comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice...
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Life Sciences in Space Research 1 (2014) 74–79
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Life Sciences in Space Research
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Comparison of space flight and heavy ion radiation inducedgenomic/epigenomic mutations in rice (Oryza sativa)
Jinming Shi a,1, Weihong Lu b,1, Yeqing Sun c,∗a College of Life Science, Northeast Forestry University, Harbin, PR Chinab Institute of Extreme Environment Nutrition and Protection, School of Food Science and Engineering, Harbin Institute of Technology, Harbin, PR Chinac Institute of Environmental Systems Biology, Dalian Maritime University, Dalian, PR China
a r t i c l e i n f o a b s t r a c t
Article history:Received 7 June 2013Received in revised form 15 January 2014Accepted 23 January 2014
Keywords:Space flightHeavy ion radiationRiceMutationEpigenetic effectDNA methylation
Rice seeds, after space flight and low dose heavy ion radiation treatment were cultured on ground.Leaves of the mature plants were obtained for examination of genomic/epigenomic mutations by usingamplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism(MSAP) method, respectively. The mutation sites were identified by fragment recovery and sequencing.The heritability of the mutations was detected in the next generation. Results showed that both spaceflight and low dose heavy ion radiation can induce significant alterations on rice genome and epigenome(P < 0.05). For both genetic and epigenetic assays, while there was no significant difference in mutationrates and their ability to be inherited to the next generation, the site of mutations differed betweenthe space flight and radiation treated groups. More than 50% of the mutation sites were shared bytwo radiation treated groups, radiated with different LET value and dose, while only about 20% of themutation sites were shared by space flight group and radiation treated group. Moreover, in space flightgroup, we found that DNA methylation changes were more prone to occur on CNG sequence than CGsequence. Sequencing results proved that both space flight and heavy ion radiation induced mutationswere widely spread on rice genome including coding region and repeated region. Our study described andcompared the characters of space flight and low dose heavy ion radiation induced genomic/epigenomicmutations. Our data revealed the mechanisms of application of space environment for mutagenesis andcrop breeding. Furthermore, this work implicated that the nature of mutations induced under space flightconditions may involve factors beyond ion radiation.
© 2014 Published by Elsevier Ltd on behalf of The Committee on Space Research (COSPAR).
1. Introduction
In space, there are many factors including microgravity, electro-magnetic radiation, particle radiation and magnetism, which caninduce multiple biological damages (Adams et al., 1981). Amongthose factors, charged particle is considered to be the primary mu-tagen because it often penetrates living tissues and causes bothdirect and indirect damages on biological molecules (Koturbash etal., 2006; Filkowski et al., 2004; Zhang et al., 2006; Naito et al.,2005; Shikazono et al., 2001). Radiation induced damages on bio-logical molecules, especially DNA, is one of the main threat to hu-man health during space exploration. Previous researches provedthat space flight can induce both morphological injury on chromo-some and base mutation on DNA molecule of humans and plants
* Corresponding author. Tel.: +86 411 84723633 888; fax: +86 411 84725675.E-mail address: [email protected] (Y. Sun).
1 Contributed equally to the research.
http://dx.doi.org/10.1016/j.lssr.2014.02.0072214-5524/© 2014 Published by Elsevier Ltd on behalf of The Committee on Space Rese
(Kranz, 1986; Yang et al., 1997; Gao et al., 2009). Moreover, spaceflight induced mutations are prone to happen on some specific“hot spots” on genome (Li et al., 2007). These studies suggest thatthe mutagenesis effect of space flight is not random. In previ-ous studies, ground simulated radiation, using accelerated particleswere considered as a valid approach to investigate the charactersof biological effects of space radiation (Wei et al., 1995; Shi et al.,2010). However, whether the simulated radiation induced muta-tion has the same characters and ”hot spots” as space radiationneeds to be investigated.
Besides genome, epigenome of both mammals and plants arealso the target of radiation induced damage in living organisms. Inthe past two decades, epigenetic mutations especially DNA methy-lation changes induced by radiation were well studied. It has beenreported that genome-wide DNA methylation changes can be in-duced in mammalian cells by γ -rays (Kalinich et al., 1989) andX-rays (Tawa et al., 1998) exposure. Further studies proved thatgene specific and tissue specific alterations in methylation werein response to both acute and chronic low dose X-rays irradiation
arch (COSPAR).
J. Shi et al. / Life Sciences in Space Research 1 (2014) 74–79 75
(Kovalchuk et al., 2004). Recent studies also found that 18 daysspace flight could cause significant alterations in DNA methyla-tion in rice (Ou et al., 2009). Besides DNA methylation, microRNApath ways were also proved to be associated with heavy-ion radia-tion induced damages in rice (Zhang et al., 2011). All these studiesshowed that epigenetic mechanisms affect living organisms underradiation stress.
In the present study, we scanned and identified the mutationson both genome and epigenome of rice after space flight and lowdose heavy ion radiation treatment. The mutagenic effects of spaceradiation and ground simulated radiation were compared.
2. Materials and methods
2.1. Plant material
2.1.1. Space flightSeeds of rice (Oryza sativa, Japonica) were aboard 20th recover-
able satellite of China for an 18-day (from September 27 to October15, 2004) space flight. A total of 200 rice seeds were packed incotton sack and fixed inside the satellite. The same number ofrice seeds were also packed in cotton sack and kept under nor-mal ambient conditions, to be used as ground control. After spaceflight, the seeds were germinated under standard conditions to-gether with the controls.
2.1.2. Heavy ion radiationSeeds of rice (Oryza sativa, Japonica) were positioned in the
chamber which was fixed to the irradiation equipment at theHeavy Ion Research Facility in Lanzhou (HIRFL) (Wei et al., 1991).Rice seeds were radiated with 12C6+ ion beam. For the first ra-diation group (R1), 150 rice seeds were treated and the meanlinear energy transfer (LET) within the seeds was calculated tobe 62.2 KeV/μm. The dose and dose rate were 200 mGy and0.2 Gy/min. For the second radiation group (R2), 150 rice seedswere treated and the mean LET within the seeds was calculated tobe 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 onthe same condition with dose of 0 Gy were used as controls.
2.1.3. Plant cultureAfter space flight and ground radiation treatment, rice seeds
were germinated under standard conditions and then cultured ontrial field for 18 weeks. The controls were cultured at the sameconditions. Leaves of the mature plants were obtained and storedat −80 ◦C, until further use. For heredity examination, the selectedindividuals were tagged and analyzed later. Panicles of the taggedplants were bagged, and seeds were collected from each individualplant and designated as the progenies. The progenies were plantedon the same trial field next year.
2.2. DNA extraction
Total genomic DNA was extracted from the leaves by a modifiedCTAB method and purified by phenol extractions (Chen and Ronald,1999).
2.3. Methylation-sensitive amplification polymorphism (MSAP) assay
The MSAP was adapted from (Xu et al., 2000). Aliquots (200 ng)of DNA were digested for 2 h at 37 ◦C and 70 ◦C for 15 min with5 U each of EcoRI and HpaII (New England Biolabs) in 10 μl buffersolution. In another reaction, the same amount of rice genomicDNA was digested with EcoRI and MspI (New England Biolabs) in10 μl buffer solution. The DNA fragments from the two reactions
were added separately to an equal volume of the adapter/liga-tion solution, and the ligation reaction was allowed to proceedovernight at 20 ◦C. The ligation mixture was then diluted 1:10dilution with TE, and used as the template for the preselectiveamplification. The reaction was performed for 25 cycles of 30 s de-naturation at 94 ◦C, 30 s annealing at 56 ◦C, and 1 min extensionat 72 ◦C. The product was diluted 20 fold (v:v) with TE buffer, andused as the template for the selective amplification reaction. In thisstep, EcoRI and HpaII/MspI primers with three additional selectivenucleotides were used. The selective PCR was performed in a fi-nal volume of 25 μl following the protocol of Vos et al. (1995). Theproducts of selective amplification were resolved by electrophore-sis on 6% denaturing polyacrylamide sequencing gels and stainedwith silver (Chalhoub et al., 1997).
2.4. Amplified fragment length polymorphism (AFLP) assay
AFLP Core Reagent Kit (Invitrogen) was used for this assay. 200ng DNA were digested for 2 h at 37 ◦C and 70 ◦C for 15 min with5 U each of EcoRI/MseI. The DNA fragments from the reactionswere added to an equal volume of the adapter/ligation solution,and the ligation reaction was allowed to maintain 2 h at 20 ◦C. Theligation mixture was then diluted 1:10 dilution with TE, and usedas the template for the preselective amplification. The procedureof preselective amplification, selective PCR and stain were same tothe MSAP assay. In selective PCR step, EcoRI and MseI primers withtwo additional selective nucleotides were used.
2.5. Data analysis
A mixture of eight individual controls was used as a reference(C0). Each plant in the same group was compared with C0 atthe differential mapping sites. Each repeatable differential mappingsite was defined as a polymorphic band of the plant. The polymor-phic rate of each plant was calculated according to the formulabelow:
Polymorphic rate = (number of polymorphic bands/number oftotal bands) ×100%
2.6. Isolation and characterization of amplified fragments
Amplified fragments were excised from the gel with a cleanrazor blade. The gel slices were grinded and then hydrated in80 μl of water and incubated at 95 ◦C for 15 min. The elutedDNA was amplified with the same primers under the conditionsused for selective amplification. Sequence information was ob-tained by cloning the fragments in the pMD18-T Simple Vector(TaKaRa) and sequencing individual clones. The sequences obtainedwere compared with nucleotide sequences in the publicly availabledatabases.
2.7. Statistical analysis
Significant differences between the means were determined us-ing the t-test. Data were shown as mean ± standard deviation(SD). The values of P < 0.05 were considered statistically signifi-cant.
3. Results
3.1. Mutation rate on genome/epigenome after space flight and heavyion radiation
Eight individuals were randomly chosen from space flight group(SP) and two radiation groups (R1 and R2). The same number of
76 J. Shi et al. / Life Sciences in Space Research 1 (2014) 74–79
Fig. 1. Selective amplification examples of MSAP (A) and AFLP (B) of the mixed con-trol (C0), eight randomly selected individual plants (1–8). The arrows indicated thepolymorphic bands.
Fig. 2. Polymorphic rates of eight plants of controls, and eight plants from threetreated groups: space flight group (SP), first radiation treated group (R1) and secondradiation group (R2), respectively. Each point is the average polymorphic rate andthe error bar is standard deviation.
control plants of each group were also randomly chosen for mu-tation rate examination using AFLP and MSAP method (Fig. 1).For AFLP assay, the average polymorphic rates of SP, R1 and R2were 2.9%, 3.4% and 3.2%, respectively. Compared with their con-trols, significant increasing of the polymorphic rate in all the threegroups was detected (P < 0.05). There were no significant differ-ences among the three treated groups (Fig. 2A). For MSAP assay,the polymorphic rates of the three treated groups SP, R1 and R2were 5.6%, 4.6% and 4.2%, respectively. Significant difference werefound between the treated group and their controls (P = 0.01).
There were no significant differences among the three treatedgroups (Fig. 2B).
3.2. Different MSAP patterns induced by space flight and heavy ionradiation
In MSAP assay, the polymorphic sites that appeared only intreated group but not control group were identified as mutationsites. According to the digestion pattern of HpaII and MspI, themutation sites were divided into four types: hyper-methylation onCG site, hyper-methylation on CNG site, hypo-methylation on CGsite and hypo-methylation on CNG site. The number and percent-age of the mutation sites in each type were listed in Table 1. Thedata showed that the above four mutation types were all exist inboth space flight and radiation groups. In radiation groups, morethan half of the mutations were happened on CG site. However, inspace flight group, more CNG mutations were founded. The aboveresults suggested that space flight induced epigenomic mutationswere different from that induced by radiation.
3.3. Identification of characteristic mutations on genome andepigenome
For both space flight and radiation assays, eight single plantsof each group (SP, R1 and R2) were detected for mutation sitescreening. Mutation sites appear in three or more than three sin-gle plants were recorded and identified as characteristic mutationsites of the group. The characteristic mutation sites of space ra-diation and heavy ion radiation were compared in Table 2. Datashowed that the mutation sites shared by R1 and R2 were morethan that shared by SP and R1/R2. This result indicated that themutation sites were differed between space flight and radiationgroups.
Sequence information of the characteristic mutation sites ofboth space radiation and heavy ion radiation were obtained byband recovery, sequencing and blasting. The results were listed inTable 3 (epigenomic mutation sites) and Table 4 (genomic muta-tion sites). The mutation sites were wide spread on rice genomeincluding coding region and repeat sequence.
3.4. Heredity of genomic/epigenomic mutations
Three offspring of each individual plant in treated group wererandomly chosen for examining the heredity of genomic and epige-nomic mutations. The mutation sites on genome and epigenomeof the offspring were detected and compared with their parent.Results indicated that 48 of the 50 detected mutation sites are her-itable (data not show).
4. Discussion
Although space environment consist of multiple harmfulsources, ionizing radiation is still the main factor to cause geneticmutation. In this study, we investigated the genomic/epigenomicmutations induced by both space radiation and ground heavyion radiation. Each radiation treatment has its own parameters.The total dose of the 18-day space flight was calculated to be2 mGy, while the doses of the on ground radiation treatment were200 mGy and 2 Gy respectively. The LET value and dose rate ofthe two radiation assays were also different. Although the radia-tion conditions were not same, the genomic/epigenomic mutationrates of the three groups were not significantly different (Fig. 2).The data showed that space flight can induced significant mu-tations even at dose as low as 2 mGy. The ability of the spaceenvironment to induced mutation in plants was shown by previ-ous researches (Yu et al., 2007; Ou et al., 2009; Long et al., 2009).
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Table 1Epigenomic mutations in rice induced by space flight and heavy ion radiation detected by MSAP method.
Groupa No. of total recorded sites No. of mutation sites (percentage of total mutation sites)
Hyper-methylation Hypo-methylation
CG CNG CG CNG
SP 890 5(16.7%) 13(43.3%) 4(13.3%) 8(26.7%)R1 890 8(33.3%) 5(20.8%) 8(33.3%) 3(12.5%)R2 890 7(31.8%) 5(25.0%) 6(26.5%) 4(16.7%)
a The three groups are space flight group (SP), first radiation treated group (R1) and second radiation group (R2).
Table 2Comparison of genomic/epigenomic mutations in rice induced by space flight and heavy ion radiation.
No. of mutation sites shared by two groups (percentage of total mutation sites)
Genome Epigenome
SP R1 R2 SP R1 R2
SP 18(100%)a 4(22.2%)b 5(27.8%)b 30(100%)a 8(26.7)b 7(23.3)b
R1 – 23(100%)a 12(52.2%)b – 24(100%)a 14(58.3)b
R2 – – 20(100%)a – – 22(100%)a
a Mutation sites in space flight group (SP), first radiation treated group (R1) and second radiation group (R2).b Mutation sites shared by two groups. The percentage in brackets was calculated using the No. of total mutation sites in row of table.
Table 3Identification of the epigenomic mutation sites by sequencing.
Fragmenta Length (bp) Type GenBank accession No. Sequence homology
sp-M1 151 CG ref|NM_001064208.1| Oryza sativa (japonica cultivar-group) Os06g0489500 (Os06g0489500) mRNA, complete cdsCytidine/deoxycytidylate deaminase
sp-M2 179 CG dbj|AP007205.2| japonica cultivar-group genomic DNA chromosome 6, BACclone:OSJNBa0048L03sp-M3 181 CG dbj|AP007205.2| japonica cultivar-group genomic DNA chromosome 6, BACclone:OSJNBa0048L03sp-M4 140 CNG dbj|BA000029.3| Oryza sativa (japonica cultivar-group) Ty3-gypsy subclass retrotransposon proteinsp-M5 111 CNG ref|NM_001057358.1| Oryza sativa (japonica cultivar-group) Os03g0660000 (Os03g0660000) mRNA, complete cdssp-M6 195 CNG gb|AC018727.10| Oryza sativa chromosome 10 BAC OSJNBa0056G17 genomic sequence, complete sequencesp-M7 114 CNG dbj|AP008217.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 11sp-M8* 106 CNG ref|NM_001068083.1| Oryza sativa (japonica cultivar-group) Os08g0326100 (Os08g0326100) mRNA, complete cds
Forkhead-associated domain containing proteinsp-M9 95 CNG ref|NM_001069574.1| Oryza sativa (japonica cultivar-group) Os09g0375700 (Os09g0375700) mRNA, complete cdssp-M10 181 CNG dbj|AP008212.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6sp-M11* 178 CG CH398137 Oryza sativa (indica cultivar-group) chromosome 6 scaffold000057 genomic scaffold, whole
genome shotgun sequence gi|56718110| gb|CH398137.1|r-M1 251 CNG dbj|AP008209.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 3r-M2 216 CG ref|NM_001056555.1| Oryza sativa (japonica cultivar-group) Os03g0333000 (Os03g0333000) mRNA, complete cds
Prefoldin family proteinr-M3 125 CG ref|NM_001048499.1| Oryza sativa (japonica cultivar-group) Os01g0137300 (Os01g0137300) mRNA, complete cds
Conserved hypothetical proteinr-M4* 83 CG dbj|AP008208.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 2r-M5 364 CG ref|NM_001061765.1| Oryza sativa (japonica cultivar-group) Os05g0334400 (Os05g0334400) mRNA, complete cds
Heat shock protein DnaJ family proteinr-M6 136 CG dbj|AP008209.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 3r-M7 124 CG dbj|AP008210.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 4r-M8 105 CG dbj|AP008214.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 8r-M9 196 CNG dbj|AP008218.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 12r-M10 194 CNG dbj|AP008218.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 12r-M11 115 CNG dbj|AP008217.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 11r-M12 96 CG ref|NM_001068023.1| Oryza sativa (japonica cultivar-group) Os08g0299600 (Os08g0299600) mRNA, partial cds.
Agamous-like MADS box protein AGL15r-M13 105 CG ref|NM_001060860.1| Oryza sativa (japonica cultivar-group) Os04g0686200 (Os04g0686200) mRNA, complete cds
Chromosome condensation regulator proteinr-M14 160 CNG dbj|AP008215.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 9
a Epigenomic mutations in space flight group (sp-M1-11) and radiation groups (r-M1-14).* Epigenomic mutations shared by space flight group and radiation groups.
Our results suggest that, compared with ground radiation, spaceflight may be more effective in causing mutations.
Analysis of epigenetic mutation site showed that both hyper-methylation and hypo-methylation were induced by space flightand heavy ion radiation. Moreover, methylation mutations hap-pened on both CG sites and CNG sites. Compared with heavy ionradiation groups, there were more hypo-methylations on CNG sitein space flight group (Table 2). DNA methylation on CNG site isa unique phenomenon in plants which has not been found inother creatures. Investigations concerning this subject indicated
that DNA methylation on CNG sites are important not only fornormal growth and development but also initiate stress-defensemechanisms in plants (Xiao et al., 2006). Dyachenko et al. (2006)demonstrated that high salinity conditions can induce two-foldincrease in CNG methylation level in nuclear genome of M. crys-tallinum plants. Ou et al. (2009) found that space flight can induceCNG site methylation changes on rice genome. In our results, mu-tations on CNG site induced by space flight were more than thatinduced by heavy ion radiation. Thus, CNG site methylation maybe susceptible to space environment.
78 J. Shi et al. / Life Sciences in Space Research 1 (2014) 74–79
Table 4Identification of the genomic mutation sites by sequencing.
Fragmenta Length (bp) GenBank accession No. Sequence homology
sp-A1* 176 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12. repeat regionsp-A2* 165 dbj|AP006757.2| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1, PAC clone:P0551C06sp-A3* 112 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12. repeat regionsp-A4 343 gb|AC091724.3| Oryza sativa (japonica cultivar-group) chromosome 10 clone OSJNBa0004E08sp-A5 125 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12sp-A6 135 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12.sp-A7 132 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12. repeat regionsp-A8 102 dbj|AP008214.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 8sp-A9 130 – Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1-12. repeat regionsp-A10 122 CH398199 Oryza sativa (indica cultivar-group) chromosome 11 scaffold000119 genomic scaffold, whole genome
shotgun sequence gi|56718048|gb|CH398199.1|[56718048]sp-A11 200 CH398168 Oryza sativa (indica cultivar-group) chromosome 8 scaffold000088 genomic scaffold, whole genome
shotgun sequence. gi|56718079|gb|CH398168.1|[56718079]sp-A12 130 dbj|AP008215.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 9sp-A13 115 CH398202 Oryza sativa (indica cultivar-group) chromosome 11 scaffold000122 genomic scaffold, whole genome
shotgun sequence. gi|56718045|gb|CH398202.1|[56718045]sp-A14 93 dbj|AP008211.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 5r-A1 157 CH398144 Oryza sativa (indica cultivar-group) chromosome 6 scaffold000064 genomic scaffold, whole genome
shotgun sequencer-A2 187 gb|AY522330.1| Oryza sativa (japonica cultivar-group) cultivar Nipponbare chloroplast, complete genome RNA
polymerase beta chainr-A3 123 ref|NM_001061765.1| Oryza sativa (japonica cultivar-group) Os05g0334400 (Os05g0334400) mRNA, complete cds Heat shock
protein DnaJ family proteinr-A4 137 dbj|AP008216.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 10 Putative receptive receptor-like
protein kinaser-A5 99 dbj|AP008211.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 5r-A6 128 dbj|AP008207.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 1r-A7 147 ref|NM_001069796.1| Oryza sativa (japonica cultivar-group) Os09g0436700 (Os09g0436700) mRNA, complete cdsr-A8 206 gb|EF555581.1| Oryza sativa (japonica cultivar-group) terminal-repeat retrotransposon in miniature ZO6, complete
sequencer-A9 249 dbj|AP008216.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 10r-A10 312 dbj|AP008209.1| Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 3
a Genomic mutations in space flight group (sp-A1-14) and radiation groups (r-A1-10).* Genomic mutations shared by space flight group and radiation groups.
Although the mutation rate in space flight group and two radi-ation groups were not significantly different, the locations of themutation sites on rice genome were not same. In Table 2, the mu-tation sites on both genome and epigenome shared by two groupswere listed. The data showed that there were more mutation sitesshared by R1 and R2 than that shared by R1/R2 and SP. Althoughthe radiation parameters were not same, more than half of the mu-tation sites of one ground radiation group appeared in the otherground radiation cohort. However, only about 1/4 of the muta-tion sites in space radiation group were shared with each groundradiation group. The detections on genome and epigenome drawthe same conclusion. These results suggested that the “hot spots”of space radiation and ground heavy ion radiation were not same.This difference might be due to the following aspects. Firstly, theradiation sources in space were more complex than that in groundsimulated radiation. Space environment consists of the multipleradiation sources such as protons, ions, electrons, and charged par-ticles. The ions in space environment are also diverse, includingcarbon, neon, iron and so on (Maalouf et al., 2011). In groundsimulated radiation assay, only carbon ions were used. Previous re-searches showed that different radiation sources have different bi-ological effects (Wei et al., 2006). So, the different radiation sourcesmay be one reason of the different mutagenic effects betweenspace flight and heavy ion radiation. Secondly, in space, other fac-tors such as microgravity and magnetism may effect the action ofradiation sources on living organisms. Recent researches reportedthat both simulated microgravity and carbon ion irradiation caninduce cell apoptosis and DNA damage (Li et al., 2013). Moreover,simulated microgravity can increase radiation induced damages onimmune system (Sanzari et al., 2013). In the present study, wecompared mutations induced by space environment and groundradiation. We found that the mutations induced by space flightwere differed from that of ground radiation. The results strongly
suggested that the nature of mutations induced by space flight in-volve factors beyond ion radiation.
Sequencing results showed that DNA methylation mutationswere related to genes coding functional proteins such as signaltransduction related protein (sp-M8), transcription factor (r-M12),metabolism related protein (sp-M1) and the proteins related tostress response including heat shock protein (r-M5) and prefoldinprotein (r-M2). Moreover, r-M13 shows homology to cell cycle reg-ulator chromosome condensation regulator protein which was alsoknown as guanine nucleotide exchange factor for the small GTPaseRan. And, sp-M4 shows homology to Ty3-gypsy subclass retro-transposon protein. For AFLP assay, many of the mutation sitesshowed homology to retrotransposon sequences (sp-A1, sp-A3,sp-A5, sp-A6, sp-A7, sp-A9 and r-A8). All of those sequences hadrelationship with pseudogene. There were also mutations showcoding genes such as heat shock protein (r-A3) and receptor-likeprotein kinase (r-A4). These results suggested that those genesand the related proteins might be responsers under the radiationstress.
It is generally believed that the DNA methylation patterns inplants are stably inherited over transgeneration. During sexual gen-erations, plants stably keep their methylation patterns instead of“erasure and reset” it as mammals. In plants, DNA methylationchanges often caused by severe circumstance, and inherited bytheir offspring (Kovalchuk et al., 2004; Grant-Downton and Dickin-son, 2005; Long et al., 2006; Boyko et al., 2007). In this study, theheredity of the mutation sites on genome and epigenome wereinvestigated. Data showed that most of the mutation sites werefaithfully inherited by the next generation (Table 2). Our resultsdraw the same conclusion that, unlike mammals, plant faithfullyinherited the epigenomic mutations of their parents. This charactermay encourage the application of space environment for epigeneticmutagenesis and epigenetic breeding.
J. Shi et al. / Life Sciences in Space Research 1 (2014) 74–79 79
Acknowledgements
We are grateful to Dr. Wenjian-Li and Xicun-Dong in Insti-tute of Modern Physics for radiation experiment. This researchwas supported by the Fundamental Research Funds for the Cen-tral Universities (DL11BA16), the Natural Science Foundation ofthe Heilongjiang Province (C201108) and National Natural ScienceFoundation of China (31270903).
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