PCR-based amplicon length polymorphisms (ALPs) at microsatellite loci and indels from non-coding DNA regions of cloned genes as a means of authenticating commercial Japanese green teas

We have developed PCR-based DNA markers with the objective of authenticating, and discouraging the fraudulent commercialization of, the forty-four most cultivated Japanese green teas. For this purpose we have generated amplicon length polymorphisms (ALPs) by targeting microsatellite loci using nucleotide information from other species and indels located in the non-coding regions of phenylalanine ammonia-lyase (PAL) and dihydroflavonol 4-reductase (DFR) genes in tea. One useful indel was detected in the unique intron of PAL and three others in introns 2, 3 and 5 of the DFR gene. All four nuclear microsatellite loci were polymorphic showing three to four alleles per locus, while only one out of the four chloroplast loci was variable with two distinct haplotypes. PCR primers were designed to amplify small but variable PCR fragments in such a way that they were applicable to low amounts of highly degraded DNA. The markers devised here were sufficient to differentiate all the teas, although a combination of several loci was necessary for most of them. This single-step PCR-based methodology is more effective than the PCR-RFLP technique commonly used for identifying food materials and will be useful for the certification of commercial Japanese green teas.