Diversity of individuals' methylation patterns to different moisture regimes in Einkorn wheat revealed by CRED-RA technique

Among the wild relatives of wheat, diploid species with desirable characteristics have high potential in crop modi�cation. One of the important epigenetic changes that is associated with the pattern of gene expression and causes genetic instability is DNA methylation. The coupled restriction enzyme digestion-random ampli�cation (CRED-RA) technique and �ve RAPD primers were used to study methylation changes in two Einkorn wheat genotypes including S10 and H9 genotypes and four soil water availability treatments included control, mild, moderate and severe stress (irrigation at 100%, 75%, 50% and 25% of �eld capacity, respectively) in two stages (14 days after water de�ciency and 14 days after re-watering). The experiment was laid out as 2×4 factorial experiment within completely randomized design replicated three times. Measured physiological traits included relative leaf water content, Fv / Fm and stomatal conductance. After 14 days of exposure to drought stress, the results showed that the lowest relative water content of leaves, Fv/Fm and stomatal conductance were related to severe stress treatment. Also, S10 genotype was superior to H9 after application of moisture stress treatments and after re-watering. Different patterns of methylation were observed in different replications of each treatments in response to severe water de�ciency, which can be due to the diversity of individuals' response to stress and the speci�city of epigenetics changes. Most of the fragments that showed a change in methylation pattern due to water de�ciency, after stress removal, returned to the state before stress, which indicates the reversibility of methylation changes in the genome.


Introduction
Plants are uncovered to different environmental stresses during development in natural and agricultural conditions.Drought is one of the most extreme environmental stresses that in uences plant productivity.
Around 80 to 95% of plant biomass is composed of water, which plays a crucial part in different physiological processes, including many aspects of plant growth, development and metabolism (Seleiman et al. 2021).In addition, some consider drought as the most important environmental factor for various plants, especially in drought-prone areas (Diatta et al. 2020) and the most important threat to future world food security ( Okorie et al. 2019).Drought stress is expanding due to erratic precipitation and changes in climate patterns.Rapid increase in atmospheric temperature has increased plant exposure to drought stress (Umair Hassan et al. 2020).On the other hand, the severity of drought stress depends on numerous components such as distribution and sum of precipitation, evapotranspiration and soil capacity to store moisture (Saud et al. 2016).
Wheat (Triticum aestivum L.) is one of the most important crops for humans, providing about 19% of food calories and more than 20% of the protein consumed by the world's population (Zhang et al. 2021).
Wheat is the staple food of about 35% of the world's population and the demand for its expansion is higher than any other crop (Shiferaw et al. 2013).The distribution of wild wheat relatives in the world is mainly limited to a number of countries located in the Fertile Crescent.Iran, as one of the main habitats of wild relatives of wheat, is one of the richest centers of diversity of hereditary reserves of this germplasm in the world (Weide et al. 2015).Wild relatives of wheat are closely related species that have a long history of growing mainly to withstand abiotic stress.These species are recognized as vital resources needed to maintain the global food supply (Pour-Aboughadareh et al. 2021).T. monococcum, T. boeoticum and T. urartu are considered as Einkorn wheat.Einkorn wheat (2n = 2x = 14, AA) are diploid and self-pollinating species known as close wild relatives of bread and durum wheat (Liu et al. 2020).
Wild wheat species have incredible potential as a source of hereditary characteristics to move forward the drought resistance of wheat cultivars since wild wheat species are profoundly tolerant to drought stress (Budak et al. 2013).Wild wheat species Triticum boeoticum is more drought tolerant than other wheat species such as Triticum dicoccoides, Triticum araraticum and common wheat cultivars (Sultan et al. 2012;Hui et al. 2015).
Plants use a variety of mechanisms, such as avoidance or tolerance, to cope with environmental stresses, allowing them to adapt to and defend themselves against stress.Such adaptation strategies are at the morphological, anatomical, biochemical, and molecular levels (Devi et al. 2017).Recently, epigenetics symptoms have been considered as important factors in gene control associated with abiotic stresses (Gallo-Franco et al. 2020).However, plants can respond to environmental stresses, including drought stress, through epigenetics regulatory strategies such as DNA methylation and histone modi cations (Duan et al. 2020;Arslan et al. 2019).DNA methylation means that under the effect of methylase, the DNA sequence of genes does not change, but the function of genes in response to external environmental stimuli changes (Sun et al. 2021).DNA methylation, which adds the methyl group to the C-5 cytosine (5meC) position, is a common epigenetics marker in many eukaryotes and occurs predominantly in CpG dinucleotides (Liu et al. 2021).Restriction enzymes with CpG detection sequences can distinguish between methylated and non-methylated states (Harshitha and Nair, 2020).CRED-RA is one of the DNA methylation detection techniques that has been studied by many researchers (Bakir andGüleray, 2021, Falahi et al. 2021).This modi cation technique uses the randomly ampli ed polymorphic DNA (RAPD) method, which uses methylation-sensitive enzymes such as HpaII and MspI to detect methylation changes between different tissues and different stages of growth after enzymatic digestion of DNA (Altunkaynak et al. 2016;Erturk et al. 2015).In this method, DNA, after ampli cation by random primers, is cut separately by HpaII and MspI enzymes, which have the same identi cation site with different sensitivity to DNA methylation, and polymorphisms between the products show differences in the methylation pattern (Lima and Bull, 2008).Taspinar et al. (2018) using CRED-RA method in corn showed that aluminum stress increased the ratio of DNA methylation.Erturk et al. (2015) used the CRED-RA technique to investigate the effects of heavy metals such as zinc on corn and concluded that different doses of zinc altered the methylation pattern in this plant.Bakir and Güleray (2021) showed that all doses of vanadium caused DNA damage and reduced the genomic stability pattern by studying the wheat genome treated with vanadium and boric acid solutions using ISSR marker and CRED-RA method.Rai et al. (2018) when investigate on beans high temperature stress and application of salicylic acid and Sodium nitroprusside utilizing CRED-RA watched that these compounds direct the methylation patterns and activity of genes included in streess and can offer assistance plants to endure high temperatures by "synchronizing" development with "physiological" adjustment.Ogutcu et al. (2014) in the study of the antagonistic effect of calcium against cadmium in beans using CRED-RA technique showed that different doses of these substances change the methylation pattern.
The targets of this study were, determination of methylation pattern in two Einkorn wheat genotypes under different soil water availability treatments and the stability of DNA methylation changes after relief from drought stress.

Plant materials and experimental design
The 2×4 factorial experiment was conducted within completely randomized design with three replications in the greenhouse of the Campus of Agriculture and Natural Resources of Razi University, Kermanshah, Iran during 2019-2020.The factors of the experiment included four soil water availability treatments included control, mild stress, moderate stress and severe stress (irrigation at 100%, 75%, 50% and 25% of eld capacity, respectively) and two Einkorn wheat genotypes collected from Kermanshah province (Genotype H9, collected from Ravansar region with geographical characteristics N, 34 ° 46.987; E, 46 °47.233) and Kurdistan province (Genotype S10, collected from Sanandaj region with geographical characteristics of N, 35 ° 22.572; E, 46 ° 16.137), both from the west of Iran.The investigation was carried out in two stages, 14 day after water de ciency and 14 day after re-watering.After two weeks of water de ciency, the plants were irrigated again and the eld capacity was obtained immediately.Five plants were randomly selected from each treatment and physiological traits were measured.

Relative leaf water content (RWC)
To measure RWC, 0.5 g leaf (FW) was selected from youngest leaf developed from each plant.Then placed them in distilled water for 4 hours until saturated with water and their weight was measured (TW).
The samples were dried for 48 hours at 70 C and their dry weight (DW) was measured and the relative leaf water content was calculated with the following equation: RWC% = [FW -DW ∕ TW -DW] × 100 (Egert and Tevini, 2002).

Stomatal Conductance
A prometer was used to determine the stomatal conductance of the leaf and the measurement was performed in the early hours of the day (between 10 am and 12 am).The middle part of the leaf was placed in the device and after 10 seconds the reading was performed.
Photochemical E ciency Of Photosystem Ii (Fv / Fm) The clamps of the chlorophyll measurement device (PEA Pocket -Hansatech model) were attached to the leaves.These clamps have a special shape on which the diode of the device is easily xed.The clamps have valves that close the part of the leaf in darkness.After half an hour, the diode is connected to the clamp and then the valve of the clamp is opened by pressing the key on the diode, the uorescence parameters appear on the display screen of the device.In this way, the Fm / Fv index was read on the device.The Fm/Fv index is obtained from the Fm/Fo-Fm formula, in this formula, Fm is the maximum chlorophyll uorescence and Fo is the minimum chlorophyll uorescence in leaves accustomed to darkness.

Molecular Studies
Isolation of DNA DNA extraction was done by Dellaporta et al. (1983) method for each sample in the laboratory.The quality and quantity of extracted DNA was evaluated using 0.8% agarose gel and Thermo Scientifc nanodrop, respectively.

Polymerase Chain Reaction Using Rapd Primers
Polymerase chain reaction (PCR) was performed in a volume of 20 microliters contained, 10 pmoles primer, 1x Masterix (Sinaclone) and 30 ng DNA template (Williams et al. 1990).Three replicates of the experiment were analyzed separately.The characteristics of the primers used in this study are shown in the Table 1.

Cred-ra Assay
Extracted DNA was subjected to enzymatic digestion using MspI (Fermentase, ER0541) and HpaII (Fermentase, ER0511) enzymes in accordance with the instructions of the manufacturer.The restriction enzyme digestion products were ampli ed individually by ve RAPD primers.The ampli ed PCR products were electrophoresed on 1.5% agarose gel and then stained with ethidium bromide under UV light.

Data analysis
Data analysis was performed with SAS 9.1 software after performing the test of normality for residuals.
Mean comparison of the traits whose F test was signi cant in the analysis of variance was done with the help of Least Signi cant Difference (LSD) method.Scoring and interpretation in CRED-RA assay based Aina et al. ( 2004) method.

Analysis of physiological traits under different soil water availability
The results showed that the two Einkorn genotypes had signi cant differences (p < 05.0) for stomatal conductance and photochemical e ciency of photosystem II (Table 2) after 14 days of water de ciency.
RWC, stomatal conductance and Fv/Fm were signi cantly different among different soil water availability treatments (Table 2).The results of mean comparisons of two Einkorn genotypes after 14 days of water de ciency showed the highest values of stomatal conductance and Fv/Fm was shown in S10 genotype (Table 2).In addition, the results of LSD test of four soil water availability treatments after 14 days of water de ciency showed that the lowest RWC, stomatal conductance Fv/Fm were related to the severe stress (Table 2).

Responses Of Physiological Traits After Re-watering
Analysis of variance of measured physiological traits in two Einkorn genotypes after 14 days of rewatering showed that H9 and S10 genotypes signi cantly were different for stomatal conductance and Fv/Fm.Signi cant difference observed between soil water availability treatments for all measured traits (Table 3).The results LSD test for two Einkorn wheat genotypes after re-watering showed that S10 genotype was signi cantly superior to H9 in terms of all traits (Table 3).Also, the results of LSD test for measured traits in the four soil water availability treatments after re-watering showed that RWC in severe stress treatment was signi cantly lower than other treatments.The highest and lowest stomatal conduction rates were obtained in control and severe stress treatments, respectively (Table 7).

Results Of Molecular Analyses
In molecular studies, four soil water availability treatments in two stages included two weeks of water de ciency and two weeks after re-watering in two Einkorn wheat genotypes (S10 and H9) in three replications were examined individually using ve RAPD primers and CRED-RA assay.
CRED-RA assay's results in two Einkorn wheat genotypes after two weeks water de ciency and two weeks re-watering

Opc-12 Primer
Ampli cation of cleaved products with HpaII and MspI enzymes in S10 genotype show different patterns of methylation (including methylation, methylation in external cytosine and methylation in internal cytosine) in different replications of four soil water availability treatments (Fig. 1).This difference in different replications can be attributed to the variability in the response of individuals to stress and the speci city of the methylation pattern in different individuals.Cleavage with methylation-sensitive restriction enzymes in H9 genotype leading to fragments ampli cation that show different methylation patterns (including methylation, change of methylation pattern from complete methylation to methylation in external cytosine and methylation in internal cytosine and methylation of genome) in different replications of moderate and severe stress treatments and in response to severe moisture stress.
The results of cleavage with HpaII and MspI enzymes in S10 genotype after re-watering showed the return and emergence of deleted fragments in irrigation treatments at moderate and severe stress treatments, except for one fragment of about 250 bp in the rst replication of moderate and severe stress treatments is still cleaved during digestion with HpaII enzyme and shows methylation in external cytosine.Methylation changes are reversible as the stress disappears.Also, the results of cleavage with HpaII and MspI enzymes in H9 genotype indicated that the methylation changes observed after 14 days of water de ciency, were reversible.

Opa-019 Primer
Cleavage results with restriction enzymes (MspI and HpaII) in S10 genotype after after 14 days of water de ciency showed that the only difference between the treatments was 900 bp fragment, which was observed in one of the replications in control treatment.Therefore, it was not considered as a change in methylation pattern in response to water de ciency (Fig. 2).Ampli cation of cleaved products with HpaII and MspI in H9 genotype after 14 days of water de ciency, showed removal of bands in the sizes of 230 and 250 bp in severe stress treatment during digestion with HpaII enzyme.Also, 550 bp fragment in moderate and severe stress treatments was cleaved by HpaII enzyme and converted from hypermethylation to methylation in foreign cytosine (Bolukbasi and Aras, 2016).These results indicate the response of H9 genotype to severe stress.
PCR-ampli ed DNA after endonuclease digestion after re-watering in S10 genotype showed that the removed fragment, when treated with MspI enzyme, which was observed in some replications after 14 days of water de ciency, appeared after re-watering.It seems that this fragment is related to a speci c growth stage in the plant and after reaching this stage of cytosine external methylation growth, it has become completely methylated.In addition, the results of cleavage with restriction methylation-sensitive enzymes after re-watering in H9 genotype indicate the reappearance of deleted fragments of 230, 250 and 550 bp after stress relief and reversibility of genome methylation changes.

Opc-08 Primer
The results of digestion with HpaII enzyme in S10 genotype after 14 days of water de ciency indicated the presence of a fragment of about 2000 bp in moderate stress treatment and change of methylation pattern from complete methylation to methylation of external cytosine (Bolukbasi and Aras 2016).This fragment was removed by MspI and HpaII enzymes in severe stress treatment, which indicates its methylation in response to severe water de ciency.A fragment of 1700 bp in severs stress treatment was removed by HpaII enzyme and showed the change of methylation pattern to methylation of foreign cytosine in irrigation treatments in moderate and sever stress treatments and in response to severe water de ciency.
The results of CRED-RA assay for H9 genotype in different treatments of moisture stress, showed the difference a 1400 bp completely de-methylated in severe stress treatment (irrigation at 25% of eld capacity) but in two replications of irrigation treatment in moderate stress treatment and one replication of mild stress treatment when cut with MspI enzyme showed methylation in external cytosine (Fig. 3).
The results of cleavage with HpaII and MspI enzymes after re-watering in S10 genotype show a stable genomic pattern and no polymorphism was observed among the ampli cation fragments.On the other hand, digestion with HpaII and MspI enzymes after re-watering in H9 genotype indicates the emergence of a 1400 bp fragment in severe stress treatment, but the remarkable point is the methylation pattern in internal cytosine in most repetitions in the mild and moderate stress treatments, so that it seems that the methylation pattern of this fragment can be related to other unknown factors.
Ops-07 Primer CRED-RA assay in S10 genotype after two weeks water de ciency showed a fragment of 900 bp that was completely removed in the control treatment, and in mild stress treatment by the MspI was removed and shows a pattern of methylation in internal cytosine, whereas this fragment was not digested in moderate and severe stress treatments in three replications when treated with any of the enzymes and showed a complete methylation pattern.A fragment of 1500 bp was also removed by both MspI and HpaII in severe stress treatment (Fig. 4).Cleavage with methylation-sensitive enzymes in H9 genotype after two weeks water de ciency show that a 750 bp fragment was removed both MspI and HpaII enzymes and demethylated by in severe stress treatment.It is from changing the methylation pattern to de-methylation.
PCR-ampli ed DNA after methylation-sensitive cleavage after re-watering in S10 genotype showed that 250 and 1500 bp fragments removed in severe stress treatment.While a 900 bp fragment in control and mild stress treatments is removed by both enzymes and in moderate and severe stress treatments when digestion with MspI enzyme is removed and shows the methylation pattern in internal cytosine.The genomic region appears to be completely methylated in response to severe moisture stress, and the methylation pattern changes due to milder moisture stress.Probably after re-watering, the effects of stress on moisture treatment are still observed in severe stress treatment.The methylation investigation results in H9 genotype show that the 750 bp fragment removed in severe stress treatment after two weeks water de ciency, re-emerged and the methylation pattern changed from methylation to hypermethylation.

Opb-07 Primer
The results of CRED-RA assay after two weeks water de ciency show that a fragment with a size of about 1000 bp in moderate stress treatment was digested by HpaII enzyme, which indicates a change in methylation pattern from hypermethylation status to methylation in external cytosine.This fragment was removed and completely methylated in the severe stress treatment by MspI and HpaII.The change of methylation pattern from complete methylation to de-methylation seems to have taken place in response to intense moisture stress.
The results of digestion with methylation-sensitive enzymes in the stress application stage in H9 genotype showed that a fragment of 760 bp in severe stress treatment was removed by both MspI and HpaII and completely methylated indicates the change of methylation pattern in severe stress from hypermethylation to de-methylation (Fig. 5).
The results of CRED-RA technique in S10 genotype after re-watering show that all removed fragments reappeared in response to moisture stress, no digestion and elimination took place and the stability of the genome pattern at this stage is 100%.Also, the results of CRED-RA technique after re-watering in H9 genotype showed the absence of 760 bp fragment in severe stress treatment during cutting with MspI and HpaII, which indicates the maintenance of methylation pattern in severe stress after relieving stress and recording changes in plant memory.

Discussion
Lack of soil water is one of the important aspects of soil irrigation and management that contributes to water stress in plants and therefore has a negative impact on crop productivity (Alordzinu et al. 2021).
Results from investigation of physiological traits in Einkorn genotypes showed that control treatment due to absorb more water from the soil has the highest amount of RWC (73.10%), which is consistent with the results of Omidian et al. (2021).In similar studies, it was observed that with increasing drought stress, the amount of RWC of black mung bean leaf (Vigna mungo) decreased and cultivars that were able to maintain more RWC of leaves under reduced soil water content were more resistant to water loss.In other words, they had a higher ability to absorb and retain water (Khorsand et al. 2021).Also, in the study of Khorsand et al. (2019), increasing drought stress reduced the RWC of maize, but some plants that are more tolerant reduce their water consumption by reducing transpiration or management.The lowest stomatal conductance and Fv/Fm were obtained from severe stress treatment.One of the rst reactions of plants under water stress is to close the pores to reduce water loss, which leads to a decrease in pore conductance and photosynthesis (Verma et al. 2021).In addition, water stress affects the yield of the whole plant and disrupts the growth and development of plants by changing stomatal factors.So that with decreasing water in the leaves, the conduction of the stomata under water stress decreases (Thruppoyil and Ksiksi, 2020).Sha q et al. (2021) reported that drought stress reduced the rate of photosynthesis, transpiration and stomatal conduction of sun ower by closing the stomatal pore.
Another effect of water stress on plants is to reduce the photochemical e ciency of photosystem II.
Decreased in Fv/Fm is evidence that the photosynthetic performance of the plant is compromised.This decrease could also be likely due to increased light energy loss in photosystem II (Martins et al. 2015).Khademian et al. (2019) reported that severe drought stress disrupted photochemical e ciency of photosystem II activity in sa ower leaves and reduced Fv / Fm.This nding indicates the occurrence of light damage to the reaction centers of photosystem II and the reduction of the maximum photochemical e ciency of photosystem II.
In this study, 14 days of water de ciency, the plants were irrigated again and the eld capacity was obtained immediately.The results of measuring physiological traits in two Einkorn genotypes showed after 14 days of re-watering and the plants still have symptoms of stress and have not fully recovered.Therefore, it seems that more time and amount of water is needed to completely eliminate the symptoms of stress.Therefore, for a more detailed study, we used the CRED-RA technique to investigate the DNA methylation pattern in the leaves of two Einkorn genotypes grown under water de ciency stress.
The results of CRED-RA technique from used ve primers showed the removal of different fragments by enzymatic digestion in soil water availability treatments in S10 and H9 genotypes, which may indicate a different methylation pattern of these two genotypes in response to water de ciency.Comparison of methylation pattern in different water de ciency treatments in both Einkorn genotypes showed more severe changes in mild and severe stress treatments than other treatments, which con rms the effect of water de ciency on methylation pattern in plants.Zhang et al. (2016) in a study on the root structure of bread wheat using RAPD marker and CRED-RA method examined the effect of EGTA.They reported that the levels of genomic DNA polymorphism and DNA methylation pattern showed a signi cant increase.
Individual differences were observed between the three replications of each genotype in terms of methylation pattern in response to water de ciency.Guangyuan et al. (2007) in the study of canola methylation pattern in response to salinity stress, reported different methylation patterns of different individuals.Most fragments that showed methylation pattern in both Einkorn wheat genotypes due to water de ciency, returned to the pre-stress state after re-watering, which indicates the reversibility of methylation changes in the wheat genome.It also seems that the methylation pattern changed from demethylation to hypermethylation in response to moisture stress.Throughout the life cycles of eukaryotic cells, including plants, the pattern of methylation is dynamic and reversible (Gehring and Henikoff, 2007).

Conclusion
The methylation changes in the both Einkorn wheat's genome were reversibility to the pre-stress state after re-watering.In general, the results of physiological traits measurement in two Einkorn wheat genotypes after water stress and re-watering showed the general superiority of S10 genotype over H9 in terms of measured traits.The most changes in the methylation pattern were observed in mild and severe stress treatments, respectively, which was an expected result and shows the effects of water de ciency on gene expression processes in plants.Most of changes in methylation pattern due to moisture stress, returned to the state before the stress after re-watering, which indicates the reversibility of methylation changes in the genome.It seems that de-methylation plays a more important role than methylation in the genome of einkorn wheat during water de ciency stress.

Falahi
et al. (2021) examined three barley cultivars under water stress using CRED-RA method and observed that the most methylation modi cations in three barley cultivars were full methylation conversion to external cytosine methylation.In clover and ax plants, the distribution patterns of methylated sites before and after metal stress relief were similar.This suggests that changes in the methylation pattern may be caused by stress and disappear after stress relief(Peng and Zhang, 2009).

Table 1
Name, sequence and annealing temperature of RAPD primers

Table 2
Analysis of variance and mean comparison of measured traits in two Einkorn wheat genotypes under four soil water availability treatments after application of soil water availability treatments ns, *, and ** indicate non-signi cant, signi cant at P < 0.05 and P < 0.01, respectively.Values with different letters in superscript in a column indicate signi cant difference at P ≤ 0.05.

Table 3
Analysis of variance and mean comparison of measured traits in two Einkorn wheat genotypes under four soil water availability treatments after re-watering ns, *, and ** indicate non-signi cant, signi cant at P < 0.05 and P < 0.01, respectively.Values with different letters in superscript in a column indicate signi cant difference at P ≤ 0.05.
Hosseinpour et al. (2020)the study of the effects of glyphosate herbicide at different concentrations on bread wheat by CRED-RA method showed that the rate of methylation of the type of hypermethylation In our study, it seems that de-methylation of genomic regions during water stress plays an important role in dealing with water stress.DNA methylation can be considered as an important epigenetics regulatory mechanism in wheat for adaptation to stress conditions(Duan et al. 2020).Many fragments that showed a change in the methylation patterns due to water de ciency stress returned to their original state after re-watering; there were few fragments that remained constant in the plant's genomic memory.Sapna et al. (2020)reported that drought stress changed DNA methylation pattern in rice.They suggested that increased drought methylation may be one of the main mechanisms associated with adaptation reactions in crops such as rice.Temel and Gozukirmizi (2012) using CRED-RA technique showed that culture conditions cause genetic diversity and epigenetics in barley's mature embryos.Hosseinpour et al. (2020)investigated the effects of zinc oxide and lactobacillus nanoparticles (PGPBs) on methylation changes in tomato seedlings under salinity stress using RAPD marker and CRED-RA method.Their results showed that zinc oxide nanoparticles were effective in increasing the stability of the genome and reducing its polymorphism.
Aydin et al. (2021).9%.Aydin et al. (2021)in the study of the effects of β-estradiol in reducing the effects of salinity stress on bread wheat by CRED-RA method showed that the use of beta-estradiol in different concentrations reduced the effects of salinity stress resulting in hypermethylation in DNA and the results showed that this hormone can play an important role in protecting wheat seedlings in stress tolerance.