Transcriptomic Analysis of Cyclamen persicum to Identify Invovled Genes in Triterpene Secondary Metabolites Pathway.

Saponins are considered as a diverse group of natural active compounds, which are widely found in crops. Mevalonate pathway (MVA) is regarded as the main pathway for synthesis of saponins in crops. This study aims to compare transcriptome of the leaf with tuber of crop including tubers and roots. First, more than 166 million reads were generated. The existence of 36,678 unigenes in the two samples out of 48,936 assembled ones showed a significant difference in expression. Finally, 310 and 290 highly up-regulated genes in leaf and tuber were selected for the next analysis. In addition, the expression profiles of 13 key genes in the MVA pathway were compared in RNA sequencing and reverse transcription-quantitative polymerase chain reaction analysis. The results indicated that cyclamen tuber has a higher level of expression of MVA pathway genes. The topological analysis for gene co-expression network involved in triterpenoid synthesis represented that the genes at the beginning of such pathway play a critical role so that the reduction of their expression challenges triterpenoid synthesis severely. The tuber of the cyclamen appears to be the major site of triterpene synthesis, and transfer of excess Isopentenyl pyrophosphate (IPP) from tuber to leaf activates downstream genes in leaf of crop.

Transcriptomic analysis of alternative splicing events for different stages of growth and development in Sistan Yaghooti grape clusters.

Sistan Yaghooti grape variety, despite characteristics such as early ripening, is vulnerable to cluster rot due to small berries and dense clusters. In this regard, AS may serve as a regulatory mechanism during developmental processes and in response to environmental signals. RNA-Seq analysis was performed to measure gene expression and the extent of AS events in the cluster growth and development stages of Sistan Yaghooti grape. The number of AS events increased during stages, suggesting that it contributes to the grapevine's adaptability to various stresses. In addition, DEG and DAS genes showed little overlap in cluster growth stages. Functional analysis of 19,194 DAS -gene sets showed that VIT_06s0004g06670 gene is involved in the activation of calcium channels (Ca2+) through the activation of 5 PLC biosynthetic pathways. Among the 27,229 DEG -sets, VIT_07s0005g05320 gene showed higher expression. Interestingly, this gene is involved in the synthesis of an EF -hand domain-containing protein capable of binding to Ca2+ by activating 4 biochemical pathways. These genes increase cytosolic Ca2+ concentration, enhancing plant stress tolerance and resistance to cracking. These results show that AS can respond independently to different types of stress. Among the other DAS genes, the GA2ox gene (VvGA2ox) showed an increase in AS events during cluster development. This gene is critical for initiating the degradation process of GA and plays a crucial role in different stages of seed development. Therefore, it is very likely that this gene is one of the main factors responsible for the density and seedlessness of Sistan Yaghooti grape.

Expression of BMP2-Hydrophobin fusion protein in the tobacco plant and molecular dynamic evaluation of its simulated model.

Plants are one of the ideal models for therapeutic protein production, however the recombinant protein purification problems in them must be overcome. Bone Morphogenetic Protein2 (BMP2) is employed for the restoration and construction of bone tissues. Hydrophobin is a fungal based protein with high hydrophobic characteristics. Due to this specificity, it is suitable for the purification of chimer protein from complex solutions when is fused to a protein utilizing an aqueous two-phase (A2P) technique. The plant optimized mature human BMP2 gene was designed and evaluated by in silico method. This process involves simulating molecular dynamics using the RMSD, RMSF and Gyration radius indexes. The synthesized Hyd-BMP2 gene was cloned into a pTRAkc-ERH plasmid and Transferred into Agrobacterium (Gv3101). The Nicotiana benthamiana plant leaves were co-agroinfiltrated with HA-Hyd-BMP2 and P19-pCambia1304 containing silencing suppressor. After purification of plant extract utilizing the A2P method, the sample was subjected to SDS-PAGE and Western-blot. By in silico study, the simulated fusion protein profitably shows reasonable protein compactness and the effect of amino acid substitution on protein-protein interaction is not remarkable. Western-blotting using anti HA tag has shown that the A2P technique partially purified the two 22 kDa and 44 kDa forms of Hydrophobin-BMP2. These results confirmed the presence of monomer and dimer forms of Hydrophobin-BMP2 proteins. Moreover, the expression level of the protein using P19 silencing suppressor increased six times and to 0.018% as shown by ELISA. This study presents a fast and easy technique for the purification of transient expressed pharmaceutical proteins from plants.

Association analysis between agronomic traits and AFLP markers in a wide germplasm of proso millet (Panicum miliaceum L.) under normal and salinity stress conditions.

BACKGROUND: Proso millet is a highly nutritious cereal considered an essential component of processed foods. It is also recognized with high water-use efficiency as well as short growing seasons. This research was primarily aimed at investigating the genetic diversity among genotypes based on evaluating those important traits proposed in previous researches under both normal and salinity- stress conditions. Use of Amplified fragment length polymorphism (AFLP) molecular markers as well as evaluating the association between markers and the investigated traits under both conditions was also another purpose of this research. RESULTS: According to the phenotypic correlation coefficients, the seed yield had the highest correlation with the forage and biological yields under both conditions. By disintegrating those traits investigated under normal and salinity-stress conditions into principal component analysis, it was found that the first four principal components justified more than 59.94 and 62.48% of the whole variance, respectively. The dendrogram obtained by cluster analysis displayed three groups of genotypes under both normal and salinity- stress conditions. Then, association analyses were conducted on 143 proso millet genotypes and 15 agronomic traits as well as 514 polymorphic AFLP markers (out of 866 created bands) generated by 11 primer combinations (out of the initial 20 primer combinations) EcoRI/MseI. The results obtained by mixed linear model (MLM) indicated that under normal conditions, the M14/E10-45 and M14/E10-60 markers had strong associations with seed yield. A similar trend was also observed for M14/E10-45 and M14/E11-44 markers in relation to forage yield. On the other hand, M14/E10-14, M14/E10-64 markers (for seed yield) and M14/E10-64 marker (for forage yield), had significant and stable association in all environments under salinity-stress conditions. Moreover, a number of markers showed considerable associations and stability under both normal and salinity stress conditions. CONCLUSIONS: According to the analysis of phenotypic data, the wide germplasm of Iranian proso millet has significant variation in terms of measured traits. It can be concluded that markers showing strong associations with traits under salinity-stress conditions are suitable candidates to be used in future marker-assisted selection (MAS) studies to improve salinity-resistance genotypes of Panicum miliaceum in arid and semiarid areas.

Evaluation of drought tolerance indices for barley landraces under irrigated and dry conditions / Avaliação de índices de tolerância seca para terraços de cevada em condições irrigadas e secas

Barley cultivation for drought areas requires a reliable assessment of drought tolerance variability among the breeding germplasms. Hence, 121 barley landraces, advanced breeding lines, and varieties were evaluated under both moisture non-stress and stress field conditions using a lattice square (11×11) design with two replications for each set of the trials. Twelve drought tolerance indices (SSI, TOL, MP, GMP, STI, YI, YSI, HM, SDI, DI, RDI, and SSPI) were used based on grain yield under normal (Yp) and drought (Ys) conditions. Analysis of variance showed a significant genetic variation among genotypes for all indices except for TOL and SSPI indices. Yp had a very strong association with Ys (r = 0.92**) that indicates high yield potential under non-stress can predict better yield under stress conditions. Yp and Ys were positively and significantly correlated with MP, GMP, STI, YI, HM, and DI indices, whereas they were negatively correlated with SSI and SDI. In principal component analysis (PCA), the first PC explained 64% of total variation with Yp, Ys, MP, GMP, STI, YI, HM, and DI. The second PC explained 35.6% of the total variation and had a positive correlation with SSI, TOL, SDI, and SSPI. It can be concluded that MP, GMP, STI, YI, HM and DI indices with the most positive and significant correlation with the yield at both non-stress and stress environments would be better indices to screen barley genotypes, although none of the indices could undoubtedly identify high yield genotypes under both conditions.

O cultivo de cevada para áreas secas exige uma avaliação confiável da variabilidade da tolerância à seca entre os germoplasmas reprodutores. Assim, 121 linhagens crioulas de cevada (linhas de reprodução avançada e variedades) foram avaliadas em campo sob condições sem estresse e com estresse de umidade do solo, utilizando-se para isso um arranjo experimental de malha quadrada (11×11), com duas repetições para cada conjunto de ensaios. Foram utilizados 12 índices de tolerância à seca (SSI, TOL, MP, GMP, STI, YI, YSI, HM, SDI, DI, RDI e SSPI), com base no rendimento de grãos sob condições normais sem estresse (Yp) e com estresse de seca (Ys). A análise de variância mostrou uma variação genética significativa entre os genótipos para todos os índices, com exceção dos índices TOL e SSPI. Yp teve uma associação muito forte com Ys (r = 0,92**), o que indica que o potencial de alto rendimento sob condições sem estresse pode prever melhor rendimento sob condições de estresse. Yp e Ys foram positivamente e significativamente correlacionados com os índices MP, GMP, STI, YI, HM e DI, enquanto, foram correlacionados negativamente com os índices SSI e SDI. Na análise de componentes principais (PCA), o primeiro PC explicou 64% da variação total com Yp, Ys, MP, GMP, STI, YI, HM e DI. O segundo PC explicou 35,6% da variação total e apresentou correlação positiva com SSI, TOL, SDI e SSPI. Pode-se concluir que, os índices MP, GMP, STI, YI, HM e DI com a correlação mais positiva e significativa com a produção nos ambientes sem estresse e com estresse seriam melhores índices para a seleção de genótipos de cevada, embora nenhum dos índices pudesse concretamente identificar genótipos de alto rendimento sob ambas as condições.

Correlation between gene expression levels under drought stress and synonymous codon usage in rice plant by in-silico study.

We studied the correlation of synonymous codon usage (SCU) on gene expression levels under drought stress in rice. Sixty genes related to drought stress (with high, intermediate and low expression) were selected from rice meta-analysis data and various codon usage indices such as the effective number of codon usage (ENC), codon adaptation index (CAI) and relative synonymous codon usage (RSCU) were calculated. We found that in genes highly expressing under drought 1) GC content was higher, 2) ENC value was lower, 3) the preferred codons of some amino acids changed and 4) the RSCU ratio of GC-end codons relative to AT-end codons for 18 amino acids increased significantly compared with those in other genes. We introduce ARSCU as the Average ratio of RSCUs of GC-end codons to AT-end codons in each gene that could significantly separate high-expression genes under drought from low-expression genes. ARSCU is calculated using the program ARSCU-Calculator developed by our group to help predicting expression level of rice genes under drought. An index above ARSCU threshold is expected to indicate that the gene under study may belong to the "high expression group under drought". This information may be applied for codon optimization of genes for rice genetic engineering. To validate these findings, we further used 60 other genes (randomly selected subset of 43233 genes studied for their response to drought stress). ARSCU value was able to predict the level of expression at 88.33% of the cases. Using third set of 60 genes selected amongst high expressing genes not related to drought, only 31.65% of the genes showed ARSCU value of higher than the set threshold. This indicates that the phenomenon we described in this report may be unique for drought related genes. To justify the observed correlation between CUB and high expressing genes under drought, possible role of tRNA post transcriptional modification and tRFs was hypothesized as possible underlying biological mechanism.

Gibberellin causes wide transcriptional modifications in the early stage of grape cluster development.

Yaghooti grape of Sistan is seedless, early ripening but has compact clusters. To study gibberellin effect on cluster compactness of Yaghooti grape, it has been studied transcriptomic changes in three developmental stages (cluster formation, berry formation and final size of cluster). We found out that 5409 of 22,756 genes in cluster tissue showed significant changes under gibberellin. Finally, it was showed that 2855, 2862 and 497 genes have critically important role on above developmental stages, respectively. GO enrichment analysis showed that gibberellin enhances biochemical pathways activity. Moreover, genes involved in ribosomal structure and photosynthesis rate in cluster tissue were up- and down- regulated, respectively. In addition, we observed location of metabolomic activities was transferred from nucleus to cytoplasm and from cytoplasm to cell wall and intercellular spaces during cluster development; but there is not such situation in gibberellin treated samples.

Distribution and possible biosynthetic pathway of non-protein sulfur amino acids in legumes.

Some grain legumes store sulfur in the form of non-protein amino acids in seed. γ-Glutamyl-S-methylcysteine is found in Phaseolus and several Vigna species. γ-Glutamyl-S-ethenylcysteine, an antinutritional compound, is present in Vicia narbonensis. In P. vulgaris, free S-methylcysteine levels are higher at early stages of seed development followed by a decline. γ-Glutamyl-S-methylcysteine accumulates later, in two phases, with a lag during reserve accumulation. The concentration of total S-methylcysteine, quantified after acid hydrolysis, is positively regulated by sulfate nutrition. The levels of both γ-glutamyl-S-methylcysteine and γ-glutamyl-S-ethenylcysteine are modulated in response to changes in seed protein composition. A model is proposed whereby the majority of γ-glutamyl-S-methylcysteine in P. vulgaris is synthesized via the intermediate S-methylhomoglutathione. Knowledge of the biosynthesis of non-protein sulfur amino acids is required for metabolic engineering approaches, in conjunction with manipulation of the protein sink, to increase the concentration of nutritionally essential methionine and cysteine. This would improve protein quality of some important legume crops.

Common Bean (Phaseolus vulgaris L.) Accumulates Most S-Methylcysteine as Its γ-Glutamyl Dipeptide.

The common bean (Phaseolus vulgaris) constitutes an excellent source of vegetable dietary protein. However, there are sub-optimal levels of the essential amino acids, methionine and cysteine. On the other hand, P. vulgaris accumulates large amounts of the γ-glutamyl dipeptide of S-methylcysteine, and lower levels of free S-methylcysteine and S-methylhomoglutathione. Past results suggest two distinct metabolite pools. Free S-methylcysteine levels are high at the beginning of seed development and decline at mid-maturation, while there is a biphasic accumulation of γ-glutamyl-S-methylcysteine, at early cotyledon and maturation stages. A possible model involves the formation of S-methylcysteine by cysteine synthase from O-acetylserine and methanethiol, whereas the majority of γ-glutamyl-S-methylcysteine may arise from S-methylhomoglutathione. Metabolite profiling during development and in genotypes differing in total S-methylcysteine accumulation showed that γ-glutamyl-S-methylcysteine accounts for most of the total S-methylcysteine in mature seed. Profiling of transcripts for candidate biosynthetic genes indicated that BSAS4;1 expression is correlated with both the developmental timing and levels of free S-methylcysteine accumulated, while homoglutathione synthetase (hGS) expression was correlated with the levels of γ-glutamyl-S-methylcysteine. Analysis of S-methylated phytochelatins by liquid chromatography and high resolution tandem mass spectrometry revealed only small amounts of homophytochelatin-2 with a single S-methylcysteine. The mitochondrial localization of phytochelatin synthase 2-predominant in seed, determined by confocal microscopy of a fusion with the yellow fluorescent protein-and its spatial separation from S-methylhomoglutathione may explain the lack of significant accumulation of S-methylated phytochelatins.

Salinity-associated microRNAs and their potential roles in mediating salt tolerance in rice colonized by the endophytic root fungus Piriformospora indica.

Piriformospora indica (P. indica), an endophytic root fungus, supports the growth and enhanced tolerance of plants to biotic and abiotic stresses. Several recent studies showed the significant role of small RNA (sRNA) molecules including microRNAs (miRNAs) in plant adaption to environmental stress, but little is known concerning the symbiosis-mediated salt stress tolerance regulated at miRNAs level. The overarching goal of this research is to elucidate the impact of miRNAs in regulating the P. indica-mediated salt tolerance in rice. Applying sRNA-seq analysis led to identify a set of 547 differentially abundant miRNAs in response to P. indica inoculation and salt stress. These included 206 rice-specific and 341 previously known miRNAs from other plant species. In silico analysis of miRNAs predictions of the differentially abundant miRNAs led to identifying of 193 putatively target genes, most of which were encoded either genes or transcription factors involved in nutrient uptake, sodium ion transporters, growth regulators, and auxin- responsive proteins. The rice-specific miRNAs targeted the transcription factors involved in the import of potassium ions into the root cells, the export of sodium ions, and plant growth and development. Interestingly, P. indica affected the differential abundance of miRNAs regulated genes and transcription factors linked to salt stress tolerance. Our data helps to understand the molecular basis of salt stress tolerance mediated by symbionts in plant and the potential impact of miRNAs for genetic improvement of rice varieties for tolerance to salt stress.

Mapping QTL controlling agronomic traits in a doubled haploid population of winter oilseed rape (Brassica napus L.).

Identification of superior alleles for agronomic traits in genetic resources of oilseed rape (Brassica napus L.) would be useful for improving the performance of locally adapted cultivars in Iran. The objective of the present work was to analyse the genetic variation and inheritance of important agronomic traits in a doubled haploid population derived from a cross between two German oilseed rape cultivars, Sansibar and Oase. Field experiments were performed in 2016-2017 with 200 doubled haploid lines and the parental genotypes applying an alpha-lattice design with two replicates. Phenological traits were recorded during the cultivation period and at maturity, seed yield, yield components and seed quality traits were determined. Significant genetic variation was found in most of the traits and heritabilities ranged from medium (48.5%) for days to end of flowering to high (92.6%) for oil content. A molecular marker linkage map was used to map 36 QTL for different traits on 17 linkage groups. Between three and four QTL were identified for each seed yield, seed weight, oil and protein content. Some of the plant material and positive QTL alleles identified for agronomic traits may be useful for improving those characters in locally adapted cultivars in Iran.

Mapping QTLs for physiological and biochemical traits related to grain yield under control and terminal heat stress conditions in bread wheat (Triticum aestivum L.).

In order to detect genomic regions with different effects for some of the physiological and biochemical traits of wheat, four experiments were conducted at Research Farm of Agricultural and Natural Resources Research Center of Zabol in 2015-2016 and 2016-2017 growing seasons. The experiments were carried out using four alpha lattice designs with two replications under non-stress and terminal heat stress conditions. Plant materials used in this study included 167 recombinant inbred lines and their parents ('SeriM82' and 'Babax'). Six traits including grain yield (GY), proline content (PRO), water soluble carbohydrates (WSC), maximum efficiency of photosystem II (Fv/Fm), cytoplasmic membrane stability (CMS) and chlorophyll content (CHL) were evaluated. Genetic linkage map consisted of 211 AFLP marker, 120 SSR marker and 144 DArT markers with 1864 cm length and 4.4 cm mean distance. QTL analysis was carried out using a mixed-model-based composite interval mapping (MCIM) method. By the combined analysis of normal phenotypic values, 27 additive QTLs and five pairs of epistatic effects were identified for studied traits, among which two additive and one epistatic QTL showed significant QTL × environment interactions. By the combined analysis of stress phenotypic values, a total of 26 QTLs with additive effects and 5 epistatic QTLs were detected, among which one additive and one epistatic QTL showed QTL × environment interactions. Six QTLs with major effects (QGY-2B, QGY-2D, QPro-5B, QWSC-4A, QFv/Fm-6A and QCMS-4B), which were common between two conditions could be useful for marker-assisted selection (MAS) in order to develop heat tolerant and high-performance wheat varieties.

Unraveling the transcriptional complexity of compactness in sistan grape cluster.

Yaghooti grape of Sistan is the earliest ripening grape in Iran, harvested every May annually. It is adapted to dry conditions in Sistan region and its water requirement is less than the other grape cultivars. The transcriptional complexity of this grape was studied in three stages of cluster development. Totally, 24121 genes were expressed in different cluster development steps (step 1: cluster formation, step 2: berry formation, step 3: final size of cluster) of which 3040 genes in the first stage, 2381 genes in the second stage and 2400 genes in the third stage showed a significant increase in expression. GO analysis showed that when the clusters are ripening, the activity of the nucleus, cytoplasmic, cytosol, membrane and chloroplast genes in the cluster architecture cells decreases. In contrast, the activity of the endoplasmic reticulum, vacuole and extracellular region genes enhances. When Yaghooti grape is growing and developing, some of metabolic pathways were activated in the response to biotic and abiotic stresses. Gene co-expression network reconstruction showed that AGAMOUS is a key gene in compactness of Sistan grape cluster, because it influences on expression of GA gene which leads to increase cluster length and berries size.

Genetic diversity of Iranian Aegilops tauschii Coss. using microsatellite molecular markers and morphological traits.

Aegilops tauschii Coss. is a diploid (2n = 2x = 14,DD) goat grass species which has contributed the D genome in common wheat. Genetic variations in 28 accessions of Aegilops tauschii belonged to different provinces of Iran, were evaluated using 16 morphological traits and 19 SSR markers. In number of spikelet per spike and plant height, there was a high variation in ssp. tauschii and ssp. strangulata respectively and for days to mature a low variation in both subspecies was found. Discriminant function analysis showed that 67.9% of original grouped cases correctly classified. Factor analysis indicated that three factor explain 66.49% of total variation. The three clusters revealed by the cluster analysis were not consistent with their geographical distributions. We determined 208 alleles using 19 microsatellites. Average of alleles for every locus was 10.94. The total average of PIC was 0.267. 2261 bands produced for total of genotypes and Chinese Spring had the highest bands (95 alleles). The range of similarity coefficients was between 0.23 and 0.73. Genotypes were clustered using UPGMA method. The accessions did not match according to morphological cluster and geographical regions. 51.2% of total variations were related to 9 principle components.

Separation of DNA for molecular markers analysis from leaves of the Vitis vinifera.

In the present study, three DNA extraction procedures were examined to determine which might yield DNA from Grape leaves suitable for molecular analysis for RAPD, SSR. AFLP and etc analysis. The three methods examined were: the miniprep procedure and the modified CTAB for difficult species and protocol CTAB. Only the modified CTAB method consistently yielded DNA suitable for Polymerase Chain Reaction (PCR) amplification, regardless of plant growing conditions or leaf age. The quality and quantity of extracted genomic DNA gained from these methods are deliberated by means UV biophotometer, electrophoresis in 1.2% agarose gel and PCR. In this regard, application chosen for young and mature leaves, the most value of qualified DNA, is extracted from fully expanded leave when PVP was added to the extraction buffer. This same procedure also yielded PCR-amplifiable DNA from various other perennial, woody species and from other fruit species such as apple (Malus domestica), cherry (Prunus avium), peach (Prunuspersica), plum (Prunus domestica). DNA yield from this procedure is high (up to 1 mg g(-1) of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the Polymerase Chain Reaction (PCR).

Polymorphism of some native Sistan grapes assessed by long and short primers for RAPD markers.

Grapevines have Bronze ages archive in Sistan area of Iran. In order to study the genetic variation and taxonomic relationships between 6 cultivars of the Sistan grapevines (Vitis vinifera L.) at molecular level, random amplified polymorphic DNA (RAPD) markers were used. The data were subjected to statistical analyses and genetic resemblance was calculated using Dice similarity index. The grapevines related to the different geographic areas of Sistan were assessed by 50 short (10 mer) and long (15-21 mer) primers. Out of 50 primers which were tested, 21 primers gave reproducible results. Selected primers created 497 bands. Resulting profiles showed that the produced bands varied in size from 300 to 3500 base pairs. The numbers of reliable polymorphic fragments for short and long primers were 86 and 334 bands, respectively. In multiplication reaction the items in the size area of 564 to 1904 base pair resulted for short primers and 564 to 4277 base pair for long primers. From the bands calculated a matrix that was analyzed by the unweighted pair group method on arithmetic averages to draw a dendrogram. The population was classified in 4 main groups in which Red Yaghooti and White Yaghooti had the maximum and Red Yaghooti and Laal had the minimum similarity coefficients. In our study, by comparing the results gained from technique long and short primers in RAPD, the potential value of long primers for the production ofpolymorphism in grapes was identified.