The Potential Role of Plastid DNA as a Biomarker and Biosensor Using Real-time Quantitative Polymerase Chain Reaction

Plastids are semiautonomous eukaryotic organelles specific to plants performing many crucial and critical functions including photosynthesis, energy generation, development, stress perception, storage, flowering, and fruit ripening. Studies have shown that the number of chloroplast, a plastid subtype is affected by biotic and abiotic stresses and the Plastome Copy Number (PCN), circular DNA molecules present in the plastids are regulated by developmental stage and environmental stimuli. This study for the first time proposes the use of PCN as a biomarker and biosensor based on parsing real-time qPCR data obtained over a period of two years by using plastid tRNA specific primers from leaves and lint samples of cotton and seed samples of cotton, rice, soybean, maize, and sesame. PCN can be used because as opposed to nuclear DNA the PCN is modulated by both internal and external factors, this method does not require a prior knowledge of plastome sequence as the primers used are universal primers targeted to plastome of all the plant genus, the PCN is much higher than the nuclear, and PCN can be determined rapidly by qPCR assays. Analysis of mean Ct values, mean log PCN values and range of log PCN values of cotton seed and cotton raw, which are seed containing samples of cotton showed that of the samples processed and obtained from different sources over a period of two years the PCN was highly invariable within the group showing a difference of up to 4 plastomes i.e. log 0.58 between different samples, indicating a potential use of PCN as biomarker for plant genus determination. Moreover, observation of lower mean PCN (log 2.9) and higher range PCN (log 3.09) for broken dehusked rice (RS) compared to higher mean PCN (log 3.86) and lower range PCN (log 0.05) for hull covered Rice Paddy (RP), where the hull protects the rice seed during post-harvest processing and storage, suggests that PCN be an indicator of seed quality. To support our assumption that PCN can be modulated by abiotic factors rice seeds were treated with high temperature and moisture, the seeds exposed to high temperature exhibited higher mean Ct values when compared to untreated seeds, indicating a decrease in PCN. In conclusion, PCN can be potentially used as a biomarker and a biosensor.