## 4 Materials and Methods
### RT-qPCR Analysis  
The expression profiles of the genes and miRNAs considered were determined using relative real-time quantitative PCR (RT-qPCR) The amplification was carried out using the SYBR Green(r) kit (Takara Holdings Inc., Shimogyoku, Kyoto, Japan) with the Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) on 96-well plates (PCR-96M2-HS-C(r), Avygen Scientific--part of Thermo Fisher Scientific, Waltham, MA, USA).  
For the analysis of genes, RT-qPCR was performed in a total volume of 10 \(\upmu\)L containing 2 \(\upmu\)L of DNA (5 ng/\(\upmu\)L) and 8 \(\upmu\)L of master mix composed of 5 \(\upmu\)L of TB Green Prmix Ex TaqMan (Tli RNaseH Plus, Takara Holdings Inc.), 0.5 \(\upmu\)L (1 \(\upmu\)M) of forward and reverse primers, 0.5 \(\upmu\)L of ROX Reference Dye, and 1.5 \(\upmu\)L of nuclease-free H\({}_{2}\)O for each sample. The amplification program comprised an initial incubation at 95 \({}^{\circ}\)C for 30 s, followed by 40 cycles of amplification (95 \({}^{\circ}\)C for 5 s, 60 \({}^{\circ}\)C for 30 s). A dissociation cycle was carried out at 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 1 min, and 95 \({}^{\circ}\)C for 15 s, followed by increasing the temperature stepwise by 0.3 \({}^{\circ}\)C.  
For the analysis of miRNAs, RT-qPCR was carried out in a total volume of 10 \(\upmu\)L containing 2 \(\upmu\)L of cDNA (1 ng/\(\upmu\)L) and 8 \(\upmu\)L of master mix composed of 5 \(\upmu\)L of TB Green Prmix Ex TaqMan (Tli RNaseH Plus) (Takara Holdings Inc.), 1 \(\upmu\)L of miRCURY LNA miRNA (QIAGEN SpA), 0.5 \(\upmu\)L of ROX Reference Dye, and 1.5 \(\upmu\)L of nuclease-free H\({}_{2}\)O for each sample. The amplification program comprised an initial incubation at 95 \({}^{\circ}\)C for 30 s, followed by 40 amplification cycles (95 \({}^{\circ}\)C for 5 s, 60 \({}^{\circ}\)C for 31 s). A dissociation cycle was carried out at 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 1 min, and 95 \({}^{\circ}\)C for 15 s, followed by increasing the temperature stepwise by 0.3 \({}^{\circ}\)C.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Name** & **a.n.** & **miRNA Sequence (5’-3’)** & **Reference** \\ \hline _ata-miRR166c-5p_ & MIMAT0037248 & ggaacguuggcuccagg & [PERSON] et al., 2013 [56] \\ \hline _ath-miR_R396b & MIMAT0000945 & uuccacaguuuucuugaacu & [PERSON] et al., 2004 [50] \\ \hline _ath-miR847_ & MIMAT0004278 & uaccuccuucuucuucuugaug & [PERSON] et al., 2006 [51] \\ \hline _gma-miR_R396f & MIMAT0021069 & agcuuucuugaacuucuuaugc [PERSON] & [PERSON] et al., 2011 [54] \\ \hline _gra-miR_R7486c & MIMAT0034235 & uuuuguccagguaacagaaaacgc & [PERSON] et al., 2013 [57] \\ \hline _gra-miR_R8759 & MIMAT0034189 & uguggaaguauuuggcccgg & [PERSON] et al., 2013 [57] \\ \hline _osa-miR_R395f & MIMAT000974 & gugaauuuguggggggaac & [PERSON] et al., 2004 [50] \\ \hline _osa-miR_R5538 & MIMAT0022174 & acugaacucaaucaugcugc & [PERSON] et al., 2011 [55] \\ \hline _osa-miR_R66a-5p_ & MIMAT0022855 & ggaaugauguugucucagg & [PERSON] et al., 2011 [53] \\ \hline _osa-miR_R166d-5p_ & MIMAT0022858 & ggaaugauguugucuggcuccagg & [PERSON] et al., 2011 [53] \\ \hline _osa-miR_R2099-5p & MIMAT0010062 & ugaaauguuguuguugacagcuuu & [PERSON] et al., 2009 [52] \\ \hline _osa-miR_R396a-5p & MIMAT0000977 & uuccacaguuuucuugaacug & [PERSON] et al., 2011 [53] \\ \hline \hline \end{tabular}  
* _—Agglips tauschii_ Cons.; _áth—Arabiidopsis thaliana_ (L) Heynh.; gma—_Glycine max_ (L) Merr; gra—_Gossgtrium raimondii_ Ubr.; osa—_Oryza sativa_ L; a.n.: miRbase accession number.  
\end{table}
Table 3: miRNAs selected for expression analysis in _Echinochla orgzicola._The threshold values (Ct) were determined by means of the 7300 Real-Time PCR System's on-board software. Each sample was tested in triplicate.