Identification of WA-type three-line hybrid rice with real-time Polymerase Chain Reaction (PCR) method

摘要

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R _(2-630)WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R _(2-630) WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P _3, P _4) and TaqMan fluorescence probe (P _(3-14)) were designed based on the R _(2-630) DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F _1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60°C and the Mg ~(2+) concentration is 3.5 mmol/L.