Chloroplast diversity indicates two independent maternal lineages in cultivated grapevine (Vitis...

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Genetic Resources and CropEvolutionAn International Journal ISSN 0925-9864 Genet Resour Crop EvolDOI 10.1007/s10722-014-0169-3

Chloroplast diversity indicates twoindependent maternal lineages incultivated grapevine (Vitis vinifera L.subsp. vinifera)

Rita Lózsa, Ning Xia, Tamás Deák &György Dénes Bisztray

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RESEARCH ARTICLE

Chloroplast diversity indicates two independent maternallineages in cultivated grapevine (Vitis vinifera L. subsp.vinifera)

Rita Lozsa • Ning Xia • Tamas Deak •

Gyorgy Denes Bisztray

Received: 7 February 2014 / Accepted: 25 August 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Chloroplast markers are powerful tools for

research into relationships among grapevine (Vitis

vinifera L. subsp. vinifera) cultivars. However, the

high number of regions common to the chloroplast and

mitochondrion genomes described in grapevine could

interfere with correct phylogeny reconstruction. In this

study, we established a unique chloroplast marker

(UCM) set and investigated the chloroplast haplotype

diversity of 17 grapevine cultivars and seven Asian

Vitis species. Sequencing of four UCMs revealed four

haplotype groups in grapevine based on three nucle-

otide substitutions, three simple sequence repeats, and

one 54 nucleotide (nt) deletion in the trnC-petN

region. The constructed molecular-variance parsi-

mony and statistical parsimony networks indicated

that two independent maternal lineages of grapevine

cultivars originated from a core group of Asian Vitis

species. The presence and absence of a newly

discovered 54 nt deletion corresponded to the two

maternal lineages: deletion (D) and non-deletion (ND)

lineages. The D lineage consisted of three haplotypes,

represented by ‘Furmint’, ‘Gouais blanc’, and ‘Sau-

vignon blanc’. Ancient noble cultivars, such as ‘Pinot

noir’, ‘Riesling’, and ‘Afus ali’, belong to a single

haplotype group (ND) in which the deletion was

absent. Our results provide compelling evidence for

the dual maternal origin of grapevine, and suggest the

occurrence of an ancient hybridization event.

Keywords Chloroplast marker � Domestication �Haplotype � Grapevine � Vitis vinifera

Introduction

Grapevine (Vitis vinifera L. subsp. vinifera [syn. V.

vinifera subsp. sativa Hegi]; Robinson et al. 2012) is

among the most ancient domesticated plants and

has a history associated with humankind since the

Neolithicum (Forni 2012). Currently, several thousand

grapevine cultivars exist, exhibiting extreme morpho-

logical and genetic diversity and also a high hetero-

zygosity that has been maintained because of

vegetative propagation since ancient times (This

et al. 2006). Polymorphism of grapevine is so

prominent that a Russian ampelographer, Alexander

Mikhailovich Negrul, established three main groups

(proles) based on the morphology, physiology, and

geographic origin of the cultivars: proles orientalis

(Armenia, Azerbaijan, Central Asia, and Iran), proles

pontica (Balkan Peninsula, Georgia, Hungary, Mol-

dova, Romania, and Turkey), and proles occidentalis

(Western Europe) (Robinson et al. 2012). This

Electronic supplementary material The online version ofthis article (doi:10.1007/s10722-014-0169-3) contains supple-mentary material, which is available to authorized users.

R. Lozsa � N. Xia � T. Deak � G. D. Bisztray (&)

Department of Viticulture, Corvinus University of

Budapest, Villanyi str. 29-43, Budapest 1118, Hungary

e-mail: [email protected]

123

Genet Resour Crop Evol

DOI 10.1007/s10722-014-0169-3

Author's personal copy

http://dx.doi.org/10.1007/s10722-014-0169-3

grouping is supported to some extent by genetic data

(Myles et al. 2011; Bacilieri et al. 2013).

Some cultivars, such as ‘Gouais blanc’, ‘Pinot

noir’, ‘Riesling’, and ‘Afus ali’, likely have been

preserved through historical periods and some of them

developed high clonal variation owing to human

selection (Myles et al. 2011; Bacilieri et al. 2013). As a

result of long-term vegetative propagation, the genetic

distance between modern grapevine cultivars and the

wild form might represent separation for only a few

generations, unlike herbaceous plants where propaga-

tion is usually via sexually reproduced seeds (Janick

2005).

Derivation of a phylogeny from chloroplast

genome (plastome) data is a popular approach to

reconstruct evolutionary relationships. The methods

that use organellar data for phylogeny reconstruction

are based on the hypothesis that the input data are

haploid and inherited uniparentally. In grapevine,

more than 40 % of the chloroplast genome is also

present in the mitochondrion genome (chondriome) at

least in part owing to lateral gene transfer between the

organelles. Theoretically, this could lead to an inac-

curate phylogeny reconstruction if unique chloroplast

markers (UCMs) are not used, because paralogous

regions evolve independently and could have higher

‘‘ploidity’’ and polymorphy (Goremykin et al. 2009).

Single nucleotide polymorphisms (SNPs), inser-

tions/deletions, and inversions in the chloroplast

genome are potentially informative characters for the

phylogenetic relationships of a given taxa (Shaw et al.

2005), but may be rare between closely related species

or at the infra-specific level because of the plastome’s

low mutation rate. Hypervariable chloroplast regions,

such as simple sequence repeats (cpSSRs), show

higher polymorphism at lower taxonomic levels, but

the risk of homoplasy is higher, especially when only

length polymorphism is examined. In V. vinifera,

cpSSRs are mostly mononucleotide (T or A) repeats

that have been extensively used in phylogenetic and

phylogeographic studies (Imazio et al. 2006; Arroyo-

Garcıa et al. 2006; Grassi et al. 2006; Peros et al. 2011;

Riahi et al. 2011).

In this work, we identified UCMs in silico and used

them to investigate relationships among ancient

grapevine cultivars and with Asian Vitis species that

are the closest relatives of V. vinifera (Zecca et al.

2012). Surprisingly, our results discriminated two

maternal lineages among grapevine cultivars that

originated independently from Asian Vitis species.

On the basis of our results, an ancient hybridization

event and dual maternal origin of grapevine are

hypothesized.

Materials and methods

Plant material

We sampled 163 grapevine cultivars originating from

several locations worldwide (Table 1) and seven

Asian Vitis species (Vitis amurensis Rupr., Vitis

armata Diels et Gilg, Vitis coignetiae Pulliat ex

Planch., Vitis flexuosa Thunb., Vitis piasezkii Maxim.,

Vitis romanetii Rom. Caill., and Vitis yeshanensis J.

X. Chen), and selected Muscadinia rotundifolia

Michx. as an outgroup. All accessions were sampled

from the germplasm collection of the Research

Institute for Viticulture and Oenology, University of

Pecs, Hungary, except for V. flexuosa and V. piasezkii

(obtained from the Botanical Garden of Corvinus

University of Budapest, Soroksar, Hungary), ‘Fur-

mint’ (T85 clone from Tokaj-Hetsz}ol}o Ltd, Tokaj,

Hungary), ‘Carina’, ‘Durif’, ‘Syrah’, ‘Dureza’,

‘Mondeuse blanche’, ‘Nebbiolo’, ‘Peloursin’, and

‘Roubinoy de Magaratch’ (from the INRA collection

of Vassal, Montpellier, France), and ‘Roubine’ (Julius

Kuhn Institute, Siebeldingen, Germany).

DNA extraction

Total DNA was extracted from leaf tissue based on the

method applied for Rosa roxburghii Tratt. (Xu et al.

2004) with minor modifications. Fresh or frozen leaf

tissue (50 mg) was homogenized in 1 mL washing

buffer [100 mM Tris–HCl (pH 8.0), 5 mM EDTA

(pH 8.0), 350 mM glucose, 2 % PVP, 0.4 %

b-mercaptoethanol (freshly added)] and kept on ice

for 30 min. Samples were centrifuged at 3,000 g in a

tabletop centrifuge for 10 min and then the superna-

tant was discarded. Next, 600 lL extraction buffer

[100 mM Tris–HCl (pH 8.0), 1.5 M NaCl, 50 mM

EDTA (pH 8.0), 3 % CTAB, 0.4 % b-mercap-

toethanol (freshly added)] supplemented with 1 lL

of 1 mg/mL RNAse was added to the pellet, vortexed,

and incubated at 65 �C for 30 min, then 60 lL of 5 M

potassium-acetate and 600 lL chloroform-isoamylal-

cohol were added. The samples were vortexed


123


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http://www.vivc.de

vigorously and then centrifuged for 10 min at 12,000 g.

The supernatant (600 lL) was aspirated and extracted

again with 600 lL chloroform-isoamylalcohol. The

supernatant (500 lL) was precipitated with 500 lL

isopropanol and 50 lL of 3 M sodium-acetate. DNA

was pelleted and washed twice with 70 % ethanol.

Pellets were resuspended in 50 lL distilled water.

Selection of chloroplast-specific markers

Given the high number of regions common to the

grapevine chloroplast and mitochondrion genomes

(Goremykin et al. 2009), the marker set was selected

only from predicted unique plastome regions. The

complete grapevine chloroplast genome (DQ424856)

(Jansen et al. 2006) and mitochondrion genomes

(NC_012119.1) (Goremykin et al. 2009) were aligned

online with PipMaker Advanced v2009-03-25-01

(Schwartz et al. 2000) and positions of the primer

sites of 17 chloroplast markers previously used in Vitis

were localized in the alignment (Fig. 1). The markers

comprised trnH-psbA, trnL-trnF, trnC-petN (Ren et al.

2011), ccmp5, ccmp3, ccmp10 (Arroyo-Garcıa et al.

2002), ccSSR-23, ccSSR-9 (Arroyo-Garcıa et al.

2006), VVCP14789, VVCP28926, VVCP50403,

VVCP13285, VVCP32585, VVCP67629,

VVCP69871, VVCP121638, and VVCP123308

(Peros et al. 2011). Those markers were predicted to

be UCMs that potentially amplify only one fragment

solely from the plastome. Figure 1 was generated from

the .pip file with Circos v0.64 (Krzywinski et al. 2009)

to visualize the marker positions. Ultimately, four

markers were selected for our study: trnC-petN (Shaw

et al. 2005), VVCP14789, VVCP28926, and

VVCP50403 (Peros et al. 2011) (see Table S1 for

additional details).

DNA amplification

Primers for the cpSSR markers VVCP14789,

VVCP28926, and VVCP50403 were described by

Peros et al. (2011) (see Table S1). Amplification was

performed with the proofreading Phusion High-Fidelity

DNA Polymerase (Thermo Scientific, Waltham, MA,

USA). Following the initial denaturation (98 �C for

30 s), amplification was performed in 35 cycles of

98 �C for 5 s, 53 �C for 10 s, and 72 �C for 10 s, with

final extension for 7 min at 72 �C. The trnC-petN

region was amplified with grapevine-specific primers

based on those used by Shaw et al. (2005) (For:

CAGTTCAAATCCGGGTG; Rev: CCAAGCGAG

ACTTACTATATCC). Polymerase chain reaction

(PCR) was conducted with Taq polymerase (MBI

Fermentas, Vilnius, Lithuania). After 3 min initial

denaturation at 94 �C, 35 cycles were performed with

the following conditions: 30 s at 94 �C, 30 s at 60 �C,

and 1 min at 72 �C, with final extension for 7 min at

72 �C. After PCR, one volume of 20 % polyethylene

glycol 6,000 (PEG; Sigma-Aldrich, Budapest, Hun-

gary) and 2.5 M NaCl (Molar Chemicals, Budapest,

Hungary) was added to the PCR reaction solution for

purification of the amplified fragments from the

primers prior to commercial direct sequencing

(Macrogen, Amsterdam, The Netherlands, and Base-

Clear, Leiden, The Netherlands) (Lundin et al. 2010).

The mixtures were incubated for 20 min at room

temperature and then centrifuged at 20,0009g for

20 min at 4 �C. The pellets were washed twice with

70 % ethanol, dried, and resuspended in 25 lL

distilled water.

Sequence analysis

Sequence assembly and raw alignments were per-

formed using CLC Sequence Viewer 6.7.1. (CLC bio

A/S, Aarhus, Denmark). Alignments were corrected

manually using BioEdit 7.1.3.0. (Hall 1999). Obtained

sequences are available in GenBank (accession num-

bers: KC962565–KC962664).

For character-based sequence analysis a matrix was

constructed, in which indels in complex regions were

treated as single mutational events (one character), but

in mononucleotide repeat regions (SSRs) every gain or

loss of a base was counted as a single mutational event

(stepwise mutation model) (Banfer et al. 2006).

Although at higher taxonomic levels hypervariable

regions are excluded from analyses (Zecca et al.

2012), at the subspecies level, inclusion of

mononucleotide repeats is commonly accepted

(Arroyo-Garcıa et al. 2006; Imazio et al. 2006). This

allowed resolution of two haplotype groups, but did

not alter the network’s topology obtained from only

SNP and indel data (data not shown).

A minimum spanning network based on molecular-

variance parsimony was constructed with Arlequin 3.5

(Excoffier et al. 2005). A statistical parsimony

network was constructed with TCS 1.21 (Clement

et al. 2000). The same data matrix was used in both


123


analyses. Gaps were treated as a fifth character and the

connection length was set to 20 steps. The obtained

haplotype networks were redrawn manually.

For phylogenetic analysis the optimal evolutionary

model was estimated with jModelTest v2.1.5 (Guin-

don and Gascuel 2003; Darriba et al. 2012) after

exclusion of indels from the alignments. All decision

theory methods suggested the same substitution model

for all loci. Phylogenetic congruence of the chloroplast

loci was tested and verified using Concaterpillar

v1.7.2 (Leigh et al. 2008) and therefore the concate-

nated sequence set was used for subsequent analyses.

Bayesian analysis was carried out with Beast v1.8.0

(Bouckaert et al. 2014). Data were partitioned into two

sets. First, all indels were excluded leaving one partition

of sequences containing only SNP variations. The

second partition contained indel (insertions, deletions,

and SSRs) data transcoded to a binary matrix. Indels in

complex regions were treated as single mutational

events (one character), but in mononucleotide repeat

regions (SSRs) every gain or loss of a base was counted

as a single mutational event (stepwise mutation model)

(Banfer et al. 2006). Bayesian inference analysis was

carried out using the F81 substitution model for the SNP

data and a simple stochastic Dollo model for binary

(indel) data. Bayesian inference analysis was carried out

in three independent runs each of 10,000,000 genera-

tions with trees sampled at an interval of 1,000

generations. The final target tree with posterior proba-

bility (PP) values for the clades was calculated with

TreeAnnotator v1.8.1 (included with Beast) with 10 %

of the trees defined as burn-in, and was visualized with

FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

Maximum parsimony (MP) analysis was carried out

using the Phylip v3.69 software package (Felsenstein

1989), using the ‘seqboot’ program to generate 1,000

bootstrapped input data matrices and ‘dnapenny’ for MP

analysis. A 50 % majority rule consensus tree was cal-

culated from the generated MP trees using the ‘con-

sense’ program of the Phylip package.

Detection of deletion in the trnC-petN region

A 54 nt deletion was identified in sequences of the

trnC-petN region in numerous cultivars (Table 1)

(accession numbers: KJ857084–KJ857228). This

ccSSR-23 §

trnH

-psb

A #

trnL-trnF # ccmp10 @

Fig. 1 Circular

representation of

homologous regions

between grapevine plastome

and chondriome, and

localization of commonly

used markers on the

chloroplast genome. The

organelle genomes are

depicted on the periphery of

the figure, homologous

regions are connected with

light gray links. Nucleotide

positions are given for every

10 and 200 kb for the

plastome and chondriome,

respectively. Note that the

sizes of the genomes are not

proportional. Location of

the 17 widely used markers

are indicated on the

plastome. IR Inverted repeat

region, SSC small single

copy region, LSC large

single copy region. @

(Arroyo-Garcıa et al. 2002),

§ (Arroyo-Garcıa et al.

2006), * (Peros et al. 2011),

# (Ren et al. 2011)


123


http://tree.bio.ed.ac.uk/software/figtree/

deletion was large enough for it to be screened by

agarose gel electrophoresis. The PCR fragments

obtained with the trnC-petN primers were resolved on

a 2 % agarose gel in 1 9 TBE at 0.8 V/cm. Gels were

stained with ethidium bromide and size differences

among bands were detected visually in relation to

appropriate control samples.

Results

Selection of UCMs

In this study, we focused on the chloroplast diversity

of grapevine with consideration of the high amount

of genetic content in common between the organellar

genomes. We predicted regions that are specific to

the plastome by aligning the available reference

genomes of the chloroplast from ‘Syrah’ (Jansen

et al. 2006) and the mitochondrion from ‘Pinot noir’

(Goremykin et al. 2009) (Fig. 1). We examined

whether 17 markers (Peros et al. 2011; Ren et al.

2011) were located in a unique chloroplast segment

(Fig. 1). If a region surrounding the marker was

transferred to the chondriome, we also verified if the

transfer occurred in one piece, potentially allowing

amplification of the same region from both

organelles. If the transfer occurred in several pieces

the PCR reaction could be hindered.

Eight markers were predicted to lie in chloroplast-

specific regions in one copy (trnH-psbA, VVCP14789,

ccmp5, VVCP28926, trnC-petN, ccSSR-9,

VVCP50403, and trnL-trnF). An additional two

markers were located on the border of a unique and

a non-unique region (ccmp3 and ccSSR-23), therefore

a single PCR product could be obtained in these cases.

These markers were specified as UCMs.

The ccmp10 marker is located in the inverted repeat

(IR) region, thus two loci could be amplified from the

chloroplast (Fig. 1). Therefore, this marker was

excluded from the UCMs.

The remaining six markers (VVCP13285,

VVCP32585, VVCP67629, VVCP69871, VVCP121638,

and VVCP123308) were transferred to the mitochon-

drion genome in one piece. Therefore, these markers

were also excluded from further analysis.

Based on the in silico predictions we chose four

UCMs for detailed investigation: one that was used

successfully in Vitis (trnC-petN) (Ren et al. 2011;

Liu et al. 2013) and three hypervariable cpSSR

markers that were specifically designed for Vitis

(VVCP14789 [atpF-atpH], VVCP28926 [rpoB-trnC],

and VVCP50403 [trnT-trnL]) (Peros et al. 2011).

Establishing haplotype groups

We chose 17 widely cultivated and/or ancient cultivars

with diverse geographic origins (Table 1, bold acces-

sions), which are ancestors of many other cultivars

(http://www.vivc.de), and seven Asian Vitis species

and M. rotundifolia as an outgroup for investigation of

the four selected UCMs (trnC-petN, VVCP14789,

VVCP28926, and VVCP50403). We sequenced in

total 1,680 nt of the four noncoding intergenic spacers

of the plastome for the 17 representative accessions.

We detected several polymorphisms in V. vinifera

[three SNPs, three SSRs (mononucleotide repeats),

and a 54 nt deletion] and additional SNPs in the other

species (Fig. 2). Four haplotype groups were resolved

in V. vinifera, and an additional four groups among

the Vitis spp. and M. rotundifolia. Five species (V.

amurensis, V. armata, V. flexuosa, V. piasezkii, and V.

yeshanensis) were indistinguishable from each other

with this marker set.

A minimum spanning network was constructed for

the haplotype groups with molecular-variance parsi-

mony using the software Arlequin. Most Asian Vitis

species formed a core group, except for V. romanetii

and V. coignetiae, which each differed in one SNP

from the central haplotype (Fig. 2a). Surprisingly, the

four haplogroups of V. vinifera were descended from

this core group in two independent lineages. Because

the two lineages were easily distinguished by the pre-

sence or absence of a 54 nt deletion in the trnC-petN

region, we designated the deletion-containing haplo-

groups as D1, D2, and D3, and the group lacking the

deletion as non-deletion (ND) (Fig. 2).

Eastern (countries to the east and south of Asia

Minor) cultivars (‘Afus ali’ and ‘Tsolikovri’) and

Western (countries from Austria to Portugal) cultivars

(‘Carignan blanc’, ‘Pinot’, ‘Riesling’, and ‘Syrah’)

were classified in the ND group (Table 1, cf.

Figure 2). Eastern European–Balkanian (EEB; Mol-

dova, Ukraine, Hungary, and countries of the Balkan

Peninsula except Turkey) cultivars were absent from

the ND group.

In the deletion (D) lineage, the haplotype group D1

differed from the core group in a 54 nt deletion in the


123


http://www.vivc.de

trnC-petN region, one SNP, and one mononucleotide

repeat position (SSR). This group comprised ancient

French cultivars (‘Cabernet franc’, ‘Chenin’, ‘Dureza’,

‘Sauvignon blanc’, and ‘Tannat’). The D2 group was

derived from D1 based on two SSR loci containing

three polymorphic characters (repeat units). The D2

group contained both EEB (‘Batuta neagra’, ‘Furmint’,

and ‘Rkatsiteli’) and Western cultivars (‘Folle

V. amurensis V. armata

V. flexuosa V. piasezkii

V. yeshanensis

Afus ali Carignan blanc

Pinot noir Riesling

Syrah Tsolikovri

V. coignetiae V. romanetii

M. rotundifolia

Batuta neagra Folle blanche

Furmint Merlot

Rkatsiteli

Gouais blanc

Cabernet franc Chenin Dureza

Sauvignon blanc Tannat

V. vinifera

Afus ali Carignan blanc

Pinot noir Riesling

Syrah Tsolikovri

Cabernet franc Chenin Dureza

Sauvignon blanc Tannat

Batuta neagra Folle blanche

Furmint Merlot

Rkatsiteli

Gouais blanc

M. rotundifolia

V. romanetii V. coignetiae

V. amurensis V. armata

V. flexuosa V. piasezkii

V. yeshanensis

SNP 14669 SNP 30119

SNP 14793

SNP 50627

DEL 30133-30186

SSR 14789-14800

SSR 28926-28938 SSR 28926-28938

SSR 28926-28938

SSR 28926-28938

SSR 50408-50419

SNP 28707

V. vinifera

25

A B

Fig. 2 Parsimony-based haplotype networks of grapevine

cultivars and Vitis species based on SNPs, indels, and cpSSRs.

a Molecular-variance-based minimum spanning network, in

which M. rotundifolia served as the outgroup. Differences are

indicated by the connections between groups, except for the

outgroup, for which the length is 25 characters. b A statistical

parsimony-based network, in which the most probable outgroup

is the Vitis spp. haplotype (represented by the square box), thus

M. rotundifolia is not connected to the network. Missing

haplotypes are indicated by small circles


123


blanche’ and ‘Merlot’). Containing the haplotype most

distinct from the core group, the D3 group differed

from the D2 group in one SNP and was represented by

‘Gouais blanc’, an ancient French cultivar.

A statistical parsimony network was generated

using the software TCS (Fig. 2b). The network

contained no loops, thus indicating the absence of

homoplasy. Based on this analysis, the Asian Vitis core

group was the most probable outgroup, thus M. rotun-

difolia was not connected in the network. The ND and

D lineages were well separated and originated inde-

pendently from the Asian core group, thus supporting

the results of the molecular-variance parsimony ana-

lysis. The D lineage included three missing haplotypes

and was not linear, as the D1 group was separated from

D2 and D3. The ND lineage contained only one

missing haplotype. The ND haplotype group was

closer than D1 or D2 to the core group.

Phylogenetic analysis

We conducted Bayesian inference and MP analyses on

the eight haplotype groups and generated a Bayesian

tree with PP values complemented with bootstrap (BS)

values (Fig. 3). Indels were deleted from the align-

ments, coded as a binary character for the Bayesian

inference analysis and inserted as a separate partition

in the sequence data set for the analyses.

Consistent with the parsimony networks, the D and

ND lineages of V. vinifera were resolved in the

Bayesian tree. The highest PP value (0.99) was

obtained for the D group, which did not contain the

ND haplotype. D1 was separated from D2 and D3

(both PP = 0.75). This result was in accordance with

the statistical parsimony network (Fig. 2b), in which

the same topology was observed. All Asian Vitis

species were grouped (PP = 0.88). The ND haplotype

grouped with the Asian Vitis clade, although with little

support (PP = 0.51). In the MP analysis all BS values

were less than 95 %.

A novel easy-to-use deletion marker

The 54 nt deletion (from nt 30,133 to 30,186) in

the trnC-petN plastome region resulted in a size

difference between PCR products and enabled

screening of samples by agarose gel electrophoresis.

We screened 163 cultivars including ancient Eastern,

EEB, and Western accessions for the absence or

presence of the deletion (Table 1). Approximately

85 % of the samples yielded a short fragment length.

Sequencing of the fragments for all 163 accessions

(Table 1) proved that all of the short fragments

contained the same deletion, thus excluding possible

homoplasy.

Eastern and Western cultivars were present in both

groups [see ‘Asyl kara’ (ND), ‘Sultanina’ (D), ‘Tein-

turier’ (ND), and ‘Traminer’ (D)], whereas EEB

cultivars (e.g., ‘Heftakilo’, ‘Kovidinka’, ‘Papsapka’,

‘Tsitsa kaprei’, and ‘Varnenska gimza’) were present

exclusively in the D group. Interestingly, some

cultivars that belonged to the same conculta (colour

variants of the same cultivar; e.g., ‘Gamay blanc’ and

‘Gamay noir’, ‘Carignan blanc’, and ‘Carignan noir’)

were placed in different lineages.

Discussion

The literature on grapevine cultivar classification into

chloroplast haplotype groups is often controversial. It

is a common phenomenon that different studies

classify the same cultivar into different haplotype

groups, even if the same marker set was used (e.g.,

Arroyo-Garcıa et al. 2006 vs Peros et al. 2011). Biases

in plant collections, sampling, data handling, and

homoplasy are among the many possible reasons

behind this phenomenon. Additional complications

could arise when usage of non-unique (multilocus)

markers results in non-haploid data, given that the

plastome data are treated a priori as haploid in

M. rotundifolia

V. coignetiae

V. romanetii

V. amurensis

ND

D3

D2

D1

0,75

0,99

0,79

0,51

0,88

0,35

87

51

100

<50

Fig. 3 Bayesian tree of the eight haplotypes with PP comple-

mented with bootstrap (BS) values from MP analysis where

applicable. PP values are between 0 and 1, BS values are

between 1 and 100


123


experimental procedures and bioinformatic analysis

pipelines. In grapevine, the high amount of genetic

content common to the chloroplast and mitochondrion

genomes raises the possibility of such a problem

(Goremykin et al. 2009). Hence, we chose UCMs to

investigate chloroplast diversity of grapevine cultivars

and their relationship with Vitis species.

We checked in silico 17 plastid markers previously

used in Vitis and found that some of the markers

indeed were located in regions that have been

transferred to the chondriome, and in addition one

marker (ccmp10) comprised two copies in the plas-

tome. Thus, PCR products for these markers likely

come from at least two loci. For example, the ccmp10

marker, which is located in the rps19–rp12 intergenic

spacer in the IR region, is highly polymorphic

(Arroyo-Garcıa et al. 2002) possibly owing to its

duplication. Interestingly, this marker was originally

designed (Weising and Gardner 1999) based on the

chloroplast genome of Nicotiana tabacum L. (Shino-

zaki et al. 1986), where this marker is on the border of

the large single copy and IR regions, potentially

yielding a single PCR product. Based on our predic-

tions, we recommend using non-UCMs with reserva-

tions or omitting them from grapevine studies.

We sequenced in total 1,680 nt of four noncoding

chloroplast intergenic spacers for eight Vitis species

and 17 grapevine cultivars. The cultivars were chosen

to represent ancient grapevines from several

geographic regions. For example, ‘Pinot noir’,

‘Rkatsiteli’, ‘Gouais blanc’, and ‘Cabernet franc’ are

all very ancient and are parents of numerous important

cultivars (Robinson et al. 2012).

Grapevine is commonly considered to have been

domesticated from Vitis vinifera L. subsp. sylvestris

(C.C.Gmel.) Hegi, which we did not analysed. This is

a highly diverse taxon that comprises morphologically

and genetically distinct geographic populations and its

taxonomic status remains uncertain (Ekhvaia and

Akhalkatsi 2010; Myles et al. 2011; Zecca et al. 2012).

We identified four haplogroups among V. vinifera

cultivars that could be distinguished by the most

polymorphic VVCP28926 marker alone. This marker

lies in the rpoB-trnC region, which is highly informa-

tive in angiosperms, even among closely related

species (Shaw et al. 2005). The four grapevine

haplogroups belong to two maternal lineages, desig-

nated ND and D. The ND group was uniform with

regard to the markers used and was identical to the

‘Syrah’ reference genome sequence (Jansen et al. 2006).

In contrast, the D group comprised three haplotypes: D1,

D2, and D3. D3, which harbored one additional SNP,

was represented by ‘Gouais blanc’ only.

According to character-based parsimony analyses,

the ND and D maternal lineages are related indepen-

dently to a central group of Asian Vitis species (Fig. 2),

which could indicate an ancient hybridization event

prior to domestication. Interspecific hybridization has

played an important role in the history of some

domesticated plant species, such as rice, apple, and

potato (Coart and Van Glabeke 2006; Takahashi et al.

2008; Gavrilenko et al. 2013). The high heterozygosity

of grapevine (Myles et al. 2011; Bacilieri et al. 2013)

and the finding that both maternal lineages are

represented among ancient cultivars from the center

of domestication (‘Rkatsiteli’ and ‘Tsolikouri’) also

support the hybridization theory. Given that we

identified more subgroups in the D lineage relative to

the ND lineage, despite the limited number of cultivars

analyzed, it is possible that the primary genetic pool

carried the deletion and that the ND haplotype

originated from a single introgression event. Lineage

D could have originated from V. v. subsp. sylvestris or

an unsampled population of an ancestral Vitis species

or species complex, not necessarily an extant popula-

tion. Another, but less likely, explanation is that

chloroplast capture (Stegemann et al. 2012), when

plastids are transmitted from one species to another

during grafting, could conceivably occur naturally

between sympatric species, although this phenomenon

has not been described in Vitis.

The phylogenetic trees obtained by Bayesian

inference and MP analyses were in agreement with

the statistical parsimony network in terms of the D

clade, for which the same topology was observed

(Figs. 2b, 3). The ND group was consistently sepa-

rated from the D clade, although its relatedness to the

other Vitis species is uncertain on account of the low

statistical support. However, it must be borne in mind

that the applicability of our marker set is limited for

evolutionary model-based phylogenetic assessment

because it is mostly based on hypervariable regions

that evolve faster than other regions, and furthermore

it resolves V. vinifera but not the other species, leaving

the latter conclusions somewhat speculative.

In the trnC-petN region, we identified a 54 nt

deletion for the first time in V. vinifera. This deletion

was absent in the investigated Vitis species and in


123


other Vitis species investigated in previous studies

(Ren et al. 2011). This region tends to contain

relatively large deletions (Shaw et al. 2005), and the

deletions are more than twice as rare in angiosperms as

nucleotide substitutions (Shaw et al. 2007). The

identified deletion can be easily screened by agarose

gel electrophoresis. We tested the utility of the trnC-

petN marker by screening 163 grapevine cultivars for

the deletion and classified them into the D or ND

lineages. We excluded possible homoplasy by

sequencing this region in all accessions. We found

that only 15 % of the sampled cultivars belonged to

the ND group, whereas 85 % contained the deletion.

The 54 nt deletion in the trnC-petN region is

suitable for assessment of certain pedigrees or of

relationships between cultivars. For example, the ND

group contains several related cultivars, e.g., ‘Pinot

noir’, ‘Mondeause blanche’, ‘Syrah’, ‘Durif’, and

‘Carina’ belong to the same family; ‘Goldriesling’,

‘Perlriesling’, and ‘Rieslingtraminer’ are offspring of

‘Riesling’. We also observed some interesting

pedigrees by screening for the deletion: ‘Gamay noir’

and ‘Gamay blanc’ are siblings (‘Gouais blanc’ 9

‘Pinot’) (Robinson et al. 2012), but they were classi-

fied to different lineages in the present study. This

could mean that the mother was ‘Gouais blanc’ for

‘Gamay noir’ and ‘Pinot’ for ‘Gamay blanc’. ‘Zwei-

gelt’ is known to have been raised from the cross of

‘Blaufrankisch’ and ‘St Laurent’ (Maletic et al. 1999);

however, both parents were classified in the D group,

whereas ‘Zweigelt’ carries an ND haplotype. ‘Gouais

blanc’ (haplotype D3) is proven to be a parent of

‘Riesling’ (Robinson et al. 2012); our results show that

it could be the paternal parent of ‘Riesling’ as the two

cultivars belong to different chloroplast lineages. This

finding is particularly interesting, because ‘Gouais’—

on the basis of a single chloroplast SNP— is proven to

be the maternal parent of several important varieties

(Hunt et al. 2010), which we found to be classified in

the D lineage (‘Chardonnay’, ‘Gamay noir’, and

‘Aligote’) (Table 1). Although these results could be

the outcome of errors in germplasm collections or

sampling, they could also provide important informa-

tion on sometimes incorrect pedigrees.

To determine the history of domesticated plants is a

difficult task, and current findings usually complicate

existing models and result in additional uncertainties.

Our work is the first that examines the plastome

diversity with consideration of inter-organellar gene

transfer and indicates possible ambiguities in chloro-

plast marker studies in grapevine. The results provide

convincing evidence for the existence of two inde-

pendent maternal lineages in grapevine and suggest

the occurrence of an ancient hybridization event.

Acknowledgments Funding was provided by the Corvinus

University of Budapest and by the New Szechenyi Plan of the

Hungarian Government (project KTIA_AIK_12-1-2013-0001).

We thank Endre Sebestyen, Anita Lozsa, and Peter Bodor for

their help and useful comments and Pal Kozma, Sandrine Lalet,

Adam Guthermut, and Erika Maul for providing materials.

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