RESUMEN
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 R2â630 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 R2â630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3â14) were designed based on the R2â630 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 Mg²âº concentration is 3.5 mmol/L.
Asunto(s)
ADN Mitocondrial/genética , ADN de Plantas/genética , Genes de Plantas , Oryza/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuencia de Bases , Quimera/genética , Cartilla de ADN/química , Cartilla de ADN/genética , ADN Mitocondrial/clasificación , Fluorescencia , Genotipo , Datos de Secuencia Molecular , Tipificación Molecular , Oryza/clasificación , ReproducciónRESUMEN
ABSTRACT The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg(2+) concentration, and extension temperature were 400 nM, 3.0 mM, and 60 degrees C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.