Estimation of the homoplasmy degree for transplastomic tobacco using quantitative real-time PCR

Estimation of the homoplasmy of transgene in chloroplast is a necessary step in chloroplast transformation. This task is usually achieved by Southern blot analysis, which is laborious, time-consuming, requires large amounts of plant materials and needs hazardous radioisotopes in some cases. To develop a fast, sensitive, stable and effective technique for determining the homoplasmy of transgene in chloroplast, one real-time PCR system based on TaqMan probe technique was developed for evaluating the homoplasmy degree. In the real-time PCR system, one assay targets the exogenous faeG gene, and another one the tobacco chloroplast reference gene, maturase gene within the trnK intron (matK). The homoplasmy of the transgene was determined by the comparison of copy number of faeG and matK. The analyzed results of 17 transplastomic tobacco lines using the developed real-time PCR system were consistent with those from Southern blot analysis, indicating that the real-time PCR method is suitable for estimating the homoplasmy degree of transplastomic plant with the advantages of high efficiency and throughput, low cost and saving time.