Experimental reconstruction of double‐stranded break ... · Mendelian inheritance of the...

Experimental reconstruction of double-stranded breakrepair-mediated plastid DNA insertion into the tobacconucleus

Dong Wang1,2,*, Jinbao Gu2, Rakesh David3, Zhen Wang2, Songtao Yang4, Iain R. Searle3, Jian-Kang Zhu2,5 and

Jeremy N. Timmis3

1Key Laboratory of Molecular Biology and Gene Engineering in Jiangxi Province, College of Life Science, Nanchang

University, Jiangxi 330031, China,2Shanghai Center for Plant Stress Biology and Center of Excellence in Molecular Plant Sciences, Chinese Academy of

Sciences, Chinese Academy of Sciences, Shanghai 200032, China,3Department of Genetics and Evolution, School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005,

Australia,4Crop Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China, and5Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA

Received 25 August 2017; revised 26 October 2017; accepted 30 October 2017; published online 20 November 2017.

*For Correspondence (e-mail: [email protected]).

SUMMARY

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the

endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the

nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible

for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we

introduced double-stranded DNA breaks into the nuclear genome of tobacco through inducible expression

of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through

double-stranded DNA break repair. Microhomology-mediated linking of disparate segments of chloroplast

DNA occurs frequently during healing of induced nuclear double-stranded breaks (DSB) but the resulting

nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable marker (neo),

used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the

screen. The instability of these de novo nuclear insertions of plastid DNA (nupts) was shown to be associ-

ated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of

transfer. This deletion of pre-existing nuclear DNA suggests that the genetic impact of organellar DNA trans-

fer to the nucleus is potentially far greater than previously thought.

Keywords: endosymbiotic gene transfer, chloroplast, nuclear insertions of plastid DNA, double-strand DNA

break repair, microhomology, genetic instability, Nicotiana tabacum.

INTRODUCTION

A pivotal feature of the evolution of eukaryotes is the for-

mation of mitochondria and plastids, the cytoplasmic orga-

nelles which originated from endosymbiotic prokaryotes.

DNA from the endosymbionts has bombarded nuclei since

the ancestral prokaryotes were engulfed by a precursor of

the eukaryotic host (Timmis et al., 2004; Bock and Timmis,

2008). This resulted in the relocation of many organellar

genes to the nucleus, and numerous nuclear sequences

were created by insertion of organelle DNA (Martin et al.,

2002; Noutsos et al., 2007). While functional gene transfers

from organelle to nucleus are relatively rare, non-

functional sequence transfer happens very often and most

nuclear genomes are extensively punctuated with orga-

nelle genome sequences (Matsuo et al., 2005). Real-time

DNA migration from organelle to nucleus has been

demonstrated in yeast (Thorsness and Fox, 1990) and

tobacco (Huang et al., 2003; Stegemann et al., 2003; Shep-

pard et al., 2008), and the frequency of this movement is

unexpectedly high. Organellar DNA fragments incorporate

into nuclear DNA through double-stranded break (DSB)

repair in yeast (Ricchetti et al., 1999) and tobacco (Wang

et al., 2012) but the molecular mechanisms responsible for

© 2017 The AuthorsThe Plant Journal © 2017 John Wiley & Sons Ltd

227

The Plant Journal (2018) 93, 227–234 doi: 10.1111/tpj.13769

mailto:[email protected]

insertion remain unclear. Bioinformatic analysis suggested

that non-homologous end joining (NHEJ) is involved in

chloroplast DNA incorporation during DSBs repair (Wang

and Timmis, 2013), but direct experimental evidence was

lacking.

De novo experimental nuclear DNA integrants originat-

ing from plastid genomes (nupts) are often unstable. Non-

Mendelian inheritance of the selectable marker (neo) used

to identify ptDNA transfers was observed in the progeny of

about half of the lines emerging from screens (Huang

et al., 2003; Wang et al., 2012). A detailed analysis demon-

strated the physical loss of the neo gene as the cause of

instability of kanamycin resistance (Sheppard and Timmis,

2009). No explanations of instability other than deletion

have been forthcoming.

In this study, we constructed a reporter system to exam-

ine whether DSB repair was involved in chloroplast DNA

insertion into the nuclear genome of tobacco. Three inde-

pendent chloroplast integrants were identified at repaired

nuclear DSBs. DNA found at these loci contained multiple

disparate DNA sequences from the chloroplast genome

linked together by a process involving microhomology.

Furthermore, the instability observed at these de novo

nupts was shown to be associated with deletion, within

one generation of chloroplast DNA transfer. Deletions

occurred not only in the insert itself but also included

flanking native nuclear DNA.

RESULTS

A genetic screen for nuclear DSB repair events that

incorporate chloroplast DNA

In a pilot experiment to investigate the effect of induced

DSBs on the frequency of plastid DNA migration, the

transplastomic tobacco (Nicotiana tabacum) line (tpGUS)

containing a GUS reporter gene (iudA) driven by the 35S

promoter (Sheppard et al., 2008) was irradiated with UV-C.

In tpGUS, the reporter GUS gene is expressed only when it

transfers to the nucleus and this event can be monitored

readily by histochemical staining to detect the GUS prod-

uct. The number of blue sectors resulting from nuclear

GUS expression observed in seedlings treated with UV-C

was greater than that of the control seedlings (Table S1).

This tentatively suggested that increasing DSB frequency

increased plastid DNA insertion into the nucleus but the

lack of significance necessitated further experimentation.

Therefore, to detect DSB repairs that mediate chloroplast

DNA insertion, an experimental system was developed in

tobacco (Figure 1a). The transplastomic line tp-neo (Huang

et al., 2003) contains, in its plastid genome, a neo gene dri-

ven by a eukaryotic promoter. It was therefore poorly

expressed in chloroplasts but strongly activated after trans-

fer to the nuclear genome, conferring kanamycin resis-

tance. To the nucleus of this line was added an inducible

DSB system (Lloyd et al., 2012; Wang et al., 2012). In this

added cassette, the gene encoding the rare cutting

endonuclease I-SceI was controlled by an alcA:35S pro-

moter expressed after ethanol treatment to cut two adja-

cent engineered target sites (Figure 1b). Consequently, the

plant line containing both nuclear and plastid transgenes

was named pISceI-neo. Two lines of pISceI-neo: pISceI-

neo-9 and pISceI-neo-10, containing low copy numbers of

T-DNA monitored by bar gene quantification (see Fig-

ure S1), were used in a screen to monitor insertion of neo

from the transplastome at cut and repaired I-SceI sites (Fig-

ure 1). In pISceI-neo-10, it was also determined that there

was only a single nuclear locus containing the two I-SceI

sites (Table S2).

To evaluate the impact of DSB repair on the frequency

of plastid-to-nucleus DNA transfer, pISceI-neo-13 harbour-

ing more than one locus was also used in the screen

(Table S2). Leaf explants of wild type (WT) tobacco were

used as negative controls. These were all bleached within

2 months when grown on regeneration medium supple-

mented with 400 mg/L kanamycin (Figure 1c). Two-week-

old pISceI-neo seedlings were sprayed with 0.7 M ethanol

and 4 days were allowed for I-SceI expression to generate

DSBs and DSB repair (Lloyd et al., 2012). After that, 340

and 400 leaf explants (~25 mm2) from pISceI-neo-9 and

pISceI-neo-10 respectively were placed on regeneration

medium containing 400 mg/L kanamycin (Stegemann

et al., 2003). In contrast to the control plants, kanamycin-

resistant shoots appeared in explants of pISceI-neo leaves

(Figure 1c). Six and eight resistant shoots were generated

from pISceI-neo-9 and pISceI-neo-10 lines respectively

(Table 1) and these shoots were designated the kr7 series.

Based on cell number estimations (Lloyd and Timmis,

2011), the occurrence of kanamycin-resistant shoots in the

screen indicated one neo transfer to the nucleus in approx-

imately 2.1 million and 1.9 million cells in ISceI-neo-9 and

pISceI-neo-10 lines respectively, consistent with the plas-

tid-to-nucleus DNA transfer frequency observed in somatic

cells from previous studies (Wang et al., 2012). For pISceI-

neo-13, 11 resistant shoots were generated in 440 leaf

explants, indicating a neo transfer to the nucleus of one in

1.5 million cells (Table 1). This frequency is slightly higher

than that in pISceI-neo-10, suggesting that the significant

increase results from the higher incidence of DSBs in the

pISceI-neo-13 nuclear genome.

Chloroplast DNA incorporation at nuclear DSB sites

Considering that earlier experimental chloroplast DNA inte-

grants were characteristically large (tens of kilobases or

more in size) and consequently too large to be amplified

by the conventional PCR (Huang et al., 2004; Lloyd and

Timmis, 2011), genome walking based on the nuclear

sequences near I-SceI site (Figure 1b) was carried out to

characterise DSB repairs. As expected, associated with the

© 2017 The AuthorsThe Plant Journal © 2017 John Wiley & Sons Ltd, The Plant Journal, (2018), 93, 227–234

228 Dong Wang et al.

acquisition of kanamycin resistance, transplastomic DNA

was present after I-SceI cleavage and DSB repair in three

independent plants obtained in the screen (Figures 2a, 3

and 4a).

Sequences adjacent to the repaired I-SceI junctions in

kr7.1, kr7.2 and kr7.3 lines (1092, 588 and 1209 nucleotides

respectively) revealed complex mosaics of diverse chloro-

plast fragments with perfect sequence conservation com-

pared with the plastome or transplastome (Figure 2a). This

sequence fidelity is consistent with the hypothesis that

escaped chloroplast DNAs from pISceI-neo integrated into

the nuclear genome at cleaved I-SceI sites during DSB

repair. Besides chloroplast DNA fragments, some unknown

sequences presumed to originate from nuclear DNA were

also observed at two of the three repaired sites (Figure 2a).

An intact 35S promoter and part of the neo gene was pre-

sent in kr7.3 that clearly originated from the pISceI-neo

transplastome (Figure 2a). To exclude the possibility that

the other two chloroplastic integrants from kr7.1 and kr7.2

originated from pre-existing nupts, PCR amplification was

carried out with the primer pairs described in Figure 2(a).

Corresponding amplicons were generated only from DNA

templates from seedlings containing the chloroplast inte-

grants and not from WT plants (Figure 2b), confirming that

fragments of chloroplast DNAs had inserted de novo into

the nuclear genome at experimentally induced DSBs.

Upon close examination, the fusion points between

chloroplast DNA sequences inside the de novo nupts sug-

gested that short microhomology was involved in their for-

mation (Figure 3). Microhomology was observed at both

chloroplast/chloroplast and chloroplast/nuclear junction

sites, consistent with bioinformatic analyses (Wang and

Timmis, 2013).

Instability of chloroplast integrants

In previous experiments, genetic instability was observed

within one generation of insertion in approximately half

Table 1 Frequency of DNA transfer from plastid to nucleusobserved in pISceI-neo-9, pISceI-neo-10 and pISceI-neo-13 lines

Leaf explants Resistant shoots Estimated transfer rate

pISceI-neo-9 340 6–2.1 9 106

pISceI-neo-10 400 8–1.9 9 106

pISceI-neo-13 440 11–1.5 9 106

Figure 1. A genetic screen for DNA transfer from

chloroplast to nucleus through DSB repair. (a)

Structure of tp-neo. The neo and aadA genes are

under CaMV 35S and psbA promoter control,

respectively (Huang et al., 2003). The black box rep-

resents the STLS2 intron. (b) Schematic representa-

tion of the DSB system in the nucleus. The top

panel presents vector T-DNA harbouring a hygro-

mycin selectable marker gene (hyg), an AlcR gene

and the I-SceI gene driven by an ethanol inducible

promoter (Lloyd et al., 2012). The bottom panel

shows vector T-DNA holding a herbicide resistant

gene (bar) and a stuffer fragment of known

sequence flanked by two I-SceI target sites. LB, left

border; RB, right border of the Agrobacterium

transformation system. (c) Stable plastid-to-nucleus

transfer. Leaf explants (~25 mm2) were grown on

regeneration medium supplemented with 400 mg/L

kanamycin. As resistant plants emerged (as pre-

sented in the right hand panel), they were trans-

ferred to half-strength MS medium to root and

subsequently moved in soil. After 2 months, all leaf

explants from Petite Havana (WT) were totally

bleached, and the screen ended at this time point.

Plates used were 11 cm Petri dishes. [Colour figure

can be viewed at wileyonlinelibrary.com]


NUPTs resulting from DSB repair 229

the kanamycin-resistant lines in a similar screen (Sheppard

and Timmis, 2009). Therefore, the stability of kanamycin

resistance was tested in the three kr7 lines reported here

by backcrossing them to WT females to replace their

transplastomes with WT and growing the progeny of indi-

vidual capsules on medium containing kanamycin. Only

one of the three lines (kr7.3) exhibited, in all eight capsules

tested, the 1:1 ratio of resistant:sensitive expected for a

nuclear hemizygote (Table 2). All capsules of kr7.1 and four

of six capsules of kr7.2 presented a very substantial paucity

of kanamycin-resistant seedlings (Table 2). No capsules

showed an excess of resistant progeny.

Figure 2. Chloroplast DNAs inserted at repaired I-SceI sites. (a) Schematic representations of partial chloroplast integrants at DSB repair junctions. DNA from

three seedlings contained mosaic integrants comprised of different chloroplast and transplastomic DNA fragments (different shades of green, shades are not

scale). Numbers in the green boxes indicate corresponding nucleotides of the native tobacco chloroplast genome (Shinozaki et al., 1986). Grey boxes represent

presumed nuclear DNA. The T-DNA vector flanking sequence of I-SceI restriction site is shown in black. The three primer pairs shown (P1F and P1R, P2F and

P2R, P3F and P3R for kr7.1, kr7.2 and kr7.3, respectively) were designed to amplify specific chloroplast integrants in the nucleus. (b) The de novo experimental

nupts are not present as nupts elsewhere in WT tobacco. One section of the tobacco nuclear genome amplified by primers from a recent study (Lloyd and Tim-

mis, 2011) was used as a positive control. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Microhomology involved in nupt-mediated DSB repair. Alignments show nupts in three kr7 lines, their corresponding nuclear sequence in pISceI-neo

(pISceI-neo_N), and the chloroplast DNA sequences (tabacum_pt) or transplastomic DNA sequence (pISceI-neo_tp). Short microhomology at fusion points is

highlighted in red, green is used to mark DNA originating from chloroplast or transplastome, and the unidentified DNA sequence in nupts is shown in blue.

Numbers inside two slashes indicate nucleotides in nupts that are identical to chloroplast DNA or unidentified DNA origins. [Colour figure can be viewed at

wileyonlinelibrary.com]



In a previous study, deletion of neo rather than epige-

netic silencing was found to be responsible for the loss of

resistance (Sheppard and Timmis, 2009). Therefore, the

integrity of neo in the kr7 backcross progenies was first

checked by employing a PCR primer pair targeting the neo

gene. As a positive control, the offspring of kr7.3, the plant

that showed consistent Mendelian segregation, was anal-

ysed first. Because half of its backcross progeny were

expected to be kanamycin sensitive, 12 seeds from a cap-

sule characterised genetically were grown on medium

without selection and their DNAs assayed individually by

PCR. Eight of the 12 samples yielded an amplicon (Fig-

ure 4b), consistent with a 1:1 ratio (P = 0.24813). A similar

result was observed (P = 0.24813, Figure 4(c) top panel),

for seedlings from the fourth capsule of kr7.2 (Table 2). In

contrast, DNA from seedlings from a capsule of kr7.1,

which showed high instability of kanamycin resistance,

yielded no amplicons of neo (Figure 4d). These results are

consistent with the previous study that determined dele-

tion of all or part of neo as the cause of loss of kanamycin

resistance (Sheppard and Timmis, 2009).

The presence of the engineered I-SceI sites and their

known flanking sequences allowed us to examine the

cause of instability of these de novo nupts in greater detail

than has been achieved in previous studies. Primers bar FP

and bar RP; ST FP and ST RP (Figure 4a), designed to

amplify neighbouring regions at chloroplast DNA integra-

tion sites in nuclear DNA, were used to assess the extent

of sequence deletion when neo was lost. Seedlings from

capsules of kr7.3 and kr7.2 presenting normal segregation

of kanamycin resistance gave the expected products from

PCR targeted to sections adjacent to the insertion site (Fig-

ure 4a–c top panel), confirming the stability of sequences

of both the chloroplast DNA integrant and its nuclear flank-

ing region. In contrast, seedlings from the fourth capsule

of kr7.1 (Table 2), which showed a complete lack of kana-

mycin-resistant progeny, yielded no PCR products using

these same primers (Figure 4a,d). Surprisingly, the

sequence missing revealed in the eighth seedling from the

fifth capsule of kr7.2 (Table 2) was found in regions neigh-

bouring the chloroplast DNA insertion site rather than in

the neo gene itself (Figure 4a,c bottom panel). To explore

the loss of nucleotides in the nupt lacking neo from kr7.2,

one primer located inside the chloroplast integrant com-

bined with a primer positioned in its nuclear flanking

region were employed (Figure 4a middle panel, P3R for

Figure 4. PCR analysis of kr7 lines showing genetic instability for kanamycin resistance. (a) Schematic representations of nupt flanking sections. Primers used

are labelled and their direction shown arrowed. Primer pairs bar FP and bar RP, ST FP and ST RP cover most of the bar gene and the stuffer fragment, respec-

tively. The primer pair DSBFP and DSBRP amplifies a region that includes the two I-SceI sites. P3R and P2R are primers specific for chloroplast integrants in

kr7.3 and kr7.2, respectively. Pnos and Tnos signify the nos promoter and nos terminator respectively. (b–d) PCR analysis of backcross progeny from three kr7

lines. DNAs prepared from the corresponding pISceI-neo seedlings were applied in the reaction. The abbreviations kr7.3_K+, kr7.2_K+ and kr7.3_K+ stand for

kanamycin-resistant seedlings from each line. The primer pair used as a control is described in Figure 2(b). Ve�, no template.



kr7.3 and P2R for kr7.2), and no products were forthcoming

(Figure 4c bottom panel). In contrast, amplicons generated

by this strategy showed the same pattern as neo and other

adjacent sequences present in capsules exhibiting stable

kanamycin resistance (Figure 4a–c top panel). These

results indicate that deletion occurs not only inside the

chloroplast DNA integrant, but also in the nuclear regions

nearby. Finally, one more primer pair (DSBFP and DSBRP)

encompassing the positions of both I-SceI sites was used

to further explore the nuclear instability near nupt (Fig-

ure 4a). In this strategy, amplicons were expected only in

pISceI-neo lines, not in plants of the kr7 lines showing

kanamycin resistance, emphasizing again that the sizes of

these de novo nupts are extremely large. As for seedlings

showing no amplicons of neo or its nearby region, no PCR

products were observed with the primer pair DSBFP and

DSBRP (Figure 4c bottom panel, and d) suggesting, in

common with all other PCR results, that large deletions are

possible in the nuclear regions flanking some nupts.

DISCUSSION

In this study, we demonstrate that chloroplast DNA inserts

into the nuclear genome during DSB repair in tobacco and

these inserts are comprised predominantly of complex

mixtures of disparate regions from the chloroplast gen-

ome. The integrants may include other, presumed nuclear

DNA sequences and all are joined together through micro-

homology (Figures 2a and 3). These findings support and

extend the previous suggestion that end joining of linear

DNA fragments creates mosaic integrants prior to, or dur-

ing, insertion into the nuclear genome (Noutsos et al.,

2005). These novel chloroplast integrants are large such

that they cannot be amplified in their entirety by standard

PCR methods (Figure 4b–d). In addition, three chloroplast

integrants inserted into an I-SceI site, and an intact I-SceI

site remained unchanged (Figure 4a). This observation is

different from DSB repair-meditated mitochondrial DNA

insertion events (Wang et al., 2012), in that HincII was not

used to remove the stuffer that contains three HincII sites

here as it was in the previous study (Lloyd et al., 2012).

After insertion, sequence loss may occur within the de

novo nupts and also in flanking nuclear DNA segments

within one generation of their formation, explaining the

genetic instability that characterises about half of the de

novo nupts.

It appears that primary insertions of organelle DNA are

initially large but, in many cases, they are reduced to

shorter stretches over very short time periods. However, it

remains a possibility that the large nupts are a feature of

the experimental selection for the entire neo gene and may

not represent the full spectrum of events.

Nucleotide substitutions were not detected as all the

nupt sequences remain identical to the corresponding

regions of the plastome. However, some nupts clearly sur-

vive for long periods of evolutionary time and substitu-

tions are seen at these older loci (Ayliffe and Timmis, 1992;

Richly and Leister, 2004; Michalovova et al., 2013; Yoshida

et al., 2014; Chen et al., 2015). Our results suggest that

scrambled nuclear organellar DNAs are generated through

deletion during the early stages of post-integration rather

than by longer term sequence decay. Of course, several to

many somatic cell divisions must have occurred before our

attempts to characterise nupt sequences and organization.

Thus we may be reporting their organization after an initial

period of great flux.

The novel demonstration that incorporation of chloro-

plast DNA can be accompanied by deletion of adjacent

non-nupt DNA, indicates that the process must be an

important player in dynamic nuclear genome evolution.

Because analyses of instability within nupts in a previous

study relied on the kanamycin resistance provided by neo

(Sheppard and Timmis, 2009), deletion of non-nupt DNA

also indicates that the frequency of deletion occurring in

nupts was underestimated. Considering the high frequency

of DNA movement from plastid to nucleus (one event in

between 11 000 or 16 000 pollen grains (Huang et al.,

2003; Sheppard et al., 2008)), high incidence of sequence

deletion within nupts not only counterbalances the result-

ing problem of increasing genome size, but also provides

more chance for prokaryotic plastid genes to gain the func-

tional elements required for nuclear expression. Moreover,

the intraspecific variation of nupts observed in the progeny

Table 2 Segregation of kanamycin resistance of progenies fromthe three kr7 lines backcrossed to female wild-type plants

Lines Capsule Resistant Sensitive P

kr7.1 1 2 168 ***2 3 88 ***3 7 176 ***4 1 164 ***5 5 76 ***

kr7.2 1 21 96 ***2 50 143 ***3 29 110 ***4 85 83 NS5 9 53 ***6 22 30 NS

kr7.3 1 58 55 NS2 21 24 NS3 47 43 NS4 50 51 NS5 49 43 NS6 59 57 NS7 44 42 NS8 56 52 NS

Seeds from individual capsules were grown on half-strength MSmedium supplemented with 150 mg/L of kanamycin. The P-valuessignify deviation from a 1:1 ratio of resistant:sensitive (v2 test).NS, not significant; ***P < 0.001.



(Figure 4c bottom panel) suggests that the majority of

sequence deletion occurs during mitosis rather than meio-

sis. It is formally possible that these deletions may be dif-

ferent in each seed of a capsule exhibiting kanamycin

sensitivity (Figure 4d), but it is more likely that local

sequence contexts determine and orchestrate a restricted

range of deletion outcomes.

Although the extent of deletion in nupts identified here

was characterized, the underlying mechanisms are still

obscure. Direct repeats are thought to be associated with

deletions occurring in nupt DNA (Noutsos et al., 2005), and

candidate short repeats were found in kr7.3 (Figure 2a).

Third generation sequencing technology with long read

lengths is required for a thorough understanding of the

instability of these nascent nuclear organelle DNAs.

EXPERIMENTAL PROCEDURES

Plant growth conditions

Nicotiana tabacum plants were grown with 16 h light/8 h darkcycle at 25°C in jars or 11 cm Petri dishes containing half-strengthMS medium. Soil grown plants were grown in a growth roomwith a 14 h light/10 h dark cycle and 25°C day/18°C night regime.UV-C treatment was performed as previously described (Rosaet al., 2013).

Generation of pISceI-neo

pISceI-neo contains a previously described chloroplast genomeinsert that is expressed as kanamycin resistance only after transferto the nucleus (Huang et al., 2003) and an inducible DSB systemin the nucleus (Figure 1a). The nuclear inducible DSB system wasas described previously (Lloyd et al., 2012) with the neo genereplaced by a selectable marker bar and a stuffer fragment ofknown sequence (Figure 1b).

Experimental induction of DSBs and kanamycin selection

Induction of I-SceI was described previously (Lloyd et al., 2012).Briefly, 2-week-old seedlings grown in jars were sprayed with0.7 M ethanol and for 4 days allowed to produce DSBs. Leafexplants (~25 mm2) were then placed on 11 cm Petri dishes con-taining regeneration medium containing 400 mg/L kanamycin.Resistant shoots were moved to half-strength MS medium to rootand later transferred to soil.

Seedling selection was performed by growing surface-steri-lized seeds on half-strength MS medium supplemented with150 mg/L kanamycin (Wang et al., 2012), and plates were placedat 25°C with a 16 h light/8 h dark cycle. The significance of devi-ation from the expected Mendelian ratio was calculated using av2 test.

DNA preparation, PCR assays and genome walking

DNA was prepared employing a DNeasy Plant Mini Kit (Qiagen,Shanghai, China), and PCR amplification was performed usingTaq DNA polymerase (TaKaRa, Beijing, China) according tostandard protocols. TaqMan real-time PCR was performed asdescribed previously (Bubner and Baldwin, 2004). Genomewalking utilized a GenomeWalker Universal Kit (Clontech, Moun-tain View, CA, USA) in accordance with the manufacturer’s

instructions. Details of the primers used are listed in Table S3.Sequence data for this article can be found in the GenBank datalibraries under accession numbers from KU240015 to KU240017.

ACKNOWLEDGEMENTS

We thank Mathieu Rousseau-Gueutin and Yuan Li for helping withplant transformation and TaqMan real-time PCR, respectively. Thiswork was funded by the National Science Foundation of China(31401077), Natural Science Foundation of Jiangxi Province(20171ACB20001) and a Nanchang University Seed Grant for Bio-medicine (9202–0210210807).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

D.W. and J.N.T conceived the study, supervised the experi-

ments, and wrote the article; D.W., J.B.G., Z.W., S.T.Y. and

R.D. performed the experiments; I.R.S., D.W., J.K.Z and

J.N.T. contributed new reagents and analytic tools; D.W.,

J.B.G. and J.N.T analysed data.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. The comparative copy number of bar in pISceI-neolines as determined by real-time quantitative PCR. The least valueof copy number is appointed as one.

Table S1. Plastid-to-nucleus DNA transfer in tpGUS seedlings.

Table S2. Segregation of herbicide Basta resistance of self-crossedpISceI-neo-9, pISceI-neo-10 and pISceI-neo-13 lines.

Table S3. Primers used in this study.

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