Monitoring foreign gene incorporation into the plastome of Chlamydomonas reinhardtii by multiplex qPCR

The genetic material of the Chlamydomonas reinhardtii chloroplast can be easily manipulated and creation of transgenic plastomes is of interest for both photosynthetic research and for biofuel and biomass production. Because multiple copies of the chloroplast genome are present, it is important to understand whether, following the introduction of a foreign gene, the resulting transgenic plastome is homoplasmic or heteroplasmic. By quantitative PCR together with a simple DNA extraction procedure and a series of DNA oligonucleotides the following protocol will determine the extent of foreign gene incorporation into a host chloroplast plastome. This approach is used to follow the degree of heteroplasmy following biolistic transformation of several transgenic strains. The approach used is quick, simple to set up, and gives an accurate quantitation of foreign genes within of the chloroplast plastome. Possible future uses of the technique are discussed.