Malvasia nera di Brindisi/Lecce'grapevine cultivar (Vitis vinifera L.) originated from 'Negroamaro'and 'Malvasia bianca lunga'

Abstract

Malvasia nera di Brindisi' and 'Malvasia nera di Lecce' are two of the few Malvasias with black berries and belong to the Apulian ampelographic assortment (South Italy). Their presumed synonymy has been re-cently ascertained with SSR markers and therefore these two black 'Malvasias' can be considered as an unique variety. We discovered that this cultivar is the cross between 'Malvasia bianca lunga' alias 'Malvasia del Chianti' and 'Negroamaro' by using 42 nuclear SSR. Both parents belong to the Apulian varietal resources, since centuries. So far, 'Malvasia nera di Brindisi/Lec-ce' origin has been obscure; now we may assert that this cultivar was born right in Apulia. Three sets of chloro-plast SSR loci were used to determine the female and the male parent: 6 ccmp loci, already used in previous pedigree studies, 15 ccSSR loci and 2 NTCP loci, derived from tobacco. The second set of loci was sequenced in order to compare the length of the markers with the reference species where they were originally obtained: in 4 cases no microsatellite motives were detected and in other 4 cases the perfect repetition found in tobacco was not maintained in grape. Unfortunately, the three sets of markers failed to show any polymorphism. A detailed comparison of the black Malvasia morphol-ogy with its two parents showed a closer similarity to 'Negroamaro'. Also the anthocyanin profile is in agree-ment with that of the black parent; its varietal aroma presents interesting levels of free and bound 2-phenyl-ethanol, responsible for rose flavor, and of bound lina-lool compounds. K e y w o r d s : anthocyanin, chloroplast SSR, flavor, nuclear SSR, pedigree.

Full text

Vitis 47 (4), 205–212 (2008)
‘Malvasia nera di Brindisi/Lecce’ grapevine cultivar (Vitis vinifera L.)
originated from ‘Negroamaro’ and ‘Malvasia bianca lunga’
M. CRESPAN1), A. COLETTA2), P. CRUPI2), S. GIANNETTO1) and D. ANTONACCI2)
1) C.R.A. Centro di Ricerca per la Viticoltura, Susegana, Italy
2) C.R.A. Unità di Ricerca per l’Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Turi, Italy
Correspondence to: Dr. M. CRESPAN, C.R.A. Centro di Ricerca per la Viticoltura, Via Casoni, 13/A, 31058 Susegana (TV), Italy. Fax:
+39-438-738-489. E-mail: manna.crespan@entecra.it
Summary
‘Malvasia nera di Brindisi’ and ‘Malvasia nera di
Lecce’ are two of the few Malvasias with black berries
and belong to the Apulian ampelographic assortment
(South Italy). Their presumed synonymy has been re-
cently ascertained with SSR markers and therefore
these two black 'Malvasias' can be considered as an
unique variety. We discovered that this cultivar is the
cross between ‘Malvasia bianca lunga’ alias ‘Malvasia
del Chianti’ and ‘Negroamaro’ by using 42 nuclear SSR.
Both parents belong to the Apulian varietal resources,
since centuries. So far, ‘Malvasia nera di Brindisi/Lec-
ce’ origin has been obscure; now we may assert that this
cultivar was born right in Apulia. Three sets of chloro-
plast SSR loci were used to determine the female and
the male parent: 6 ccmp loci, already used in previous
pedigree studies, 15 ccSSR loci and 2 NTCP loci, derived
from tobacco. The second set of loci was sequenced in
order to compare the length of the markers with the
reference species where they were originally obtained:
in 4 cases no microsatellite motives were detected and
in other 4 cases the perfect repetition found in tobacco
was not maintained in grape. Unfortunately, the three
sets of markers failed to show any polymorphism. A
detailed comparison of the black Malvasia morphol-
ogy with its two parents showed a closer similarity to
‘Negroamaro’. Also the anthocyanin profile is in agree-
ment with that of the black parent; its varietal aroma
presents interesting levels of free and bound 2-phenyl-
ethanol, responsible for rose flavor, and of bound lina-
lool compounds.
K e y w o r d s : anthocyanin, chloroplast SSR, flavor,
nuclear SSR, pedigree.
Introduction
Pedigree reconstruction studies with SSR markers
display very interesting information on the relationships
among grapevine cultivars and explain their chronological
order of appearance (BOWERS and MEREDITH 1997, BOWERS
et al. 1999, VOUILLAMOZ et al. 2003, MALETIĆ et al. 2004,
DI VECCHI STARAZ et al. 2007, SCHNEIDER et al. 2008, CRE-
SPAN et al. 2008). This kind of research has not only a cul-
tural interest, since it provides new insights into evolution
and nature of the present ampelographic assortment, cul-
tivars identity, variability development and it will be use-
ful for further studies on linkage disequilibrium (BARNAUD
et al. 2006).
'Malvasias' belong to a numerous and heterogeneous
population of varieties growing in many European coun-
tries and their history is an intriguing enigma. In Italy, at
the present moment, 'Malvasias' are spread from North
to South and 17 'Malvasia' cultivars are registered in the
Italian National Catalogue. Among them ‘Malvasia delle
Lipari’ and ‘Malvasia di Sardegna’ have been shown to
be synonyms of the same cultivar (CRESPAN et al. 2006 a);
moreover, ‘Malvasia rosa’ is a pink mutant for the berry
color of the white ‘Malvasia di Candia aromatica’ (FREGONI
et al. 2005).
In Italy there are few Malvasias with black berries,
mostly growing in Piedmont (North West Italy): ‘Malvasia
di Casorzo’, ‘Malvasia nera lunga’ and ‘Malvasia di Schi-
erano’; other two, ‘Malvasia nera di Brindisi’ and ‘Malva-
sia nera di Lecce’, contribute to the Salento oenological
production (Apulia, Southern Italy). So far the two Apulian
Malvasias have been considered different varieties in the
official documents, on the basis of the following charac-
ters: ‘Malvasia nera di Lecce’ should have a more dense
and heavy bunch compared to ‘Malvasia nera di Brindisi’,
that should produce aromatic berries. However not all the
experts agree with this distinction, since the observed dif-
ferences may be due to the environment influence on the
same genotype instead of being different varieties.
In a recent study we collected ‘Malvasia nera di
Brindisi’ and ‘Malvasia nera di Lecce’ accessions from
different vineyards in the Salento traditional cultivation
area and compared them analyzing eleven SSR loci. These
two Malvasias have been shown to be the same cultivar,
since they have the same SSR profile (COLETTA et al. 2006).
Additional more recent SSR profiling, performed on new
samples from other vineyards in Apulia, confirmed the
published results (GASPARRO et al. 2008); thus these two
Malvasias can be considered as a unique variety, hereafter
called ‘Malvasia nera di Brindisi/Lecce’ (MnBR/LE).
Comparing a first set of nuclear SSR loci we found that
MnBR/LE could be the progeny of ‘Malvasia bianca lun-
ga’ alias ‘Malvasia del Chianti’ (MBL) and ‘Negroamaro’
(NA). Actually these three varieties have been sharing the
same cultivation area for a very long time. Salento region
is a renowned viticultural area, where once vineyards con-
sisted of few predominant varieties (‘Negroamaro’, ‘Mal-

206 M. CRESPAN et al.
lowing conditions. The PCR reaction mixture (25 µl final
volume) contained 20 ng total DNA, 10 µl Eppendorf Hot-
MasterMix (2.5 x) and 10 pmoles of each primer. The PCR
was performed in an AB 9700 thermal cycler with the fol-
lowing steps: 1 min 30 s at 94 °C; 35 cycler at 94 °C for
30 s, 55° C for 30 s, 65 °C for 30 s; 65 °C for 7 min and
a final step of at least 10 min at 8 °C to stop the reaction.
Five µl of the PCR product were tested on 2 % agarose
gel. On the basis of signal intensity, 0.1-0.2 µl of amplified
DNA were used for electrophoresis with a sequencing gel
(5 % polyacrylamide, TBE 1 x, urea 7 M). Gel bands were
revealed by silver staining, as reported in CRESPAN and MI-
LANI (2001). Gels were visually scored at least twice.
c c S S R s e q u e n c i n g : ccSSR amplifica-
tion products obtained from MnBR/LE were sequenced
by BMR Genomics, official spin-off of the University of
Padova (Italy); sample preparation was performed follow-
ing the instruction given by the service, for allele length
determination and microsatellite motif survey.
A m p e l o g r a p h i c c o m p a r i s o n : The
morphological comparison among the three varieties was
performed by means of primary and secondary descriptors,
as indicated in the frame of the Genres081 project (http://
www.genres.de/eccdb/vitis/).
F l a v o r c o m p o s i t i o n a n a l y s i s
F r e e a r o m a c o m p o u n d s e x t r a c t i o n
f r o m b e r r y : Three samples of 450 g were col-
lected for each variety, added with 25 μl of BHA in EtOH
(12.66 mg·ml-1), homogenized for 5 min and centrifuged at
4,000 g for 3 min at 20 °C. The supernatant, subdivided in
two volumes of about 200 ml, was extracted with CH2Cl2
(30 ml) in agitation for 1 h at 10 °C, whereas the precipi-
tate was kept in maceration in CH2Cl2 (30 ml) for 4 h at
10 °C. The organic fractions, pooled together, dehydrated
with Na2SO4 and supplied with 2 μl 2-methyl-pentanol,
were first concentrated with rotavapor to about 5 ml and
then with a nitrogen flux to reach 1 ml volume.
G C - M S D a n a l y s i s : GC analyses were performed
with an Agilent 6890 N gas chromatograph, equipped with
split-splitless injector, a capillary column DB-Wax (60 m
x 0.25 mm i.d., 0.5 μm film thickness; J&W Scient. Inc.),
interfaced with a detector MS Agilent 5973N.
The temperature program was: 40 °C for 5 min with a
ramp of 2 °C·min-1, up to 200 °C for 15 min, with 1 s ramp
of 1 °C·min-1 up to 250 °C, both for qualitative analysis in
full scan and quantitative analysis in SIM. The flow speed
of carriage gas (He) was 1 ml·min-1. The mass spectra for
full scan analysis were registered in the 28-300 m/z inter-
val. Compound identification was done by comparison of
their retention times and mass spectra with those of pure
standard references.
The Principal Component Analysis (PCA) was carried
out using the values of free aroma compounds detected in
at least one of the three varieties, with STATISTICA soft-
ware version 6.0.
A n t h o c y a n i n c o m p o s i t i o n a n a l y s i s :
This analysis was performed only on the two colored varie-
ties, NA and MnBR/LE.
A n t h o c y a n i n e x t r a c t i o n f r o m s k i n s :
Three groups of ten berries were collected randomly from
vasia nera’, ‘Susumaniello’, ‘Malvasia bianca’ and some
others less important). MBL has been growing in Apulia
for centuries; NA is considered to have an even longer
presence in the same region and it is one of the most impor-
tant and popular wine varieties of Salento area (ANTONACCI
2006). MnBR/LE is bound to the same area, since no infor-
mation about its presence in other Italian places or in other
Countries have been found. So we decided to investigate
this potential kinship by extending the molecular analyses
to 42 nSSR loci and the initial hypothesis has been con-
firmed.
In order to distinguish the male and the female parent,
we performed chloroplast microsatellite markers analy-
sis, since the chloroplasts are inherited in grapevine from
the mother (STREFELER et al. 1992, ARROYO-GARCIA et al.
2002). We used 6 ccmp SSR loci from WEISING and GARD-
NER (1999), already successfully applied in grapevine for
analogous pedigree studies (CRESPAN et al. 2006 b), and
other microsatellite markers detected in tobacco: 15 ccSSR
loci found by CHUNG and STAUB (2003) and 2 NTCP loci by
BRYAN et al. (1999).
Morphological and chemical analyses were also per-
formed to evaluate similarities and differences about ber-
ry flavor composition and anthocyanin profile between
MnBR/LE and the two putative parents.
Material and Methods
P l a n t m a t e r i a l : Mn BR/LE, MBL and NA
samples came from the Istituto Sperimentale per la Viti-
coltura (ISV) collections of Conegliano (Treviso) and Turi
(Bari), Italy.
N u c l e a r S S R m a r k e r s : The same thirty nSSR
used in CRESPAN et al. (2006 b) were analysed, plus twelve
additional: VMC4c6, VMC2h4, VMC1b11 and VMC4f3
from Vitis Microsatellite Consortium, scu05 (SCOTT et al.
2000), UCH11 (LEFORT et al. 2002) and VVIb09, VVIi51,
VVIp31, VVIp77, VVIp37 and VVIv36 (MERDINOGLU
et al. 2005). The additional loci were singly amplified and
analyzed using the same procedure described in CRESPAN
et al. 2006 a.
L i k e l i h o o d r a t i o s o f n S S R d a t a : Mo-
lecular data diversity was estimated with SEFC and WAG-
NER (1999) Identity 1.0 program, using nSSR profiles of
wine and table grape cultivars of Centro di Ricerca per la
Viticoltura molecular database at the first 36 loci in Tab. 1.
The number of cultivars used ranged from 400 (the maxi-
mum imposed by the program) to 61, with a mean of 230
cultivars per locus. Among them, 120 were table and 280
wine varieties; moreover, 160 cultivars were presumably
of Italian origin, the others were more or less spread in
Europe. nSSR data available for less than 50 different vari-
eties were not used in this computation.
C h l o r o p l a s t S S R l o c i a n a l y s i s :
Twenty-three chloroplast microsatellite loci were analyzed
by using the consensus primer pairs designed by WEIS-
ING and GARDNER (1999) for 6 ccmp, by CHUNG and STAUB
(2003) for 15 ccSSR loci, and by BRYAN et al. (1999) for
2 NTCP loci. They were singly amplified, using the fol-

‘Malvasia nera di Brindisi/Lecce’ grapevine cultivar 207
5 clusters for each cultivar, weighed and peeled. After a
brief drying, skins were soaked in 25 ml ethanol hydro-
chloric and kept in infusion for at least 12 h. The extract
was separated from skins by settling, filtered with regener-
ated cellulose filters of 0.45 μm and directly analyzed by
HPLC.
H P L C - D A D a n a l y s i s : The chromatographic
separation was done on Zorbax SB-C18 Agilent (4,6 x 250
mm, 5 μm) supplied with a “guard column” Phenomen-
ex; the injection volume was 5 μl. For mobile phase, ace-
tonitrile (solvent A) and formic acid 10 % (solvent B) were
used. The elution program provided a constant flow of
0.7 ml·min-1 and a gradient: 0 min, 5 % A-95 % B; 10 min,
13 % A-87 % B; 20 min, 15 % A-85 % B; 30 min, 22 %
A-78 % B; 50 min, 22 % A-78 % B; 55 min, 5 % A-95 %
B. Rebalancing period for next sample charge was 10 min
with the solvent mixture used at time zero.
The analysis was performed by monitoring the absorb-
ance signal at 520 nm. The peaks identification was ob-
tained by comparison of retention times and absorbance
spectra of analogous compounds reported in literature
(GARCÍA-BENEYTEZ et al. 2003) with those of pure chemi-
cals.
Results and Discussion
N u c l e a r S S R d a t a : Among the nSSR loci used,
only three are not yet mapped and the others are spread
over all chromosomes except LG9, LG13 and LG15. This
choice was suggested by the opportunity to validate the
possible first degree relationship with markers independ-
ently inherited, therefore more informative than linked
markers. MnBR/LE exhibited one allele derived from each
of the presumed parents at all fourty-two nuclear loci, sup-
porting the preliminary hypothesis about a parent-progeny
relationship among these cultivars (Tab. 1).
Cumulative likelihood ratios of MnBR/LE being the
progeny of MBL (1) and NA (2), versus alternative parents,
including close relatives are reported in Tab. 2. These es-
timates showed that the indicated parents were more than
1021 times more probable than other combinations of two
T a b l e 1
Molecular profiles of ‘Malvasia nera di Brindisi/Lecce’ and its presumed parents, ‘Malvasia bianca lunga’ and
‘Negroamaro’ at 42 nSSR loci. Three reference varieties profiles are also provided for easier data comparison
SSR nuclear loci
(linkage group)* Malvasia bianca lunga Malvasia nera di
Brindisi/Lecce Negroamaro Moscato bianco Cabernet
Sauvignon Sultanina
VVMD26 (1) 249:251 251:251 251:251 251:251 249:251 249:251
VVS29 (1) 171:171 171:171 171:171 171:171 179:181 171:179
ISV3 (2) 133:139 133:139 139:145 133:139 133:139 133:139
VVMD28 (3) 251:257 237:251 237:239 249:271 239:237 221:247
VVMD36 (3) 254:254 254:270 244:270 244:264 254:264 250:268
VVIp37 (4) 118:134 118:118 118:138 138:138 150:150 134:138
VVIp77 (4) 173:185 181:185 181:191 189:191 181:185 173:199
VVMD32 (4) 253:257 253:253 253:273 273:265 241:241 251:251
VrZAG 21 (4) 204:206 202:206 202:206 206:206 200:206 190:202
VMC4C6 (5) 160:163 157:163 157:163 157:157 163:175 151:157
VMC6E10 (5) 95: 97 97:117 97:117 111:113 111:121 121:127
VVMD27 (5) 179:179 179:181 179:181 179:194 175:189 181:194
VrZAG 79 (5) 242:250 250:258 258:258 250:254 246:246 246:258
VMC2G2 (6) 119:119 119:119 119:125 123:125 121:125 125:125
VMC2H9 (6) 117:117 117:117 117:123 121:123 119:123 123:123
VMC4G6 (6) 133:143 129:143 129:143 127:143 127:133 125:129
VMCNG4B9 (6) 150:176 150:158 158:168 158:166 168:176 138:158
VVMD21 (6) 249:249 249:249 249:249 249:266 249:258 249:256
VVIv36 (7) 149:155 149:155 149:155 149:153 149:155 155:155
VVMD7 (7) 239:253 239:249 249:249 233:249 239:239 239:253
VVMD31 (7) 212:214 212:212 212:212 212:216 206:210 212:212
VrZAG 62 (7) 195:199 195:201 189:201 185:195 187:193 187:187
VMC1B11 (8) 174:198 168:174 168:172 186:190 186:186 168:186
VrZAG 83 (8) 190:194 194:194 188:194 188:188 200:200 188:194
VMC3D7 (10) 163:171 163:163 159:163 161:163 159:161 161:163
VrZAG 64 (10) 137:159 143:159 137:143 141:159 139:159 143:159
ISV4 (11) 177:177 177:187 169:187 169:187 169:191 191:193
VVMD25 (11) 243:245 245:245 245:267 245:253 243:253 243:253
VVS2 (11) 145:145 145:145 145:151 133:133 139:151 145:151
VMC2H4 (12) 198:200 200:214 206:214 200:214 212:220 204:214
scu 05 (12) 163:165 163:165 165:169 160:165 165:168 165:171
ISV2 (14) 143:165 143:143 141:143 141:143 141:165 143:143
VMC1E12 (14) 250:260 250:260 250:250 260:260 240:250 244:260
VVMD24 (14) 210:210 210:214 214:214 214:219 210:219 210:219
VVMD5 (16) 226:240 226:236 226:236 228:236 232:240 234:234
VVIb09 (17) 275:279 269:275 269:279 269:279 275:277 277:279
VVMD17 (18) 221:222 222:222 222:222 220:222 221:222 222:222
VVIp31 (19) 171:175 171:187 181:187 181:185 187:187 177:181
VVS1 (20) 180:181 181:190 181:190 181:181 181:181 181:188
UCH11 242:262 242:248 244:248 244:248 244:262 242:244
VMC4F3 168:206 168:168 168:168 168:206 174:180 190:192
VVIi51 246:260 246:248 246:248 246:260 260:260 246:248
* The linkage group numbers, where available, refer to RIAZ et al. (2004) reference map.

208 M. CRESPAN et al.
random cultivars (more than 1016 with 95 % upper confi-
dence limit of allele frequencies). These ratios remained
far higher also compared to the values calculated when one
of the suggested parents was assumed and the other parent
was a close relative of the second suggested parent.
C h l o r o p l a s t S S R d a t a : Three sets of chlo-
roplast SSR loci were used to determine the sexual role
of each parent of MnBR/LE. The 6 ccmp chloroplast mi-
crosatellites did not show any polymorphism among the
three varieties and their haplotype was the same as 'Rab-
oso Piave' and 'Raboso veronese' (CRESPAN et al. 2006 b).
A set of 15 ccSSR loci, designed by CHUNG and STAUB
(2003), was used in the second analysis. In this case all
primer pairs gave a strong amplification product between
180 and 360 bp, nevertheless none detecting length differ-
ences. An analysis of ccSSR allele size was performed by
sequencing of MnBR/LE amplicons and comparison with
those obtained in tobacco, as reported in Tab. 3. In four
cases no microsatellite motives were identified (ccSSR12,
ccSSR15, ccSSR18 and ccSSR22), in other cases the per-
fect repetition found in tobacco was interrupted in grape
(ccSSR9, ccSSR17, ccSSR19 and ccSSR21); in general,
the repeated stretches were rather short and this could be
the reason for length polymorphism absence in the grape
cultivars analyzed in this study.
NTCP8 and NTCP12 loci (BRYAN et al. 1999) were
also analysed. Only the first one, identified in the Solanace-
ae family, amplified efficiently in grapevine and showed
to be polymorphic in a preliminary screening on a limited
number of varieties, but without useful results in our three
samples. As NTCP8 PCR product was not sequenced, its
length was estimated around 250 bp, with a 100 bp lad-
der on a 2 % agarose gel. In summary, the three cultivars
showed the same haplotype and the absence of any poly-
morphism did not allow to define the male and the female
parent.
A m p e l o g r a p h i c c o m p a r i s o n : MnBR/
LE showed a very higher resemblance with NA than MBL,
sharing as many as 24 characters exclusively with the black
parent, but only 10 with MBL (Tab. 4). On the whole, how-
ever, the morphological similarity of MnBR/LE came up
with both parents, since as many as 23 characters were in
common with the same expression level (Tab. 5). Only 8
characters showed expression levels different from those
of the two parents (Tab. 6).
C h e m i c a l b e r r y c o m p o s i t i o n
c o m p a r i s o n : In this study some secondary metabo-
lites, aromatic and anthocyanin compounds, were quanti-
fied due to their importance for cultivar characterization.
F r e e a r o m a c o m p o u n d s : Aromatic com-
pounds of MBL (STORCHI et al. 2005) were compared with
those of NA and MnBR/LE obtained from the analysis of
clusters coming from Turi (Bari, Italy). The total amount of
lipid derivatives was highly similar between the two black
varieties. Benzyl compounds had a higher concentration,
from 4 to 6 times, in the offspring than in the two par-
ents. Only MnBR/LE showed high levels of free monoter-
penols (160 ± 30 µg/kg): we found the relevant presence
of cis-furan-linalool-oxide and trans-furan-linalool-oxide,
not detectable in the two parents, neither as traces. On the
contrary, HO-trienol and geraniol were not detected in the
black Malvasia, even if they were present as traces in NA
and in MBL. It was noteworthy the absence of many ter-
pene compounds, such as linalool, nerol and α-terpineol,
in the three cultivars studied, which is a common trend for
non aromatic varieties (BORSA et al. 2005).
The PCA analysis was carried out with the 14 com-
pounds listed in Tab. 7. It allowed to find three well defined
T a b l e 2
Cumulative likelihood ratios of 'Malvasia nera di Brindisi/Lecce' being the progeny of 'Malvasia bianca lunga' (1)
and 'Negroamaro' (2), versus alternative parents, including close relatives, combined over 36 nuclear SSR loci
Parents combinations 1 x 2 (1) x X rel (2) x (1) (2) x X rel (1) x (2)
with observed allele frequencies 3,49 x 1021 3,74 x 1014 1,01 x 1052,88 x 1011 8,62 x 103
with 95 % upper confidence limit 8,01 x 1016 1.25 x 1012 2,04 x 104 1,52 x 1091,59 x 103
T a b l e 3
Chloroplastic SSR allele length (bp) in grape compared with tobacco (after CHUNG and STAUB 2003)
and microsatellite motif found in ‘Malvasia nera di Brindisi/Lecce’
Locus name SSR motif in grape SSR motif in
tobacco
expected size in
tobacco
size in Vitis
vinifera L.
ccSSR4 (T)8 (T)8 205 279
ccSSR6 (T)10 (T)8 299 299
ccSSR7 (T)11 (T)11 349 359
ccSSR9 (A)3T(A)10 (A)13 173 167
ccSSR12 no microsatellite motives found (A)8 249 236
ccSSR13 (T)8 (T)9 264 279
ccSSR15 no microsatellite motives found (T)9 264 264
ccSSR16 (T)11 (T)7C(T)2 123 356
ccSSR17 (A)5G(A)7 (A)13 236 227
ccSSR18 no microsatellite motives found (A)8 264 264
ccSSR19 (T)3C(T)7 (T)8 335 359
ccSSR20 (A)13 (A)8 311 329
ccSSR21 (A)11 - (T)7C(T)5 (T)13 280 281
ccSSR22 no microsatellite motives found (T)8 190 185
ccSSR23 (A)7 - (A)15 - (A)9 (A)14 217 281

‘Malvasia nera di Brindisi/Lecce’ grapevine cultivar 209
T a b l e 4
‘Malvasia nera di Brindisi/Lecce’ characters in common only with one parent
Negroamaro Malvasia bianca lunga
Character
code Description Expression
level
Character
code Description Expression
level
3 Young shoot: intensity of anthocyanin
coloration on prostrate hairs of the tip
7 76 Mature leaf: shape of teeth 5
8 Shoot: color of ventral side of internodes 2 452 Leaf: degree of resistance to
Plasmopara
3
015_2 Shoot: intensity of anthocyanin
coloration on the bud scales
5 606 Mature leaf: length petiole sinus to
lower leaf sinus
3
74 Mature leaf: profile 3 613 Mature leaf: width of teeth N2 5
79 Mature leaf: opening/overlapping of
petiole sinus
2 459 Cluster: degree of resistance to
Botrytis
5
455 Leaf: degree of resistance to Oidium 7 223 Berry shape 5
601 Mature leaf: length of vein N1 5 236 Berry particular flavor 5
602 Mature leaf: length of vein N2 5 617 Mature leaf: length between the tooth
tip of N2 and the tooth tip of the first
secondary vein of N2
7
066_5 Mature leaf: vein N3, length petiole
sinus to vein N4
5 066_4 Mature leaf length of vein N5 3
87 Mature leaf: density of erect hairs on the
main veins (lower side)
5 84 Mature leaf: density of prostrate
hairs beween the main veins (lower
side)
5
079_1 Mature leaf: opening/overlapping of
petiole sinus
1
202 Bunch: length 5
206 Bunch: length of peduncle 1
208 Bunch: shape 1
209 Bunch: number of wings 3
609 Mature leaf: angle between N3 and N4 3
615 Mature leaf: width of teeth N4 3
616 Mature leaf: number of teeth between
the tooth tip of N2 and the tooth tip of
the first secondary vein of N2 including
the limits
9
220 Berry: length 5
225 Berry: color of skin 6
502 Bunch: weight of a single bunch 3
503 Berry: single berry weight 5
505 Sugar content of must 9
508 pH of must 3
T a b l e 5
'Malvasia nera di Brindisi/Lecce' characters in common with both parents
Character
code Description Expression
level
1 Young shoot: shape of the tip 7
4 Young shoot: density of prostrate hairs of the tip 7
6 Shoot: attitude 7
16 Tendrils: distribution on the shoot 2
70 Mature leaf: anthocyanin coloration of the main veins on the upper side of the blade 1
72 Mature leaf: goffering of blade 1
603 Mature leaf: length of vein N3 5
604 Mature leaf: length of vein N4 7
605 Mature leaf: length petiole sinus to upper leaf sinus 3
612 Mature leaf: length of teeth N2 3
614 Mature leaf: length of teeth N4 3
51 Young leaf: color of the upper side (leaf 4) 1
53 Young leaf: density of prostrate hairs between veins at the lower side of leaf 7
80 Mature leaf: shape of base of petiole sinus 1
081_1 Mature leaf: presence of teeth in the petiole sinus 1
081_2 Mature leaf: petiole sinus limited by veins 1
083_2 Mature leaf: presence of teeth at the base of the upper leaf sinuses 1
67 Mature leaf: shape of blade 3
68 Mature leaf: number of lobes 3
204 Bunch: density 7
230 Berry: color of flesh 1
235 Berry: degree of firmness of flesh 5
241 Berry: presence of seeds 3
and no overlapping groups (Fig. 1), one for each variety.
The variables giving a very high contribution to the first
component, that explains 50.79 % of total data set vari-
ance, are n-hexanol, hexanoic acid, benzyl alcohol, 2-phe-
nyl-ethanol, cis- and trans-linalool-oxide. The second
principal component (40.94 % of total variability) was in-
fluenced by trans-2-hexenal, capronaldehyde, trans-2-hex-
enol, cis-3-hexenol, HO-trienol and geraniol.

210 M. CRESPAN et al.
NA musts were characterized by compounds associated
with positive values of the two first principal components:
trans-2-hexenal, capronaldehyde and HO-trienol; MBL
musts were characterized, primarily, by cis-3-hexenol and
geraniol; finally, MnBR/LE samples by n-hexanol, benzyl
alcohol, 2-phenyl-ethanol, cis- and trans-linalool-oxide.
B o u n d a r o m a c o m p o u n d s : Benzyl and
terpene compounds were examined by putting together
the data produced by DI STEFANO et al. (1997) and BORSA
et al. (2005); their results were reported in Tab. 8. The total
bound benzyl aromas of black Malvasia had a concentra-
tion in-between the two parents and closer to NA. Interest-
ingly, 2-phenyl-ethanol level was very similar to that of the
white Malvasia. The same intermediate trend was shown
by terpene compounds quantities. As NA, the black Mal-
vasia was characterized by a varietal terpene aroma, with
compounds belonging mainly to the linalool class.
A n t h o c y a n i n c h a r a c t e r i z a t i o n :
The total amount of anthocyanins showed that MnBR/LE
produced about half color than NA, 319 and 639 mg·kg-1
respectively. The relative percentage of each pigment, and
therefore the anthocyanin profile, was almost the same in
both cultivars (Fig. 2).
Conclusions
We showed that MnBR/LE could be the progeny of
MBL and NA. Molecular data and available informa-
tion on the historical distribution of MnBR/LE led to the
conclusion that this variety originated in Apulia, where it
found a suitable environment for its success. Therefore it
was not imported in this region from Greece or from other
East Mediterranean Countries, as generally hypothesized
for Malvasia varieties (GALET 2000, CALÒ et al. 2001).
Our study proved that transferability of chloroplast
markers from plant species very distant from grape is also
possible, but their SSR polymorphism may be very low, or
absent. Moreover, the repeated motif found in the reference
species may be lacking in the new one analyzed. For this
reason it is difficult to identify the sexual role of each par-
ent in spontaneous plant breeding, as in the case reported
here. MnBR/LE shows a greater resemblance with NA
than with MBL, and actually it is likely to confuse the two
varieties. Analogous cases, now explainable with parent-
offspring relationship, are well known: for example, the
‘Chardonnay’ is morphologically very similar to the white
phenotype of the parent ‘Pinot’ and in Italy they have been
confused for long time; the same difficulty is found in dis-
tinguishing ‘Raboso veronese’ from its mother, ‘Raboso
Piave’.
T a b l e 6
‘Malvasia nera di Brindisi/Lecce’ characters different from both parents
Character
code Description
Malvasia
bianca
lunga
Malvasia nera
di Brindisi/
Lecce
Negroamaro
Expression level
7 Shoot: color of dorsal side of internodes 1 3 2
015_1 Shoot: distribution of the anthocyanin coloration on the bud scales 1 9 5
75 Mature leaf: blistering of upper side 5 3 5
607 Mature leaf:angle between N1 and N2 measured at the first ramification 9 3 5
608 Mature leaf:angle between N2 and N3 measured at the first ramification 7 3 5
610 Mature leaf:angle between N3 and the tangent between petiol point and the tooth tip of N5 9 7 9
221 Berry width 3 5 3
506 Total acid content of must 1 3 7
T a b l e 7
Free aroma compounds used for Principal Component Analysis
and factor coordinates of these variables
Variables Factor 1 Factor 2
trans-2-Hexenal 0,927606
Capron-aldehyde 0,976376
n-Hexanol -0,997165
trans-2-Hexenol -0,820160
cis-3-Hexenol -0,964508
Hexanoic acid -0,938463
Benzyl-alcohol -0,973926
Phenyl-acetaldehyde 0,769587
Benzyl-aldehyde 0,61868
2-Phenyl-ethanol -0,994165
cis-Furan-linalool-oxide -0,986958
trans-Furan-linalool-oxide -0,978152
Ho-trienol 0,778596
Geraniol -0,802948
Fig. 1: Principal component diagram of 'Malvasia bianca lunga'
(MBL), 'Malvasia nera di Brindisi/Lecce' (MnBR/LE) and 'Ne-
groamaro' (NA) as a function of 14 variables for 9 must samples.
Factor score plot 1-2: axis 1 and 2 account for 91.7 % of the total
variance.

‘Malvasia nera di Brindisi/Lecce’ grapevine cultivar 211
Acknowledgements
This work was supported by “Provit” and “Vitivin-valut”
projects financed by Ministero delle Politiche Agricole, Alimen-
tari e Forestali.
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T a b l e 8
Concentration of the glycosidically bound aroma compounds released by enzymatic
hydrolysis. Values are expressed as μg·kg-1 of berry fresh weight
Bound aromatic compounds Negroamaro Malvasia nera di
Brindisi/Lecce
Malvasia
bianca lunga
Benzilic
compounds
Benzylalcohol 232 n.d. n.d.
Acetovanillone 24 130 37
Zingerone 29 26 4
Vanillin 5 14 26
2-Phenyl-ethanol 166 239 235
Total 456 409 302
Terpenes
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trans- Furan-linalool-oxide 12 33 4
trans-8-OH-linalool 43 92 18
cis-8-OH-linalool 124 51 74
Linalool 20 9 9
Geraniol 39 8 24
Geranic acid 6 22 11
Nerol 7 4 6
α-Terpineol 3 3 65
Total 257 251 215
Fig. 2: Anthocyanic profile of ‘Negroamaro’ and ‘Malvasia nera
di Brindisi/Lecce’.

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