Development and Application of SCAR Markers for Discriminating between CMS lines and Their Maintainer Lines in Indica Rice (Oryza sativa L.)

The DNA fragments about 1 600 bp were amplified using random amplified polymorphism DNA (RAPD) primer OPA12 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region (SCAR) primers were then developed to discriminate the cytoplasmic male sterile (CMS) lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissue of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could be also amplified from the total DNA of green leaf of other two CMS-WA lines, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID line Ⅱ-32 A, but not inⅡ-32 B, and the specific fragment was amplified from DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting genetic purity of a man-made mixture seed lot of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. In all, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at seedling stage.