Genetic Analysis And Gene Mapping Of Two Glume Color Mutants In Rice

Pigments in rice not only have important physiological effects on their own development,but also have been widely used in rice breeding,landscape gardening,and so on.A golden hull and internode rice mutant,gh881,was isolated from an elite Japonica cultivar Changligeng(CLG)using EMS mutagenesis strategy.Compared with wild type,gh881 exhibited golden hull and internode at the mature stage.Except for no signif icant difference in the number of panicles per plant,there were signif icant reduction in plant height,the seed setting-rate,the number of spikelets per panicle,the number of grains per panicle and 1000-grain weight between WT and gh881.Genetic analysis and gene fine-mapping results suggested that gh881 was controlled by a single recessive gene and the mutant gene was mapped to a region of 33.2 kb in which four open reading flames(ORFs)existed on the short arm of chromosome 2 between markers FH-13 and RH-25.Sequencing analysis revealed that a single base mutation(G to A)was at the site 3563 bp of OsCAD2(Os02g0187800)encoding a Cinnamyl-Alcohol Dehydrogenase(CAD),which led to a G297 D mutation(codon GGC to GAC).This implied that gh881 might carry a novel allele of OsCAD2.The quantitative real-time PCR analys is showed that the relative expression level of OsCAD2 decreased significantly in the internode,while it almost increased signif icantly in the f lag leaf sheath and panicle,and the related-genes also almost changed signif icantly.These results demonstrated that OsCAD2 is an important gene involved to lignin metabolic pathway,and may regulate other related-genes expression feedback.The clg-642 mutant also from the mutant library of EMS-treated Japanica cultivar Changligeng(CLG)showed brown glume and grain chalkiness.Compared with wild type,clg-642 exhibited brown glume and it had more grian chalkiness.Endosperm scanning electron microscopy and amylose content measurement showed that the endosperm from the grains of clg-642 developed abnormally as a result of loosely packed and spherical starch granules,and amylose content of its endosperm had decreased by 17.5%.Except for no significant difference in the number of panicles per plant and plant height,there were significant reduction in the seed setting-rate,the number of spikelets per panicle,the number of grains per panicle and 1000-grain weight between WT and clg-642.Genetic analysis suggested that clg-642 was controlled by a single recessive gene.We obtained 2.52 Mb candidate regions on the long arm of chromosome 4 by MutMap method,where we selected reliable 19 SNPs as candidate SNPs located to 12 canditate genes.Sequencing analysis and restriction endonuclease Hae Ⅱ digestion revealed that a SNP was at the site 1659 bp of the third exon of gene OsCIN2 encoding a cell-wall glycosyl hydrolase leading to an A159 T mutation.This implied that clg-642 might carry a novel allele of OsCIN2.The quantitative real-time PCR analys is demonstrated that OsCIN2 began to play an important role in the ear ly stage of rice grain filling,and affected starch synthsis and metabolism-related gene expression level at various stages of endosperm development.