Single nucleotide polymorphism genotyping and its application on mapping and marker-assisted plant breeding

The nucleotide diversity across a genome is the source of most phenotypic variation. Such DNA polymorphism is the basis for the development of molecular markers, an indispensable tool in geneticmapping studies. In general, the high resolution fine mapping of genes is often limited by lack of sufficient number of polymorphic molecular markers. This problem is compounded with traits controlled by multi-genes because in several such studies, QTL cannot be resolved to a workable resolution that could be feasible for predicting the candidate gene(s) associated with traits of interests. The availability of abundant, high-throughput sequence-based markers is the key for detailed genomewide trait analysis. Single-nucleotide polymorphisms (SNP) are the most common sequence variation and a significant amount of effort has been invested in re-sequencing alleles to discover SNPs. In fully sequenced small-genome model organisms, SNP discovery is relatively straight forward, although highthroughputSNP discovery in natural populations remains both expensive and time-consuming. Here five central biochemical reaction principles that underlie SNP-genotyping methods specifically for large panel sizes and an intermediate number of SNPs are reviewed

Mapping QTLs for submergence tolerance during germination in rice

To understand the genetic bases of tolerance and to identify relevant quantitative trait loci (QTLs), KHAIYAN (Aus type) was crossed with IR64, a semi-dwarf, modern variety moderately sensitive toanaerobic conditions during germination. Results of screening of BC2F2 lines showed that survival percentage in IR64/KHAIYAN//IR64 population ranges from 0 to 68% with an average of 30%. A linkagemap was constructed with 155 polymorphic SSR markers, resulting in a map of 1483.5 cM with a mean inter-marker distance of 9.6. Four putative QTLs were detected in this population, one each onchromosomes 1 (qAG-1), 2 (qAG-2-1), 11 (qAG-11), and 12 (qAG-12). The LOD value of these QTLs ranged from 3.66 to 5.71 with phenotypic variance in the range of 12 to 29.24%. Total phenotypicvariation explained by the four QTLs was about 51.4%. The additive value of the QTL on chromosome 1 was negative, indicating that the allele from KHAIYAN increased tolerance to anaerobic conditionsduring germination. However, the QTLs on chromosome 2, 11, and 12 were positive indicating contribution of the alleles from IR64

Association between polymorphism at 3׳UTR of urokinase gene and risk of calcium kidney stones

Background: Kidney stone is a common multifactorial disease in Iran. Environmental and genetic factors including single nucleotide polymorphism (SNP) affect the incidence of kidney stones. Objective: The aim of this study was to determine the association of +4065 T/C polymorphism at 3′untranslated region (3'UTR) of urokinase gene and calcium kidney stones. Methods: This case-control study was conducted on 70 patients with history of calcium kidney stones as case group and 70 healthy subjects as control group in the Baqiyatallah hospital in 2013. The polymorphism was assessed using the Allele Specific PCR (AS-PCR) method. Allele and genotype frequencies of the two groups were compared using 2x2 contingency tables. HardyWeinberg equilibrium was compared between the two groups using Chi-square test. Findings: Of 70 cases, 10 (15%) were heterozygous and 24 (34%) were homozygous for the polymorphism. Of 70 controls, 25 (35%) were heterozygous for the polymorphism. The frequency of mutant T allele was 41% in the case group and 18% in the control group. The frequency of mutant C allele was 59% in the case group and 82% in the control group. The risk of calcium kidney stones in carriers of the mutant allele was 1.7 times higher than non-carriers (OR: 1.7). Conclusion: With regards to the results, it seems that there is a significant association between the polymorphism at 3׳UTR of urokinase gene and formation of calcium kidney stones. Urokinase gene polymorphism may be introduced as a candidate gene involved in calcium stone formation.Keywords: Single Nucleotide Polymorphism, 3' Untranslated Regions, Polymerase Chain Reactio

Association between polymorphism at 3 ׳UTR of urokinase gene and risk of calcium

Background: Kidney stone is a common multifactorial disease in Iran. Environmental and genetic factors including single nucleotide polymorphism (SNP) affect the incidence of kidney stones. Objective: The aim of this study was to determine the association of +4065 T/C polymorphism at 3′untranslated region (3'UTR) of urokinase gene and calcium kidney stones. Methods: This case-control study was conducted on 70 patients with history of calcium kidney stones as case group and 70 healthy subjects as control group in the Baqiyatallah hospital in 2013. The polymorphism was assessed using the Allele Specific PCR (AS-PCR) method. Allele and genotype frequencies of the two groups were compared using 2x2 contingency tables. Hardy-Weinberg equilibrium was compared between the two groups using Chi-square test. Findings: Of 70 cases, 10 (15%) were heterozygous and 24 (34%) were homozygous for the polymorphism. Of 70 controls, 25 (35%) were heterozygous for the polymorphism. The frequency of mutant T allele was 41% in the case group and 18% in the control group. The frequency of mutant C allele was 59% in the case group and 82% in the control group. The risk of calcium kidney stones in carriers of the mutant allele was 1.7 times higher than non-carriers (OR: 1.7). Conclusion: With regards to the results, it seems that there is a significant association between the polymorphism at 3 ׳UTR of urokinase gene and formation of calcium kidney stones. Urokinase gene polymorphism may be introduced as a candidate gene involved in calcium stone formation

The Effect of Propiconazole and Protective Effects of Selenium Gene Expression Profile of Caspase 9 in the Testicular Tissue of Male Sprague Dawley (SD) Rats

Background & aim: Conazoles including imidazoles or triazoles are anti- fungal agents widely used to prevent fungal growth and their infections. Propiconazole placed in this group is a systemic fungicide used widely for detoxification of cereal seeds especially rice in Iran and other countries. This fungicide were designed to inhibit a specific cytochrome P450, CYP51 (lanosterol-14-α- demethylase), a critical step in the biosynthesis of ergosterol, a steroid required for the formation of the fungal cell wall. In the present experimental study, the effect of propiconazole on Caspase 9 gene expression profile as an initiator of apoptotic process and protective effect of selenium were investigated. Methods: Forty SD rats were divided into 10 groups of 4,  including : control , sham1 (solvent of propiconazole, distilled water), sham 2 (solvent of selenium, normal saline) and 1 group received 0.5 mg/kg selenium ,3 groups received propiconazole in doses of 10,50,75  mg/kg and 3 groups received propiconazole in doses of 10,50,75 mg/kg propiconazole with 0.5 mg/kg of selenium. Injections were intrapritoneal for two weeks in alternate days. Then, using RT-PCR and Total Lab program gene expression of caspase-9 testicular of all groups were studied. Data were analyzed using descriptive statistics. Results:  A significant increase of caspase 9 expression were observed among all experimental groups compared to control and sham groups. These findings indicated that 0.5 mg/kg selenium is not a suitable dose to create protection in this experimental study.  Conclusion: The significant increase in Caspase 9 gene expression profile observed in all experimental groups as compared to control suggests activation of apoptosis and inefficacy of selenium to protect the testis against induced damages

Construction of pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus

Background: Infection with Echinococcus granulosus causes hydatidosis in human and ruminants. With regards to the high prevalence of hydatidosis in Iran, dealing with this disease is important in terms of public health. Objective: The aim of this study was to construct pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus Methods: This experimental study was conducted in the Razi Vaccine & Serum Research Institute, Karaj in 2013. Initially, epitopes stimulating the host immune response were predicted by IEDB Database and the coding sequences were made. The sequences were amplified by PCR. The PCR products were cloned into pEGFP-N1 vector after digestion with XhoI restriction enzyme. The bacteria containing recombinant plasmid were evaluated using Colony PCR, agarose gel electrophoresis and sequencing methods. Findings: Four peptides with 10 linear epitopes were predicted in EgTrp antigen. The nucleotide sequence coding ChEgTrp was amplified by PCR using specific primers and a 270 bp fragment was obtained. This fragment was cloned into pEGFP-N1 vector and the recombinant plasmid was confirmed by Colony PCR and agarose gel electrophoresis. For final confirmation, the recombinant plasmid was sequenced and the pEGFP-ChEgTrp was constructed. Conclusion: The ChEgTrp was successfully cloned into the pEGFP-N1 vector and this plasmid can be used to design DNA vaccines

Prediction and cloning linear Tcell epitopes of P14-3-3 antigen into pEGFP–N1 as a DNA vaccine model to induse immuno response against hydatidosis and it\'s expression in CHO cell line

ABSTRACT Background & purpose: Hydatidosis is a zoonotic disease that caused by infection with the larvae of Echinococcus granulosus. Different antigens produced in larval stage of this parasite that recombinant vaccine base these antigens created significant immunity in infected animals. One of the important antigens is p14-3-3 that it's recombinant antigen created considerable immunity in mouse models. In this study according to the high immunity of antigen epitopes region the coding sequence of T-cell epitopes of P14-3-3 was cloned into pEGFP-N1vector in order to produce an effective DNA vaccine model to stimulate high level of Th1 immune response.   Material and method: In this study bioinformatics tools were used to prediction of linear T-Cell epitopes of Echinococcus granulosus P14-3-3 &zeta antigen. The nucleotide coding sequence of these epitopes was synthesized by PCR. the ampliqon was digested with XhoI restriction enzyme and cloned into pEGFP–N1 vector That has been purificated by modified sambrook method with CaCl2 and PEG6000..Positive colony was selected by direct colony PCR and confirmed by the sequencing.and evaluation of it's expression in Eukaryotic cells was done by transformed to CHO cell line with electroporation. Results: Linear T-cell epitopes of Echinococcus granulosus P14-3-3 after prediction,synthesis and amplification wae successfully cloned into pEGFP-N1 vector that purificated by new method with maximum vector and minimum RNA concentration.The expression of new constract in CHO cell line as a eukaryotic cells achivment by fluorescent microscope and will be used as a DNA vaccine model to evaluation immuno response in mouse models.   Discussion: Successfully cloning of The linear T-cell epitppes coding sequence of Echinococcus granulosus P14-3-3&zeta antigen into pEGFP-N1 verificated by sequencing and fluorscent microscope images demonstrated expression of recombinant protein in CHO cell lin