Download Insertional mutagenesis using Tnt1

Plant Physiology Preview. Published on July 29, 2013, as DOI:10.1104/pp.113.221903
Insertional mutagenesis using Tnt1 retrotransposon in potato
Saowapa Duangpan1, Wenli Zhang1, Yufang Wu1, Shelley H. Jansky1,2, and Jiming Jiang1,*
1
2
Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA
United States Department of Agriculture-Agricultural Research Service, Vegetable Crops Research Unit, Madison,
WI 53606, USA
______________________________________
*Corresponding author; e-mail [email protected]
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Copyright 2013 by the American Society of Plant Biologists
ABSTRACT
Insertional mutagenesis using T-DNA or transposable elements, which is an important
tool in functional genomics and is well established in several crops, has not been developed in
potato. Here we report the application of the Tnt1 retrotransposon as an insertional mutagen in
potato. The Tnt1 retrotransposon was introduced into a highly homozygous and self-compatible
clone, 523-3, of the diploid wild potato species Solanum chacoense. Transposition of the Tnt1
elements introduced into 523-3 can be efficiently induced by tissue culture. Tnt1 preferentially
inserted into genic regions in the potato genome and the insertions were stable during sexual
reproduction, making Tnt1 an ideal mutagen in potato. Several distinct phenotypes associated
with plant stature and leaf morphology were discovered in mutation screening from a total of 38
families derived from Tnt1-containing lines. We demonstrate that the insertional mutagenesis
system based on Tnt1 and the 523-3 clone can be expanded to the genome-wide level to
potentially tag every gene in the potato genome.
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INTRODUCTION
Insertional mutagenesis is one of the most important tools in plant functional genomics.
Application of T-DNA, the transfer DNA of the Ti plasmid of Agrobacterium tumefaciens, was
the first and the most successful methodology of genome-wide insertional mutagenesis in plants.
Many T-DNA lines were developed in two of the most important model plant species,
Arabidopsis thaliana and rice (Oryza sativa) (Krysan et al., 1999; Jeon et al., 2000; Alonso et al.,
2003; Sallaud et al., 2004). These T-DNA stocks have served as the foundation for the
identification and characterization of numerous genes in these two species. A major limitation of
the T-DNA based technique is the requirement for a highly efficient transformation system to
generate a large number of transgenic lines. Unfortunately, in many plant species,
Agrobacterium-based transformation is either not yet developed or is not efficient enough to
produce a sufficient number of T-DNA lines that would allow a genome-wide gene tagging.
Several transposable elements (TEs) have been used for insertional mutagenesis in plants,
including the Activator (Ac) and Mutator (Mu) transposons in maize (Zea mays) (Walbot, 1992),
the Tam3 transposon in Antirrhinum majus (Luo et al., 1991), the Tos17 retrotransposon in rice
(Hirochika, 2010), and the LORE1 retrotransposon in Lotus japonicus (Fukai et al., 2012). Most
remarkably, the Tnt1 retrotransposon, originally identified in tobacco (Nicotiana tabacum)
(Grandbastien et al., 1989), has been successfully used in insertional mutagenesis in several
heterologous species, including A. thaliana (Courtial et al., 2001), Medicago truncatula
(d'Erfurth et al., 2003), lettuce (Lactuca sativa) (Mazier et al., 2007), and soybean (Glycine max)
(Cui et al., 2013). Tnt1 was used to generate ~12,000 independent lines which represent over
300,000 insertions in M. truncatula (Tadege et al., 2008; Cheng et al., 2011). Many M.
truncatula genes have been identified through forward and reverse genetics approaches using the
Tnt1 retrotransposon insertion population (Pang et al., 2009; Zhao et al., 2010; Laurie et al.,
2011; Tadege et al., 2011; Zhou et al., 2011; Cheng et al., 2012; Bourcy et al., 2013).
Potato (Solanum tuberosum) is one of the most important food crops in the world.
Cultivated potato is an autotetraploid (2n=4x=48) with a highly heterozygous genome. These
characteristics make potato a poor model for both forward and reverse genetics research. Very
few potato genes have been cloned using the traditional map-based cloning strategy. The lack of
fertile homozygous clones makes insertional mutagenesis infeasible in potato. However, the
recent sequencing of the potato genome (The Potato Genome Sequencing Consortium, 2011) has
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significantly changed the status of potato genetics and genomics research. Highly efficient
genotyping systems based on DNA microarrays or DNA sequencing will make genetic mapping
and association mapping much more efficient in potato (Hamilton et al., 2011; Felcher et al.,
2012). We expect an acceleration of forward genetics-based gene identification in potato. A
genome-wide mutagenesis system is urgently needed for the potato research community. Here
we report the development of an insertional mutagenesis system in potato using the Tnt1
retrotransposon. This system is built on a highly homozygous and self-compatible clone (523-3)
of the diploid potato species Solanum chacoense (2n=2x=24), one of the most widespread wild
Solanum species (Miller and Spooner, 1996). S. chacoense is sexually compatible with diploid
cultivated potato (Leue and Peloquin, 1980; Hermundstad and Peloquin, 1985). It has been an
important germplasm source for the improvement of potato cultivars, especially those for use in
the processing market (Love et al., 1998). We demonstrate that this system can be used to
potentially tag every potato gene and serve as an important foundation for potato functional
genomics research.
RESULTS
Transposition of Tnt1 occurred during in vitro transformation of potato
A self-compatible clone of S. chacoense was first reported by Hosaka and Hanneman in
1998 (Hosaka and Hanneman, 1998, 1998). An S7 clone from the Hanneman program, 523-3,
was derived from seven generations of selfing from the original S. chacoense clone. The high
level of homozygosity of 523-3 was confirmed by a genome-wide SNP-based genotyping
(unpublished data) and the uniform phenotype of the progenies derived from selfing 523-3. The
523-3 clone readily self-pollinates and can generate an average of 100 seeds per fruit. More
importantly, this clone can produce tubers in both greenhouse and field conditions.
We developed several transgenic 523-3 lines by transforming internode explants using
Agrobacterium strain GV3101 containing an autonomous copy of Tnt1 in plasmid Tnk23
(d'Erfurth et al., 2003) (Figure 1A). A total of 17 transgenic plants (T0) was generated and
confirmed by polymerase chain reaction (PCR) for the presence of both nptII gene and Tnt1specific sequences. Genomic DNA was isolated from the T0 plants, double digested with HindIII
(three sites in construct, Figure 1A) and EcoRI (no site in construct), and hybridized sequentially
by a nptII gene probe (Figure 1B) and a Tnt1-specific probe (Figure 1C), respectively.
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All T0 lines contained at least one copy of the transgenic Tnt1 element, based on the
number of bands hybridized to the nptII gene. The numbers of transgenic Tnt1 elements among
the T0 lines ranged from one to six, based on the number of nptII gene-specific bands on the
Southern blots (Figure 1B). The Tnt1-specific probe hybridized to two DNA fragments derived
from each Tnt1 element, a common 879-bp fragment and another fragment that represents an
individual Tnt1 element (Figure 1C). The 879-bp fragment was detected in every T0 line. The
numbers of total Tnt1 elements among the T0 lines ranged from one to >20, based on Tnt1specific hybridization bands (Figure 1C). The total numbers of Tnt1 elements in all T0 lines,
except line CT1, were more than the numbers of the original transgenic Tnt1 elements. These
results suggest that some of the transgenic Tnt1 elements transposed immediately after
integrating into the 523-3 genome, resulting in additional Tnt1 elements that are not associated
with the nptII gene.
Tnt1 transposition can be induced by in vitro regeneration
Tissue culture-induced retrotransposition of Tnt1 was previously reported in several plant
species (Courtial et al., 2001; d'Erfurth et al., 2003; Mazier et al., 2007; Cui et al., 2013). We
wanted to confirm whether tissue culture can also induce Tnt1 transposition in the potato
genome. The T0 line CT1, which contains a single copy of Tnt1 (Figure 1), was selected for in
vitro regeneration experiments. Internode explants were cut from in vitro CT1 plants and were
transferred to regeneration media. The explants were kept in an incubator and were transferred
to new media every two weeks. Regenerated shoots were then transferred to root-inducing
medium. We developed a total of 13 regenerated T1 plants (CT1:1 to CT1:13). Southern blot
hybridization of the regenerated plants revealed that each of the 13 T1 plants contains at least one
additional copy of Tnt1 compared to CT1 (Figure 2). The numbers of additional Tnt1 elements
ranged from one to >20 among the 13 plants. These results showed that tissue culture can
effectively induce Tnt1 transpositions in potato.
Tnt1 insertions are stable during sexual reproduction
Reverse genetics using TE as a mutagen involves self-pollination to develop homozygous
insertional mutants. Therefore, the stability of inserted TEs during sexual reproduction is
essential for maintaining the mutation. To investigate the stability of the Tnt1 elements in the
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potato genome, we conducted Southern blot hybridization analysis of 80 selfed progenies
derived from each of four T1 lines (CT1:3, CT1:4, CT1:5 and CT1:7). For each T1 line, DNA
samples isolated from 20 selfed progenies were pooled, digested with HindIII and EcoRI, and
hybridized with the Tnt1-specific probe. We did not observe any additional hybridization bands
in these progenies compared to the parental T1 lines (Figure 3A), suggesting that the Tnt1
elements in these four T1 lines were stable during sexual reproduction.
To confirm that the potentially re-transposed Tnt1 element(s) is detectable in this pooled
hybridization approach, we developed a blot by mixing an equal amount of DNA from each of
19 progenies from CT1:3 with DNA from CT1:4. The Tnt1 elements associated with CT1:4 can
be unambiguously detected in the mixed DNA sample (Figure 3B).
Retrotransposition efficiency does not depend on number of Tnt1 elements
We were interested to know if the average number of transpositions during tissue culture
can be controlled by using lines with different copy numbers of the Tnt1 element. We conducted
regeneration experiments using four lines (CT1, CT1:4, CT1:3 and CT6), which contain 1, 3, 8
and 9 Tnt1 copies, respectively. The Tnt1 copy numbers of 10 regenerated progenies from each
of these four lines were determined by Southern blot hybridization. Interestingly, we did not
observe a correlation between initial Tnt1 copy number and the average Tnt1 copy number in the
regenerated progenies (Table 1). For example, CT1 contains a single Tnt1 element, while the 10
regenerated progenies from CT1 contained an average of 10.4 Tnt1 elements, resulting in an
average of 9.4 new insertions. In comparison, CT6 contains 9 copies of Tnt1, and the 10
progenies from CT6 contained an average of 13.5 Tnt1 elements, resulting in only an average of
4.5 new insertions.
All progenies from CT1 (one Tnt1 copy) and CT1:4 (three Tnt1 copies) contained more
Tnt1 copies than the parental lines, suggesting that retrotransposition occurred in every plant. In
contrast, three plants from CT1:3 (8 Tnt1 copies) and three plants from CT6 (9 Tnt1 copies)
contained the same numbers of Tnt1 as parental lines. These results suggest that most of the
Tnt1 elements in CT1:3 and CT6 were not activated by tissue culture. Thus, the
retrotransposition efficiency of a line is likely dependent on how easily the Tnt1 element(s) can
be activated rather than the number of original Tnt1 elements.
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Tnt1 preferentially inserts into genic regions in potato genome
We developed a Tnt1-seq method to map the genomic positions of a large number of
Tnt1 insertions (Figure 4). Genomic DNA was isolated from 70 T0 and T1 lines. Tnt1-flanking
DNA sequences were isolated by two rounds of DNA walking using PCR. The PCR products
were then pooled and used for preparation of a paired-end Tnt1-seq library (Figure 4). To check
the quality of the library, we cloned and sequenced 20 randomly selected DNA fragments from
this library. All clones contained the expected left- and right-board adapters. A total of nine
independent inserts were identified (some inserts were identical). This library was sequenced
using the Illumina MiSeq platform, resulting in a total of 5,931,429 paired-end sequence reads
(150 bp). The sequences from the adapter immediately adjacent to partial Tnt1 sequence were
found to contain sequence errors. Thus, only the sequences adjacent to the left adapter were used
in mapping. We mapped 474,424 sequences to the DM1-3 reference genome
(PGSC_DM_v3_2.1.11) (The Potato Genome Sequencing Consortium, 2011), corresponding to
1,667 insertion sites, including all nine inserts identified by manual cloning and sequencing.
Thus, the 70 different T0 and T1 lines contain an average of 24 Tnt1 insertions. Mapping of the
1,667 insertion sites revealed a near random distribution on all 12 potato chromosomes (Figure
5).
A total of 533 insertions (33%) were associated with genic regions in the DM1-3
reference genome, including 15% in exons, 13% in introns, 3% in 3’ UTR, and 2% in 5’UTR.
The genic sequences account for approximately 14% of the potato genome, thus, Tnt1 elements
preferentially insert in genic regions. In comparison, 78.6% of the Tnt1-flanking sequences
matched coding sequence in M. truncatula (Tadege et al., 2008). The difference in the
frequencies of Tnt1 insertions into genic regions in the two species may be caused by the
differences in the accuracy of annotations of the two genomes, and the lengths of the Tnt1flanking sequences generated for analysis. The DM1-3 genome was based on the cultivated
diploid species Solanum tuberosum Group Phureja (The Potato Genome Sequencing
Consortium, 2011). The level of sequence divergence between S. phureja and S. chacoense is
unknown. The sequence divergence between the two species may prevent accurate mapping of
the relatively short Illumina sequence reads derived from S. chacoense to the DM1-3 genome.
Preliminary screening of mutations caused by Tnt1 insertions
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To investigate the potential of Tnt1 as an insertional mutagen, we developed 38 families
by selfing independent T0 and T1 lines. We sowed 30 seeds from each family and then grew 15
randomly-selected plants in a greenhouse, along with progenies from wild type 523-3. The
germination rate and all visible phenotypes were recorded.
Plants from five families showed seven phenotypes that were unambiguously
distinguishable from the wild type 523-3 (Table 2, Figure 6). The germination rate of wild type
523-3 seeds was 90%. In contrast, seeds from the CT4:33 family showed a much lower
germination rate of 60%. A Tnt1 element in CT4:33 possibly landed in a gene that is essential to
embryo/seed development, thus, progenies homozygous for this insertion may not be viable.
Extreme dwarf plants were observed in the CT4:20 family. These plants showed a severely
stunted growth and died approximately one month after being transplanted into individual pots.
Plants in several families showed distinctive leaf shapes. Plants in the CT4:20 family had
large, round leaves that curled inward (Figure 6D). Some plants from CT4:50 and CT4:33 had
short internodes and as a consequence were bushy (Figures 6A, 6B). Small and inward-curling
leaves were observed in plants from the CT41 family (Figure 6C).
DISCUSSION
A mutation caused by a T-DNA or TE insertion into a gene will interrupt only one of the
two homologues in diploid species. Selfing of the heterozygous mutation line is required to
identify the homozygous recessive mutant. Therefore, insertional mutagenesis is most
appropriately applied only in homozygous and self-compatible inbred lines. Most asexually
propagated species, such as potato, contain highly heterozygous genomes and are often selfincompatible, thus, cannot benefit from insertional mutagenesis technology. Ishizaki and Kato
(2005) introduced the Tto1 retrotransposon from tobacco into a sterile diploid potato clone.
However, transposition of Tto1 was not induced by tissue culture in potato (Ishizaki and Kato,
2005), although Tto1 can be activated by tissue culture in rice and Arabidopsis (Hirochika et al.,
1996; Okamoto and Hirochika, 2000).
An alternative approach in plant species with a heterozygous genome is to develop
activation tagging lines by transforming a construct containing a strong promoter (Weigel et al.,
2000). Insertion of a strong promoter may result in an over-expression of an adjacent gene or a
gain-of-function phenotype for the gene. Such a dominant phenotype can be observed in T0
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transgenic lines, avoiding the selfing process. The Canadian Potato Genome Project generated
10,000 activation-tagged potato lines (Regan et al., 2006). However, the phenotype-inducing
efficiency of the activation-tagging approach is not known in potato. In addition, each tagging
line has to be individually transformed. Thus, the value of activation-tagging technology
remains to be seen in potato.
We have demonstrated that the Tnt1-based insertional mutagenesis system using S.
chacoense clone 523-3 can be used to tag individual potato genes. This is because: (1) The 5233 clone can be easily transformed and regenerated. Thus, many regenerated lines from a selected
starter line can be readily developed, and it will be feasible to develop a large number of Tnt1
lines to saturate the entire potato genome (see below); (2) Sufficient amount of seeds from each
regenerated line can be readily produced by self pollination. Each plant can produce dozens of
berries, each of which typically contains about 100 seeds. Potato seeds are small and can be
stored in small size tubes or envelopes in a -20°C freezer for decades; (3) The 523-3 plants are
much smaller than typical tetraploid potato cultivars, and can be grown in small size pots for
large scale screening in greenhouses; (4) The 523-3 clone tuberizes well in both greenhouses and
field, even under long photoperiod conditions (Kittipadukal et al., 2012). Thus, tuber-related
traits can be screened.
Tnt1-based genome-wide mutagenesis has already been well demonstrated in M.
truncatula (Tadege et al., 2008; Cheng et al., 2011). The Tnt1 element in potato showed similar
activity and behavior as that documented in other plant species, including activation by tissue
culture, stability during sexual reproduction, and preferential transposition into genic regions
(Courtial et al., 2001; d'Erfurth et al., 2003; Mazier et al., 2007; Cui et al., 2013). We examined
several factors that may affect the Tnt1 retrotransposition. We observed that the Tnt1 copy
numbers in the new regenerants do not correlate with the length of time that the callus was
grown on the regeneration medium (data not shown). This is consistent with a similar study in
M. truncatula (Tadege et al., 2008). The new Tnt1 copy numbers in regenerants were not
correlated with the original Tnt1 copy numbers in the parental lines (Table 1). It was
demonstrated in A. thaliana that the transcription of Tnt1 was often silenced in plants containing
numerous copies. The silencing of Tnt1 was associated with 24-nucleotide short-interfering
RNAs (siRNAs) targeting the promoter localized in the LTR region and with the non-CG site
methylation of these sequences (Perez-Hormaeche et al., 2008). Our data are in agreement with
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those in A. thaliana. Potato lines with more copies of Tnt1 generated fewer new Tnt1 copies
during regeneration than CT1, a T0 line containing a single copy of Tnt1 (Table 1). Thus, most
Tnt1 copies in lines with numerous insertions are likely silenced and cannot be activated by
tissue culture.
Our preliminary mutation screening revealed the potential of the Tnt1-based system to
generate potato mutants. Seven distinct phenotypic changes were observed in five of the 38
T0/T1 families. Each phenotype was observed in 3-5 plants in a particular family (Table 2),
indicating that each change was likely caused by a recessive mutation of a single gene. More
subtle changes were observed in several additional families. In the screening of the M.
truncatula mutant population, approximately 30% of the lines showed new recognizable
phenotypes (Tadege et al., 2008). However, the screening involved inoculation with arbuscular
mycorrhizal fungi and Sinorhizobium meliloti and was performed under low-nitrogen and lowphosphorus conditions. In the current study, only a few families were screened, under regular
greenhouse growing conditions, but the Tnt1-based system appears to have a similar phenotypechanging efficiency as in M. truncatula.
Our ultimate goal is to develop a 523-3 population that is large enough to tag every
potato gene with a Tnt1 element. The average number of Tnt1 insertions will directly affect the
number of Tnt1-tagged 523-3 lines needed for mutation saturation. The transposed Tnt1 copy
numbers of the regenerants derived from CT1 ranged from 1 to more than 20, which is similar to
the numbers observed in other plant species. The transposition events ranged from 4 to more
than 30 in M. truncatula (d'Erfurth et al., 2003), 0 to 26 in A. thaliana (Courtial et al., 2001), and
4 to 19 in soybean (Cui et al., 2013). A total of 1,667 insertions was recovered from 70 Tnt1habouring lines, averaging 24 insertions per line. The possibility of finding an insertion in a
given gene can be estimated by P = 1-(1-[X/G])n , where P = The possibility of finding an
insertion in a given gene, X = the length of the gene in kilobases, G = genome size in kilobases,
and n = number of insertions in the population (Krysan et al., 1999). Considering that the size of
the potato genome is 845 Mb and the average gene size is 2.5 kb, we estimate that approximately
one million insertions are needed to achieve 95% genome saturation. If each Tnt1 tagging line
contains 24 insertions, then 42,000 lines will be required. However, this equation assumes the
random insertion of Tnt1. Therefore, the number of mutants needed to saturate the potato gene
complement would be significantly reduced due to the preferential insertions of Tnt1 in genic
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regions. In a comparison, the genome size of M. truncatula is approximately 500 Mb,
approximately 90% of the M. truncatula genes were covered by a population of ~12,000 Tnt1
insertion lines (Cheng et al., 2011).
MATERIALS AND METHODS
Plant material
S. chacoense clone 523-3, developed by Robert Hanneman in Madison, Wisconsin
(Hosaka and Hanneman, 1998, 1998), was used for transformation. For germination, seeds were
soaked in 1500 ppm gibberellic acid for 24 h to break dormancy. The seeds were then sown in
square pots (10 cm x 10 cm) using soil-less potting mix. After 4 weeks, the seedlings were
transplanted to individual pots and maintained in a greenhouse. Growth conditions were a 16
hour photoperiod and 18°C and 15°C day and night temperatures, respectively.
Potato transformation and regeneration
A. tumefaciens strain LBA4404 was transformed with the Tnk23 vector (Lucas et al.,
1995). Internode explants from in vitro 523-3 plantlets were then transformed using the
LBA4404 strain following standard Agrobacterium-mediated potato transformation (Bhaskar et
al., 2008). The transformed shoots were transferred to rooting medium without antibiotics. In
vitro regeneration was performed by cutting the internodes of each Tnt1-containing plant into
explants approximately 5 mm long. The explants were then placed on medium used for
transformation but without any antibiotics and transferred to new medium every 2 weeks.
Regenerated shoots were transferred to rooting medium.
Identification of plants containing Tnt1 elements
Genomic DNA was extracted from leaf tissue of transgenic plants grown in greenhouses
using Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, California). The oligonucleotides
KAN1 5’-CCAACGCTATGTCCTGATAG-3’ and KAN2 5’-TTTGTCAAGACCGACCTGTC3’ were used to verify the presence of nptII gene, and oligonucleotides LTR1 5’ATGTCCATCTCATTGAAGAAGTA-3’ and LTR2 5’-GGGAATAAACCCCTTACCAAAA-3’
were used to verify the presence of Tnt1 element. PCR conditions for both primer pairs were
94°C, 30 sec; 55°C, 30 sec;72°C, 1 min, 30 cycles.
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For Southern blot hybridization, approximately 20 μg of genomic DNA was digested
with HindIII and EcoRI, and then blotted on a membrane. The blots were probed with a 528-bp
fragment complementary to nucleotides 141 to 669 of nptII gene or with 194-bp fragment
complementary to nucleotides 1 to 194 of Tnt1. The blots were hybridized overnight at 65°C
using standard protocols (Sambrook et al., 2001).
Identification of Tnt1 flanking region
DNA walking on pooled genomic DNA from T0 and T1 lines was performed using DNA
walking SpeedUPTM Kit II (Seegene, Seoul, South Korea). Tnt1-specific primers used in the first
and second PCR were TSP1 5’-CCCGAGAGGAGCAACTGATA-3’ and TSP2 5’AAGAAATGAGAGTTGAAGCTCTCC-3’, respectively. The PCR products from the second
round of DNA walking were used for preparation of paired-end Tnt1-seq library following the
standard protocol provided by Illumina. Briefly, the protocol includes end blunting, adding an
“A” tail, ligation of paired-end adaptors and enrichment of the flanking region of Tnt1 insertions.
The flanking sequences immediately adjacent to Tnt1 were amplified using the Tnt1 enrichmentspecific primer 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCC
CTACACGACGCTCTTCCGATCTCACATGCCTAATACTTCTTCAATGAG-3’ and adapterspecific primer 5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCT
GCTGAACCGCTCTTCCGATCT-3’. The amplification programs were as follows: 30s, 98°C;
18 cycles of 10s, 98°C, 30s, 65°C, 30s, 72°C; 5 min, 72°C. DNA fragments between 300-550 bp
were cut from a 2% agarose gel and purified using MinElute PCR purification columns (Qiagen,
Valencia, California). The quality of purified DNA (Tnt1-seq library) was checked by cloning
into pCR™4-TOPO® TA vector (Invitrogen, Carlsbad, California). Twenty clones were
randomly selected and sequenced by Sanger sequencing. Tnt1-seq library was then quantified
using an Agilent Bioanalyzer 2100 and sequenced on the Illumina MiSeq platform. The
sequence reads were mapped to the DM1-3 reference genome using BLAT (Kent, 2002).
ACKNOWLEDGMENTS
We thank Marie-Angele Grandbastien and Pascal Ratet for sharing the Tnk23 vector, thank
Kiran Mysore for sharing his experience on Tnt1-mediated mutagenesis in Medicago. We are
grateful to Scott Jackson and Christian Hans for helping on the Illumina MiSeq sequencing. S.D.
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was supported by the Strategic Scholarships for Frontier Research Network of Thailand’s
Commission on Higher Education. This work was supported by grant IOS-1237969 from the
National Science Foundation and Hatch funds to J.J.
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Figure legends
Figure 1. Transpositions of Tnt1 elements in transgenic potato lines. (A) Structure of the Tnk23
T-DNA plasmid. The two small horizontal arrows point to the Tnt1-specific probe designed
from the LTR region. Vertical black arrows point to the three HindIII sites within the Tnt1
element. Each Tnt1 element will result in a 879-bp fragment after HindIII digestion. (B)
Southern blot hybridization of five T0 lines using an nptII-specific probe. Genomic DNAs were
double digested with EcoRI and HindIII. Each hybridization band represents a single transgenic
Tnt1 site. (C) Southern blot hybridization of the same blot as (B) using the Tnt1-specific probe.
The expected 879-bp fragment, as indicated by black arrow, was observed in every line. Each of
the remaining bands represents an independent Tnt1 element.
Figure 2. Tissue culture-induced transposition of Tnt1 in potato. DNAs from the original
transgenic line CT1 and four regenerated lines from CT1 were digested with both EcoRI and
HindIII and hybridized with the Tnt1-specific probe. Arrows point to the two bands derived
from the original Tnt1 element from CT1. Other bands represent new Tnt1 elements
retrotransposed from the original copy.
Figure 3. Stability of Tnt1 insertions during sexual reproduction. (A) Genomic DNAs were
pooled from 20 selfed progenies from one of the four lines (CT1:3, CT1:4, CT1:5, CT1:7),
digested with EcoRI and HindIII, and hybridized to a Tnt1-specific probe. An 879-bp fragment
can be observed in each lane. The hybridization patterns associated with the selfed progenies are
identical to the patterns of the parental lines. (B) Southern blot hybridization analysis of two
DNA samples. Lane 1: DNA isolated from CT1:3; Lane 2: pooled DNA from 19 progenies of
CT1:3 and genomic DNA from CT1:4. Arrows point to the two bands associated with CT1:4.
Figure 4. Development of Tnt1-seq library. DNA walking was performed on pooled genomic
DNA from 70 T0 and T1 lines. Each of the ACP primers (DW-ACP1, DW-ACP2, DW-ACP3,
DW-ACP4) and the TSP1 (Tnt1-specific primer 1) primer were used to amplify the target region
from pooled genomic DNAs in the first PCRs. The second PCR reaction using the DW-ACP-N
primer and the TSP2 (Tnt1-specific primer 2) primer was performed using the first PCR product
as a template. The PCR products were then combined and subjected to DNA end blunting,
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adding adenine (A) to 3’ end, and paired-end adaptor ligation. The third PCR reaction was
conducted to enrich the Tnt1-flanking sequences. Each DNA fragment in the Tnt1-seq library
contains an adaptor-specific primer (black), DW-ACP-N primer (red), Tnt1 flanking region
(white), Tnt1 sequence from amplification (green), synthesized Tnt1 sequence, which is part of
the Tnt1 enrichment specific primer (purple) and part of the Tnt1 enrichment specific primer
which was used for Illumina sequencing (sky blue).
Figure 5. Distribution of Tnt1 insertion sites in the DM1-3 genome (PGSC_DM_v3_2.1.11).
Vertical lines above or below the line represent the positions of Tnt1 insertions in the forward or
reverse strand of the DM1-3 reference genome. Thick horizontal bars associated with
chromosomes 1, 2, 5, and 12 indicate large physical gaps in the pseudomolecules.
Figure 6. Visible phenotypes observed in selfed progenies from some T0 and T1 families
compared to wild type 523-3 plants. (A) Short internodes of a plant from the CT4:50 family
(left) and internodes from a wild type 523-3 plant (right). (B) Bushy phenotype of a plant from
the CT4:33 family (left) and a wild type 523-3 plant (right). (C) A plant with small, inwardcurling leaves from the CT41 family (left) and a wild type 523-3 plant (right). (D) Rounded,
inward-curling leaves of plants from the CT4:20 family (left) and wild type leaves (right).
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Table 1. Tnt1 copy numbers in progenies derived from four start lines
Progenies
Total Tnt1 Progenies Total Tnt1
Progenies
Total Tnt1
Progenies
Total Tnt1
from CT1
copies
from CT6
copies
from CT1:3
copies
from CT1:4
copies
CT1:1
>201
CT6:5
11
CT1:3:1
8
CT1:4:2
15
CT1:3
9
CT6:7
16
CT1:3:7
>20
CT1:4:3
>20
CT1:4
3
CT6:9
9
CT1:3:5
14
CT1:4:4
8
CT1:5
2
CT6:11
16
CT1:3:13
12
CT1:4:5
7
CT1:6
2
CT6:15
13
CT1:3:12
10
CT1:4:8
8
CT1:7
10
CT6:17
9
CT1:3:11
8
CT1:4:12
>20
CT1:10
>20
CT6:22
15
CT1:3:10
8
CT1:4:13
12
CT1:12
16
CT6:23
9
CT1:3:6
>20
CT1:4:15
16
CT1:14
>20
CT6:24
18
CT1:3:4
11
CT1:4:16
8
CT1:16
2
CT6:29
19
CT1:3:2
>20
CT1:4:42
9
Average2
10.4
13.5
13.1
12.3
Parental Tnt1
1
9
8
3
copies
New Tnt1
9.4
4.5
5.1
9.3
copies
1
>20: If a plant contains more than 20 Tnt1 elements, the number cannot be accurately counted based on Southern blot
hybridization.
2
Average: 20 were used in calculation if a plant contained >20 copies.
Table 2. Putative mutation phenotypes associated with families selfed from T0 and T1 lines
Phenotype
Family
Number of plants showing
the phenotype1
Low germination rate
CT4:33
60% germination rate
Extreme dwarf and stunted growth
CT4:20
5
Bushy, short internode
CT4:50, CT4:33
4, 4
Small leaf
CT1:13
3
Small and curling-up leaf
CT41
3
Round and curling-up leaf
CT4:20
3
1
Number of plants from a total of 15 in each family.
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