Epigenetic changes and transposon reactivationin Thai rice hybrids

Laksana Kantama • Supaporn Junbuathong •

Janejira Sakulkoo • Hans de Jong •

Somsak Apisitwanich

Received: 9 August 2012 / Accepted: 17 January 2013 / Published online: 31 January 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Inter- or intraspecific hybridization is the

first step in transferring exogenous traits to the

germplasm of a recipient crop. One of the complicat-

ing factors is the occurrence of epigenetic modifica-

tions of the hybrids, which in turn can change their

gene expression and phenotype. In this study we

present an analysis of epigenome changes in rice

hybrids that were obtained by crossing rice cultivars,

most of them of indica type and Thai origin.

Comparing amplified fragment length polymorphism

(AFLP) fingerprints of twenty-four cultivars, we

calculated Nei’s indexes for measuring genetic rela-

tionships. Epigenetic changes in their hybrids were

established using methylation-sensitive AFLP finger-

printing and transposon display of the rice transpos-

able elements (TEs) Stowaway Os-1 and Mashu,

leading to the question whether the relationship

between parental genomes is a predictor of epigenome

changes, TE reactivation and changes in TE methyl-

ation. Our study now reveals that the genetic relation-

ship between the parents and DNA methylation

changes in their hybrids is not significantly correlated.

Moreover, genetic distance correlates only weakly

with Mashu reactivation, whereas it does not correlate

with Stowaway Os-1 reactivation. Our observations

also suggest that epigenome changes in the hybrids are

localized events affecting specific chromosomal

regions and transposons rather than affecting the

genomic methylation landscape as a whole. The weak

correlation between genetic distance and Mashu

Electronic supplementary material The online version ofthis article (doi:10.1007/s11032-013-9836-x) containssupplementary material, which is available to authorized users.

L. Kantama

Faculty of Liberal Arts and Science, Kasetsart University,

Kamphaengsaen Campus, Nakorn Pathom 73140,

Thailand

e-mail: faaslsk@ku.ac.th

S. Junbuathong

Pathumthani Rice Research Centre, Bureau of the

Research and Development, Rice Department, Minister of

Agriculture and Cooperation, Pathum Thani, Thailand

e-mail: supaporn@brrd.mail.go.th

J. Sakulkoo � S. Apisitwanich (&)

Department of Genetics, Faculty of Science, Kasetsart

University, Bangkhen Campus, Bangkok 10900, Thailand

e-mail: fscissa@ku.ac.th

H. de Jong

Laboratory of Genetics, Wageningen University, P.O.

Box 309, 6700 AH Wageningen, The Netherlands

e-mail: hans.dejong@wur.nl

S. Apisitwanich

Center of Advanced studies for Tropical Natural

Resources, Kasetsart University, Bangkok 10900,

Thailand

123

Mol Breeding (2013) 31:815–827

DOI 10.1007/s11032-013-9836-x

methylation and reactivation points at only limited

influence of genetic background on the epigenetic

status of the transposon. Our study further demon-

strates that hybridizations between and among specific

japonica and indica cultivars induce both genomic

DNA methylation and reactivation/methylation

change in the Stowaway Os-1 and Mashu transposons.

The observed epigenetic changes seem to affect the

transposons in a clear manner, partly driven by

stochastic processes, which may account for a broader

phenotypic plasticity of the hybrids. A better under-

standing of the epigenome changes leading to such

transposon activation can lead to the development of

novel tools for more variability in future rice breeding.

Keywords Oryza sativa � Intraspecific hybridization �DNA methylation � Transposon � Epigenetics

Abbreviations

AFLP Amplified fragment length polymorphism

CTAB Hexadecyl trimethyl-ammonium bromide

EDTA Ethylene diamine tetra-acetic acid

IR Rice accession from IRRI, Philippines

MITE Miniature inverted-repeat transposable

element

MSAP Methylation-sensitive amplified

polymorphism

RD Rice department accession number

TD Transposon display

TE Tris-EDTA buffer

TMD Transposon methylation display

UPGMA Unweighted pair group method with

arithmetic mean

Introduction

Plant breeders explore various methods to broaden the

genetic base of their crops. When the primary gene

pool is insufficient, desired traits like drought toler-

ance, disease resistance and favourable fruit or plant

shapes are screened for in related taxa. A suitable

relative will then be crossed with the crop in order to

transfer the desired trait to the recipient crop. Such

interspecific crosses followed by consecutive back-

crossing and selection rounds are known as introgres-

sive hybridization, a process that is time-consuming

and can be troublesome (reviewed in Anderson 1953;

Rieseberg and Carney 1998). Major bottlenecks

include incompatibilities (Blakeslee 1945; Stebbins

1958), complex epistatic interactions, linkage drag

(Young et al. 1988), transgressive segregation

(Rieseberg et al. 1999; Stelkens and Seehausen

2009) and epigenetic phenomena (Grant-Downton

and Dickinson 2006). In addition, interspecific hybrids

may display traits that either or both of the parents

lack, and so can adapt to a broader ecological niche

(Rieseberg et al. 2003; Donovan et al. 2010). Such

changes, sometimes considered unexplainable, are

now more often interpreted in the light of epigenetic

changes that effect expression of genes in hybrids and

their progenies. McClintock (1984) first described the

existence of what she called ‘genome shock’, the total

of unpredicted large-scale genome changes that lead to

transposon activation and other structural modifica-

tions of the chromosomes. Her ideas were substanti-

ated when studies revealed the molecular base of these

genomic changes (Liu and Wendel 2000; Comai et al.

2003; Madlung and Comai 2004; Michalak 2009).

Epigenome mutations are induced by changes in

cytosine methylation patterns, which can be regulated

by small interfering (si)RNAs (Shapiro 2010) leading

to up- and down-regulation of gene expression and

reactivation of transposable elements resulting in the

transposition, deletion, insertion and amplification of

their sequences. Epialleles can also be induced by

chemical treatment or in a genotype background

devoid of methylation maintenance. Such alleles

characterized by hypo- or hypermethylation can lead

to constitutive changes in gene expression and stable

inheritance (Akimoto et al. 2007).

This study presents the results of an analysis of

epigenome changes in rice hybrids of various cultivars

and landraces. A selection of 24 rice cultivars were

used, most of them indica type and grown in Thailand.

Genetic distances were calculated by Nei’s index

based on band sharing of amplified fragment length

polymorphism (AFLP) fingerprints. Six cultivars with

different Nei’s values were selected for in-depth

analysis of DNA methylation changes of their hybrids.

Our first interest was focused on the question whether

genetic distance of the parents could explain the

changes in DNA methylation upon hybridization.

If so, we expected to find a correlation between genetic

distance of the parental lines and DNA methyla-

tion alterations and transposon methylation and

816 Mol Breeding (2013) 31:815–827

123

transposable element mobilization. To this end meth-

ylation-sensitive amplification polymorphism (MSAP)

and transposon display (TD) with transposon methyl-

ation display (TMD) protocols were used to establish

activity of Stowaway Os-1 and Mashu, two transpo-

sons that occur in high copy numbers in the rice

genome. Both transposons were chosen because of

their high prevalence in the rice genome, sufficient

clear-cut polymorphic bands between the varieties and

different potential for mobilization (Kishima et al.

2005; Takagi et al. 2003).

Materials and methods

Rice (Oryza sativa L.) seeds were obtained from the

Thai Rice Department and the Thai Department of

Agriculture, Ministry of Agriculture and Coopera-

tives. The varieties were (1) O. sativa ssp. indica var.

Kae Tom Klang; (2) Hawm Indok; (3) Pathum Thani1;

(4) Suphan Buri1 (SPR 1); (5) Chai Nat1; (6) Niaw

Phrae1; (7) RD 7; (8) RD 10; (9) RD 17; (10) Nam

Roo; (11) Jao Sawuey; (12) Puang Mahlai; (13) Look

Daeng Ruang Yao; (14) Goo Meuang Luang; (15)

Khao Tah Haeng; (16) Pah-tawng Daw; (17) Hawm

Pae; (18) Hawm Mali 105; (19) Khao Dawk Mali 105

(KDML 105); (20) Leuang Pratew 123; (21) Lon

Yung; (22) Kaw Diaw; (23) IR 36; (24) O. sativa ssp.

japonica var. Nipponbare. Following a genetic dis-

tance analysis, six varieties (4, 10, 13, 19, 23, 24) were

selected as parents (see Suppl. Table 1 for their sub-

species and phenotypic characteristics) and grown in a

greenhouse under controlled photoperiodicity for

precisely controlling flowering time. Thirty reciprocal

crosses were made using the method of emasculated

pollination panicles. Twenty-five F1 hybrids were

obtained and they were grown in 8-L pots under Thai

greenhouse conditions. For each hybrid we extracted

genomic DNA from the second and third leaves of

plants that were 2 months old. Four plants per hybrid

were used for the analysis.

DNA extraction

DNA was isolated using a modified version of the

CTAB method (Saghai-Maroof et al. 1984). Three

grams of liquid nitrogen-ground leaf material was

mixed with 10 mL of CTAB buffer (100 mM Tris, pH

8.0, 1.4 M NaCl, 20 mM EDTA, 2 % hexadecyl

trimethyl-ammonium bromide and 0.1 % 2-mercapto-

ethanol) and was incubated at 60 �C for 50 min. The

mixture was extracted two times with chloroform:iso-

amyl alcohol (24:1). Two volumes of CTAB precipi-

tation buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1 %

hexadecyl trimethyl-ammonium bromide) was added

to the aqueous phase and left for 5 min at 20 �C. All

mixture samples were centrifuged for DNA collection.

Each DNA sample was dissolved in 500 lL 1.5 M NaC

and, 5 lg/mL RNase and incubated for 15 min at

50 �C. The DNA was then precipitated with two

volumes of absolute ethanol and washed with 70 %

ethanol, and finally dissolved in 0.2 mL TE buffer.

Amplified fragment length polymorphism (AFLP)

DNA polymorphisms in the 24 rice varieties were

investigated by AFLP fingerprinting. The procedure

was based on Vos et al. (1995) with a few small

modifications. All DNA samples were digested with

EcoRI and MseI, followed by ligation with EcoRI and

MseI adapters. For the pre-amplification step two

selective nucleotide primers were used for MseI and

one selective nucleotide for EcoRI, of which we used

the eight primer combinations showing most poly-

morphisms (Suppl. Table 2). Pre-amplification was

performed in forty cycles of 94 �C for 30 s, 56 �C for

60 s, and 72 �C for 60 s. Selective amplification was

carried out consisting of one cycle of 94 �C for 30 s,

65 �C for 60 s and 72 �C for 60 s as first step; the

second step was a touch-down PCR with twelve

cycles, while the annealing temperature was decreased

in steps of 0.7 �C for each consecutive cycle from

65 �C to 56.6 �C. The last amplification step com-

prised 23 cycles of 94 �C for 30 s, 56 �C for 60 s and

72 �C for 60 s. The amplification products were

separated by electrophoresis on 6 % denaturing poly-

acrylamide gel and stained with 6 mM silver nitrate

plus 0.056 % formaldehyde silver nitrate for 30 min

(Echt et al. 1996). DNA bands in the range of

200–700 bp were scored for further analyses.

Genetic distance analysis

AFLP fingerprints were scored for calculating genetic

distances using Nei’s method (Nei 1973). NTSYSpc

software, version 2.10t (Rohlf 2001) was used for

cluster analyses according to the unweighted pair

group method with arithmetic mean (UPGMA).

Mol Breeding (2013) 31:815–827 817

123

Methylation-sensitive amplification

polymorphism (MSAP)

The DNA methylation status of parents and F1 hybrids

was compared using the MSAP (methylation-sensitive

amplification polymorphism) method of Xiong et al.

(1999). The technique is a modification of the AFLP

fingerprinting in which, next to the EcoRI restriction

enzyme, two more methylation-sensitive HpaII and

MspI isoschizomers were used. The pre-amplification

with one pair of primers was followed by selective

amplification with ten primer pairs (Suppl. Table 3).

Details of the pre-amplifi-cation, selective amplifica-

tion, gel electrophoresis and staining were the same as

the AFLP method described above. The MSAP

fingerprints of F1 hybrids were analyzed for altera-

tions in the banding patterns compared to those of the

parents according to patterns of additivity (Jin et al.

2008; Shaked et al. 2001).

Transposon display (TD)

We used transposon display (TD), a specific modifica-

tion of the AFLP fingerprinting, for detecting DNA

polymorphisms resulting from transpositions (Casa et al.

2000). The restriction enzyme used was EcoRI. Digested

fragments were ligated with EcoRI adapters as used for

the AFLP protocol. The pre-amplification primers were

EcoRI primer and transposable element specific primers

for mPing, Stowaway Os-1, Mashu and Dasheng

(Ngezahayo et al. 2009; Takagi et al. 2003; Kashkush

and Khasdan 2007). In the selective amplification step,

one selective nucleotide for EcoRI primers was used

with two selective nucleotides of transposable element-

specific primers (Suppl. Table 3). Details of pre-ampli-

fication, selective amplification, gel electrophoresis and

staining were the same as for the aforementioned AFLP

method. The transposon fragments identified from F1

hybrids were analyzed with those of their parents

according to the pattern of additivity.

Transposon methylation display (TMD)

A transposon methylation display (TMD) was used

for establishing methylated transposable element

sequences and their flanking sequence polymorphisms

(Takagi et al. 2003, Kashkush and Khasdan 2007). The

method is a modification of the TD in the sense that

restriction enzymes are now the HapII and MseI

isoschizomers along with HpaII/MseI adapters, and

primers for pre-amplification were HpaII/MspI prim-

ers, along with transposable element-specific primers,

the same as TD. The conditions for pre-amplification,

selective amplification, gel electrophoresis and stain-

ing were similar to MSAP and TD (Suppl. Table 3).

The polymorphic methylated fragments between par-

ents and their F1 hybrid were scored as described for

MSAP.

Results

Genetic distances between the cultivars

We used 198 polymorphic AFLP bands for establish-

ing Nei’s index values between 24 selected rice

varieties. Their genetic distance ranges from 1.34 for

Nipponbare—Indica var. RD 7, to 0.20 for Lon

Yung—Gaw Diaw (Fig. 1). The dendrogram based

on Nei’s index values revealed a clear separate cluster

of the japonica type Nipponbare and indica var. Nam

Roo (Suppl. Figure. 1). According to its phenotype,

Nam Roo is an upland landrace variety, previously

classified as a tropical japonica. In order to study

whether genetic distances of parents relate to the

extent of epigenetic change in the hybrids, we selected

six varieties as parents with genetic distances, increas-

ing at 0.1 intervals (Suppl. Table 4).

DNA methylation alteration in 25 families

DNA methylation changes between parental lines and

their hybrids were evaluated by MSAP fingerprinting.

We focused on the gel regions containing DNA

fragments of 150–350 base pairs, which displayed

19–33 bands. Bands with hemi-, complete or no CG

methylation in the offspring plants were compared with

their parents for establishing DNA methylation changes

(Fig. 2). Twenty-five hybrids, of which the parents’

genetic distances ranged from 0.35 to 1.04, showed

average methylation changes of 12.1–51.2 % (Table 1).

The highest rate of DNA methylation change was found

in the Nam Roo 9 IR 36 hybrid (51.2 %), whose

parental genetic distance is 0.87, whereas the lowest

change (12.1 %) was observed in the IR 36 9 Nippon-

bare and Nipponbare 9 IR 36 hybrids with a parental

genetic distance of 0.78. The parents with the highest

genetic distance, Nam Roo 9 KDML 105 and KDML

818 Mol Breeding (2013) 31:815–827

123

Kae T

om K

lang

Chai Nat

1RD 7

Hawm

Indok RD 17

RD 10

Nam R

ooJa

o Saw

uey Puang M

ahlai Look D

aeng R

uang

Yao

Goo Meu

ang L

uang

Khao T

ah H

aeng

Pah-ta

wng Daw Haw

m P

aeNiaw

Phr

Hawm

Mali

105

Leuan

g Pra

tew 12

3

Khao D

awk M

ali 10

5

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e Patum

Than

i 1 Suphan B

uri 1

IR 36

Lon Yung

Gaw D

iaw

Kae

Tom

Kla

ng**

**

Cha

i Nat

10.

526

****

RD

70.

393

0.63

4**

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awm

Indo

k0.

384

0.55

90.

604

****

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17

0.45

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578

0.49

0.58

****

RD

10

0.43

80.

456

0.59

70.

463

0.52

6**

**

Nam

Roo

0.92

81.

135

1.00

31.

119

0.86

40.

847

****

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uey

0.48

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575

0.56

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20.

736

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ng M

ahla

i0.

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0.62

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60.

518

0.58

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451

0.77

70.

212

****

Look

Dae

ng R

uang

Yao

0.58

90.

453

0.63

80.

596

0.53

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496

0.65

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0.47

3**

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Meu

ang

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g0.

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614

0.89

50.

784

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40.

702

****

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o T

ah H

aeng

0.65

0.48

10.

763

0.35

60.

723

0.43

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182

0.64

90.

489

0.56

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****

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aw0.

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0.50

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0.51

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113

0.73

50.

530.

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0.41

2**

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Haw

m P

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ali 1

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569

0.39

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362

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70.

327

****

Kha

o D

awk

Mal

i 105

0.50

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0.64

40.

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90.

566

0.55

10.

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han

Bur

i 10.

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10.

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904

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60.

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30.

619

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40.

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40.

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90.

511

0.51

80.

570.

573

0.39

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442

0.49

60.

520.

589

0.77

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419

0.47

4**

**

Lon

Yun

g0.

465

0.53

0.56

90.

446

0.6

0.48

30.

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0.47

0.40

50.

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0.61

80.

461

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40.

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10.

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0.47

6**

**

Gaw

Dia

w0.

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0.68

30.

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0.49

40.

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0.55

40.

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0.53

70.

413

0.61

80.

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0.32

40.

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0.50

20.

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40.

556

0.97

60.

573

0.41

70.

595

0.2

****

Fig. 1 Nei’s (1973) genetic

distances of 24 rice varieties

in this study. The heatmap

displays the level of genetic

distance between the

cultivars, ranging from 0.2

(blue) to 1.4 (red). The

relatively large distances

between the japonica type

rice (NamRoo and

Nipponbare), and the indicatype rice are obvious

Mol Breeding (2013) 31:815–827 819

123

105 9 Nam Roo (1.04), showed methylation changes

of 43.5 and 48.1 %, respectively, whereas the parental

combinations with the lowest genetic distance, Nippon-

bare 9 Nam Roo and Nam Roo 9 Nipponbare (0.35),

showed methylation changes of 38.9 % and 47.2 %.

The Pearson product-moment correlation coefficient of

the genetic distance (r) and DNA methylation is –0.16

(Suppl. Figure 2A) and is not significant (P = 0.46).

Clearly, the genetic distances of their parents cannot

explain the rate of DNA methylation alterations in the

hybrids. Suppl. Table 5 presents the effect of the gender

of the variety on the level of DNA methylation changes

in the hybrid. The range of the alteration is different

between the varieties, e.g., IR 36 plant as paternal plant

could give a broader range of 12.1–51.2 % and Look

Daeng Ruang Yao as maternal parent gave the narrow

range of 32.5–43.5 %. The broarder range of the

alteration indicates that either variety being mother or

father alone does not influence the methylation change,

but more likely the specific combination of the parents

used. As an example, the hybrid of IR 36 as mother

displays 12.1 % methylation change, if father is

Nipponbare, whereas the methylation change is

48.7 % if the father is Nam Roo.

Transposon display of the rice hybrids

For the assessment of transposable element activation,

several class I and II transposable elements were tested,

including the LTR retrotransposon Dasheng and the

mPing, Stowaway and Mashu transposons. The latter

two were selected for further analyses because of their

high genomic copy number, clear DNA fingerprints

and abundance of DNA polymorphisms.

The first part of this study involved the transposon

display (TD) analysis of 25 hybrids. For each hybrid,

the transposon fingerprints of four different plants

were compared. Clear changes in the DNA finger-

prints were considered reactivation of the transposon

and the bands with different position compared to their

parents were used for calculating reactivation fre-

quencies. For Stowaway Os-1, only two plants were

found with a clear alteration in the DNA banding

pattern. In the IR36 9 Suphan Buri 1 and IR36 9

Nipponbare hybrids reactivation was detected in only

one out of the four plants (Table 2; Fig. 3). In one IR

36 9 Nipponbare hybrid plant 15.4 % Stowaway Os-

1 transposition was observed, whereas for one IR

36 9 Suphan Buri 1 plant this was 39.3 %.

The TD of the Mashu transposon showed that this

transposon has a far more dramatic effect on the

genomes of the rice hybrids. The highest average

change of bands in the hybrid plants of cross Nam

Roo 9 Nipponbare amounted to 36.1 % (Table 2).

The TD fingerprints in only one plant of the IR

36 9 Suphan Buri 1 cross did not change at all.

Correlation between this transposon reactivation and

genetic distance was weak and not significant

(r = 0.38, with P = 8 %). The four plants of the

same cross gave similar DNA fingerprints in 11 of the

25 hybrids, whereas the 14 remaining crosses pro-

duced plants with clearly very different finger-

prints, suggesting that these changes were stochastic

Fig. 2 a DNA methylation changes in the MSAP pattern of

IR36 9 SPR1 family. M = Marker; H = HpaII; M = MspI.

F1.1, F1.2, … are the respective individual F1 plants. The whitearrowheads indicate sites of demethylation change in the

progeny, whereas the black arrowheads show the methylation

change

820 Mol Breeding (2013) 31:815–827

123

(Table 2). Reactivation of Mashu was not correlated

with Stowaway Os-1 reactivation, except for one plant of

the IR 36 9 Suphan Buri1 hybrids in which similar

levels of reactivation were observed in both Stowaway

Os-1 and Mashu, measuring 39.3 and 40.0 %

respectively.

Transposon methylation display (TMD)

We also performed a transposon methylation display

(TMD) for Stowaway Os-1 and Mashu, asking the

question whether transposon methylation affects their

reactivation. Methylation in Stowaway Os-1 did not

change except in the IR 36 9 Suphanburi 1 hybrid that

showed methylation changes of 11.1 % (Table 2). In the

IR 36 9 Nipponbare hybrid, the transposon reactiva-

tion did not coincide with DNA methylation change. For

the flanking DNA sequence of Mashu, we observed

methylation alterations varying from 2.9 to 75.6 %,

according to additivity inheritance (Table 2; Fig. 4).

In hybrids of some crosses (e.g., IR 36 9 Look

Daeng Ruang Yao, Look Daeng Ruang Yao 9 IR 36

and IR 36 9 Nipponbare) similar TMD patterns

between the four plants were observed, while in other

crosses (e.g., Nam Roo 9 IR 36) Mashu methylation

changes varied dramatically between the plants, i.e.,

13.6–84.6 %. The hybrid plants obtained from the

Khao Dawk Mali 105 9 Nipponbare cross do not only

have the highest methylation change (75.6 %) but also

displayed a higher rate of Mashu mobilization (35.7 %).

Table 1 Overview of

crosses between rice

cultivars, their genetic

distances and methylation

changes, with average

values and ranges

The bracketed numbers

display the range of

percentage of methylation

change. NA not available,

SPR1 Suphan Buri 1,

KDML 105 Kwan Dawk

Mali 105

* The single numbers are

the average percentage of

methylation change

Cross Genetic distance Methylation change (%)*

Nipponbare 9 Nam Roo 0.35 38.9

Nam Roo 9 Nipponbare 0.35 47.2

Look Daeng Ruang Yao 9 SPR 1 0.44 39.3

SPR 1 9 Look Dang Rung Yao 0.44 46.4 (42.9–50.0)

IR 36 9 SPR 1 0.47 40.3 (27.8–50.0)

SPR 1 9 IR 36 0.47 28.4 (27.3–31.8)

IR 36 9 Look Dang Rung Yao 0.51 47.8 (39.1–52.2)

Look Daeng Ruang Yao 9 IR 36 0.51 43.5

Look Daeng Ruang Yao 9 KDML 105 0.57 NA

KDML 105 9 Look Daeng Ruang Yao 0.57 NA

IR 36 9 KDML 105 0.59 40.0

KDML 105 9 IR 36 0.59 27.2 (17.4–34.8)

SPR 1 9 KDML 105 0.64 20.0

KDML 105 9 SPR 1 0.64 NA

Look Daeng Ruang Yao 9 Nam Roo 0.65 32.5 (30.0–35.0)

Nam Roo 9 Look Daeng Ruang Yao 0.65 38.7 (30.0–50.0)

Look Daeng Ruang Yao 9 Nipponbare 0.76 NA

Nipponbare 9 Look Daeng Ruang Yao 0.76 NA

IR 36 9 Nipponbare 0.78 12.1

Nipponbare 9 IR 36 0.78 12.1

Nipponbare 9 KDML 105 0.83 29.6 (27.8–33.3)

KDML 105 9 Nipponbare 0.83 29.2 (27.8–33.3)

IR 36 9 Nam Roo 0.87 48.7 (35.0–60.0)

Nam Roo 9 IR 36 0.87 51.2 (45.0–55.0)

Nam Roo 9 SPR1 0.90 22.6 (21.4–25.0)

SPR1 9 Nam Roo 0.90 23.2 (21.4–25.0)

SPR 1 9 Nipponbare 0.98 39.8 (36.4–45.4)

Nipponbare 9 SPR 1 0.98 31.8 (27.3–36.4)

Nam Roo 9 KDML 105 1.04 43.5 (41.7–44.4)

KDML 105 9 Nam Roo 1.04 48.1 (44.4–55.6)

Mol Breeding (2013) 31:815–827 821

123

In addition, the overall correlation of Mashu methylation

and its reactivation amount r = 0.40 (P \ 0.05, Suppl.

Figure 2B) and that of DNA methylation change and

genetic distance r = 0.43 (P \ 0.05).

Discussion

Our study clearly shows that intraspecific F1 hybrids

can undergo epigenetic changes while the selfed

progeny of their parents do not. We also demonstrated

that this epigenome alteration is not proportional to

genetic relationship; rather, it seems that specific

combinations of parents lead to such changes in the

progeny. Previous reports have described different

extents of DNA methylation change for both inter-and

intraspecific rice hybrids. Jin et al. (2008) observed

7.9 % DNA methylation changes in Oryza sati-

va 9 O. officinalis F1 and BC1 progeny, whereas

Xiong et al. (1999) reported a 1.5 % gain or loss of

Table 2 Percentages of transposon reactivation and methylation changes in rice hybrids

Cross Stowaway Os-1* Mashu*

Reactivation (%) Methylation

change (%)

Reactivation (%) Methylation

change (%)

Nipponbare 9 Nam Roo 0 0 33.3 32.1

Nam Roo 9 Nipponbare 0 0 36.1 32.1

Look Daeng Ruang Yao 9 Suphanburi 1 0 0 3.1 4.8 (2.9–5.7)

Suphanburi1 9 Look Dang Rung Yao 0 0 4.6 (3.1–6.1) 2.9

IR 36 9 Suphanburi1 9.8 (0–39.3) 2.8 (0–11.1) 10.9 (0–40.0) 20.2 (16.0–24.0)

Suphanburi1 9 IR 36 0 0 13.9 20.0

IR 36 9 Look Dang Rung Yao 0 0 6.7 10.5

Look Daeng Ruang Yao 9 IR 36 0 0 6.7 10.5

Look Daeng Ruang Yao 9 KDML 105 NA NA NA NA

KDML 105 9 Look Daeng Ruang Yao 0 0 10.5 52.9

IR 36 9 KDML 105 0 0 27.5 50.0

KDML 105 9 IR 36 0 0 22.5 (15.0–27.5) 51.2 (50–55)

Suphanburi1 9 KDML 105 NA NA NA NA

KDML 105 9 Suphanburi1 NA NA NA NA

Look Daeng Ruang Yao 9 Nam Roo 0 0 15.3 (12.0–18.5) 39.3 (35.7–42.9)

Nam Roo 9 Look Daeng Ruang Yao 0 0 10.8 (8.3–12.0) 8.3 (7.1–10.7)

Look Daeng Ruang Yao 9 Nipponbare NA NA NA NA

Nipponbare 9 Look Daeng Ruang Yao NA NA NA NA

IR 36 9 Nipponbare 3.85 (0–15.4) 0 4.5 (3.1–8.6) 46.4

Nipponbare 9 IR 36 0 0 4.8 (2.9–5.7) 46.4

Nipponbare 9 KDML105 0 0 21.9 (10.5–29.2) 32.1 (30.8–34.6)

KDML105 9 Nipponbare 0 0 28.8 (22.7–34.6) 75.6 (73.1–76.9)

IR 36 9 Nam Roo 0 0 16.7 (10.0–26.1) 17.4

Nam Roo 9 IR 36 0 0 25.7 (18.2–35.7) 32.4 (13.6–86.4)

Nam Roo 9 SPR1 0 0 15.1 (8.3–18.5) 15.1 (13.6–22.7)

SPR1 9 Nam Roo 0 0 19.6 (15.3–21.4) 19.3 (18.2–22.7)

SPR 1 9 Nipponbare 0 0 10.8 35.0

Nipponbare 9 SPR 1 0 0 10.8 35.0

Nam Roo 9 KDML 105 0 0 10.3 (7.1–13.3) 66.7 (67.3–73.0)

KDML 105 9 Nam Roo 0 0 15.6 66.2 (54.1–75.7)

* Single numbers are average values. Bracketed values refer to their range. NA not available, SPR1 Suphanburi1, KDML 105 Khaw

Dawk Mali 105

822 Mol Breeding (2013) 31:815–827

123

cytosine methylation in the Chinese Shanyou 63 elite

rice hybrid, compared to its parental lines (both O.

sativa). Their results suggest that crosses between

more distant parents (interspecific crosses) have a

more dramatic effect on DNA methylation than

intraspecific crosses (Moghaddam et al. 2010). In

our study we showed that intra-subspecies crosses

induce methylation changes in the range of

20.0–51.2 %, which is close to such changes in

inter-subspecies (japonica and indica) crosses

(12.1–47.2 %). In addition, the highest methylation

change of 51.2 % was found in the Nam Roo 9 IR 36

F1 whose parental genetic distance was 0.87, while the

parents Nam Roo and KDML 105 with the highest

genetic distance of 1.04 produced hybrids with 48.1 %

change (Table 1). The results, although still small in

number, favour the hypothesis that genetic difference

between parents in rice crosses is not the major

determinant of DNA methylation changes.

Additional information on the complex relationship

of genetic distance and DNA methylation alterations

came from a study of a Zizania latifolia introgression

to O. sativa, in which dramatic changes were detected

in introgression lines of rice with\0.1 % of the wild

rice DNA (Dong et al. 2006). The authors hypothe-

sized that the induction of DNA methylation changes

is the consequence of disturbances of chromatin states

caused by a combination of alien chromatin insertion,

introduction of exogenous trans-acting methylation-

modifying factors and/or genomic rearrangements.

Therefore, the induction factors for epigenome

changes are more likely to depend on specific com-

binations of parental lines, maternal or paternal effects

or interplay of cytoplasmic factors rather than the

overall level of DNA sequence divergence.

Fig. 3 Transposon display of Stowaway Os-1. Lanes 1.1–1.4

are the F1 s of Suphan Buri 1 9 IR 36; lanes 2.1–2.4 the F1 s of

IR36 9 SPR1. Most F1 s of both families showed no reactiva-

tion of Stowaway Os-1, except one hybrid of IR 36 9 SPR 1,

F12.1. The arrows point at examples where transposons have

mobilized (black = new band; white = missing band). The

weak contrast in the second lane of the parental IR-36 has been

improved digitally

Fig. 4 Transposon methylation display of Mashu in a hybrid of

Nipponbare 9 Suphan Buri 1 (nos. 1.1–1.3) and Suphan Buri

1 9 Nipponbare (nos. 2.1–2.4). The black arrowheads point at

cases of disappearance of parental Nipponbare bands in the F1 s,

indicating that the sequences around Mashu became methylated

Mol Breeding (2013) 31:815–827 823

123

The level of DNA methylation alteration

and reactivation of the transposons

DNA methylation makes up part of the plant’s defence

system by silencing transposable elements in the

genome and DNA methylation changes may cause

transposon reactivation. Here we examined two

members of the miniature inverted repeat transposable

elements (MITEs): Stowaway Os-1 and Mashu.

MITEs are non-autonomous transposons, which are

highly polymorphic and prevalent, especially in

single-copy regions in the genome of higher plants

(Feschotte et al. 2003; Jiang et al. 2004; Nagano et al.

2002). Stowaway Os-1 elements display high

sequence divergence and are thought to be relatively

old, inactive elements. They contrast with Mashu

transposons whose uniform sequences and high var-

iability of insertion sites between AA_genome vari-

eties suggest a younger and more active element.

(Takagi et al. 2003). In our TD study we demonstrate

that Stowaway Os-1, in spite of its silent nature can—

in specific cases—also be reactivated in intraspecific

hybrids.

Our TMD analysis on the flanking sequences of

Stowaway Os-1 reveals epigenome effects in only one

of the hybrid plants of the IR 36 9 Suphanburi 1

cross. This plant has a methylation change of 44.4 %,

while one of the other plants from the same cross with

an overall 50 % methylation change did not show

Stowaway Os-1 methylation change at all. So, the

TMD of Stowaway Os-1 and MSAP data clearly

indicate that genomic and Stowaway Os-1 DNA

methylation changes are here not related. Further-

more, our data also show that the IR 36 9 Nipponbare

hybrid with 15.4 % reactivation of transposons expe-

riences no concurrent changes in its DNA methylation,

suggesting that regulation of transposon activation is

orchestrated by one of a combination of different

mechanisms, e.g., CG and non-CG methylation,

histone modification, small RNAs, chromatin struc-

ture and gap repair (Rigal and Mathieu 2011; Wang

et al. 2009; Takagi et al. 2003). We also speculate that

the low rate of Stowaway Os-1 reactivation (2 % of all

hybrids) is likely a stochastic phenomenon, rather than

being caused by the hybridization. To prove this

hypothesis, a far larger number of different rice hybrid

families needs to be analyzed.

TD and TMD of the younger and possibly more

active MITE Mashu transposon showed a very

different picture. The TD analysis for reactivation of

the transposon was found in all hybrids, except one for

IR 36 9 Suphanburi 1. The TMD fingerprints revealed

methylation changes in DNA flanking Mashu transpo-

sons in all hybrid plants. The rate of Mashu was

significantly correlated (P = 0.047) with methylation

changes, as well as with methylation changes and

genetic distances of their parents (P = 0.030). Mashu

reactivation versus genetic distance was not clearly

correlated (P = 0.053) and not significant. Even

though Stowaway Os-1 and Mashu reactivation differ

in their correlation to genetic distance, our observa-

tions suggest that epigenetic regulation of transposon

methylation and reactivation are not directly deter-

mined by genetic distances and/or their interaction, but

are more likely under the control of unknown factors.

Consequently the genomic shock leading to epigenetic

change might be a combination of hybridization and

interaction of parental factors. Also, the differences in

response to transposons between the hybrid families

might be explained in terms of differences in trans-

posable element expression conditions in the evolu-

tionary and environmental history of each host

(Grandbastien et al. 2005).

Genome stress by hybridization can induce trans-

poson activation, but not all transposons react simi-

larly, as was shown by the differences between

Stowaway Os-1 and Mashu. This observation suggests

that hybridization is not a general causal factor

explaining mobilization of all transposon families.

Wang et al. (2009) revealed that transposon reactiva-

tion differs between transposons for different stresses.

For instance, incompatible cross-pollination in rice

activated mPing, Osr7, Tos17 and Osr23, but not Osr2,

Osr3, Osr35, Osr42, Os19, Ping and Pong. This

specificity probably depends on how the small motif

in the regulatory region of the transposon responds to

stress signal molecules and how such a motif can be

part of the specific inducible enhancers which are

present and accumulate in the regulatory region

(Grandbastien 1998; McDonald et al. 1997). Stow-

away Os-1 and Mashu are MITEs whose transposi-

tions depend on transposases of related autonomous

elements. The former belongs to the Stowaway

superfamily, whereas Mashu is a Tourist-like and

belongs to the PIF-Harbinger superfamily member

(Zhang et al. 2004), and so both have their own

evolutionary history. The regulatory motifs may

therefore differ in their response to hybridization

824 Mol Breeding (2013) 31:815–827

123

stress. In addition, Osmars, whose transposase are

likely capable of Stowaway transpositions, occur to a

great extent in deleted forms (Yang et al. 2009) and so

may explain the low reactivation of this transposon in

an altered epigenetic landscape.

Kawakami et al. (2011), who analyzed natural

hybrids of wild sunflower (Helianthus spp.), suggested

that element depression can be an alternative expla-

nation for reactivation of transposable elements by

hybridization. The authors argued that control of

reactivation and expansion acts as a posttranscrip-

tional mechanism of repression. Cross-hybridization

can trigger epigenome changes and reactivation of

transposons to different extents, depending on the

specific responses of regulatory mechanisms, of the

transposon to stress signals, host-specific expression

conditions in the evolutionary and environmental

history and other unknown factors (Grandbastien

et al. 2005), as well as depression by posttranscrip-

tional mechanisms. This complex amalgam of factors

may explain why mobilization and methylation of

transposons following inter- and intraspecific hybrid-

ization does not clearly relate to genetic distances and

relatedness of parental lines.

Stochastic reactivation and methylation

of Stowaway Os-1 and Mashu

A specific aspect of epigenetic variation is the

occurrence of stochastic accumulation of epigenetic

alterations. In our rice experiments we were able to

establish such changes in plants of the same family in

which methylation changes at specific loci are random.

Stochastic epigenetic changes were most frequently

observed in Mashu, typically in families in which

methylation changes range widely, e.g. in the Nam

Roo 9 IR 36 offspring (cf. Table 2). Comparing

Mashu and Stowaway Os-1 reactivation shows that

Stowaway Os-1 has a lower rate (2/25) of transposon

reactivation among hybrids in the same family than

Mashu, which showed more random changes in 14 of

the 25 plant families. In addition, Stowaway-Os1

scored much lower than Mashu in the flanking DNA

methylation. Apparently these stochastic changes in

transposon reactivation and methylation are both

transposon- and family-specific.

Stochastic epigenetic changes have been studied in

both plants and animals (Wang et al. 2004; Singer

et al. 2011; Xie et al. 2011; Reinders et al. 2009) and

are known to occur at different loci, cell types and

individuals (Reiss et al. 2010; Reiss and Mager 2007).

Pfeifer et al. (1990) stated that each locus had a certain

efficiency of methylation maintenance and mainte-

nance failure, and so could differ in either response to

transposon type, plant hybrid family or offspring

individual. What causes this stochastic variation of

DNA methylation is still not known. Szyf (2011)

postulated that stochastic epigenetic changes might be

caused by an array of some agents/factors that do not

target main factors controlling methylation or activa-

tion of transposons, but can trigger epigenetic

changes. Another explanation underlying stochastic

epigenetic changes in transposons was suggested by

Reiss and Mager (2007), who postulated a molecular

conflict between cis- and trans-acting factors of

chromatin remodelling. In a cancer study in mouse,

the factors significantly related to DNA methylation

variability were aging and environment (Christensen

et al. 2009), which implies internal and external

effects. In our study, stochastic fragments were found

in the range of 0–84 % in the hybrid plants, all of

which were reared under the same greenhouse condi-

tions, suggesting that non-environmental factors play

a more prominent role in this variation. One can

speculate that if stochastic epigenetic changes play a

role in adaptation or generation of new phenotypes,

then they may play a significant role in long-term

processes of adaptation, selection and evolution. In

that respect, the young and dynamic transposon types

like the Mashu family may provide a high potential for

application in breeding programs.

We have demonstrated in our comparative study on

rice hybrids that intra-subspecific and intersubspecific

crosses between indica and japonica cultivars can

cause epigenetic changes, both specific and in sto-

chastic patterns to various extents. We have learned

that sequence divergence of the parents does not

explain such changes, but that DNA methylation

changes rather rely on the combination of the parents,

whereas transposon methylation and activation can

only be observed in specific genome parts in each

family. Such epigenetic alterations are potentially

interesting to geneticists and breeders as they may

provide more plasticity for organisms under stress

conditions. Support came from a study of Reinders

et al. (2009) on a population of recombinant inbred

lines of Arabidopsis, which were crossed to a

hypomethylated met1 parent. New phenotypes were

Mol Breeding (2013) 31:815–827 825

123

generated by randomly combining epi-alleles which

allowed the creation of new epigenetic phenotypes for

organisms with a narrow genetic base (Akimoto et al.

2007; Sakthivel et al. 2010). In our study, all families

were found to induce epigenetic changes to different

extents. However one can assume that part of these

changes can be beneficial for the individual, and so

may provide potential application in breeding pro-

grammes of rice.

Acknowledgments We thank our colleagues of the Rice

Department and Department of Agriculture, Ministry of

Agriculture and Cooperative, Thailand for providing the rice

seeds. This work was supported by grant no. MRG5080196 from

the Thailand Research Fund and by the Kasetsart University

Research and Development Institute, Bangkok, Thailand. The

authors also thank Dr. Erik Wijnker for extensive critical

remarks on the manuscript.

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