فصلنامه ژنتیک نوین
سال پانزدهم شماره 4 (پیاپی 63، زمستان 1399)

Beta-thalassemia is a group of inherited blood disorders with autosomal recessive inheritance. The most effective strategy is to reduce the incidence of this genetic disease, which can be accomplished by utilizing appropriate prenatal diagnosis in clinical practice. Aggressive biopsy during the pregnancy is associated with an abortion risk of approximately 1% for the fetus. Free fetal DNA in maternal plasma is an excellent source of genetic material for prenatal molecular diagnoses. Relative haplotype dosage (RHDO), a haplotype‐based approach, has shown promise as an application for noninvasive prenatal diagnosis. This study was conducted to investigate beta thalassemiain the fetus through maternal blood with NGS technique and RHDO analysis. Total of 5 beta-thalassemia carrier couples (minor) were genotyped by ARMS-PCR for IVSII-I G>A mutation. During the pregnancy, 10 ml of blood was collected from pregnant women, A sample of saliva was also taken from their Husbands. Samples were sent to the Genoma Center of Italy for NGS examination. Finally, results were compared with those of the invasion method.The results of NGS technique with RHDO analysis without having to analyze the affected person by haplotypic block analysis have 100% accuracy. The results with cffDNA were confirmed by invasive methods according to the national protocol for beta-thalassemia. A total of 5 fetal samples belong to above couples were genotyped for IVSII-I G>A mutation, in which 1 fetus were affected, 2 fetus were normal and 2 fetus were carrier of beta-thalassemia. Sensitivity and specificity of NGS test were equal to 100% and 100% respectively. Also, positive and negative predictive values were obtained as 100% and 100%, respectively.Targeted sequencing of maternal plasma DNA for NIPD of  beta- thalassemia  and  using  SHAPEIT software  for RHDO analysis is feasible, proved that it is applicable to construct parental haplotypes without information from other family members.

Triticum and Aegilops genera are the main gene pool of bread wheat due to their breeding potential such as tolerance to various abiotic and biotic stresses. The main objective of this study was to evaluate the genetic diversity in 95 landraces and wild relative accessions belonging to T. aestivum and Ae. tauschii using microsatellite (SSR) markers. A total of 49 polymorphic fragments were amplified using 25 tested primers. The average values of number of alleles, polymorphic information content (PIC) and gene diversity (H) were 1.96, 0.32 and 0.41, respectively. The results of molecular variance analysis (AMOVA) showed the highest portion of genetic variance referred to within species compared to between them. According to genetic variation parameters, the highest values of variation parameters were estimated for Ae. tauschii than T. aestivum accessions. Cluster analysis based on Jaccard’s coefficients and neighbor-joining algorithm grouped all investigated accessions into two main clusters, so that some of accessions from each species were placed in the same sub-cluster. The result of principal coordinate analysis (PCoA) revealed that the first two components accounted for 50.38% of the total molecular variation. The biplot rendered based on the first two components was accordance with the grouping pattern obtained from cluster analysis. The population structure analysis grouped all investigated individuals into four main subpopulations so that the average of FST index among them was 0.50. In general, our results revealed a high level of genetic diversity within T. aestivum and Ae. tauschii species as well as the used markers were capable well to present of this diversity.

Goats are highly adapted domesticated species and are considered as genetic resources for human beings around the world. Protecting and identifying genetic diversity in goat populations is imperative because of the risk of a severe decline. The Adani dairy goat is one of the most important indigenous goat breeds of Iran and is reared in the coastal area of the Persian Gulf in Boushehr province. In spite of high temperatures, humidity and low quality and quantity pastures, the breed has been well adapted to the harsh environmental conditions. Considering the importance of conservation of indigenous breeds adapted to harsh environmental conditions, the objective of the current study was to determine the maternal line and the phylogenetic relationship Adani goat breed using hyper variable region 1 (HVR 1). For this purpose, blood samples were collected from 12 Adani goats of Bushehr province. After DNA extraction, a 968 base pair fragment was amplified by specific primers using the PCR method and sequenced. Analysis of HVR1 sequences showed 10 haplotypes with 42 different positions. Moreover, the haplotype and nucleotide diversity based on HVR1 region were estimated 0.954 and 0.0123, respectively. These results indicate high genetic variation in Adani goat. The phylogenetic tree pattern revealed that the Adani goat was clustered with Afar breed from Ethiopia and also, was classified into haplogroup A. This could be due to the geographical location of Bushehr province and its location next to the Persian Gulf and the Sea of Oman.

Sesame (Sesamum indicum L.) is an important crop for the production of useful compounds such as sesamin and sesamolin. Sesamin, a furofuran class lignin, is widespread in vascular plants and represented by Sesamum spp. Sesamin has been of rapidly growing interest because of its beneficial biological effects in mammals. In this regard, in the last decade, there have been attempts to increase the production of this substance through metabolic engineering, gaining more knowledge of biosynthetic pathways and the genes involved in them and increasing their expression using different elicitors. In this study, the effect of silver nitrate and zinc nanoparticles on expression level of a key gene involved in sesamin biosynthesis pathway CYP81Q1, was detected using optimized sesame cell suspension culture. Different concentrations of the elicitors including 0.25, 0.5 and 1 mg/l were added to the 18 day-old cell suspension culture. Sampling was carried out after 0, 2, 8 and 24 hours post treatment. Cell samples frozen with liquid nitrogen were used to elucidate the expression level of genes by QRT-PCR technique.Our data showed that CYP81Q1 gene reached maximum levels at 8 h post-elicitation with 0.25 mg/l zinc nanoparticles. Expression level of CYP81Q1 was detected in the cell treated with 0.5 and 1 mg/l silver nitrate and showed peak at 24 and 8 h post-elicitation respectively. We observed that in the cells treated with silver nitrate and zinc nanoparticles, expression level of CYP81Q1 key gene involved in sesamin biosynthesis was increased. This increase was generally dependent on the elicitors amount and time post-elicitation. The results showed both elicitors made an increase in gene expression.

Genetic diversity is the basis of plant breeding that allows selection and improvement of plants with desirable traits and characteristics. Aegilops spp. is one of the wild relatives of bread wheat and has a large distribution in the Middle East and West Asia, that Iran covering a large part of this region. In this study, 75 accessions of Aegilops triuncialis species, stored in the National Genetic Bank of Iran, were evaluated for the diversity of high molecular weight glutenin subunits (HMW-GS) using SDS-PAGE method. Generally 10 protein bands in 20 different patterns were observed among the samples. The highest band frequency was for bands h and i with frequency of 0.93 and the least frequency was related to band k with frequency of 0.026 that was observed only in Qazvin and Golestan provinces. The highest corrected genetic variation (uh) was observed in Markazi province (0.4) and the least corrected genetic variation was for Kurdistan province (0.13). The genetic distance matrix of the provinces based on Nei index showed that the highest distance was between Golestan and North Khorasan provinces with West Azarbaijan province, and the lowest genetic distances observed between Khorasan Razavi and Semnan provinces as well as North Khorasan and Mazandaran provinces. Cluster analysis using UPGMA method divides the studied provinces into three groups. There was a clear logical relationship between genetic diversity and geographical variation.

This research was performed to investigate the possible genetic change in micropropagation of Damask Rose (Rosa damascena Mill.) using start codon targeted polymorphism (SCoT) and CAAT-box derived polymorphism (CBDP) markers as gene targeted markers. The shoot single node segments included lateral buds were used as the explants. After sterilization of the explants, a solid MS medium was used for the establishment stage of explants. After 4 weeks, regenerated shoots  were subcultured in VS medium supplemented with different combinations Auxins and Cytokinins for multiplication stage. For in vitro rooting, the plantlets were subcultured in MS medium supplemented with 2 mg/L IAA(Indole AceticAcid)  and 2 mg/L IBA(Indole ButyricAcid). After 8 weeks, the rooted plants were transferred ex vitro and acclimated in the greenhouse. Start codon targeted (SCoT) and CAAT-box derived polymorphism (CBDP)markers were used for analyzing the genetic diversity of 8 Damask Rose plantlets obtained from media with  different combinations of plant growth regulators. Fifteen CBDP and fifteen SCoT primers amplified a total of 173 scorable bands, of which 74 were polymorphic. The average of polymorphic information content(PIC) for SCoT and CBDP primers were 0.32 and 0.35 respectively which revealed a good efficiency of both markers in analyzing genetic diversity. The dendrogram generated based on SCoT data separated the individuals into two groups as well as clustering based on CBDP data. Although, our findings revealed a low level of genetic variability among samples, the results of cluster analysis showed that the use of 2,4-D  in particular in higher concentrations in medium, can increase the rate of genetic changes. These results also, indicated that SCoT and CBDP techniques can be used for detection of genetic variation in in vitro cultures.

In this study, phylogenetic and evolutionary relationships between wild and cultivated species of  Triticum L. and Aegilops L., were studied using karyotype analysis. For each population, three metaphase cells was prepared and karyotype traits were calculated. A significant difference was observed between the species for all the traits evaluated. The results showed that the Triticum L. and Ae. speltoides have higher chromosomes than other species.  Ae. umbllulata revealed the highest ratio of the arms and the lowest centromeric index. Cluster analysis of species based on karyotypic traits was able to distinguish species of the Aegilops L. and Triticum L. Although Ae. speltoides were placed in Triticum L. cluster. The species were classified in different subgroups according to their genome. Based on discriminant analysis, genotypes of Ae. tauschii, Ae. umbllulata, Ae. triuncialis, Ae. cylindrical, T. aestivum  and T. turgidum were completely separated. In general, Aegilops species had more developed karyotypes.

Rye cereal Secale is one of the most important crops in Iran and belongs to the family Poaceae Understanding the Rye genetic diversity is essential for breeding programs of this plant. In this study, AFLP molecular markers were used to determine genetic diversity among 39 populations of Rye (from different regions of Iran, the United States and Russia). Thirteen primers were used, which could create 188 detectable and repeatable bands and 94% of primers showed polymorphism (177 bands) among different populations. The highest PIC was for the ECC 3N 4S 1 starter pair and the highest MI was for the ECC 3N 4S 5 starter pair. To determine the similarity between populations, the Dissse similarity coefficients were used. Maximum similarity (0.971) between the two populations Montanum of Park Jhan nama and Cereal of Fereydoun Shahr, and the minimum similarity (0.386) between Sermont of Arak and Sermont of the United States was observed. The cluster analysis was performed using the UPGMA algorithm based on the similarity matrix. Cluster analysis classified different populations of Rye in five main groups, which relevant to the grouping of principal components analysis. The results of this study showed noticeable genetic diversity among Rye populations of Iran, besides the results of the analysis of molecular variance show that intra-species diversity is higher than inter-species diversity, the average Shannon (I) index in rye species was 0.48, which indicates a relatively good diversity within species.

LEA proteins as cumulative proteins were identified in the late embryonic period to cope with environmental stresses for the first time in wheat and cotton Dehydrogenase proteins are the second group of LEA family of highly hydrated proteins that are resistant to drought. In order to identify and evaluate the molecular evolution of the nucleotide sequence of the DHN5 gene, this gene was derived from three wild species of diploids (Aegilops speltoides, Aegilops tauschii and Triticum urartu), two durum wheat cultivars of tetraploid (Behrang and Shabrang) and two wheat cultivars of hexaploid (Bolani And Sistan) were isolated and sequenced. This is the first report of detection of DHN5 gene in wheat.  Results of gene sequencing in wheat cultivars and its ancestors were recorded in the NCBI. The results showed that transient succession was more than interchanging substitution and the ratio of dN / dS was 1.13, which shows the trend of positive selection of DHN5 gene among wheat cultivars and wildlife during evolution. Protected areas form a small part of the DHN5 gene sequence, which indicates its susceptibility to nucleotide and mutational changes. The phylogenetic tree of the wheat genes sequence, along with 28 other plant varieties, was plotted using the Neighbor-joining method, which indicates the various evolutionary pathways of the DHN5 gene. Phylogenetic analysis showed that Sistan cultivar suffered from many mutations because of its compatibility with drought stress conditions, therefore the sequence of this gene has changed greatly, which makes it possible to separate from other wheat in the phylogenetic studies. According to the results obtained in all of the studied cultivars, it can be concluded that this gene is probably located on all three genomes A, B and D of wheat.

In this research work, Intra-species karyotypic diversity for 17 populations of Silybum marianum from different geographical regions of Iran along with one population from Hungary were investigated. To prepare the appropriate chromosomal specimen, one and a half centimeters of root tip were isolated, after using fixation of pre-treatment, the picture were taken from the karyotypes which prepared and the chromosome base number of all of them was  x = 17, therefore the number of chromosomes obtained 2n = 34. In terms of karyotypic symmetry indices DRL, S%, TF%, A1 and A2, there were significant differences between ecotypes at probability level of 1%, which indicates that there were genetic diversity between the studied ecotypes. Regarding these results, the Ramhormoz ecotype and then the Shushtar ecotype were considered as the most asymmetric and most advanced ecotypes and the ecotype of Najaf-Abad and ecotype of Karaj-2 were considered as the most symmetrical and incomplete ecotypes. Based on the cluster analysis, the karyotypic characters of the populations were divided into three groups. The greatest difference was observed between the two Ramhormoz and Najafabad ecotypes. To determine the contribution of each karyotypic trait in the creation of diversity among ecotypes, factor analysis was carried out and the first, second and third factors accounted for more than 87% of the variation among the populations. The results of karyotypic analysis showed that there is a high karyotypic variation among Silybum marianum ecotypes in Iran, so that they can be used in breeding programs and widening genetic diversity in gene pools.

The objective of this study was to determine the effect of the dopamine D1 receptor gene on egg production traits and body weight in West Azerbaijani native chicken by PCR-SSCP. 180 blood samples were taken from native chickens in a poultry breeding station in West Azerbaijan, Iran. DNA was extracted from blood samples using the salting-out procedure. Specific primers were designed for PCR amplification of the 283 base segment of the dopamine D1 receptor gene. In order to demonstration of gene polymorphism, Single-stranded conformation polymorphism followed by sequencing was performed. Four samples from each banding pattern were sent for sequencing. We detected three genotypes in DRD1 gene. The frequencies of three genotypes were 0.42 (AA), 0.49 (AG), and 0.09 (GG), respectively. This finding indicates that the dopamine D1 receptor gene was polymorphic and could be a candidate locus or linked to a major gene that influences reproductive traits in chickens.