Primers for 130 regions of the chloroplast genome were evaluated in four species for which two or more sequences per species are available in GenBank (Fragaria versa, Gossypium herbaceum, Olea europaea, and Oryza sativa) by alignment in Sequencher. The primers of Shaw et al. (2005, 2007), Scarcelli et al. (2011) and Dong et al. (2012) were evaluated, along with primers posted here. Primers that failed to align automatically (in Sequencher), or that aligned incorrectly, were re-aligned manually whenever possible (guided by the GenBank annotations).
Two factors were evaluated:
Likelihood of amplification success, and
Variation in the sequence within species was inferred under three criteria: total number of variable characters (substitutions, indels, and inversions), percent sequence divergence (substitutions only) and a rank combining substitutions, indels, and inversions as a function of sequence length.
See the excel spread sheet (APPS-D-14-00085_AppendixS2.xlsx) on the Primer Utility page for the list of regions examined, amplification success predictions, information on the fastest evolving regions (by taxon), genomic diversity statistics, and taxon specific details. The excel spread sheet below compares sequence variability within species.