## 4 Materials and Methods
### Analyzed Genes and miRNA Prediction and Validation  
Genes involved in herbicide detoxification were selected on the basis of previously published studies [16; 17; 18; 21; 23; 61]. We analyzed the expression of nine cytochromes P450 monooxygenase (_CYP81A12_, _CYP81A21_, _CYP81A22_, _CYP81A24_, _CYP81A63_, _CYP81A6_, _CYP71 AK2_, _CYP72A122_, and _CYP72A254_), two glutathione S-transferase (_EcGST_ and _GSTF1_), and the eukaryotic translation initiation factor 4B (_elF4B_). The sequences of the most conserved DNA regions of each gene were obtained in Gene Bank [62] and in GrainGenes--A Database for Triticeae and Avena [63]. The primer sequences of the selected genes were designed using Primer-BLAST [64]. The primer selection was based on default parameters, with the exception of annealing temperature, which was set at 60 \({}^{\circ}\)C; the primer length was set around 20 bp, and the expected PCR product size was set from 70 bp to 150 bp.  
Table 2 lists the primers used to analyze the expression of CYP450, GST, and _elF4B_ genes in _E. oryzaeola_.  
According to the methodology of [PERSON] et al., 2024 [65], miRNA prediction was carried out in silico using the psRNATarget: A Plant Small RNA Target Analysis web  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Gene ID** & **Primer Sequence (5’-3’)** & **Reference** \\ \hline _CYP81A12_ & F: tgagcctctcctgctg & [PERSON] et al., 2014 [18] \\  & R: tacttttttggcagctccgct & \\ \hline _CYP81A21_ & F: tagcatcatcatcacagagcgc & [PERSON] et al., 2014 [18] \\  & R: tacacgtctaccagcgcat & \\ \hline _CYP81A22_ & F: cgcgcgcgctgctcgtt & [PERSON] et al., 2014 [18] \\  & R: tacatgagcagttccagcg & \\ \hline _CYP81A24_ & F: gaggtttacaccgatgccac & [PERSON] et al., 2014 [18] \\  & R: cttttgagcttccggggt & \\ \hline _CYP81A63_ & F: gaggcactcgctcagacca & [PERSON] et al., 2020 [13] \\  & R: atetftgttcctcacgcgcga & [PERSON] et al., 2019 [17] \\ \hline _CYP81A6_ & F: gactatctaacccggggcgat & [PERSON] et al., 2022 [61] \\  & R: caggtttgcgcgcagag & \\ \hline _CYP71 AK2_ & F: acgtgtgtgggacaagttcctg & [PERSON] et al., 2014 [21] \\  & R: ggctttgagcgctgctg & \\ \hline _CYP72A122_ & F: agttcacggggaggttc & [PERSON] et al., 2014 [21] \\  & R: cactttggcttcacagcgcg & \\ \hline _CYP72A254_ & F: tacagagtactccggctgt & [PERSON] et al., 2014 [21] \\  & R: gtcagggctctggtgtaatgt & \\ \hline _EcGST_ & F: gccgaggaggacctgaagac & [PERSON] et al., 2013 [16] \\  & R: gtgactcacagagagctctccgt & \\ \hline _GSTF1_ & F: tgccttctaacccccatgat & [PERSON] et al., 2018 [23] \\  & R: agtgactcgtgtcggggagag & \\ \hline _elF4B1_ & F: cgagcgcttaacaggact & [PERSON] et al., 2018 [23] \\  & R: gtggttccataccaccaga & \\ \hline \hline \end{tabular}
\end{table}
Table 2: Nucleotide sequences of the primers used for RT-qPCR analysis of CYP450, GST, and _elF4B_ gene expression in _Echinochla oyzzicola_.  
server (Schema V2, 2017 release) [42; 43; 44]. Starting from the complete coding sequence of genes, in \".fasta\" format (Table 2), optimal alignment between miRNAs and target genes was assessed by applying the Smith-Waterman algorithm [66] using the default parameters. Mature miRNAs with a sequence complementarity rate \(\geq\) 80% with the target gene and annotated in miRbase [67] for other plants were selected (Table 3). According to [PERSON] et al., 2011 [68], the predicted miRNAs were finally validated in _E. orgzicola_ via RT-qPCR.