Genetic Analysis And Fine-mapping Of Two Leaf-color Mutants In Rice(oryza Sativa L.)

For the global importance of rice, together with its small genome size and genetic relations to other major cereals, efforts have been undertaken to sequence the entire genomes of the two subspecies of rice—indica and japonica, which makes rice as a monocot model plant with great importance for rice breeders. Leaf is an important organ for photosynthesis in rice which plays a significant role in rice growth and development. As a photosynthetic trait, leaf color is closely related with the rice yield. Mutations of the gene conditioning leaf color would affect the leaf color and photosynthesis, thus influencing the accumulation of organism and decreasing rice yield ultimately. Therefore, it is necessary to elaborate the molecular mechanisms of leaf color mutation, in order to fulfill the super high-yielding rice breeding.From a large number of rice mutants in our laboratory, we identified two leaf color-related mutants and used map-based cloning method to map the genes. The study shed light on the molecular mechanism of photosynthesis. The results of our studies are as follows:1) A new leaf color mutant (yellow leaf, yl) derived from a japonica cultivar Nipponbare with Ethylmethylsulfone (EMS) treatment was used in this study. yl showed yellow leaf at the seedling stage, and the leaf color stayed yellow during the whole growth stage. To identify whether the yl mutant was controlled by a single gene, genetic analysis on reciprocal crosses between yl and japonica cultivars NPB, ZH11 and CJ06 were conducted, the individuals of the F1 of all the cross showed green leaf which indicated that yl was a recessive mutant, based on the individuals of the F2 segregation population, all the ratio of normal leaf color and yellow leaf individuals fit into 3: 1 (χ2<χ20.05,1=3.84), indicating that the yl mutant was controlled by a single recessive gene. Besides, at the tillering stage, measurement of chlorophyll content of functional leaves in yl and the wild-type revealed that both chlorophyll content and carotenoid content drastically decreased in yl, but the ratio of Chla and Chlb was much higher than that in the wild-type. In addition, the electron microscopic analysis was conducted to detect the condition of chloroplast development. The result showed that the mutant had underdeveloped or retrograded thylakoid membranes and few grana compared with the wild-type. Furthermore, the indicators of the photosynthesis determined at the full heading stage indicated decreased photosynthetic rate and transpiration rate, which probably resulted from the lower chlorophyll content. 144 F2 mutagenic individuals were screened for the primary mapping, and the YL was located on the short arm of chromosome 3 between the two markers RM468 and RM3684. Then the candidate region was delimited to a 134 kb physical distance between the YL7 and YL14 (these markers were on the BAC OSJNBa0028F23 and OSJNBb0033J23, respectively).2) In addition, we also obtained a negative mutant (yellow-green leaf 4, ygl4) from T-DNA insert bank, which showed yellow-green leaf from the seedling stage to the full heading stage. However, after the full heading stage, the leaf color returned normal. Allelic tests between ygl4 and the other two mutants(yellow-green leaf 3 and yellow-green leaf 5, ygl3 and ygl5) which discovered in our laboratory showed that ygl3,ygl4 and ygl5were controlled by different genes. The agronomic and photosynthetic traits showed that the tillering number was decreased significantly, which maintained three tillerings till productive stage, and compared with the wild-type, the photosynthetic rate also reduced. From the seedling stage to productive stage, we investigated the chlorophyll content which showed a dynamic change: with the progress of growth stage, the chlorophyll content of the mutant increased gradually. At the productive stage, the content reached the general level and the leaf returned normal. During the dynamic changes, the rangeability of the chlorophyll a was in accordance with chlorophyll b. Based on the result of previous primary mapping, the candidate region was delimited to a 68 kb physical distance in the BAC B1131G08. The forecast websites predicted that there are eight open reading frames in the region. Currently, we are selectively sequencing the genes related to the mutation.