DNA barcoding

Phase 2 protocols

Regions and Primers

Five plastid regions were chosen to continue into phase 2, with a sixth region, ndhJ, being added during the pilot stage of phase 2.   Each region had an initial set of two forward and two reverse primers:

Following the initial phase 2 pilot screening, where the six regions were tested in representative taxa from each of the study groups for ease of amplification and level of variability, the decision was taken to exclude YCF5 from the remainder of phase 2. Whilst YCF5 was potentially useful for some plants, it failed to yield sufficiently high quality sequences for certain taxonomic groups and has apparently been deleted from the plastid genome of bryophytes. It was therefore felt that YCF5 did not reach the requirements for a suitable barcode for all land plants.

Amplification Conditions

PCR reaction: PCR mix Buffer X 1 Mg2+ 1.5mM dNTPs 0.2mM FW test primer 1micromolar RE test primer 1micromolar Taq DNA polymerase 2 units BSA 0.1mg/ml Template (variable) Water to 20 microlitre	Thermal cycler program: PCR 94ºC 1min 1 cycle 94ºC    30sec Variable up to 40 cycles 53ºC   40sec 72ºC   40sec 72ºC   5min 1 cycle

Cycle sequencing is being performed according to the protocols of each partner institute on an extensive sampling of the groups listed.

During the course of phase 2, design of additional primer pairs and modification of the standard protocols outlined above was necessary for some regions. These details are now available as part of the phase 2 update.