IC4R007-Epigenomic-2014-26432592

Project Title

• Comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice(Oryzasativa)

The Background of This Project

• Radiation induced damages on biological molecules, especially DNA, is one of the main threat to human health during space exploration. Previous researches proved that spacef light can induce both morphological injury on chromosome and base mutation on DNA molecule of humans and plants. Moreover, space flight induced mutations are prone to happen on some specific “hot spots” on genome. These studies suggest that the mutagenesis effect of space flight is not random.
• Besides genome, epigenome of both mammals and plants are also the target of radiation induced damage in living organisms. In the past two decades, epigenetic mutations especially DNA methylation changes induced by radiation were well studied.
• In the present study, the researchers scanned and identified the mutations on both genome and epigenome of rice after space flight and low dose heavy ion radiation treatment. The mutagenic effects of space radiation and ground simulated radiation were compared.

Plant Materials & Treatment

• Seeds of rice(Oryzasativa,Japonica) were aboard 20th recoverable satellite of China for an 18 day(from September 27 to October 15, 2004) space flight. A total of 200 rice seeds were packed in cotton sack and fixed inside the satellite. The same number of rice seeds were also packed in cotton sack and kept under nor-mal ambient conditions, to be used as ground control. After space flight, the seeds were germinated under standard conditions together with the controls.
• Seeds of rice(Oryzasativa,Japonica) were positioned in the chamber which was fixed to the irradiation equipment at the Heavy Ion Research Facility in Lanzhou (HIRFL). Rice seeds were radiated with 12C6+ ion beam. For the first radiation group (R1), 150 rice seeds were treated and the mean linear energy transfer (LET)within the seeds was calculated to be 62.2 KeV/μm. The dose and dose rate were 200 mGy and 0.2 Gy/min. For the second radiation group (R2), 150 rice seeds were treated and the mean LET within the seeds was calculated to be 27.4 KeV/μm. The dose and dose rate were 2 Gy and 0.1 Gy/min. For each radiation assay, the same number of rice seed placed on the same condition with dose of 0 Gy were used as controls.
• After space flight and ground radiation treatment, rice seeds were germinated under standard conditions and then cultured on trial field for 18 weeks. The controls were cultured at the same conditions. Leaves of the mature plants were obtained and stored at −80℃, until further use. For heredity examination, the selected individuals were tagged and analyzed later. Panicles of the tagged plants were bagged, and seeds were collected from each individual plant and designated as the progenies. The progenies were planted on the same trial field next year.

Research Findings

• Eight individuals were randomly chosen from space flight group (SP) and two radiation groups (R1 and R2). The same number of control plants of each group were also randomly chosen for mutation rate examination using AFLP and MSAP method (Fig.1). For AFLP assay, the average polymorphicrates of SP,R1 and R2 were 2.9%, 3.4% and 3.2%, respectively. Compared with their controls, significant increasing of the polymorphic rate in all the three groups was detected (P<0.05). There were no significant differences among the three treated groups (Fig.2A). For MSAP assay, the polymorphic rates of the three treated groups SP, R1 and R2 were 5.6%, 4.6% and 4.2%, respectively. Significant difference were found between the treated group and their controls (P=0.01). There were no significant differences among the three treated groups (Fig.2B).

Figure 1 Selective amplification examples of MSAP(A) and AFLP(B) of the mixed control(C0), eight randomly selected individual plants(1–8). The arrows indicated the polymorphic bands.

Figure 2 Polymorphicrates of eight plants of controls, and eight plants from three treated groups: space flight group (SP), first radiation treated group (R1) and second radiation group (R2), respectively. Each point is theaverage polymorphicrate and the error bar is standard deviation.

• For both genetic and epigenetic assays, while there was no significant difference in mutation rates and their ability to be inherited to the next generation, the site of mutations differed between the space flight and radiation treated groups. More than 50% of the mutation sites were shared by two radiation treated groups, radiated with different LET value and dose, while only about 20% of the mutation sites were shared by space flight group and radiation treated group.

• In MSAP assay, the polymorphic sites that appeared only in treated group but not control group were identified as mutation sites. According to the digestion pattern of HpaII and MspI, the mutation sites were divided into four types: hyper-methylation on CG site, hyper-methylation on CNG site, hypo-methylation on CG site and hypo-methylation on CNGsite. The number and percentage of the mutation sites in each type were listed in Table 1. The data showed that the above four mutation types were all exist in both space flight and radiation groups. In radiation groups, more than half of the mutations were happened on CG site. However, inspace flight group,more CNG mutations were founded. The above result ssuggested that space flight induced epigenomic mutations were different from that induced by radiation.

Table 1 Epigenomic mutations in rice induced by space flight and heavy ion radiation detected by MSAP method.

Labs working on this Project

• CollegeofLifeScience,NortheastForestryUniversity,Harbin,PRChina
• InstituteofExtremeEnvironmentNutritionandProtection,SchoolofFoodScienceandEngineering,HarbinInstituteofTechnology,Harbin,PRChina
• InstituteofEnvironmentalSystemsBiology,DalianMaritimeUniversity,Dalian,PRChina

Corresponding Author

• Y.Sun:yqsun@dlmu.edu.cn