Comparison of the specificity, stability, and PCR efficiency of six rice endogenous sequences for detection analyses of genetically modified rice

Species-specific endogenous reference sequences are indispensable in the development of methods to detect genetically modified (GM) crops and food/feed. We evaluated and compared the applicability of 6 rice (Oryza sativa) endogenous sequences, including 5 previously reported sequences; SPS1 derived from the sucrose phosphate synthase (SPS) gene, PLD1 and PLD2 derived from the phospholipase D (PLD) gene, GOS9 derived from the root-specific gene gos9, and ppi-PPF derived from the ppi-phosphofructokinase (ppi-PPF) gene, as well as a newly designed sequence, SPS2 in the rice SPS gene promoter region. PCR efficiency and stability were evaluated with 28 rice cultivars, and species specificity was evaluated using gDNAs isolated from major crops and rice-related species. SPS1 and GOS9 were less easy to be amplified and showed lower PCR amplification stabilities than the other sequences among rice cultivars. On the other hand, PLD1 showed high PCR efficiency and stability but low specificity against rice. Meanwhile, ppi-PPF was moderate in all evaluated characteristics. SPS2 and PLD2 showed higher PCR efficiencies and stabilities than those of other sequences, and also had acceptable species specificities. We conclude that SPS2 and PLD2 are ideal endogenous sequences for use in the development of methods to detect and quantify GM rice.