Internal transcribed spacer primer evaluation for vascular plant metabarcoding

The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has potential provide a powerful tool for accurate and rapid monitoring. Unfortunately, many cases, lack universal standards undermines widespread application metabarcoding. One most important considerations plants, aside from selecting potent barcode marker, is primer choice. Our study evaluates published ITS primers silico vitro , through mock communities presents newly designed primers. We were able show that large proportion previously available have unfavourable attributes. combined results support recommendation introduced ITS-3p62plF1 ITS-4unR1 as best current plant specific ITS2 combination. also found PCR optimisation, such addition 5% DMSO, essential obtain meaningful Finally, we conclude continuous quality assurance indispensable reliable results.