# Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden
## 3 Results and Discussion  
### Detection of AsMaV and RT-PCR-RFLP  
RT-PCR was performed using detection primers for AsMaV [5] on total RNA extracted from collected plant material, resulting in the amplification of expected products of approximately 317, 288, and 672 bp for AsMaV RNA3-5, respectively, in 34 samples from Sweden and 49 samples from Finland. Using the primer set, AsMaV-R3-18-s/AsMaV-R3-end-as and AanI restriction enzyme, electrophoresis of digested RT-PCR products revealed seven different restrictotypes (Table 1). Restrictotype VII was the most prevalent and dominant type found in all tested areas, except in Mattinen-Tervola (Finland) and Skede-Krakshult (Sweden), where only Type IV and Type VI profiles were found, respectively. Notably, Kuninkaansaari-Helsinki and Vallisaari-Helsinki (Finland), the two small islands connected by a narrow bridge, showed five different restrictotypes (Type II, IV, VI, and VII). Similarly, Kisa (Sweden) showed three different restrictotypes (Type III, V, and VII). Identical restrictotypes were identified in both countries, suggesting the fixation of these mutations in RNA3. At least three samples from each location and each of restrictotypes were selected for sequencing and further analysis (in total 27 isolates). There was no correlation between geographical origin and restrictotype, and the distribution of restrictotypes was not uniform across all studied geographical regions.  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Restrictotype**} & \multirow{2}{*}{**Position of AanI Site on cRNA3**} & \multirow{2}{*}{**No. Cut 1**} & \multirow{2}{*}{**No. Fr. 2**} & \multicolumn{4}{c}{**Frequency of**} \\  & & & & & & **Restrictotypes (\%) 3** \\ \hline Type I & & & 0 & 1 & 1587 to 1589 & 1.2 & 6.1 & 7.3 \\ Type II & & & 1 & 2 & 1474, and 115 & 0.0 & 1.2 & 1.2 \\ Type III & & & 1 & 2 & 962, and 627 & 2.4 & 3.7 & 6.1 \\ Type IV & & & 2 & 3 & 627, 521, and 442 & 0.0 & 4.9 & 4.9 \\ Type V & & & 3 & 4 & 627, 515, 266, and 182 & 1.2 & 2.4 & 3.7 \\ Type VI & & & 3 & 4 & 627, 442, 338, and 182 & 3.7 & 1.2 & 4.9 \\ Type VII & & & 4 & 5 & 627, 442, 264, 182, and 73 & 32.9 & 39.0 & 72.0 \\ \hline \hline \end{tabular}  
* number of DNA restriction enzyme sites; 2 number of fragments recorded on electrophoresis after digestion of the RT-PCR with AanI restriction enzyme; 3 the percentages of each restrictotype were calculated based on the total number of collected isolates in this study, the list of which, along with their corresponding restrictotype groups, is presented in Supplementary Table S1 and the geographical distribution of restrictotypes is presented in the Supplementary Figure S1.  
\end{table}
Table 1: Position and frequency of AanI restriction site on AsMaV-RNA3 of 34, 47, and 81 collected samples from Finland (F), Sweden (S), and in total (T), respectively. The position of the nucleotides is based on AsMaV-RNA3 sequence of isolate E55089 (GenBank accession number LR742463). The black line represents the genomic RNA and orange rectangle shows the position of the ORF3, and the vertical red lines are denoting the position of AanI restriction site on the RNA3 (nucleotides: 627, 1069, 1142, 1406, 1474).