Mol Gen Genomics (2004) 271: 91–97
DOI 10.1007/s00438-003-0960-x
O R I GI N A L P A P E R
R. A. Queen Æ B. M. Gribbon Æ C. James Æ P. Jack
A. J. Flavell
Retrotransposon-based molecular markers for linkage and genetic
diversity analysis in wheat
Received: 11 May 2003 / Accepted: 10 November 2003 / Published online: 2 December 2003
Springer-Verlag 2003
Abstract Retrotransposon-based molecular markers
have been developed to study bread wheat (Triticum
aestivum) and its wild relatives. SSAP (Sequence-Specific
Amplification Polymorphism) markers based on the
BARE-1/ Wis-2-1A retrotransposons were assigned to
T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are
distributed among all wheat chromosomes, with the
lowest proportion being assigned the D wheat genome.
SSAP markers for BARE-1/ Wis-2-1A and three other
wheat retrotransposons, Thv19, Tagermina and Tar1,
are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both
showing approximately 13% of bands polymorphic in a
mapping population, Tagermina showing approximately
17% SSAP band polymorphism and Tar1 roughly 18%.
This suggests that Tagermina and Tar1 have been more
transpositionally active in the recent evolutionary past,
and are potentially the more useful source of molecular
markers in wheat. Lastly, BARE-1 / Wis-2-1A markers
have also been used to characterise the genetic diversity
among a set of 35 diploid and tetraploid wheat species
including 26 Aegilops and 9 Triticum accessions. The
SSAP-based diversity tree for Aegilops species agrees
well with current classifications, though the Triticum tree
shows several significant differences, which may be
associated with polyploidy in this genus.
Communicated by M.-A. Grandbastien
R. A. Queen Æ B. M. Gribbon Æ A. J. Flavell (&)
Plant Research Unit, University of Dundee at SCRI,
Invergowrie, Dundee, DD2 5DA, UK
E-mail: a.j.fl[email protected]
Tel.: +44-1382-562731
Fax: +44-1382-568587
C. James Æ P. Jack
Monsanto plc, Maris Lane, Trumpington, Cambridge,
CB2 2LQ, UK
Keywords Retrotransposon Æ Transposable element Æ
Triticum aestivum Æ copia
Introduction
Retrotransposons are mobile genetic elements which
transpose replicatively through RNA intermediates.
They are found in all major eukaryote divisions and
comprise major fractions of the genomes of plants
(SanMiguel et al. 1996; Pearce et al. 1996). The two long
terminal repeat (LTR)-containing retrotransposon
groups (Ty1-copia group and the gypsy group) and the
non-LTR retrotransposons (also known as LINE elements) are all present in plant genomes (Flavell et al.
1992a; Voytas et al. 1992; Kubis et al. 1998; Suoniemi
et al. 1998).
In both monocot and dicot angiosperms, LTR retrotransposons comprise highly heterogeneous populations, whose members frequently span different genera
(Konieczny et al. 1991; Flavell et al. 1992a, 1992b;
Voytas et al. 1992). The BARE-1 element is a good
example of this; BARE-1 was originally isolated from
barley, and has since been shown to have close homologues in wheat, oat and rye (Pearce et al. 1997, Gribbon
et al. 1999).
Several retrotransposons have been shown to be highly
polymorphic for insert location within plant species
(Waugh et al. 1997; Ellis et al. 1998, Kalendar et al. 1999,
Porceddu et al. 2002). These properties have been
exploited in several molecular marker systems for genetic
analysis in a range of cereal grass and grain legume species
(Waugh et al. 1997; Ellis et al. 1998; Kalendar et al. 1999;
Yu and Wise 2000; Porceddu et al. 2002).
The most popular transposon-based marker method
is the Sequence-Specific Amplification Polymorphism
approach (SSAP; Waugh et al. 1997), which has also
been called transposon display (Casa et al. 2000). SSAP
markers have been developed and deployed in barley
(Waugh et al. 1997, Gribbon et al. 1999), pea (Ellis et al.
92
1998), oat (Yu and Wise 2000) maize (Casa et al. 2000)
and Medicago (Porceddu et al. 2002).
Bread wheat ( Triticum aestivum)is an allohexaploid
which probably arose by hybridisation events between
three different ancestral Triticum species during early
cultivation approximately 10, 000 years ago. Modern T.
aestivum cultivars tend to show very low levels of polymorphism between each other, making marker-assisted
studies difficult. There is therefore a need for sensitive
marker methodologies that can detect differences between very closely related T. aestivum lines. In a previous
study (Gribbon et al. 1999), we showed that molecular
markers derived from barley retrotransposons reveal
genetic diversity in wheat. Here we describe the development, comparison and use of SSAP molecular
markers derived from four different LTR retrotransposons for mapping and diversity studies in a broad variety
of wheat species.
the Human Genome Mapping Project (HGMP) Facility, Hinxton,
Cambridge, UK). Construction of marker-based phylogenetic trees
used the PAUP programme (version 4.0; Swofford 1998). A heuristic random search created four equally possible trees, which were
then combined to create a consensus tree.
Genetic linkage maps for retrotransposon SSAP markers in the
Opata85 · Synthetic W-7984 mapping population (Van Deynze
et al.. 1995) were constructed using JOINMAP (Stam and van Coijen
1993; http://www.kyazma.nl/index1.php). SSAP markers were
mapped on a framework of RFLP markers, spaced approximately
10 cM apart (http://www.graingenes.org/). The RFLP markers were
pre-screened visually for their plausibility by checking the numbers
of recombination break points deduced for them in the mapping
population; any marker showing an unrealistically high number was
discarded. The marker score data were analysed using Joinmap32.
The program JMSLA32.exe was used to look for any markers which
differed significantly from the 50:50 ratio. These markers were then
removed from further stages of analysis. The remaining markers were
grouped using JMGRP.exe with a LOD threshold of 4.0. Markers
which failed to fall into linkage groups were discarded. The markers
were then assigned to map positions with JMAP32.exe at a LOD
score of 0.001 and a recombination value of 0.499.
Analysis of Thv19 and BARE-1 insertional polymorphism
Materials and methods
Wheat samples
Triticum and Aegilops seeds (Table 1) were supplied from the John
Innes Wheat Germplasm Collection by Dr. Steve Reader, John
Innes Centre (Norwich, UK). DNA was prepared from mature
leaves by the Qiagen DNAeasy Plant 96 method.
Ty1- copia group retrotransposon marker systems
used in this study
SSAP marker systems based on the BARE-1 and Thv19 retrotransposons have been described previously (Waugh et al. 1997;
Gribbon et al. 1999). Retrotransposon-specific SSAP primers were
also derived from the LTRs of Tar1 (Genbank Accession
No. AB008772) and Tagermina (GenBank Accession No. M63224).
The basic protocol of Waugh et al. (1997) was used throughout,
and the following selective primer combinations were employed for
this study: Pst-AAA, Pst-AAT, Pst-ACG, Pst-CGA, Pst-CAT and
Pst-CTG with BARE-1-A; and Pst-GT, Pst-CC, Pst-GC with
BARE-1-AA, BARE-1-AC and BARE-1-AG, respectively. PstCTA, Pst-CAG and Pst-ATT performed poorly and were discarded
from the study. The BARE-1-specific primers were based on the
first 19 nucleotides of the BARE-1 LTR (Manninen and Schulman
1983, Accession No. Z17327), which is identical to the corresponding region of the wheat element Wis-2-1A (Accession No.
X63184), facing outwards from the 5¢ LTR (CTAGGGCATAATTCCAACA), plus one or two selective bases at the 3¢ end to
inhibit internal priming within the retrotransposons from the
3¢ LTR. The selective bases used were A, AA, AC and AG. Thv19
SSAP analysis used 5¢-GCCCAACCGACCAGGTTGTTACAG3¢, Gribbon et al. 1999), corresponding to bases 48–25 of the Thv19
LTR (Accession No. AJ241330). The Tagermina and Tar1 primers
used were 5¢-AGAGGAGGATATCCCAACAT-3¢ and 5¢-CTCCCAGTTGACCAACAA-3¢, respectively.
Computer analysis
The Tagermina retrotransposon was discovered by plant genome
database searches using TFASTA (Wisconsin Package Version
10.0, Genetics Computer Group (GCG), Madison, Wisconsin) at
Table 1 Wheat accessions used in this study
Species name
Accession No.
Species name
Accession No.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
Ae.
2060001
2060002
2090001
2090003
2050001
2110001
2110002
2100003
2100004
2150001
2150003
2130005
2170001
2170002
2140002
2140004
2220001
Ae tauschii
Ae. triaristata
Ae. triaristata
Ae. triuncialis
Ae. triuncialis
Ae. variabilis
Ae. variabilis
Ae. ventricosa
Ae. ventricosa
T. dicoccoides
T. dicoccum
T. durum
T. monococcum
T. polonicum
T. turgidum
T. urartu
T. urartu
2220015
2030001
2030002
2080001
2080008
2070002
2070003
2070001
2270002
1060025
1070024
1180111
1040005
1100001
1080057
1010001
1010006
biuncialis
biuncialis
caudata
caudata
columnaris
comosa
comosa
cylindrica
cylindrica
longissima
longissima
mutica
sharonensis
sharonensis
speltoides
speltoides
tauschii
Results
Genomic distribution of Triticum aestivum
retrotransposons
SSAP is an anchored AFLP approach that amplifies the
region between a retrotransposon-specific primer and a
nearby cleaved restriction site, to which an oligonucleotide adaptor has been added (Waugh et al. 1997).
The BARE-1 and Wis2A-1A elements share the same 19bp sequence at their 5¢ ends, so SSAP markers using this
sequence can be derived from either retrotransposon.
There are approximately 1000 Wis2A retrotransposons
in the wheat genome (Moore et al. 1991) and an unknown number of BARE-1 retrotransposons, so four
selective bases were used to reduce the band numbers to
a usable level, one base was added to the transposonspecific primer and three to the adaptor primer. Several
different restriction enyzyme/adaptor combinations were
tried. In general the highest levels of polymorphism were
obtained using Pst I/MseI pre-amplification, followed
93
by Pst I selective amplification (data not shown), so this
combination was used thereafter.
To identify the chromosomes to which the BARE-1/
Wis-2-1A SSAP markers belong, a set of nullisomictetrasomic wheat chromosome substitution lines was
used. A representative SSAP gel autoradiograph is
shown in Fig. 1. A marker was assigned to an individual
chromosome if it was absent from a single nullisomictetrasomic line and present in the other 20 lines, representing the other 20 wheat chromosomes. A summary of
the data obtained is shown in Table 2. In all, 101
markers were assigned to individual wheat chromosomes based on their absence from one of the nullisomic-tetrasomic lines. Markers were found on all
chromosomes. The A genome yielded 35 markers, the B
genome generated 36 and the D genome yielded 18.
Twelve markers could not distinguish between chromosomes 2B and 2D for unknown reasons. Chromosomes 4 and 7 appear to carry somewhat fewer BARE-1/
Wis-2-1A markers than the other five chromosomes, but
the small number of markers scored means that the
significance of this observation is questionable.
There are many different retrotransposons in plant
genomes, and it is desirable to analyse a variety of different elements because these can show differing transposition histories in different plant lineages, which can
collectively improve the overall resolution of the genetic
structure of a species (Pearce et al. 2000). Therefore,
three additional Ty1- copia group retrotransposons were
selected and analysed for SSAP polymorphism. The first
of these, Thv19 , was originally isolated from barley, and
homologues are present in rye and wheat (Gribbon et al.
1999). In barley Thv19 shows lower rates of polymorphism than BARE-1. The second retrotransposon, TAGERMINA, is a previously undescribed Ty1-copia
group element residing in a wheat germin locus (Lane
et al. 1991), which we discovered by database searching.
The last is the Tar1 retrotransposon, described by
Matsuoka and Tsunewaki (1997).
Retrotransposon-specific SSAP primers were designed for these three elements, and the associated
polymorphisms, together with those associated with
BARE-1/Wis-2-1A markers, were scored in a recombinant inbred mapping population derived from a
cross between the lines Opata and Synthetic W-798.
Examples of SSAP autoradiographs for these retrotransposons, with BARE-1/Wis-2-1A for comparison,
are shown in Fig. 2, and the polymorphism data are
Fig. 1 SSAP retrotransposon markers segregating in wheat nullisomic-tetrasomic lines. Markers assigned to wheat A, B or D
chromosomes are indicated (12 of the 21 wheat lines are shown).
For each line, duplicate SSAP products from independent SSAP
reactions were loaded in adjacent lanes to control for reproducibility. cs, the control hexaploid wheat, cv. Chinese Spring
Table 2 Chromosome assignments of SSAP markers in T. aestivum
Genome
A
B
D
Total
a
Chromosome
1
2
3
4
5
6
7
Total
8
11
2
21
5
4
8
4
16
6
2
1
9
1
7
4
12
10
6
4
20
1
2
3
6
35
36a
18a
101
a
a
17a
12 markers gave identical results for nullisomic 2B and nullisomic
2D lines
Fig. 2 SSAP markers derived from four wheat retrotransposons
segregating in individual lines from the Opata and Synthetic
W-7984 mapping population
94
Table 3 SSAP polymorphism levels for four retrotransposons in a
wheat mapping population
Retrotransposon
Total number Number of
Polymorphism
of bands
polymorphic levela
bands
BARE-1/Wis-2-1A
Thv19
Tar1
TAGERMINA
209
62
96
102
a
27
8
17
17
0.13
0.13
0.18
0.17
The proportion of polymorphic bands per total bands
summarised in Table 3. Approximately 13% of BARE1/Wis-2-1A SSAP bands are polymorphic in this population. Thv19 shows a similar polymorphism level
(13%), and both Tar1 and TAGERMINA display higher
polymorphism (18% and 17%, respectively). Tar1
detects a lower number of bands per gel than the other
three retrotransposons. The same number of selective
bases was used in all cases, suggesting that Tar1 is
present in lower copy number in the T. aestivum genome
than are the other three retrotransposons. For all four
marker types, approximately 50% of the bands derived
from each parent, and no reproducible non-parental
bands, were observed in the mapping population.
The segregating SSAP markers derived from the four
wheat retrotranspososns were then mapped against a
framework of RFLP markers (Fig. 3). The markers map
broadly across the linkage groups, with some evidence
for clustering of BARE-1/Wis-2-1A markers (prefixed
with Br in the Figure) at sites on linkage groups 3B,
4A and 5B. There is no evidence that any of the
other retrotransposon markers (markers prefixed Tv,
Tr, TG, corresponding to Thv19, Tar1 and TAGERMINA
respectively) cluster, either by themselves or with each
other.
Analysis of wheat genetic diversity using BARE-1/
Wis-2-1A markers
Retrotransposons have proven valuable as markers for
the definition of genetic diversity in pea (Ellis et al. 1998;
Pearce et al. 2000). To test their performance in wheat,
BARE-1/Wis-2-1A SSAP markers were next used to
characterise the genetic diversity among a set of 22
wheat samples, including 14 Aegilops and 8 Triticum
accessions. In all, 270 polymorphic markers were scored
and the relationship tree deduced from these data is
shown in Fig. 4.
The tree derived from the retrotransposon SSAP data
shares many consistencies with known relationships
among the accessions. First, the Aegilops and Triticum
species are effectively distinguished from each other.
Second, all five U chromosome-containing Aegilops
species (Ae. biuncalis, Ae. columnaris, Ae. neglecta,
Ae. triuncalis, Ae. variabilis) group together; Third, the
three D genome-containing Aegilops species, comprising
the two members of Section Vertebrata (Ae. ventricosa,
Ae. tauschii; van Slageren 1994) and Ae. cylindrica form
a clade. Fourth, the three Aegilops species with S genomes group together (Ae, longissima, Ae sharonensis ,
Ae. speltoides) and the close relationship between Ae.
longissima and Ae. sharonensis is consistent with current
classifications (Kellogg et al. 1996, Sasanuma et al.
1996). Fifth, Ae. comosa andAe. mutica group together,
in agreement with a previous study based upon plastid
sequences (Wang et al. 1997). A few apparent inconsistencies are also seen for Aegilops species; for example, M
chromosome-bearing accessions (Ae. biuncialis, Ae. columnaris, Ae. neglecta, Ae. comosa) are not well grouped,
nor are accessions containing C chromosomes (Ae.
caudata, Ae. triuncialis, Ae. cylindrica).
All the Triticum accessions are grouped into a single
clade, but the internal structure of the clade shows several apparent inconsistencies with the known cytogenetic
relationships. Neither the A chromosome-containing
accessions (T. monococcum, T urartu), nor the AB
chromosome-bearing accessions (T. dicoccoides, T. dicoccum, T. durum, T. polonicum, T. turgidum) group
together. However, T. aestivum groups closer to
T. dicoccoides than to T. dicoccum, in agreement with the
plastid-based study of Wang et al. (1997).
Discussion
Retrotransposon SSAP markers based upon the BARE1 element of barley have been used previously in Hordeum (Waugh et al. 1997), Avena (Yu and Wise 2000)
and, to a lesser extent, in wheat (Gribbon et al. 1999).
This study has shown that such markers collectively
show a broad distribution in the wheat genome and
should prove effective for linkage analysis. There
appears to be some clustering of BARE-1/Wis-2-1A
markers on the linkage map (Fig. 3), but no clustering is
observed for the other retrotransposons, either with
themselves or any other element. The D genome appears
perhaps to carry somewhat lower amounts of BARE-1/
Wis-2-1A markers than the A and B genomes (Table 2)
but we cannot be certain if this also is true for the other
three retrotransposons used in this study, because too
few markers were mapped. The nullisomic-tetrasomic
experiments indicate that many BARE-1/Wis-2-1A
SSAP bands (we estimate approximately one half of the
bands) are shared between two or more chromosomes.
This suggests that many insertions of this retrotransposon occurred before the divergence of the progenitor
species.
Polymorphism of different Ty1-copia group
retrotransposons within Triticeae species
Our data indicate that different Ty1- copia group retrotransposons show different levels of polymorphism in
wheat. This is not surprising, as the transposition
95
Fig. 3 Genetic map of wheat
retrotransposon SSAP markers.
Retrotransposon markers are
indicated by shaded boxes;
BARE-1/ Wis-2-1A markers are
designated by a Br prefix,
Thv19 markers by Tv, Tar1 by
Tr and TAGERMINA by TG.
SSAP markers were mapped in
relation to a framework of
RFLP markers ( unshaded; see
http://www.graingenes.org/)
96
Fig. 4 Consensus phylogenetic tree for 22 Triticeae species,
deduced from SSAP retrotransposon marker data using the PAUP
parsimony-based analysis method (Swofford 1998). Confidence
values for the nodes are shown
activity of retrotransposons is known to vary in maize
and barley (SanMiguel et al. 1998; Gribbon et al. 1999).
It is intriguing that BARE-1/Wis-2-1A and Thv19 elements show lower polymorphism levels than Tar1 or
TAGERMINA. The former two were originally isolated
from barley, where the BARE-1 element shows high
levels of polymorphism. In contrast, Tar1 and
TAGERMINA were first described in the wheat genome,
where they show higher polyorphism levels than BARE1/Wis-2-1A and Thv19. This may be a coincidence, but it
is nevertheless an interesting observation.
Ty1- copia group retrotransposons as markers
for genetic relationships between Triticeae species
Our retrotransposon-based SSAP markers are very
effective in determining genetic relationships among wild
wheat species (Fig. 4). For 14 Aegilops species the SSAPbased diversity tree agrees well with known cytogenetic
relationships and with other DNA-based diversity
analyses. The SSAP tree appears to reflect the genetic
diversity of Aegilops more accurately than a previous
study based on plastid DNA sequence divergence (Wang
et al. 1997). These two methods for phylogeny estimation may turn out to have complementary strengths, for
the following reasons. First, plastids are maternally
inherited, so a plastid sequence-based phylogeny will
provide a more accurate description of one half of the
evolutionary story for the genus. Second, retrotransposons show different polymorphism levels (Pearce et al.
2000) and copy numbers (Kalendar et al. 2000) in different plant lineages. SSAP markers may therefore
provide different levels of discrimination for the different
ancestral genomes which have combined to make up the
modern tetraploid and hexaploid wheats. It is clear from
the diversity tree (Fig. 4) that the U, D and S genomes
form well defined groups but the C and M genomes do
not. Perhaps the BARE-1/Wis-2-1A element family can
tell us more about the evolution of the U, D and S
genomes, because it is in these genomes that this retrotransposon type has generated the greater proportion of
its transpositions. If this turns out to be true, it may be
possible to define the genetic relationships among the
wheat species containing the C and M genomes by
choosing a different retrotransposon from those used in
this study.
The retrotransposon markers used here have proven
to be less convincing in resolving the phylogeny in the
Triticum genus. A contributory factor to this may be the
difficulty in comparing species with different ploidy
levels. For example, an AB allotetraploid is 50% different to either of its A and B progenitors, and may not
cluster with either in a DNA-based diversity tree. Nevertheless, we would expect species containing the A
genome and the AB genome to each form a separate
clade, but this is not the case. This could be explained by
a high level of diversity within the A genome, which,
together with multiple independent hybridisation events,
could create a variety of very different AB genomes.
Clearly, more work is needed to resolve this issue.
Comparison between wheat SSAP retrotransposon
markers and other marker types
The ideal marker method should access a very large
number of polymorphisms which are broadly distributed
within the genome under analysis. The markers should
be cheap to establish and assay, and the analytical
method should be easy to perform, reproducible and
inexpensive. Retrotransposon-based SSAP markers fulfil
these criteria very well. Technically, there is very little
difference between AFLP and SSAP marker technology,
and SSAP should be considered as an alternative to this
marker method. The disadvantage of SSAP for a newly
studied species is the need to find retrotransposon sequences for primer design. This is no longer a problem
for wheat and, for other species, rapid and effective
methods have been developed (Pearce et al. 1999). The
data quality for SSAP closely resembles that of AFLP.
One advantage of SSAP over AFLP is that higher levels
of polymorphism are accessible (Waugh et al. 1997; Ellis
et al. 1998), so that fewer experiments are needed to
generate the desired numbers of markers.
97
Another advantage of SSAP over AFLP is that it
performs better in the analysis of genetic diversity.
Retrotransposon markers generate taxonomic data that
are more consistent with geographical and morphological criteria than do AFLP-based markers (Ellis et al.
1998). The reason for this is not clear, but it may reflect
two facts. (1) Retrotransposon insertion is a single type
of biological event, whereas the different mutations
which are detected as AFLP polymorphism can arise in
any type of DNA sequence, and a polymorphism in, for
example, a highly repetitious sequence is unlikely to
shown the same phylogenetic properties as an insertion
in a gene. (2) Retrotransposon insertions are irreversible.
Therefore, the presence of an insertion (occupied site) is
a derived state, while the absence of the insertion is the
ancestral condition. Actually, SSAP bands might be lost
by sequence change at restriction or primer sites but a
component of the polymorphism is nevertheless irreversible and this may contribute to the enhanced performance of this method relative to AFLP.
Acknowledgements RAQ and BMG were supported by CASE Ph.
D. Research Studentships from the BBSRC. We thank Steve
Reader for providing seeds, Amar Kumar for advice and help with
growing plants, Andreas Meier and Simone Gauch for help and
advice with Qiagen DNAeasy methodology, and David Marshall
and Naeem Syed for much helpful advice on genetic map construction. This work was supported in part by Grant QLRT-200031502 (TEGERM) from the European Commission under the
Framework V Program
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