# Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden
## 3 Results and Discussion
### Genetic Diversity and Phylogeny of AsMaV Isolates Based on RNA3
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{3}{*}{**Origin of Isolates**} & \multicolumn{8}{c}{**Genomic Region**} \\ \cline{2-10}  & & \multicolumn{3}{c}{**RNA3**} & \multicolumn{3}{c}{**RNA4**} & \multicolumn{3}{c}{**Partial RNA1**} \\ \cline{2-10}  & & **5\({}^{\prime}\) UTR** & **ORF3** & **3\({}^{\prime}\) UTR** & **5\({}^{\prime}\) UTR** & **ORF4** & **3\({}^{\prime}\) UTR** & **5\({}^{\prime}\) UTR** & **ORF1** \\ \hline \multirow{2}{*}{Finland} & nt & 95–100 & 88.9–100 & 80.8–98.8 & 97.6–100 & 95.9–99.9 & 82.4–100 & 96.5–100 & 85.2–99.7 \\  & aa & & **88.4–100** & & & **96.9–100** & & **96.6–100** \\ \hline \multirow{2}{*}{Sweden} & nt & 94–100 & 89.4–99.6 & 80–98.8 & 100 & 95.9–99.9 & 80–98.8 & 97.6–100 & 87.4–98.7 \\  & aa & & **91.8–100** & & & **97.5–100** & & **97.1–99.7** \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The identities of the RNA3, RNA4 and partial RNA1 at nucleotide (nt) and amino acid (aa) levels among the 18 Finnish and seven to eight Swedish isolates sequenced in this study. in the long 3' UTR based on the minimum free energy (MFE) and Centroid secondary structure with the minimum base pair distance prediction ([[http://rna.tbi.univie.ac.at/](http://rna.tbi.univie.ac.at/)]([http://rna.tbi.univie.ac.at/](http://rna.tbi.univie.ac.at/)) /cgi-bin/RNAWebSuite/RNAfold.cgi, 12 April 2023) in this region showed the potential complementary regions forming stem-loop structures. Interestingly, nt 1272-1358 in the 3' UTR was the most conserved AT-rich region. This region did not harbor any secondary structure which might explain the toleration of mutations resulting in variations in this region. A similar sequence was not found when we applied BLAST search. The substitution models of T92 + G and TN93 + G + I were selected as the best substitution models for AsMaV RNA3-4 and the partial RNA1, respectively, using the Find best DNA/Protein fit model test implemented in MEGA11 [49]. Similar tree topologies were obtained with both neighbor-joining and parsimony methods. Therefore, we only present and discuss the parsimony-generated trees. Based on the maximum parsimony tree generated from almost full-length 26 (18 Finnish and 8 Swedish) AsMaV RNA3 sequenced in this study and one previously reported isolate (LR742463) at the nucleotide level, AsMaV isolates clustered into four main groups. The first group, R3-G1, included E57260, E58627 (both from Finland), and E58213 (Sweden), and the second group, R3-G2, consisted of E58632 from Finland and E58227 from Sweden, group 3 (R3-G3) included the rest of the Swedish isolates (E58191, E58209, E58299, E58247, E58306, and E55089 with the E58491 isolate from Finland, and finally the rest of the Finnish isolates were placed in group 4 (R3-G4), i.e., E58377, E58385, E58386, E58397, E58398, E58405, E58413, E58421, E58433, E58434, and E58426. The identical grouping profile was documented when the tree was drawn based on the coding region of RNA3 (ORF3) at the nucleotide level. A similar tree, but with one difference was obtained when the phylogeny was based on the deduced amino acid sequences. In the latter tree, isolates E58227 (Sweden) and E58632 remained ungrouped. In general, isolates from Finnish and Swedish locations were separated based on their  
Figure 1: Nucleotide sequence alignment of AsMaV ORF3 termini reveals the presence of three distinct stop codons in the sequenced isolates. To highlight these stop codons, they have been marked with gray rectangular regions accompanied by the label “Stop” at the top. Notably, an indel event involving the insertion of three nucleotides, specifically “GAG”, has been observed in isolates E58213, E58627, and E57260, occurring immediately prior to the termination codon. These significant positions have been duly identified using asterisks. Furthermore, experimental confirmation of the AanI restriction site downstream of the respective stop codons has been obtained, and their corresponding positions in the alignment are indicated as AnanI site.