Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 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 tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, 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 （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, 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 the seedling stage.

Estimating Out-Crossing Rate of Bg 379-2 Using Morphological Markers and Confirmation by Molecular Markers

Rice is largely self-fertilized and accordingly a field population of rice is completely composed of near homozygotes. Due to the emergence of off-types, homozygosity will be affected. With the time, this will cause the reduction of genetic purity in some rice varieties. One of the reasons has been suspected to be the high out-crossing frequencies of such varieties. Studies were conducted at the Rice Research and Development Institute, Batalagoda, Sri Lanka to estimate the out-crossing rate of Bg 379-2, a variety having the problem of maintaining genetic purity. Bg 379-2 was allowed to out-cross with Bg 450 and the number of out- crossed plants were counted using dominant morphological markers such as short-round grain and purple culm of pollen donor. A molecular confirmation of out-crossing was also performed using sequence tagged site （STS） molecular marker pTA248. The variety Bg 379-2 showed a potential out-crossing rate of 3.41% and an average out-crossing rate of 1.29% using dominant morphological markers. Polymorphism was cleady detected between parents and out-crossed plants as well as selfed plants of Bg 379-2 using their banding patterns. A similar study can be performed to determine the out-crossing rates of other varieties which show high percentage of off-types in the population for the better understanding of the breeding behavior of the varieties.