مجله بیوتکنولوژی کشاورزی
سال پنجم شماره 4 (پیاپی 12، زمستان 1392)

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, is one of the most devastating disease of wheat worldwide. Due to time and rate of defense genes expression, cultivar reaction to disease is different. So time and rate of defense genes expression can lead to develop an in vitro test for screening relative resistance of cultivars against pathogen. Forty wheat genotypes were screened by artificial infection and Marvdasht and Chamran as a resistant and Darab2 and Tajan as a susceptible cultivar were selected. Two treatments including scratched and not scratched leaves were considered for each cultivar. The soluble components were extracted 24 and 48 h after scratching the leaves and effect of extracts on sporulation and germination of M.graminicola spores was investigated. Increment of spore population in resistant cultivars was lower than susceptible ones. Also germination percent on media with resistant cultivars extract was lower as compared with media with extracts of very susceptible cultivars extract. The cDNA-AFLP experiment showed that expression of peroxidase 3 and 8 genes was increased in Chamran and Marvdasht cultivars respectively 48 hour after scratching the leaves. So it seems that we can use in vitro plant extract for screening relative resistance of wheat cultivars against M.graminicola.

The existence of genetic variation of genotypes is very interesting in reducing genetic vulnerability and lead to stable control of production. Proline and glutamine-rich wheat seed endosperm proteins are collectively referred to as prolamins. HMW-GS are major determinants of gluten elasticity. The inheritance of glutenin subunits follows Mendelian genetics with multiple alleles in each locus. Identification of the banding patterns of glutenin subunits could be used as an estimate for screening high quality wheat germplasm. In this study 116 genotypes of Triticum turgidum originating from different geographical areas of Iran and six countries, Portuguese, Italy, Yugoslavia, Bulgaria, Iraq and Afghanistan, were evaluated for variation in high molecular weight glutenin subunit. Glutenin extracted from seeds using SDS-PAGE electrophoresis and scoring bands were determined. 16 allele combinations were identified. In pure lines of durum wheat, the Null allele was observed more frequently than the 1 and * alleles. In% 21/6% of genotypes 1allele and 12.5%, 2* allele was observed. The locus GluB1, 7 type alleles was detected. The locus GluB1 20 allele had the highest frequency and was observed in 42 genotype. 20 genotypes were having 17+18 allele. The 7+8, 6+8 and 7 alleles were observed in 21, 12 and 7 genotypes respectively. Each of the 22 and 7+9 alleles also was observed in one genotype. In the one line of native country of Portugal, observed unique allele at the GluB1 locus, which conventional scoring models did not match. The tested genotypes were classified in nine groups according to the linkage distance analysis. The genetic variability in Glu-A1and Glu- B1 locus Were respectively, 0.42 and 0.81. HMW glutenin Glu-1 quality scores are in the range between 2 and 6.

In vitro culture of plant cells is an ideal method for commercial production of large amounts of several important secondary metabolites. There are some wild apples (Malus sp.) native to Central Asia and Siberia that can produce anthocyanin in their various organs even in vitro and contain compounds such as flavonoids and polyphenols, which have antioxidant activity. There are numerous factors affecting anthocyanin production in in vitro condition. The aim of this study was to determine the best source and optimum concentration of carbohydrates including sucrose, glucose, fructose, maltose and mannitol to produce anthocyanin in callus culture of an in vitro grown anthocyanin producing wild apple. The results showed that the mannitol concentration of 3% plus 3% sucrose was the most effective carbohydrate treatment for anthocyanin production. Increasing mannitol concentration to more than 3% resulted in reduced anthocyanin production. Increasing mannitol concentration, decreased callus growth index and callus fresh weight; while, callus dry weight increased. The highest anthocyanin production and the lowest callus growth index were observed in 6% among the other sucrose concentrations. Glucose, fructose and maltose had weaker effects on anthocyanin content.

In this study, a 254 bp fragment containing exon 6 and a part of introns 5 and 6 of ovine CAST gene were amplified in 169 Lori-Bakhtiari lamb from Lori -Bakhtiari sheep breeding station by polymerase chain reactions. The PCR-SSCP method and vertical electrophoresis of PCR products on 12% acrylamide gel at 4 C° and silver-staining were used for genotyping of amplified fragments. Ten genotypic patterns containing AA, BB, AB, AC, AD, BE, AF, AG, AH and AJ were identified with frequencies of 0.029, 0.195, 0.065, 0.166, 0.024, 0.08, 0.325, 0.053, 0.012 and 0.042, respectively. Association analysis with growth traits demonstrated different genotypes significantly associated with birth weight, weaning weight and daily gain from birth to weaning (P<0.05). The best genotypes for birth weight were AB and AC and for weaning weight and daily gain were AJ and AB. It seems that sequencing of this gene and identification of probable SNPs in the next projects and also association studies at larger population, can determine favorable alleles and genotypes of CAST gene for growth traits with very high accuracy and use them in Marker Assisted Selection (MAS).

The objective of the current study was analysis of the genetic structure of Iranian native dogs in comparison with the exotic breeds. To follow this, blood samples of 93 dogs were obtained from diverse areas of Iran and, in addition, 655 sequences of canine D-loop region obtained from GenBank. Total DNA of the samples were extracted by salting out procedure and applied as template for amplification and sequencing of the D-loop region of mtDNA. Sequence analysis of the 582-bp D-loop region of mtDNA in all samples defined a total of 25 haplotypes. The total nucleotide diversity was 0.01354± 0.00704 for Iranian native dogs. Analysis of molecular variance (AMOVA) showed significant percent for the variance between the studied populations (ΦST=0.029, P=0.0000). Fixation index values using FStatistics method ranged from -0.02671 to 0.3748. Analysis of molecular variance displayed significant difference between some of these populations. The variation was especially observed between Tazi and Sarabi, Bakhtiari, Alborz native and Western regions populations or between Sarabi population with Kurdi, Sangsari, Alborz native and Western regions populations. Bakhtiari population showed a significant difference (P < 0.05) with Alborz native and Western regions populations, and finally European breeds exhibited a difference with South West Asia breeds. In addition Tazi, Sangsari and Western regions populations displayed significant difference with European breeds and South West Asia breeds (P < 0.05). Overall, the results of this study showed an acceptable genetic variation and genetic similarity in some Iranian dog populations.

The objective of the present study was to determine single nucleotide polymorphisms (SNP) located in the promoter region of the bovine heat shock protein 70 (Hsp70) gene. And evaluate associations between Hsp70 SNP with conception rates in Holstein and Sarabi cows. Blood samples were collected from 124 cows. The Cows were Holstein (n=72) and Sarabi (n=52). Genomic DNA was extracted using modified salting-out method. The quantity and quality of extracted DNA were examined with spectrophotometery and gel electrophoresis. Specific primers were designed for PCR amplification of a 539 base segment of the bovine Hsp70 promoter. The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method was used to scan for mutations within the amplified regions. The polymorphisms were confirmed by direct sequencing. Seven single nucleotide polymorphisms were detected; one deletion at base position 895 four transitions (G1045A, G1117A, 1134C and T1204C), and two transversions (A1125C, G1128T). And also four haplotypes were detected (CAAAGCC, DGGCTTC, CDGGCTTT, CGGAGTT). Statistical analysis showed polymorphism in the promoter region of the bovine heat shock protein 70 region significantly correlated with conception rate (P <0.0001). Interestingly in Holstein breed the haplotype (CGGAGTT) of the significant mean conception rate heavier than had haplotypes (DGGCTTC, CDGGCTTT) and in Sarabi breed the haplotypes (CGGAGTT,CAAAGCC) of the significant mean conception rate heavier than had haplotype (DGGCTTC) (P< 0.01). The result of present study verified candidate SNPs within the promoter region of the bovine Hsp70 gene may be useful in selecting cows with a greater fertility.

IGF-1 hormone transports by connecting to IGF-binding proteins (often IGFBP-3) in the biological fluids. This protein acts as an IGF1 carrier and helps to prolong half-life of hormone and can increase or decrease biological activity of hormone on cellular surface. The purpose of the research was to study the polymorphisms of IGFBP-3 gene and its association with carcass traits and fatty acids profiles in Zel sheep as a thin-tailed breed using PCR SSCP. In this study we used 130 Zel sheep randomly and recorded their traits measure we wanted. to determine of fatty acids, we took 6ml blood in venojects containing EDTA and immediately centrifuged them for 10 minutes by 3000g (5200 rpm) to separate the plasma. DNA was extracted from muscle tissue by Biotech company kit. Then the Polymerase chain reaction (PCR) used to amplify of 654 bp fragment of exon 3 of IGFBP-3 gene. PCR products were electrophoresed on polyacrylamide gel (method SSCP) and stained with silver nitrate method to distinguish different patterns. Results indicated the different patterns, may be due to polymorphism in this fragment. Frequency of patterns 1, 2, 3, 4 and 5 were 67/66, 33/3, 67/6, 33/13 and 10 percent respectively. Trait carcass weight (P <0.05), back fat thickness (P <0.01) and crude protein in meat (P <0.01) had significantly differences with different patterns. Significant association observed by Heptadecanoic acid (C17: 0) with different patterns of IGFBP3 gene. No other association was observed by IGFBP3 patterns with other traits and fatty acids profiles.

Understanding phylogenic relationships and genetic diversity in citrus is considered to be important in clarifying their genetic relationships, germplasm characterization and the registration of new varieties. There are some Citrus accessions in the country citrus collections which have been classified merely based on their morphological traits and much genetic research has not been done. Molecular markers would help to infer their relations with known cultivars. In the present research work, phylogenic relationships among 55 citrus genotypes including 43 unknown local genotypes and 12 commercially important citrus varieties were investigated through ISSR markers. In total, 10 ISSR primers produced 92 polymorphic bands with average of 9.2 bands per primer. Polymorphism percent of each primer varied from 75 in primer BDB(CA)7C to 100 in primer (AC)8YT. Genetic similarities among accessions were calculated according to Jaccard Similarity Index and used to construct a dendrogram based on the unweighted pair groups method arithmetic averages (UPGMA), which put the 55 samples into nine major groups i.e. (A, B, C, D, E, F, G, H and I). Orange, mandarin and 25 unknown genotypes into group A, grapefruit and 9 unknown genotypes into group B, Pummelo, Darabi and seven unknown genotypes into group C, sour orange and an unknown genotype into groups D & E, citron into group F, Eureka lemon into group G, an unknown genotype into group H and sweet lime and Mexican lime into group I were clustered. Genetic diversity and phylogenetic analysis in citrus, provide useful information for further breeding programs, collection, preservation and utilization.

In order to detect polymorphism in TGF-b3 loci and effect of this gene on phenotypic and breeding values of body weight traits, blood samples were collected randomly from 120 hens of Fars native fowls. DNA was extracted using standard kit and a DNA fragment of 294 bp from TGF-b3 loci was amplified. After PCR, the amplified fragment digested using BsrI enzyme and revealed two alleles T and t with the frequency of 0.29 and 0.71, respectively. The frequencies of TT, Tt and tt genotypes were achieved 0.15, 0.275 and 0.575 respectively. Values for Shanon Index, Effective number of alleles, observed heterozygosity and expected heterozygosity for TGFb3 were 0.60, 1.69, 0.28 and 0.41 respectively. Results showed that this population has a good genetic diversity and must try to conserve it to use these valuable gene pools. T allele had significant effect on body weight gain at 1 day and 8 weeks (P 0.05). Our results demonstrated that this population has a good genetic diversity, thus these valuable gene pools should be conserved to be used in the future if needed. TGF-b3 gene can also consider as a candidate gene in breeding of this animal.

The pomegranate (Punica granatum L.) is native horticultural plants of Iran which have been cultivated from ancient times for its economic, ornamental and medicinal properties globally. Now Iran is one of the largest manufacturers and exporters of pomegranate in the world. Although the number of Punica species is very low, but there is a high morphological diversity have been observed within the existing varieties and genotypes in country. Therefore, the use of appropriate markers in order to separate the rich pomegranate germplasm of country is necessary. In this study, six out of 50 microsatellite markers (SSR) were polymorphic on 194 samples of the sweet pomegranate germplasm. Total numbers of observed alleles were 24, the number of alleles per locus have ranged between 2 to 8. Average polymorphism information content and heterozygosity of these primers were 0.746 and 0.778 respectively, have proved strong nature of microsatellite markers in genetic diversity studies. In order to assess of genetic relationships and population structure, Cluster analysis was performed by methods UPGMA and NJ with MEGA4 software, structure analysis modelbased Bayesian by STRUCTURE2.3 software and principal coordinate analysis (PCoA) with NTSYS software. Results of all mentioned methods have shown that cultivars and genotypes are generally clustered independently from their geographical origin and their proposed denomination suggesting that severe admixture in studied samples. Survey of our study indicated that there is a high genetic diversity among of sweet pomegranate germplasm in Iran that this result can be used for purpose of breeding.

This experiment was conducted to investigate the PNOC and NPY mRNA expression levels in broiler chicks fed with purslane seed extracts. Two-hundred-fifty day-old broiler chicks were used and randomly allotted equally into five experimental groups with five replications comprised 10 chicks in each replication.The effect of various levels of purslane seed extract investigated on mRNA expression levels of PNOC and NPY genes during a day period, Dietary treatments included a control basal diet (without adding purslane seed), basal diet + 50, 100,150 and 200 mg/kg purslane seed. Livak method was used to determine mRNA expression levels of PNOC and NPY genes. At 42 days of age two bird of each replicate were randomly selected, slaughtered and their brain stem tissue were used to assess the effect of treatments on PNOC and NPY mRNA expression levels. The mRNA expression of NPY in treatments containing purslane seed extract was not significant in compare with control treatment, although it was higher in treatment containing 200 mg/kg purslane seed. The mRNA expression of PNOC in treatment containing 200 mg/kg purslane seed extract was significantly higher than the control treatment (p>0.01); meanwhile the other treatments had no effect in compare with control. The results showed that use of 200 mg/kg levels portulaca oleracea extract led to the significant increase of PNOC mRNA expression and non significant increase of NPY mRNA expression. Therefore, portulaca oleracea extract could be effective on the mRNA level expression of accelerated neuropeptides of appetite in broiler chicks.

In rice breeding programs through hybridization, information on genetic distances and similarities between varieties is necessary for development of new lines. This study conducted with the aim of evaluating genetic distances between sixteen rice varieties using Random Amplified Polymorphic DNA (RAPD) analysis. Eight out of fifteen primers generated polymorphism and the number of amplification products generated by each primer varied from 4 (OPB-04) to 11 (OPD-11) with an average of 7.75 bands per primer. Out of 62 bands, 4 (69.35 percent) were found to be polymorphic between varieties. Cluster analysis grouped genotypes into 5 clusters. Results of the study showed that varieties Hashemi and Pouya had more genetic distance from each other and from the rest varieties. The information obtained in this study will be helpful in selecting genetically diverse parents for designing rice breeding program in Iran.