# Rapid Molecular Identification of Scolytinae (_Coleopterara: Curculionidae_)
## 3 Discussion
### Primer Pairs for Molecular Identification of Diverse Genera  
Markers COI and EF-1a are recommended for initial molecular identification attempts based on the ability of the respective primers to amplify DNA from multiple genera (_Coptoborus_, _Xyleborus_, _Hypothenemus_, _Theoborus_, and _Araptus_) and the ability of the resulting sequences to provide accurate and unambiguous matches in GenBank. BLASTn analysis of EF-1a sequences correctly identified four out of the five genera and COI sequences identified at least one sample of every genus tested and was the only primer pair to correctly identify _Araptus_ specimens. The low number of EF-1a (1), 28S (7), and ITS2 (0) sequences from _Araptus_ individuals present in GenBank compared with COI (137) is likely the reason the latter marker was the only one capable of identifying members of this genus.  
Primer pair D2F1/D3R2 amplified the best and generated high-quality sequences capable of identifying _Coptoborus_, _Xyleborus_, and _Theoborus_, however, neither bark beetle genus (_Hypothenemus_ or _Araptus_) could be identified using these primers. Although six 28S sequences from _Hypothenemus_ are available in GenBank, no _Hypothenemus_ sp. accessions were within the 100 best matches. A single 28S sequence from _Araptus_ sp. is available in GenBank (AF375297); however, 87% of the identity it shares with the sequences generated in this study is lower than the 91-93% shared with _Ips duplicatus_ accessions. This may be due to misidentification, or high variability in the genus. Other _Araptus_ species may have greater homology with the specimens in this study. As more sequences from confirmed _Araptus_ species are deposited in GenBank, molecular sequence identification may become viable for members of this genus. Due to inconclusive results following BLASTn analyses, identification of J1, J2, and J4 was performed by [PERSON] (personal communication, 28 October 2019).  
The COI gene is considered the ideal gene for species identification of insects [28] due to its popularity and variety of different samples in GenBank. Sequences generated in this study based on the COI gene led to correct genus level identifications for all five genera analyzed. However, only five published studies were found where primer pair LCO1490/HCO2198 was used for Scolytinae [20; 24; 25; 29; 30]. These primers showed inconsistent amplification and low-quality sequences were generated inthis study (even with high concentrations of amplified PCR product), indicating that LCO1490/HCO2198 may not be the best primer pair for amplifying COI in these organisms. Primers developed by [PERSON] et al. [31] have been used successfully in Scolytinae beetles and may be a good alternative.  
ITS2 sequences were insufficient to identify any of the samples tested, BLASTn results showed all sequence matches having query coverages of less than 50% with three out of the four sequences having a query coverage of less than 30% which may be due to low representation in GenBank. Additionally, it was difficult to obtain amplification for a wide variety of Scolytinae.