Table 2 Universal genetic markers used for DNA-based analysis in apiary studies

Cytochrome c oxidase complex

Cytochrome oxidase I (COI)

COI has been the predominant metabarcoding marker for distinguishing between metazoans species¹²⁴. As such, it is mainly utilised for determining entomological sources, such as differentiation of honey bees from different geographical regions¹²⁵, as well as traces of Hemiptera species as honeydew sources⁷².

Ribosomal subunits

18 S ribosomal RNA (18 S)

16 S ribosomal RNA (16 S)

18 S has been widely used as a universal primer for all eukaryotes in environmental samples¹²⁶, but honey-based studies appear to prefer more biologically relevant targets such as chloroplast genes for plants and COI for metazoans. In comparison, 16 S rRNA remains the most widely used universal primer for prokaryotes across different environmental samples¹²⁷ and has been used to identify bacterial species in honey to identify bee gut microbiota, microbes via interactions with the surrounding environment, as well as potential pathogens¹¹.

Internal transcribed spacer

ITS1

ITS2

Markers for both ITS1 and ITS2 regions have been used extensively to classify both fungal¹²⁸ and plant¹²⁹ species in DNA-based studies. ITS2 alongside rbcL was able to successfully identify over 900 plant species from honey samples¹³⁰, and when compared to a melissopalynological approach was able to conclude the same botanical species in an easier fashion⁹⁷. Fungal communities in honey were tested using ITS2 to ascertain microbial quality control¹³¹, and via the complete ITS region to delineate honey samples from different geographical regions¹¹.

Plant chloroplast

Ribulose bisphosphate carboxylase (rbcL)

Chloroplast tRNA gene intron (trnL)

Maturase K (matK)

trnH-psbA intergenic spacer region (trnH-psbA)

Plastid-based markers from the plant chloroplast are used to differentiate species, although there are still debates on which provides optimal plant identification, especially for use in honey samples¹³². Many honey sample studies use more than one set of markers, with plastid-based markers such as trnH-psbA¹³³, matK¹³⁴, rbcL¹³⁰ or trnL¹³⁵ used alongside the nuclear ribosomal ITS2 for increased resolution in delineating species.