Study On The Dormancy Molecular Mechanisms Using Rice Cv.N22 With Strong Seed Dormancy And Its Less Dormant Mutants

The dormancy of rice is a very important agronomic trait.In breeding application it's better to cultivate varieties which have moderate dormancy.If the dormancy of the cultivated varieties is too high it will result in uneven field seedling which is not conducive to plant.And if the dormancy of the cultivated varieties is too low it will result in per-harvest sprouting in rainy days and thus greatly reduce the rice yield and qualities.So it's very important to find the dormancy related genes and study the mechanism of seed dormancy.N22 is a rice variety with very high dormancy.With 400Gy 60Co radiation mutagenesis treatment of N22 seeds we got two weak dormant mutants named Q4359 and Q4646.In this study we observe the phenotypic traits of the two mutants.Microarray-based gene expression analysis was made using N22 and the two mutants at 24 days after heading(DAH).We analyze the gene expression data and try to find molecular mechanisms leading to dormancy weakened.The main results of this study are as follows:1.The wild type germination rate is generally 0%,while the mutants Q4359 and Q4646 have different germination rates due to the impact of different environmental in different years.In recent years the germination rates of mutants Q4359 and Q4646 are about 40%,so the dormancy of the mutations is weaker than N22.The two mutants have proximity dormancy.And the dormancy of Q4359 is slightly weaker than the Q4646.Compared to wild-type N22,mutants Q4359 and Q4646 can break dormancy faster.And Q4359 break dormancy slightly faster than the Q4646.Mutants and N22 germination rate significantly improved with the treatment of peeling the seed shell.The dormancy of N22 may be due to the seed poor permeability ability to water or air.The water absorption rates of mutants and N22 were detected.The results show that the water absorption rates of N22 are weaker than the mutant Q4359.We examined seed a-amylase activity and hydrogen peroxide content of Q4359,Q4646 wild type N22 at 24 and 28 days after heading(DAH).The results show that the a-amylase activity of mutant Q4359 and Q4646 is 3-5 times the N22.And hydrogen peroxide content of mutant Q4359 is higher than N22 at 24 days after heading(DAH)2.There is no significant ABA content difference between wild type N22 and mutant Q4359 seed.This shows that Q4359 is not a ABA deficient mutant.Comparing with N22,mutants Q4359 and Q4646 decreased sensitivity to ABA.The difference in sensitivity of ABA is not obvious between mutants and wild type at low concentrations of ABA treatment.But Q4359 and Q4646 obviously reduced sensitivity to ABA at 20?M concentrations of ABA treatment.Real-time PCR was performed to study the expression profile of ABI3 and ABI5 both of which are important genes in ABA signaling pathway.The results show that gene expression levels of the two mutants was significantly lower than that of wild-type N22.The ABA signaling pathway in mutants may be hindered.3.Genetic analysis indicated that dormancy trait was caused by a single recessive allele of a nuclear gene in both mutants.The germination rate of Q4359/N22 F1 and Q4646/N22 F1 is similar.The germination rate of Q4359/N22 F2 and Q4646/N22 F2 which stored 50 days exhibits a bimodal distribution.In Q4359/N22 F2,153 plants performed wild type and 50 plants performed mutant type segregation ratios of which is in line with 3:1(x2=0.21<x20.05=3.84).In Q4646/N22 F2,149 plants performed wild type and 53 plants performed mutant type segregation ratios of which is in line with 3:1(x2=0.24<x20.05=3.84).4.Microarray-based gene expression analysis was made using N22 and the two mutants at 24 days after heading(DAH).In total,6,605 genes differed by more than 1.5-fold and 2,897 genes differed by more than 2-fold between Q4359 and N22.Likewise,2,138 genes differed by more than 1.5-fold and 773 genes differed by more than 2-fold between Q4646 and N22.In Q4359,the reduced seed dormancy phenotype was correlated with decreased ABA signaling and increased GA biosynthesis and signaling.Besides reactive oxygen species(ROS)related genes' expression levels are higher in Q4359 which leading to the ROS content is higher than N22.And then the changed ROS content lead to the germination rates of Q4359 higher.We have proved these results with treatments of adding ROS scavengers and donors respectively.