Screening Of The Rolled Leaf Mutants And Fine Mapping Of The Rolled Leaf Gene Rl_t In Rice

Semi-rolled leaf is one of the most important morphological characters in plant breeding. Some high yield varieties or their hybrids were semi-rolled leaf cultivars. Studies on effects of leaf rolling on photosynthetic physiology, population ecology and economical traits showed that semi-rolled leaf had some upstanding effects, for instance making leaf erect, optimizing canopy light transmission, increasing effective leaf area per unit land and improving the quality of population in late growth stage. However, most of rolled leaf materials found in past research have different leaf rolling degrees and most of them could not be applied in rice breeding. Therefore, identifying more useful rolled-leaf genes is still an important goal for practical rice breeding. On the other hand, rice has become a model plant of monocots, so the function research for it on rolled leaf genes will provide good chances for elucidating leaf development mechanism in rice.Some rolled leaf mutants were obtained via screening the T-DNA inserted mutant pool and one of them was confirmed to be co-segregated with T-DNA. A rolled gene, rl(t), with high value for breeding, was fine mapped and its putative function was preliminarily analysed and identified.1. T-DNA tagging is a high throughput method for identifying and cloning novel genes. The sequence flanking the T-DNA insertion site could be obtained according to T-DNA tagging, then the gene tagged by T-DNA could be discovered based on the flanking sequence position in the rice genome. Twelve rolled leaf mutants were obtained via screening 4416 rice T1 tagged lines. These lines were tested by PCR using primers amplifying special T-DNA segment and 3 co-segregated lines were selected. Mutants were PCR positive and several plants with natural phenotype were PCR negative in the co-segregated lines. Seeds from natural plants and mutants grew out T2 generation. The co-segregation criterion of T2 generation was as follows, in the progeny blocks of mutants all plants had mutant phenotype and were PCR positive, moreover, in the progeny blocks of some narural plants, the ratio of narural plants to mutants fit 3:1 and all mutants of them were PCR positive. Line 1345 was co-segregaed according to this criterion. The results indicated line 1345 in T1 generation had two copy T-DNA insertion and one resulted in mutant phenotype. The mutant lines with single copy T-DNA were obtained. The flanking sequence from the line with targeted copy was amplied by TAIL-PCR, but its sequencing result was false positive (detail in chapter 4). We would separate the flanking sequence by plasmid rescue. The measure of separating flanking sequence and the co-segregation test were discussed.2. A applied method of designing molecular markers was brought forward in the process of fine mapping the rolled leaf rl(t) in rice. The parents of map-based cloning have japonica-indica diversity, so markers could be designed based on the sequence diversity between the genome of Nipponbare and that of 93-11. Nipponbare BAC in the mapping region were downloaded from IRGSP, and then these BAC were aligned with the 93-11 genome in NCBI. InDel and SNP could be discovered according to the alignment result. Some InDel and SNP were screened out to design markers (about 15 kb interval when region was longer than 100 kb, and 5 to 10 kb when shorter than 100 kb). SNP could be transferred into CAPS, dCAPS and AS-PCR markers (detail in chapter 5). Forty seven markers were designed in this research. Twenty four InDel markers and 14 SNP markers have polymorphism. How to improve the efficiency of marker design was discussed.3. Research and application for rolled leaf genes were important for plant type breeding in rice. The rolled gene rl(t) was valuable in rice breeding and primarily mapped in our past research. Two great segregated population were developed to fine map rl(t). The strategy of fine mapping was as follows. First, marker genotypes of population were tested by double primers PCR method. Two primers of this method should be flanked to the region covering rl(t). Then, recombinants were chosen out and analyzed by markers in the region. Finally, the co-segregation relationship between marker genotype and plant phenotype was analyzed in all recombinants, and the precise region was confirmed. Two recombinants were found, whose recombined sites were flanked to rl(t) and very close to each other. The result indicated that the rolled gene rl(t) was located between marker P95053 and P113.6. The physical distance from marker P95053 to P113.6 was 11 kb, and only one candidate gene lay in this region. All conclusion laid a foundation to clone rl(t) and research the regulation mechanism of rl(t). How to improve efficiency of fine mapping was discussed.4. Only one predicted gene in the 11 kb region was discovered from rice automated annotation database (RAD) and rice annotation project database (RAP-DB). The putative gene belonged to HD-GL2 type transcription factor. Alignment between the putative gene and HD-GL2 type genes indicated that the amino acid sequence of rl(t) showed high similarity to that of ZmOCL1 (85%) and ANL2 (60%). The HD-GL2 type genes showed L1-layer or protoderm-specifc expression in the SAM during the shoot and flower development. Thirteen pairs sequencing primers were designed according to Nipponbare BAC(AP005885) to amplify the DNA of QMX and NIL-rl(t). The PCR product were sequenced and the result indicated that a nucleotide substitution lay in the 3' UTR of rl(t). It was suggested that the point mutant in 3' UTR of rl(t) could result in change of mRNA stability or translation and induce rolled leaf.5. Post-transcriptional silencing of plant genes could identify the gene function effectively. The RNAi technique could efficiently silence genes in this way. Three interference segments were designed from different position of the candidate gene. Subsequently, they were constructed into RNAi vector by BP reaction. Finally, the constructed vectors were transformed into Zhonghua 11. The targeted gene would be silenced to identify function.