Analysis Of Differential DNA Methylation Sites In Photo-thermo Sensitive Genic Male Sterile Rice Peiai 64S

The pollen fertility of PTGMS rice is regulated by both light and temperature.Under natural conditions,the length of light is determined by geographical latitude and season,which is regular and predictable,while the change of temperature is random.Therefore,the research on the regulation and mechanism of temperature on fertility is more important.The mechanism of action between temperature and pollen fertility of PTGMS rice is still unclear.In this study,near isogenic lines(PA2364S,PA2864S)of PA64 S with different temperature sensitivity to fertility were used as materials.Under long light and different temperature treatments,5-Aza as a methylation inhibitor,was sprayed on the leaves at the end of secondary branch and glume primordium differentiation stage(stage III)of young panicle differentiation.In order to explore different temperature treatments about the relationship between the level of DNA methylation and fertility,the whole genome methylation sequencing of young panicles was carried out at the stage of pollen mother cell formation(stage V).The main results are as follows:1.There was significant difference in temperature response of fertility between near isogenic lines PA2364 S and PA2864 S.Under the treatment of 14 h-21 ℃(light length average temperature),both were fertile;under the treatment of 14 h-25 ℃,PA2364 S was sterile,PA2864 S was fertile;under the treatment of natural long light and high temperature(> 28 ℃),both were sterile.2.The pollen fertility of sterile condition could be partially restored by methylated inhibitor 5-Aza.The results showed that the fertility iodine rate of PA2364 S was 0.00 %at natural temperature and 25 ℃,3.45 % and 15.10 % after spraying 5-Aza respectively;the iodine rate of PA2864 S was 0.00% and 6.56 % after spraying 5-Aza.3.Genome wide methylation analysis of PA2364 S and PA2864 S at V stage showed that there were three kinds of methylation sequences CG,CHG and CHH(H = A,C,or T),and the order from high to low was CG > CHG > CHH.4.The methylation level of CG and CHG sequences decreased significantly by spraying 5-Aza,and the methylation level of CG sequence decreased from 56.13 % to53.58 %;the methylation level of CHG sequence decreased from 28.82 % to 28.20 %.5.In CG and CHG sequences,the methylation extent of sterile plants was higher than that of fertile plants.The methylation levels of CG and CHG sequences in male sterile plants of PA2364 S were 56.13 % and 28.82 % respectively,and those in fertile plants were 53.89 % and 27.01 % respectively;the methylation levels of CG and CHG sequences in male sterile plants of PA2864 S were 51.84 % and 27.28 % respectively,and those in fertile plants were 48.25 % and 25.60 % respectively.6.Twenty-one methylation genes were selected from PA2364 S and PA2864 S under different temperature treatments,and they were all responsive to different fertility;Seven of them responded to the difference between PA2364 S and PA2864S;eleven of them responded to 5-Aza treatment;os04g0556300,os04g0457500,os01g0337900 and os12g0264500 were found to respond to fertility,strain and 5-aza treatment.os04g0556300,os04g0457500,os01g0337900 and os12g0264500 can participate in the regulation of ROS.In this study,we obtained a lot of information through the whole genome bisulfite sequencing(wgbs)technology.The methylation level of CG and CHG sequences is related to pollen fertility.Spraying 5-aza can reduce the methylation level,which has the effect of partial fertility restoration.It is preliminarily clear that there is a certain relationship between temperature,DNA methylation level and pollen fertility,and some of them may participate in pollen fertility The analysis of the expression level of sex regulated methylated genes can be used as the basis for further study of fertility mechanism.