نشریه تحقیقات ژنتیک و اصلاح گیاهان مرتعی و جنگلی ایران
سال بیست و نهم شماره 2 (پیاپی 58، پاییز و زمستان 1400)

Aegilops tauschii Coss is an annual and diploid plant (2n = 2n = 14, DD) that grows as a wild weed in the highlands or plains near the shores of the high seas, from Turkey to China. Since the northern regions of Iran are considered as one of the most important centers of origin and diversity of this species, it is very important to identify sources of resistance to brown rust (wheat leaves) in these regions. In the present study, the resistance of rangeland species, Aegilops tauchii Coss., to six isolates of brown rust was evaluated in seedling and adult plant stages. SSR and EST-SSR markers with proper coverage on D genomes of wheat were applied to identify marker-trait association (MTA). Analysis of population genetic structure showed that the 100 investigated genotypes separated in two distinct sub-populations. Association analysis among markers and phenotypic data was conducted out based on the Kinship coefficients using generalized linear and mixed linear models. Significant associations among markers and traits obtained for amplified fragments of Xgwm2, Xgwm44 and Xgwm30 primers correspondingly located on 3D, 7D & 2D chromosomes, respectively. Furthermore, EST-SSR primers SWES186 and SWES92 correspondingly located on 2D & 7D chromosomes, respectively. These associated markers have the highest R2 coefficients for resistance level to leaf rust disease. Informative markers with association to most isolates could be exploited for genomic selection and early screening of genotypes for resistance to leaf rust.

MicroRNAs (miRNAs) are one of the small and non-coding regulatory molecules, which regulate gene expression by transcriptional cleavage or translational suppression. miRNAs are involved in regulating a wide range of metabolic and physiological processes in plants.The mint family plants, especially Satureja khuzistanica, are well known herbs for its flavor, fragrance and medicinal properties. To date, no miRNAs have been identified in Satureja species. In present study, a computational approach based on homology search was used to identify miRNAs and their targets of Satureja khuzistanica. Non-coding unigenes were identified and considered for candidates of miRNAs precursor. After evaluating the general parameters such as GC percentage, minimum folding free energy (MFE), minimum free energy index (MFEI) and secondary structure, 58 miRNAs were identified, which among them, by applying plant specific parameters and filtring the miRNAs from several transcripts, overall ten miRNAs were identified. Then, 930 target transcripts were predicted using psRNATarget website and the annotation of them was performed by using BLASTx tools of NCBI Blast+ software (v2.6.0). Examination of target genes showed that auxin-responsive genes, GRAS, AGO2 and LAC family genes are the main targets of the identified miRNAs in S. khuzistanica. Pathway enrichment analysis of the target genes revealed that the secondary metabolic pathway is significantly among the targets of the identified miRNAs. This is the first study describing miRNAs and their role in the regulation of target genes in S. khuzistanica.

Peppermint (Mentha piperita) is one of the most important medicinal plants producing secondary metabolites. Menthol is an important component of peppermint essential oil monoterpenes, which is widely used in pharmaceutical and industrial applications. This monoterpene is produced by the Menthone Menthol Reductase (MMR) enzyme. Plant growth as well as the expression of this gene are affected by environmental stresses. In this study, peppermint rhizomes were first surface-sterilized and cultured in MS medium supplemented with sodium chloride (zero, 50, and 100 mM) and mannitol (zero, 50, 100, and 150 mM). Then they were placed in growth chambers at 23, 26, and 29°C. Three weeks after applying stresses, the expression of this gene in the leaves of the plants was measured by Real-Time PCR, and the obtained data were analyzed. According to the results, the expression of the MMR gene decreased at low concentrations of sodium chloride but increased by 97% at high concentration of 100 mM compared to the control. However, the expression of this gene has reached a minimum level of expression at high concentrations of mannitol as well as at high temperatures. Due to the importance of peppermint in the pharmaceutical industry, increasing the production of menthol through applying different treatments is important. Based on the obtained results, the treatment with mannitol (zero mM) and sodium chloride (50 mM) at 23°C increased the expression of the MMR gene by 93% compared to the control and it seems that the menthol production has reached its maximum level.

Aegilops range grasses (Poaceae family) as one of the most important ancestors of wheat is a rich source of stresses tolerance genes. For this, the fingerprinting of genetic relationships in accessions belonging to eight species of Aegilops was studied using targeted genes-related CoRAP molecular markers. In designing the fixed primers, six genes CAT, MnSoD, SoS1, miR398, miR160b and miR169gR responsible for abiotic stresses tolerance were used. The lowest and highest polymorphic information contents were related to CoRAP2 and CoRAP10 primers with 0.92 and 0.96, respectively. The marker index varied from 6.89 in CoRAP7 primer to 13.49 in CoRAP9 primer. Low gene flow (Nm) and high differentiation (Gst) was observed between species. Also, the Fst index equal to 0.45 showed that the studied populations were completely separated. Due to the high values of the effective allele number and Nei genetic diversity, two species Ae. cylandrica and Ae. caudata showed high intra-specific diversity. The greatest similarity was observed between two species Ae. truncialis and Ae. umbelulata as well as Ae. neglecta and Ae. cylanrica. Cluster analysis appropriately divided species into distinct groups, and principal coordinate analysis confirmed the results of cluster analysis. Using structure analysis of the population, the mode of gene flow and genetic intermixture among species were in accordance with the grouping results of cluster analysis and PCoA biplot.

Allium includes more than 920 species worldwide. Iran is the main habitat of many wild Allium species. In this study, onion tissue of two species A. stipitatum and A. scabriscapum from two subspecies Melanocrommyum and Reticulatobulbosa were used to evaluate and identify the expression pattern of storage proteins obtained by the SDS-PAGE method. Two-dimensional gel electrophoresis and MALDI TOF/TOF MS mass spectrometry were used to identify protein spots and to observe the expression changes of the two species. Analysis of gels by two-dimensional electrophoresis revealed 138 protein spots. The results showed a slight similarity between the gels and a different expression pattern between the samples, based on which a small number of similar spots were obtained in both gels. The results showed that there were significant differences in the expression profile of onion tissue proteins of these samples. Using the T-test, 9 protein spots with the most significant changes were identified at the 5% probability level, and among them, 3 different spots were identified using Mass spectrometry was selected for identification. The results of mass spectrometry experiments and matching of the obtained data with NCBI and Uniprot databases and bioinformatics tools showed that the tested spots were consistent with Maturasek proteins, Agmatine coumaroyltransferase-1, and a protein with the unknown function called Hypothetical Protein. The findings of this study showed that since the samples belonging to A.stipitatum and A.scabriscapum are of the same genus, but the expression pattern of their stored proteins in onion tissue is very different from each other.

Savory (Satureja spp.) with different species and valuable compounds such as thymol and carvacrol, has special importance among medicinal plants. In this study, the seeds of eight populations of three different species of Satureja (S. hortensis, S. mutica, and S. boissieri), as well as the seeds of two improved German savory cultivars (S. hortensis cv. Aromag) and (S. hortensis cv. Saturn), were evaluated cytogenetically. Karyotypic results showed that eight populations of S. hortensis (S1P1-S1P8) were diploid (2n = 2x = 48) and two populations of S. boissieri and S. mutica (S2P1 and S3P1) were tetraploid (2n = 4x = 60) with different basic chromosome numbers of 24 and 15, respectively. The average chromosome length in all the populations was 1.46 µm (ranged from 1.19 to 1.71 µm). Propidium iodide (PI) flourochrome and standard parsley plant (Petroselinum crispum, 2C DNA = 4.45 pg) were used to measure genome size using flow cytometry. Data were statistically analyzed in a completely randomized design with five and three replications for chromosome length and genome size, respectively. The results showed a significant difference among the populations based on the two statistics methods (p<0.01). The mean value of 2C DNA for all the diploids was 3.52 pg (ranged from 3.29 to 3.78 pg) and for the two tetraploid populations was 2.38 pg (ranged from 2.25 to 2.51 pg). In the present study, the amount of genome size was reported for the first time for the mentioned species, which along with other measured traits can contribute to the richness of karyological knowledge in Satureja

Ferulago (Ferulago ssp.) medicinal plant belongs to Apiaceae family and is endemic of Iran. Species of this genus are extremely endangered, considering the seed dormancy, excess harvesting and environmental stresses such as drought. By considering this plant as a valuable medicinal-rangeland plant, its production and reclamation is an essential task. This investigation was done to optimize micropropagation of two endemic species of Ferulago, included F. angulate and F. subvelutina. Maximum percentage of callus induction in F. angulata specie was reached as 100%, using rootlet explant (under treatments of 0 BAP + 1 NAA, 0 BAP + 1.5 NAA and 0 BAP + 2 NAA mg/l) and also true leaf explants (under treatments of 0.5 BAP + 0.5 NAA and 1 BAP + 2 NAA mg/l), and in F. subvelutina specie using true leaf explants (under treatments of 0.5 BAP + 0.5 NAA and 1 BAP + 2 NAA mg/l) respectively as 72.33 and 71.66%. The combined application of the cytokinin and auxin phytohormones has been shown to be effective in regenerating Ferulago species; so that the maximum regeneration was reached as 53.33 and 46.66% in F. angulata respectively under treatments of 0.5 BAP + 0.2 NAA mg/l and 0.5 BAP + 0.4 NAA mg/l and so on as 53.33 and 60 percent in F. subvelutina respectively under treatments of 1 BAP + 0.2 NAA mg/l and 1 BAP + 0.4 NAA mg/l. The best rooting treatment in both species was selected as the ½ MS cultural medium included 0.5 mg/l IBA. The adaptation stage was done in a mixture of soil, peat soil and sand with a weight ratio of 1: 1: 1, in which the total of 80% of regenerated plants from tissue culture, were successfully adapted to the outside environment.

Protecting the germplasm of native tree species of the Caspian hyrcanian forests, especially the Caspian locust (Gleditschia capsica Desf.) is a priority of the research. In this study, somaclonal diversity in regenerated plants from different explants of Caspian locust was investigated using the Inter Simple Sequence Repeat (ISSR) marker under in vitro conditions. Mother leaf (natural) and stem, root and cotyledon explants from the regenerated plants as studied genotypes were selected and they were cultured in MS culture medium containing 0.5 mgL-1 TDZ and 0.5 mgl1 2,4-D. Somaclonal diversity was examined after six subculture inoculations. Polymorphism (PIC), Nei and Shannon diversity traits were used to investigate the diversity between genotypes and then clustering of genotypes was performed using UPMGA algorithm and Principal Component Analysis (PCA). The polymorphism percentage was 87.9% using 10 primers. Molecular Analysis of Variance (AMOVA) showed 16% diversity between genotypes and maternal origin. The results of cluster analysis grouped the genotypes into three distinct categories, which was also confirmed by principal component analysis (PCA). Accordingly, the mother basal and stem genotypes were grouped in a group. The mother basal had the highest and lowest similarity coefficients with stem and root genotypes, respectively. Therefore, it was suggested to use stem explants that have high genetic similarity with the mother genome for studies of Caspian locust tissue culture. In general, the results showed that somaclonal diversity among regenerated plants will increase with increasing number of subcultured.

Narcissus is a member of the Amaryllidaceae family. This specie is very important in the world as an ornamental plant. In order to study the diversity between narcissus genotypes, an experiment was conducted in a completely randomized block design with 13 genotypes and 4 replications at Agricultural Sciences and Natural Resources University of Khuzestan. In this study, effective indicators on cut flower quality include: number of flowers, stem height, stem and flowers weight, solution uptake, visual quality, chlorophyll of leaf, florets proline, soluble carbohydrates of florets, total phenols of florets, malondialdehyde content of florets, protein, activity of peroxidase and catalase enzymes activities were measured. The results showed that based on the cluster analysis of physiological data, genotypes were divided into two main groups. The first group included the genotypes of Kazerun, Behbahan, Jahrom, Ilam, Shiraz and Kerman. In this group, important traits such as total chlorophyll, solution absorption and total phenol had the highest amount. The second group was divided into two subgroups. Subgroup A included Khosf, Ghaemshahr and Khorramabad genotypes and subgroup B included Ahvaz, Abdanan, Gachsaran and Mehran. Subgroup A, traits such as proline content, malondia aldehyde, antioxidant enzymes peroxidase and catalase were effective factors in this group. Subgroup B had the highest total protein and soluble carbohydrate properties. It was found that the populations of Ahvaz, Abdanan, Gachsaran and Mehran, having the highest amount of total protein and soluble carbohydrates at the end of the experiment, and the content of proline, malondialdehyde, antioxidant enzymes peroxidase and catalase were at a low level, after 10 days, therefore, they introduced as superior populations for cut flowers. In general, the study showed that there is a high morphophysiological diversity in the native genus of narcissus, which can be the result of very different climatic diversity in Iran, as well as mutation and sexual reproduction by seeds in this plant. In general, understanding such a high diversity is useful in the management and protection of the germplasm of this plant and helps the breeder in determining the strategies of exploitation, breeding and domestication and cultivation of this plant.

Orchardgrass (Dactylis glomerata L.) is a perennial forage grass, wind-pollinated and out-crossing that belongs to the Poa ceae family, which is used for pasture, rangeland renovation and hay production. The aim of this study was to investigate the genetic diversity of D. glomerata populations using morphological markers to identify high yielding populations. For this purpose, 25 populations of D. glomerata were sown on a randomized complete block design with three replications in the research farm of the Agricultural Biotechnology Research Institute in Tabriz in the 2018-2019 cropping year. The results obtained from the analysis of variance showed a significant difference in the studied traits, indicating high genetic diversity among populations. Based on the mean comparison results, G2, G5, G14, G16, G18, G19, G24 and G25 populations with 312-342 g/plant in terms of forage yield and G7, G8, G2 and G16 with 27-35 g/plant in terms of seed yield were identified as the best populations. Therefore, G2 population from Shahroud and G16 population from Isfahan were the two top populations in terms of seed and forage yield. Cluster analysis was performed and populations were classified into four groups based on the studied traits. The cluster3 with seven members included most of the top populations. In this study, the highest genetic distance was related to the populations of clusters 1 and 3. In order to produce synthetic cultivars, the parental plants for polycross should be selected from these groups.

This study was executed to compare forage yield and leaf area index of different alfalfa genotypes for two years and also, to estimate of LAI via leaf weight and plant height during 2011 and 2012. Twenty local and forigen alfalfa genotypes were planted in a RCB design with three replications in spring, 2011 at Seed and Plant Research Institute (SPII), Karaj, Iran. Two-years analysis of variance indicated significant diferent among genotype's means for fresh and dry forage yield in which Bami x Yazdi, Mesa sersa and KFA17 genotypes with 41.99, 39.84 and 39.15 (t ha-1) fresh forage yield and 9.75, 9.41 and 9.16 (t ha-1) dry forage yield were the most forage production, respectively. Despite the significanat difference among genotypes for leaf to stem ratio, there was no significance difference for LAI. The results showed that the fast, accurate and low-cost estimation of LAI is possible through leaf weight and plant height. In this study, leaf weight trait (especially the fresh leaf weight) had more efficiency than plant height in estimating leaf area index through a linear model (without constant) with high coefficient of determination (R2 ≥ 94). It was resulted that every 100 gr of fresh and dry leaf and 10 cm of plant height of alfalfa had 0.33, 1.51 and 0.25 m2 leaf area, respectively.