The Plant Journal (1999) 19(6), 711±717
TECHNICAL ADVANCE
Rapid isolation of plant Ty1-copia group retrotransposon
LTR sequences for molecular marker studies
Stephen R. Pearce1,2,*,², Caroline Stuart-Rogers2,
Maggie R. Knox3, Amar Kumar1, T.H. Noel Ellis3 and
Andrew J. Flavell2
1
Scottish Crop Research Institute, Invergowrie, Dundee
DD2 5DA, UK,
2
Department of Biochemistry, University of Dundee,
Dundee DD1 4HN, UK, and
3
John Innes Centre, Colney, Norwich NR4 7UH, UK
Summary
The terminal sequences of long-terminal repeat (LTR)
retrotransposons are a source of powerful molecular
markers for linkage mapping and biodiversity studies.
The major factor limiting the widespread application of
LTR retrotransposon-based molecular markers is the
availability of new retrotransposon terminal sequences.
We describe a PCR-based method for the rapid isolation
of LTR sequences of Ty1-copia group retrotransposons
from the genomic DNA of potentially any higher plant
species. To demonstrate the utility of this technique, we
have identi®ed a variety of new retrotransposon LTR
sequences from pea, broad bean and Norway spruce.
Primers speci®c for three pea LTRs have been used to
reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
Introduction
Retrotransposons are ubiquitous in plant genomes (Flavell
et al., 1992a; Kunze et al., 1997; Voytas et al., 1992). They are
classi®ed into two major families, based on the presence or
absence of long-terminal repeats (LTRs) at the transposon
ends (Boeke and Corces, 1989). LTR retrotransposons are
present as large heterogeneous populations within plants
(Flavell et al., 1992b; Matsuoka and Tsunewaki, 1996; Pearce
et al., 1997; Vanderwiel et al., 1993). These retrotransposon
populations are highly plastic, showing great variation in
copy number and genomic localization between even
closely related species (Casacuberta et al., 1997; Kumar
et al., 1997; Pearce et al., 1996a; Pearce et al., 1996b). This
Received 2 February 1999; revised 16 July 1999; accepted 19 July 1999.
*For correspondence (fax +44 1273 606755;
e-mail [email protected]).
²
Present address: School of Biological Sciences, University of Sussex,
Falmer, Brighton BN1 9QG, UK.
ã 1999 Blackwell Science Ltd
variation has been exploited in retrotransposon-based
molecular marker systems in pea and barley (Ellis et al.,
1998; Flavell et al., 1998; Kalendar et al., 1999; Waugh et al.,
1997). Several of these methods yield signi®cant improvements over current marker technologies (Ellis et al., 1998;
Waugh et al., 1997).
Retrotransposon-based markers require sequence information from the terminal regions of the mobile element.
This is only available for a few of these mobile elements in
a small number of plant species (reviewed by Kunze et al.,
1997). The scarcity of LTR terminal sequence is therefore
the main obstacle preventing the extension of this
approach to all plants. In this report, we describe a method
which rapidly delivers Ty1-copia retrotransposon LTR
sequences directly from the genomic DNA of any higher
plant. Using this technique, we have isolated 23 novel LTR
sequences from two dicotyledonous plants (pea and broad
bean) and a gymnosperm (Norway spruce). Primers
speci®c to four of these new pea elements have been
used to generate polymorphic SSAP markers in Pisum.
Results
General outline of the technique
This technique was devised to enable the rapid isolation of
Ty1-copia retrotransposon terminal sequences. The LTR
sequences of Ty1-copia retrotransposons are present at
each end of the retrotransposon and are identical
(Figure 1). The LTRs contain no conserved motifs which
would allow their direct ampli®cation by PCR. The nearest
conserved sequence motifs to the 3¢ LTR is within the
adjacent RNaseH gene. We isolate and characterize PCR
fragments between the RNaseH motif and a restriction site
in the adjacent LTR sequence. Sequence analysis of these
fragments identi®es LTR terminal sequences which can
then be used to design primers for retrotransposon-based
marker analysis (Ellis et al., 1998; Kalendar et al., 1999;
Waugh et al., 1997).
The method is shown schematically in Figure 2. Genomic
DNA is digested partially with MseI, a four-cutter restriction
endonuclease, to generate DNA fragments of an average
size of 1 kb. The LTR of most Ty1-copia retrotransposons is
within 500 bp of the RNaseH motif (Figure 1). Partial
digestion therefore increases the number of fragments
within this size range. Adapters are ligated to the MseI
fragments and PCR carried out using a biotinylated RNaseH
711
712 Stephen R. Pearce et al.
Figure 1. Ty1-copia retrotransposons: structural features of RNaseH, PPT
and LTR terminal sequences.
Sequence alignment of RNaseH genes, polypurine tracts (PPT) and 3¢ LTR
terminal sequences of several Ty1-copia group retrotransposons. Dots
indicate intervening sequence of the size indicated. A hyphen indicates a
gap. RNaseH motifs 1 and 2 are shown in bold, along with the
degenerate primer design based on these conserved sequences.
motif 1 primer and a primer homologous to the adapter
(Figure 2). The adapters are unphosphorylated; this means
that only one strand of the adapter is ligated to each
restriction fragment. An initial denaturation step removes
the unligated strand of the adapter, with the result that the
ampli®cation of non-speci®c products is greatly reduced.
The RNaseH motif can lie at any position on the 1 kb
template and so the biotinylated RNaseH products are on
average 500 bp long. These fragments are isolated by
af®nity selection using streptavidin paramagnetic beads
and recovered by another round of PCR with a nested
primer to a second RNaseH motif (Figure 2). This produces a
mixed PCR product which is subcloned and sequenced to
identify the population of RNaseH-LTR terminal sequences.
Figure 2. Isolation of SSAP fragments containing Ty1-copia group
retrotransposon LTR sequences.
Genomic DNA is partially digested with restriction enzyme (E). Adapters
are then ligated and PCR carried out with a biotinylated motif 1 primer
and the adapter primer to generate the RNaseH/PPT/LTR fragments.
These are then af®nity-puri®ed and recovered by PCR with a nested
RNaseH motif 2 primer and adapter primer, prior to subcloning and
sequence determination.
Identi®cation of Ty1-copia retrotransposon RNaseH-LTR
sequences
Figure 3 shows the sequences obtained from three plant
species, pea, broad bean and Norway spruce, using this
method. Each sequence has features characteristic of Ty1copia retrotransposons (Figure 1). Following motif 2, the
RNaseH open reading frame, containing some or all of the
seven conserved amino acid residues, continues for
approximately 60 bp. This is followed by a polypurine tract
(PPT) and a (T)TG-TG sequence motif characteristic of the 5¢
end of a retrotransposon LTR. Beyond the LTR end there is
very little sequence conservation except between very
closely related sequences such as Tpa2 and Tpa3 and
Tvf5 and Tvf6. The variation within the LTRs is shown
clearly by the way in which two similar sequences from the
RNaseH sequence, Tpa5 and Tpa6, are quite different in the
LTR sequence.
Figure 3. Ty1-copia group retrotransposon RNaseH/PPT/LTR junction
sequences from pea, broad bean and Norway spruce.
Alignment of RNaseH encoded peptide sequences followed by nucleotide
sequences of polypurine tracts (PPT) and 3¢ LTR terminal sequences.
Intervening sequence is of the size indicated in brackets. Consensus
sequence is from Figure 1.
Typically, 95% of the sequences obtained contain most
or all of the consensus RNaseH-PPT and LTR terminal
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 711±717
Rapid isolation of plant Ty1-copia LTR sequences
713
Molecular markers from LTRs of pea Ty1-copia group
retrotransposons
Figure 4. Sequence-speci®c ampli®cation polymorphisms of new LTR
sequences in Pisum genus.
SSAP polymorphism produced by retrotransposon primers to the
terminal sequences of Tps7 with selective adapter M(C), Tps6 with
selective adapter primer M(AC) and Tps3 with the selective primer M(AC).
Pea lines tested are: lane (a) JI157 and lane (b) JI281 (two accessions of
P. sativum); lanes (c±f) are other Pisum species (c) JI225 (P.
abyssinicum); (d) JI1006 (P. fulvum); (e) JI2055 (P. elatius); (f) JI1794 (P.
humile). Major bands indicate insertions of each individual element in
each accession.
structural features (Figure 3). Few duplicates or near
duplicates were obtained, and so it is likely that many
more new Ty1-copia group retrotransposon LTRs could be
obtained by the characterization of more subclones. We
conclude that this technique is highly ef®cient at revealing
the LTR sequences of a diverse range of new Ty1-copia
retrotransposon sequences from a wide range of higher
plant species.
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 711±717
The pea LTR retrotransposon PDR1 has been used
successfully as a source of highly polymorphic molecular markers using sequence-speci®c ampli®cation
polymorphism (SSAP) (Ellis et al., 1998). To test the
potential of the new P. sativum LTRs identi®ed in this
study, primers were designed from the LTR terminal
sequences of three P. sativum elements (Tps3, Tps6 and
Tps7) and SSAP analysis carried out on six Pisum
accessions (Figure 4).
Each of the three primers produce bands on the SSAP
gel for each accession, showing that each transposon is
present within all the species. The banding pattern
produced in each accession relates to the number and
position of each retrotransposon insertion in each
accession. The number of shared bands between each
accession is related to the phylogenetic relationships of
these accessions. The ®rst six lanes in Figure 4 represent Tps7 insertions. Lanes (a) and (b) are identical,
indicating that JI157 and JI281 are closely related. When
each of the other accessions is compared, we see that
as well as a number of shared bands there are also a
number which are unique to each species. The two
other retrotransposon primers Tps6 and Tps3 each give
a distinct pattern, but the banding patterns in each set
show more similarity between the P. sativum accessions
(lanes a and b) than between the other Pisum species
(lanes c±f). Although the data set is too small to draw
any detailed conclusions, these ®ndings are consistent
with existing ampli®ed fragment length polymorphisms
(AFLP) and SSAP data based on the pea retrotransposon PDR1 (Ellis et al., 1998). The overall polymorphism
level (the relative proportion of unshared bands) for
each primer in these accessions is 77% for Tps3, 85%
for Tps6 and 70% for Tps7. The differences in percentage indicate that each primer has a characteristic level
of transposition in these accessions. The comparable
polymorphism level for PDR1 in these same accessions
is 93% (Ellis et al., 1998). This demonstrates that each of
the three newly isolated retrotransposon markers has
distinctive polymorphism characteristics, resulting from
its individual transposition properties. The potential
usefulness of using several markers simultaneously is
discussed below.
Distribution and inheritance of Tps10 insertions in pea
To test the distribution and inheritance of the LTR
markers, Tps10 SSAP analysis was performed on
a recombinant inbred cross of two P. sativum
accessions JI15 and JI399 and 42 progeny (Figure 5).
Tps10 was chosen as of all the markers it was most
714 Stephen R. Pearce et al.
Figure 5. Distribution and inheritance of Tps10 insertions in pea.
Segregation of Tps10 LTR + T(ATT)-derived sequence-speci®c ampli®cation polymorphisms in 42 progeny of a cross between two parental lines JI15 and
JI399 (lanes 43 and 44). Four bands (Tps10/1±4, arrowed) which are polymorphic between the parental lines segregate in the progeny (lanes 1±42).
polymorphic between these lines. A number of bands
are polymorphic between the two parental lines, four
of these bands (Tps10/1±4) all behave as dominant
markers within the progeny (lanes 1±42), and map to
dispersed locations in the pea genome (data not
shown).
Discussion
Molecular markers based on retrotransposons have
proved to be more informative than non-transposon-based
marker methods in the few cases tested to date (Ellis et al.,
1998; Kalendar et al., 1999; Waugh et al., 1997).
Unfortunately, retrotransposon-based markers are only
available for a small number of well-characterized mobile
elements in relatively few species groups. The method
described here allows the rapid extension of this approach
to include every higher plant species, including those for
which no genomic information is available. We demonstrate this by the generation of heterogeneous populations
of LTR sequences from two dicot angiosperms (pea and
broad bean) and a gymnosperm (Norway spruce); additionally this approach has also yielded positive results in
wheat, a monocot (B. Gribbon, P. Jack, A. Kumar and A.J.
Flavell, unpublished data). As it is PCR-based, the method
can yield sequence data for primer design in several days,
enabling retrotransposon-based marker technologies to be
employed immediately.
This technique not only makes retrotransposon-based
marker analysis accessible for all plant species, it also
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 711±717
Rapid isolation of plant Ty1-copia LTR sequences
can increase the power of this approach by providing
several different retrotransposon markers per species. A
great variety of different Ty1-copia retrotransposons
exist in most higher plants (Flavell et al., 1992a; Flavell
et al., 1992b; Matsuoka and Tsunewaki, 1996; Pearce
et al., 1996a; Pearce et al., 1996b;, Pearce et al., 1997;
SanMiguel et al., 1996; Vanderwiel et al., 1993; Voytas
et al., 1992). Some of these, which have been transposing in the recent past, are extremely polymorphic within
species and may be used as markers for linkage
analysis and intra-speci®c diversity studies (Ellis et al.,
1998; Waugh et al., 1997). Other retrotransposons which
were active several million years ago (SanMiguel et al.,
1998), should be more useful in elucidating phylogenetic relationships between species. Lastly, some transposon types are more active in one species group than
another (Casacuberta et al., 1997; Pearce et al., 1996a;
Pearce et al., 1997). By combining data from several
retrotransposons, a clearer picture can be obtained of
the inter-relationships among complex species groups
than is possible from studies involving a single element
(S. Pearce, M. Knox, A. Flavell, N. Ellis and A. Kumar,
unpublished results).
Although the usefulness of the particular retrotransposon LTR sequences and primers identi®ed in this
study remains to be determined, the preliminary marker
data for the three new pea primers is encouraging
(Figure 4). First, each primer produces polymorphism
data across the Pisum genus which are in broad
agreement with equivalent results using AFLP and
PDR1-based SSAP (Ellis et al., 1998). Second, each of
the three primers has a distinctive level of polymorphism and all three are lower than PDR1 (Ellis et al., 1998).
These lower levels of polymorphism, caused by the
higher number of shared bands between species, may
make these elements particularly useful for clarifying
older phylogenetic relationships within the complex
Pisum genus. The behaviour of Ty1-copia retrotransposon markers is already well established in barley
(Waugh et al., 1997) and pea (Ellis et al., 1998). To
establish the potential of the markers isolated here, we
examined the segregation of Tps10 in pea recombinant
inbred lines derived from a cross between two accessions JI15 and JI399 (Figure 5). The four Tps10 insertions Tps10/1±4 behaved as dominant markers. These
insertions are located at dispersed locations within the
pea genome representing different linkage groups (data
not shown).
This study shows that a wide variety of retrotransposon
markers can be rapidly generated from any plant species
using this approach. Screening these markers for their
polymorphism parameters by SSAP on a reference set of
accessions is simple. This should allow the selection of
particular retrotransposon marker types with performance
ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 711±717
715
characteristics required for particular aspects of plant
genome analysis.
Experimental procedures
Plant materials
Pisum lines JI157, JI281, JI15 and JI399 (P. sativum), JI225 (P.
abyssinicum), JI1006 (P. fulvum), JI2055 (P. elatius) and JI1794 (P.
humile) were selected from the John Innes Germplasm core
collection (Matthews and Ambrose, 1994). Recombinant inbred
(RI) lines from the cross JI15 3 JI399 have been described
previously (Ellis et al., 1992). Pisum DNA was isolated as
described by Ellis (1994). Vicia faba (cv. Troy) was from the
Vicia germplasm collections of the Scottish Crop Research
Institute. V. faba DNA was isolated by the method of SaghaiMaroof et al. (1984). Picea abies (Norway spruce) was obtained
from the University of Dundee Botanical Garden and DNA was
isolated using Phytopure (Amersham Pharmacia Biotech,
Amersham, UK).
Computer analysis
Computer analysis was carried out using the SEQNET facilities at
the BBSRC Computing Centre, Daresbury, UK, using the GCG
program (Version 8, August 1994; Genetics Computer Group,
Madison, Wisconsin, USA).
Generation and characterization of retrotransposon
sequences
Pisum sativum (accession number JI15) total genomic DNA
(2.5 mg) was partially restricted to a mean fragment length of
1 kb with 2 units MseI (New England Biolabs) in 100 ml restriction
ligation (RL) buffer (Vos et al., 1995) at 37°C. Size optimization was
achieved by taking 20 ml aliquots of the enzyme digestion at 2, 5,
10, 20 and 40 min, terminating the digestion by incubation at 80°C
for 10 min. Of these aliquots, 5 ml was visualized by 1% TBE
agarose gel electrophoresis (Sambrook et al., 1989). The sample
with a mean size of 1 kb was chosen for template preparation.
Template DNA was prepared by ligating 2 pmol MseI adapters
[Mse1, 5¢-GACGATGGATCCTGAG-3¢; Mse2 5¢-TACTCAGGATCCAT-3¢] to the digested DNA in RL buffer containing 1 mM
ATP and 0.1 units T4 DNA ligase. The samples were incubated at
20°C for 2 h, then stored at ±20°C. The ligated template was
puri®ed using a Qiaquick PCR puri®cation column (Qiagen), and
50 ng of the template was PCR-ampli®ed with 0.8 mg biotinylated
RNaseH primer 1 (5¢-MGNACNAARCAYATHGA-3¢; the biotin is
attached to the 5¢ end of the primer via a hexylene glycol linkage
to reduce steric hindrance of DNA elongation) and 0.15 mg MseI
adapter primer (5¢-GATGGATCCTGAGTAA-3¢) in 50 ml 0.2 mM
deoxynucleoside triphosphates (dNTPs), 13 PCR reaction buffer
(Promega), 0.25 units Taq DNA polymerase (Promega) on a
Techne Genius instrument. Taq polymerase was added to
samples after an initial incubation step of 94°C (120 sec),
immediately prior to 30 cycles of 94°C for 60 sec; 45°C for 90 sec;
72°C for 90 sec. Biotinylated products were size-fractionated by
agarose gel electrophoresis, and products in the 0.4±1 kb size
range recovered by electroelution (Sambrook et al., 1989) followed by QIAquick PCR puri®cation (Qiagen) and eluted in a 40 ml
volume. Biotinylated products were af®nity puri®ed using streptavidin magnespheres (Promega): 30 ml magnespheres were
716 Stephen R. Pearce et al.
washed in 0.23 SSC, resuspended in 30 ml 0.23 SSC and 20 ml
(50%) of eluate added. This was incubated for 10 min at 20°C with
occasional shaking. The magnespheres were washed in 100 ml
0.23 SSC twice, then in 100 ml 13 Pfu reaction buffer (Stratagene)
twice. The magnespheres with the bound PCR product were
resuspended in 50 ml 1 3 Pfu buffer, and 2 ml of the suspension
was PCR-ampli®ed with 0.8 mg of RNaseH motif 2 primer (5¢GCNGAYATNYTNACNAA-3¢), 0.15 mg MseI adapter primer, in
50 ml Pfu reaction buffer containing 0.2 mM dNTPs and 1 unit Pfu
polymerase (Stratagene). The product was puri®ed by Qiaquick
PCR puri®cation column (Qiagen) and digested with BamHI
restriction endonuclease. The digest was again puri®ed on a
Qiaquick column and the product 5¢ phosphorylated using T4
polynucleotide kinase then ligated into a phosphatase-treated
M13mp18 vector which had been digested with BamHI and HincII.
The recombinants were transformed into XL1Blue supercompetent cells (Stratagene), the insert sizes con®rmed by PCR and
sequences determined using Sequenase (Amersham).
Sequence-speci®c ampli®cation polymorphism (SSAP)
marker analysis
SSAP analysis was carried out as described by Waugh et al.
(1997), except that the template DNA was digested with MseI and
adapters ligated. The template was pre-ampli®ed with primer M
homologous to the adapter sequence (M = 5¢-GATGAGTCCTGAGTAA) with 30 cycles of 94°C for 30 sec, 45°C for 30 sec
and 72°C for 60 sec.
Selective ampli®cation was carried out on 1 ml of diluted preampli®cation with 1 ml of either Tps3-, Tps6- or Tps7-labelled LTR
oligonucleotide (Tps3 = 5¢-CCTTTGGGATATTAACCACAC-3¢; Tps6
= 5¢-GTGAGATAGTTATATGTC-3¢; Tps7 = 5¢-CTATAATACATAACAAGC-3¢), with either 50 ng primer M, M(C) (5¢-GATGAGTCCTGAGTAAC-3') or M(AC) (5¢-GATGAGTCCTGAGTAAAC-3¢) (as shown
in Figure 4) with 10 cycles of 94°C for 30 sec, 55°C (reducing by 1°C
for each successive cycle) for 30 sec and 72°C for 60 sec. This was
followed by 20 cycles of 94°C for 30 sec, 45°C for 30 sec and 72°C
for 60 sec.
Tps10 SSAP was carried out using a Tps10 LTR primer (5¢GGAATGATAGGCCTTGCC-3¢) and a TaqI adapter primer with
three bases of selection T(ATT) under conditions as described by
Ellis et al. (1998).
Acknowledgements
S.R.P. is supported by EC Biotechnology project BIO4 CT96-0508
under Framework IV. C.S.-R. is funded by a BBSRC earmark
studentship. A.K. is funded by the Scottish Of®ce Agriculture,
Environment and Fisheries Department. We wish to thank Mark
Taylor and Barbara Gribbon for useful discussions.
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ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 711±717
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