# Rapid Molecular Identification of Scolytinae (_Coleopterara: Curculionidae_)
## 2 Results
### Primer Pairs for Molecular Identification of Diverse Genera
Primer pair ets149/efa754 amplified in all genera except _Araptus_, but yielded fewer high-quality sequences in _Theoborus_ and _Coptoborus_, compared to the previous primer pair. For _Hypothenemus_ individuals, two of the four samples generated sequences with quality scores above 89% and were at least 98% identical to _Hypothenemus_ nr. _eruditus_ sp. (JX264092) (Table 5). One of the four produced a low-quality sequence (26%) which shared 85.5% identity to _Phloeocleptus cristatus_ sp. (KY805879) but this is not included in the results table due to the low quality of the input sequence. Primer pair ITS2F/ITS2R amplified well in _Xyleborus_ and _Coptoborus_ and produced decent quality sequences (85.2-97.7%); however, a BLASTn analysis showed these sequences to be uninformative. Sequences in both groups (samples B7b and B4) were 100% identical to _Pityogenes chalcographus_ (JQ066311) and _Galapaganus_ spp. (EU748796) (Table 6), which are morphologically distinct from the samples processed as seen in the microscopy images for each of these samples. In addition, sequences obtained from two additional _Coptoborus_ samples yielded no significant similarities for any matches in GenBank regardless of their qualities being \(<97\%\) and having lengths of 299 bp and 679 bp. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & & **28S (D2F1/D3R2)** & & \\ \hline \multirow{2}{*}{**Sample**} & \multirow{2}{*}{**Genus**} & **PCR** & & **Accession** & \\  & & **Concentration** & **GenBank Match** & **Number** & **\% Identical** \\  & & **(ng/uL)** & & **Number** & **\% Identical** \\ \hline B2 & _Coptoborus pseudotemuis_ & 44.6 & _Coptoborus pseudotemuis_ & HM099689 & 100.0\% \\ B4 & _C. pseudotemuis_ & 45 & _Coptoborus pseudotemuis_ & HM099689 & 100.0\% \\ B6 & _C. pseudotemuis_ & 16.6 & _Coptoborus pseudotemuis_ & HM099689 & 100.0\% \\ B7a & _C. pseudotemuis_ & 46.5 & _Coptoborus pseudotemuis_ & HM099689 & 100.0\% \\ B7b & _C. pseudotemuis_ & 38.2 & _Coptoborus pseudotemuis_ & HM099689 & 100.0\% \\ \hline \multirow{2}{*}{B3} & \multirow{2}{*}{_Xyleborus vorlulus_} & HM099763 & 100.0\% \\  & & & _X. perforans_ & HM099747 & 100.0\% \\  & & & _X. bispinatus_ & HM099741 & 100.0\% \\  & & & _X. affinis_ & GU808581 & 100.0\% \\ \hline B14 & _Theoborus_ sp. & 39.2 & _Theoborus_ sp. & HM099718 & 99.7\% \\ BC1 & _Theoborus_ sp. & 23 & _Theoborus_ sp. & HM099718 & 99.7\% \\ BC9 & _Theoborus_ sp. & 42.8 & _Theoborus_ sp. & HM099718 & 99.7\% \\ BC13 & _Theoborus_ sp. & 37.7 & _Theoborus_ sp. & HM099718 & 100.0\% \\ \hline B16 & _Hypothenemus_ sp. & 2.31 & _Theoborus_ sp. & HM099718 & 99.7\% \\ B17 & _Hypothenemus_ sp. & 6.07 & _Theoborus_ sp. & HM099718 & 99.7\% \\ B19 & _Hypothenemus_ sp. & 4.74 & _Theoborus_ sp. & HM099718 & 99.7\% \\ B20 & _Hypothenemus_ sp. & & No amplification & & \\ \hline J1 & _Araptus_ sp. & 4.91 & _Ips duplicatus_ & JX263733 & 93.2\% \\  & & & _Araptus_ sp. & AF375297 & 87.9\% \\ J2 & _Araptus_ sp. & 9.32 & _Ips duplicatus_ & JX263733 & 91.4\% \\  & & & _Araptus_ sp. & AF375297 & 87.0\% \\ J4 & _Araptus_ sp. & 8.08 & _Ips duplicatus_. & JX263733 & 91.5\% \\  & & & _Araptus_ sp. & AF375297 & 87.0\% \\ \hline \hline \end{tabular}
\end{table} 
Table 3: BLASTn results for 28S sequences amplified and sequenced with primers D2F1 and D3R2. Concentration of PCR product sent for sequencing also shown to the left of the GenBank match. Primer pair LCO1490/HCO2198 amplified well in all groups tested, but only about half of these resulted in good quality sequences (defined as quality greater than 40% by authors). For _Xyleborus_, _Theoborus_, _Hypothenemus_, and _Araptus_, the best sequence matches in GenBank were from individuals within the correct genus. However, identification of _Coptoborus_ was ambiguous as the generated sequences had greater % identity to _Coptodrays_ sp. (HM064072) than to accessions of the correct genus,