An identification system using multiplex allele-specific PCR assay in the Japanese black pine (Pinus thunbergii Parl.)

A compatible individual-identification system with the multiplex SNuPE assay system, which was constructed of eighteen highly polymorphic and codominant single nucleotide polymorphisms (SNPs), was developed in the Japanese black pine (Pinus thunbergii Parl.). The eighteen SNPs were transformed into dominant markers by employing an allele-specific PCR (ASP) assay. Three multiplex PCR sets, each containing six ASP markers, were designed to increase cost saving and assay simplicity. Analyses of statistical parameters of 18 ASP markers revealed that gene diversity (H) and polymorphic information contents (PIC) were 0.331 to 0.500 (average of 0.425), and 0.375 to 0.500 (average of 0.473), respectively. The cumulative confusion probability (Ccumulative) of the system was extremely low (3.14 × 10−6), roughly one chance in 318,000 genotypes. The cumulative discrimination power (Dcumulative) was exceedingly high (0.999997), which means that the identification system is significantly powerful to discriminate the unrelated individuals.