A Tri-primer Multiplex PCR Method for the Exogenous Genes in Bt Rice T1C-19

T1C-19 is a transgenic rice possessing Bacillus thuringiensis (Bt) insecticide crystal protein encoding gene cry1C, which has a good resistance to leaf folder and stem borer, and can be used as an elite germplasm in the breeding of insect-resistant rice. The genotype identification of cry1C could greatly improve the breeding efficiency. This research aimed at developing a tri-primer multiplex PCR method to identify the genotype of cry1C in T1C-19 and its relative lines. Three primers of C1, C2 and C3 were designed according to the genomic sequence T1C-19. C2 was located on the T-DNA sequence where cry1C was located, and C1 and C3 were located on two flanking rice genomes of the T-DNA, respectively. The quantity of primers in multiplex PCR system was optimized. The result indicated that the tri-primer multiplex PCR system with 0.5 μL 10 μmol/L C1+0.4 μL 10 μmol/L C2+0.2 μL 10 μmol/L C3 could correctly identify three genotypes of cry1C. Only one DNA band was amplified in the homozygote and the negative, the DNA band was 512bp in the homozygote, but 386bp in the negative. The heterozygote had both two DNA bands above. A few varieties and two breeding populations from T1C-19 were used to verify the accuracy of the tri-primer multiplex PCR system, the result confirmed that the tri-primer multiplex PCR system could provide a convenient and effective method for the identification of cry1C gene in the breeding of insect-resistant rice with T1C-19 as a parent. Keywords Transgenic rice; Multiplex PCR; Co-dominant marker; cry1C; T1C-19