# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 3 Results
### MSAP Results  
The two varieties tested (Chaironia and Lamia) presented different patterns of DNA methylation in response to drought stress. In fact, according to the mean value of polymorphic alleles that were provided from the R software with the MSAP_calc program for each category of alleles (h, m and u), Lamia controls presented a 68.57% percentage of total methylation, while a decrease of the total methylation percentage was observed in Lamia D1D2 (61.92%) and CD2 (62.31%). On the other hand, the total methylation in the leaves of Chaironia controls reached a percentage of 71.12%, whereas a decrease of total methylation was noticed in Chaironia CD2 treatment (58.23%), but not in the case of Chaironia D1D2 (72.36%) (Table 3, Figures 6 and 7). Figure 4: Shoot water content expressed as g of water per g of dry shoot weight. Bars show the mean of the biological replicates \(\pm\) SE on day 133; lower case letters indicate significant differences between the means (\(p<0.05\)). Figure 5: Root water content expressed as g of water per g of dry root weight. Bars show the mean of the biological replicates \(\pm\) SE on day 133. There were no significant differences among the means. Furthermore, according to the Kruskal-Wallis test for h alleles for lepi and hepi, very strong evidence of the difference was provided (\(p<0.001\)) between the mean ranks of at least one pair of groups. For m alleles and total methylation (h + m alleles), the Kruskal-Wallis tests also found significant differences between groups for lepi and hepi (\(p<0.001\)). Only for uninformative alleles (u alleles), no significant differences were found according to the Kruskal-Wallis test (\(p=0.917\)). According to [PERSON]'s post hoc tests with Bonferroni correction for lepi and hepi on each pair of the group, we found that:  
1. For h alleles, no significant differences were found between Lamia treatments, while for Chiaronia significant differences were found between CC/CD2, and CD2/D1D2 treatments. 2. For m alleles, significant differences were found for Lamia CC/Lamia D1D2 and for Chiaronia CC/Chaironia CD2, Chiaronia CD2/Chaironia D1D2. 3. For total methylation (h+m), significant differences were found between Lamia CC/Lamia D1D2, Chiaronia CC/Chaironia CD2, Chiaronia D1D2. More specifically for lepi, Lamia variety exhibited a statistically significant decrease in total (h- and m-alleles) DNA methylation after the second drought episode (D1D2) as compared to the controls (Table 4). In addition, both CD2 and D1D2 treatments showed a decrease in DNA methylation as compared to the initial non-stressed control (CC), although there is no statistically significant difference between total methylation of CD2 and CC. On the other hand, for lepi and regarding the DNA methylation status of the Chiaronia variety, the total methylation (h and m alleles) of CD2 is \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Polymorphic Markers**} & **Lamia** & **Lamia** & **Lamia** & **Chaironia** & **Chaironia** & **Chaironia** \\  & **CC** & **CD2** & **D1D2** & **CC** & **CD2** & **D1D2** \\ \hline
**h** methylation & 114 & 87 & 79 & 120 & 79 & 133 \\
**m** methylation & 102 & 80 & 69 & 146 & 66 & 134 \\ Uninformative & 99 & 101 & 91 & 108 & 104 & 102 \\ Total polymorphic markers & 315 & 268 & 239 & 374 & 249 & 369 \\ Total methylation (h + m) & 216 & 167 & 148 & 266 & 145 & 267 \\ Percentage of total methylation \% & 68.57 & 62.31 & 61.92 & 71.12 & 58.23 & 72.36 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: DNA methylation polymorphic markers in control (CC) and the two drought treatments (CD2 and D1D2) for the two varieties of _M. sativa_. Figure 6: Methylation patterns (demonstrated as distinct h/m/u markers) of all treatments tested. Y-axis represents total methylation (h + m alleles). significantly lower compared to CC, whereas in D1D2 plants, the total methylation has increased significantly as compared to CD2.