Genetic Analysis And Gene Mapping Of A Dominant Rolled-leaf Mutant In Rice(Oryza Sativa L.)

Rice is one of the most important cereals in the world. It is of urgency to improve rice production for fulfilling people’s need of daily life and food. Rice leaf known as the main organ for photosynthesis, is an important componentin plant-type breeding.Previously, it has been shown that moderate leaf rollingis very useful to keep leaf erect without drooping, increase lower part transmittance for improving light conditions in rice population, which is helpful to make rice productivity progress. In this project, a dominant rolled-leaf mutant, named 70-36, obtained from Nipponbare(Oryza sativa L. ssp. japonica)mutagenized by ethylmethylsulfone(EMS), was used as the starting material for analysis.The mutant 70-36 was characterized by genetical, cytological, physiological and biochemical analyzation.The mutation locus was mapped by molecular marker analysis and the candidate gene was screened, and the possible mechanism of rolled-leaf was discussed.The main results were as follows:At the beginning of six-leaf stage, the leaf phenotype of 70-36 was significantly different from Nipponbare, the wild-type variety. The mutant’s leaves appeared rolling inward and this appearance remained unchanged in subsequent growth period. As compared to the wild-type, the rolling index of the last 1stand 2ndleaf of 70-36 were both increased by about 49%, leaf length increased by approximately 23% and 38%, leaf width increased by approximately 13% and 14%, respectively, in addition, the plant height and 100-grain weight were increased by about 19% and 8%, respectively, however, the seed setting rate and the number of effective tillers were reduced by about 29% and 13%, respectively.By using hand-held slicing and semi-thin sectioning, the flag leaf in booting stage was observed to investigate cytological variations, and the results showed that vascular bundle development demonstrated abnormalities at the beginning of six-leaf stagein 70-36,for instance, not only did the leaf vascular sclerenchyma on the abaxial side of the midriband lateral veins demonstrate developmental defects, the number of small vascular veins on the midrib also increased when compared with the wild type. Using TEM for further observation, it was shown that the cell-wall thickness of sclerenchyma on the abaxial side of vascular veins reduced in the mutant.By using Fourier Transform Infrared Spectroscopy(FT-IR) qualitative analysis, the midrib cell-wall composition of flag leaf at the booting stage wasmeasured, the results showed that the content of cellulose, hemicellulose and lignin all significantly declined in70-36. By spectrophotometric method, chlorophyll and carotenoid content of the flag leaf at booting stage were measured, the results showed that the chlorophyll a in leaf blade increased about 12.5%, while the content of other pigments remained unchanged in the mutant when compared with the wild type.The mutant 70-36, as female parent, was crossed with a wide compatible variety,Jingxian 89 with flat-leaf phenotype, to investigate genetic characteristics. The plants of F1 generantion all demonstrated a similar leaf phenotype to 70-36; in F2 population, the ratio of rolled-leaf plants to flat-leaf plants was 3.20 :1(χ2= 0.47 < χ20.05= 3.84), which was completely in agreement with the Mendelian segregation law upon one pair of alleles,indicating that the rolled leaf mutation was genetically controlled by a single dominant gene.Molecular marker mapping was performed by using a total of 164 recessive plants with flat leaf phenotype in F2 segregating population, then the genetic locus of rolled-leaf gene was localized in an about 1.7-Mb region between two In Del makers, Ind3 and Ind6,on chromosome 3 in rice. Subsequent re-sequencing in the region demonstrated that base variations occurred in four genes, including Os03g0386000, Os03g0389100,Os03g0389900 and Os03g0395100. By real-time PCR technique, the expression of these genes was detected and the results showed that one of them, Os03g0395100, had a significant up-regulation expression, but other genes had no remarkable changes, at the five-leaf stage in comparison with the wild-type, therefore, Os03g0395100 was finally selected as the candidate gene responsible for leaf-rolling mutation in 70-36.The results of transcriptome sequencing(RNA-seq) analysis showed that a total of 207 genes with significantly differential expression were detected between the wild type and themutant, including 162 genes up-regulated and 45 genes down-regulated in the mutant.Functional classification and PATHWAY analysis of these genes was performed, the results indicated that remarkable variations occurred on the expression level of some important genes related to leaf polarity establishment, bulliform cell development, sclerenchyma development, etc., in 70-36. In addition, the mutation of 70-36 was probably tightly associated with the expression alteration of some genes involved in signal transduction,secondary metabolic pathways, etc. Real-time PCR was utilized to validate these differentially expressed genes, the results showed that two genesrelated to leaf polarization establishment, Os YABBY1 and Os YABBY7, and one gene related to sclerenchyma development, SLL1, were significantly up-regulated, however, two genes related to lignin biosynthesis, Dirigent1 and 4CL3, was significantly down-regulated, in 70-36. These results indicated that the mutation in 70-36 was strongly influenced byexpression changes of the genes related to leaf polarity development, the sclerenchyma development and lignin biosynthesis. However, whether these differentially expressed genes are directly regulated by the candidate gene, Os03g0395100, thus leading to leaf rolling mutation, further research will be needed in order to elucidate its detailed molecular mechanism.