Khorasan Razavi (Mashhad) and Markazi (Mahallat) provinces samples which have been showed the symptoms were collected. Primary identification of CERV tested by Indirect enzyme-linked immunosorbent assay. Total DNA was extracted from fresh young leaf tissue by CTAB buffer. Polymerase Chain Reaction (PCR) was carried out using specific primers and fragments of 1500 bp were amplified by PCR. Comparisons were made between these isolates and related sequences of other caulimoviruses available in Genbank. The phylogenetic tree of inclusion body (IB) gene of CERV was drown, by MEGA5 software using Neighbor joining method. The results showed that the maximum similarity of Mahallat isolate was 99.4% to Indian isolate (AJ853858). Comparision of nucleotide sequences was shown the homology of two Iranian isolates together (92.2%) and with the other isolates (90-99.4%). Maximum amino acid changes were indicated in Holland isolate. Although there was a significant difference (60.6-92.9%) in amino acid level between the sequences of the IB region of different caulimovirus.

The wolf (Canis lupus) in Iran is widely distributed all over the country, except in the deserts. Recent studies showed that dogs (Canis lupus familiaris) were domesticated by managing small packs of wolves in south-west and south-east of Asia, and consequently, they are very similar genetically. We used mitochondrial DNA control region (D-Loop) to compare the nucleotide parameters between wolf and dog populations in the country, since it shows a high mutation rate and is known as a good marker to identify intra-group variations. Our results revealed that there were no significant differences in the frequencies of adenine, cytosine, thymine and guanine bases of the control region between wolf and dog populations. Furthermore, nucleotide substitution patterns of this region uncovered a large value of transitions and transversions in both species. The results also demonstrated that wolf populations in Iran have maintained favorable genetic diversity, but habitat destruction is very likely to affect them in near future.

Maintaining redox homeostasis in the cell is critical for different cellular metabolisms and signal transduction pathways. In plants different molecular mechanisms are involved in maintaining cellular redox homeostasis. NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR) and thioredoxin plays important role as electron donor to disulfide bonds in many cellular proteins. In this study, the gene encoding one of NTR isoform from rice, namely OsNTRB was cloned in pET28a as fusion with His6-tag and transferred to Roseta (DE3), a strain of Escherichia coli. Considerable amount of His-OsNTRB was produced after induction of bacteria culture with IPTG and purified using affinity chromatography. Heterologous expression and purification of recombinant form of OsNTRB enabled us to study the interaction of this protein with thioredoxin from barley (HvTrxh1) and E. coli (Ec Trx). The results showed that recombinant form of OsNTRB is active and can reduce both HvTrxh1 and EcTrx in vitro. However the rates of reaction with these two Trx were significantly different.

According to the importance of studies of abiotic stress, present study was done in order to identify of molecular markers with significant association to flooding stress tolerance in barley. This study carried out using of AFLP markers and 40 barley genotypes. Generally 22 variables in both normal and flooded, eight characters including shoot dry weight, length, volume, wet weight, dry weight, leaf area, root diameter and density tolerance was assessed with six indicators (SSI, STI, GMP, TOL, YI and YSI). Seven primer combinations of EcoRI and MseI a total of 245 bands were produced, of which, 227 bands were polymorphic and had an average of 92.37 percent polymorphism. Three combinations E90-M160, E100-M160 and E90-M150 had higher values of genetic variation compared to other combinations and were more effective in distinguish of genotypes. Association analysis was performed using structure matrix and statistical models of GLM and MLM by using of TASSEL software for 22 variables. The MLM model in 5 percent probability level identified 87 markers related to evaluated traits. According to the results of association analysis in normal condition the highest of coefficient of determination was for E100M16027 with explanation of 32 percent of root diameter variation and for tolerance indices and stress condition the highest coefficient of determination were for E80M1501 and E100M15022 with 24 percent explanation of variation of GMP index and shoot dry weight respectively.

The control of flowering time requires activation of a cascade of successive genes which are affected by several internal and external factors. Some of these genes, such as Curly Leaf (CLF), have inhibitory effect on flowering. Thus, by silencing of these genes, initiation of flowering can be accelerated. In this research, the RNA Silencing technology is used to investigate the possibility of production of early-flowering plants via CLF silencing. First, in order to produce hairpin structure, a fragment of CLF gene of Arabidopsis thaliana was amplified by PCR and then cloned in two sense and antisense directions, into pFGC5941 vector. The resulted recombinant vector was then transferred into Arabidopsis plants via Agrobacterium method. As expected, initiation of flowering was accelerated in transgenic plants in comparison with the wild-type plants. In agreement to the obtained results, RT-qPCR analysis showed that CLF expression in transgenic plants was decreased in comparison with the wild-type plants. In contrast, FT (a gene which induces flowering) expression was increased in transgenic plants. These results show that RNA silencing as a novel and time-saving biotechnological method could be applied to generate early-flowering plants.

Remobilization of water soluble carbohydrates of wheat stem especially fructan play an important role in grain filling and yield production under stress. To study the effect of salt stress on the key genes involved in this mechanism, 1-sst and 6-sft genes contributed in fructan biosynthesis, 1-feh and ivr genes involved in degradation of fructan and sucrose, respectively, and sut1 as a sucrose transporter gene were examined in Bam as salt-tolerant and Ghods as salt-sensitive varieties using Real-Time PCR. Salt stress was applied since anthesis by irrigation water with EC of 15dSm-1. The experiment was done in greenhouse with three replicates using completely randomized design with factorial arrangement. Sampling was done for stem fructan content at five points with 7-day intervals during seed-filling period and for measurement of prolin and relative water content (RWC) from leaf and gene analysis from stem and seed at day 21 after anthesis. Fructan remobilization was estimated by subtraction of maximum and minimum of fructan content. Results showed that salt stress had a significant influence on RWC and prolin content and induced fructan remobilization along with the stem 1-sst, 1-feh and ivr genes as well as seed sut1 gene in Bam. There was a significant positive correlation between the 1-feh and ivr expression and fructan remobilization under salinity. Based on the obtained results, Bam had higher capacity to hydrolase and remobilize fructan by up-regulation of the critical genes during seed filling, so it was more efficient to use stem reserve carbohydrates and produced higher grain yield under salt stress.

Germination vigour is one of the most important factors for plant stablishing under drought stress. In order to detect of quantitative trait loci related to drought tolerance in germination stage a genetic map was provided by 74 SSR marker and 192 individuals of F2:4 derived from cross between Tarom mahalli´Khazar population. The hundred seeds of 20 families were used for recording of germination rate, radical length and plumule length. Seventy four SSR markers grouped in 12 linkage groups. Thirteen QTLs were detected for traits. Three QTLs were mapped on chromosome 1 that increased drought tolerance in germination stage in rice. Out of these QTLs, qGR-1a distinguished as major effect and explained 26.27% of the total variation. Correlations between traits and overlapping QTLs in RM466-RM259 interval provided a good region for gene pyramiding and marker assisted programs.

Orobanche spp. is holoparasitic plant, parasitising roots of different crops. Genetic polymorphism was investigated among and within 3 Orobanche species collected from different regions of northwest Iran, using ISSR markers. Out of 34 ISSR primers tested, 20 were found to be polymorphic and produced clear bands. 261 discernible bands were generated with 254 (94%) being polymorphic. Among studied species, "O. aegyptiaca" showed 2 unique bands. Clustering algorithm was divided collected Orobanche specimens into 6 main groups. It was obvious that genetic relationships among studied landraces did not have force tendency to associate with their geographic origins. According to AMOVA, 100% of the total variation was partitioned within species. Such variability is important for any attempt to develop resistant host crops against parasite.

To study of fungi and symptoms associated with vine trunk diseases, various vineyards in Kerman province were inspected during spring and summer of 2012. Samples showing decline symptoms were collected from trunk and branches of vines. Fungal isolation from affected tissues were done on malt extract agar medium (MEA) supplemented with 1 g 1–1 streptomycin sulphate (MEAS). A total of 476 fungal isolates from diseased grapevines were obtained. Fungal isolates were identified based on their morphological and molecular characteristics. Morphological identifications of Phaeoacremonium spp. were confirmed by sequence analysis of partial b-tubulin gene (BT) sequences amplification using primers T1 and Bt2b. Botryosphaeria isolates were also confirmed by amplification and sequence of the internal transcribed spacer (ITS) region using the primers ITS4 and ITS5. The most common fungi isolated from vineyards were Phaeoacremonium aleophilum and Phaeomoniella chlamydospora with 103 and 30 isolates, respectively. This is the first report of molecular identification of Pm. aleophilum, Pa. chlamydospora, Pm. parasiticum and Botryosphaeria dothidea associated with different internal symptoms of vine decline in Kerman province.

The objective of this sudy was to detection of polymorphis in IGF-1 gene and its association with milk producton and growth traits in Mahabadi goats using PCR-SSCP method. For this purpose, blood samples were taken from 140 (89 mature goats and 51 kiddings) Mahabadi goats reared in the farm of department of animal Scince Tehran University (Karaj). Genomic DNA was extracted from whole blood using modified salting out method. The Polymerase chain reaction (PCR) used to amplify of 326bp fragment of exon 4 of IGF-I gene. PCR products were electrophoresed on polyacrylamide gel (method SSCP) and stained with silver nitrate method to distinguish different patterns. The results revealed two band patterns. The frequency of two Patterns is 65.7% and 34.2% respectively. The polymorphism of the IGF-I gene were associated with milk production(p<0.05), But fat percent, protein percent, somatic cell count and also birth weight, weaning weight, daily gain, feed intake and final weight among two genotypes were showen not significantly.

Thymus is one of the famous medicine herbs of Lamiaceae genus. Due to the high potential of hybridization and introgression among thymus species, they have high genetic variability and study of genetic variability is difficult in this genus. According to the existing problems in the field of breeding of this medicine herb, use of molecular markers could be a valuable tool for evaluation and exploitation of germplasm. In this study, for assessment of genetic diversity among 70 Iranian Thymus, 30 semi-random ISJ (Intron-exon Splicing Junction) primers were used. Total primers produced 694 bands that 683 bands were polymorphic. Average band number per primer and per genotype was 23.13 and 9.91, respectively. Cluster analysis using DARwin5 software and UPGMA method based on Dice's similarity matrix divided accessions into 6 clusters. The highest similarity was estimated between T. kotschyanus and T. transcaucasius and lowest similarity was estimated between T. lancifolius and T. fedtschenkoi. The highest and lowest of polymorphic information content (PIC) revealed by IT15-36 and IT18-2 primers, respectively. The highest and lowest marker index (MI) included ET15-33 and ET18-6 primers, respectively. Results showed that clustering based on cluster analysis partly adapted with geographical origin dispersion. Totally, application of semi-random primers could be useful in assessment of genetic diversity of Thymus accessions.

Follicle stimulating hormone receptor (FSHR) and growth differentiation factor 9 (GDF9) both are the most important proteins that affect reproduction process in mammalian. Polymorphism in these genes and their possible relationship with twining trait in Zel (n=150) sheep are investigated in the present study. Blood samples were collected randomly via vein puncture. PCR-RFLP technique for FSHR marker site showed two A and B alleles with the frequency of 93.33 and 6.67 and two genotypes of AA and BB with the frequency of 93.33 and 6.67, respectively. However, the heterozygote AB genotype was not observed in studied samples. There was no significant association between FSHR marker site and twining (lambing per parity). Detection of genetic variation of GDF9 was carried out by PCR-RFLP and PCR-SSCP technique, respectively. All studied samples had similar banding pattern in both genetic markers and showed wild type homozygote AA genotype. Our finding in Zel breed in the present study along with pervious investigations on other major genes, It can be concluded that the common and major gene affecting reproduction trait are not present in Zel sheep. Therefore, further study is necessary to find a functional gene(s) in this breed.

Assignment test is applied to identify the origin of a specific individual, population differentiation and medical forensic cases. Therefore, this study conducted to analyze some genetic variation criteria, comparison of the different assignment methods and assignment of individuals to eight populations of Iranian native dogs using 13 autosomal microsatellite markers (C26.73320; CXX.6727,10,18; FH20609,20; FH20169,20; FH2790; FH2795; FH2914; FH3053; REN59H07; REN87O21; REN126A15; REN144M10 and REN86G15). The blood and tissue samples were taken from the dogs and total DNAs of the samples were extracted using salting out or enzymatic digestion methods. The results indicated that the least and the most diversity for Sangsari population (0.53) and Kurdish population (0.72), respectively. The individual’s assignment was done using 7 different methods. Among the methods base on the likelihood, the Baudouin and Lebrun method and among the methods base on the genetic distance, Nei minimum method showed the highest accuracy. Totally, markers used in this study could assign the individuals to their source population with 73 % accuracy.

Maintain genetic diversity in population of native chickens is very important. They are as a national investment and the small size of their populations. Studying cytochrome b region of mitochondrial genome in between or within breeds can give useful information about genetic diversity of the population to be studied. The purpose of current study was genetic and phylogenetic investigate cytochrome b region of mitochondrial genome in native chicken of Khorasan. In order to, blood samples were collected from 5 native chicken of Khorasan. After extracting DNA, fragment 858bp of cytochrome b region genome mitochondrial amplified by primers was designed and fragment amplified after purification was sequenced. Two different haplotypes were determined based on two single nucleotide polymorphism sequence and the final sequences of each haplotype with length approximate 780bp which includes 26/25% adenine, 13/57% guanine, 36/36% cytosine and 23/82% thymine. Using the UPGM phylogenetic test results showed that the native chicken of Khorasan is in group of Asia chicken and has the lowest genetic distance with native chicken of china.

This study was carried out to detect QTLs affecting milk production traits in Iranian Holstein population using 10 microsatellite markers including: ILSTS039, CSSM066, ILSTS011, CBDIKM002, CBDIKM004, DIK5080, DIK4884, DIK4361, DIK258 and BM1508. Samples were collected from 10 sire families including 233 milking cows from dairy farms of ten provinces in Iran (Isfahan, Central province, Khorasan, Fars, Yazd, Qazvin and Zanjan). Genomic DNA extracted using standard kit. Genotyping of the samples was performed with capillary electrophoresis. Genotypic and phenotypic data were statistically analyesed using DMU software. Most probable places for detecting QTL in which LRT was meaningful at 5% for milk fat percentage were observed at 3cM, for milk production at 54cM and also QTL of milk fat production was detected in the position 54cM on chromosome 14.