Abstract: 【Objective】Rice leaf is not only a vital site for photosynthesis, but also an important factor for ideal plant architecture in rice. This study is useful in map-based cloning of target gene and marker-assisted transferring rolled gene in rice breeding programs. 【Method】A mutant rl16(t) (rolled leaf 16) with rolled leaves was derived from the Indica type rice 9311(wild -type WT) via the radiation of ⁶⁰Co-γ. Then, it was planted for several generations to test the stability of mutant phenotype in the field. At heading stage, rl16(t) and WT were randomly selected 10 strains to measure the main agronomic traits including leaf rolling index, respectively.The same part of leaf of rl16(t) and WT was taken and fixed by FAA, then dehydrated in series ethanol and paraffin embedding. After that, the fixed leaf was incised to 10 microns slices and these slices were stained by sarranine. Finally, the bulliform cells were observed, the number of them was counted and their area was measured under the microscope. Chlorophyll content of rl16(t) and WT were tested at tilling stage. In addition, the leaf phenotype of the F₁ plants and F₂ population were investigated, and the mode of rolled gene inheritance was analyzed by chi-square test. To fine-map the Rl16(t), an F₂ population was generated by crossing between the rl16(t) mutant and a Japonica type rice Dianjingyou. Target gene was limited by simple sequence repeat (SSR) and InDel markers. Three known domain structure genes in the mapped region were analyzed by gene quantitative expression. 【Result】Compared with wild-type, the mutant had not only typically adaxially rolled leaves, but also decreased height and panicle length, as well as the seed setting percentage. The whole plant of rl16(t) appeared leaf longitudinally rolled into the approximate tubular phenotype at seedling stage (three leaves and one new), then kept it at the rest of time. On the contrary, WT had flat leaves in the entire life. Paraffin section of flag leaf indicated that the number and area of bulliform cell of rl16(t) were lower than that of WT. The number of bulliform cell in WT was (385.1±43.6) cells/mm², while that in the rl16(t) was(1059.5±254.4) cells/mm². However, for the bulliform cell, there were no significant and obvious changes of rl16(t) and WT. The carotenoid content of rl16(t) was lower than that of WT, but the chlorophyll a, chlorophyll b and total chlorophyll content were significantly higher than that of WT. All of the F₁ hybrid between rl16(t) and WT indicated flat leaves, meanwhile, in an F₂ population, rolled and flat leaf plants segregated in a 3﹕1 ratio (97 rolled versus 326 flat leaves, chi-square = 0.86＜chi-square 0.05 = 3.84). Cytological analysis indicated that the rolled leaf phenotype might be caused by the change of number and size of bulliform cells. Genetic analysis indicated that the rolled leaf characters were controlled by a recessive nuclear gene. Using SSR markers, Rl16(t) was initially mapped in the region between the RM23769 and RM23916 on the long arm of chromosome 9. Furthermore, with enlarged population and more developed InDel markers, Rl16(t) was finally delimited to a 51 kb region governed by the InDel marker DF70 and SSR marker RM23818. Three known structure coding genes (LOC_Os09g09320, LOC_Os09g09360, LOC_Os09g09370) were predicted in the limited interval. The expression of LOC_Os09g09320 and LOC_Os09g09370 in rl16(t) showed no obvious differences with that of WT, but the expression of LOC_Os09g09360 in rl16(t) was only the half of WT.As for quantitative expression analysis of the eight genes that contributed to the rolled leaf, seven of them (SLL1, ROC5, RL14, SRL1, ACL1, NRL1, NAL7)had a decline in the rl16(t) mutant, while OSZHD1 appeared a rise.【Conclusion】The rolled leaf of rl16(t) was due to reduced number and area of bulliform cells. In addition, thisgene would be a putative novel rolled leaf gene. As the result of quantitative gene expression, LOC_Os09g09360 may be the target genes.

Key words: rice (Oryza sativa L.), rolled leaf traits, genetic analysis, fine mapping