ORIGINAL PAPER

A novel T-DNA integration in rice involving twointerchromosomal translocations

Bharat Bhusan Majhi • Jasmine M. Shah •

Karuppannan Veluthambi

Received: 13 October 2013 / Revised: 10 December 2013 / Accepted: 14 January 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Key message A male sterile transgenic rice plant TC-19

harboured a novel T-DNA integration in chromosome 8

with two interchromosomal translocations of 6.55 kb

chromosome 3 and 29.8 kb chromosome 9 segments.

Abstract We report a complex Agrobacterium T-DNA

integration in rice (Oryza sativa) associated with two

interchromosomal translocations. The T-DNA-tagged rice

mutant TC-19, which harboured a single copy of the

T-DNA, displayed male sterile phenotype in the homozy-

gous condition. Analysis of the junctions between the

T-DNA ends and the rice genome by genome walking

showed that the right border is flanked by a chromosome 3

sequence and the left border is flanked by a chromosome 9

sequence. Upon further walking on chromosome 3, a

chromosome 3/chromosome 8 fusion was detected. Gen-

ome walking from the opposite end of the chromosome 8

break point revealed a chromosome 8/chromosome 9

fusion. Our findings revealed that the T-DNA, together

with a 6.55-kb region of chromosome 3 and a 29.8-kb

region of chromosome 9, was translocated to chromosome

8. Southern blot analysis of the homozygous TC-19 mutant

revealed that the native sequences of chromosome 3 and 9

were restored but the disruption of chromosome 8 in the

first intron of the gene Os08g0152500 was not restored.

The integration of the complex T-DNA in chromosome 8

caused male sterility.

Keywords Agrobacterium � Genome walking �Interchromosomal translocation � Male sterility �Rice � T-DNA integration

Abbreviations

T-DNA Transferred DNA

MS Murashige and Skoog

hph Hygromycin phosphotransferase gene

cht42 Chitinase gene of Trichoderma virens

TT Homozygous transgenic event

Tt Hemizygous transgenic event

LB T-DNA left border

RB T-DNA right border

BLAST Basic local alignment search tool

GSP Gene-specific primer

AP Adaptor primer

IRGSP International rice genome sequencing project

NCBI National Center for Biotechnology Information

Introduction

Agrobacterium T-DNA integration in plants is a very

complex process, which is still not fully understood. Many

proteins synthesized in both Agrobacterium and plant cell

contribute to successful T-DNA integration (Gelvin 2010).

Precise integration of the T-DNA, delimited by right border

(RB) and left border (LB), is an advantage of Agrobacte-

rium-mediated transformation (Gelvin 2003; Tinland

Communicated by H. Jones.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-014-1572-0) contains supplementarymaterial, which is available to authorized users.

B. B. Majhi � J. M. Shah � K. Veluthambi (&)

Department of Plant Biotechnology, School of Biotechnology,

Madurai Kamaraj University, Madurai 625 021,

Tamil Nadu, India

e-mail: kveluthambi@rediffmail.com

123

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DOI 10.1007/s00299-014-1572-0

1996). Nevertheless, T-DNA integration is often associated

with localized disturbances of the target site. Small dele-

tions, duplications and filler sequences of unknown origin

are often observed at T-DNA integration sites in both

dicots and monocots (Gheysen et al. 1987; Kim et al. 2003;

Mayerhofer et al. 1991; Ohba et al. 1995; Sha et al. 2004;

Windels et al. 2003; Zambryski et al. 1982). These

observations led to the proposal that T-DNA integration is

mediated through illegitimate recombination (Gheysen

et al. 1991; Mayerhofer et al. 1991). Two models have

been proposed for T-DNA integration into the plant gen-

ome. The first model, referred to as the single-stranded gap

repair (SSGR) model, envisages the requirement of a pre-

existing nick or gap on one DNA strand in the plant gen-

ome (Gheysen et al. 1991; Mayerhofer et al. 1991). The

second is the double-stranded break and repair (DSBR)

model (De Neve et al. 1997; Gheysen et al. 1991; Maye-

rhofer et al. 1991; Salomon and Puchta 1998; Tzfira et al.

2003).

In addition to the commonly observed minor localized

rearrangements, major chromosomal rearrangements were

also associated with T-DNA integration. Many T-DNA

insertion lines of Arabidopsis thaliana have been identified

to carry such complex chromosomal rearrangements. In

many cases, the chromosomal rearrangement was found to

be due to reciprocal translocation between two non-

homologous chromosomes post T-DNA integration (Clark

and Krysan 2010; Curtis et al. 2009; Forsbach et al. 2003;

Guan et al. 2003; Lafleuriel et al. 2004; Nacry et al. 1998;

Ray et al. 1997; Yuen et al. 2005). In two cases, large

paracentric inversions were observed at the T-DNA inser-

tion sites (Laufs et al. 1999; Nacry et al. 1998). Two

T-DNA insertion lines were shown to carry interchromo-

somal translocations as well as duplications of the trans-

located chromosomal fragments (Tax and Vernon 2001). In

both the lines, translocated chromosomal fragments were

linked to the LB. Clark and Krysan (2010) reported two

transgenic Arabidopsis lines that displayed ‘T-DNA bor-

ders separate’ phenomenon, where the T-DNA-flanking

sequences at RB and LB were genetically unlinked.

Though interchromosomal translocations could have con-

tributed to such events, the chromosomal rearrangements

were not fully characterized in those lines. The frequency

of chromosomal rearrangements in collections of the

T-DNA-tagged Arabidopsis lines was estimated to be

around 19–20 % (Castle et al. 1993; Clark and Krysan

2010). Based on the above observations, Clark and Krysan

(2010) proposed that chromosomal translocations are a

common phenomenon in A. thaliana T-DNA insertion

lines. Chromosomal translocation associated with T-DNA

integration has not been reported in rice. In one rare report,

transgenic rice plants which were generated by calcium

phosphate method were found to have chromosomal

translocations, duplications and deletions (Takano et al.

1997).

Most of the above reports have analysed the T-DNA-

associated chromosomal translocations in Arabidopsis by

genetic mapping using molecular markers. In a majority of

the studies, the transgenic plants were propagated for

several generations prior to the analysis (Clark and Krysan

2010; Guan et al. 2003; Tax and Vernon 2001; Yuen et al.

2005). Therefore, it is difficult to determine whether the

chromosomal rearrangement happened during the process

of T-DNA integration or in subsequent generations. The

break points of the translocated fragments at the nucleotide

level were not reported. The exact sizes of the translocated

chromosomal fragments were not reported.

In this study, we report the determination of transloca-

tion breakpoints at the nucleotide sequence level in a

transgenic rice line TC-19 in which a novel T-DNA inte-

gration was associated with two interchromosomal trans-

locations. TC-19 harboured a single copy of the T-DNA

and displayed male sterile phenotype in the homozygous

condition. Analysis of genomic sequences flanking the

T-DNA showed that the T-DNA is flanked by a chromo-

some 3 sequence at the RB and a chromosome 9 sequence

at the LB. Further detailed characterization revealed that

the T-DNA, together with a 6.55-kb region of chromosome

3 and a 29.8-kb region of chromosome 9, was translocated

to a locus in chromosome 8. The translocated chromosomal

parts were found to be restored by duplications in the

native chromosomes 3 and 9. This is the first example of

complete characterization of a complex T-DNA-associated

rearrangement in rice which involved three chromosomes,

where two chromosomal fragments were translocated along

with the T-DNA into a third chromosome. Insertion of the

T-DNA with the flanking translocated fragments in chro-

mosome 8 disrupted an essential gene which resulted in

male sterility in the homozygous condition.

Materials and methods

Plant materials and growth conditions

Untransformed rice (Oryza sativa L. subsp. indica cv Pusa

Basmati1) and transgenic TC-19 (Shah et al. 2008) T2 seeds

(from a selfed, T1 hemizygous plant) were dehusked, surface

sterilized and germinated axenically on half-strength Mu-

rashige and Skoog (MS) medium (Murashige and Skoog

1962) without any antibiotic for 2 days in dark. To identify

transgenic T2 progeny, the germinated rice seedlings were

transferred to half-strength MS medium with 50 mg hygro-

mycin/L and placed under 16 h light (100 lE m-2 s-1)/8 h

dark photoperiod for 2 weeks, and then transferred to pots

and grown to maturity in a greenhouse.

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Light microscopy

Morphological features of control and TC-19 mutant

flowers were observed under a Nikon (C-DSS230) micro-

scope (Tokyo, Japan).

Southern hybridization analysis

Genomic DNA was extracted from fresh leaves using

cetyltrimethylammonium bromide (Rogers and Bendich

1988) and estimated in a fluorometer (Dyna Quant 200,

Hoefer Inc., San Francisco, USA) using the Hoechst dye

33258. Plant DNA (2.5 lg) from control and TC-19 plants

was digested with restriction enzymes and electrophoresed

in a 0.8 % agarose gel in 1X Tris–borate-EDTA buffer. After

depurination, denaturation and neutralization, DNA was

transferred to the Zeta-probe membrane (Biorad, Hercules,

USA). The probe DNA was labelled with [a-32P]dCTP

(Board of Radiation and Isotope Technology, Mumbai,

India) using MegaprimeTM labelling kit (GE Healthcare UK

Limited, Little Chalfont, UK). Hybridization and washes

were done as described earlier (Ramanathan and Veluthambi

1995). Densitometry analyses of the bands in autoradio-

grams were done using the AlphaEaseTM software (version

5.5, Alpha Innotech Corporation, San Leandro, USA) and

integrated density values (IDV) of bands were determined.

Genome walking

Genomic DNA from the TC-19 homozygous plant was

used for genome walking (Siebert et al. 1995) using the

GenomewalkerTM Universal kit (Clonetech, Mountain

View, USA).

PCR primers and amplification conditions

The gene-specific primers (GSP1 and GSP2) for genome

walking and primers for genomic DNA amplification were

designed using the PERLPRIMER software (http://perlpri

mer.sourceforge.net). The sequences of the primers are

listed in Table 1. Primary and secondary amplifications for

genome walking were carried out under conditions rec-

ommended by the manufacturer (Clonetech, Mountain

View, USA). PCR for genomic DNA was carried out in a

volume of 25 ll in the presence of 100 ng genomic DNA,

1.5 mM MgCl2, 200 lM of each dNTP, 0.2 lM of each

primer and 1 unit of Taq DNA polymerase.

DNA sequencing and data analysis

PCR amplified fragments were gel extracted using the

QIAquick gel extraction kit (Qiagen, Hilden, Germany),

and were either sequenced directly using the respective

gene-specific primer and the adaptor primer 2 (AP2) or

cloned into the pGEM-T vector (Promega, Madison, USA)

and sequenced using the universal M13 forward and

reverse primers. The locations of the T-DNA flanking

sequences on the rice genome were determined through

BLASTn homology searches (Altschul et al. 1990) using

the NCBI database of Oryza sativa L. subsp. japonica cv

Nipponbare (www.ncbi.nlm.nih.gov/BLAST).

Results

TC-19, a transgenic rice line transformed with the Trich-

oderma virens endochitinase gene (cht42), harbours a sin-

gle copy of the T-DNA (Shah et al. 2008). Upon selfing,

the segregation ratio of hygromycin-resistant (Hygr) and -

sensitive (Hygs) progenies was 3:1. While the hemizygous

and null T1 plants were fertile, none of the homozygous T1

plants set seeds (Shah et al. 2008).

T-DNA insertion in TC-19 caused male sterility

Fifteen Hygr T2 plants of a hemizygous TC-19 T1 plant

were grown to maturity in a greenhouse. As a control,

Table 1 Sequences of primers used for characterizing the TC-19

T-DNA insertion line

Name Sequence

h-GSP1 50-CAGGCTCTCGATGAGCTGATGCTTTGGG-30

h-GSP2 50-TCTATCAGAGCTTGGTTGACGGCAATTTCG-30

c-GSP1 50-GGGAAGCCTCAGCCGATAAGAAAGGAACTG-30

c-GSP2 50-CAGAACCTGCTGAGCTACCCCAACTCCAAG-30

AP1 50-GTAATACGACTCACTATAGGGC-30

AP2 50-ACTATAGGGCACGCGTGGT-30

C3F 50-ACTTAGGTGACAACTGAAATACAAGGACC-30

C3R 50-GTTGTTGATGTTTAACCATGCTTTGC-30

C9F 50-TGACATTTGCGCCATTTGTTACTACAATCC-30

C9R 50-ACCACAGGGCCATGTTCATCCTTTAACAC-30

chr3-GSP1 50-GGATGAGGCGGACGATGACAAGGCTGATG-30

chr3-GSP2 50-CGTTGCTGAGCAGACCAAGGACAAGGGTG-30

chr8-GSP1 50-CTGTGGCCATCTGAGAGTGGATGCCTTCTGC-30

chr8-GSP2 50-TTACGGCTGCTTCCTCCGTCGGTTGTGCCC-30

C8F 50-TCAGAATATTTATTAGCTCTGGACTTG-30

C8R 50-TCGATTTCTCCTCCTCCTCCTCTCTCTCG-30

RBF 50-GCTGTGTAGAAGTACTCGCCGATAGTG-30

C3R1 50-GCATTGCAGCCATTCATCTCATCAAATCC-30

LBF 50-GGGGATCCTCTAGAGTCGACCTGCAGG-30

C9R1 50-TTTCATCATTCTGGCCTCAAGATCATCTG-30

C3F1 50-TTGCTGAGCAGACCAAGGACAAGGG-30

C8R1 50-GATTTCTCCTCCTCCTCCTCTCTCTC-30

C8F1 50-AGAATATTTATTAGCTCTGGACTTG-30

C9R2 50-CTTTTATAAAAGTATTTTTCAAGAA-30

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non-transformed plants were grown simultaneously. DNA

blot analysis was done to identify the homozygous (TT)

plants (Sridevi et al. 2006). Plant DNA was digested with

EcoRI and Southern blot analysis was performed with both

hph and cht42 probes (Fig. 1). The expected junction

fragments of 2.9 kb (hph probe) and 6.6 kb (cht42 probe)

were detected in all fifteen T2 plants (Fig. 2a, b). The

hybridization signals with both hph and cht42 probes were

twice as intense for the T2 plants 4, 8, 9 and 10 than for the

other plants. Densitometry was done and integrated density

values (IDV) were determined to quantitate the intensity of

bands that hybridized to the hph probe. The IDV for the

plants 4, 8, 9 and 10 were 51,282, 50,544, 49,096 and

50,616, respectively. For the plants 1, 2, 3, 5, 6, 7, 11, 12,

13, 14 and 15 the IDV were 26,460, 26,180, 24,975,

25,200, 21,645, 22,972, 36,620, 23,970, 28,710, 29,860 and

33,264, respectively. Therefore, the T2 plants 4, 8, 9 and 10

were predicted to be homozygous (TT) for T-DNA inte-

gration. Other T2 plants were inferred as hemizygous. The

IDV of the homozygous plants were twice higher than

those of the hemizygous plants.

All homozygous (TT) plants displayed a 3-week delay

in flowering in comparison to hemizygous (Tt) and

untransformed control (C) plants (Fig. 3a). The florets were

found to be cleistogamous in the homozygous (TT) plants

(Fig. 3b). The anthers of the homozygous (TT) plants were

half the size of the hemizygous (Tt) and control (C) plants

(Fig. 3c) and did not contain mature pollen grains. All

homozygous (TT) plants were male sterile. However, the

carpels of the homozygous (TT) plants were phenotypi-

cally normal (Fig. 3d). Upon crossing the female

homozygous plants (TC-19) with a male fertile homozy-

gous transgenic rice plant RM1, which harboured the bar

(for phosphinothricin resistance) and rice chitinase (chi11)

Fig. 1 The T-DNA region of the binary vector pCAM1300-35S-

cht42-4C. It harbours the Trichoderma virens chitinase gene (cht42)

driven by the Cauliflower mosaic virus (CaMV) 35S promoter in

pCAMBIA1300. It has the hph gene as the plant selectable marker. A

left border (LB) junction fragment of [2.1 kb (the distance between

EcoRI and LB) released upon EcoRI digestion hybridizes to the hph

probe. The right border (RB) junction fragment of [2.7 kb (the

distance between EcoRI and RB) released upon EcoRI digestion

hybridizes to the cht42 probe. The regions used as probes (hph, cht42)

are marked in bold lines. The junction fragments, which hybridize to

the probes, are marked by broken lines with arrows. The internal

T-DNA fragments which are released upon XhoI digestion (1.1, 1.8

and 1.3 kb) are marked by dotted lines. P35S, Cauliflower mosaic

virus 35S promoter; 35S 30, Cauliflower mosaic virus polyadenylation

signal; hph, hygromycin phosphotransferase gene; cht42, Tricho-

derma virens chitinase gene. Recognition sites for restriction endo-

nucleases BamHI (B), HindIII (H), EcoRI (E), PstI (P), SacI (S), SalI

(Sa), SphI (Sp), SmaI (Sm), XbaI (Xb) and XhoI (X) are indicated.

Positions of primers used for genome walking (h-GSP1, h-GSP2,

c-GSP1 and c-GSP2) are marked in filled arrows. Scale (1.0 kb) is

marked

Fig. 2 Zygosity analysis of the T2 plants of the TC-19 transgenic rice

line by Southern blotting with (a) hph and (b) cht42 probes. a and

b Plant DNA (2.5 lg) from 15 T2 plants was digested with EcoRI and

loaded in each lane. The blot was probed with [a-32P]dCTP-labelled

probes. Lane C EcoRI-digested DNA from the untransformed control

plant; Lane U undigested DNA from TC-19-1. The numbers on the

top represent T2 plant numbers of the line TC-19. A portion of

the ethidium bromide-stained gel in 23-to 9-kb region is shown in the

lower panels to reflect equal loading of plant DNA in all lanes. The

sizes of the k/HindIII fragments are positioned on the left

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genes, seed setting was normal in the F1 hybrid plants (TC-

19 X RM1). The hybrid plants were confirmed by Southern

blot analysis of the respective junction fragments (results

not shown). Therefore, TC-19 homozygous (TT) plants

were inferred as male sterile and female fertile. Thus, a

gene important for male fertility is disrupted in TC-19 upon

T-DNA insertion.

Characterization of the T-DNA insertion site in the rice

genome in TC-19

To identify the gene disrupted in the TC-19 mutant, the

flanking sequences at the right and left T-DNA borders

were cloned by genome walking (Siebert et al. 1995). Four

genome walking libraries of TC-19 were constructed by

DraI, EcoRV, PvuII and StuI digestions. To isolate the

T-DNA right border (RB) and plant genome junction,

cht42 gene-specific primers (c-GSP1 and c-GSP2) were

used (Fig. 1). Genome walking at the RB yielded a major

amplified product of 1,771 bp in the EcoRV library, which

was cloned and sequenced. The sequence contained 546 bp

of the T-DNA right border region and 1,225 bp of the

flanking rice genomic sequence. Nucleotide BLAST ana-

lysis with the NCBI Oryza sativa database revealed that the

RB-flanking sequence showed complete homology to the

rice genome sequence on chromosome 3 (nucleotide

position 35808433 to 35809658 of RefSeq accession

NC_008396.2). The insertion site of the T-DNA was at the

nucleotide position 35809658 on chromosome 3. The

breakpoint was located in the first intron of Os03g0832300,

which encodes a 127-amino acid conserved hypothetical

protein. A 6-bp deletion of the T-DNA RB end and

insertion of a 7-bp filler sequence 50TGGATGA at the

junction were observed. A 3-bp microhomology was

observed between the T-DNA RB end with the rice

sequence flanking the breakpoint.

To map the LB insertion site, gene-specific primers from

the hph gene (h-GSP1 and h-GSP2) were used (Fig. 1). A

1,046-bp PCR product obtained in the DraI library was

cloned and sequenced. It contained 450 bp of the T-DNA

LB region and 596 bp of rice genomic sequence, which

displayed complete homology to the rice genome sequence

on chromosome 9 (nucleotide position 20295498 to

20296094 of RefSeq accession NC_008402.2). The LB

region in the T-DNA lost a stretch of 72-bp and a 6-bp filler

sequence 50TCGGGA was inserted at the junction. The

T-DNA insertion was at the nucleotide position 20295498

on chromosome 9. The insertion was located 1,678 bp

upstream of the start codon of Os09g0507100, which

encodes a putative SQUAMOSA promoter binding

Fig. 3 Morphological

characteristics of the T-DNA

insertion mutant TC-19.

a Comparison of the control (C),

hemizygous (Tt) and

homozygous (TT) T2 plants of

TC-19. The numbers at the

bottom represent T2 plant

numbers of TC-19. Comparison

of the appearances of the mature

florets (b), dissected anthers

(c) and dissected carpels (d) of

control (C) and homozygous

(TT) plants of TC-19

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protein-like (SBP) transcription factor. Unexpectedly, the

T-DNA in TC-19 was flanked by a chromosome 3

sequence at the RB and a chromosome 9 sequence at the

LB.

The TC-19 rice line carries a single intact copy

of the T-DNA

The results presented above raised the doubt as to whether

the line TC-19 harboured two truncated T-DNA integra-

tions, one at chromosome 3 and another at chromosome 9.

To check the above possibility, Southern blot analysis was

performed with a mixture of three T-DNA-specific probes

of 1.8, 1.3 and 1.1 kb, obtained by digesting the binary

vector pCAMBIA1300-35S-cht42-4C with XhoI (Fig. 1).

The plant DNA was digested with XhoI. If a single com-

plete T-DNA was integrated, three bands of 1.8, 1.3 and

1.1 kb were expected to hybridize. On the other hand, if

TC-19 rice line harboured two truncated T-DNAs,

hybridization signals of additional junction fragments were

expected. DNA from two representative TC-19 T2 plants

displayed hybridization to the three internal T-DNA frag-

ments of 1.8, 1.3 and 1.1 kb (Fig. 4a). The results sug-

gested that a single copy of intact T-DNA is integrated in

TC-19. In a second experiment, the TC-19 plant DNA was

digested with SacII, which does not cut within the T-DNA.

The blot was probed with a mixture of three XhoI frag-

ments of the T-DNA. One hybridization signal of 19.5 kb

was observed in two TC-19 T2 plants (Fig. 4b). The results

confirmed that the TC-19 rice line harbours only a single

copy of intact T-DNA. The possibility of two independent

T-DNA integrations was ruled out. It was inferred that the

T-DNA integration in TC-19 is complex and interchro-

mosomal rearrangement such as translocation may have

occurred.

T-DNA integration in TC-19 is associated

with translocations of segments of chromosomes 3

and 9

The nature of chromosomal rearrangement proximal to the

RB at the T-DNA integration site was studied by Southern

blotting. The control (C), TC-19 hemizygous (Tt) and

homozygous (TT) plant DNA samples were digested with

EcoRI, and the blot was hybridized with the 1,117-bp

chromosome 3 segment (nucleotide position 35808512 to

35809629 of RefSeq accession NC_008396.2) present

immediately flanking the RB. Hybridization was seen to an

expected band of 4.9 kb in the control (C) plant DNA

(Fig. 5a, c) (two EcoRI sites are present in the T-DNA

integration site in rice chromosome 3 at nucleotide posi-

tions 35806271 and 35811206 of RefSeq accession

NC_008396.2). In the TC-19 hemizygous (Tt) plant DNA,

the chromosome 3 probe detected the 4.9-kb band expected

from the native chromosome 3 plus the expected T-DNA

RB-chromosome 3 junction fragment of 6.6 kb (Fig. 5a, c).

As expected, the size of the junction fragment was the

same as the one which hybridized to the cht42 probe

Fig. 4 Southern blot analyses to check the presence of a single intact

T-DNA in TC-19 using a mixture of three T-DNA-specific probes

(1.8-kb, 1.3-kb and 1.1-kb XhoI fragments). a Analysis of XhoI-

digested plant DNA. b Analysis of SacII-digested plant DNA. a and

b Plant DNA (2.5 lg) from a control plant (C) and two TC-19

homozygous (TT) plants (TC-19-4 and TC-19-8) digested with XhoI

or SacII was loaded in each lane. The blot was probed with a mixture

of three [a-32P]dCTP-labelled T-DNA fragments (1.8-kb, 1.3-kb and

1.1-kb XhoI fragments). Lane BI 50 pg of XhoI-digested pCAM1300-

35S-cht42-4C (a) or 50 pg of SacII-digested pCAM1300-35S-cht42-

4C (b). The sizes of the k/HindIII fragments are positioned on the left

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(Fig. 2b). The homozygous (TT) plant DNA was expected

to display hybridization only to the 6.6-kb junction frag-

ment. However, homozygous (TT) plant DNA displayed

hybridization to the 4.9-kb band corresponding to the

native chromosome 3 and to the 6.6-kb junction fragment

(Fig. 5a, c). As expected, the hybridization signal for the

junction fragment (6.6 kb) was twice as intense in the

homozygous (TT) plant DNA (IDV 41,600) than in that of

the hemizygous (Tt) plant (IDV 20,616) (Fig. 5a). From

these results, we inferred that the predicted chromosome 3

sequence did form a junction to the T-DNA RB, but the

native chromosome 3 sequence remained intact. Disruption

of native chromosome 3 locus by T-DNA insertion would

have reduced the intensity of the 4.9-kb band by half in the

hemizygous (Tt) plant DNA in comparison to the control

(C) plant DNA, and it should be absent in the homozygous

(TT) plant DNA. Unexpectedly, the native chromosome 3

locus appeared intact in both hemizygous (Tt) and homo-

zygous (TT) TC-19 plants. These findings suggested that a

segment of chromosome 3, which formed a junction with

the T-DNA in TC-19, is translocated to some other part of

the genome.

The chromosome 9 and T-DNA LB junction was ana-

lysed by hybridization with the 526-bp chromosome 9

fragment (nucleotide position 20295525 to 20296051 of

RefSeq accession NC_008402.2) present next to the LB in

TC-19. EcoRI-digested control (C), TC-19 hemizygous

(Tt) and homozygous (TT) plant DNA samples were used

for Southern blotting. As expected, hybridization to a sin-

gle band of 4.5 kb in control (C) plant DNA was observed

Fig. 5 Southern blot analyses to study the chromosomal rearrange-

ment associated with the T-DNA integration in TC-19. a Analysis

with the [a-32P]dCTP-labelled 1,117-bp chromosome 3 fragment

flanking the RB as probe. b Analysis with the [a-32P]dCTP-labelled

526-bp chromosome 9 fragment flanking the LB as probe. a and

b Plant DNA (5 lg) from control (C), TC-19 hemizygous (Tt) and

homozygous (TT) plants was digested with EcoRI and loaded in each

lane. Positions of the bands corresponding to the native chromosomes

(chr 3 or chr 9) and the junction fragments (T-DNA RB-chr 3 or

T-DNA LB-chr 9) are marked with arrows. The sizes of k/HindIII

fragments are positioned on the left. A portion of the ethidium

bromide-stained gel in 23- to 9-kb region is shown in the lower panels

to reflect equal loading of plant DNA in all lanes. c Deduced

restriction maps of chromosome 3 (dotted box), chromosome 9

(striped box) native loci and the organization of the T-DNA and the

flanking sequences (T-DNA locus) in TC-19. Positions of the EcoRI

(E) sites are indicated. Positions of the chromosome 3 and 9 probes

are indicated in boxes below the maps. The sizes of DNA fragments,

which hybridize to the probes, are marked in dashed lines. The

predicted T-DNA insertion sites in chromosome 3 and 9 native loci

are marked with vertical arrows. Positions of primers used for

amplifying the native chromosomes 3 and 9 (C3F/C3R and C9F/C9R)

are marked in filled arrows on either side of the predicted T-DNA

insertion sites. Scale (1.0 kb) is marked

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(two EcoRI sites are present in the T-DNA integration site

in rice chromosome 9 at nucleotide positions 20291526 and

20296035 of RefSeq accession NC_008402.2) (Fig. 5b, c).

In the DNA isolated from the TC-19 hemizygous (Tt)

plant, the LB-flanking chromosome 9 probe displayed

hybridization to the 4.5-kb chromosome 9 fragment

expected from the native chromosome 9 plus the T-DNA

LB-chromosome 9 junction fragment of 2.9 kb (Fig. 5b, c).

It is to be noted that the 2.9-kb junction fragment hybrid-

ized to the 526-bp chromosome 9 fragment probe as well as

to the hph probe (Fig. 2a). Surprisingly, the chromosome 9

fragment probe hybridized to the 4.5-kb native chromo-

some 9 band and the 2.9-kb junction fragment in the

homozygous (TT) plant DNA (Fig. 5b). The results con-

firmed that the predicted chromosome 9 sequence formed a

junction to the T-DNA LB, but surprisingly, the native

chromosome 9 locus was intact.

Southern blot analyses with probes derived from the

fragments of chromosome 3 and 9 revealed that the T-DNA

in TC-19 is flanked by chromosome 3 and 9 sequences at

the RB and LB, respectively. The result also indicated that

the native chromosome 3 and 9 sequences were intact in

the native loci. To confirm the restoration of the native

chromosome 3 and 9 sequences, we PCR amplified the

chromosome 3 and 9 sequences corresponding to the

T-DNA integration sites using forward and reverse primers

(C3F/C3R and C9F/C9R) on either side of the T-DNA

insertion sites (Fig. 5c). We obtained the expected ampli-

fication of a 0.7-kb fragment for native chromosome 3 and

a 1.0-kb fragment for native chromosome 9 in control

(C) and TC-19 hemizygous (Tt) and homozygous (TT)

plant DNA samples (Supplementary Fig. S1a, b). The

sequences of the PCR products confirmed the intactness of

the native chromosome 3 and 9 sequences in all three

categories of plants.

These results suggested a possible event in which the

T-DNA along with a segment of chromosome 3 at the RB,

and a segment of chromosome 9 at the LB had translocated

into a new chromosomal locus.

Search for the new locus of integration of the T-DNA

with the flanking segments of chromosome 3 and 9

If the T-DNA, along with segments of chromosome 3 and

9, was translocated and integrated into a new chromosomal

locus, there must be two more junctions formed between

the two translocated chromosomal segments with a yet

unidentified chromosomal locus. To identify whether a

breakpoint appears on chromosome 3 sequence which

flanks the T-DNA RB in TC-19, a Southern blotting-based

extended walking was done. Control (C) and homozygous

(TT) TC-19 plant DNA samples were digested with dif-

ferent restriction enzymes which cut within the T-DNA and

cut the chromosome 3 sequence progressively away from

the T-DNA. The blot was hybridized with the chromosome

3 (1,117 bp) sequence that flanks the RB in TC-19. The

sizes of the native chromosomal fragments and the junction

fragments, which are expected to hybridize to the probe,

were determined from the rice genomic database (RefSeq

accessions NC_008396.2) and listed in Table 2. With all

eight restriction enzymes, the chromosome 3 probe dis-

played hybridization corresponding to the expected sizes of

native chromosomal fragments in the control (C) plant

DNA (Fig. 6a, b; Table 2). In the TC-19 homozygous (TT)

plant DNA, digestion with the first four restriction enzymes

PstI, XbaI, EcoRI and HindIII released the expected

junction fragments (Fig. 6a, b; Table 2). However, diges-

tion with the next four restriction enzymes SacI, XhoI, SalI

and SphI released junction fragments that differed from the

expected sizes (Fig. 6a, b; Table 2). This indicated that the

translocated chromosome 3 sequence is not continuous

beyond the HindIII site (nucleotide position 35803167 of

RefSeq accession NC_008396.2). Therefore, the break-

point of the translocated chromosome 3 segment in TC-19

was predicted between the HindIII and SacI sites. Extended

walking with restriction enzymes into chromosome 9 up to

22.6-kb distance did not reveal any break point (results not

shown). This suggested that the translocated chromosome 9

fragment is longer than 22.6 kb.

A 6.55-kb chromosome 3 segment is translocated

along with the T-DNA into chromosome 8

Genome walking was done to isolate the unknown chro-

mosome sequence present adjacent to the predicted break-

point on the chromosome 3 segment in TC-19. Gene-

specific primers (chr3-GSP1 and chr3-GSP2) were designed

from the chromosome 3 sequence before the HindIII site

Table 2 Sizes of expected restriction fragments versus observed

sizes in Southern blotting-based extended walking using the 1,117-bp

chromosome 3 fragment as the probe

Restriction

enzymes

Sizes of DNA fragments (bp)

Control (C) TC-19 homozygous (TT)

Expected Observed Expected JF Observed JF

PstI 1,936 1,936 1,904 1,904

XbaI 3,269 3,269 3,301 3,301

EcoRI 4,944 4,944 6,643 6,643

HindIII 17,803 17,803 6,745 6,745

SacI 9,780 9,780 7,511 11,900

XhoI 12,381 12,381 10,500 12,225

SalI 14,052 14,052 12,916 11,750

SphI 16,076 16,076 15,310 7,230

JF junction fragment

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(Fig. 6b). DraI, EcoRV and StuI libraries yielded two

amplified products in each, because the gene-specific

primers could anneal to both native chromosome 3

sequence and to the translocated chromosome 3 segment.

All PCR products were sequenced using the gene-specific

primer (chr3-GSP2) and the adaptor primer 2 (AP2). A

major 1,571-bp product obtained in the EcoRV library

included a 297-bp part of translocated chromosome 3

sequence (nucleotide position 35803103 to 35803400 of

RefSeq accession NC_008396.2). The remaining 1,274-bp

of sequence interestingly matched with a chromosome 8

sequence (nucleotide position 3010735 to 3012009 of

RefSeq accession NC_008401.2). The chromosome 3

breakpoint (nucleotide position 35803103) was located

176-bp upstream of the start codon of Os03g0832200,

which encodes a putative calcium-binding protein precursor

(calreticulin). The breakpoint of chromosome 3 (nucleotide

position 35803103 of RefSeq accession NC_008396.2) is

6.55 kb away from the point of T-DNA RB integration

(nucleotide position 35809658 of RefSeq accession

NC_008396.2). The chromosome 8 breakpoint (nucleotide

position 3010735 of RefSeq accession NC_008401.2) was

located in the first intron of the gene Os08g0152500, which

encodes a 268-amino acid conserved hypothetical protein.

Thus, as represented in Fig. 7c, T-DNA along with a 6.55-

kb translocated segment of chromosome 3 is integrated into

chromosome 8.

The chromosome 3 and 8 junction was analysed by

Southern blotting. The control (C), TC-19 hemizygous (Tt)

and homozygous (TT) plant DNA samples were digested

with BamHI or HindIII and the blots were hybridized to the

1,571-bp genome walking fragment (comprising 297-bp of

chromosome 3 and 1,274-bp of chromosome 8). As

expected, upon BamHI digestion of the control (C) plant

DNA, 11.4-kb and 5.3-kb bands lighted up (Fig. 7a). The

1,274-bp chromosome 8 sequence in the probe is expected

to hybridize intensely to a 11.4-kb fragment in the native

chromosome 8 (two BamHI sites are present in the inser-

tion site in rice chromosome 8 at nucleotide positions

3010672 and 3022048 of RefSeq accession NC_008401.2).

Similarly, the 297-bp chromosome 3 sequence in the probe

is expected to hybridize weakly to a 5.3-kb fragment in the

Fig. 6 Southern blotting-based extended walking in TC-19 DNA

using the RB-flanking chromosome 3 sequence (1,117 bp) as a probe

to locate the breakpoint on chromosome 3. a Plant DNA (5 lg) from

the control (C) and TC-19 homozygous (TT) plants was digested with

the marked restriction enzymes and loaded in the respective lanes.

The blot was probed with the [a-32P]dCTP-labelled 1,117-bp

chromosome 3 fragment. The sizes of the k/HindIII fragments are

positioned on the left. b Deduced restriction map of the chromosome

3 (dotted box) native locus and the organization of the T-DNA and the

flanking sequences (T-DNA locus) in TC-19. Recognition sites for

PstI (P), XbaI (Xb), EcoRI (E), HindIII (H), SacI (S), XhoI (X), SalI

(Sa) and SphI (Sp) are indicated. Position of the chromosome 3 probe

is indicated in boxes below the maps. The predicted T-DNA insertion

site in chromosome 3 native locus is marked with a vertical arrow.

Positions of primers used for genome walking (chr3-GSP1 and chr3-

GSP2) are marked in filled arrows. Scale (1.0 kb) is marked

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native chromosome 3 (two BamHI sites are present at the

detected breakpoint in rice chromosome 3 at nucleotide

positions 35799795 and 35805090 of RefSeq accession

NC_008396.2) (Fig. 7a, c). Similarly, upon HindIII

digestion, one intense band of 10.9 kb (corresponding to

native chromosome 8) and a weak band of 17.8 kb (cor-

responding to native chromosome 3) lighted up in the

control (C) plant DNA (Fig. 7b, c). In the hemizygous (Tt)

plant DNA, the probe hybridized to both native chromo-

some 8 (11.4-kb with BamHI digestion and 10.9-kb with

HindIII digestion) and chromosome 3 (5.3-kb with BamHI

digestion and 17.8-kb with HindIII digestion) fragments,

plus the expected chromosome 3–8 junction fragment

(13.4-kb with BamHI digestion and 5.4-kb with HindIII

digestion) (Fig. 7a–c). As expected of a typical T-DNA

integration, the hybridization signal for the native chro-

mosome 8 fragment (11.4-kb in BamHI and 10.9-kb in

HindIII digestions) reduced to half the intensity in the

hemizygous (Tt) plant DNA (BamHI-IDV 15,456; HindIII-

IDV 29,120) in comparison to the control (C) plant DNA

(BamHI-IDV 31,600; HindIII-IDV 56,648). In the TC-19

homozygous (TT) plant DNA, the probe displayed

hybridization to the native chromosome 3 fragment (5.3 kb

upon BamHI digestion and 17.8 kb upon HindIII digestion)

and to the chromosome 3–8 junction fragment (13.4 kb

upon BamHI digestion and 5.4 kb upon HindIII digestion)

Fig. 7 Southern blot analyses to study the chromosome 3–8 junction

with the 1,571-bp fragment (comprising a 297-bp [0.3 kb] portion of

chromosome 3 and a 1,274-bp [1.3 kb] sequence of chromosome 8) as

the probe. a Analysis of BamHI-digested plant DNA. b Analysis of

HindIII-digested plant DNA. a and b Plant DNA (5 lg) from the

control (C), TC-19 hemizygous (Tt) and homozygous (TT) plants was

digested with BamHI or HindIII and loaded in each lane. The blot was

probed with [a-32P]dCTP-labelled 1,571-bp (1.6 kb) chromosome

3–8 junction sequence. Positions of bands corresponding to the native

chromosome 8 (chr 8), native chromosome 3 (chr 3) and the

chromosome 3-chromosome 8 junction fragment (chr 3-chr 8) are

marked with arrows. The sizes of k/HindIII fragments are positioned

on the left. A portion of the ethidium bromide-stained gel in 23-to

9-kb region is shown in the lower panels to reflect equal loading of

plant DNA in all lanes. c Deduced restriction maps of the

chromosome 8 (filled box), chromosome 3 (dotted box) native loci

and the organization of the T-DNA and the flanking sequences (T-

DNA locus) in TC-19. Positions of the HindIII (H) and BamHI

(B) sites are indicated. Positions of the probes are indicated in boxes

below the maps. The sizes of DNA fragments from different loci,

which hybridize to the probe, are marked in dashed lines. The

predicted breakpoint in native chromosome 3 and the predicted

insertion site in native chromosome 8 locus are marked with vertical

arrows. Positions of primers used for genome walking (chr8-GSP1

and chr8-GSP2) are marked in filled arrows. Scale (1.0 kb) is marked

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(Fig. 7a, b). As expected, the hybridization signal for the

junction fragment was twice as intense in the homozygous

(TT) plant DNA (BamHI-IDV 34,884; HindIII-IDV

56,056) than that of the hemizygous (Tt) plant DNA

(BamHI-IDV 17,224; HindIII-IDV 30,608) in both diges-

tions. Due to disruption of the chromosome 8 locus, the

hybridization signal for the native chromosome 8 fragment

(11.4-kb in BamHI digestion and 10.9-kb in HindIII

digestion) was totally absent in the homozygous (TT) plant

DNA (Fig. 7a, b). The intensity of the hybridization signal

for the native chromosome 3 fragment (5.3-kb in BamHI

digestion and 17.8-kb in HindIII digestion) was equal in

control (C), hemizygous (Tt) and homozygous (TT) plant

DNA. These results confirmed that the predicted chromo-

some 8 sequence is actually present adjacent to the chro-

mosome 3 segment. This also confirmed that the

chromosome 8 locus was disrupted due to insertion of the

T-DNA along with the chromosome 3 segment. The 6.55-

kb chromosome 3 region was found to be intact in TC-19 in

the native chromosome 3.

A 29.8-kb chromosome 9 segment is translocated

along with the T-DNA to chromosome 8 in TC-19

The breakpoint of chromosome 8 identified in the above

experiment could be interpreted as the final site of inte-

gration of the complex T-DNA comprising the translo-

cated segments of chromosomes 3 and 9. On this basis, a

genome walking experiment was done to walk from the

opposite end of the chromosome 8 break point to chro-

mosome 9. Gene-specific primers (chr8-GSP1 and chr8-

GSP2) on the chromosome 8 sequence were used for

genome walking (Fig. 7c). A 2,186-bp major PCR prod-

uct obtained in the PvuII library contained 1,373 bp of the

chromosome 8 sequence (nucleotide position 3009322 to

3010695 of RefSeq accession NC_008401.2). The

remaining 813-bp sequence showed 100 % identity to the

chromosome 9 sequence (nucleotide position 20324487 to

20325300 of RefSeq accession NC_008402.2). The

chromosome 9 breakpoint at nucleotide position

20325300 was located in the first exon of Os09g0507400,

which encodes a conserved hypothetical protein. The

results showed that a 29.8-kb chromosome 9 segment is

translocated along with the T-DNA into the chromosome

8 locus. The chromosome 8 breakpoint was located in the

predicted first intron of Os08g0152500. A detailed ana-

lysis of nucleotide sequences at both chromosome 8

junctions (chromosome 3–8 and chromosome 9–8)

revealed a 37-bp deletion at the chromosome 8 locus. A

3-bp filler sequence 50CCA is present at the junction of

chromosome 8 and 3, and a dinucleotide filler sequence

GA is present at the junction of chromosome 8 and 9. No

microhomology was observed between both junctions.

The chromosome 8 and 9 junction was analysed by

Southern blotting using the 2,186-bp genome walking

fragment (containing 1,373 bp of chromosome 8 and

813 bp of chromosome 9) as the probe. Control (C), TC-19

hemizygous (Tt) and homozygous (TT) plant DNA sam-

ples were digested with EcoRI or HindIII. Upon EcoRI

digestion of the control (C) plant DNA, the probe hybrid-

ized to a 5.7-kb native chromosome 8 fragment (two EcoRI

sites are present in the insertion site in rice chromosome 8

at nucleotide positions 3005441 and 3011127 of RefSeq

accession NC_008401.2) and to a 3.1-kb native chromo-

some 9 fragment (two EcoRI sites are present in the

chromosome 9 at nucleotide positions 20322046 and

20325173 of RefSeq accession NC_008402.2) (Fig. 8a, c).

Similarly, upon HindIII digestion, hybridization signals for

a 10.9-kb fragment in the native chromosome 8, and a 8.9-

kb native chromosome 9 fragment (these sizes in our indica

variety differed from the japonica-derived database

sequence) were observed (Fig. 8b, c). As expected, in the

hemizygous (Tt) plant DNA, the probe hybridized to native

chromosome 8 and 9 fragments, plus an expected fragment

of 5.2 kb upon EcoRI digestion and a fragment of 7.3 kb

upon HindIII digestion, representing the chromosome 8–

chromosome 9 junction (Fig. 8a–c). As expected of a

typical T-DNA integration, hybridization signal for the

native chromosome 8 fragment (5.7 kb in EcoRI digestion

and 10.9 kb in HindIII digestion) reduced by half the

intensity in the hemizygous (Tt) plant DNA (EcoRI-IDV

64,260; HindIII-IDV 59,040) in comparison to the control

(C) plant DNA (EcoRI-IDV 127,208; HindIII-IDV

113,740). In the homozygous (TT) plant DNA, the probe

did not detect the native chromosome 8 fragments of 5.7 kb

(EcoRI digestion) and 10.9 kb (HindIII digestion) but

detected the native chromosome 9 fragment (3.1 kb in

EcoRI and 8.9 kb in HindIII digestions) and the chromo-

some 8–9 junction fragment (5.2 kb in EcoRI and 7.3 kb in

HindIII digestions) (Fig. 8a, b). As expected, the hybrid-

ization signal for the junction fragment was twice as

intense in the homozygous plant (TT) DNA (EcoRI-IDV

122,408; HindIII-IDV 123,165) than in that of the hemi-

zygous (Tt) plant DNA (EcoRI-IDV 62,888; HindIII-IDV

63,600) in both digestions (Fig. 8a, b).

Thus, we confirmed that the T-DNA with a 6.55-kb seg-

ment of chromosome 3 at the RB and a 29.8-kb segment of

chromosome 9 at the LB is inserted into chromosome 8. The

chromosome 8 locus was found disrupted, but the translocated

chromosome 3 and 9 loci were restored to the native status.

The disruption of the chromosome 8 locus was studied

by PCR. Amplification of native chromosome 8 DNA with

forward and reverse primers (C8F/C8R) from either side of

the chromosome 8 breakpoint (Fig. 8c) gave the desired

amplification of 920-bp fragment in the control (C) and

hemizygous (Tt) plant DNA (Fig. 9). This amplification

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did not happen when DNA from the homozygous (TT)

plant was used as the template (Fig. 9). This confirmed that

the chromosome 8 locus is disrupted in the homozygous

plant due to integration of the T-DNA along with segments

of chromosome 3 and 9.

Amplification of all four junctions in the TC-19

transgenic plant

To provide the final evidence that the T-DNA with seg-

ments of chromosome 3 and 9 is inserted in chromosome 8,

PCR analysis was carried out using primer sets specific for

the junctions of T-DNA RB/chromosome 3 (RBF/C3R1),

T-DNA LB/chromosome 9 (LBF/C9R1), chromosome 3/8

(C3F1/C8R1) and chromosome 8/9 (C8F1/C9R2) to

amplify all four junctions in the TC-19 rice line (Fig. 10a,

b). As expected, no amplification was seen in the control

(C) plant DNA with different primer sets. PCR amplifica-

tion with DNA of the TC-19 homozygous (TT) plant

yielded the expected products of 1.2 kb (with RBF/C3R1),

0.5 kb (with LBF/C9R1), 1.1 kb (with C3F1/C8R1) and

0.65 kb (with C8F1/C9R2) (Fig. 10a). These results are

Fig. 8 Southern blot analyses to study the chromosome 8–9 junction

with the 2,186-bp fragment (comprising a 1,373-bp [1.4 kb] portion

of chromosome 8 and a 813-bp [0.8 kb] sequence of chromosome 9)

as the probe. a Analysis of EcoRI-digested plant DNA. b Analysis of

HindIII-digested plant DNA. a and b Plant DNA (5 lg) from the

control (C), TC-19 hemizygous (Tt) and homozygous (TT) plants was

digested with EcoRI or HindIII and loaded in each lane. The blot was

probed with [a-32P]dCTP-labelled 2,186-bp (2.2 kb) chromosome

8–9 junction sequence. The positions of the bands corresponding to

the native chromosome 8 (chr 8), native chromosome 9 (chr 9) and

the chromosome 8–chromosome 9 junction fragment (chr 8-chr 9) are

marked with arrows. The sizes of k/HindIII fragments are positioned

on the left. A portion of the ethidium bromide-stained gel in 23- to

9-kb region is shown in the lower panel to reflect equal loading of

plant DNA in all lanes. c Deduced restriction maps of the native

chromosome 8 locus (filled box), native chromosome 9 locus (striped

box) and the organization of the T-DNA and the flanking sequences

(T-DNA locus) in TC-19. Positions of the EcoRI (E) and HindIII

(H) sites are indicated. Positions of the probes are indicated in boxes

below the maps. The sizes of DNA fragments from different loci,

which hybridize to the probe, are marked by dashed lines. The

predicted breakpoints in chromosome 8 and 9 native loci are marked

with vertical arrows. Positions of primers used for amplifying the

native chromosome 8 sequence (C8F/C8R) are marked in filled

arrows on either side of the T-DNA insertion site. Scale (1.0-kb) is

marked

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consistent with the interchromosomal translocation asso-

ciated with the complex T-DNA integration in TC-19.

Discussion

We report a novel T-DNA integration in rice which is

associated with two interchromosomal translocations. In

Arabidopsis, four types of large chromosomal rearrange-

ments associated with the T-DNA integration have been

reported: (1) reciprocal translocation between two non-

homologous chromosomes (Clark and Krysan 2010; Curtis

et al. 2009; Guan et al. 2003; Forsbach et al. 2003; Laf-

leuriel et al. 2004; Ray et al. 1997; Yuen et al. 2005), (2)

reciprocal translocation associated with inversion (Nacry

et al. 1998), (3) paracentric inversion (Laufs et al. 1999)

and (4) interchromosomal translocation associated with

duplication (Tax and Vernon 2001).

The interchromosomal translocation associated with the

T-DNA integration in TC-19 in our study is similar to the

fourth type. Tax and Vernon (2001) identified two Arabi-

dopsis mutant lines emb88 and LRRPK-TKO. In both

cases, linkage mapping, along with molecular analysis of

the mutated gene flanking the T-DNA revealed that the

flanking DNA had originated from a locus unrelated to the

site of T-DNA integration. Wild-type chromosomes of

these translocated fragments also remained intact at the

native locations. T-DNA integration in the emb88 mutant

had caused translocation/duplication of a large fragment

([40 kb) of chromosome V into a chromosome I locus. In

the LRRPK-TKO mutant, the T-DNA is associated with

translocation/duplication of a chromosome IV segment into

a chromosome V locus. Both lines have translocated

sequences only adjacent to the LB of the T-DNA. The

possibility of presence of translocated sequences adjacent

to the RB cannot be ruled out because detailed character-

ization of the RB-flanking DNA was not carried out in both

lines. A similar kind of interchromosomal translocation,

associated with T-DNA integration, was reported in the

mutant Arabidopsis line atTOC33 (Gutensohn et al. 2004).

The T-DNA integration in this line had caused transloca-

tion of a fragment of chromosome V linked to LB into the

atTOC33 gene on chromosome I. The atTOC33 gene locus

was confirmed to be disrupted by Southern hybridization

analysis. However, whether the translocation was accom-

panied by duplication at the native locus or a deletion was

caused was not characterized in the mutant. The chromo-

somal breakpoints at nucleotide sequence level and the

exact sizes of the translocated chromosomal fragments

were not deduced in the three mutants discussed above.

In the TC-19 rice line, we have described for the first

time a very rare example of T-DNA-associated interchro-

mosomal translocations/duplications that involved three

chromosomes. The T-DNA with a 6.55-kb chromosome 3

segment adjacent to the RB and a 29.8-kb chromosome 9

segment adjacent to the LB was translocated into a chro-

mosome 8 locus. Translocations of segments of the chro-

mosome 3 and 9 occurred without alterations of the native

loci. The chromosome 8 locus was found to be disrupted.

The availability of complete rice genome sequence

(International Rice Genome Sequencing Project 2005)

enabled us to determine the breakpoints accurately. The

exact sizes of the translocated fragments were determined.

The basis of the T-DNA-associated translocations in

TC-19 is not very clear. The possibility that the translo-

cations occurred due to recombination between multiple

T-DNAs, as observed previously (Curtis et al. 2009;

Forsbach et al. 2003; Laufs et al. 1999; Nacry et al. 1998),

can be ruled out in TC-19. The TC-19 rice line harboured a

single copy of intact T-DNA, and there were no other

T-DNA integrations in the genome. Two previously stud-

ied examples of Arabidopsis mutant lines C and BNP23,

harbouring single copies of T-DNAs, carried translocations

(Forsbach et al. 2003; Lafleuriel et al. 2004). The following

events could have led to the interchromosomal transloca-

tions in TC-19. The RB and the LB of one T-DNA were

integrated into chromosome 3 and 9 simultaneously, which

were physically very close in the nucleus when the T-DNA

was inserted. This could have formed a transient T-DNA

Fig. 9 PCR-based detection of the native chromosome 8 sequence in

the control (C), TC-19 hemizygous (Tt) and homozygous (TT) plants

using chromosome 8-specific forward (C8F) and reverse (C8R)

primers designed on either side of the predicted breakpoint. Positions

of primers are indicated on the chromosome 8 native locus in Fig. 8c.

Template DNA (100 ng) from control (C), TC-19 hemizygous (Tt)

and homozygous (TT) plants was used for amplification. Lane M 1-kb

ladder. The sizes of 1-kb ladder are positioned on the right. The

expected amplicon of 920-bp is indicated with an arrow

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bridge between the two chromosomes. Subsequently, the

aberrant T-DNA structure was excised from that site with

portions of chromosome 3 and 9 at the RB and LB,

respectively, and became integrated into the chromosome 8

locus. Alternatively, the presence of the translocated

chromosome 3 and 9 sequences at the T-DNA integration

site was caused by duplications of those chromosomal

segments prior to T-DNA integration. In this model, the

native chromosome 3 and 9 sequences would have never

been altered, as found in this report. A third possibility is

that the T-DNA was excised twice before the final suc-

cessful integration in chromosome 8. The T-DNA may

have been accompanied with a fragment of each of the

previous target chromosomes in which the integration was

unsuccessful. Abortive T-DNA integrations are common in

T-DNA mutagenized populations. For instance, Azpiroz-

Leehan and Feldmann (1997) reported that in a T-DNA-

mutagenized population, approximately 65 % of the

mutant phenotypes were not linked to T-DNA insertions,

suggesting that abortive T-DNA integrations could result in

deletions, additions and base substitutions (Nacry et al.

1998; Negruk et al. 1996).

The exact mechanism of T-DNA integration into plant

genome remains unknown. Two proposed models for the

T-DNA integration into plant genome are the SSGR and

DSBR models. Microhomology between the T-DNA ends

and the plant DNA plays a central role in the SSGR model

(Tinland and Hohn 1995). However, except the T-DNA RB

and chromosome 3 integration site in TC-19, we did not

observe any homology in other junctions. The DSBR

model involving the non-homologous end joining (NHEJ)

mechanism better explains the integration of the T-DNA in

the TC-19 rice line. It has been shown previously that

double-stranded breaks (DSBs) are ‘‘hot spots’’ for double-

stranded T-DNA integration (Chilton and Que 2003; Sal-

omon and Puchta 1998; Tzfira et al. 2003). Recently,

Singer et al. (2012) suggested that prior to T-DNA inte-

gration, a single-stranded T-DNA (T-strand) gets converted

into a double-stranded (dsT-DNA) form. The plant’s DSB

DNA repair machinery recognizes the dsT-DNA as a

substrate to repair the break. How the dsT-DNA ligates into

the DSBs is still not clear. Based on the DSBR model, we

propose that the single-stranded T-DNA in TC-19 was

converted into a double-stranded molecule inside the

Fig. 10 PCR-based confirmation of the four junctions in TC-19.

a PCR analysis of the four junctions. Genomic DNA template and

primer combinations used in each reaction are labelled above each

lane. Lane C control plant DNA; Lane T TC-19 homozygous plant

DNA; Lane M 1-kb ladder. The sizes of 1-kb ladder are positioned on

the right. b Deduced organization of the T-DNA integrated locus in

TC-19. Positions of the primer sets (RBF/C3R1, LBF/C9R1, C3F1/

C8R1 and C8F1/C9R2) used to amplify the four junctions are

indicated with filled arrows

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nucleus. Then two independent DSBs, one on chromosome

3 and the other on chromosome 9 present physically close

in the nucleus might have captured the two ends of the

T-DNA to repair the breaks. This could have generated a

transient T-DNA bridge. Subsequently, the excised dsT-

DNA, with segments of chromosome 3 and 9, may have

become integrated into a DSB in chromosome 8 through

the DSBR process using the NHEJ mechanism. The DSBR

process, generally results in addition or removal of a few

base pairs from the host genome and from both ends of the

T-DNA. We observed 72-bp and 6-bp deletions of the

T-DNA LB and RB, respectively. A deletion of 37-bp on

the chromosome 8 locus was also observed. Filler DNA is a

pattern found at genomic DSBR sites in plants (Chilton and

Que 2003; Singer et al. 2012; Salomon and Puchta 1998;

Tzfira et al. 2003; Windels et al. 2003). We found filler

sequences in all the four junctions present in TC-19. Our

observations also support a previous report (Clark and

Krysan 2010) which proposes that cells which are ame-

nable to T-DNA insertion have a high potential for the

generation of chromosomal translocations due to the

enhanced presence of DSBs in those cells.

The translocations in TC-19 occurred without altering

the native sequences in chromosomes 3 and 9. Large

deletions are likely to be lethal for the gametes. We suggest

that deletions corresponding to the chromosome 3 and 9

translocated regions could have been present in the primary

transformant (T0) of TC-19. Chromosomes 3 and 9 con-

taining deletions might have segregated away in the sub-

sequent generation, and the recovered plants may have

retained only the normal wild-type chromosomes. Some

T-DNA mutants in Arabidopsis like hosoba toge toge

(Kaya et al. 2000) and salade (Zhao et al. 2009) which had

very large chromosomal deletions (75.8 kb and 11.4 kb)

did not have duplications. An explanation was given that

the 14 genes present in the 75.8-kb deleted region in the

hosoba toge toge mutant had homologous genes in other

chromosomes. Consequently, gametes bearing the large

deletion might have survived. In the rice mutant TC-19,

two genes present in the translocated region of chromo-

some 3 and four genes present in the translocated region of

chromosome 9 do not have any homologous genes else-

where in the genome. Therefore, gametes bearing deletions

in chromosome 3 and 9 might not have survived. An

alternative explanation can be that the two deletion mutants

described above in Arabidopsis were not associated with

any chromosomal translocations, whereas TC-19 rice line

has interchromosomal translocations. Thus, as observed by

Tax and Vernon (2001), interchromosomal translocations

caused due to T-DNA integration may generally be

accompanied by duplications. More studies are needed to

understand the mechanism by which the complex T-DNA

locus in TC-19 was formed.

This is the first report of interchromosomal translocation

associated with T-DNA integration in rice. Our findings

suggest that large chromosomal rearrangements can occur

in transgenic rice plants harbouring even single-copy

T-DNA insertions. This is also the first report which

describes two interchromosomal translocations associated

with a single T-DNA integration. Our results emphasize the

importance of detailed analysis of plant DNA sequences

that flank both T-DNA borders to ensure that T-DNA

integration is clean.

Acknowledgments We thank Dr. K. Dharmalingam, School of

Biotechnology, Madurai Kamaraj University for his permission to use

the radioisotope facility. This work was supported by Department of

Biotechnology, Ministry of Science & Technology, Government of

India [Project entitled ‘‘Functional Analysis of Gene Regulatory

Networks During Flower and Seed development in rice’’, Project No.

BT/AB/FG-I(PH-II)(5)2009].

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