Genetic And Physio-biochemical Analysis Of Two Mutants In Rice (Oryza Sativa L.)

Rice is one of the most important food crops in the world.As the significant materials of functional genomics studying,rice mutants have been obtained mianly by spontaneous mutation,physical mutagenesis,chemical mutagenesis and insertional mutagenesis.Among chemical mutagens,ethylmethane sulfonate?EMS?mutagenesis and N-methyl-N-nitrosourea?MNU?mutagenesis are commonly used.In the present study,there were seven mutants?M15,M50,M85,M89,M96,M117 and M270?isolated from a mutant bank which was generated by ethylmethane sulfonate?EMS?mutagenesis of indica rice accession ZJ100.The mutants showed accelerated leaf senescence phenotype on leaves from the tillering stage?M85 M89 and M117?or heading stage?M15,M50,and M270?to final maturation.However,the M96 mutant shows prostrate growth mutant-phenotype.The agronomic traits contained plant height,spike number,spike length,1,000-grain weight and seed-setting rate showed difference to some extent compared with WT.That indicated the leave premature senescence of mutants caused some negative effects on agronomic traits and then influenced the yield and quanity of mutants.Furthermore,the discrepancies of agronomic traits caused by leave premature senescence in these six mutants displayed different levels,but the M85,M89 and M117mutants showed most drastic changes on agronomic traits compared to WT and other mutants.At the heading stage,these mutants exhibited a significant increased MDA content but decreased chlorophyll content.Enzyme of reactive oxygen removal system various indicated that the ROS scavenging system has been broken in these six mutants.And we found photosynthesis rate and strength weakened in the mutants because of the leave premature senescence by measuring the photosynthetic parameters.In contrast to WT,the numbers of chloroplast and grana thylakoid were decreased obviously in all mutants and severe chloroplast impairing occurred in M50 mutant leaves at the heading stage by transmission electron microscopy.There were more starch granules and it showed bigger size in M85and M89 mutants compared to WT.While in M15,M117 and M270 chloroplasts,we found there were more osmiophilic granules in mutants than in WT.All these indicated that the chloroplast development and structure were abnormal in all six mutants.We used the six mutants as the female parents to hybrid with different male parents,then observed the phenotype of F₁ plants and analyzed the segregation ratio of F₂ groups.Genetic analysis indicated that these six mutants?M15,M50,M85,M89,M117 and M270?were controlled by a single recessive gene,respectively.We employed a map-based cloning approach to map the candidate gene of M85 mutant in a 321-kb region between the markers RM7153 and RM1186 on the short arm of chromosome 7.Further studies of histochemical analysis and dark treatment indicated that the cell death and ROS accumulation presented in M85 mutant but there were no respond to dark induction in M85 mutant.The M96 mutant was characterized as prostrate growth at the beginning of germination,and the prostrate growth phenotype ran through the whole life duration.Tiller angle and tiller number of M96increased significantly in comparison with the wild type.And tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96.We mapped the target gene of M96 to a region of 506 kb flanked by markers RM5960 and InDel8 on the long arm of chromosome 11.Subsequently sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840.Additionally,real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96.Furthermore,the expression level was more than four times in M96 stem than in the wild type stem.