Genetic diversity of Vitis vinifera in Georgia: relationships between local cultivars and wild grapevine, V. vinifera L. subsp. sylvestris

  • Published on
    24-Jan-2017

  • View
    219

  • Download
    0

Transcript

  • RESEARCH ARTICLE

    Genetic diversity of Vitis vinifera in Georgia: relationshipsbetween local cultivars and wild grapevine, V. vinifera L.subsp. sylvestris

    Jana Ekhvaia Maia Gurushidze

    Frank R. Blattner Maia Akhalkatsi

    Received: 29 October 2013 / Accepted: 14 April 2014

    Springer Science+Business Media Dordrecht 2014

    Abstract The Caucasus and Middle East regions are

    considered to be theprimary centre of origin ofcultivated

    grapevine, and, as confirmed by archaeobotanical,

    archaeological, and cultural evidence, Georgia belongs

    to this earliest centre of winemaking. This study aims to

    investigate the genetic diversity, population structure

    and relationships of local autochthonous wine cultivars

    and wild grapevine, Vitis vinifera subsp. sylvestris.

    Multiple accessions of 15 Georgian aboriginal cultivars

    and 42 individuals of wild grapevine from different

    regions of Georgia and adjacent Turkey were genotyped

    at 17 nuclear microsatellite loci. A total of 160 alleles

    were detected with a mean number of 9.41 alleles and the

    effective number of 4.6 alleles (r) per locus, indicating

    that the SSRs were highly informative. Despite high

    genetic diversity, the level of genetic differentiation

    among defined wild and cultivated populations is low

    (Fst = 0,05; P***), which together with the outcome of

    model-based cluster analyses and genetic assignment

    methods point to gene flow among wild populations, as

    well as among cultivated and wild accessions. Besides,

    the data presented here suggest that local cultivars

    Saperavi and Tavkveri are independently derived

    from different local wild populations, while the majority

    of Georgian cultivars seem to have a single origin.

    Overall, the present study takes important steps for better

    characterization of Georgian cultivated and wild grape-

    vines, and supports Georgia as one of the important

    centres of grapevine domestication still harbouring

    valuable genetic resources for grapevine breeding.

    Keywords Domestication Genetic diversity Genetic similarity Georgian grape cultivars Microsatellites Vitis vinifera subsp. sylvestris V. vinifera subsp. vinifera

    Introduction

    Grapevine (Vitis vinifera L.) is one of the most valuable

    crops worldwide and consists of two forms, domesti-

    cated V. vinifera L. subsp. vinifera and wild V. vinifera

    L. subsp. sylvestris (C.C.Gmel.) Hegi. The Caucasus

    region is considered to be within the primary centre of

    origin of domesticated grapevine, with high relevance

    Electronic supplementary material The online version ofthis article (doi:10.1007/s10722-014-0125-2) contains supple-mentary material, which is available to authorized users.

    J. Ekhvaia

    Institute of Ecology, Ilia State University, Tbilisi, Georgia

    M. Gurushidze (&) F. R. BlattnerLeibniz Institute of Plant Genetics and Crop Research

    (IPK), 06466 Gatersleben, Germany

    e-mail: maiko@ipk-gatersleben.de

    F. R. Blattner

    German Centre for Integrative Biodiversity Research

    (iDiv), 04103 Leipzig, Germany

    M. Akhalkatsi

    Department of Plant Genetic Resources, Institute of

    Botany, Ilia State University, Tbilisi, Georgia

    123

    Genet Resour Crop Evol

    DOI 10.1007/s10722-014-0125-2

    http://dx.doi.org/10.1007/s10722-014-0125-2

  • for the further distribution of the crop throughout the

    Mediterranean basin and for the development of

    modern European cultivars (De Candolle 1885; Poteb-

    nia 1911; Negrul 1946; Mullins et al. 1992; Jackson

    1994; Damania et al. 1997; Sefc et al. 2003; Constan-

    tini 2004; Forni 2006; This et al. 2006; Vouillamoz

    et al. 2006). Grapevine was among the first fruits to be

    cultivated in Georgia (Javakhishvili 1930). Confirma-

    tions for long-lasting cultivation of grapevine in

    Georgia stem from archaeological remains of berries

    and seeds of domesticated grapes dated *8,000 yearsbefore present (yBP) in southeastern Georgia (Ra-

    mishvili 1988; McGovern et al. 1997). Other archae-

    ological evidences of prehistoric winemaking are

    found in near proximity of the Caucasian region in

    northern Iran at the Hajji Firuz Tepe site in the northern

    Zagros Mountains dated to about 7,4007,000 yBP

    (McGovern 2003) and in the Levant where archaeo-

    logical findings are dated to *6,000 yBP (Zohary andSpiegel-Roy 1975; Zohary and Hopf 2000).

    Another indicator of a possible origin of cultivated

    grapevine in the Caucasus region is high genetic and

    morphological diversity of both wild and cultivated

    grapes in this area (Vavilov 1931; Grassi et al. 2006;

    Ekhvaia and Akhalkatsi 2010). About 500 names of

    autochthonous grapevine varieties, including the cen-

    turies-old cultivars Rkatsiteli, Ojaleshi, and Sa-

    peravi, are known from Georgia (Javakhishvili 1930;

    Ketskhoveli et al. 1960). They show great ampelo-

    metric variability and broad adaptability to different

    climates and soils (Vinogradov-Nikitin 1929; Negrul

    1946; Ketskhoveli et al. 1960; Ramishvili 1970;

    Tsertsvadze 1989; Ekhvaia and Akhalkatsi 2006).

    However, only half of these cultivars have been

    conserved in some national collections, and today only

    a small number of local varieties are still cultivated

    (Chkhartishvili and Tsertsvadze 2004). This causes

    genetic erosion on this rich ampelographic heritage,

    involving loss of a valuable gene pool before it could

    be evaluated.

    Wild grapevine occurs in the Caucasus region

    mainly in riparian forests and reaches upper vegetation

    zones such as oak-hornbeam, beech and spruce forests

    at up to 900 m a.s.l. (Ramishvili 1988). Like in other

    parts of the world, the distribution area of the

    subspecies was dramatically reduced in Georgia due

    to human activities. Therefore, investigation and

    preservation of genetic variability of wild grapevine

    populations has become a priority to avoid genetic

    erosion and maintain invaluable genetic resources for

    cultivated grapevines (Arnold et al. 1998).

    In recent years, particular attention has been paid to

    elucidate the domestication history of cultivated grape

    (Grassi et al. 2003; Sefc et al. 2003; Arroyo-Garcia et al.

    2006; Myles et al. 2011). Myles et al. (2011) supposed a

    Near East origin of subsp. vinifera, while others argue

    that at least a second independent domestication centre

    exists in the Mediterranean (Grassi et al. 2003; Sefc et al.

    2003; Arroyo-Garcia et al. 2006; Lopes et al. 2009).

    Special emphasis has been given to evaluation and

    genetic characterization of different cultivars and/or

    determination of the main events that enabled the

    morphological transformation from wild subsp. sylves-

    tris to cultivated grapevine (Aradhya et al. 2003;

    Vouillamoz et al. 2006; Imazio et al. 2006; This et al.

    2006; DOnofrio et al. 2010; Bacilieri et al. 2013).

    Unfortunately, the above-mentioned studies included

    none or only a few autochthonous varieties from the

    Caucasus region. A recent work of Imazio et al. (2013)

    evaluated the genetic diversity of Georgian cultivars

    from European germplasm repositories and investigated

    their relationships with some worldwide distributed,

    mainly European varieties. However, there is very

    limited knowledge about genetic diversity and popula-

    tion structure of Georgian wild grapevine. Conse-

    quently, it is of high importance to study aboriginal

    grape varieties from the supposed domestication area

    and determine their genetic relationships to the local

    wild populations. In addition, knowledge about genetic

    variation in natural populations may provide relevant

    information for biodiversity conservation strategies of

    the region. In the present study, we have performed a

    comprehensive comparative study of the native popula-

    tions of wild subsp. sylvestris and the local autochtho-

    nous cultivars. We employed 17 nuclear microsatellite

    (SSR) markers to (1) study genetic diversity of Georgian

    wild and domesticated grape germplasm and (2) assess

    the relationships among autochthonous cultivars and

    wild grapevine populations in order to shed light on the

    origin of the locally cultivated varieties.

    Materials and methods

    Plant material

    A total of 99 samples representing 57 accessions were

    studied: 15 Georgian autochthonous cultivars (V.

    Genet Resour Crop Evol

    123

  • vinifera subsp. vinifera) and 42 individuals of wild V.

    vinifera subsp. sylvestris from different regions of

    Georgia and adjacent territory of Turkey. The wild

    grapevine was represented by six populations located in

    river basins: Ajaristskali population (n = 4), Alasan-Iori

    population (n = 10), Aragvi population (n = 5), Chor-

    okhi population (n = 8), Mtkvari population (n = 13),

    and Tskhenistskali population (n = 2) (Table 1; Fig. 1).

    All wild individuals were sampled from locations

    characterized by environmental conditions typical for

    wild grapevine habitats: riparian and floodplain forests

    with a high degree of humidity and abundant tree species

    on which grapevines grow as lianas. All individuals

    within the studied populations of wild grape were

    identified as dioecious plants, which clearly separates

    them from the monoecious cultivated form.

    Table 1 List of cultivated and wild accessions, colour ofberries (Rred, Wwhite); sex of individuals (Ffemale,

    Mmale), location and source institution name (MSEM

    Martvili State Ethnographical Museum collection, Martvili,

    West Georgia; VOIViticulture and Oenology Institute col-

    lection, Tbilisi, East Georgia)

    N Variety and synonymy Sample number Colour Region of origin Source

    Cultivated group

    1 Aleksandrouli 4 R Racha-Lechkhumi reg., Georgia VOI

    2 Avshiluri 4 R Samegrelo reg., Georgia VOI

    3 Chodi 4 R Guria reg., Georgia MSEM

    4 Chvitiluri 4 R Samegrelo reg., Georgia VOI

    5 Kachichi 4 R Abkhazeti reg., Georgia VOI

    6 Kamuri Tetri 4 W Guria re., Georgia MSEM

    7 Khojishtoli, Kharistvala Kolkhuri 4 R Ajara, Guria, Samegrelo reg., Georgia VOI

    8 Mujretuli 4 R Racha-Lechkhumi reg., Georgia VOI

    9 Ojaleshi 4 R Samegrelo reg., Georgia MSEM

    10 Rkatsiteli 4 W Kakheti reg., Georgia VOI

    11 Saperavi 4 R Kakheti rek., Georgia VOI

    12 Shkhucheshi 4 W Samegrelo reg., Georgia MSEM

    13 Shonuri, Svanuri 4 R Svaneti reg., Georgia MSEM

    14 Tavkveri 4 R Shida Kartli reg., Georgia VOI

    15 Uchakhardani 4 R Samegrelo reg., Georgia VOI

    N Population Individuals number Sex Location

    Wild group

    1 Ajaristskali 4 1M/3F Near vil. Godgadzeebi, Khulo distr., Ajara reg., Georgia

    2 Alasan-Iori 10 7M/3F Near vil. Khalatsani, Akhmeta distr., Kakheti reg., Georgia

    Lagodekhi Nature preserve, Lagodekhi distr., Kakheti reg., Georgia

    Jumas Kure Nature preserve, Dedoplis Tskaro distr., Kakheti reg.,

    Georgia

    Iori Nature preserve, Sagarejo distr., Kakheti reg., Georgia

    3 Aragvi 5 1M/4F Near vil. Zhinvali, Dusheti distr., Mtskheta-Mtianeti reg., Georgia

    4 Chorokhi 8 5M/3F Near Artvin HES, Artvin, Turkey

    5 Mtkvari 13 7M/6F Betw. vv. Atskuri and Likani, Borjomi distr., Samtskhe-Javakheti

    reg., Georgia

    Gardabani forest-park,Gardabani distr., Kvemo Kartli reg., Georgia

    Near vil. Sakorintlo, Kaspi distr., Shida Kartli reg., Georgia; Suburb

    of Tbilisi, Georgia

    6 Tskhenistskali 2 2F Jonoula gorge, Tsageri distr., Racha-Lechkhumi reg., Georgia

    Tskhaltubo-Tsageri road, Tsageri distr., Racha-Lechkhumi reg.,

    Georgia

    Genet Resour Crop Evol

    123

  • Molecular methods

    Genomic DNA was extracted with Qiagen DNeasy

    Plant Mini Kit according to the manual provided by

    the manufacturer or according to Lodhi et al. (1994)

    from silica-dried leaves. 17 nuclear microsatellite loci,

    well characterized in previous studies, were used:

    VVS2, VVS4 (Thomas and Scott 1993); VVMD7,

    VVMD24, VVMD25, VVMD27, VVMD28,

    VVMD32, VVMD34 (Bowers et al. 1996, 1999);

    scu04vv, scu14vv (Scott et al. 2000) used in studies on

    V. vinifera, and VrZAG21, VrZAG47, VrZAG62,

    VrZAG64, VrZAG79, VrZAG83 (Sefc et al. 1999),

    originally identified in V. riparia Michx. Eight of these

    markers (VrZAG62, VrZAG79, VVMD7, VVMD25,

    VVMD27, VVMD28, VVMD32, VVS2) had been

    previously selected by the European GENRES con-

    sortium as the core set for genotyping grapevine

    collections (Costantini et al. 2005). PCR amplification

    was performed in 10 lL final volume containing about10 ng template DNA, 0.2 lM of each dNTP, 0.2 lMof each primer (one primer from each pair was

    fluorescently labelled), 1.5 mM MgCl2 and 0.2 U Taq

    DNA polymerase in 109 reaction buffer (QIAGEN).

    The thermocycler (PE Biosystems, Gene Amp 9700)

    was programmed for an initial step of 2 min at 94 Cfollowed by 40 cycles at 92 C for 30 s, 5056 C for1 min, and 70 C for 2 min, and a final extension stepat 72 C for 10 min.

    Length polymorphisms of the amplified products

    were determined on a MegaBACE 1000 DNA

    sequencer (Amersham Biosciences). Fragment lengths

    were estimated in relation to an internal size standard

    (Amersham Biosciences). In each run, we have

    included 14 reference cultivars (Table 2), approved

    by the two European projects GENRES-081 (Maul

    and This 2008) and GrapeGen06 (Bacilieri 2007).

    They served as standards in order to have consistent

    allele sizes over all runs and they allowed allele size

    comparison of our study with other published data.

    Data analysis

    Genotypes showing one or two alleles for each locus

    were scored as homozygous and heterozygous, respec-

    tively. Genetic polymorphism for each population

    was assessed by calculating the mean number of

    alleles per locus (MNA), the observed heterozygosity

    Fig. 1 Geographical distribution of the studied wild V. vinifera subsp. sylvestris populations in Georgia and Turkey

    Genet Resour Crop Evol

    123

  • (Ho) (Brookfield 1996), expected heterozygosity (He)

    under HardyWeinberg equilibrium (Nei et al. 1983),

    probability of identity (PI) (Paetkau et al. 1995), and

    estimation of null allele frequency from the heterozy-

    gote deficiency (No) (Brookfield 1996) were calcu-

    lated using genetic analysis package IDENTITY 4.0

    (Wagner and Sefc 1999). The effective number of

    alleles (r) was obtained according to the formula

    Ne =P

    (pi2)-1 (Morgante et al. 1994). The discrim-

    ination power (D), which is an estimation of the

    probability that two randomly sampled accessions

    could be distinguished by their SSR profiles (Tessier

    et al. 1999), was calculated as D = 1 - C, where C is

    probability of coincidence, or the probability that two

    varieties match by chance at one locus (C =P

    pi2,

    where pi is the frequency of different genotypes for a

    given locus). The polymorphism information content

    (PIC), which provides an estimate of the discrimina-

    tory power of each SSR locus, was calculated as 1 -P

    pi2 -

    PP2pi

    2pj2, where pi equals the frequency of

    the ith allele and pj the frequency of the (i ? 1)th

    allele (Botstein et al. 1980).

    Population comparisons were made using measures

    based on allele or genotype identity (exact tests, Fst)

    because Gaggiotti and Excoffier (2000) have shown

    that Fst is always the better estimator of gene flow/

    population divergence when the sample sizes are small

    to moderate. Allele frequencies were compared using

    the probability test for genic differentiation described

    by Raymond and Rousset (1995). Excess and defi-

    ciency of heterozygotes, deviations from Hardy

    Weinberg equilibrium, genic and genotypic differen-

    tiation were compared using GENEPOP, version 3.4

    (Raymond and Rousset 1995) or GENEPOP on the Web

    (...

Recommended

View more >