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Combining Microsatellite Markers and Ampelography for Better Management of Romanian Grapevine Germplasm Collections

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  • National Research and Development Institute for Biotechnology in Horticulture Stefanesti, Arges, Romania

Abstract and Figures

Aiming to investigate the correspondence of phenotypic variability and molecular markers, 50 varieties of Vitis vinifera ssp. vinifera, ancient grapevine varieties grown on Romanian territory, were selected to be analysed. All varieties were subjected to ampelographic analyses with OIV descriptors and also to molecular analyses with 13 microsatellites. The morphology description with recommended methods corresponded with the reference literature, proving the correct registration of the varieties at the moment of acquisition. Markers efficiency for assessing the genetic diversity among studied varieties was evaluated by computation of statistic parameters referring to the polymorphisms found. Among the internationally recommended SSR markers, ISV4 and VMCNG4b9 markers displayed interesting PIC and PI values. By comparing the SSR profiles of the 50 Romanian grapevine varieties with those in the Eu database, the following can be concluded: confirmation of 10 synonymies mentioned in old documents, while 3 synonymies mentioned in old reference literature were not confirmed, new synonyms were found for 4 accessions, additional synonyms were found for 4 accessions, which are to be added to the previous ones already mentioned in the literature, the synonymies in three groups of accessions were documented for the first time in the specialised literature, the unicity of three Romanian accessions was proved (ʻMoroştinăʼ, ʻNegru mareʼ and ʻRomânieʼ) and the genetic profiles of 15 varieties were identical to those already recorded in the investigated databases
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Original Article
Combining Microsatellite Markers and Ampelography for Better
Management of Romanian Grapevine Germplasm Collections
Carmen F. POPESCU1*, Manna CRESPAN2
1
National Research and Development Institute for Biotechnology in Horticulture (NRDIBH) Stefanesti-Arges, Sos. Bucuresti-Pitesti, no. 37,
Stefanesti, CP 117715, County Arges, Romania; carm3n_popescu@yahoo.com (*corresponding author)
2
Council for Agricultural Research and Economics - Centre of Viticulture and Enology Research, Via XXVIII Aprile 26, 31015 Conegliano (TV),
Italy; manna.crespan@crea.gov.it
Abstract
Aiming to investigate the correspondence of phenotypic variability and molecular markers, 50 varieties of
Vitis vinifera
ssp.
vinifera, ancient grapevine varieties grown on Romanian territory, were selected to be analysed. All varieties were subjected to
ampelographic analyses with OIV descriptors and also to molecular analyses with 13 microsatellites. The morphology
description with recommended methods corresponded with the reference literature, proving the correct registration of the
varieties at the moment of acquisition. Markers efficiency for assessing the genetic diversity among studied varieties was
evaluated by computation of statistic parameters referring to the polymorphisms found. Among the internationally
recommended SSR markers, ISV4 and VMCNG4b9 markers displayed interesting PIC and PI values. By comparing the SSR
profiles of the 50 Romanian grapevine varieties with those in the EU database, the following can be concluded: confirmation
of 10 synonymies mentioned in old documents, while 3 synonymies mentioned in old reference literature were not confirmed,
new synonyms were found for 4 accessions, additional synonyms were found for 4 accessions, which are to be added to the
previous ones already mentioned in the literature, the synonymies in three groups of accessions were documented for the first
time in the specialised literature, the unicity of three Romanian accessions was proved (ʻMoroştinăʼ, ʻNegru mareʼ and
ʻRomânieʼ) and the genetic profiles of 15 varieties were identical to those already recorded in the investigated databases.
Keywords: autochthonous cultivars; genotyping; homonyms; synonyms; SSR markers
AcademicPres
Available online: www.notulaebiologicae.ro
Notulae Scientia Biologicae
Print ISSN 2067-3205; Electronic 2067-3264
Not Sci Biol, 2017, 10(2):193-198. DOI: 10.25835/nsb10210297
Introduction
A comprehensive characterization of accessions
preserved in germplasm collections enta
ils ampelographic
description with standardized descriptors and molecular
analyses with an internationally shared set of SSR markers.
The two approaches ensure accurate identification and
guarantee the authenticity of plant material used in research
work,
establishing new vineyards. The first international standard
for the classification of grapevine based on morphological
characters was established in 1873 by the International
Ampelography Committee in
Vienna. Permanently
improved, this system for describing and characterizing
species and varieties of Vitis
genus comprises today 150
descriptors, of which 48 are essential.
Since the mid-
1990s, in addition to the descriptor
system, microsatellite markers
have been used for the
genetic characterization of grapevine varieties, as a
useful
tool complementing ampelography. Thomas
et al
. (1993)
first applied the SSR analysis for identifying grapevine
varieties and their results showed that the microsatellite
s
equences are: a) abundant in the genome; b) highly
informative for identifying Vitis vinifera
varieties; c)
transmitted to descendants following the Mendelian laws of
heredity (co-
dominant); d) suitable for genetic mapping and
investigation of the degree o
f genetic relatedness among
genotypes. The main advantage of microsatellite sequences
is the high reproducibility, which allows the exchange of
data between laboratories around the world, the final result
being a unique genetic profile for each variety and
,
implicitly, unambiguous characterization of any variety.
These features were considered underpinned for the
OIV decision to recommend in 2009 the use of six SSR loci
to identify Vitis
genotypes (OIV, 2009). Subsequently, after
testing improved methods o
n a number of over 2,000
genotypes, belonging to 30 germplasm collections, nine SSR
markers were finally recommended (Maul et al
., 2012).
Thus, identifying grapevine varieties using at least 9 SSRs
R
eceived: 10 May 2018. Received in revised form: 27 Jun 2018. Accepted: 28 Jun 2018. Published online: 29 June 2018.
Popescu CF and Crespan M / Not Sci Biol, 2018, 10(2):193-198
following the protocol described by Migliaro
et al
. (2013),
usi
ng fluorescent primers and an ABI3130xl genetic
analyser (Applied Biosystems, Foster City, CA).
Statistical analysis
Data analysis: statistics on scored alleles were computed,
such as the number of alleles (Na), the number of effective
alleles (Ne), obse
rved heterozygosity (Ho), expected
heterozygosity (He), polymorphism information content
(PIC), the probability of identity (PI), and the probability
of null alleles (F(null) using the freely available Cervus 3.0.7
and GenAlEx softwares.
Results and Discussion
Ampelographic description
From the total of fifty (50) varieties, five (5) were very
well appreciated and grown in many other countries with
long time viticulture tradition, seven (7) are major local
cultivars, extensively grown in Romania and two (2)
are
minor local cultivars, fairly utilized in wine production
(Table 1).
Most of the studied varieties (36) are considered
autochthonous, grown for centuries on the study territory,
but today rarely notified in private households, or preserved
in few germ
plasm collections. Valuable information with
detailed ampelographic de
scriptions was found for 44
varieties in old documents, so it was possible to obtain a
preliminary con
firmation of the authenticity of the
corresponding accessions. No information was
found for
ʻBraghină albăʼ, ʻGalbe maruntă'ʼ, ʻMoroștiʼ
,
ʻRomânieʼ, ʻȚâța caprei neagʼ and ʻȚâța vacii neagʼ
, so
it was not adequate to establish their possible authenticity
only on the basis of our morphological characterization.
Ampelographic description
of all varieties was
performed at specific phenophases (shoot tip and young
leaf, during spring; mature leaf after flowering; bunch and
berry between ripening and harvesting), on target organs, in
specific position on the vine and the right notifications w
ere
recorded for expression level of each character. All features
and their descriptor records were compared with those in
the reference literature, presenting comprehensive
information for each variety.
Some characters were slightly
different, such as: in
tensity of anthocyanin coloration of the
shoot tip (OIV 003), colour of upper side of the young leaf
(OIV 051), shape of the mature leaf (OIV 067), degree of
opening/overlapping of petiole sinus (OIV 079), shape of
petiole sinus (OIV 080), density of prost
rate hairs between
main veins on lower side of mature leaf (OIV 084), bunch
density (OIV 204), bunch shape (OIV 208), berry shape
(OIV 223), sugar content of must (OIV 505) and total
acidity of must (OIV 506). These differences are often
explained as diffe
rent response of each variety to variable
cultural conditions, healthy status of the plants, observer
subjectivity (Fatahi et al., 2003; Carimi et al
., 2010; Tassie,
2010).
In some instances, were noted varieties that were
morphologically very similar vari
eties, so that they cannot be
distinguished by visual comparison, but had different DNA
profiles, and, on the contrary, clones of the same cultivar
showed different phenotypes, even if their DNA profile was
identical.
has become a common practice for synonyms and
homonyms det
ection, preliminary investigation on genetic
relatedness between or among varieties, and identification
of plant materials of unknown origin by comparing their
profiles with reference genotypes collected in the
international databases.
Grapevine germplas
m collections in Romania have a
wide range of varieties, an overwhelming majority having a
long tradition of vineyar
d culture. The series of seven
“Ampelografia of the Romanian People’s Republic
published between 1959 and 1966 with the detailed
descriptio
n of the old grapevine varieties and the works of
Indreas and Visan (2001) and Rotaru (2009) about those
considered of economic importance, are the reference
literature for the field of ampelography. In the last two
decades, for many of the vine varieties
considered valuable
and important for the production of grapes and wine, the
ampelographic description was completed with molecular
characterization (Bodea et al., 2009; Butiuc-Keul et al
.,
2010; Coste et al., 2010; Gheorghe et al., 2008; Gheţea
et
al
., 2010 and 2012; Popescu et al., 2017).
The objectives of this study were: 1) to complete the
identification of some grapevine varieties, supposed to be
autochthonous, on the base of 48 OIV descriptors and 13
SSR markers; 2) to evalua
te the genetic diversity of the
analysed Romanian grapevine varieties.
Materials and Methods
Biological material
Table 1 presents the fifty (50) varieties of grapevine used
in the present study and selected because considered
indigenous and cultivated fo
r many centuries in our
country. All of them are growing in the ex situ
germplasm
collection held by NRDIBH Stefanesti.
Morphological description
For ampelographic description, 48 standardized OIV
descriptors for grapevine (OIV, 2009) were applied,
follo
wing the recommended methodology; they refer to:
young shoot (OIV 001, 003, 004, 006, 007, 008 and 016),
young and mature leaf (OIV 051, 053, 067, 068, 070, 072,
074, 075, 076, 079, 080, 081-1, 081-2, 083-
2, 084, 087 and
094), type of flower, bunch and ber
ry aspects (OIV 151,
155, 202, 204, 206, 208, 209, 220, 221, 223, 225, 231, 235,
236 and 241), phenology, growth, quality and quantity of
grape yield (OIV 301, 303, 351, 502, 503, 504, 505, 506 and
508).
Protocol for molecular analyses
For molecular analy
ses, the DNA extraction was
performed from 100 mg fresh leaf with Qia
gen DNeasy
Plant mini-
kit (Qiagen, Hilden, Germany). For genotyping
were used 13 SSR markers: nine of them belonged to the set
of those internationally recommended (Maul et al
., 2012) as
a standard for grapevine (VVS2, VVMD5, VVMD7,
VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62
and VrZAG79), plus additional four, namely ISV2, ISV3,
ISV4 and VMCNG4b9. The SSR analyses were performed
194
Popescu CF and Crespan M / Not Sci Biol, 2018, 10(2):193-198
195
Table 1. Grapevine cultivars characterized using OIV descriptors and SSR markers
Accession name Accession number Berry colour Use Distribution*
Description -
Constantinescu et al.
(1959-1966)
VIVC
no**
ʻArdeleancăʼ ROM051-237 green wine 360 1959 (p. 115-126) 904
ʻBăbească neagrăʼ ROM051-238 blue black wine 320 1959 (p. 127-154) 843
ʻBacatorʼ ROM051-011 rose wine 360 1959 (p. 155-168) 905
ʻBăşicatăʼ ROM051-239 green wine 360 1961 (p. 121-13 6) 1022
ʻBătută neagrăʼ (1) ROM045-025 blue-black wine/table 360 1959 (p. 169-182) 1042
ʻNegru românescʼ (1) ROM045-172 black wine 360 1966 (p. 93-104) 1042
ʻBerbecelʼ ROM051-240 green wine 360 1959 (p. 183-194) 1148
ʻBusuioacă de Bohotinʼ ROM051-035 red wine 320 1960 (p. 615-628) 8248
ʻIordanăʼ ROM051-113 green wine 320 1960 (p. 7-18) 5544
ʻGordanʼ ROM051-257 green wine 360 1961 (p. 565-576) 5544
ʻZemoasăʼ ROM051-273 green wine 360 1962 (p. 669-680) 5544
ʻBraghină albăʼ (1) ROM045-037 green wine 360 - 1645
ʻBraghină rozʼ (1) ROM045-036 rose wine 360 1959 (p. 213-232) 1644
ʻCadarcăʼ ROM051-047 black wine 320 1959 (p. 273-290) 5898
ʻCârcioasăʼ (1) ROM045-048 green wine 360 1965 (p. 177-188) 935
ʻCârlogancăʼ ROM051-241 green wine/table 330 1959 (p. 485-509) 3237
ʻCeauş albʼ (1) ROM045-051 green table 360 1959 (p. 291-306) 10196
ʻCeauş rozʼ ROM051-242 rose table 360 1959 (p. 307-320) 2507
ʻCioinicʼ ROM051-243 green wine/table 360 1961 (p. 327-340) 2674
ʻCoada oilorʼ ROM051-244 green wine 360 1962 (p. 363-375) 5852
ʻCoarnă albăʼ ROM051-246 green wine/table 360 1959 (p. 433-452) 2724
ʻCoarnă neagrăʼ ROM051-247 black-red table 320 1959 (p. 453-469) 2726
ʻCoarnă roşieʼ ROM051-248 red table 360 1961 (p. 377-390) 2728
ʻCreaţăʼ ROM051-072 green wine/table 360
ʻCreaţă de Banatʼ ROM051-249 green wine/table 360 1959 (p. 470-484) 6501
ʻCruciuliţăʼ ROM051-250 green wine 360 1961 (p. 415-426) 3267
ʻFetească albăʼ ROM051-251 green wine 310 1959 (p. 523-554) 4119
ʻFetească neagrăʼ ROM051-252 black wine 310 1959 (p. 555-570) 4120
ʻFetească regalăʼ ROM051-253 green wine 310 1959 (p. 627-64 4) 4121
ʻFrâncuşeʼ ROM051-254 green wine 320 1959 (p. 571-592) 4221
ʻGalbenă de Odobeştiʼ ROM051-255 green wine 320 1959 (p. 645-666)
ʻZghihară de Huşiʼ ROM051-274 green wine 320 1960 (p. 667-678) 12727
ʻGalbenă uriaşăʼ ROM051-256 green wine 360 1961 (p. 512-523) 4322
ʻGalbenă măruntăʼ (1) ROM045-100 green wine 360 - 5920
ʻGordinʼ ROM051-258 green wine 360 1959 (p. 681-698) 4901
ʻGrasă de Cotnariʼ ROM051-106 green wine 310 1959 (p. 699-719) 4948
ʻMajarcă albăʼ ROM051-259 green-rose wine 360 1960 (p. 65-76) 11866
ʻMoroştinăʼ (1) ROM045-146 green wine 360 - 8007
ʻNegru mareʼ ROM051-261 black wine/table 360 1962 (p. 267-278) 2452
ʻNegru moaleʼ ROM051-262 black wine 360 1960 (p. 215-238) 8464
ʻNegru vârtosʼ ROM051-263 black wine 360 1960 (p. 239-262) 7540
ʻPârciuʼ ROM051-265 green wine 360 1962 (p. 423-432) 9300
ʻPlăvaieʼ ROM051-166 green wine 330 1960 (p. 353-366) 9553
ʻRomânieʼ (1) ROM045-206 green wine 360 - 10177
ʻTămâioasă româneascăʼ(2) ROM06-0134 green wine 310 1960 (p. 585-614) 25546
ʻTâţa caprei albăʼ (1) ROM045-234 green table 360 1962 (p. 617-630) 16449
ʻTâţa caprei neagrăʼ ROM051-268 black table 360 - 5423
ʻTâţa vacii albăʼ ROM051-269 green table 360 1962 (p. 631-644) 6419
ʻTâţa vacii neagrăʼ (1) ROM045-237 black table 360 - 25547
ʻVulpeaʼ ROM051-272 black wine/table 360 1962 (657-668) 13186
(1) Acc essions under evaluation received from Research and Development Station for Viticulture and Oenology Drăgășani; (2) Accession under eval uation received
from University of Agronomic Sciences and Veterinary Medicine of Bucharest
*310 = local cultivar, spread all over, international cultivar; 320 = major local cultivar, of local importa nce, but extensively grown; 330 = minor local cultivar, of local
importance, fairly utilized; 360 = local neglected cultivar, at risk of extinction
**
V
IVC The
Vitis
Intern
ational Variety Catalogue is a database of various species and varieties/cultivars of grapev ine.
Popescu CF and Crespan M / Not Sci Biol, 2018, 10(2):193-198
SSR profiles for identifying synonyms,
homonyms,
misnomers and unique genotypes
In the last 30 years, in Europe, the interest of grapevine
growers and wine producers for old and autochthonous
varieties has increased and therefore it became necessary to
correctly identify the varieties. It is v
ery well known that the
same variety may be grown in a number of countries or
regions under different names (synonyms) or different
varieties may be known under the same name (homonyms).
Part of this information is found in Romanian most
representative lit
erature in ampelography domain. The
results obtained with SSR profiles comparison provided
valuable and sometimes unexpected information for the
identification of varieties and revealed new or different
synonyms and homonyms. SSR profiles also allowed the
detection of unique varieties and accessions incorrectly
identified by means of ampelographic descriptors.
The genetic profile of each accession was obtained
(Popescu et al.
, 2017) and the results were certified after
comparison with three databases: Vitis
International Variety
Catalogue (VIVC) (http://www.vivc.de/), CREA
-
Viticulture and Enology molecular database (partially
published) and INRA database. Forty-
five different SSR
profiles were obtained. Corroborating the genetic profiles
for 13 SSR markers
with reference literature information
describing each accession, the authors came to the following
conclusions:
A. The synonymies mentioned in old documents were
confirmed for: ʻArdeleancăʼ and ʻBakator belyiʼ; ʻCreaţă
ʼ
and ʻCreaţă de Banatʼ, and ʻKreacaʼ; ʻBacatorʼ
and
ʻBakator rozʼ; ʻBraghină rozʼ and ʻBraghină roşie rarăʼ
;
ʻCadarcăʼ and ʻKadarka Kekʼ; ʻCeauş albʼ and ʻ
Chaouch
blancʼ; ʻMajarca albăʼ and ʻSlankamenka belaʼ; ʻNeg
ru
vârtos
ʼ and ʻMavrud Varnenskiiʼ; ʻPlăvaieʼ and ʻPlavayʼ
;
ʻTâţa caprei albăʼ and ʻTsitsa kapreiʼ.
B. Some synonymies presented in reference old literature
were not confirmed and new ones or different from
previous statements were found:
- ʻBusuioacă de Bohotinʼ
had the same SSR profile with
ʻMuscat à petits grains blancsʼ
, but a different colour of the
berries; thus, it can be concluded that ʻBusuioa
de
Bohotinʼ
is the red somatic variant for berry colour of
ʻMuscat à petits grains blancsʼ and not a syn
onym with
ʻMuscat rouge de Madereʼ as mentioned Constantinescu
et
al
. (1960);
- ʻGalbenă uriaşăʼ
SSR profile matched that of
ʻMirkovacaʼ
, an endangered Croatian variety described by
Maletet al
. (1999); so, the present results proved that
ʻGalbenă uriaşăʼ is not a clonal variant of ʻGalbenă
de
Odobeştiʼ as mentioned Constantinescu et al. (1961);
- ʻmâioasă româneascăʼ
accession, one of the most
important and appre
ciated grapevine varieties in Romania,
was proven not to share the same SSR profile of
ʻ
Muscat à
petits grains blancsʼ
, and therefore not to be a synonym with
it, as mentioned by Constantinescu et al
. (1960). Because
the investigated accession seemed to be similar to those from
other collections, it is supposed the existence of a mixture of
at least two varieties of ʻTămâioasă românesʼ
in
Romanian vineyards and, accordingly, Popescu et al
. (2017)
recommended the study of the mix of varie
ties in old
ʻMuscat à petits grains blancsʼ
vineyards all over the
country.
196
C. Synonyms were found for some ac
cessions for which
there is no information in the Romanian ampelographic
literature, such as:
- ʻGalbenă mărunʼ
SSR profile matched that of
ʻKakotrygisʼ
, a Greek variety, so it is a synonym with this
one;
- ʻŢâţa caprei neagrăʼ SSR profile matched that o
f
ʻHoraʼ, a well-known Bulgarian variety;
- ʻTâţa vacii neagrăʼ matched that of ʻ
Kozi Cici
cherveniʼ, also a Bulgarian variety;
- ʻBraghină albăʼ and ʻBraghi rozʼ
showed two
different molecular profiles, so they are different varieties,
not somatic varia
nts for berry colour and with no
relatedness with ʻBakatorʼ
as is supposed by Constantinescu
et al. (1959).
D. New synonyms, which will be added to the previous
mentioned in the literature, were proven for some varieties:
- ʻCeauş rozʼ has the same profile with ʻ
Chaouch
rozovyiʼ from Turkey;
- ʻCoada oilorʼ is synonym with ʻJuhfarkʼ
from
Hungary;
- ʻŢâţa vacii albăʼ
showed the same SSR profile as
ʻHalholyagʼ from Ukraine;
- ʻCârcioasăʼ shared the same genotype with ʻ
Balint
weiss
ʼ from Hungary.
E. For the fi
rst time in Romanian literature, synonymies
were documented for the following varieties
:
- ʻBătută neagrăʼ shares the same SSR profile of ʻ
Negru
românescʼ
, though they were considered different varieties
and were described separately by Constantinescu et al
. in
1959 and 1966, respectively;
- ʻGordanʼ, ʻIordanăʼ and ʻZemoasăʼ
accessions showed
identical microsatellite profiles, proving to be synonyms;
- ʻZghihară de Huşiʼ and ʻGalbenă de Odobeşti
ʼ
varieties present in many germplasm collections, grown on
lar
ge areas and used in wine producing, have the same SSR
profiles, and proven to be synonyms.
F. The different genetic profiles obtained for
ʻMoroştinăʼ, ʻNegru mareʼ and ʻRomânieʼ
varieties, proved
their uniqueness in the European Vitis
germplasm
collections.
G. Fifteen varieties showed genetic profiles identical to
those already recorded for their respective names in the
databases; they are: ʻBăbească neagrăʼ, ʻBăşicatăʼ, ʻBerbecelʼ
,
ʻCârloganʼ, ʻCioinicʼ, ʻCoarnă albăʼ, ʻCoarnă neagrăʼ
,
ʻCoar roşieʼ, ʻCruciuliţăʼ, ʻFeteas albăʼ, ʻ
Fetească
neagrăʼ, ʻFetească regalăʼ, ʻFrâncuşeʼ, ʻGrasă de Cotnariʼ
,
ʻVulpeaʼ.
The results obtained and presented in the hereby paper
prove the usefulness of genotyping as a precious and
convenient tool for supporting ampelog
raphy in the correct
identification of varieties managed in the germplasm
collections.
This way, there were found similarities among
accessions from Romanian collections and the varieties
cultivated in collections from other countries, discovered
incorrect records of the accessions, and
were able to rename
the misnomered ones.
Molecular methods provide scientific
support for documenting inconsistencies and inaccuracies
in ampelographic descriptions and complete the picture for
description of all accessions
present in germplasm
collections.
Popescu CF and Crespan M / Not Sci Biol, 2018, 10(2):193-198
197
SSR markers for assessing genetic diversity in Romanian
grapevine varieties
Statistics about the discriminatory efficiency of the 13
SSRs markers used are presented in Table 2.
The high
degree of genetic variability among
the 50 cultivars is proven
by the high number (112) of different alleles. With analysed
accessions, the number of different alleles per locus ranged
from 6 for VVMD25 and ISV4
to 13 for VVMD28, with
an average of 8.62, comparable to the results obtained i
n
other laboratories (Westman et al., 1997; Salayeva et al
.,
2010). The highest Ne was for VVMD28 locus (8.16), while
the lowest for ISV3 locus (2.74) with the mean value of 4.84.
Mean observed heterozygosity (82.9%) showed to be higher
than that reported
for other grapevine germplasm collections,
like Iran with 76% (Fatahi et al
., 2003) and Spain with 70.7%
(Ibanez et al
., 2003), and comparable to the values obtained
by Carimi et al. (2010) and Lacombe et al. (2013).
For microsatellite markers efficiency
were considered
observed and expected heterozygosity (Ho, He)
to evaluate
the genetic variability among analysed grapevine. Ho ranged
from 0.64 for VVS2 to 0.93 for VVMD5; m
ean Ho was
slightly higher (0.829) than the expected one (0.784
), with
great differences locus by locus.
The ability of microsatellites to identify grapevine
varieties is linked to the polymorphic information content
(PIC) (Weber, 1990) and the probability of identity (PI)
(Paetkau et al., 1995).
The correspondence between these
paramet
ers is the following: an SSR effective in discriminating
among genotypes has high levels of Ho, He and PIC, and low
PI values (Salayeva et al., 2010).
Among the
four additional SSR markers
tested in the current study, were
remarked the ISV4 and VMCNG4b9 ma
rkers
which
displayed high diversity levels regarding the expected
heterozygosity (higher than mean value of 0,784
), high values
of PIC (higher than mean value of 0,742)
"and low values of
PI (in comparison with other markers).
In the present case, the PI values
ranging from 0.028 to
0.200 and the combined PI value of 3.39E-
15, classified all
the 13 markers as effective in discriminating the analysed
varieties and highly informative for proving genetic diversity
among studied grapevine varieties. These valu
es are in
concordance with those presented by Lopes
et al.
(1999
) and
Sefc et al. (2000).
Null-allele frequency
F (null) showed to be very
interesting, because it was negative for 11 out of 13 SSRs.
This parameter is valuable to reveal some problems
with
allele amplification during genotyping or a possible deletion
in the target sequence.
In the hereby study were obtained
positive values for F (null) with VVS2 and VVMD28
markers indicating possibility of the presence of null alleles.
With these two markers
, were registered lower values of the
observed heterozygosity in comparison with the expected
ones, also probably due to the occurrence of null alleles at
VVS2 and VVMD28 locus.
Between these two markers,
VVS2 was a little bit critical, despite the high nu
mber of
alleles.
Conclusions
Variety identification is important to breeders, curators,
grape growers, winemakers and grape and wine consumers
and, also it is a proof of a good traceability system
throughout producing of grapevine planting material and
wi
nemaking process.
Beside ampelographic description, the
DNA analyses are necessary to verify grapevine identity
before acquisition for germplasm collection, distribution or
exchange plant material, planting and establishing a
vineyard or investing in wine
production.
The efficiency of
the two methods used for the complete characterization of
the autochthonous grapevine varieties represents
overwhelming evidence for the necessity of further
investigation of other accessions existing in germplasm
collections.
These data with entire documentation of plant
material authenticity are essential for improving the
knowledge on Romanian grape varieties, completing
information on the existence of synonyms with other
varieties in European germplasm collections, as well
as
identifying unique genotypes that require particular
attention for preservation.
The complete description and
compliance with the requirements for European germplasm
collections of each accession is the primary requirement for
the exchange of genetic ma
terial among germplasm
collections
.
Table 2. Genetic markers (13 microsatellite loci) and parameters applied to analyze their effectiveness to highlight genetic variability from the
grapevine germplasm collection
Locus Na Ne Ho He PIC PI F (null)
VVS2 9 3,99 0,644 0,758 0,710 0,102 +0.0831
VVMD5 8 7,23 0,933 0,871 0,846 0,035 -0.0413
VVMD7 8 3,27 0,733 0,703 0,651 0,137 -0.0197
VVMD25 6 4,09 0,822 0,765 0,713 0,103 -0.0426
VVMD27 8 4,57 0,844 0,790 0,748 0,082 -0.0400
VVMD28 13 8,16 0,867 0,887 0,865 0,028 +0.0048
VVMD32 11 5,09 0,911 0,813 0,780 0,062 -0.0660
VrZAG62 8 4,51 0,867 0,787 0,746 0,082 -0.0534
VrZAG79 10 5,13 0,822 0,814 0,784 0,059 -0.0118
ISV2 8 3,57 0,822 0,729 0,673 0,125 -0.0721
ISV3 7 2,74 0,800 0,642 0,568 0,200 -0.1233
ISV4 6 5,03 0,889 0,810 0,771 0,070 -0.0522
VMCNG4B9 10 5,48 0,822 0,827 0,794 0,057 -0.0024
Mean values 8,62 4,84 0,829 0,784 0,742
Combined values
3,39E-15
Na: number of alleles; Ne: number of effective alleles, Ho: observed heterozygosity; He: expected heterozygosity; PIC: Polymorphism information content, PI:
probability of identity, F(null): probability of null alleles
Popescu CF and Crespan M / Not Sci Biol, 2018, 10(2):193-198
198
Acknowledgements
The results were obtained within the framework of the
COST project - Action FA1003 "East-
West Collaboration
for Grapevine Diversity Exploration and Mobilization of
Adaptive Traits for Breeding". Analyzed
plant material was
used to establish the grapevine ex situ collecti
on through
ADER 3.1.3. project.
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