Utilization of single nucleotide polymorphism-based allele-specific PCR to Identify shiikuwasha (citrus depressa hayata) and calamondin (Citrus madurensis Lour.) in processed juice

To develop a method for the identification of shiikuwasha (Citrus depressa Hayata) and calamondin (Citrus madurensis Lour.), trnL-trnF and trnT-trnL intergenic spacer regions of their chloroplast DNA were amplified using PCR and the nucleotide sequences were determined. In each region, a single nucleotide polymorphism (SNP) site specific to the respective citrus species (shiikuwasha and calamondin) was found. For species discrimination using PCR, two forward primers containing the allele-specific SNP site at the 3’-end and a mismatched nucleotide at the 3 ^rd base from the 3’-end were designed. The allele-specific forward primers specific to shiikuwasha and calamondin were respectively designated CiDeLF-F and CiMaTL-F. To confirm the specificity of the designed primers, PCR was carried out with DNA prepared from citrus peel or hand-squeezed juice as the template. Results showed that shiikuwasha and calamondin fruits and juices were identifiable by PCR using the allele-specific primers. Furthermore, this allele-specific PCR method can be applied to industrially processed and concentrated juice by amplifying DNA in advance.