# Identification of Seasonal Honey Based on Quantitative Detection of Typical Pollen DNA
## 4 Discussion  
The _Prunus_ plants mainly provide honeybees with nectar and pollen after the critical winter season in South Korea. Consequently, a high quantity of _Prunus_ pollen was accumulated in the honey sample collected in April (Figure 1). Therefore, the identification of cherry blossom (_Prunus_ sp.) honey based on quantification of its pollen was easier compared to that of the other honey samples collected from May to July owing to the diverse sources of pollen accumulated in the honeycomb in these months (Figure 1). Such accumulation could lead to little pollen collection in the following months [30,31]. In addition, honeybees tend to consume the newly stored pollens [32]. Therefore, the older accumulated pollens could be remained in the hive for a long time. These accumulation leads to the difficulty of monofloral honey identification by melissopalynology method.  
Figure 2: Quantitative detection of pollen DNA of four targeted plants in commercial honey samples. The quantity of DNA corresponding to _Prunus_ sp., _R. pseudoacacia_, _Castanea_ sp., and _Kalopanax_ sp. was calculated from the monofloral honey samples of cherry blossom, false acacia, chestnut, and avalia, respectively, purchased in Korea. The result was calculated from triplicate PCRs (mean + standard deviation).  
Although the pollen spectrum of each specific monofloral honey sample is different, which ranges from 15% in lavernder honey to >45% in chestnut honey [33; 34; 35; 36], such pollen deposition makes the identification of dominant pollen present in the honey difficult and influences the accurate identification of botanical origin of honey based on the total collected pollens [37; 38]. Therefore, pollen-based parameters are not sufficient to accurately identify monofloral honey samples. A combination of pollen determination with other techniques is necessary [39], such as analysis of specific protein patterns [12], mineral content, physicochemical parameters [40], or aroma compounds by dynamic headspace extraction and gas chromatography-mass spectrometry [41]. Furthermore, identification of total pollen DNA in combination with bacterial and fungal taxa using DNA metabarcoding and metagenomic differentiation could be helpful [42; 43].  
The presence of plant species detected in the collected samples confirmed that the flowering periods of the four target plants were consistent with those proposed by [PERSON] and [PERSON] 2007 [25], and the presence of a plant species in the honey sample that has a different flowering season could be a simple and rapid method for identification of adulterated monofloral honey, such as the presence of _Kalopanax_ sp. (blooming in July) in the false acacia honey (_R. pesudacacai_, blooming in May). However, the honey produced in June and July contain the pollens of diverse species due to the accumulation of pollens. Therefore, the standard index of pollen spectrum and other physicochemical parameters of these monofloral honey should be determined in further study.  
DNA markers was used as one of the important methods for identification of the valuable manuka monofloral honey [44]. However, detection of pollen DNA of a single plant species was not sufficient to determine the monofloral due to the stored pollens present in honeycomb after the flowering season or adulteration by mixing targeted pollen with pure honey [39]. Therefore, multiplex qPCR using primers targeting on major nectar plants that typically bloom in successive months in a particular region could be helpful to identify the time of honey production and monofloral honey.