**5. Conclusions**

Three species-specific primer sets targeting the *NADH dehydrogenase 2* (*ND2*) region of mtDNA were designed and successfully applied to trace the entomological origin of honey produced by different honeybees, *A. cerana*, *A. dorsata* and *A. mellifera*. In addition, the *A. mellifera* specific primer set is applicable in honey fraud detection. The possibility of using melting curve analysis in discrimination of the origin of honey using the same primer sets is also confirmed. Our preliminary studies indicated the impossibility of providing species-specific primers with a smaller size of PCR product in the mitochondrial DNA (except the one provided by Soares et al. [7] for ACH). However, further studies targeting nuclear DNA are required. PCR-based method using species-specific primers provides a rapid and cost-effective method to screen the entomological origin of honey. Therefore, the development of new primer sets to identify honey produced by other species of honeybees will be valuable. On the other hand, more studies are needed to understand the pace of DNA degradation in honey and the applicability and limitations of using molecular methods in the authentication of older honey samples.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11070928/s1, Table S1: The honey samples and their labels and localities used for analysis. Table S2: Mitochondrial sequences of honeybees (*A. cerana*, *A. dorsata* and *A. mellifera*) used to design species-specific primers. Figure S1: Preliminary specificity test of ACF/ACR (Suarez et al., 2018) using DNA extracts of honeybees [39–57].

**Author Contributions:** S.M.N.: Visualization, methodology, design primers, Sequence assembling and blast, investigation, writing the original draft. F.Y.: Visualization, writing the original draft, investigation, methodology. H.W.C.: Project administration, supervision, review and editing. C.J.: Supervision, funding acquisition, resources, review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the BSRP through the National Research Foundation of Korea (NRF), ministry of Education (grant number NRF-2018R1A6A1A03024862), and Rural Development Administration (RDA agenda PJ01574604 on honeybee pollination).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the sequences generated in this research, have been deposited in GenBank under accession numbers MW660861-MW660880.

**Acknowledgments:** We are grateful to Bajaree Chuttong, Chiang Mai University who provided us with honey and bee samples from Thailand. We also thank Ratna Thapa for facilitating SMA and CJ's expedition trip to Nepal. This study was supported by the BSRP through the National Research Foundation of Korea (NRF), ministry of Education (grant number NRF-2018R1A6A1A03024862).

**Conflicts of Interest:** S.M.N. and F.Y. are a research professor and Ph.D. student at ANU, respectively, and received a full-time salary for this work. C.J. has received research grants from NRF, and supervised the work. H.W.C. has served on advisory for the project. All declare no conflicts of interest on this paper.
