JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2015 | Volume:6 | Issue:4|Papers Count:13

Determine genetic diversity in rice genetic resources is the first step toward the development of rice breeding programs. In this study genetic diversity of fifty cultivars of rice were analyzed using forty microsatellite markers linked to iron and zinc loci. Molecular analysis results showed the number of observed alleles per locus markers with average of 5.27 was varied from 2 to 10. The polymorphic information content values of loci was varied from 0.24 (RM19675) to 0.83 (RM276, RM402), respectively. The average of polymorphic information contents was estimated 0.63. RM276 marker was showed the highest genetic diversity and Shannon Index. Cultivars were classified into two sub-population groups according to analysis of population structure including landraces as first group and improved and foreign cultivars as second group. Based on the analysis of molecular variance, intrapopulation variance was higher than inter-population variance and the minimum and maximum genetic distance was between improved and foreign cultivars and landraces and foreign cultivars, respectively. Based on the cluster analysis, landraces cultivars were separate group than other cultivars. The results of this research could be useful in breeding programs of grain iron and zinc and expanding the genetic bases of rice cultivars.

‘Candidatus Phytoplasma aurantifolia’ has been reported as causal agent of witches’ broom disease in Mexican lime (Citrus aurantifolia L.), Bakraee (Citrus sp.) and Grapefruit (Citrus paradisi Macfad) trees in Iran. Almost all of the lime orchards in Hormozgan, Sistan & Baluchistan and Kerman provinces are infected by this pathogen now. The pathogen is not naturally associated with other citrus varieties in Iran. During 2010, a several number of Citron (Citrus medica L) trees in commercial citrus orchards in Southern Kerman, Iran with witches’ broom symptoms were found. In order to detection and identification of the associated phytoplasma, nucleic acid was extracted from leaf midribs of symptomatic citron plants showing witches’ broom symptoms. Healthy citron plants were used as negative control. Mexican lime trees infected with ‘Ca. Phytoplasma aurantifolia’ were used as positive control. Nested PCR analysis of symptomatic citron as well as mexican lime trees showing witches’ broom symptoms produced an amplicon of the expected size using phytoplasma universal p1/p7 primer pair in first step and 16F2n/16R2 in second step. The healthy citron trees did not. Sequences obtained from all the amplicons were identical and consisted of 1250 bp nucleotide which had a high sequence similarity (99.8%) with ‘Ca. Phytoplasma aurantifolia’. This is the first report of a natural phytoplasma infection on citron in Iran.

Interspecies hybridization is considered as the most important tools for improving genetic characteristics of Agaricus bisporus, which require having compatible homokaryons to cross. One challenge is low percentage of homokaryons among the basidiospores because of the secondary homothalism of this mushroom. Therefore, selection and confirmation of homokaryons among single spore isolates has always been involved with laborious efforts and researchers struggle with it. In this study, we attempted to develop a high-throughput method with high accuracy for screening and confirmation of homokaryons. We used 10 SSR markers -that represented nine linkage map groups of Agaricus bisporus-. Of 71 isolates, several isolates showed two bands as the same as those that heterokaryotic parents did, while some of them showed only one band. We were able to divide the isolates into two main groups: homoallelic and heteroallelic. The homoallelic group included 23 isolates that were monoallelic in the whole sites; which were thus confirmed as homokaryons. Further, the findings of the molecular markers were compared to the observations of a fruiting test. The results revealed that isolates that were confirmed by SSR to be heterokaryotic did not produce fruit bodies in the fruiting test. These findings obviously suggested that fruiting tests are less reliable than SSR markers. Genetic variation among 23 putative homokaryons was also calculated with the NTSYSpc software. The genetic similarity was variable between 0.3-1 among the isolates. The isolates were divided into two main groups by dendrogram. The results showed that these 10 SSR markers are able to detect homokaryons with a probability rate over 99 percent.

Rheb (Ras homolog enriched in brain) gene is originally identified as immediate early gene (IEG) in 1994, encoding 184 amino acids with a deduced molecular mass of 20 497 Da in hippocampus. Rheb belongs to Ras family that encodes a carboxylterminal CAAX box indicating that the protein may undergo post-translational farnesylation. Rheb is an upstream regulatory factor in mTOR signaling pathway and the Rheb-GTP can active mTOR that inducing strong phosphorylation of endogenous S6K1 at residues Thr389, Thr421 and Ser424. Overexpression of Rheb stimulates cell growth while knockdown of Rheb expression inhibits protein synthesis and cell growth in insects. Over expression of Rheb-GAP inhibits mTOR activation and reduces fiber cross-sectional area in mammalian skeletal muscle. There is cross-talk between Mstn and mTOR signaling pathways in mammalian skeletal muscles.Tissues including brain, heart, lung, pancreatic, spleen, kidney, liver and testis were collected from the Raini Cashmir goat after slaughter. Extracted RNA were immediately stored at - 80°C. Quality and quantity of RNA were evaluated and cDNA was synthesized and PCR was performed. PCR Products were electrophoresed on 1.5% agarose gel and were evaluated different levels of expression in studied different tissues. Results showed that the Rheb gene was expressed in all the tested tissues and the highest level of expression was observed in kidney and the lowest level was detected in pancreatic and testis. Results of SPSS analyziz demonstrated that expression of Rheb gene in Raini cashmir goat significantly (P£0.01) is different in various tissues. Hence, can suggest that Rheb has probably role in goat cells and must detect in future investigations.

The association of genes polymorphism of DGAT1 and SCD1 has proved with milk nutritive value. The aim of the present study was the comparison of genetic structure of DGAT1 and SCD1 loci between Holstein and Simmental population.102 Mazandaran Simmental and Holstein cows were randomly selected and done blooding of neck vein. In digestion of SCD1 PCR products (725bp) with AluI enzyme, two alleles V and A were revealed with the frequency of 79 and 21 percent and 92 and 8 percent in Holstein and Simmental breeds, respectively. In digestion of the 325bp fragment of this gene with NcoI enzyme, two alleles of C and T were detected with the frequency of 40 and 60 percent and 45 and 55 percent in Holstein and Simmental breed, respectively. Two alleles of A and K were detected from amplification of DGAT1 locus with the frequency of 60 and 40 percent and 57 and 43 percent in Holstein and Simmental population, respectively. The result of statistical comparison of genotype and allele frequency in studied loci between two breeds indicates that DGAT1locus and second locus of SCD1 gene are same as gene frequency between two breeds and are different as genotype frequency in DGAT1 locus and are same as genotype frequency in SCD1 locus. According to observed polymorphisms, we can result that studied loci are fit for selection of animals with favorite genotype for increasing of production of milk useful fatty acids.

In recent year species of Artiodactyla have suffered rather decrease in populations as result of poaching. In many cases detecting the organs and tissues obtained from arrested poachers is not possible visually therefore it makes difficult to affirm the occurred violation.The aim of this study was to provide a molecular technique based on mtDNA marker and universal primer for indentifying 8 species of Cervidae, Bovidae and Suidae families. Tissues samples of eight ungulate species (Cervus elaphus maral, Capreolus capreolus, Gazella bennettii, Gazella subgutturosa, Cervus dama mesopotamica, Sus scrofa, Ovis vignei, Capra aegagrus) from different wild populations were examined and identified. The results showed that the mitochondrial control region (D-loop) is highly polymorphic in these species (Cervus elaphus maral 569 bp, Capreolus capreolus 573 bp, Gazella bennettii 598 bp, Gazella subgutturosa 644 bp, Cervus dama mesopotamica 578 bp, Sus scrofa 566 bp, Ovis vignei 1062 bp, Capra aegagrus 990 bp). Accordingly Identification of species from tissues is accessible by this technique. The advantage of this technique is to make genetic evidence for the courts concerning poaching violations. In addition by means of this technique could discover the presence of the rare species in wide habitats which improves their ecological studies.

Tomato ring spot virus (ToRSV) is one of the most important viruses threatening vegetable production worldwide. During the years 2010 to 2012, a total of 110 asymptomatic and symptomatic pepper leaf samples with viral affecting like symptoms were randomly collected from fields and greenhouses of Tehran province. Plant samples were tested for the presence of ToRSV using Double Antibody Sandwich Elisa (DAS-ELISA) and Dot immunobinding assay (DBIA). Results indicated that 22% of the samples were infected with ToRSV. Bioassay was performed with the mechanical inoculation of herbaceous indicator plants to evaluate the biological properties of the detected isolates. The morphological features of the virus isolates were studied using Immunosorbent electron microscopy (ISEM). Specific pair of primers designed on the basis of a portion of the RdRP gene sequence and the presence of the ToRSV was confirmed with the reverse transcription-polymerase chain reaction (RT-PCR) for serologically detected samples. An PCR amplicon of a sequence related to the RdRP gene from a representative isolate, was selected and cloned into the pTZ57R/T plasmid vector.Recombinant plasmid contained ToRSV-RdRP gene was transformed into Escherichia coli DH5α. Recombinanat plasmid was isolated and the inserted DNA fragment was sequenced and submitted to NCBI with accession number (JQ972695). At the deduced amino acid and nucleotide sequence levels of the RdRP gene, Iranian ToRSV isolate showed 94-98% and 87- 94% identity to corresponded American isolates respectively. Results of this study could be useful for ToRSV detection, host range determination, distribution condition and designing of control management strategies against ToRSV in pepper fields of Iran.

Safflower (Carthamustinctorius L.) belongs to the Asteraceae familyand its high resistance to environmental stresses, cased it can be used as a model plant for investigatingthe molecular basis of stress tolerance. There is a major mechanism in the plants to transfer cytosol Na+reducing its toxic effects. In this mechanism the excess of Na+was pumped in the vacuoles or tissues with less sensitive. This displacement was carried by vacuole antiporter (NHX). In this study, the pattern of NHX expression was studied in safflower under salt stress by using semiquantitative RT-PCR. In this experiment, 14-days-old plants of resistant variety of safflower, PBR-321, were subjected to NaCl treatment (with five concentrations of 50, 100, 150 and 200mM). Sampling of control and treated plants were performed at different time points (6, 12, 24 and 48 hours) after all four salt treatments. The results showed that expression rate of NHX geneincreased in all concentrations at different times than control plant. Maximum expression levels at different timeswere observed in 150mM of Sodium chloride. Totally, NHX antiporterhas important role in response to salt stress is safflower.

Recently, new advanced high-throughput sequencing technology as a novel tool has opened the way to study of genomic variants and functional information stored within farm animals. The Caspian horse is one of the valuable horses ever exist in the world. Hence, propose of this study was to investigate genetic variants of single nucleotide polymorphisms, insertion and deletions and copy number variations within the genome of Caspian horse and their involved biological pathways. Using high-throughput sequencing technology, we generated 108 Gb (Average depth of 45.8) of DNA sequence from three Caspian horse mares resulting in an average of 14.41X coverage and 76.4% covered with reference genome. Using a stringent filtering method, we identified 1666717 single nucleotide polymorphisms, 358020 insertion and deletions, and 3109 copy number variations. Functional clustering analysis of genic variants revealed that most of the genetic variants in the Caspian horse’s genome were enriched in nervous system, GTP-related signal transduction, cellular morphogenesis, cytoskeleton organization, vascular development and cellular movement. Moreover, we have detected structural variations as like as inversion, intraand inter-chromosomal translocations, large insertion and deletions which could be useful for marker based population genetic investigation.

In this study, genetic fingerprinting of 44 Iranian native apple cultivars were done using 16 simple sequence repeat pairs of primers and then the association of these markers with 16 morphological and biochemical traits were inspected. Based on marker data, 45 alleles were identified using 16 SSR pairs of primers. The number of allele per SSR locus was varied between 2 to 5 with mean of 2.8. Here in, the mean of number of effective allele was 2.2.Polymorphism information content ranged from 0.18 to 0.76 with mean of 0.49. To identify positive markers associated with studied traits, step wise regression was done among marker data as independent variables and studied traits as dependent variables. Results revealed that there is not any linked marker for traits including chlorophyll index, angle of branch and trunk cross sectional area. Considering to association analysis, thirteen traits include fruit volume, fruit diameter, fruit weight, fruit length, fruit firmness, days to ripening, organic acid content, total soluble solids, vitamin C, pH, leaf size, internodes length and tree height had linked SSR markers. The maximum number of allele (6 alleles) was seen for days to ripening and minimum number of allele (1 allele) was seen for traits including PH, fruit weight, fruit firmness and tree height. Regarding linkage between some SSR alleles with some traits, this is possible to use these markers in apple breeding programs effectively.

Eleven common genotypes belonging to two different groups (cranberry bean and lima bean) were submitted to drought stress during vegetative and reproductive stage under controlled glasshouse condition. Drought stress was induced at vegetative stage with the appearance of the third trifoliate and at reproductive stage when flower buds were passing through meiosis. Then proline level and D-1-proline -5-carboxylate synthetase (P5CS) gene expression were analyzed. In all genotypes free proline accumulated under drought stress, however proline levels increased earlier in drought-tolerant genotypes compared to more susceptible ones and so the content of free proline in bean flower buds was 10-fold higher than leaves under both conditions of drought and control. The expression of key gene in proline metabolism (P5CS), was studied in the leaves and flower buds of experimental plants by semi-quantitative RT-PCR under drought stress. This abiotic stress caused significant upregulation of the expression of P5CS. An increase of expression of P5CS was observed in drought-resistant genotypes of bean, compared with sensitive ones. This may be resulted from an increase of the level of final product of the gene. Expression of P5CS gene correlates with the proline levels found in leaf and flower buds tissue.

The purpose of this study was to identify genomic regions affecting weight and percentage of internal organs on chromosome 1 in an F2 population of Japanese quail. A three-generation resource population was developed by using two distinct Japanese quail strains, wild (meat type) and white (layer type). Eight pairs of white and wild birds were crossed reciprocally and 34 F1 birds were produced. The F1 birds were intercrossed to generate 422 F2 offspring. Phenotypic data including weight of organs were collected on F2 birds. All of the animals from three generations (472 birds) were genotyped for eight microsatellite markers on chromosome 1. QTL analysis was performed with least square regression interval mapping method fitting three various statistical models. Significant QTL were identified for pre-stomach weight, uropygial gland weight, bursa of fabricius weight, gizzard weight, percentage of pre-stomach, percentage of intestine, percentage of uropygial gland and percentage of bursa of fabricius. The proportion of the F2 phenotypic variation explained by the significant additive, dominance and imprinted QTL effects ranged from 4.06 to 7.29%, 1.96 to 4.65% and 2.7 to 6.25%, respectively. The results of this study show that there are quantitative trait loci associated with weight and percentage of internal organs on chromosome 1 in Japanese quail, but more studies are needed to better understand genetic structure of these traits.

Lactose determines the milk volume of mammary gland due to osmotic property. Glucose is essential precursor for lactose synthesis, which is mainly absorbed by glucose transporter 1 from blood to mammary gland. In the current study, the transcription levels of GLUT1 have been investigated during prenatal, milking and drying times in mammary glands of Adani goats having high and low breeding values. At the first, breeding values of the animals were estimated by multi-trait random regression model. Then, the samples were taken by biopsy gun and Real-Time PCR method was applied to study the expression of GLUT1. The results indicated that all fixed factors including breeding value groups, sampling times and interaction between them were significant (p<0.005). In addition, the expression of the gene only was different at milking times between two breeding value groups, so that the high breeding value group showed more transcription levels compared with the other group. The expression pattern of this gene in mammary gland was also different between two breeding value groups. Minimum expression of GLUT1 was observed at the prenatal time in high breeding value group, however low breeding value group presented the lowest expression at the milking time. The expression differences observed between the two groups in milking time could be due to the nucleotide variations in transcription factor or miRNA binding sites.Therefore, analysis of nucleotide polymorphisms need to be investigated in coding and promoter regions of this gene in further studies on Adani goats.