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enetic diversity and clonal variation within the main Sicilian oliveultivars based on morphological traits and microsatellite markers
. Caruso, F.P. Marra ∗, F. Costa, G. Campisi, L. Macaluso, A. Marcheseepartment of Agricultural and Forest Sciences (SAF), University of Palermo, Viale delle Scienze, Edificio 4 Ingresso H, 90128 Palermo, Italy
r t i c l e i n f o
rticle history:eceived 23 July 2014eceived in revised form 8 October 2014ccepted 11 October 2014
The richness of Olea europaea (L.) genetic resources in Sicily is well documented. In the last 30 years, mostof the local cultivars, landraces and ecotypes have been gathered together in a large ex-situ collection,containing more than 300 genotypes. In this study, 45 putative clones of the main Sicilian olive cultivarswere characterized morphologically using microsatellite markers to unambiguously identify possiblesuperior genotypes. The microsatellites employed were polymorphic (observed heterozygosity = 0.71;polymorphic information content = 0.59), discriminated 52% of the genotypes and enabled the detectionof intra-cultivar polymorphism, derived from both somatic mutations, indicating the presence of poly-clonal cultivars, or from gametic origin, thus suggesting the presence of cultivar-populations. A high levelof genetic variability was detected within the ‘Biancolilla’, ‘Giarraffa’ and ‘Moresca’ genotypes, whereaslow variation was found within the ‘Cerasuola’ and ‘Tonda Iblea’ genotypes. The combination of UPGMAcluster analysis of data obtained from microsatellite analysis, with canonical discriminant analysis (CDA),based on 18 morphological variables, measured under the same conditions, enabled intra-cultivar diver-
sity, attributable to genetic factors rather than to environmental ones to be identified. The goodness offit between microsatellite profiles and the CDA analysis was significantly supported by the Mantel test(r = 0.3; p < 0.001). Genotypes and clonal variants with superior traits (larger fruit size; compact tree habit,apt for high density planting; higher oleic acid content) were identified, suitable for enlarging their areaof cultivation.
Olive (Olea europaea L.) is one of the most economically impor-ant tree species in the Mediterranean basin, cultivated for its fruitnd oil. Italy is the world’s second largest olive producer after SpainFaostat, 2013). Among the Italian regions, Sicily has a significantlace in the olive and olive oil production, industry and exporta-ion, being the third largest region of production after Apulia andalabria (ISTAT, 2013).
Olives have been cultivated in Sicily since antiquity; the Phoeni-ians brought the olive to Sicily in the sixth century BC and theomans continued the expansion of olive cultivation throughout
he Mediterranean by developing grafting techniques (Zohary andopf, 1994; Besnard et al., 2001; Rugini et al., 2011). The genetic
ichness of Sicilian olive germplasm is well documented (Bottari
and Spina, 1952; La Mantia et al., 2005; Caruso et al., 2007; Marraet al., 2013; Besnard et al., 2013) and represents a heritage patri-mony of economical and scientific value, particularly for breedingprogrammes. Parentage studies performed by Marra et al. (2013)demonstrated that both somatic mutations and new genotypesderived through sexual crosses created the rich diversification ofthe Sicilian olive germplasm.
The first historical investigation on indigenous Sicilian heritageolive was conducted in the second half of the XIX century by Caruso(1883). Sicilian farmers probably selected late ripening cultivars,since long, mild and wet autumns allow for olive oil accumulationand complete ripening. It is likely that vigorous and drought resis-tant trees, with large-sized fruits were selected by local farmersover the centuries.
Since the 1980s, the Department of Agricultural and ForestSciences (SAF) of Palermo has worked extensively on the character-
ization and the conservation of the main local olive cultivars andhas studied their efficiency in yield and in oil quality (La Mantiaet al., 2005; Caruso et al., 2007; Marra et al., 2013), following thefirst survey conducted in the middle of the XX century by Bottari
nd Spina (1952). In 2002, a large olive germplasm collection wasstablished in Sicily, which today contains eight well-known andxtensively grown cultivars, 17 minor or neglected cultivars and22 native genotypes.
Currently in the island, olive oil production is based mainlyn the cultivars ‘Biancolilla’, ‘Cerasuola’, ‘Moresca’, ‘Nocellara delelice’, ‘Nocellara Etnea’, ‘Ogliarola Messinese’, ‘Santagatese’ and
Tonda Iblea’ (Caruso et al., 2007). The table olive industry is alsoignificant (8% of total olive production) and based mainly on theultivar ‘Nocellara del Belice’ and, to a minor extent, on ‘Nocellaratnea’, ‘Ogliarola Messinese’ and ‘Moresca’ producing large-sizedruits of high commercial value (Caruso et al., 2007). As most vari-ties are often known under different names, according to thereas of cultivation, many cases of homonymy and synonymy occur,omplicating cultivar identification. However, a certain amount oforphological variability has been observed within each cultivar,
uggesting that the name of a single cultivar could encompass aool of different genotypes (cultivar populations) and/or popula-ions of clones (mixture of clonal variants). Generations of farmersave played a role in selecting, conserving and genetic improv-
ng of the Sicilian olive germplasm. Many of the morphologicalypes of each of the main cultivars, identified thanks to reportsf millers, growers and nurserymen, have been collected ex situn a regional repository and observed over the years concern-ng their canopy architecture, time-course of phenological phases,ree crop efficiency, and fruit, leaf, inflorescence and endocarpraits.
It is largely accepted that the olive cultivar discriminationased on morphological descriptions is not completely reliableBelaj et al., 2001) therefore DNA molecular markers, particularly
icrosatellites (SSRs; simple sequence repeats), are today widelysed (Bracci et al., 2011) to complement morphological analysesnd to unambiguously identify the accessions held in collections.enetic variation has been reported among naturally occurringlive clones in literature with molecular markers. Clones were iden-ified with RAPD and ISSR (Gemas et al., 2004; Gomes et al., 2008;
artins-Lopes et al., 2009), with AFLP (Frane et al., 2010), andicrosatellites (Lopes et al., 2004; Muzzalupo et al., 2010; Zaher
t al., 2011; Albertini et al., 2011; Ipek et al., 2012). Although cur-ently there is intense research to develop reliable techniques foretecting mutations in genes, clone identification is still predom-
nantly based on the study of phenotypic traits, integrated witholecular analyses.In this study, intra-cultivar variation of the most widely
istributed Sicilian olive cultivars, was identified through mor-hological and molecular characterization, employing a reliableet of SSRs (reviewed in Baldoni et al., 2009), to detect newlonal variants and to confirm the suspected existence of poly-lonal cultivars and cultivar-populations. Novel insights on oliveenetic diversity were acquired, that will be useful for conservationnd breeding purposes, tree nursery genetic certification and oilraceability.
. Material and methods
.1. Plant material
A total of 45 putative clones belonging to eight standardultivars were studied. These comprised: 10 putative clones ofBiancolilla’, six of ‘Cerasuola’, three of ‘Giarraffa’, eight of ‘Moresca’,1 of ‘Nocellara del Belice’, two of ‘Ogliarola Messinese’, four of
Tonda Iblea’ and two genotypes of ‘Nocellara Messinese’, a minorultivar the fruits of which could play a role in the further devel-pment of the Sicilian table industry. The cultivar ‘Nocellara Etnea’as included in the analysis as an internal control. All genotypes
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were grown at the Sicilian olive regional repository established in1991 in the ‘Azienda Carboj E.S.A.’—Castelvetrano (37.30 Lat N.;Sicily). In that collection, there were nine plants of each putativeclone, divided into three replicates of three plants, placed at a dis-tance of 5 × 7 m and trained to free vase shape.
Sicilian cultivars chosen as standards for genetic and phytosani-tary certification purposes (Caruso et al., 2007) were planted in thesame farm; they were distributed in the field according to the sameexperimental scheme adopted for the putative clones, divided intothree replicates of three plants, in order enable the comparison withtheir putative clones and to exclude the influence of environmentalfactors on the phenotype.
In the text the standard cultivars were indicated as STD.
2.2. Morphological traits and biostatistical analysis
Morphological observations were carried out on all cultivarsand their putative clones in 2012–2013. Eighteen morphologicalvariables, interval and quantitative were selected for the character-ization of the putative clones among the range of characters definedas the main descriptors of the olive tree (Bottari and Spina, 1952;Barranco et al., 2000; Bartolini et al., 2005; COI, 1997; Caruso et al.,2007). The choice of these traits was guided by previous studies onItalian olive genetic resources (Marra et al., 2013); the interval clas-sification was obtained using the classes reported by Caruso et al.(2007).
Canonical discriminant analysis (CDA) based on the18 mor-phological variables was performed using the Systat statisticalprogram (SYSTAT Software Inc., Chicago, IL). The power of discrim-ination of the canonical discriminant functions (CDFs) generatedwas tested by the “Jackknife misclassification method”, a procedurewhich omits one observation, elaborates the classification func-tion employing all other observations (n1 + n2 − 1) and uses theclassification function to classify the excluded observation. Thisprocess is reiterated for each of the observations (Osuji et al.,2013).
Means of standardized canonical scores of major CDFs wereplotted. Two-dimensional CDFs plots were created in order tovisualize the groups of putative clones in relation to the cultivarconsidered as standard. Ellipses marked the 68% confidence levelfor the analyzed groups.
2.3. DNA extraction and microsatellite evaluation
Genomic DNA was extracted from young leaves according to theprotocol developed by Doyle and Doyle (1987). DNA was amplifiedwith nine fluorescently labeled SSR primer pairs, five of which werecombined in two multiplexed primer sets as follow DCA: 03, 05, 18(Sefc et al., 2000); Gapu: 45, 71b (Carriero et al., 2002); and fourused in single PCR reactions: DCA13 (Sefc et al., 2000); EMO90 andEMOL (De la Rosa et al., 2002); UDO43 (Cipriani et al., 2002). Thenumber of alleles for each SSR locus, the expected heterozygosity(He), the observed heterozygosity (Ho) and PIC (polymorphic infor-mation content) were calculate with PowerMarker v3.25 software(Liu and Muse, 2005). Genotypes that were not distinguishableat the molecular level and/or those showing extra alleles wereexcluded for the calculation of SSR genetic parameters. A UPGMAdendrogram was also constructed using the PowerMarker V3.25software employing the coefficient of similarity Nei (1973). AMantel (1967) test was also computed with PowerMarker (Liu and
Muse, 2005) to check the goodness of fit between genetic profilesand CDA analyses, based on morphological traits, of all genotypesanalyzed, by using Simple Matching dissimilarity matrixes; p valueswere also calculated.
The olive genotypes under study were evaluated for 18 morpho-ogical and biometric traits following the descriptors reported byaruso et al. (2007).
The morphological variability found among ‘Biancolilla’ putativelones was high. Regarding the leaf, the predominant shape waslliptic-lanceolate (4< length-to-width ratio <6); the leaf lengthmm) varied between 46.54 (clone 9) and 65.31 (clone 4), and theeaf width (mm) between 10.61 (clone 6) and 13.72 (clone 8). Fruithape was spherical (length-to-width ratio <1.25) in clones 7 and, unlike the remaining clones and the STD, in which the shapef the fruit was elliptical (1.25< length-to-width ratio <1.50). Theeight of the fruit (g) varied between 2.3 (clone 6) and 6.8 (clone
1), the latter had the highest fruit weight among all clones. Aarge proportion of the clones presented elliptical pit shape (1.9<ength-to-width ratio <2.2), except clone 7, showing oval pit (1.4<ength-to-width ratio <1.8). Clone 11 had the largest pit.
Among the ‘Cerasuola’ group, the leaf shape was elliptical-anceolate in all clones; whilst the STD displayed an ellipticalhape (length-to-width ratio <4), the length (mm) of the leaf variedetween 57.81 (clone 1) and 65.49 (clone 6), the leaf width (mm)aried from 13.73 (clone 3) to 16.9 STD. The fruit shape was spheri-al, pit shape was oval and the weight of the fruit (g) varied between.36 (clone 1) and 4.41 (clone 4).
Regarding the putative clones of ‘Giarraffa’, the prevalent formf the leaf was elliptical-lanceolate, the leaf length (mm) rangedrom 47.80 (clone 2) to 60.88 (clone 3), the leaf width (mm) var-ed from 12.18 (clone 2) to 13.78 (STD). The shape of the fruit waslliptical and the highest weight of the fruit (g) was found in clone
(7.49) and in the STD (7.33). All analyzed putative clones and STDresented elliptical pit shape.
Among the putative clones of ‘Moresca’, the predominant shapef the leaf was elliptical, except for clones 1 and 10, which dis-layed an elliptical-lanceolate leaf shape; the leaf length (mm)aried between 54.19 (clone 3) and 62.45 (clone 6), the smallesteaf width (mm) was found in clone 10 (11.71). The fruit shape waslliptical in all putative clones; clone 10 showed fruit weight (g)reater than 6; most of the putative clones presented an ellipti-al pit shape, except the clone 10, which had elongated pit shapelength-to-width ratio >2.2).
In all putative clones of ‘Nocellara del Belice’, the shape of theeaf was elliptic-lanceolate, leaf length (mm) varied between 52.15clone 2) and 74.85 (clone 8), the leaf width (mm) ranged from 12.29clone 1) to 17.68 (clone 7). The fruit shape was mainly spherical,xcept clones 1 and 11, which had elliptical fruit shape; the highesteight of the fruit (g) was found in clone 10 (10.68 g), while the
owest was found in clone 11 (3.72 g); most of the putative clonesresented oval pit shape, except clones 1 and 11, which presentedlliptical pit shape.
Concerning ‘Nocellara Messinese’, putative clone 1 presentedlliptic-lanceolate leaf, fruit and endocarp elliptical as the standard.owever, fruit and pit weight was smaller compared to STD.
All putative clones of ‘Ogliarola Messinese’ and the STD pre-ented elliptic-lanceolate leaf shape, fruit elliptical and elongatedit, except for the clone 3, having elliptical pits.
‘Tonda Iblea’ putative clones were quite similar among them.hey presented leaf shape elliptical, spherical fruit shape and ovalit shape.
.2. Biostatistical analyses of morphological traits
The 18 morphological and biometric data were statistically ana-yzed by CDA and results were represented in Fig. 1. As reported in
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Table 1, the CDF1 explained 24% of the total variance present in thestudied genotypes, the CDF2 however, explained 16% of the totalvariance. CDF3 and CDF4 contributed an additional 12% and 10% ofthe total variance, respectively. The combination of the first fourCDFs explained 62% of the total variance. Total canonical struc-ture was computed for CDF1/CDF2, explaining 40.3% of the totalvariance, and for CDF1/CDF4, which explained 33% of the total vari-ance. For CDF1, the parameters with higher discriminating abilitywere: shape, width and length of the fruit. Concerning CDF2, leafshape, area and length were found to have the greatest discrimina-tory power. For CDF3: the weight of the fruit and the length of thepit (or endocarp) were the factors with the greatest discriminatorypower. Finally, for CDF4: length, width and shape of the pit; weight,length, width and shape of the fruit and the flesh to pit ratio had thegreatest discriminatory power. In Fig. 1a, many putative clones of‘Biancolilla’ showed a high degree of dispersion from the genotypestandard. Putative clones 4, 7, 9, 11 and 12 mainly differed alongthe CDF2, on which morphological parameters related to the leafcarried greater weight. Putative clones 4, 5 and 9 differed also alongCDF1 and CDF4, on which fruit and pit weight loaded more; clones6, 10, 11 and 12 placed along the CDF4.
The putative clones of ‘Cerasuola’ showed a low degree of dis-persion with respect to their STD along CDF1 and CDF2 (Fig. 1b).However, along the components CDF4 clones 3, 5 and 6 were dis-criminated.
All three putative clones of ‘Giarraffa’ were scattered comparedto the genotype standard (Fig. 1c).
Clones 5, 6 and 10 of ‘Moresca’ were rather dispersed from thestandard (Fig. 1d). In particular, clone 10 showed marked differ-ences from the other for all three components examined.
Concerning putative clones of ‘Nocellara del Belice’, 1, 5, 10 and11 were all dispersed from the standard genotype (Fig. 1e). In detail,clones 1, 10 and 11 separated along components CDF1, CDF2 andCDF4. Clone 5 differentiated along component 4.
All three putative clones of ‘Ogliarola Messinese’ were differ-entiated for all components (Fig. 1f), as well as the putative clone1 of ‘Nocellara Messinese’ differing for all components from thestandard (data not shown).
All putative clones of ‘Tonda Iblea’ did not differ significantlyfrom the standard (Fig. 1 g). However, clone 1 showed some differ-ences from the standard along the component CDF2, while clone2 differed along CDF4. Overall the correct classification matrixaverage was 72% (data not shown) and 65% with the Jackknifeclassification matrix. The genotypes showing the highest correctJackknife classification (>70%) are presented in Table 3. On thewhole 44% of the genotypes were correctly classified with the Jack-knife method. The discrepancies between the classification matrixand the Jackknife classification matrix depended on the differentclassification functions used by the two methods. As expected, theJackknife classification matrix presented lower values, since theJackknife procedure provides conservative estimates of the actualprobability of misclassification, especially when numerous vari-ables are included in the analysis.
3.3. SSR genetic parameters and clustering analysis
The number of alleles per locus ranged from three with Gapu45to 11 with UDO43, with an average of 5.7. The average expected het-erozygosity was 0.62. The average observed heterozygosity valuewas 0.71, ranging from 0.32 (EMOL) to 0.96 (DCA13 and DCA03).The PIC (polymorphic information content) varied from 0.25 withEMOL to 0.81 with DCA18, with an average value of 0.59 (Table 2).
Excluding the genotype employed as a control, this set of SSRmarkers discriminated 52% of the genotypes. The UPGMA dendro-gram (Fig. 2) showed the undistinguishable putative clones fromthe reference cultivars. In most of the cases these SSRs enabled the
T. Caruso et al. / Scientia Horticulturae 180 (2014) 130–138 133
F and fom esca’ (i
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a
ig. 1. Plots of first and second discriminant functions (CDF1/CDF2) and of first
easured in putative clones of: ‘Biancolilla’ (a); ‘Cerasuola’ (b); ‘Giarraffa’ (c); ‘Morndicates the 68% confidence interval.
istinction of somatic mutants, differing only in one or two SSRlleles from their STD or other closest genotypes (similarity index
0.95), from the siblings, sharing at least one allele for each SSRocus.
All genotypes belonging to the group ‘Biancolilla’ represented clear case of cultivar - population (Fig. 2). Putative clones 4 and
urth discriminant functions (CDF1/CDF4) calculated on the basis of 18 variablesd); ‘Nocellara del Belice’ (e); ‘Ogliarola Messinese (f)’; ‘Tonda Iblea’ (g). The ellipse
11 showed very narrow genetic relationships and could be somaticmutants of each other, but they were determined to be an unrelated
individual from ‘Biancolilla’ STD. Clones 7, 8 and 12 had a closerrelationship between each other but they could not be consideredsiblings of the STD. Putative clones 5 and 9 can be considered gen-uine somatic mutants of the cultivar; while ‘Biancolilla’ 12 shared
134 T. Caruso et al. / Scientia Horticulturae 180 (2014) 130–138
(Cont
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Fig. 1.
ibling relationships with the STD. The molecular identity of theBiancolilla’ clones 1, 6 and 10 with the STD was also supportedy the morphological similarity depicted also by the CDA analysisFig. 1a).
Putative clones of ‘Cerasuola’ 1, 4 and 5 were found to be somatic
utants of the STD (Fig. 2), indicating that they represent a mix-
ure of clonal variants; this was confirmed from the morphologicalimilarity (Fig. 1b). The remaining clones presented the same SSRrofiles than the STD.
inued ).
Genotypes 1 and 2 of ‘Giarraffa’ were revealed as possible sib-lings of the STD, while ‘Giarraffa 3’ is genetically close but not asibling, however this evidence needs to be confirmed by furtherinvestigations. Genetic relationships emerged between the ‘Giar-raffa’ genotypes with ‘Ogliarola Messinese’ STD and its clones 2
and 3; ‘Nocellara del Belice’ clone 1; and ‘Moresca 10’ (Fig. 2).
Concerning the cultivar ‘Moresca’, genotypes 5 and 10 wereclearly different from their STD. In particular, the SSR analysisshowed that the ‘Moresca’ 5 shared a genetic relationship with
T. Caruso et al. / Scientia Horticulturae 180 (2014) 130–138 135
Table 1Standardized canonical coefficients of the first four canonical discriminant functions(CDF) of 18 morphological variables and relative % of variance explained.
lones 4 and 5 of the cultivar ‘Cerasuola’. In the case of ‘Moresca’0 with ‘Giarraffa 3’ (Fig. 2) the genetic relationship is confirmedy the substantial morphological similarity (Fig. 1c and d) that mayepresent a case of misidentification which deserves further inves-igation. Putative clones 1 and 9 were found to be genuine somatic
utants of the STD, while the remaining genotypes 2, 3, 4, 6 weredentical to the STD for their SSR profiles (Fig. 2), suggesting thatMoresca’ is a polyclonal cultivar.
Regarding ‘Nocellara del Belice’, while clones 6 and 10 did notiffer from the STD, all the remaining clones showed moderateolymorphism (Fig. 2). Clones 1 and 2 of ‘Nocellara del Belice’ cane considered siblings of the STD, while the other clones can beegarded as somatic mutants. Therefore, ‘Nocellara del Belice’ coulde regarded as a cultivar-population in which also clonal variantsre present.
All ‘Tonda Iblea’ putative clones revealed identical molecularrofiles (Fig. 2); this was confirmed morphologically, except forlones 1 and 2 (Fig. 1g). Thus, ‘Tonda Iblea’ can be considered aolyclonal cultivar.
Concerning the minor cultivar ‘Nocellara Messinese’ genotype 1,epresented a case of sibling, which separated from the STD in thePGMA dendrogram of similarity, based on SSRs (Fig. 2). Finally,
he differences found among ‘Ogliarola Messinese STD’ and thelone 3 can be attributed to somatic mutations, indicating that its polyclonal cultivar.
able 2. of alleles, observed heterozygosity (Ho), expected heterozygosity (He); polymor-hic information content (PIC) in eight STD cultivar and 23 putative clones.
Fig. 2. UPGMA dendrogram showing the genetic diversity of 45 putative clones ofeight Sicilian standard cultivars (STD), based on microsatellite markers. ‘NocellaraEtnea STD’ was included in the analysis as an internal control.
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36 T. Caruso et al. / Scientia Ho
. Discussion
In the present study morphological traits and microsatellitearkers were used to detect for the first time the intra-cultivar vari-
bility of the main economically important olive cultivars grown inicily and to identify new promising genotypes or superior cloneshrough clonal selection.
Both clonal selection and cross-breeding programmes are inrogress in many countries to select new cultivars. Today desir-ble traits in novel olive cultivars are quite different respect tohose chosen in the recent past. These include early bearing, annualroduction, low vigour for intensive cultivation and canopy archi-ecture suitable for mechanical harvesting with straddle or canopyontact machines, fruits having high olive oil content and produc-ion oil with high rate of bioactive substances beneficial for theuman health (Fabbri et al., 2009).
.1. Morphological diversity
A high degree of polymorphism was observed morphologicallymong the putative clones, confirming the observations of localarmers, millers and growers. The CDA analysis was performed todentify patterns of variability among the putative clones and theirelative standards. The combination of the first four CDFs explained2% of the total variance and was considered statistically satisfac-ory in order to interpret our experimental results, since valuesver 50% of the total variance are considered effective for culti-ar discrimination in other fruit tree species (Barone et al., 1996).he other canonical functions, from CDF5 to CDF17, explaining theemaining 38% of the total variance, were less important for theiscrimination of putative clones and they were excluded for thelot construction. All resulting CDFs were significant and statisti-ally supported by p-value <0.001 and Wilks’s Lambda test. Theharacters with greater discriminating ability were those relat-ng to the fruit, to the leaf and to the pit (Table 1). Thus, theseharacters should be preferred and prioritized in the complex activ-ty of morphological characterization of the accession of the oliveree. Belaj et al. (2011) highlighted the importance of the symme-ry of the fruit, the shape of the pit, the flesh to pit ratio and oilontent for characterization of wild olive genotypes, finding thathese morphological traits discriminated 95% of the wild genotypes.’Imperio et al. (2011) demonstrated the importance of morpho-
ogical traits of the pit in the discrimination of three Italian oliveultivars from Molise and their relative somatic mutants. In bothorks, the morphological characteristics of the leaf were only mod-
rately useful in identifying cultivars. In the present study, the traitshat have assumed great importance as a source of variability inhe first four principal components were partially different fromhose detected by Belaj et al. (2011) and D’Imperio et al. (2011),ince parameters concerning the morphology of the leaf were foundlso significant in the discrimination of putative clones of key oliveultivars grown on the island. Leaf morphological traits are consid-red highly influenced by climatic and environmental conditionsnd are therefore often excluded from analysis. In the context inhich our investigation was conducted, however, the environ-ental influence on the variability of the leaves can be excluded
ecause all genotypes were grown at the same site and subjectedo the same cultural practices. Thus the morphological differencesbserved could be attributable solely to genetic factors. Distribu-ions of the genotypes in the obtained CDA plots were in agreementith morphological and biometric differences observed.
.2. SSR genetic parameters and genetic polymorphisms
The majority of the SSR markers amplified a single locus and themplification of extra alleles was only sporadically observed with
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the primer pair DCA13. The average observed heterozygosity valuewas higher than the expected heterozygosity value, similar to thefinding of Baldoni et al. (2009). The most informative markers wereDCA18 and UDO43, in accordance to Baldoni et al. (2009), while theleast polymorphic markers were EMOL and DCA05. EMOL was dis-carded by Baldoni et al. (2009) since it presented two drawbacks; ahigh occurrence of null alleles and allele homoplasy. The list of themost useful primers in olive reported by Baldoni et al. (2009) shouldbe slightly updated or modified, considering that other recom-mended primers amplified two loci in the Sicilian germplasm, suchas GAPU101, amplifying alleles, ranging from 112–147 bp (data notshown). Our data can be compared to those published by Baldoniet al. (2009) when examining four cultivars (‘Arbequina’, ‘Carolea’,‘Koroneiki’ and ‘Leccino’), used as control in our PCR reactions, andsix microsatellites in common (DCA:3, 5, 18; UDO43; EMO90 andGapu71b), but allelic length differences and allele drop out occurred(data not shown).
Apart from the morphological traits, a high degree of polymor-phism was also found at the molecular level. The chosen SSRsrevealed intra-cultivar genetic diversity, caused by somatic muta-tions, leading to polyclonal cultivars, or by sexual reproduction,originated from natural crossing and subsequent seed dissem-ination leading to cultivar-populations. ‘Biancolilla’, ‘Giarraffa’,‘Nocellara del Belice’, ‘Nocellara Messinese’ and ‘Moresca’ repre-sented a clear case of cultivar–populations, in which also clonalvariants were present, whereas ‘Cerasuola’, ‘Ogliarola Messinese’and ‘Tonda Iblea’ resulted a mixture of clonal variants. In the litera-ture, high level of intra-cultivar genetic diversity has been reported.Clones were identified by means of RAPD and ISSR markers inthe Portuguese cultivar ‘Galeca’ (Gemas et al., 2004), in ‘Verdeal-Transmontana’ (Gomes et al., 2008) and in the cultivar ‘Cobranc osa’(Martins-Lopes et al., 2009). Frane et al. (2010), using amplifiedfragment length polymorphisms, identified intra-cultivar polymor-phism in a pool of genotypes of the Croatian cultivar ‘Oblica’ grownin the same agro-climatic conditions. Muzzalupo et al. (2010) foundgenetic variability in ‘Carolea’ using SSR markers, which the authorsattributed to the occurrence of somatic mutations that had accumu-lated over the centuries of cultivation of this genotype. High levelsof genetic polymorphism were also detected with SSR markers in‘Picual’, ‘Conserva de Elvas’, ‘Verde Verdelho’, ‘Ascolana’, ‘Coratina’and ‘Picholine’ (Lopes et al., 2004); within ‘Picholine Marocaine’genotypes (Zaher et al., 2011); and among ‘Gemlik’ genotypes (Ipeket al., 2012).
The goodness of fit between SSR profiles and CDA analysis inthis investigation was confirmed by the Mantel test (1967) (r = 0.3;p < 0.001). We performed a test to better understand the unex-pected identity in the molecular profiles of the following threecases (Fig. 2): (a) ‘Giarraffa’ 1 and 2 versus ‘Ogliarola Messinese’STD versus ‘Ogliarola Messinese’ 2; (b) ‘Giarraffa’ 3 versus ‘Moresca’10; (c) ‘Giarraffa’ STD versus ‘Nocellara del Belice’ 1; and to verifywhether these genetic relationships fitted the observed morpho-logical similarity through the CDA (data not shown). High levelsof morphological similarity were found in agreement with themolecular data. In general, it seems possible that some of thesegenotypes have been wrongly identified and named. Fruits of ‘Gia-rraffa’, ‘Ogliarola Messinese’ and ‘Moresca’ tend to be large, earlyripening, completely black when fully ripened and in Sicily are tra-ditionally used as table olives, generically called “passuluna” (fadedfruits) in the local dialect. Therefore, it is likely that the name “Giar-raffa” may have been wrongly attributed to other genotypes whichfruits can be used, at black ripening stage, as table olives; ‘Giar-raffa 1’, ‘Giarraffa 2’ and ‘Ogliarola Messinese STD’, showing the
same SSR profiles, could be sibling of ‘Giarraffa STD’, since theyshared one allele for each SSR locus. This is also confirmed bythe allelic patterns of ‘Giarraffa STD’ and ‘Ogliarola Messinese STD’found by Marra et al. (2013) for 11 SSR loci, with the exception
f the locus Gapu103, where an allele drop out possibly occurred.Moresca 10’ and ‘Giarraffa 3’ could represent further cases of erro-eous attribution of the name, although they are genetically closeo the ‘Giarraffa’ group, while, ‘Nocellara Belice 1’ may be a sibling ofNocellara Belice STD’ and ‘Giarraffa STD’, which shared morpholog-cal similarity with the latter cultivar. The integration of differenttatistical methods applied to the analysis of morphological andolecular data does not always permit the same conclusions to be
eached about the relationships between the cultivars and clonesven within the same gene pool (Belaj et al., 2003; Hagidimitriout al., 2005). From our results, it seemed clear that the combina-ion of the CDA with the cluster analysis of the molecular profiles ishe most appropriate and reliable way to study relations amongolyclonal cultivars and cultivar-populations and thus to verify
ntra-cultivar diversity.Clonal selection, proposed since the early 1960s, for olive cul-
ivar improvement (e.g. Serrano et al., 1999; Grati Kamoun et al.,000; Oueslati et al., 2009; Tous et al., 2011) was confirmed to ben effective strategy which can assist in improving the standardf a cultivar. The results presented are indeed useful for undertak-ng selection of superior genotypes such as ‘Nocellara del Belice’lone 10 which showed the largest fruit size and the highest flesho pit ratio among the ‘Nocellara del Belice’ genotypes and thereforeeserves particular attention by the table olive industry. Likewise
Biancolilla’ clone 6 has been reported to have adaptive traits toumid environment conditions while, on the contrary, ‘Biancolilla’, has been described by local growers as tolerant to salty windnd drought stress, since it is widely spread and cultivated in theindy island of Pantelleria, a small volcanic island in the middleediterranean sea where water availability is scarce all the year
round and the soil water capacity is very low. ‘Cerasuola’ clone 6as shortened internodes resulting in a dwarfing and compact veg-tative growth habit with dense tree canopy in contrast to the other
Cerasuola’ genotypes, so it represents an interesting genotypeshat should be exploited for high-density plantings and evaluatedor trunk-shaker mechanical harvesting; in addition, the olive oil ofhis clone has an higher content of oleic acid (79%) respect to that ofhe STD one (74%), with total polyphenol content of 376 ppm. Theseenotypes could be also employed for breed new olive varieties foracing future environmental challenges or new market/consumereeds.
. Conclusion
Our research allowed the identification of the most discriminantorphological traits such as shape, width and length of the fruit;
hape, area and length of the leaf; fruit weight; length, width andhape of the endocarp, which should be prioritized in the morpho-ogical characterization. Consequently, the number of traits to betudied for olive characterization could be reduced without losingmportant information, saving time, thus decreasing cost of mor-hological analysis. The combination of traditional cluster analysisUPGMA) based on microsatellite markers and canonical discrimi-ant analysis (CDA) based on morphological variables, measuredn the same environmental conditions, was found powerful fortudying relationships among genotypes.
The ex-situ collection at the regional repository can be con-idered the only source of propagation material of Sicilian oliveenotypes and standard cultivars, from which a process of nurs-ry certification could be initiated. Traders and experts from theil and table olive production chain may benefit from genetic and
ealth certification as they can enhance the activity of the selectionf local genotypes, guarantee varietal identity and typical pro-uctions, improve plant quality, and permit product traceabilityrom plant to food. Furthermore, intra-cultivar variability can be
turae 180 (2014) 130–138 137
exploited to extend the area of cultivation of the main cultivarsin new zones, where environmental conditions are quite differentwith respect to the site of origin of the standard clones.
Results of this work can contribute to a list of the most inter-esting genotypes showing novel traits which could be tested bylocal growers in Sicily to select genotypes adapted to the variousenvironmental conditions of the Island.
Acknowledgement
This research was supported and founded by Dieta Mediter-ranea e Salute (DI.ME.SA)—Sicilian Cluster on Agro-Industry andFishery, PON0200451 3361785, Palermo, Italy. We thank Dr. DanielJ. Sargent for English revision.
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