Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

Identification of a RAPD marker linked to a blast resistance gene in Oryza sativa L

Marker-aided selection has received more attention in recent years. This relies on the exploitation of dose linkage between molecular markers and target gene(s). We report here a randomly amplified polymorphic DNA (RAPD) marker tightly linked to the blast resistance gene Pi-ll(t) derived from Hongjiaozhan, which confers the resistance to race ZB1 of Pyricularia oryzae Cav.

Pyramiding of Pi46 and Pita to improve blast resistance and to evaluate the resistance effect of the two R genes

Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179（HH179） using the marker-assisted backcross breeding（MABB） procedure. As a result, three improved lines（e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita） were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines（NILs）. The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179（23.5%）. Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety （Digu） by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 （pZH01-tp309） and three different expression constructs, pCB-Pi-d25.3kb （pCB5.3kb）, pCB-Pi-d26.3kb （pCB6.3kb） and pZH01-Pi-d22.72kb （pZH01-2.72kb） of Pi-d2, driven by Pi-d2 gene’s own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.

Cultivar Mixture Cooperation(CMC)of rice varieties(lines) with different blast resistant gene

We studied Cultivar Mixture Cooperation(CMC)of rice varieties(lines)by screening mixture ofrice varieties which possess different genes,1983-1996.

Characterization and mapping of a novel light-dependent lesion mimic mutant lmm6 in rice(Oryza sativa L.)

A novel rice lesion mimic mutant (LMM) was isolated from an ethane methyl sulfonate (EMS)-induced 02428 mutant bank. The mutant, tentatively designated as lmm6, develops necrotic lesions in the whole growth period along with changes in several important agronomic traits. We found that the initiation of the lesions was induced by light and cel death occurred in lmm6 accompanied with accumulation of reactive oxygen species (ROS). The lower chlorophyl content, soluble protein content and superoxide dismutase (SOD) activity, the higher malondialdehyde (MDA) content were detected in lmm6 than in the wild type (WT). Moreover, the observation by transmission electronic microscope (TEM) demonstrated that some organel es were damaged and the stroma lamel a of chloroplast was irregular and loose in mesophyl cel of lmm6. In addition, lmm6 was more resistant than WT to rice blast fungus Magnaporthe grisea infection, which was consistent with increased expression of four genes involved in the defense-related reaction. Genetic analysis showed that mutant trait of lmm6 is inherited as a monogenic recessive nuclear gene located on the long arm of chromosome 6. Using simple sequence repeat (SSR) markers, the target gene was ifnal y delimited to an interval of 80.8 kb between markers MM2359 and MM2370, containing 7 annotated genes. Taken together, our results provide the information to identify a new gene involved in rice lesion mimic, which wil be helpful in clarifying the mechanism of cel death and disease resistance in rice.

A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice （Oryza sativa L.） blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat （CC-NBS-LRR） protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.

Comparative Analysis of the 100 kb Region Containing the Pi-k^h Locus Between indica and japonica Rice Lines

We have recently cloned a pathogen inducible blast resistance gene Pi-k^h from the indica rice line Tetep using a positional cloning approach. In this study, we carried out structural organization analysis of the Pi-k^h locus in both indica and japonica rice lines. A 100 kb region containing 50 kb upstream and 50 kb downstream sequences flanking to the Pi-k^h locus was selected for the investigation. A total of 16 genes in indica and 15 genes in japonica were predicted and annotated in this region. The average GC content of indica and japonica genes in this region was 53.15% and 49.3%, respectiveIy. Both indica and japonica sequences were polymorphic for simple sequence repeats having mono-, di-, tri-, tetra-, and pentanucleotides. Sequence analysis of the specific blast resistant Pi-k^h allele of Tetep and the susceptible Pi-k^h allele of the japonica rice line Nipponbare showed differences in the number and distribution of motifs involved in phosphorylation, resulting in the resistance phenotype in Tetep.