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DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost.

Food security and nutrition concerns are putting an ancient, climate-smart grain back on our plates; Farm to fork, there has been a revival of interest in millet. Foxtail millet, as a multi-purpose product, has nutritious and medicinal potentials. This research is aimed at identifying combined markers as well as stable associations between such markers and the investigated agronomic traits in a wide range of foxtail millet germplasms under normal and salinity-stress conditions. In this context, association analysis has been conducted among 14 agronomic traits and 331 polymorphic AFLP markers generated by 12 primer combinations in 134 foxtail millet genotypes. Based on the analysis of population structure, the foxtail millet genotypes were divided into six subpopulations. The results showed that a number of markers had stable and significant associations under both normal and salinity-stress conditions with agronomic traits. The primer combinations had high polymorphic percentage, diversity indices were highly reliable and revealed significant genetic variability among the genotypes. Their PIC, MI and Shannon’s indices were also highly reliable and revealed significant genetic variability among the genotypes. Since the markers introduced in this research have stable and strong associations with the investigated traits under normal and salinity stress conditions, they can be suitable candidates’ in future marker-assisted breeding to improve salinity- resistance genotypes of foxtail millet in arid and semiarid areas.

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.

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.

Verticillium wilt is a major disease of olive (Olea europaea) orchards worldwide. To control the disease, use of a reliable method for pre-planting screening of new planting stocks for infection by V. dahliae is essential. In this study, 20 symptomatic and asymptomatic olive trees, sampled from Zahak region of Sistan in Iran, were analyzed with real-time and nested PCR for in planta detection of V. dahliae. We could confirm the presence of V. dahliae in 12 out of 20 (60%) sampled trees using real-time PCR, whereas nested PCR produced amplicons for all tested symptomatic and asymptomatic samples (100% of tested trees), indicating that nested PCR was more efficient method than real-time PCR for in planta detection of V. dahliae. We used the designed nested PCR to determine also the pathotype of V. dahliae using Vdf2/Vdr2 primer set at the second round, which could produce the 297-bp amplicon for all tested samples infected with non-defoliating pathotype of V. dahliae. We showed that although the Verticillium wilt is spread in the olive cultivation areas in Zahak region of Sistan, nested PCR can be deployed for the timely detection and thereby prevention of the further spread of V. dahliae in the olive cultivation areas.

Background Kochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread, environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na+ concentrations. Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na+/H+ antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na+ concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts. Results Coding sequence of plasma membrane Na+/H+ antiporter (SOS1) and vacuole membrane Na+/H+ antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologous in salicornia brachiate and suede maritime, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control. Conclusion The results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops.

Kochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread. environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na+ concentrations. Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na+/H+ antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na+ concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts. Coding sequence of plasma membrane Na +/H + antiporter (SOS1) and vacuole membrane Na /+H + antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologue in salicornia brachiata and suadea maritima, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control. The results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops.