TAXON 56 (2) • May 2007: 493–504
Schlüter & al. • A screen of low-copy nuclear genes
A screen of low-copy nuclear genes reveals the LFY gene as phylogenetically
informative in closely related species of orchids (Ophrys)
Philipp M. Schlüter1,2,*, Gudrun Kohl1, Tod F. Stuessy1 & Hannes F. Paulus2
Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, 1030 Vienna,
Austria. [email protected] (author for correspondence)
2
Department of Evolutionary Biology, University of Vienna, Althanstraße 14, 1090 Vienna, Austria
* Current address: Ecological Plant Genetics, Swiss Federal Institute of Technology Zürich (ETH),
CHN G29, Universitätsstrasse 16, 8092 Zürich, Switzerland
1
This paper presents PCR primers and PCR conditions for low-copy nuclear genes in Ophrys and related
orchid genera identified via screening of both published and newly designed primers. For Ophrys, the most
useful markers identified in this screen are the LFY/FLO gene which contains an intron of 2 kb size and the
MADS-box PI/GLO gene whose 2 first introns contain single nucleotide polymorphisms with variation at the
populational level. In the taxa tested, our PCR primers amplified single-copy regions. Phylogenetic analysis
of closely related taxa of Ophrys section Pseudophrys, based on LFY, revealed the following groups that are
delimited by morphology: O. lutea s.l.; O. omegaifera s.l. with O. iricolor nested in this group; the two O.
fusca s.l. taxa, O. leucadica and O. bilunulata; and the O. fusca s.l. taxon O. cinereophila together with a
group of endemics from Crete.
KEYWORDS: LEAFY/FLORICAULA (LFY/FLO), low-copy nuclear sequence markers, Ophrys fusca s.l.,
Ophrys section Pseudophrys, PISTILLATA/GLOBOSA (PI/GLO), sexually deceptive orchids
INTRODUCTION
The European orchid genus Ophrys is remarkable
for its pollination by sexual deception which makes it an
interesting system for evolutionary studies (Kullenberg,
1961; Paulus & Gack, 1990). However, the reconstruction
of relationships within Ophrys, especially among very
closely related species, has been hindered by the lack of
resolution obtained with standard chloroplast or nuclear
ribosomal internal transcribed spacer (ITS) sequence
markers (Pridgeon & al., 1997; Aceto & al., 1999; Soliva &
al., 2001; Bateman & al., 2003). The availability of highly
variable sequence markers is therefore highly desirable
to address the question of species relationships within
Ophrys.
Ophrys section Pseudophrys represents a monophyletic group within which standard sequence markers do not
provide any resolution (Soliva & al., 2001; Bateman & al.,
2003; Bernardos & al., 2005). This section is characterised
by attachment of pollinia to a pollinator’s abdomen rather
than its head. Section Pseudophrys contains the morphologically readily distinguishable O. lutea s.l., O. fusca s.l.
and O. omegaifera s.l. complexes, and the O. iricolor/O.
mesaritica species group which has often been considered
to be a sub-group of the O. fusca s.l. complex (Paulus
& al., 1990; Paulus, 1998). The relationships among and
within these complexes have so far been amenable only
to speculation based upon morphology, chromosomal
data not permitting additional insights apart from the
identification of tetraploid taxa in the east Mediterranean
(Greilhuber & Ehrendorfer, 1975; Bernardos & al., 2003;
D’Emerico & al., 2005).
The present study therefore seeks to evaluate nuclear
low-copy genes, to identify sequence markers that are
phylogenetically informative and can be used to infer
relationships within Ophrys at a fine level, using sect.
Pseudophrys as a model system. The usefulness of lowcopy nuclear sequence markers is becoming increasingly
recognised since they frequently outperform ITS and
plastid markers (e.g., Bailey & Doyle, 1999; Emshwiller & Doyle, 1999; Lewis & Doyle, 2002; Sang, 2002;
Oh & Potter, 2003; Howarth & Baum, 2005). We have
screened a large number of available PCR primers for
nuclear genes to identify gene regions that may be useful
within Ophrys and related orchids and in addition, have
designed novel primers from sequences in the sequence
databases.
MATERIALS AND METHODS
Plant material and DNA extraction. — Ophrys
plant material (Table 1) was collected in the field and
leaves preserved in silica gel. Non-Ophrys material
was from Orchis italica, Serapias cf. bergonii, Himantoglossum hircinum and Himantoglossum (syn. Barlia)
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TAXON 56 (2) • May 2007: 493–504
Schlüter & al. • A screen of low-copy nuclear genes
Table 1. Taxa used and EMBL sequence database accession numbers for LFY.
Taxon
O. atlantica Munby
O. basilissa Alibertis & Reinhard
O. basilissa Alibertis & Reinhard
O. bilunulata Risso
O. cinereophila Paulus & Gack
O. creticola Paulus
O. iricolor Desfontaines
O. iricolor Desfontaines
O. iricolor Desfontaines
O. “kedra” Paulus (nom. prov.)
O. leucadica Renz
O. omegaifera Fleischmann
O. “pallidula” Paulus (nom. prov.)
O. phryganae Devillers-Terschuren
& Devillers
O. sicula Tineo
O. sitiaca Paulus, Alibertis & Alibertis
O. tenthredinifera Willdenow
Species
groupa Country, Island, Locality
O
O
O
F
F
F
F
F
F
F
F
O
F
L
L
O or F
E
Dateb
Accession EMBL Sequence No.
Spain, Alhaurin de la Torre
Greece, Samos, Klima
Greece, Kos, Asklepion
Spain, Coin Las Delicias
Greece, Crete, Akoumia
Greece, Crete, Jouchtas
Greece, Crete, Kato Horio
Greece, Crete, Ag. Paraskies
Greece, Athens
Greece, Crete, Spili/Gerakari
Greece, Kos, Kephalos
Greece, Crete, Thripti
Greece, Crete, Thripti
08.04.2004
21.02.2004
27.02.2002
09.04.2004
02.04.2003
30.03.2003
29.03.2003
30.03.2003
26.03.2004
07.05.2003
01.03.2002
25.03.2002
04.05.2003
196A
174A
66A
198A
114A
104A
100C
106A
208A
150A
67A
37A
145C
AM489434
AM489432
AM489423
AM489435
AM489427, AM489428
AM489426
AM489425
AM489419
AM489436
AM489431
AM489424
AM489420
AM489430
Greece, Rhodes, Kattavia
Greece, Samos, Klima
Greece, Crete, Jouchtas
Greece, Crete, Gourtinia
21.04.2003
22.02.2004
14.02.2001
??.02.2001
120A
177A
61A
56A
AM489429
AM489433
AM489422
AM489421
Note: All plants collected by HFP with vouchers in WU, except accessions 120A and 208A, collected by PMS and M. Fiedler,
respectively.
a
Putative membership of the listed taxa in morphological species groups within Ophrys sect. Pseudophrys are indicated,
where F, Ophrys fusca s.l.; L, O. lutea s.l.; O, O. omegaifera s.l.; while E is Ophrys sect. Ophrys (syn. Euophrys).
b
Dates are given in DD.MM.YYYY format.
robertianum. Additional plant material (Dendrobium,
Vanilla, Asparagus) was obtained from plants grown at
the Botanical Garden of the University of Vienna. DNA
was extracted using DNeasy Plant Mini Kit (Qiagen)
and the manufacturer’s protocol, eluting DNA in 200
µL Tris-EDTA, pH 8.0. In addition, genomic DNA from
Arabidopsis thaliana (Invitrogen, included in the AFLP
Core Reagent Kit) was used.
Primer design. — For design of new primers, we
used sequences available in the public databases and
amino-acid alignments of exons between distantly related
taxa (where available, including orchid sequences) to identify highly conserved regions and noted known intron
positions. Alignments were carried out using Clustal
X (Thompson & al., 1997) and Bioedit 7 (Hall, 2001).
Primers were then designed from nucleotide sequence
alignments such that (1) their binding sites would lie in
conserved exonic gene regions, (2) PCR would amplify
enough exonic sequence to allow gene identification by
BLAST searches and (3) also variable intronic or exonic
sequence would be amplified. In particular, PI and LFY
primer design was aided by GenBank sequence AB094985
from Orchis italica and the orchid LFY sequences obtained by Montieri & al. (2004), respectively. Primers
were checked for expected melting temperature, loops
and primer-primer interactions using Oligo Analyzer 1.0.2
software (Kuulasmaa, 2002).
Marker screening via polymerase chain reaction
(PCR). — Both published and new primers (Tables 2
494
and 3) were screened with standard PCR protocols on a
gradient PCR machine (Thermo Hybaid PX2 or Corbett
Research Palm-Cycler) using annealing temperatures
between 40°C and 65°C degrees. Initial reactions were
performed in a volume of 25 µL containing 12.5 µL REDTaq ReadyMix PCR Reaction Mix (Sigma-Aldrich), 1
µL of each, 5 µM forward and reverse primers, and c.
25 ng genomic DNA. Thermal cycling conditions were
95°C 4 min.; 38× (95°C 40 sec.; TA 40 sec.; 72°C 3 min.);
72°C 10 min.; 4°C hold, where in each PCR, annealing
temperatures (TA ) varied over a 15°C temperature gradient depending on the expected melting temperatures
of the primers used. PCR products were loaded on 1%
agarose gels in TAE (tris acetate EDTA) buffer stained
with ethidium bromide (0.28 mg/L) and photographed
under UV light using a Gel Doc 2000 system (BioRad). If
no amplification product was obtained, DNA and primer
concentrations were varied, different polymerases (e.g.,
Taq DNA polymerase, recombinant, from Fermentas)
used, and in some cases, the thermal cycling conditions
altered. PCR reactions that yielded either a smear or weak
amplification products were subjected to a two-step PCR
optimisation testing different buffer systems and PCR enhancers, using PCR Optimization Kit II (Sigma-Aldrich)
and the manufacturer’s protocol. If multiple bands were
obtained, they were separated by excision and elution from
gel using QIAquick Gel Extraction Kit (Qiagen). Amplified fragments were then sequenced directly to check a
PCR product’s identity by BLAST searches, or cloned and
TAXON 56 (2) • May 2007: 493–504
Schlüter & al. • A screen of low-copy nuclear genes
Table 2. PCR primers developed in this study.
I. Primers for LFY
Primer
Primer sequence (5′→3′)
PCR primers for amplification of LFY from genomic DNA
E1Cf
ATGGTGCTGGCCACATCGCAGCAACA
E2Gr
GAAGAGGTAATCGAGCCCGTTCTTCTTAGCYC
Nested PCR primers for LFY
E1Jf
GGAGCTAGAGGAGGTGTTCGAGG
E1Bf
GGTACTCGACGATTGCTCGG
E1Af
CGCTCTCGACGCACTTTCC
I1Df
CCGTCAGCTTGTTTGTTCCTCAC
I1Ef
CGTCTGTTCCATTGAACTTCTTGG
I1Ff
ATGTATCTTCATCCGATTTGGAATG
I1Af
AAGTCATTTCAGACAATCTTAAGTTTKG
I1Ar
CMAAACTTAAGATTGTCTGAAATGACTT
I1Gf
CGACCGCCAACACGCACCTAACAAAG
I1Gr
CTTTGTTAGGTGCGTGTTGGCGGTCG
I1Cf
GATACAGATATRCTGTTCAAAGAGC
I1Cr
GCTCTTTGAACAGCATATCTGTATC
I1Kf
ATTAGGATGAAAGCAGTAAGATTGC
I1Kr
GCAATCTTACTGCTTTCATCCTAAT
I1Lf
TTGAATATGGCTATTCGCAGTTCA
I1Lr
TGAACTGCGAATAGCCATATTCAA
I1Jr
AATAAAACAAATAGCAAAAGTGCCC
I1Br
TACTAAAATGTGCTGACAAATG
E2Ar
AGCTGCACTGGCTCCTCAG
E2Lr
CCTTTCCATCTCTCCTGCCTA
E2Kr
CCGTCGTCATCCTCATCATTCTC
Primer Binding Site
Length
1
2791
26
32
96
131
465
576
651
816
1029
1029
1241
1241
1425
1425
1714
1714
1837
1837
2064
2275
2524
2578
2739
23
20
19
23
24
25
28
28
26
26
25
25
25
25
24
24
25
22
19
21
23
II. Primers for other genes
Target genes/proteins
Primer
Primer sequence (5′→3′)
Acyl-CoA ∆ desaturase
D9Des1f
D9Des1r
D12Des2f
D12Des3r
Def4f
Def5r
AsnStAf
AsnStBr
AsnStCr
ATM1f
ATM2r
Col1f
Col2r
OCko1E2f
OCko1E3f
OCko1E3r
OCko1E4r
Ockx2f
Ockx3r
M1f
K1r
Susy7f
Susy8f
Susy11r
Susy12r
TTTCAYCAYCARTTYACIGAYWSIGA
TCRAAIGCRTGRTGRTTRTTRTGCCA
CAYMGIMGICAYCAYWSIAAYACIGG
AAIARRTGRTGIGCIACRTGIGTRTC
ARGARCTGCGCGGTCTTGAGCAA
GTYTGIGTRSYGATGATSACATGATA
TGATGATGAAGAGAATCCTTATC
GCATTCAGCATCATTCTATCAG
ACCTTTCAAAGATCATTCTGTAG
GAYGAYCTNAGRCARGAYGCNGT
CCYTGYTCRAANGCNACNCCNAGRTCDATRTG
TGYGAYGCYGAYATYCAYTCYGCYAAYCC
GCRTAYCTDATNGTYTTYTCRAA
AGCAGAGCTGATAAAGCTCAG
ATGTTCCACATCCATGGCTC
AGCCATGGATGTGGAACATC
CTGGAATTGAAGTAGACATCC
GTGTTAGGAGGTTTGGGWCARTTYGG
AGAGRTTRAGCCAWGGATGWGGAAC
AGATCAAGCGSATCGAGAAC
CTTGATCCKATCRATYTCCG
GRTGTTCAAYGTYGTYATCYTVTCYCCYCAYG
AYCAAGTICGYGCKITGGAGAAYGARATGC
CRATYTCTTGGAAIGTRCTKGTGATGATGAARTC
GASACRATRTTGAACTTIGGRTCRAAIACATC
9
Acyl-CoA ∆12 desaturase
APETALA3/DEFICIENS (AP3/DEF)
Asparagine synthetase
Ataxia telangiectasia mutated (ATM )
CONSTANS-Like (COL)
Cytokinin oxidase 1 (OCkx1)
PISTILLATA (PI)
Sucrose synthase
Length
26
26
26
26
23
26
23
22
23
23
32
29
23
21
20
20
21
26
25
20
20
32
30
34
32
Note: All primers are written as 5′→3′ sequences (where I is inosine), and the length of primers is indicated. For LFY primers,
the 5′ nucleotide of the primer binding-site is indicated; the sequence used here as a reference is that of O. iricolor (accession
106A; EMBL accession AM489419), position 1 corresponding to the first nucleotide in exon 1. LFY primers are sorted in order
of their occurrence in the gene (5′ to 3′; see also Fig. 1). The first two characters of LFY primer names indicate exon 1, 2 and intron 1, the third letter being a unique primer position within that gene region and f and r denoting forward and reverse primers.
PI primers M1f and K1r bind in the MADS and K-domains of the gene, respectively.
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Table 3. Nuclear genes screened in this study.
Results in
Ophrysb
Gene product (Acronym)
Number of primers (Ref.)a
Actin
2 (*Arab. 368)
–
Acyl-CoA ∆9 desaturase
2 (this study)
–
Acyl-CoA ∆ desaturase
2 (this study)
-
Alcohol dehydrogenase (ADH)
2 (Strand & al., 1997),
2 (Small & Wendel, 2000)
-
Apetala3/Deficiens (AP3/DEF)
2 (this study)
12
+ (multiple)
Asparagine synthetase
3 (this study)
-
Ataxia telangiectasia mutated (ATM)
2 (this study)
+
Calmodulin (CaM)
2 (Strand & al., 1997)
-
Cellulose synthase (CEL)
2 (*Rice. 313)
+
Cellulose synthase (CES)
2 (*Arab. 222)
–
Chalcone isomerase (CHI)
2 (Strand & al., 1997)
–
Chalcone synthase (CHS)
2 (Strand & al., 1997)
–
Chloroplast-expressed glutamine synthetase
2 (Emshwiller & Doyle, 1999)
-
Constans-like (COL)
2 (this study)
+ (multiple)
Cytokinin oxidase 1 (OCkx1)
6 (this study)
-
eIF2-γ
2 (*Arab. 156)
–
Glyceraldehyde 3-phosphate dehydrogrenase
(G3PDH, GAPDH, GapC locus)
2 (Strand & al., 1997)
2 (Wall, 2002), 2 (this study)
+S
Heat shock protein 70, putative (Hsp70)
2 (*Arab. 262)
Comments
– in positive control
+S in Dendrobium
Multiple bands, not further
analysed
+ in Asparagus
Multiple bands, not further
analysed
+S in Dendrobium
-
Leafy/Floricaula (LFY/FLO)
2 (+ nested primers, this study)
Malate synthase
2 (Lewis & Doyle, 2002)
+SV
+
Methionine synthase
2 (*Arab. 379)
+
Phosphoenolpyruvate carboxylase (PEPC)
2 (Gehring & al., 2001),
2 (D. Fulop, pers. comm.),
2 (*Arab. 163)
+
6-Phosphoglucose isomerase (PGI, GPI)
2 (Strand & al., 1997)
-
Phytochrome C
5 (Mathews & Donoghue, 1999)
-
Phosphoribulokinase (PRK)
7 (Lewis & Doyle, 2002)
Pistillata/Globosa (PI/GLO)
2 (this study)
+S in Vanilla
- in positive control for some
combinations
+
+SV
RNA polymerase II (RPB1)
2 (*Arab. 183)
–
Serine/Threonine protein kinase, putative
2 (*Arab. 069)
+
Splayed (SPD)
2 (*Arab. 076)
-
Sucrose synthase
4 (this study), 2 (*Arab. 185)
+S
Triose phosphate isomerase (TPI, TIM)
2 (Strand & al., 1997)
–
An asterisk (*) in the reference column identifies primers that were developed by use of the database approach of Xu & al.
(2004) and whose sequences were kindly provided by J. Padolina. For these, the primer database code is given.
b
Results in the study group are, no amplification at all (–), no clear amplification product (-), good amplification product (+),
good amplification with sequence matching target gene in BLAST searches (+S), and (+SV) as before but with sequence
variation in Ophrys fusca s.l. taxa.
a
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Schlüter & al. • A screen of low-copy nuclear genes
sequenced, as detailed below. Initial screening was on
DNA material from Ophrys and positive controls, which
were Arabidopsis thaliana where suitable, and otherwise
the organism from which the gene under consideration
was first isolated. Variability of sequences was compared
between closely related Ophrys accessions (Table 1; at
least 5 randomly chosen DNAs).
Sequencing. — Amplification products were sequenced using BigDye 3.1 (Applied Biosystems) and Dyenamic ET dye terminators (Amersham) using the manufacturers’ protocols scaled to a reaction volume of 10 µL.
Sequences were loaded on ABI 377 or ABI 3130XL DNA
sequencers (Applied Biosystems) after loading preparations
as recommended by the sequencer manufacturer.
Cloning of PCR products. — PCR products were
cloned into pGEM-T vector (Promega) and inserted into E.
coli JM109 cells (Promega) by chemical transformation,
using the manufacturer’s protocols. Cells were plated out
on LB medium containing 50 mg/L ampicillin, IPTG and
X-Gal so as to identify positive clones. Inserts were amplified from apparently positive clones by colony PCR using
M13 forward (–20) and reverse vector-located primers. At
least 16 colonies were screened for insert size variation
per cloning reaction and 5 clones of every size class were
then directly sequenced.
Cloning of the LFY genomic PCR product. — All
attempts to clone the LFY genomic PCR fragment (see below) failed, using pGEM-T (Promega), StrataClone Blunt
PCR Cloning Kit (Stratagene), TOPO TA (Invitrogen) or
TOPO Zero Blunt (Invitrogen), and the manufacturers’
protocols for cloning and preparation of PCR fragments
for cloning, i.e., blunting of PCR fragment ends using Pfu
DNA polymerase, or A-tailing using Taq DNA polymerase.
Since simple cloning proved impracticable, PCR products
were subcloned using Alu I and Rsa I-digested amplicons
in Sma I-digested pUC18 vector (enzymes, protocols and
vector from Fermentas). Inserts were then amplified by
colony PCR using M13 primers and sequenced as detailed
above.
Routine amplification conditions for PI. — PI
could be amplified reliably under a wide range of PCR con
ditions, both from genomic DNA and floral cDNA. Typical
conditions for PCR performed in 20 µL used 0.8 µL of
each, 5 µM M1f forward and K1r reverse primer (Table 2),
10 µL REDTaq ReadyMix (Sigma-Aldrich) and 1 µL 1 : 10
dilution of genomic DNA (c. 25 ng). The following PCR
programme is suitable for amplification of PI from Ophrys
and related orchids: 95°C 4 min.; 38× (95°C 30 sec.; 50°C
30 sec.; 72°C 3 min.); 72°C 10 min.; 4°C hold.
Routine amplification conditions for LFY. —
The amplification of LFY from genomic DNA was only
possible under optimised PCR conditions. Antibody
hotstart PCR was performed with primers (Table 2 and
Fig. 1) located in exons 1 and 2 of LFY. Reactions were
performed in 20 µL volume using 2 µL 10× AccuTaq
LA PCR buffer (Sigma-Aldrich; 500 mM Tris-HCl, pH
9.3, adjusted with NH4OH, 150 mM (NH4)2SO4, 25 mM
MgCl2, 1% Tween 20), 1 µL 10 mM each dNTP (Fermentas), 1.6 µL of each, 5 µM E1Cf forward and E2Gr reverse
primer, 1 µL 1 u/µL Jumpstart REDAccuTaq LA DNA
polymerase (Sigma-Aldrich) and 1 µL genomic DNA
extract (c. 250 ng). The PCR programme used was 96°C
25 sec.; 37× (94°C 10 sec.; 60°C 30 sec.; 68°C 5 min.);
68°C 15 min.; 4°C hold. Resulting PCR products were
separated on a 1% agarose-TAE gel, excised and PCR
products of ~3 kb length purified from the gel. 1 µL of
a 1 : 10 dilution of purified Ophrys LFY PCR fragment
was used as a template for each nested PCR with a different combination of nested primers (Table 2 and Fig.
1). Nested PCR was performed in 20 µL reactions using
0.8 µL of each, 5 µM forward and reverse primer, 10 µL
RedTaq ReadyMix (Sigma-Aldrich) and the following
PCR programme: 95°C 1 min.; 38× (94°C 20 sec.; 60°C
30 sec.; 72°C 3 min.); 72°C 10 min; 4°C hold. All nested
primer combinations expected to work could be amplified, typical combinations being E1Jf–I1Ar, I1Ef–I1Jr and
I1Cf–E2Kr. For routine sequencing of LFY, removal of
residual primers and nucleotides from nested PCR fragments was accomplished by cleaning them enzymatically
with E. coli exonuclease I (Fermentas) and calf intestine
alkaline phosphatase (Fermentas) using the method of
Werle & al. (1994) with slight modifications. 5–7 µL of
Fig. 1. LFY primer map showing exon 1, intron 1 and exon 2 of the gene, using a sequence from O. iricolor as reference sequence (accession 106A; EMBL accession AM489419). Major insertions and deletions found in Ophrys sect. Pseudophrys
relative to O. iricolor are indicated. The letter indicated for primer designations (see Table 2) is unique within each exon
and intron. Bold face is used for genomic PCR primers and italics for intronic primers.
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cleaned nested PCR fragments were used for sequencing
as detailed above.
PCR walking. — PCR walking was carried out
following the protocol of Siebert & al. (1995), using 1 µg
of genomic DNA for generation of adapter-ligated DNA
libraries after digestion with Dra I, Eco RV (Eco 32I), Ssp I,
Stu I (Eco147I), Pvu II, Sma I or Sca I (all enzymes from
Fermentas) and a PCR set-up as detailed for the amplification of LFY from genomic DNA. Differing from the
original protocol (Siebert & al., 1995), the short adapter
strand used was 5′-pACCTGCC-s-ddC-3′, where s indicates a phosphothiorate linkage to prevent exonucleolytic
cleavage (as suggested by Padegimas & Reichert, 1998),
and ddC is a terminal 2′,3′-dideoxy-C to prevent priming
from the oligonucleotide’s 3′ end during PCR.
Reverse transcriptase (RT)-PCR for PI. — Flowers collected in the field were dissected into lip, petals,
sepals and column, and frozen in liquid N2. Messenger
RNA was extracted with QuickPrep Micro mRNA Purification Kit (Amersham) and the manufacturer’s protocol.
All mRNA obtained (suspended in a volume of 10 µL)
was reverse transcribed using 100 pmol anchored oligodT primer (5′-pT18VN-3′), RevertAid H Minus M-MuLV
Reverse Transcriptase (Fermentas) and Ribonuclease Inhibitor (Fermentas), according to the supplier’s protocol
and PCR carried out for PI as described above, but using
Jumpstart REDAccuTaq LA Polymerase (Sigma-Aldrich)
and 68°C extension temperature.
Single-strand conformational polymorphism
(SSCP) analysis of PI PCR products. — SSCP were
performed for PI to assess the allelic variation pattern. PI
was amplified by PCR both from genomic DNA of Ophrys populations (not sequenced) and clones with known
sequence in a volume of 20 µL, as described above. Five
microlitres of PCR products were then digested with 1 u
Rsa I (Fermentas) in a reaction volume of 10 µL for 3 hrs
at 37°C, and then kept at 4°C. The restriction digest (10
µL) was then combined with 10 µL of SSCP loading dye
(10 mM NaOH, 0.03% bromophenol blue, 0.03% xylene
cyanol, in formamide abs.), denatured for 5 min. at 95°C
and immediately chilled on ice, for a minimum of 3 min.,
until loading of 5 µL on a native 12 % polyacrylamide gel
(50 : 1 acrylamide : bis-acrylamide, with 0 or 5% glycerol)
in Tris-borate EDTA (TBE) buffer. Electrophoresis was
carried out at 22°C and 50 V for 20 min. followed by
250 V for 3 hrs in a Hoefer SE 600 Electrophoresis system (Amersham) coupled to a MultiTemp Thermostatic
Circulator (Amersham). Gels were stained with PlusOne
DNA Silver Staining Kit (Amersham) and the manufacturer’s protocol, and included digested, but undenatured
PI dsDNA controls as well as undenatured Generuler 100
bp DNA ladder (Fermentas).
Phylogenetic analysis of LFY. — Sequences were
edited using SeqMan II (DNAStar Inc.) and entered into
498
the EMBL sequence database (for accession numbers see
Table 1), and aligned using Clustal X (Thompson & al.,
1997) and Bioedit 7 (Hall, 2001). Where clearly distinguishable allelic variants were encountered in a single
individual, two allelic sequences were compiled that were
maximally different. Partial intron sequences of several
individuals of the same population were checked for additional allelic variation. A model of molecular evolution
was estimated using Modeltest 3.7 (Posada & Crandall,
1998) for the entire nucleotide dataset and separately for
exon and intron sequence using MrModelTest 2.2 (Nylander, 2004). The model of evolution selected for the entire
nucleotide data was HKY + Γ in a hierarchical likelihood
ratio test (hLRT) and TVM + I using the Akaike information criterion (AIC). When exon and intron data were
treated separately, the models F81 + I + Γ or GTR + I were
selected for exon and HKY + Γ or GTR + Γ for intron data,
using hLRTs or the AIC, respectively. Maximum parsimony (MP) analysis with equal character weights was performed in PAUP* 4b10 (Swofford, 2002) using a heuristic
search with 10 random sequence addition replicates. Most
parsimonious trees were summarised by consensus tree
methods available in PAUP*. Maximum likelihood (ML)
analysis in PAUP* using a heuristic search with 10 random
sequence addition replicates were performed with both,
the model selected using hLRT and AIC. Bootstrap branch
support in ML and MP reconstructions was estimated
using 100 pseudo-replicates.
For Bayesian inference, information from insertion/
deletion (indel) characters compiled from the sequence
alignment were included, using complex indel coding
(Simmons & Ochoterena, 2000). Indel characters were
largely unambiguous so that the use of step matrices
was unnecessary. Bayesian phylogenetic inference was
carried out in MrBayes 3.1.2 (Ronquist & Huelsenbeck,
2003) on the complete nucleotide sequence combined
with the indel data matrix. Separate models of evolution
for exon and intron characters were used, as selected in
either hLRT or AIC, indel information being treated as
‘standard’ (morphological) data. Two parallel analyses
with three Markov-chain Monte Carlo (MCMC) chains
were run for 10 million generations. Results from the
first one million generations were discarded, MCMC
sampling seemingly having converged by this time in
all cases.
RESULTS
Marker screening. — The results of the PCR
marker screen are summarised in Table 3. Most primer
combinations either did not yield PCR products, yielded
PCR products that were unsuitable, or PCR products did
not contain sequences that corresponded to target loci.
TAXON 56 (2) • May 2007: 493–504
Schlüter & al. • A screen of low-copy nuclear genes
Amongst those genes that could be amplified were Adh
and Cko1 in Dendrobium, PI/GLO, LFY/FLO, AP3/DEF,
and genes for G3PDH and sucrose synthase for Ophrys.
However, lack of variability or poor sequence quality
that precluded design of more specific primers led us to
discontinue laboratory efforts for most of these, leaving
only PI and LFY for further characterisation.
The PI/GLO gene. — Based on the sequence of
the 441 bp PI PCR product, spanning the first two introns, and PCR walking experiments, the positions of
the first three introns in Ophrys thriptiensis PI (EMBL
accessions AM489437 to AM489439), compared with
the Orchis italica cDNA sequence, correspond to intron positions in Antirrhinum majus GLO (Tröbner &
al., 1992) rather than Arabidopsis thaliana PI (Goto &
Meyerowitz, 1994). In Ophrys, PI introns 1, 2 and 3 are
85, 90 and > 119 bp in length with exon-intron junctions
AC/GT..AG/GT (exon/intron/exon), AG/GT..AG/AA and
AG/GT.., respectively. Variation among PI clones was
limited, identifying two alleles in O. thriptiensis differing by two point mutations in intron 2. These, but no
additional alleles, were also found in O. cinereophila,
O. iricolor, O. creberrima and O. leucadica individuals.
Additional putative alleles were identified using SSCP
of Rsa I-digested PI PCR products from an Ophrys populational sample of the same taxa, although occurrence
of these alleles did not seem to coincide with Ophrys
populations or taxa. Because PI variation was unlikely
to be phylogenetically informative, putative SSCP alleles
were not cloned and PI not pursued further as a phylogenetic marker within Ophrys fusca s.l. Comparison of
PI sequences of exons 1–3 (266 bp) show 19 silent substitutions among Ophrys thriptiensis and Orchis italica.
PCR of cDNA from dissected Ophrys fusca s.l. flowers
showed PI to be expressed in the lateral and dorsal sepal,
petals, the lip and the column.
The LFY/FLO gene. — The ~3 kb LFY genomic PCR
product spans intron 1 and sequences can be obtained
reliably from nested PCR products. LFY was found to be
phylogenetically informative within Ophrys sect. Pseudophrys and a summary of the variability encountered in
LFY is presented in Table 4. Intron-exon boundaries of
the first Ophrys LFY intron are in good agreement with
eukaryotic consensus splice sites (Long & Deutsch, 1999;
Moore, 2000). We observed great length variation of the
LFY genomic PCR product among Ophrys and related
genera, suggesting considerable variation in intron length
(inferred approximated intron lengths are Ophrys iricolor:
2 kb, Himantoglossum hircinum: 1.5 kb, Himantoglossum
robertiamum: 1.8 kb, Serapias cf. bergonii 0.1 kb, Orchis
italica: 1 kb). Even within Ophrys, LFY intron 1 contains a
number of indels of > 30 bp length, smaller indels present
even within the closely related taxa of the O. fusca s.l.
group.
Table 4. Comparison of nucleotide and indel characters obtained from LFY (this study), and trnL and ITS data available in
the public sequence databases. Variation is shown (1) in comparison with an outgroupa taxon and (2) within the ingroupb.
Ingroup + Ophrys tenthredinifera
Nu
Ni
Nv
%Var
Nu
2847
760
2087
37
Ingroup + O.t. (Nseq=18; Ntax=14)
98
58
156
5.5%
16
3
19
2.5%
82
55
137
6.6%
17
20
37
–
Ingroup only (Nseq=17; Ntax=13)
25
57
82
2.9%
2
3
5
0.7%
23
54
77
3.7%
5
19
24
–
Total sequence
Exon sequence
Intron sequence
Indel characters
804
311
493
2
Ingroup + O.t. (Nseq=3; Ntax=3)
–
–
8
1.0%
–
–
4
1.3%
–
–
4
0.8%
–
–
2
–
Ingroup only (Nseq=2; Ntax=2)
–
–
1
0.1%
–
–
1
0.3%
–
–
1
0.2%
–
–
2
–
ITS (nuclear ribosomal DNA)
Total sequence
ITS1 spacer
5.8S rRNA gene
ITS2 spacer
Indel characters
629
237
153
239
0
Ingroup + O.t. (Nseq=12; Ntax=11)
11
0
11
1.7%
8
0
8
3.8%
0
0
0
0.0%
3
0
3
1.3%
0
0
0
–
Ingroup only (Nseq=11; Ntax=10)
3
0
3
0.5%
3
0
3
1.3%
0
0
0
0.0%
0
0
0
0.0%
0
0
0
–
Gene/region
Characters
Nt
LFY (nuclear)
Total sequence
Exon sequence
Intron sequence
Indel characters
trnL (chloroplast)
Ingroup only
Ni
Nv
%Var
Note: Column headings are as follows: Nseq, number of sequences; Ntax, number of taxa; Nt, total number of characters; Nu,
parsimony uninformative characters; Ni, parsimony informative characters; Nv, total number of variable characters; %Var,
percentage of variable nucleotide characters.
a
O. tenthridinifera was used as an outgroup taxon, and includes O. tenthredinifera LFY exon data from Montieri & al. (2004).
ITS data from Soliva & al. (2001) and Bernardos & al. (2005 and 1 unpublished sequence); trnL data from Soliva & al. (2001).
b
Ingroup refers to Ophrys sect. Pseudophrys.
499
Schlüter & al. • A screen of low-copy nuclear genes
Phylogenetic reconstructions. — The phylogeny
(Fig. 2) of closely related taxa of Ophrys sect. Pseudophrys inferred from the LFY gene is well resolved. Tree
topologies and branch lengths obtained from different
phylogenetic analyses and different models of molecular
evolution agreed well with each other, whether indel char-
TAXON 56 (2) • May 2007: 493–504
acters were included or not. In all reconstructions, we
found the O. lutea s.l. taxa, O. sicula and O. phryganae as
one group, which is sister to the group formed by morphologically very similar O. bilunulata and O. leucadica from
the west and east Mediterranean, respectively. Members
of the O. omegaifera complex including O. omegaifera,
Fig. 2. Phylogenetic reconstructions from the LFY dataset. The tree shown is a Bayesian tree with hLRT-selected models
of evolution for exon and intron data, and indel data. Posterior support is shown above branches. Bootstrap support for
maximum likelihood (hLRT-selected model) and maximum parsimony topologies, respectively, is indicated below branches, where support was greater than 50.
500
TAXON 56 (2) • May 2007: 493–504
O. basilissa, O. sitiaca and O. atlantica appeared as a
sister group to these two groups, with O. iricolor nested
in O. omegaifera s.l. A further group obtained contained
O. cinereophila and the endemic taxa from Crete, O.
creticola, O. pallidula and O. kedra.
DISCUSSION
Effectiveness of primer screening for marker
isolation. — As can be seen from the high number of
markers initially tested, screening of previously characterised markers did not prove to be a very effective means
of identifying suitable low-copy markers for use in closely
related Ophrys taxa. A more efficient approach to marker
identification may have been isolation of markers from
cDNA (Schlüter & al., 2005; Whittall & al., 2006). However, since good quality mRNA only became available
when screening efforts were nearing completion, cloning
of mRNA was not available as an alternative option. The
apparent inefficiency of identifying variable sequence
markers using a primer screening approach may in part be
due to (1) many screened markers having been developed
for different plant groups (many are for dicots) and (2)
many genes having housekeeping functions and a high
degree of sequence conservation. It is interesting to note
in this respect that the best marker identified in the present study, LFY, is a gene involved in development rather
than metabolism.
The PI/GLO gene. — The PI/GLO (PISTILLATA/
GLOBOSA) gene of eudicots is a MIKC-type B-class
MADS-box gene involved in establishing petal and
stamen organ identity, its function in monocots being
less clear (e.g., Krizek & Fletcher, 2005, and references
therein). PI expression in all parts of the Ophrys flower is
in agreement with the expression pattern reported by Tsai
& al. (2005). The limited variation encountered among
clones from PI genomic PCR products suggests that our
PCR primers pick up a single copy of the gene in Ophrys,
despite the fact that our PCR primers target conserved
regions of PI. This may indicate that a PI homologue is
present as a single copy gene in Ophrys, as has been found
in the tropical orchid Phalaenopsis (Tsai & al., 2005).
Southern blot experiments would be necessary to test this
hypothesis. PI has previously been used for phylogenetic
purposes in dicots (Bailey & Doyle, 1999). Although our
PI PCR fragment is not phylogenetically informative
within Ophrys fusca s.l., the presence of multiple alleles in
this group suggest that PI may be a useful genetic marker
for the study of Ophrys populations. Also, the number
of substitutions among Ophrys thriptiensis and Orchis
italica PI coding sequences suggest that this gene is likely
to be phylogenetically informative at the level of species
groups or genera. While the here described PCR primers
Schlüter & al. • A screen of low-copy nuclear genes
target a 5′ portion of PI, additional sequence variation
would be expected in the 3′ region of the gene, covering
PISTILLATA’s C domain.
The LFY/FLO gene. — In flowering plants, LFY
(LEAFY in Arabidopsis thaliana; FLORICAULA [FLO] in
Antirrhinum majus) is a floral meristem identity gene and
an important flowering time pathway integrator, several
genetic pathways resulting in the expression of LFY (Weigel & al., 1992; Blázquez & Weigel, 2000; Parcy, 2005;
Simpson & Dean, 2005; Yoon & Baum, 2005). The LFY
protein acts as a transcription factor and its activation in
turn leads to the activation of the floral meristem and
consequently to flowering (Blázquez & al., 1997; Wagner
& al., 2004; William & al., 2004; Maizel & al., 2005). LFY
is present as a single-copy or low-copy gene in many plant
groups (Frohlich & Meyerowitz, 1997; Frohlich & Parker,
2000; Gocal & al., 2001; Wada & al., 2002; Bomblies &
al., 2003). In Orchis and other investigated orchid genera
including Ophrys, a single copy of LFY could be identified
by Southern blotting (Montieri & al., 2004). Therefore,
at least in diploid European Orchidoideae, paralogy is
unlikely to be an issue when using LFY for phylogeny
reconstructions. LFY has been used for phylogenetic purposes in other plant groups (Oh & Potter, 2003, 2005; Grob
& al., 2004; Hoot & al., 2004; Howarth & Baum, 2005),
where the second intron of LFY typically is the longer
one (e.g., Bomblies & al., 2003). In Orchis, however, the
first intron (1 kb) is larger than the second (0.1 kb) intron
(Montieri & al., 2004), which is likely also true for Ophrys
and related genera. The observed intron length variation
among genera is also mirrored by the large number of LFY
indels within Ophrys sect. Pseudophrys, as compared to
ITS. Clearly, the overall information content is higher for
LFY than for ITS or trnL, LFY harbouring 5.8 times more
per cent variable nucleotide characters in the ingroup than
ITS. Moreover, since the amplified LFY gene region is
longer than ITS, the absolute number of characters obtainable from it is greater.
Phylogenetic inference. — The phylogeny (Fig.
2) of closely related taxa of Ophrys taxa based on LFY is
well resolved and represents a major improvement over
previous phylogenetic reconstructions (Pridgeon & al.,
1997; Aceto & al., 1999; Soliva & al., 2001; Bateman &
al., 2003; Bernardos & al., 2005). It clearly shows the
potential of the first intron of the single-copy gene LFY.
Unfortunately, the rather tedious laboratory work necessary to extract sequence information from this gene makes
it difficult to use LFY for routine sequencing with a large
number of samples.
Our phylogenetic reconstructions in part confirm
relationships of taxa based on morphology and pollination
biology. LFY data support the distinctness of O. fusca s.l.,
O. lutea s.l. and O. omegaifera s.l., although two separate groups including O. fusca s.l. taxa were identified.
501
Schlüter & al. • A screen of low-copy nuclear genes
This would suggest that an O. fusca-type species may
have been at the base of Ophrys sect. Pseudophrys. The
placement of O. sitiaca in the O. omegaifera complex is in
agreement with AFLP data (Schlüter & al., in press). However, based on morphology, O. iricolor would have been
expected to be nested in the mainly Andrena-pollinated O.
fusca complex rather than in the O. omegaifera complex,
which is pollinated by Anthophora rather than Andrena
males. Taken together, our phylogenetic reconstruction
is in good agreement with the grouping of taxa based on
pollinators and on morphology, and for the first time provides a molecular hypothesis for the relationship among
O. fusca s.l., O. lutea s.l. and O. omegaifera s.l. groups.
However, it is clear that a phylogeny based on a single
gene does not necessarily reflect organismic history (see
e.g., Sang, 2002). Particularly, recent speciation events or
hybridisation may lead to incongruence between species
and gene trees, where recent species divergence may mean
that coalescence of alleles can pre-date the establishment
of reproductive isolation among speciating populations,
especially if ancestral population size was large. Likewise, gene flow among species may lead to the presence
of additional alleles in a species, which, depending on
the amount of genetic divergence of hybridising species,
may or may not be readily distinguishable from ancestral
polymorphism. Clearly, inference of evolutionary history
in Ophrys should ideally employ multiple nuclear genes,
the highly variable single-copy gene LFY being one of the
tools required. We hope that the availability of low-copy
markers for the genus Ophrys will further our understanding of evolution in this difficult group.
ACKNOWLEDGEMENTS
We wish to thank Eva Hotwagner for help with lab work,
Daniel Fulop and Elena Kramer for access to unpublished sequence and primer information, David Baum for initial help
with primer design, Joanna Padolina for access to her primer
database, Herta Steinkellner for helpful discussions, Matthias
Fiedler for additional plant material, Eleni Maloupa for help
with collection permits, and two anonymous reviewers for
providing valuable comments. We are grateful for funding by
the Austrian Science Fund (FWF) on project P16727-B03.
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