Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method

Appl Biochem Biotechnol. 2012 Feb;166(4):819-29. doi: 10.1007/s12010-011-9471-0. Epub 2011 Dec 8.


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 R₂₋₆₃₀ 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 R₂₋₆₃₀ WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P₃, P₄) and TaqMan fluorescence probe (P₃₋₁₄) were designed based on the R₂₋₆₃₀ 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 F₁ 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 Mg²⁺ concentration is 3.5 mmol/L.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chimera / genetics
  • DNA Primers / chemistry
  • DNA Primers / genetics
  • DNA, Mitochondrial / classification
  • DNA, Mitochondrial / genetics*
  • DNA, Plant / genetics*
  • Fluorescence
  • Genes, Plant*
  • Genotype
  • Molecular Sequence Data
  • Molecular Typing
  • Oryza / classification
  • Oryza / genetics*
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproduction


  • DNA Primers
  • DNA, Mitochondrial
  • DNA, Plant