# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 2 Materials and Methods
### Data Collection and Statistical Analysis  
The dry weight of shoots, roots, their ratio, and the water content of shoots and roots were tested for normality using the Shapiro-Wilk test. All the parameters were normally distributed (\(p\) = 0.09, 0.10, 0.98, 0.48), respectively, except the water content of shoots and roots, which are log-transformed. The log-transformed water content of shoots was normally distributed (\(p\) = 0.18), while for roots was not. Additionally, the Levene test and the partial eta-squared test were applied in order to test the homogeneity of variances and the magnitudes of the main effects, respectively. Thus, the effect of treatment on the dry weight of shoot and roots was detected by two-way ANOVA. For not normally distributed data, the Kruskal-Wallis test was used. The Tukey test at 0.05 probability level was chosen for the detection of differences among means. The IBM SPSS Statistics 23 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. An AFLP Excel Macro [27] was used to convert allele size data from GeneMapper4.0 (Applied Biosystems, Foster City, CA, USA) into binary form and to indicate the presence or absence of fragments. Only reproducible fragments ranging from 150 to 500 bases were counted and further analyzed in order to reduce the impact of potential size homoplasy [28]. For MSAP analyses, a comparison of the banding patterns of _EcoRI_/_HpAll_ and _EcoRI_/_Mspl_ reactions results in four conditions of a particular fragment; I: fragments present in both profiles (1/1), indicating an unmethylated state; II: fragments present only in _EcoRI_/_Mspl_ profiles (0/1), indicating hemi- or fully methylated CG-sites; III: fragments present only in _EcoRI_/_HpAll_ profiles (1/0), indicating hemimethylated CHG-sites; and IV: the absence of fragments in both profiles (0/0), representing an uninformative state caused either by different types of methylation, or due to restriction site polymorphism [29]. The total analysis was performed in R software with MSAP_calc program and Mixed Scoring II method, which is a reliable program and method in order to access different methylation changes [29]. We further analyzed each category of methylated (h & m) and non-methylated (u) alleles with GenAlEx 6 [30] in order to compute the epigenetic Shannon Information Index (Iepi) and haploid epigenetic diversity (hepi) within and between cultivars and treatments. The Kruskal-Wallis test was performed in order to examine significant differences between different treatments and cultivars. Furthermore, we performed [PERSON]'s post hoc tests with Bonferroni correction on each pair of groups for h, m alleles, and total methylation (h+m alleles) for lepi. Due to the fact that the Shannon Diversity Index and genetic diversity are strongly correlated, and Shannon provides an alternative method of quantifying biological diversity across multiple scales [30], we further discussed results for lepi. Moreover, the Shannon Information Index (I) is widely used in ecological studies as a measure of diversity, as it calculates allele differences [29; 30] across loci of different samples. \begin{table}
\begin{tabular}{c c} \hline \hline  & **5\({}^{\prime}\) to 3\({}^{\prime}\) Sequence** \\ \hline _EcoRI_ adapter & CTCGTAGACTGCGTACC \\  & AATTGGTACGACTC \\ _Hpall/Mspl_ adapter & GACGATGCTCGAT \\  & CGATCGAGACTCAT \\ Pre-selective _EcoRI_ primer & GACTGCGTACCAAITTC-A \\ Pre-selective _Hpall/Mspl_ primer & ATGAGTCTCGATCCG-T \\  & GACTGCGTACAATTC+ATG \\ Selective _EcoRI_ primers & GACTGCGTACCAATTC+AACT \\  & GACTGCGTACCAATTC+AAG \\ Selective _Hpall/Mspl_ primer & ATGAGTCTCGCATCGG+TCA \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Adapters and primers used for the MSAP analysis. Finally, we produced a heat map presentation, which is actually a permuted data matrix of the six different treatments in a three-variable classification space (h-, m-, and u-methylation).