Development And Application Of DNA Markers For Discriminating Between Four Types Of Cms Lines And Their Maintainer Lines In Rice (Oryza Sativa L.)

Rice(Oryza sativa L.) is one of the most important crops in the world and provides the main resource of food for more than half of the world population. Hybrid rice is a successful technology to increase rice yield proved by production practice over the past three decades. China is the biggest country to produce and consume hybrid rice seeds in the world, having a huge hybrid rice seed market, and required annually about400thousand tons hybrid rice seeds. With the expanding acreage of hybrid rice, China has an increasing seed demand, and mechanical mixing, biology mixed and artificially doped with is along with process of hybrid seeds production, resulting in a serious impact to the authenticity and genetic purity of hybrid rice seeds, and then creating a severe grain cuts. Therefore, seed purity has become a prominent issue in production. Degradation of CMS lines are the main reason leading to degradation of hybrid rice seed. If the CMS line mixed with its maintainer line, maintainer line in CMS line may increase gradually due to the maintain line is self-pollination. A large number of sterile plants will be appear in hybrids in process of hybrid seeds production, which the CMS line was contaminated with maintainer line, and then bring about a serious impact to exert rice heterosis. Nowadays, however, the method for authenticity and genetic purity of hybrid rice seed testing is through identification planted in the field, it is a long time for one growth cycle and costs a lot of work, time-consuming. Thus, this method can’t meet the demand for seed market, new varieties authorization in time, and so on. Therefore, in order to develop DNA molecular markers for steadily discriminating CMS lines and their isonuclear maintainer lines in hybrid seeds production and seed purity testing of CMS lines at early stage of plant growth. On the basis of previous studies, two aspects of research are performed in this study. Firstly, the about1600bp DNA fragments amplified by RAPD primer OPA12using mitochondrial DNA of Zhenshan97A and Zhenshan97B as templates at32-35℃annealing temperature were sequenced and compared. Based on sequence information, SCAR primers were developed for discriminating between CMS lines and their maintainer lines. Secondly, by using four pairs of functional primers based on the specific chimeric genes of different CMS, DNA markers discriminating HL-type CMS and maintainer seedlings were identified using total DNA of Yuetai A and Yuetai B as templates, and DNA markers discriminating BT-type CMS and maintainer seedlings were identified using total DNA of eight pairs of BT-CMS A and B as templates.The main results obtained were as follows:1. There was no DNA polymorphism between Zhenshan97A and Zhenshan97B amplified with OPA12digested by the seven restriction enzymes(Hap II, Hae III, Acc II, Hha Ⅰ,AluⅠ,Rsa I, TagⅠ).2. There was no difference between Zhenshan97A and Zhenshan97B in full-length sequence and the base pairs of the about1600bp DNA fragment amplified by OPA12using mtDNA as templates. The precise number of base pairs was1588bp.3. A clear and specific1588bp DNA fragment was amplified by primers CHI19F2/CHI19R2, which was designed based on sequencing, when mitochondrial DNA was from Zhenshan97A, but was not observed in Zhenshan97B.4. A clear and specific1588bp DNA fragment was amplified by primers CHI20F3/CHI23R3,which was designed based on results of TAIL-PCR, when mitochondrial DNA was from Zhenshan97A, but was not observed in Zhenshan97B.5. The specific1588bp DNA fragment was also amplified by four pairs of primers, when total DNA from Zhenshan97A was used, but not in Zhenshan97B.6. The specific fragment also appeared in other two CMS-WA lines, namely Zhenpin A and Tianfeng A, but did not in their maintainer lines. By using total DNA, each of the four pairs of SCAR primers could also amplified the1588bp fragment in CMS-ID line Ⅱ-32A, but not in Ⅱ-32B. The1588bp DNA fragment appeared in all the F1and F2plants of Shanyou63.7. The results of detecting genetic purity of a man-made mixture seed lot of Zhenshan97A using CHI20F3/CHI23R3were completely consistent with the phenotypic data. These results indicated that the1588bp DNA fragment amplified by CHI20F3/CHI23R3was 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.8. A clear and specific239bp DNA fragment were amplified by three functional primers HL-orf-1, HL-orf-2, HL-orf-3in Yuetai A, but was not observed in Yuetai B and9311.9. Similarly, the clear and specific239bp DNA fragment were amplified by primer BT-orf-1, in BT-type Liuqianxin A, Wuqiang A,863A, Wuyujing3A,731A, Xu2A,9522A,18A, but not in Liuqianxin B, Wuqiang B,863B, Wuyujing3B,731B, Xu2B,9522B,18B and6427R. The results of detecting genetic purity of a man-made mixture seed lot of Wuyujing3A using BT-orf-1were completely consistent with the phenotypic data. These results indicated that the239bp DNA fragment amplified by functional markers derived from specific ORFs of CMS mitochondrial DNA, and could be used to distinguish CMS-HL and CMS-BT lines from their corresponding maintainer lines at seedling stage.