Application Of SSR Markers On Transfering Of Restoring Gene Rf-1, Identification Of F1 Purity And Discremination Of Cultivars In Japonic Rice (Oryza Sativa L.)

There are no effective restorer genes in japonica rice. Restorer lines in japonica ricewere bred by transfering restorer gene from indica rice into japonica flee. Whether a riceplant contains restorer gene must be identified by testcrossing progeny in field. Theefficency of restorer lines breeding in japonica rice will be increased by molecularmaker-assisted selection(MAS) as the process of testcrossing can be omitted. For thispurposes, SSR marker which can be used for (MAS) on Rf-1 was screened by using restorergene near-isogenic lines Liuqianxin A, Liuqianxin B and 8 iso-cytoplasmic restorer lines,and 77302-1(the restorer gene donor), F₁ of Liuqianxin A/Liuqianxin R. The consistency ofF₁ seed setting rate in field and profile amplified by the screened marker was calculated bymaking 178 F₁ combinations. The Rf-lwas transferred into a japonica conventional varietyNingjinglhao by MAS using the screened marker. Meanwhile, the identification of thepurity of hybrid rice and the genuineness of conventional variety were performed by SSRmarkers. Results are summarized as follows:1. From 12 pairs of SSR primer linked to restorer gene Rf-1 in japonica ricereported upto now, we found RM5629, which located on 10th chromosome, could amplify a band of120bp in LiuqianxinA and LiuqianxinR, but a band of 110bp among 8 iso-cytoplasmicrestorer lines and 77302-1, the Rf-1 donor. Meanwhile it amplified two bands of 110bp and120bp in hybrid F₁ of LiuqianxinA/LiuqianxinR, indicating that RM5629 was a reliableSSR marker for MAS on Rf-1.2. Six-eight lines of iso-cytoplasmic restorers with 9 types of nucleus, 8noniso-cytoplasmic restorers and Zijianwujing were crossed with 5 CMS in japonica rice andthe F₁ seed setting rate was investigated. The 77 male parents mentioned above and theCMS lines, container lines were amplified by RM5629 using total DNA as template. Bycomparing the relationship between the seed setting rate of F₁ and the profile of the maleparents, we found that, (1) in 6427R, Wuyujing3R, Liuyan189R, LiuqianxinR, 157TR,4016R, 4016LHR, all lines showed completely consistency between the profile and the restoring ability, while among the 17 lines of 16 lines showed completely consistencybetween the profile and the restoring ability, showing the consistency of 94.1ï¼…. InXiushui04R, the profile did not consistent with restoring ability.(2) 7 elite combinationswere found from the 178 testcrossing combinations. They were Wuyujing3A/4016LHR-70,Wuyujing3A/157TR-68, LiuqianxinA/157TR-68, WuqiangA/C bao, WuqiangA/Xiangqing,WuqiangA/6427R-16, WuqiangA/R254.3. A conventional cultivar Ningjinglhao resistant to rice stripe was crossed andbackcrossed with a japonica restorer line R254 used in Taihu valley widely. Thirty nineplants which had the restorer gene were identified from 89 plants in BC₁F₁ population byRM5629. Eleven plants which performed almost the same agronomic traits asNingjinglhao among the 39 plants were selected and backcrossed with Ningjinglhao. Theseed of BC₂F₁ will be used for further backcrossing.4. The purity of five hybrid japonica rice combinations which abroadly applied inJiangsu province were identified by using RM5629. RM5629 amplified a band of 120 bp inall CMS lines, a band of 110 bp in all restorer lines, two complementary bands of 110 bpand 120 bp in all Fi of the five combinations. Howere, the profile amplified by Rm5629 cannot be used to distinguish the 5combinations from each other. Therefore, additional 67 pairsof SSR primer were used for distinguish the 5 combinations and their parents. Of the 67pairs of primer 14 pairs of primer showed stable polyrnorphism between the male andfemale parents at least in one combination and amplified complementary bands in F₁. Thepolymorphism frequency among different combinations ranged from 8.8ï¼…to11.8ï¼….RM224 could distinguish 3 combinations simultaneously. In combination Changyoulhao F₁,two complementary bands of 160 bp and 130 bp were amplified. In combinations bothLiuyoulhao F₁ and 3you18 F₁ two complementary bands of 130 bp and 140 bp wereamplified. In combination Siyou422 F₁ two complementary bands of 145 bp and 160 bpwere amplified.5. The genomic DNA of 35 japonica rice varieties were amplified by 68 pairs of SSRprimers, Forty-six primers could amplify stable polymorphism among the 35 varieties. 6varieties could be identified by single SSR marker, while the other 29 varieties could beidentified by combination of different SSR f'mgerprint. All the 35 varieties could bedistinguished each other by the fingerprint map constituted by 12 pairs of core SSR primers.The genetic similarity coefficients of the 35 varieties ranged from 0.27 to 0.98.Furthermore, all the varieties were classified into 4 groups, at the level of genetic similarity coefficient 0.82 according to cluster analysis by an un-weighted pair-group average methodwith arithmetic mean. As expected, the result of cluster reflected basically the geneticrelatives based on the pedigree analysis.