Abstract
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.
Highlights
Cultivated rices in Asia were divided into Oryza sativa L. subspecies indica and Oryza sativa L. subspecies japonica [1], hsien and keng subspecies called in China for thousands of years [2, 3]
Kanno et al [23] found that indica rice had a 69 bp deletion in the ORF100 region of Pst I-12 fragment of chloroplast DNA compared with japonica rice
DNA Extraction 100 g rice seeds or eaten rice were ground into powder, 1.0 g of powder was added into 50 mL centrifuge tube, CTAB method [31] was used to extract DNA. 2 μL DNA solution were taken in the ultra-micro nucleotides determinator for examining, dilute to 100 ng/μL with TE solution, keep at -20°C
Summary
Cultivated rices in Asia were divided into Oryza sativa L. subspecies indica and Oryza sativa L. subspecies japonica [1], hsien and keng subspecies called in China for thousands of years [2, 3]. As a complicated method for the identification of indica-japonica subspecies, the previously published methods for the differentiation and identification of indica-japonica rice based on the PCR amplification polymorphism of nuclear DNA and cpDNA were presented, there are some problems such as expensive cost and low accuracy. This technique only detects one stable cpDNA site, which is accurate, efficient, economical and simple. Another primer pair and probe used to detect endogenous gene gos [32] of rice were combined with above primer pair and probe detecting cpDNA to establish duplex fluorescence PCR for cpDNA and gos gene for improving detection accuracy
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