*5.3. Amplification*

The choice of barcode marker is regarded as one of the most important considerations of DNA barcoding studies and its applications, ultimately affecting the number of taxa recovered and the level of species discrimination obtained [32]. DNA barcode markers require high universality so that a large proportion of species in a sample are amplified, but also low intra-specific and high inter-specific variation for effective species discrimination [33]. Short markers allow for amplification of degraded DNA; however, these come with a caveat of reduced taxonomic resolution [144].

There is no single marker which meets the ideal requirements for a plant barcode [31,32]. For pollen metabarcoding, five regions are commonly used: *rbcL*, ITS2, *matK*, *trnL*, and *trnH-psbA* (Table S1). A multi-locus approach is recommended to ensure the greatest number of taxa are identified [24,38,144]. The length of *matK* (800 bp), restricts its use in metabarcoding due to limitations in read length on standard sequencing platforms [32]. Therefore, it is recommended that *rbcL* and ITS2 are used for pollen metabarcoding, due to their ability to identify taxa at varying taxonomic levels along with additional taxa unique to one marker which provides accurate identification of plant species within mixed pollen samples [38,45,78].

Contamination may also occur in the laboratory; therefore, stringent cleaning procedures are required to minimise these risks. The use of controls (negative in extraction, positive and negative in PCR amplification) helps in the identification of sources of contamination and should be sequenced with samples [38,145]. If sequences occur in negative controls, the number of reads of each taxon should be removed from all samples [81].
