Identification of Indica-Japonica Subspecies Rice by Duplex Fluorescence PCR Detection for Chloroplast DNA and Endogenous gos Gene

Asia cultivated rice are classified into two subspecies, indica and japonica rice (hsien and keng rice called in China, separately). It is necessary to establish a convenient and effective method to identify two subspecies because of the different quality characteristics between the rice products of them, which lead to different processing uses and commodity values. The identification method of PCR based on the difference of chloroplast DNA is convenient and effective, to be attempted to establish. Based on 69 bp fragment deletion of chloroplast DNA (cpDNA) found in ORF100 region nucleotides within the cpDNA Pst I-12 fragment in indica rice but not in japonica, the primer pair and a probe located on the specific cpDNA fragment in japonica rice were designed to identify indica and japonica rice. Another primer pair and probe used to detect endogenous gene gos in rice were combined with above primer pair and probe for detecting cpDNA to establish duplex fluorescence PCR to amplify cpDNA and gos gene for improving detection accuracy, to avoid the false negative results caused by DNA extraction error. The duplex fluorescence PCR detection method was established using typical japonica rice (pearl rice) and typical indica rice (Taixian 11). The accuracy of the method was validated with 547 samples including 177 samples of rice seed and eaten rice known as conventional japonica varieties and japonica type hybrid combinations and 370 samples of rice seed and eaten rice known as conventional indica rice varieties and indica type hybrid combinations. In 177 japonica samples, 170 samples with both positive results of cpDNA marker and gos gene, a coincidence rate of 96.05% agreement japonica rice, and 7 samples with positive results of gos gene and negative of cpDNA marker, not agreement japonica rice with non-coincidence rate of 3.95%, were detected. Of the 370 samples of indica rice seed and eaten rice, with positive results of gos gene and negative of cpDNA marker were detected in 340 samples, the rate of coincidence to indica rice was 91.89%. Other 30 samples with both positive results of cpDNA marker and gos gene, were detected, and the non-coincidence rate with indica rice was 8.11%. These identification results were in good agreement with known indica and japonica varieties, and the coincidence rate of japonica varieties was higher than indica. This method can be used to identify indica and japonica subspecies rice, especially suitable to identify conventional rice varieties.