[Development of transformation system of rice based on transformation-competent artificial chromosome (TAC) vector].

The TAC clone (NK15) containing a ca.50 kb DNA insert was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. The NK15 was stable in Agrobacterium tumefaciens strain LBA4404 under kanamycin selection for many generations. The calli of mature embryo of Nongken58S were infected with the Agrobacterium tumefaciens strain LBA4404 carrying NK15. PCR and Southern analyses of transgenic plants indicated that the 50 kb of foreign DNA was transferred into the rice genome, and most of transgenic plants had one copy of the insertion. Genetic and PCR analyses of T1 progeny confirmed that the inserted forgein DNA was stably inherited.

[Molecular mapping of the fertility restorer gene Rf-4 for WA cytoplasmic male sterility in rice].

The cytoplasmic male sterility for wild-abortive (CMS-WA) has been wildly used for hybrid rice breeding in China. The fertility restoration of CMS-WA is controlled mainly by two independent and dominant nuclear fertility restoring genes, Rf-3 and Rf-4. To map the Rf-4 gene with molecular markers, rice YAC clones of RGP, Japan were used to create new molecular marker. YAC contigs located between RFLP markers R1877 and G2155 on chromosome 10 were confirmed by hybridization with 12 RFLP probes. Six YAC clones, Y4630, Y2670, Y4892, Y2111, Y3821 and Y5528 were identified. Chromosome DNAs of the YAC clones were prepared and separated by CHEF. A total of 119 probes were created by sub-cloning of the YAC DNAs. RFLPs were screened between Zhenshan 97A and its near-isogenic lines with Rf-4Rf-4 genotype. Two probes, Y3-8 from Y4892 and Y1-10 from Y4630, were found to be polymorphic. Using F2 populations from crosses between Zhenshan 97A and its near-isogenic lines ZSR11, Y3-8 and Y1-10 were mapped to Rf-4 locus with genetic distances of 0.9 cM and 3.2 cM, respectively.

[Chromosome mapping of the S-b locus for F1 pollen sterility in cultivated rice (Oryza sativa L.) with RAPD markers].

S-b is one locus for F1 pollen sterility in cultivated rice (Oryza sativa L.), and the genotype of Taichung 65 (abridged as T65) is Sj/Sj, while its isogenic line, TISL2 is Si/Si at this locus. The results of pollen fertility analysis showed that the pollen of T65 and TISL2 were fertile, but the F1 plant from T65 x TISL2 produced only 40.6% fertile pollens, and the type of sterile pollen was stainable abortive. In F2 population from the cross T65 x TISL2 and BC1F1 population from the cross T65/TISL2/T65, the individuals could be classified into plants with normal pollens and plants with semi-sterile pollens, and the ratio of number of these two types of plants agreed well with the Mendel segregation ratio in 1:1. A total of 53 fertile F2 plants were testcrossed with T65, and all of them showed sterility F1 pollen in. These results demonstrate that the F1 pollen sterility is controlled by a single gene locus S-b, and the allelic interaction of S-bi and S-bj causes the pollen carrying S-bj allele abortive. A total of 187 RFLP markers and 500 RAPD primers were used to screen the polymorphism between T65 and TISL2; only H08-1300 and Y09-1500, two bands amplified by RAPD primer H08 and Y09 were found to be polymorphic. Purified H08-1300 and Y09-1500 were used as probe to hybridize with DNAs from T65 and TISL2, and the results indicated that H08-1300 and Y09-1500 appeared to be single copy in the T65 and TISL2 genome, then the RAPD marker were successfully converted into RFLP marker. The two markers were then used to perform segregation analysis, the results from co-segregation analysis of the genotypes of these two markers and the phenotypes of pollen fertility with F2 population indicated that the S-b was linked to H08-1300 and Y09-1500, and the genetic distances between each marker and the locus were 1.3 cM and 6.6 cM, respectively. To determine the chromosomal position of the S-b locus, H08-1300 was cloned and its two ends were partially sequenced. The homologous comparative analysis of these sequences with published rice sequences with BLAST was performed, and 540 bp of left end sequence of H08-1300 showed 86% homologous with the sequence of rice PAC clone P0033D06 (Accession No. AC079357), and 94% homologies of 101 bp at right end were also observed. Clone P0033D06 had been anchored by RFLP markers R3166 that was located on 18.8 cM position of rice chromosome 5 by Japan Rice Genome Program, which suggested that the S-b locus was mapped on chromosome 5 and tightly linked with R3166. The gene mapping result from this study suggests that using the rice genomic sequences published to determine the chromosome position of RAPD marker, as well as linked genes, would be a useful approach in tagging new genes.