the plant journal 699–709 doi: 10.1111/tpj.12233...

Characterization of the LTR retrotransposon repertoire of aplant clade of six diploid and one tetraploid species

Mathieu Piednoel*, Greta Carrete-Vega and Susanne S. Renner

Systematic Botany and Mycology, University of Munich (LMU), Munich 80638, Germany

Received 8 January 2013; accepted 2 May 2013; published online 10 May 2013.

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

SUMMARY

Comparisons of closely related species are needed to understand the fine-scale dynamics of retrotransposon

evolution in flowering plants. Towards this goal, we classified the long terminal repeat (LTR) retrotranspo-

sons from six diploid and one tetraploid species of Orobanchaceae. The study species are the autotrophic,

non-parasitic Lindenbergia philippensis (as an out-group) and six closely related holoparasitic species of

Orobanche [O. crenata, O. cumana, O. gracilis (tetraploid) and O. pancicii] and Phelipanche (P. lavandulacea

and P. ramosa). All major plant LTR retrotransposon clades could be identified, and appear to be inherited

from a common ancestor. Species of Orobanche, but not Phelipanche, are enriched in Ty3/Gypsy retrotrans-

posons due to a diversification of elements, especially chromoviruses. This is particularly striking in O. grac-

ilis, where tetraploidization seems to have contributed to the Ty3/Gypsy enrichment and led to the

emergence of seven large species-specific families of chromoviruses. The preferential insertion of chromovi-

ruses in heterochromatin via their chromodomains might have favored their diversification and enrichment.

Our phylogenetic analyses of LTR retrotransposons from Orobanchaceae also revealed that the Bianca clade

of Ty1/Copia and the SMART-related elements are much more widely distributed among angiosperms than

previously known.

Keywords: next-generation sequencing, polyploidy, genome downsizing, transposable elements, LTR retro-

transposons, Ty3/Gypsy, Ty1/Copia, Orobanche, Phelipanche, Orobanchaceae.

INTRODUCTION

In angiosperms, nuclear genome size varies 2400-fold

(Pellicer et al., 2010), largely because of different propor-

tions of non-coding DNA, especially repetitive DNA (Leitch,

2007). Apart from whole-genome duplications (polyploidi-

zation), the main cause of genome size increase is the accu-

mulation of tandem-repeat DNA families and transposable

elements (TEs). Variation in nuclear genome size is of major

evolutionary importance because it determines key traits,

such as the duration of the cell cycle, that directly impact fit-

ness (Gregory and Hebert, 1999; Meagher and Vassiliadis,

2005; Gruner et al., 2010). The insertion and accumulation

of TEs is therefore expected to be counter-selected and

transposition activity suppressed, for example, by TE

autoregulation (Simmons and Bucholz, 1985; Lohe and

Hartl, 1996; Lohe et al., 1996), protein regulators (Adams

et al., 1997), RNA silencing (Sarot et al., 2004; Aravin et al.,

2007; Brennecke et al., 2007; Olivieri et al., 2010) and meth-

ylation (Verbsky and Richards, 2001; Bird, 2002; Slotkin and

Martienssen, 2007). Under stable genomic conditions,

transposition activity is therefore probably low.

Genomic stresses, however, can facilitate transposition

(Zeh et al., 2009), and polyploidization (which often accom-

panies hybridization) is one such stress thought to promote

the proliferation of TEs (Kashkush et al., 2002; Liu and

Wendel, 2003; Shan et al., 2005; Chen and Ni, 2006; Renny-

Byfield et al., 2011). The resulting temporary increase of

genome size is sometimes counterbalanced by rapid gen-

ome ‘downsizing’ (Bennetzen, 2002; Leitch and Bennett,

2004; Skalick�a et al., 2005; Hawkins et al., 2008; Mun et al.,

2009; Eilam et al., 2010; Renny-Byfield et al., 2011). Thus

far, the evidence for such downsizing via the loss of repeat

types, particularly Ty3/Gypsy retrotransposons, comes

mainly from the allotetraploid Nicotiana tabacum (Renny-

Byfield et al., 2011). A recent analysis of the repetitive DNA

in nine species of Orobanchaceae of different life histories

(seven holoparasitic species, one hemiparasitic species and

one autotrophic species; Piedno€el et al., 2012) also pointed

to genome downsizing in the tetraploid species included in

the sample. The genomic proportions of repetitive DNA var-

ied greatly among the nine species, ranging from 25 to

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd

699

The Plant Journal (2013) 75, 699–709 doi: 10.1111/tpj.12233

60%, with long terminal repeat (LTR) retrotransposons mak-

ing up most of the repetitive DNA; the tetraploid species dif-

fered substantially in Ty3/Gypsy families.

Retrotransposons, a TE class specific to eukaryotes,

transpose via an RNA intermediate. Based on structural

features and phylogenetic relationships, five orders of ret-

rotransposons have been defined (Wicker et al., 2007): LTR

retrotransposons; tyrosine recombinase-encoding retro-

transposons (e.g. DIRS1-like elements); Penelope elements;

long interspersed elements (LINEs); and short interspersed

elements (SINEs). The LTR retrotransposons, related to ret-

roviruses (Xiong and Eickbush, 1990), usually encode two

open reading frames (ORFs): one called gag, which

encodes a structural protein for virus-like particles, and

another called pol, which encodes enzymatic domains

involved in the transposition cycle, such as an aspartic pro-

tease (AP), a reverse transcriptase (RT), an RNase H (RH)

and an integrase (INT). The two major superfamilies of

plant LTR retrotransposons are Ty1/Copia and Ty3/Gypsy

(see Velasco et al., 2010; table S6), which differ in their pol

gene order (Capy et al., 1997; Wicker et al., 2007; Eickbush

and Jamburuthugoda, 2008): the RT and RH genes are

located upstream of the INT gene in Ty3/Gypsy, but down-

stream in Ty1/Copia.

In the present study, we characterize the TE dynamics in

Orobanchaceae at a finer scale than previously achieved in

this or any other flowering plant clade. For this purpose, we

analyzed the Ty1/Copia and Ty3/Gypsy elements phyloge-

netically, using seven of the nine species to represent clos-

est relatives and one out-group (Lindenbergia philippensis,

Orobanche crenata, Orobanche cumana, Orobanche graci-

lis, Orobanche pancicii, Phelipanche ramosa and Phelipan-

che lavandulacea; Figure 1). We wondered whether specific

elements are responsible for the Ty3/Gypsy diversification,

and we also wanted to know how the Ty1/Copia and Ty3/

Gypsy families reacted to the tetraploidization in O. gracilis.

Earlier studies have taken a similar approach, but were usu-

ally based either on a single species (Domingues et al.,

2012) or on a single clade of elements (Gao et al., 2012).

Comparative classifications of TEs from closely related spe-

cies have also been performed on species in which TEs had

previously been well characterized (Wicker and Keller, 2007;

Estep et al., 2013). Our study is the first to exhaustively

sample and classify all highly and moderately repeated

Ty1/Copia and Ty3/Gypsy families based on next-

generation sequencing data from species in which ele-

ments have not been previously well characterized. Among

the unexpected results is the wide distribution of the Bianca

clade and of SMART-related elements across angiosperms.

RESULTS

Clusters are hypothesized to be TE families, each

consisting of related elements (based on all-to-all BLAST and

graph-based clustering, see Experimental procedures).

Within each cluster, contigs were obtained by read assem-

bly, using an identity threshold of 80% over at least 40 bp.

The effect of using a single contig per family was tested

with a phylogenetic analysis of the largest Ty1/Copia fam-

ily from O. pancicii that we called Opan_CL2 (Figure 2). All

individual contigs from the Opan_CL2 family formed a

highly supported clade (99 bootstrap support). Given this

result, we subsequently included only one representative

contig per cluster (family).

Phylogenetic structure of Ty1/Copia elements in

Orobanchaceae

Phylogenetic analyses were performed for each of the

seven Orobanchaceae species using reference TEs, from

the Ty1/Copia clades found in plants: Hopscotch, Tos17,

SIRE1/Maximus, Tnt1, Angela, Tont1 and Bianca (Wicker

and Keller, 2007; Llorens et al., 2009; Hribov�a et al., 2010).

The tree composed of Ty1/Copia families from O. gracilis

is shown in Figure 3 (the other species trees are shown in

Figures S1–S6).

For each species, most of the seven Ty1/Copia clades

have a bootstrap support ≥80% (Figure 3). Only a few

Orobanche cumana

GS = 1.45 pg 2n = 38

Orobanche gracilis

GS = 2.10 pg 2n = 76

Orobanche pancicii

GS = 3.24 pg 2n = 38

Orobanche crenata

GS = 2.84 pg 2n = 38

PhelipanchelavandulaceaGS = 4.38 pg

2n = 24

Phelipancheramosa

GS = 4.34 pg 2n = 24

LindenbergiaphilippensisGS = 0.46 pg

2n = 32

Ty1/Copia 16.01% 18.41% 18.82% 21.42% 21.13% 22.83% 17.21%

Ty3/Gypsy 17.02% 28.34% 24.16% 21.44% 15.16% 15.92% 1.93%

Total repetitiveDNA 45.57% 60.13% 56.09% 54.94% 43.01% 47.02% 29.63%

Figure 1. Phylogenetic relationships and key genomic parameters of the seven Orobanchaceae species studied. Genomic proportions and species phylogeny

from Piedno€el et al. (2012), except for recalculated values for Phelipanche lavandulacea.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 75, 699–709

700 Mathieu Piednoel et al.

clades, such as Tos17 and Tont1, are weakly supported.

Five of the Ty1/Copia clades (SIRE1/Maximus, Tnt1,

Angela, Tont1 and Bianca) occur in all seven species. Hop-

scotch is restricted to Phelipanche species and Tos17 is

restricted to P. ramosa, where it makes up 0.01% of the

genome. The elements FRretro64 and OSCOPIA2, related

to the small LTR retrotransposons (SMARTs), cluster

together with the element Victim, either as the sister-group

of the Hopscotch clade (e.g. Figure 3) or nested within it

(e.g. Figure 2). We accordingly considered FRretro64,

OSCOPIA2 and Victim to belong to the Hopscotch clade.

The phylogenetic analyses also revealed that one element

from P. ramosa and two elements from P. lavandulacea

are closely related to the SMART retrotransposons.

Most Ty1/Copia families per species could be included

in the phylogenies because their contigs harbored reliable

matches with the RT domain; fewer than 13 families (per

species) could not be included. Most of these families were

instead assigned to clades using a BLAST-based classifica-

tion (see Experimental procedures); <0.2% of the genomes

remained unassigned to TE clades (Figure 4). The SIRE1/

Maximus families make up the largest proportions of Ty1/

Copia in Orobanchaceae (Figure 4), representing between

10.5% of the genome, in O. cumana, and 15.3%, in

P. ramosa. TEs from the Angela clade also are abundant:

contributing 2.2% of the genome in L. philippensis, and

4.9% in O. crenata. Families from the Hopscotch clade

make up 2.4 and 3.9% of the P. ramosa and P. lavandula-

cea genomes, whereas they were undetectable in three of

the four Orobanche, the exception being O. cumana, in

which they made up 0.04% of the genome. The Tnt1,

Tont1, and Bianca clades also occurred in low genomic

proportions (0.1–0.8%), except for Tont1, which makes up

~3.8% of the O. crenata genome.

To date, Bianca elements have been reported from only a

few species. Here we found, however, that L. philippensis

contains two Bianca families, O. gracilis and O. cumana

each contain four families, and the remaining species each

have five Bianca families. To test whether the presence of

Bianca elements in Orobanchaceae results from an under-

estimation of their distribution in angiosperms or from hori-

zontal transfer(s), we performed similarity searches against

the nr/nt database from NCBI (http://www.ncbi.nlm.nih.

gov). Low E-values were obtained for the Elote1 element

from maize and for sequences from Arachis hypogaea, Beta

vulgaris, Brassica rapa, Capsella rubella, Citrullus lanatus

and Vitis vinifera, as well as from Ipomoea trifida and Sola-

num lycopersicum. When these high-similarity elements

were included in a phylogenetic analysis, they clustered

together in a single Bianca clade (Figure 5).

Hopscotch

Tos17

SIRE1/Maximus

Tont1

Angela

Tnt1

BiancaATCOPI

A5

PDR1

SC-3

0.2

Figure 2. Phylogenetic relationships in the Opan_CL2 family, inferred from neighbour-joining analysis of the reverse transcriptase encoding domain. Contigs

from the Opan_CL2 family are indicated in dark red. Statistical support (>70%) comes from parametric bootstrapping using 100 replicates.


Fine-scale LTR retrotransposon classification 701

Phylogenetic structure of Ty3/Gypsy elements in

Orobanchaceae

Similar to our phylogenetic analyses of the six known Ty1/

Copia clades, we compared Orobanchaceae Ty3/Gypsy ele-

ments with the seven known plant clades from this TE

superfamily, Tekay, Galadriel, CRM, Reina, Athila, Ogre

and Tat (Llorens et al., 2009; Hribov�a et al., 2010). Figure 6

shows the resulting tree for O. gracilis (the other species

trees are shown in Figures S7–S12). The element clades

have bootstrap supports of ≥80%, except for Reina and Tat.

Families that could not be included in the phylogenetic

analyses (~20–30 in each species) were classified using the

BLAST-based approach (see Experimental procedures). Only

a few families, comprising less than ~1% of the genomes,

could not be assigned. Two families (Ocre_CL223 and

Lind_CL46), which turned out to be caulimoviruses instead

of Ty3/Gypsy, make up 0.01 and 0.08% of the genomes in

which they were found (O. crenata and L. philippensis).

The genomic proportion of Ty3/Gypsy in L. philippensis is

thus lower (1.85%) than previously calculated (1.93%;

Piedno€el et al., 2012).

Three of the seven plant Ty3/Gypsy clades (Tekay, CRM

and Athila) are found in all seven species (Figure 7). Tekay

is more abundant in Orobanche (>9.7%) than in Phelipan-

che (6.0–6.5%), and makes up 0.78% of the genome of the

out-group L. philippensis. CRM elements make up a lower

proportion in Phelipanche (<0.1%) than in Orobanche (from

0.3% in O. crenata up to 0.7% in O. gracilis), and the single

CRM family in L. philippensis (Lind_CL124; 0.02%) was

only detected using BLAST. Athila families, by contrast, are

more abundant in Phelipanche (5.6%) than in Orobanche

(<3.5%) and comprise a substantial proportion of the

L. philippensis genome (0.43%). Tat appears absent from

L. philippensis, but is ubiquitously distributed in the other

species, where it makes up 2.8–7.1% of the genomes. The

remaining elements are more rare, with Reina and Ogre

restricted to O. gracilis (0.05 and 0.08% of the genome,

respectively), and Galadriel restricted to O. gracilis (~0.7%of the genome), O. pancicii, O. crenata and L. philippensis

(<0.1% of their genomes).

LTR retrotransposon dynamics in the tetraploid species

O. gracilis

To better understand the genome modifications that

occurred in the tetraploid O. gracilis, we investigated in

detail both its species-specific LTR retrotransposons and

the TE families it has lost (compared with the remaining

Hopscotch

Tos17

SIRE1/Maximus

Tnt1

Angela

Tont1

Bianca

ATCOPI

A5

ATCOPIA63

0.2

Figure 3. Phylogenetic relationships in Ty1/Copia elements from Orobanche gracilis, inferred from neighbour-joining analysis of the reverse transcriptase

encoding domain. Families from O. gracilis are indicated in dark red. Families that are widely distributed in Orobanche but are lost in O. gracilis are indicated in

green. Statistical support (>70%) comes from parametric bootstrapping using 100 replicates.



Orobanche). None of its species-specific families belong to

Ty1/Copia, whereas 11 belong to Ty3/Gypsy. These 11

make up 5.85% of the O. gracilis genome (Figure 6), and

comprise seven Tekay families, two Athila families and

two Galadriel families. The seven Tekay families by them-

selves make up 5.12% of the O. gracilis genome. TE

families that are lost in O. gracilis are two Ty3/Gypsy fami-

lies (Tekay) and four Ty1/Copia families (one Angela, one

SIRE1/Maximus and two Bianca; Figures 3 and 6, and

BLAST-based classification).

DISCUSSION

This study is a fine-scale analysis of the Ty1/Copia and

Ty3/Gypsy LTR retrotransposons in closely related species

of Orobanchaceae, including a young tetraploid and a phy-

logenetically more distant species as an out-group. Most

of the TE families belong to clades commonly found in

plants (Hribov�a et al., 2010; Staton et al., 2012). Two fami-

lies (Ocre_CL223 and Lind_CL46), which we previously

classified as Ty3/Gypsy (Piedno€el et al., 2012), are in fact

caulimoviruses and make up 0.01 and 0.08% of the

genomes in which they were found (O. crenata and L. phil-

ippensis, respectively). Before this study, Bianca elements

had been reported from only a few species, including

Arabidopsis thaliana, Lotus japonicus, Medicago truncula-

ta, Oryza sativa (rice) and Triticeae (Schulman and

Kalendar, 2005; Holligan et al., 2006; Wicker and Keller,

2007; Wang and Liu, 2008), which are distantly related to

Orobanchaceae. It is now clear, however, that the Bianca

clade of Ty1/Copia, which also comprises the Elote1 ele-

ment from Zea mays (maize), is more widely distributed

across angiosperms, occurring in Brassicales (Arabidopsis

thaliana, Brassica rapa and Capsella rubella), Caryophyll-

ales (Beta vulgaris), Cucurbitales (Citrullus lanatus),

Fabales (Arachis hypogaea, Lotus japonicus and Medica-

go truncatula), Lamiales (Orobanchaceae spp.), Poales

(O. sativa and Triticeae spp.), Solanales (Ipomoea trifida

and Solanum lycopersicum) and Vitales (Vitis vinifera).

This suggests that Bianca originated early during angio-

sperm evolution, which fits the hypothesis that Bianca may

be the most ancient Ty1/Copia clade in angiosperms

(Wicker and Keller, 2007).

Considering the wide distribution of Bianca and the

poorly resolved phylogenetic relationships between ele-

ments from Orobanchaceae and the other species

(Figure 5), we hypothesize that the Bianca clade is verti-

cally inherited in the Orobanchaceae family. No uncor-

rupted full-length Bianca elements from Orobanchaceae

are known, and these elements are therefore probably no

longer active, even though the Elote1 element transposed

‘recently’ in inbred maize (Wang and Dooner, 2006). In

Musa acuminata (banana; Hribov�a et al., 2010), Saccharum

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

Gen

om

ic p

rop

ort

ion

Clades

Ty1/Copia

O. cumana

O. gracilis

O. pancicii

O. crenata

P. ramosa

P. lavandulacea

L. philippensis

Figure 4. Ty1/Copia clade distribution (%) among the seven Orobanchaceae species. NC: not classified.



officinarum (sugar cane; Domingues et al., 2012) and Gly-

cine max (soybean; Du et al., 2010), Bianca has not been

detected, possibly because of low family number, judging

from its low diversity in other angiosperms where it has

been detected (Orobanchaceae, this study; Triticae, rice

and Arabidopsis, Wicker and Keller, 2007). Likewise, our

study reveals the first elements (Plav_CL114 and Plav_180,

Figure S4; Pram_CL107, Figure S5) related to SMARTs out-

side monocotyledons (Gao et al., 2012). As P. lavandula-

ceae and P. ramosa only parasitize dicotyledon species

(http://www.farmalierganes.com/Flora/Angiospermae/Orob

anchaceae/Host_Orobanchaceae_Checklist.htm, accessed

February 2013), the presence of SMART-related elements

in these two species probably results from an underestima-

tion of the distribution of SMART elements among

angiosperms.

Our LTR retrotransposon classification shows that sev-

eral Ty1/Copia and Ty3/Gypsy clades (SIRE1/Maximus,

Tnt1, Angela, Tont1, Bianca, Tekay, CRM, Athila and Tat)

are widely distributed in Orobanchaceae (Figures 4 and 7),

and may have been present in the family’s most recent

common ancestor. We previously found that Orobanche

and Phelipanche are characterized by different TE dynam-

ics (Piedno€el et al., 2012). The present analysis further

illustrates this. For example, Hopscotch is restricted to

Phelipanche, whereas Tekay is overabundant in Oroban-

che. In addition, there are species-specific features. For

example, the L. philippensis genome has a high proportion

of Ty1/Copia (17.2%), but a very low proportion of Ty3/

Gypsy (1.9%), and O. crenata is enriched in Tont1 elements

(3.8%), compared with all other species (<0.8%). The two

closely related Phelipanche resemble each other in their

Ty1/Copia element proportions (21.1% in P. lavandulacea;

22.8% in P. ramosa), but diverge in their Ty1/Copia compo-

sition, with P. ramosa enriched for the SIRE1/Maximus ele-

ments and P. lavandulacea enriched for Hopscotch. This

highlights the need to study TE dynamics at both large and

fine scales, and in a comparative context. TE transposition

can be activated by stress (Melayah et al., 2001; Fablet and

Vieira, 2011) or the colonization of new environments

(Vieira et al., 2002), and it has been suggested that the TE

repertoire of a gene pool could promote, or be associated

with, the emergence of evolutionarily separate lines (Oliver

and Greene, 2009, 2011; Jurka et al., 2011).

The Ty3/Gypsy genome proportions are higher in Oroban-

che than in Phelipanche (Figure 1), which we have attributed

to diversification rather than a burst of transposition (Pied-

no€el et al., 2012). The present results fit that hypothesis.

Hopscotch

Tos17

SIRE1/MaximusTont1 Angela

Tnt1

Bianca

ATCOPI

A3

0.1

Figure 5. Phylogenetic relationships in the Bianca clade, inferred from neighbour-joining analysis of the reverse transcriptase encoding domain. Families from

Orobanche are indicated in dark red, families from Phelipanche are indicated in red, families from Lindenbergia philippensis are indicated in blue and the Bianca

element is indicated in pink. Additional sequences are indicated in green. Statistical support (>70%) comes from parametric bootstrapping using 100 replicates.



Firstly, Orobanche genomes comprise more diverse element

clades than Phelipanche (Galadriel, Reina and Ogre were

only detected in Orobanche). Three Galadriel families

are present in low genomic proportions in the out-group

L. philippensis (Lind_CL123, Lind_CL135 and Lind_CL137),

and thus perhaps this family as well as Reina and Ogre were

lost in the common ancestor of Phelipanche. Secondly, Tat,

Tekay and CRM are more abundant in Orobanche than in

Phelipanche, with the Tekay clade (and to a lesser extent the

Tat families) making up most of the Ty3/Gypsy enrichment

in Orobanche (Figure 7). This enrichment is accompanied

with an increase of the CRM and Tekay family number in

Orobanche (4–9 CRM and 25–33 Tekay families), compared

with Phelipanche (1–2 CRM and 19–22 Tekay families).

The Tekay elements, as well as the CRM, Galadriel and

Reina elements, are chromoviruses (Gorinsek et al., 2004;

Llorens et al., 2009). Chromoviruses are the earliest-

diverging branch of Ty3/Gypsy, and are found in plants,

fungi and animals (Kordis, 2005). They have a high geno-

mic turnover (Gorinsek et al., 2004), which may result

from their ‘strategy’ to escape repression and elimination

mechanisms (Kordis, 2005; Baucom et al., 2009; Novikov

et al., 2012). Chromoviruses differ from other Ty3/Gypsy

elements in harboring a chromodomain in their 3′ end,

which is a structural domain commonly found in proteins

associated with the remodeling and manipulation of chro-

matin (Gorinsek et al., 2004). Chromodomains are highly

constrained (Novikov et al., 2012), and may promote the

integration of TEs in heterochromatin regions (Gao et al.,

2008; but see Novikov et al., 2012 for chromoviruses in

euchromatin). In accordance with this, CRM families

appear to be centromere-specific (Luo et al., 2012). The

high genomic turnover and site-specific integration of

chromoviruses probably both contribute to their survival

and abundance, especially in plants where they often

attain large numbers of young copies (Kordis, 2005). It is

therefore not surprising that chromoviruses make up a

high proportion of the Orobanchaceae Ty3/Gypsy. There

is, however, an exception to this global pattern, with the

Athila element enrichment in Phelipanche. Once again,

this underlines the need to study TE dynamics at both

large and fine scales.

We previously showed that O. gracilis (the sequenced

plant was tetraploid, with 2n = 76) has a particular TE com-

position, probably related to its tetraploidization and subse-

quent genome downsizing (Piedno€el et al., 2012). Although

O. gracilis has one of the smallest genomes of the Orob-

anchaceae studied, it has the highest proportion of TEs,

Galadriel

CRMReina

Athila

ERIKA1 TM

01

Ogre

Ogr

a_C

L19

SP

E

0.1

Tekay Tat

Figure 6. Phylogenetic relationships in Ty3/Gypsy elements from Orobanche gracilis, inferred from neighbour-joining analysis of the reverse transcriptase

encoding domain. Families from O. gracilis are indicated in dark red, and species-specific families from this species are labeled with an ‘SPE’ tag in their name.

Families that are widely distributed in Orobanche, but are lost in O. gracilis, are indicated in green. Statistical support (>70%) comes from parametric bootstrap-

ping using 100 replicates.



especially of Ty3/Gypsy retrotransposons (Figure 1). The

present LTR retrotransposon classification allows a better

understanding of the underlying dynamics. As is character-

istic of Orobanche, Tekay elements are enriched in O. grac-

ilis: Tekay families make up 17.82% of its genome, with

seven Tekay families unique to O. gracilis and making up

5.12% of its genome. These seven families represent

almost one-third of the O. gracilis species-specific families,

and 75% of the Ty3/Gypsy genomic enrichment compared

with O. crenata and O. pancicii. Previous studies have

shown that polyploidy can be associated with selective

amplification of repetitive DNA (Parisod et al., 2010 for a

review). The O. gracilis polyploidization could thus have

promoted the proliferation of specific Tekay families.

Polyploidy may have been only one of the factors activat-

ing the Tekay elements in O. gracilis. In maize, only one of

the LTR retrotranposon amplification bursts was initiated

by polyploidy, whereas the other element activations were

not (Estep et al., 2013). Additionally, the chromodomain of

Tekay chromoviruses could have helped their persistence

and thus accumulation (as described above). Like the CRM

families, Tekay elements may be preferentially located in

centromeric and pericentromeric regions of plant chromo-

somes (Theuri et al., 2005; Domingues et al., 2012). Inter-

estingly, the tetraploid O. gracilis is the only species in our

sample that harbors all four chromovirus clades (Tekay,

Galadriel, CRM and Reina), with Galadriel elements being

especially diverse and abundant (partially via the presence

of two specific families; Figures 6 and 7).

The Ty3/Gypsy enrichment in O. gracilis might be

related to the elimination of genes, especially redundant

ones, as suggested for the triploid Brassica rapa genome,

compared with the diploid A. thaliana (Mun et al., 2009).

This could be the case because Ty3/Gypsy elements (and

more especially chromoviruses) are preferentially located

in heterochromatin, in contrast to genes, which are prefer-

entially located in euchromatin. However, this differential

location cannot be the only explanation of the TE enrich-

ment, because genome downsizing in O. gracilis also led

to the loss of TE families from various LTR retrotransposon

clades (Figures 3 and 6), including the presumably hetero-

chromatin-located Tekay elements. This loss of entire TE

families matches results from the allopolyploid Nicotiana

tabacum, in which common repeats from the parental spe-

cies are lost in the polyploid descendant (Renny-Byfield

et al., 2011).

In conclusion, we classified the Ty1/Copia and Ty3/Gypsy

LTR retrotransposons from a rapidly speciating clade of

Orobanchaceae, as indicated by the numerous morphologi-

cally poorly separated species that differ mainly in flower

color and host preferences (Carl�on et al., 2008; Pusch and

G€unther, 2009). Most of the identified LTR retrotransposon

clades appear to have been inherited from the most recent

common ancestor of Orobanchaceae. Orobanche has the

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0G

eno

mic

pro

po

rtio

n

Clades

Ty3/Gypsy

O. cumanaO. gracilis

O. pancicii

O. crenata

P. ramosaP. lavandulacea

L. philippensis

Figure 7. Ty3/Gypsy clade distribution (%) among the seven Orobanchaceae species. NC: not classified.



greatest Ty3/Gypsy element diversification, perhaps because

chromoviruses (Tekay, Galadriel, CRM and Reina) targeted

heterochromatin regions via their chromodomains, and

thus were able to persist longer. The tetraploidization

event in O. gracilis appears to have promoted the prolifera-

tion of seven species-specific Tekay families in this spe-

cies. In contrast to the Ty3/Gypsy elements, the Ty1/Copia

repertoire appears more homogeneous among Orobancha-

ceae species, although there are striking species-specific

TE dynamics. Finally, this study revealed that the Bianca

clade of Ty1/Copia elements and SMART-related elements

are more widely distributed in angiosperms than previ-

ously known.

EXPERIMENTAL PROCEDURES

Plant material

Lindenbergia philippensis (Cham. and Schltd.) Benth. (2n = 32) isa fully autotrophic species from Bangladesh, India, Burma,Thailand, Cambodia, Laos, Vietnam, tropical China and the Philip-pines. Orobanche cumana Wallr. (2n = 38) is distributed from theMediterranean region to Central Asia, and its main hosts belongto Asteraceae. Orobanche gracilis Beck (2n = 76) is found in theMediterranean, and northwards to southern Central Europe, itshosts are exclusively shrubby Fabaceae. Orobanche pancicii Beck(2n = 38) is distributed from the Balkan Peninsula, northwards tothe Eastern Alps, and its hosts are species of Knautia and Scabi-osa. Orobanche crenata Forssk. (2n = 38) is found in the Mediter-ranean region and the Near East, and its hosts are legumes,mainly annual crop species. Phelipanche lavandulacea Pomel(2n = 24) is a Mediterranean species, and its sole host is Bitumina-ria bituminosa, a perennial Fabaceae. Finally, Phelipanche ramosa(L.) Pomel (2n = 24) has been introduced worldwide, but its nativedistribution is the Mediterranean region and the Near East. Itshosts are a broad range of annual species. Chromosome numbersof the individuals studied were reported in Schneeweiss et al.(2004) and Piedno€el et al. (2012).

Sequencing data and assembly

The 454 pyrosequencing reads were obtained from the sequenceread archives (accession no. SRA047928). Filtering for plastid con-taminants resulted in 76–555 Mb of DNA sequence for each spe-cies. This amounts to ~23% of the O. cumana genome (1.42 Gb),~20% of the O. gracilis genome (2.05 Gb), ~20% of the O. crenatagenome (2.78 Gb), ~16% of the O. pancicii genome (3.17 Gb),~12% of the P. ramosa genome (4.25 Gb) and ~11% of theP. lavandulacea genome (4.29 Gb). The corresponding genomesizes were reported in Weiss-Schneeweiss et al. (2006) andPiedno€el et al. (2012).

The reads were processed as described in Piedno€el et al. (2012).Briefly, they were assembled using a graph-based clusteringapproach (Nov�ak et al., 2010), in which vertices correspond tosequence reads, and overlapping reads are connected, with edgesassociated with edge weights corresponding to their similarityscores. Clusters of frequently connected nodes represent groupsof similar sequences (hereby considered as families of genomicrepeats). The number of reads in each family is proportional to itsgenomic abundance. Within each family, the reads were assem-bled into contigs, representing chimeric consensus sequences,using TIGR Gene Indices clustering tools (Pertea et al., 2003), with

the �O′ �p80 �o40′ parameters, specifying overlap percentageidentity and minimal length cut-off for the cap3 assembler.

Family classification

For both Ty1/Copia and Ty3/Gypsy, we reconstructed phylogenetictrees including several previously classified TEs, representative ofall LTR retrotransposon clades described from plants. Referencesequences were selected from the matrices used in Hribov�a et al.(2010), plus the ATCopia28 and OSCOPIA2 elements from Arabid-opsis thaliana and rice, all deposited in Repbase (Jurka et al.,2005). Some other elements not represented in Hribov�a et al.(2010) were also added: (i) Bianca, Eninu, Opie and Victim fromthe maize TE database (http://maizetedb.org/~maize), (ii) Giepumand Ji from the Retrotransposon database (http://data.genomics.purdue.edu/~pmiguel/projects/retros); and (iii) Ale (HE774675),Araco (AC079131:14472-19329), FRetro64 (JN806224), Ivana(EF067844:429582-434664) and Kielia (EU195798) from GenBank.For each Orobanchaceae LTR retrotransposon family, one contigcovering the RT domain was then included in the phylogeneticanalyses as a representative of its entire family. The representativecontigs were selected as the most conserved considering theirsimilarity scores with known elements obtained using RPS-BLAST(reversed position-specific BLAST; Altschul et al., 1997) and threealignment profiles: pfam07727, pfam00078 and cd01650.

The RT domains of the reference sequences and representativecontigs were extracted using a custom Python script and the RTboundaries provided by the results of RPS-BLAST. All RT domainswere then translated using Traduit (http://www.snv.jussieu.fr/~wensgen/soft/doc/index.html), and the corresponding sequencesof amino acids were aligned using MAFFT (Katoh et al., 2009). Align-ments were manually curated, and ambiguously aligned siteswere filtered out using BMGE (Criscuolo and Gribaldo, 2010). Phylo-genetic analyses were carried out using the neighbour-joiningmethod, 100 bootstrap replicates and the pairwise deletion ofgaps option included in MEGA 5.0 (Tamura et al., 2011). The best-fitting model, JTT + G (Jones et al., 1992), was selected usingTOPALI 2 (Milne et al., 2009).

Some families without any reliable hit on the RT domain couldnot be included in the phylogenies. We thus classified them con-sidering their BLASTX results on the Gypsy database (Llorens et al.,2011). A family was assigned to a particular clade using two crite-ria: (i) best hits obtained on a unique clade, and (ii) an E-value dif-ference between these hits and the best hits obtained on otherclades of at least 1E�5.

Bianca clade distribution

To determine the Bianca distribution among angiosperms, similar-ity searches were performed using the AT28Copia element fromArabidopsis thaliana and elements from Orobanchaceae speciesagainst the nr/nt database. For this purpose, both BLASTN and BLASTX

were used. Several candidates from Arachis hypogaea (HQ637177),Beta vulgaris (GU057342), Brassica rapa (AC232487), Capsellarubella (DQ103594), Citrullus lanatus (JX027061), Ipomoea trifida(AH013750), Lotus japonicus (AP009656), Medicago truncaluta(AC161750), Oryza sativa japonica group (AC018929), Solanumlycopersicum (AF275345), Vitis vinifera (AM477556) and Zea mays(Elote1: DQ493648) were selected and included in a specific phylo-genetic analysis.

ACKNOWLEDGEMENTS

This work was supported by the German Science Foundation (RE603/9-1 and -2). We thank Jiri Macas from the Institute of PlantMolecular Biology in Budweis for the RepeatExplorer pipeline.



SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article.Figure S1. Phylogenetic relationships in Ty1/Copia elements fromOrobanche crenata inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S2. Phylogenetic relationships in Ty1/Copia elements fromOrobanche pancicii inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S3. Phylogenetic relationships in Ty1/Copia elements fromOrobanche cumana inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S4. Phylogenetic relationships in Ty1/Copia elements fromPhelipanche lavandulacea inferred from neighbor-joining analysisof the reverse transcriptase encoding domain.

Figure S5. Phylogenetic relationships in Ty1/Copia elements fromPhelipanche ramosa inferred from neighbor-joining analysis ofthe reverse transcriptase encoding domain.

Figure S6. Phylogenetic relationships in Ty1/Copia elements fromLindenbergia philippensis inferred from neighbor-joining analysisof the reverse transcriptase encoding domain.

Figure S7. Phylogenetic relationships in Ty3/Gypsy elements fromOrobanche crenata inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S8. Phylogenetic relationships in Ty3/Gypsy elements fromOrobanche pancicii inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S9. Phylogenetic relationships in Ty3/Gypsy elements fromOrobanche cumana inferred from neighbor-joining analysis of thereverse transcriptase encoding domain.

Figure S10. Phylogenetic relationships in Ty3/Gypsy elementsfrom Phelipanche lavandulacea inferred from neighbor-joininganalysis of the reverse transcriptase encoding domain.

Figure S11. Phylogenetic relationships in Ty3/Gypsy elementsfrom Phelipanche ramosa inferred from neighbor-joining analysisof the reverse transcriptase encoding domain.

Figure S12. Phylogenetic relationships in Ty3/Gypsy elementsfrom Lindenbergia philippensis inferred from neighbor-joininganalysis of the reverse transcriptase encoding domain.

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