Magazine
R1
Correspondence
Genome
expansion in
three hybrid
sunflower
species is
associated with
retrotransposon
proliferation
Mark C. Ungerer, Suzanne C.
Strakosh and Ying Zhen
The origin of new diploid
species through inter-specific
hybridization may be facilitated
by rapid genomic reorganization.
There is evidence that this
process was involved in the
independent origins of three
annual sunflower species in the
genus Helianthus. The three
hybrid taxa, H. anomalus, H.
deserticola and H. paradoxus, are
products of ancient hybridization
events between the same two
parental taxa, H. annuus and H.
petiolaris [1]. The hybrid species
have geographically restricted
ranges and occupy habitats that
are abiotically extreme relative to
other Helianthus species;
H. anomalus and H. deserticola
are found in desert environments,
whereas H. paradoxus is
restricted to saline marshes
[2]. In addition to several novel
karyotypic rearrangements [3],
each hybrid taxon has a nuclear
genome at least 50% larger than
that of either parental species [4].
These genome size differences
occur in spite of the fact that the
hybrid and parental species are
diploids and all possess the same
number of chromosomes (n = 17).
Because both inter-specific
hybridization and abiotic stress
have played important roles in the
evolutionary history of the hybrid
taxa, and because both have
been implicated as natural agents
of retrotransposon activation and
proliferation [5,6], we sought to
determine whether the genome
size differences associated
with hybrid speciation in these
sunflowers could be attributable
to proliferation of mobile genetic
elements in the hybrid taxa.
While multiple categories of
transposable elements exist in
eukaryotic genomes, the class I
elements known as long terminal
repeat (LTR) retrotransposons
most often have been associated
with genome size variation in
plants [7]. Based on a previously
reported Ty3/gypsy-like LTR
retrotransposon sequence in
Helianthus [8], we developed a
PCR probe (887 base pairs from
the integrase-domain-encoding
region) to use in Southern
blot experiments comparing
element abundance in the hybrid
and parental taxa. The probe
was amplified from genomic
DNA of the parental species
H. annuus. Southern blots
revealed a considerably stronger
hybridization signal for the hybrid
species relative to their parental
species (Figure 1), indicating a
higher relative abundance of Ty3/
gypsy sequences in the hybrid
species' genomes. This remained
the case whether loadings
were standardized by genome
equivalents, standardized to
1 g, or standardized to 500
ng without a restriction digest
(Figure 1).
To gain better quantitative
estimates of element copy
numbers in these sunflower
genomes, we used a quantitative
PCR strategy. Examination of
6­10 individuals per species
and two different populations of
each parental taxon confirmed a
stunning increase of Ty3/gypsy
sequences in all hybrid taxa
(Figure 2), with 5.6 to 23.6-fold
increases in copy number in
the hybrid species. Statistically
significant differences were
not observed among different
populations of parental species.
Assuming a size of 5.2 kb for a
Ty3/gypsy element in Helianthus
[9] we estimate an additional
1330 Mb, 1162 Mb and 909 Mb
of DNA in H. anomalus, H.
deserticola and H. paradoxus,
respectively, that is attributable
to Ty3/gypsy proliferation.
These estimates account
for ~73%, ~79%, and ~62% of
the differences in genome size
between the parental species H.
annuus (3000 Mb) [10] and
H. anomalus, H. deserticola and
H. paradoxus, respectively
[4]. Retrotransposons represent
ancient lineages that often
exhibit considerable sub-lineage
diversity in plant genomes. So
although estimates reported in
Figure 2 accurately reflect real
differences between the hybrid
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Current Biology
Figure 1. Southern blots probed with an 887 base pair region of the Ty3/gypsy integrase
domain.
Lanes 1 and 5: parental species H. annuus and H. petiolaris, respectively. Lanes 2­4:
hybrid species H. anomalus, H. deserticola and H. paradoxus, respectively. Blots depict
standardized loadings of (A) genome equivalents (H. annuus, 1.10 g; H. anomalus,
1.76 g; H. deserticola, 1.63 g; H. paradoxus, 1.63 g; H. petiolaris, 1.00 g); (B) 1 g
DNA; and (C) 500 ng uncut genomic DNA.
Current Biology Vol 16 No 20
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and parental taxa, they may
not reflect global estimates of
element copy number because
of the PCR-based nature of
this assay and the potential for
sequence variation at priming
sites.
Alternative explanations for
these observations such as
preferential probe hybridization/
PCR amplification in the hybrid
species and/or extra-chromosomal
DNA (retroelement cDNA) in the
hybrid species seem untenable.
Preferential probe hybridization/
PCR amplification in the hybrid
species relative to the parental
species is implausible because
the development of our Southern
blot probe and quantitative PCR
primers was based on a Ty3/
gypsy sequence derived from
the parental species H. annuus
[8]. Extra-chromosomal DNA in
the hybrid species is ruled out by
our Southern blots: a Southern
blot of undigested genomic DNA
probed with a Ty3/gypsy integrase
fragment also exhibits stronger
hybridization signal in the
hybrid species (Figure 1C), and
fails to exhibit distinct, faster
migrating cDNA transposition
intermediates [11].
Future work will explore
the potential impact of the
proliferation of retrotransposons
on the establishment and
differentiation of the hybrid
species. Given the prominent
roles played by hybridization and
abiotic stress in the evolutionary
history of all three of the hybrid
taxa, it is tempting to conclude
that one or both of these factors
may have been involved in
these proliferations. Further
experiments, however, will be
necessary in order to address
this question explicitly. Therefore,
in addition to their established
role as a model system for
investigating homoploid hybrid
speciation in plants [1,2,12],
these hybrid sunflower species
will likely emerge as an excellent
group for studying the ecological
and evolutionary dynamics of LTR
retrotransposon activation and
proliferation.
Supplemental data
Supplemental data including
experimental procedures are available
at http://www.current-biology.com/cgi/
content/full/16/20/R872/DC1/
Acknowledgments
We thank Kaitlin Stimpson for technical
assistance. This work was funded
by NSF EPSCoR and Kansas State
University.
References
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A.E., Donovan, L.A., and Lexer, C. (2003).
Major ecological transitions in wild
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(2006). Ty3/gypsy-like retrotransposon
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Division of Biology, Kansas State
University, Manhattan, Kansas 66506,
USA. E-mail: mcungere@ksu.edu
Figure 2. Copy number es-
timates (per genome) of the
Ty3/gypsy integrase domain
based on quantitative PCR.
Two different populations of
the Helianthus parental spe-
cies (H. annuus and H. peti-
olaris) and one population
each of the three diploid
hybrid species (H. anoma-
lus, H. deserticola and H.
paradoxus) were assayed.
Shown are means with one
SE. The number of indi-
viduals assayed from each
species are as follows: H.
annuus (UT), n = 9; H. an-
nuus (TX), n = 10; H. anoma-
lus, n = 10; H. deserticola,
n = 10; H. paradoxus, n = 6;
H. petiolaris (UT), n = 9; H.
petiolaris (TX), n = 9. Differ-
ent lowercase letter above
histogram bars indicate that
means are significantly dif-
ferent (Tukey-Kramer HSD).
a ab
b
c
c
c c
annuus
(U
T)
annuus
(TX)
petiolaris
(TX)
petiolaris
(U
T)
anom
alus
deserticola
paradoxus
Ty3/gypsyintdomaincopynumber
0
50000
100000
150000
200000
250000
300000
350000
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