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Scientia Horticulturae
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tility of wild germplasm in olive breeding
atjana Klepoa,∗, Raúl De la Rosaa, Zlatko Satovicb, Lorenzo Leóna, Angjelina Belaja
IFAPA Centro Alameda del Obispo, Avda. Mendéz Pidal, s/n, 14004, Córdoba, SpainFaculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia
r t i c l e i n f o
rticle history:eceived 15 June 2012eceived in revised form 3 December 2012ccepted 13 December 2012
eywords:lea europaeaild olive
reeding programsorphological traits
a b s t r a c t
Olive breeding programs are only based on commercial cultivars as genitors. However, due to its widevariability, wild olive gremplasm could represent an interesting source of genes for the obtention ofnew cultivars. With purpose of evaluating the utility of wild olives in breeding programs, two openpollinated progenies (o.p.), originated from a wild olive (Alga05) and the main Spanish olive cultivar(Picual), were compared. Both progenies were analyzed by morphological descriptors, agronomical traitsand SSR markers. The use of these 3 marker systems revealed great discrimination capacity, high level ofmorpho-agronomic and genetic diversity and their complementariness on the evaluation of these oliveprogenies. As expected, for most of morpho-agronomical traits, ‘Picual’ o.p. progeny showed superiorvalues in comparison to the wild o.p. progeny. However, ‘Alga05′ wild olive progeny was more vigorous,
gronomical traitsSR
with shorter juvenile period and more abundant flowering than ‘Picual’ o.p. For both progenies, PCAshowed strong association between different agro-morphological traits (fruit and stone dimensions inwild olive progeny and fruit trait with oil content in ‘Picual’ progeny) which could facilitate the selectionof the most appropriate traits for further evaluations, increasing thus, the efficiency of olive breedingprograms. Our results indicate that the use of wild olive as genitors in breeding programs may be usefulfor generating new genotypes with interesting characters.
Most of the olive cultivated germplasm (Olea europaea subsp.uropaea var. europaea) comes from the empiric selection madey the growers hundreds or even thousand years ago (Barrancot al., 2010; Bracci et al., 2011). Therefore, new cultivars, obtainedy systematic breeding, and well adapted to the new olive grow-
ng systems are needed (Rallo, 2005). In this way, most of thelive breeding programs are focused in obtaining new olive cul-ivars with high productivity and earliness of bearing, resistanceo biotic and abiotic stresses and adaptation to several harvest-ng mechanization systems (Baldoni and Belaj, 2009; Bellini et al.,008; Lavee and Avidan, 2011; L. Rallo et al., 2008). Those breed-
ng programs are based in both cross breeding and open pollinatedrogenies (Baldoni and Belaj, 2009). Open pollination is an easy
ay to produce segregating progenies as both cultivated and wild
lives are mainly self-incompatible (Beghé et al., 2012; Hannachind Marzouk, 2012; Pinillos and Cuevas, 2009).
∗ Corresponding author. Permanent address: Institute for Adriatic Crops and Karsteclamation, Put Duilova 11, 21000 Split, Croatia. Tel.: +385 21 434471;
Up to date, all the breeding efforts carried out in olive were basedon cultivated material, being Australia the unique case where feralolive populations (escapes from cultivated trees) have been evalu-ated for breeding purposes (Sedgley, 2000). However, wild relativeshave been successfully used in other fruit species to introduce genesfor characters such as disease or drought resistance (Fischer andFischer, 2002; Sorkheh et al., 2009, 2011; Nikoumanesh et al., 2011).
Wild olive germplasm (Olea europaea subsp. europaea var.sylvestris) contains more variability than the cultivated one(Baldoni and Belaj, 2009; Belaj et al., 2010; Lumaret et al., 2004),it is distributed throughout all the Mediterranean including areaswhere the presence of cultivated olive is absent or symbolic (Belajet al., 2007; Lumaret et al., 2004; Vargas and Kadereit, 2001). Alongwith strawberry tree (Arbutus unedo L.), lentisk (Pistacia lenticus L.),myrtle (Myrtus communis L.) and rosemary (Rosmarinus officinalisL.), wild olives form typical Mediterranean vegetation changing inhabitus from shrub to tree (Mulas, 1999; Zohary, 1994). Wild olivepopulations are found in a high diversity of environments, withdifferent altitudes and soils and may be a very important source ofresistance to abiotic stresses such as drought, salt, wind and low
temperature (Aranda et al., 2011; Baldoni et al., 2006; Meddad-Hamza et al., 2010; Mulas, 1999). They may also be interesting asa source of resistance to biotic stresses, such as Verticillum Wilt(Verticillium dahliae, Sesli et al., 2010), peacock spot (Cycloconium
leaginum, Ciccarese et al., 2002) or olive fly (Bactrocera oleae, Mkizet al., 2008).
Most of the studies carried out on wild olive germplasm haveeen focused on its variability (Belaj et al., 2007, 2010; Besnardt al., 2002; Lumaret et al., 2004) or the establishment of geneticelationships between wild and cultivated olives by means ofolecular markers (Belaj et al., 2010; Breton et al., 2006; Erre et al.,
010; Munoz-Díez et al., 2011). On the contrary, there are scarcetudies on morphological and pomological evaluation of genuineild olive populations like those carried out in Italy (Mulas and
rancesconi, 1999), Tunisia (Baccouri et al., 2011; Dabbou et al.,011; Hannachi et al., 2009) or in feral Australian (Sedgley, 2000).
In Spain, the use of wild populations in olive breeding programsas been proposed as an alternative approach to increase the diver-ity sources for its breeding. For that aim, continuous efforts forheir collection, genetic diversity evaluation and ex situ conserva-ion are being carried out. Molecular (Belaj et al., 2007, 2010) and
orfo-agronomical markers (Belaj et al., 2011; García-Donas Díaz,001) are jointly being used at both in situ and ex situ levels.
In this sense, the present work aims to investigate, the use-ulness of wild olives in a breeding program by comparing, at
orphological, agronomical and molecular levels, two open pol-inated progenies coming from a wild olive tree and the principalpanish cultivar ‘Picual’. To the best of our knowledge there is norevious report on the use of wild olive progenies for breedingurposes.
. Materials and methods
.1. Plant material
Two open pollination (o.p.) progenies from a wild olive treeAlga05) and the cultivar ‘Picual’, were used in the present study.he wild olive tree was chosen based on its regular and abundantructification as shown previously in our studies (Belaj et al., 2007,011). The wild genitor ‘Alga05′ was located in an undisturbed for-st in the province of Cadiz (Southern Spain), an area of very lowlive growing surface. ‘Picual’ is a well known Spanish olive cultivarnd was chosen for its high productivity and high oil content (Delío et al., 2005a,b) and for being one of the main genitors in the Olivereeding Program of Cordoba, Spain (León et al., 2007). The ‘Picual’ree used was located in the World Olive Germplasm Bank of IFAPAordoba. In 2006, seedlings from both progenies (‘Alga05′ o.p. and
Picual’ o.p.) were planted at 4 × 1.5 m spacing at IFAPA (Andalusiannstitute for Research in Agriculture), Center “Alameda del Obispo”n Cordoba, Spain. They were distributed in a randomized completelock design with 14 blocks and 12 genotypes per elementary plot.herefore, a total of 168 trees per progeny were planted. Trees wererained up to form the canopy at 1 m height. No pruning was per-ormed from that point in order to allow the canopy to developreely.
.2. Morpho-agronomical characterization
Thirty fully expanded leaves were randomly collected from thexternal part of the canopy of each seedling. A random sample of50 g of olives was also collected in the 107 seedlings which bearednough number of fruits in 2010 (71 from the wild o.p. progeny and6 from ‘Picual’ o.p. progeny). Thirty fruits were used for morpho-
ogical evaluation and the rest was destined to further agronomicalharacterization. Morphological and agronomical characterization
n a single year of olive progenies was considered representatives high correlation between years was observed in previous worksith both wild olives (Belaj et al., 2011) and progenies from culti-
ars (León et al., 2004b).
turae 152 (2013) 92–101 93
2.2.1. Qualitative morphological descriptionA total of 23 qualitative morphological traits were evaluated fol-
lowing a previously developed protocol for wild olives (Belaj et al.,2011; García-Donas Díaz, 2001). This included leaf (blade length,blade width and shape), fruit (Table 1) and stone (Table 2) char-acteristics. For common and comparable characterization of bothprogenies it has been necessary to modify some of the previouslyestablished traits states, related to fruit and stone (Tables 1 and 2).These modifications, which affect only trait states with overlappingranges were done in a way that each trait states corresponds to bothwild and cultivated seedling progenies.
In order to visualize the relationships, among the 23 qualitativetraits within each progeny as well as between them, three facto-rial correspondence analysis (FCA) were performed using PROCCORRESP in SAS (SAS Institute, 2004). The multivariate analysisof variance (MANOVA) based on Dice’s distance matrix was per-formed using GenAlEx package (“Genetic analysis in Excel”, Peakalland Smouse, 2006).
2.2.2. Quantitative and agronomic traitsQuantitative morphological characterization was performed in
leaves, fruits and stones through image analysis (Aequitas IA ‘Lite’Software, Skye Instruments Ltd., 2007). Perimeter, maximum andminimum diameter and area were directly measured in a sample of30 leaves, fruit and stones. In all cases, shape was calculated as therelation between maximum and minimum diameter. The volumeof fruits and stones was estimated using the formula:
V = 43
�maximum diameter
2×
(minimum diameter
2
)2
Agronomic evaluation included vigor, flowering and fruit traits.Trunk diameter was measured during winter from 2007 to 2011 at60 cm height. Flowering was also recorded in this period accord-ing to a 0–4 scale (0 – no flowers, 1 – poor flowering, 2 – lowflowering, 3 – medium flowering, 4 – abundant flowering). Phasechange from juvenile to adult was associated to the onset of thefirst flowering. Therefore, plants flowering three years after plant-ing or earlier were considered to have short juvenile period. Fruitremoval force was measured in a sample of 25 fruits per tree usinga modified hand dynamometer (Correx). Oil content, fruit weightand flesh/stone ratio in dry and wet basis was determined followingthe protocol described by Del Río and Romero (1999) with modi-fications described by León et al. (2004a). Fruit traits and trunkdiameter were transformed into categorical variables and used asqualitative traits.
Relationships between quantitative and agronomic traits. Analy-sis of variance was performed for each quantitative morphologicaltrait using Statistix Version 8 (Analytical Software, Tallahassee,FL, USA, 2003). Principal component analysis (PCA) using PROCPRINCOMP in SAS (SAS Institute, 2004) was performed to explorerelationships among the 23 quantitative traits analyzed for each ofthe two progenies. One PCA was also computed in order to inves-tigate relationships between genotypes of both progenies.
2.3. Molecular characterization
DNA from ‘Picual’ and ‘Alga05′ open pollinated seedlings and thetwo genitors was extracted from fresh leaves following the protocoldescribed by De la Rosa et al. (2002). Six microsatellite (SSR) loci(Cipriani et al., 2002; Sefc et al., 2000) were used for genotyping.
Most of these SSRs were selected from a consensus list of the bestSSR on olive fingerprinting (Baldoni et al., 2009). The PCR conditionsof amplification used in the study were previously described by Dela Rosa et al. (2002). The analysis of the PCR products was carried
Table 1Variability for the nine qualitative fruit traits evaluated in open pollination progenies of wild (‘Alga05’ o.p.) and cultivated (‘Picual’ o.p.) origin. Total number of traits states (KT), number of observed states (Kj) and number of traitstates shared (Ksh) between the two progenies are indicated. The letters W (wild olive) and C (cultivated olive) were added in some cases in order to avoid possible trait states overlapping as well as to distinguish between traitstates defined for wild and cultivated plant materials.
Weight FW Low WA Medium W High W–Low C Medium CP High C Very high C 6 2 4 0(<0.5) (0.5–1) (1–2) (2–4) (4–6) (>6)
Symmetry (A position) FS SymmetricalP Slightly asymmetricalA Asymmetrical 3 3 3 3Position of max. diameter (B position) FMD Toward base CentralAP Toward apexU 3 1M 2 0Apex shape (A position) FAS Pointed RoundedAP 2 2 2 2Base shape (A position) FBS Truncate RoundedAP 2 2 2 2Fruit/stone ratio in fresh basis FFSR Low WA Medium W High W; Very low C Low CP Medium C High C Very high C 7 3 5 1
(<1.8) (1.8–2.5) (>2.5 to <4.7) (4.7–6.2) (6.2–7.7) (7.7–9.2) (>9.2)Olive oil on wet matter OWM LowA (<10%) MediumP (10–15%) High (>15%) 3 2 2 1Olive oil on dry matter ODM Low (≤20%) Medium (20–30%) High (≥30%) 3 2 2 1
Underlined: Most frequent trait states in ‘Alga 05′ (A), ‘Picual’ (P) and in both (AP) o.p. progenies.Underlined and in bold: most frequent traits states which coincided with the description of their respective genitors.Italics: less frequent traits found in only one of the two progenies.M Monomorphic trait.U Unfound trait sate in the progenies under study.
Table 2Variability for the 11 qualitative stone traits evaluated in open pollination progenies of wild (‘Alga05’ o.p.) and cultivated (‘Picual’ o.p.) origin. Total number of traits states (KT), number of observed states (Kj) and number of traitstates shared (Ksh) between the two progenies are indicated. The letters W (wild olive) and C (cultivated olive) were added in some cases in order to avoid possible trait states overlapping as well as to distinguish between traitstates defined for wild and cultivated plant material.
Stone trait/abbreviation Trait states KT Kj Ksh
‘Alga05’ o.p. ‘Picual’ o.p.
Shape SS Spherical (L/A < 1.4) Oval (L/A 1.4–1.8) Elliptic (L/A 1.8–2.2)AP Longer (L/A >2.2) 4 2 4 2Weight SW Low W (≤0.15)A Medium W–Low C (0.15–0.3) Low W–Medium C (0.3–0.45)P High C (0.45–0.7) Very high C (>0.7) 5 2 4 1Symmetry (A position) SSA Symmetrical Slightly asymmetricalAP Asymmetrical 3 3 3 3Symmetry (B position) SSB SymmetricalAP Slightly asymmetrical 2 2 2 2Position of maximum diameter (B position) SMD Toward base CentralAP Toward apex 3 2 2 1Apex shape (A position) SASH Pointed RoundedAP 2 2 2 2Base shape (A position) SBSH Truncate RoundedAP Pointed 3 3 3 3Surface (B position) SSU SmoothA Rough KnottyP 3 2 2 1Number of grooves SNG Low W (<6) Medium W–Low C (6 to ≤7)A Medium (7–8) High W–Medium C (9–10)P High C (>10) 5 4 4 3Distribution of grooves SDG RegularP IrregularA 2 2 2 2Mucron SM PresentAP Absent 2 2 2 2
Underlined: Most frequent trait states in ‘Alga 05′ (A), ‘Picual’ (P) and in both (AP) o.p. progenies.Underlined and in bold: most frequent traits states which coincided with the description of their respective genitors.Italics: less frequent traits found in only one of the two progenies.
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ut using Genescan 3.7 and Genotyper 3.7 software from Appliediosystems.
SSR data analysis. For each microsatellite locus, the follow-ng genetic diversity parameters were calculated: total number oflleles (Na), numbers of effective alleles (Ne; Berg and Hamrick,997), Shannon information index (I; Lewontin, 1972), observedHO) and expected heterozygosity (HE; Nei, 1973) and inbreedingoefficient (FIS). The factorial correspondence analysis (FCA) waserformed to visualize the relationships between individuals fromifferent progenies. The analysis of molecular variance (AMOVA;xcoffier et al., 1992) was performed based on Dice’s distanceatrix to examine the partition of microsatellite diversity between
nd within progenies. All the calculations were carried out usingenAlEx package (Peakall and Smouse, 2006).
. Results
.1. Morfo-agronomical characterization
.1.1. Qualitative morphological traits and their relationshipsThe 23 qualitative traits analyzed in the open pollinated proge-
ies of wild olive ‘Alga05′ and cultivar ‘Picual’ showed a wide rangef morphological diversity with a number of observed states perrait ranging from 1 (monomorphic trait) to 5 (Tables 1 and 2). Threeraits, leaf blade length (LL), leaf blade width (LW) and fruit posi-ion of maximum diameter (FMD) did not show any variability inhe wild olive progeny.
No differentiation of leaf traits was observed between the tworogenies, which were mostly elliptic-lanceolate in shape (LS),edium in length and narrow in width (data not shown). In
ontrast to their respective progenies, both genitors were lance-late in shape, and the cultivar ‘Picual’ have leaves of mediumength.
Regarding the fruit traits (Table 1) the two progenies could note differentiated for the position of maximum diameter (FMD), forpex (FAS) and base shape (FBS). And no fruits with FMD towardpex were found in the two progenies. In general, fruits of ‘Alga05′
.p. progeny were larger in shape (FSH), with low weight (FW),lightly asymmetrical (FS), low to medium fruit/stone ratio in freshasis (FFSR) and low olive oil content both in wet (OWM) and dryatter (ODM). While ‘Picual’ o.p. seedlings fruits were oval in shape
FSH), medium weight (FW), symmetrical (FS), had low fruit/stoneatio in fresh basis (FFSR), medium olive oil wet content (OWM)nd high dry matter content (ODM). It is worth mentioning thatruit weight (FW), fruit/stone ratio in fresh basis (FFSR), and oliveil content on wet (OWM) as well as on dry matter (ODM) were theost discriminated traits between the two progenies. And almost
o overlapping states ranges were observed between the two prog-nies for these high discriminating traits. When compared to theirespective genitors, ‘Alga05′ o.p. progeny showed lower FFSR, OWMnd ODM values, while ‘Picual’ o.p. progeny displayed more sym-etrical fruits and lower value of FFSR.In contrast to the fruit and leaf description, no monomorphic
raits were found in stones of either progeny (Table 2). However,ve stone traits such as symmetry at both A (SSA) and B (SSB) pos-
tions, the position of maximum diameter at B position (SMD) andhe apex (SASH) and base (SBSH) shape at A position, were notfficient for discrimination between the progenies. The maximumumber of stone grooves (SNG), the weight of the stones (SW) andheir surface (SSU) were the traits which best discriminated withinnd between the two progenies. The ‘Alga05′ o.p. progeny stones
ere low weighted (SW), smooth surfaced (SSU) and with lowumber (SNG) and irregular distribution (SDG) of stone grooves. Inifference, stones of ‘Picual’ o.p. progeny were medium weightedSW), with knotty surface (SSU) and with medium number of
turae 152 (2013) 92–101 95
grooves (SNG) regularly distributed (SDG). The stones of ‘Alga05′
were longer (SS) than its descendants, with pointed apex (SASH),truncate base (SBSH), regularly distributed grooves (SDG) and with-out mucron (SM). While, in comparison to most of its seedlings,‘Picual’ had stones of higher weight (SW), with pointed apex (SASH)and medium numbers of grooves (SNG).
The first two axes of the correspondence analysis of qualitativemorphologic traits for ‘Alga05′ o.p. progeny accounted for 34.9% ofthe total variance (Fig. 1a). The first dimension explained 19.47% oftotal variation and was positively associated with fruit weight (FW),number of stone grooves (SNG) and stone surface (SSU), while neg-atively with fruit (FS) and stone (SSA) symmetry in A position andstone distribution of grooves (SDG). The second axis, that explained15.43% of variation, was positively associated with fruit/stone ratioin fresh basis (FFSR) and olive oil on dry matter (ODM) while neg-atively with stone weight (SW) and stone mucron (SM).
A different plot distribution was observed in the ‘Picual’ o.p.progeny, although the percentage of variance accumulated in thefirst two axes (40.47%) was similar to ‘Alga05′ o.p. progeny (Fig. 1b).The first axis explained 22.14% of total variance and was associatedwith shape trait states of leaf (LS), fruit (FSH), stone (SS) and stonebase (SBSH). On the other hand, the second axis assumed 18.33%of total variance and was associated with fruit (FS) and stone (SSA)symmetries in A position, stone symmetry in B position (SSB), stoneapex shape (SASH), stone mucron (SM) and fruit/stone ratio in freshbasis (FFSR).
Factorial correspondence analysis of the individuals of thetwo open pollinated progenies under study resulted in a clearseparation between the progenies (Fig. 2). The first two axesprovide 60.75% and 7.42% of total variance, respectively, showinga high degree of diversity within progenies (especially for the o.p.wild olive progeny). Moreover, multivariate analysis of variance(MANOVA) for qualitative traits has shown that 77% of total vari-ance is due to the differences within each progeny while 23% wasdue to differences between the two progenies. Differences betweenprogenies were significant (PhiPT = 0.231, P < 0.01).
3.1.2. Quantitative and agronomic traits and their relationshipsSignificant differences between the open pollinated progenies of
‘Alga05′ and ‘Picual’ were observed for the four quantitative mor-phologic measurements (perimeter, maximum diameter, volumeand shape) in fruits and stones. In fact, genitor effect representedmore than half of total sums of squares (from 52% to 77%) for allquantitative fruit traits and most stone ones. However, no dif-ferences were found among the two genitors for the leaf traitsmeasured. The most pronounced differences between ‘Alga05′ and‘Picual’ o.p. progenies were found for fruit volume (mean value444 vs. 3510 mm3 respectively). A wider range and coefficient ofvariation was observed for all characters in ‘Picual’ o.p. progeny,with the exception of the fruit shape trait (Fig. 3). Mean values inboth progenies were similar to the ones found for their respectivegenitors.
Respect to the agronomic evaluation, seedlings from ‘Alga05′
o.p. progeny showed a significantly higher trunk diameter through-out the period under study in both juvenile and adult plants (Fig. 4).This progeny also showed a higher number of genotypes with shortjuvenile period (flowering in the third year after planting or earlier),with 45.2 and 23.9% of adult seedlings for ‘Alga05′ and ‘Picual’ prog-enies, respectively. Flowering plants from ‘Alga05′ o.p. progeny alsopresented higher intensity of bearing (on a 0–4 scale) three yearsafter planting: 2.18 vs. 1.79. Significant differences between the o.p.progenies of ‘Alga05′ and ‘Picual’ were also observed for the agro-
nomic fruit traits evaluated. Genitor effect represented 61–78% oftotal sums of squares for olive oil content on dry matter (ODM),fruit/stone ratio on dry basis (DFSR) and fruit removal force/fruitweight (FRF/FW) and 20% for fruit removal force (FRF). In general,
96 T. Klepo et al. / Scientia Horticulturae 152 (2013) 92–101
Fig. 1. Correspondence analysis of 23 qualitative morphologic traits evaluated in ‘Alga05′ (a) and ‘Picual’ (b) progenies. Each group of traits is marked by a different symbol(leaf traits with a triangle, fruit traits with a square, stone traits with a circle and agronomic traits with a minus symbol). LL – leaf blade length, LW – leaf blade width, LS –leaf shape, FW – fruit weight, FSH – fruit shape, FS – fruit symmetry (position A), FAS – fruit apex shape, FBS – fruit base shape, FMD – fruit position of maximum diameter,S e symp ution
O
‘iduF
aFwpt3rsb
W – stone weight, SS – stone shape, SSA – stone symmetry (position A), SSB – stonosition of maximum diameter, SNG – stone number of grooves, SDG – stone distribDM – olive oil on dry matter, FFSR – flesh/stone ratio in fresh basis.
Picual’ o.p. progeny showed wider ranges of variation in compar-son to the wild o.p. progeny. In this sense, ‘Picual’ o.p. progenyisplayed higher mean values for ODM and DFSR, similar FRF val-es, as well as much lower mean values and variation ranges forRF/FW (Fig. 5).
PCA of the 23 quantitative traits analyzed was computed sep-rately for each open pollinated progeny (Fig. 6a,b). Similarly toCA of qualitative traits, the percentage of total variance explainedith the first two principal components (PC) was similar for bothrogenies, but there were differences in the relationships amonghe traits. For ‘Alga05′ o.p. progeny (Fig. 6a), the first PC explained
8.09% of total variance, and was mainly determined by 12 traitselated to fruit and stone dimension that clustered together. Theecond PC explained 15.97% of total variance, and was influencedy the leaf (LSHQ), fruit (FSHQ) and stone shape (SSHQ), leaf
metry (position B), SASH – stone apex shape, SBSH – stone base shape, SMD – stoneof grooves, SM – stone mucron, SSU – stone surface, OWM – olive oil on wet matter,
perimeter (LPE), leaf maximum diameter (LMXD) and trunk diam-eter in the year 2010/2011 (DT10/11). Similarly, in ‘Picual’ o.p.progeny the first PC explained 39.80% of variance, and was mainlyassociated with agronomical and morphological fruit and stonetraits including oil content (Fig. 6b). The second PC was positivelyassociated with leaf minimum diameter (LMID), area (LAR) andtrunk diameter in the year 2010/2011 (DT10/11) and negativelywith fruit moisture (HUM). Finally, a global PCA including data ofboth progenies allowed a clear separation according to the genitor(data not shown).
3.2. Molecular analysis
A total of 186 genotypes (90 of ‘Alga05′ progeny, 94 of ‘Picual’progeny and two genitors ‘Alga05′ and ‘Picual’) were analyzed with
T. Klepo et al. / Scientia Horticulturae 152 (2013) 92–101 97
Fig. 2. Individual factorial correspondence analysis (FCA) of the wild (blue dia-monds) and cultivated (red circles) open pollination progenies. The two genitors,‘Alga05′ and ‘Picual’, are indicated with a yellow and green triangle, respectively.(r
sfiovatotnwo
Fig. 4. Mean trunk diameter of open pollinated seedlings of ‘Alga05′ and ‘Picual’grouped by their short (first flowering ≤3 years after field planting) or long juvenileperiod (first flowering >3 years after field planting). Error bars represent standarderrors. *Ws – wild olive seedlings with a short juvenile period, Wl – wild oliveseedlings with a long juvenile period, Ps – ‘Picual’ seedlings with a short juvenile
For interpretation of the references to color in this figure legend, the reader iseferred to the web version of the article.)
ix microsatellites (Table 3). All of them were polymorphic and con-rmed the maternal genitor of their respective offsprings. A totalf 117 alleles (Na) were found. The number of alleles per locusaried from 4 (ssrOeUA-DCA03) to 19 (ssrOeUA-DCA16) and theverage number of alleles per locus was 9.75. For each progeny,he average numbers of alleles per locus was 11.50 for ‘Alga05′
.p. progeny and 8.00 for ‘Picual’ o.p. The average number of effec-ive alleles (Ne) for all loci and genotypes was 3.68, though this
umber for ‘Alga05′ o.p. progeny was lower (3.50) in comparisonith ‘Picual’ o.p. progeny (3.87). Slightly higher average values for
bserved (HO = 0.77) than for expected (HE = 0.70) heterozygosity
Fig. 3. Range and coefficient of variation of four quantitative morphological fruit
period, Pl – ‘Picual’ seedlings with a long juvenile period.
were obtained. Shannon information index (I) and inbreeding coef-ficient (FIS) were similar for ‘Alga05′ o.p. (1.52; −0.10) and ‘Picual’o.p. (1.53; −0.11) progenies, respectively.
The analysis of molecular variance (AMOVA) confirmed a widerange of genetic variability and significant differences among openpollinated progenies (PhiPT = 0.284; P < 0.001). Moreover, geneticvariability within progenies accounts for 72% of a total variance,while the rest (28%) is due to differences among progenies.
Finally, the factorial correspondence analysis (FCA) resulted in
a clear separation of two progenies according to genitor, ‘Alga05′
on one side and ‘Picual’ on another (data not shown). A total of
traits evaluated in the open pollinations progenies of ‘Alga05′ and ‘Picual’.
98 T. Klepo et al. / Scientia Horticulturae 152 (2013) 92–101
traits
5a
4
ramre
TPe
Fig. 5. Range and coefficient of variation of four agronomic fruit
6.93% variability was explained by the first two coordinates, whichccount for 41.37% and 15.56%, respectively.
. Discussion
The use of morphological, agronomical and SSRs markersevealed a high diversity within and between the two wild (Alga05)
nd cultivated (Picual) o.p. progenies under study. Qualitativeorphological characterization of these progenies coincides with
esults reported for genuine wild and feral olive populations (Belajt al., 2011; García-Donas Díaz, 2001; Hannachi et al., 2008;
able 3rogeny, repeat motif, the number of genotypes (N), the total number of alleles (Na), the
xpected heterozygosity (HE), inbreeding coefficient (FIS), for six microsatellite loci analy
Progeny Locus Repeat motif N
‘Alga05′ × o.p. Meana 89.50
ssrOeUA–DCA03 (GA)19 90
ssrOeUA–DCA09 (GA)23 89
ssrOeUA–DCA11 (GA)26(GGGA)4 91
UDO99–043 (GT)12 82
ssrOeUA–DCA18 (CA)4CT(CA)3(GA)19 87
ssrOeUA–DCA16 (GT)13(GA)29 87
Mean 87.67
‘Picual’ × o.p. ssrOeUA–DCA03 (GA)19 91
ssrOeUA–DCA09 (GA)23 93
ssrOeUA–DCA11 (GA)26(GGGA)4 92
UDO99–043 (GT)12 85
ssrOeUA–DCA18 (CA)4CT(CA)3(GA)19 95
ssrOeUA–DCA16 (GT)13(GA)29 92
Mean 91.33
a Mean value for all microsatellites loci analyzed in two progenies.
measured in open pollinations progenies of ‘Alga05′ and ‘Picual’.
Mulas, 1999; Sedgley, 2000), seedling evaluation from breedingprograms (León et al., 2004a,b) as well as studies on olive culti-vars from different collections (Barranco et al., 2005; Cavagnaroet al., 2001; Del Río et al., 2005b). The low levels of variabil-ity found in leaf traits within (specially the wild offspring) andbetween the two o.p. progenies indicate their low efficiency toevaluate genetic variability in olive progenies. Low discrimination
capacity of leaf traits has also been observed in wild olive popula-tions (Belaj et al., 2011), related subspecies (García-Verdugo et al.,2009, 2010) as well as in cultivated germplasm (Barranco et al.,2005).
number of effective alleles (Ne), Shannon information index (I), observed (HO) andzed in open pollinated progenies of ‘Alga05′ and ‘Picual’.
T. Klepo et al. / Scientia Horticulturae 152 (2013) 92–101 99
DT10/11
FWQFDWQ
HUM OWMQODMQ
FPE
FMXD
FMID
FVO
FAR
FSHQ
SPE
SMXD
SMID
SVOSAR
SSHQ
LPE
LMXD
LAR
LMID
LSHQ
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-0.4 -0.2 0 0.2 0.4 0.6 0.8 1
PC1 (38.09%)
(a)
(b)
PC2
(15.
97%
)
DT10 /11
FWQ
FDWQ
HUM
OWMQ
ODMQ
FPE
FMXD
FMID
FVO
FARFSHQ
SPESMXDSMIDSVO
SAR
SSHQ
LPELMXD
LAR
LMID
LSHQ
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2
PC1(39.80%)
PC2(
18.9
9%)
Fig. 6. Principal component analysis of 23 quantitative traits evaluated in ‘Alga05′ (a) and ‘Picual’ (b) progeny. Each group of traits is marked by a different symbol (leaf traitswith a triangle, fruit traits with a square, stone traits with a circle, and agronomic traits with a minus). LPE – leaf perimeter, LMXD – leaf maximum diameter, LAR – leafarea, LMID – leaf minimum diameter, LSHQ – leaf shape, FWQ – fruit weight in fresh basis, FDWQ – fruit weight in dry basis, FPE – fruit perimeter, FMXD – fruit maximumdiameter, FMID – fruit minimum diameter, FVO – fruit volume, FAR – fruit area, FSHQ – fruit shape, SPE – stone perimeter, SMXD – stone maximum diameter, SMID – stonem UM –D
mnFnot2bTi
inimum diameter, SVO – stone volume, SAR – stone area, SSHQ – stone shape, HT10/11 – trunk diameter in year 2010/2011.
On the contrary, high effectiveness of fruit and stone traits fororphological characterization and discrimination of o.p. proge-
ies was observed, in agreement with the above mentioned studies.ruit and stone variability found in ‘Alga05′ and ‘Picual’ o.p. proge-ies were comparable to the ones found in wild olive populationsf Andalusia, Southern Spain (Belaj et al., 2011), seedlings from cul-ivated progenies (León et al., 2004a,b) and cultivars (Del Río et al.,
005b). Additionally, for some leaf, fruit and stone traits differencesetween the genitors and their respective seedlings were found.his may be due to complex inheritance of morphological traits orn the open pollination origin of the two progenies evaluated.
fruit moisture, OWMQ – olive oil on wet matter, ODMQ – olive oil on dry matter,
Short juvenile period is one of the main objectives in olivebreeding programmes (Baldoni and Belaj, 2009; De la Rosa et al.,2006; Lavee et al., 1996; P. Rallo et al., 2008), but no previousreport on the length of the juvenile period of progenies from wildorigin has been reported. In this work, a higher percentage ofseedlings with short juvenile period were found in the wild thanin ‘Picual’ progeny. This could be, at least in part, related to the
higher stem diameter observed in seedlings coming from wild o.p.,as a strong negative relationship between stem diameter and thelength of the juvenile period has been previously reported (De laRosa et al., 2006; Santos-Antunes et al., 2005). The transmission of
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hort juvenile period is an interesting trait for breeding for botheducing the evaluation period of seedlings and the unproduc-ive period of the subsequent possible new cultivars (León et al.,007).
Fruit, stone and agronomic traits were strongly influenced by thewo genitors as previously reported for breeding progenies (Laveend Avidan, 2011; León et al., 2004a). As expected, values for allhe fruit traits measured were lower for the wild progeny than forPicual’ progeny, particularly for interesting agronomic traits suchs fruit weight and oil content. This seems to indicate that the usef wild relatives for breeding will be associated to a long lastingrocess requiring two, or even three generations, as reported inther fruit species (Schuster et al., 2011). This study was carriedut on a first generation progenies from wild olive and, therefore,gronomic characters of the selected genotypes could be improvedfter several cycles of breeding.
PCA showed a strong association among fruit and stone dimen-ions, which agrees with previous studies in wild (Belaj et al.,011; Hannachi et al., 2008) and cultivated trees (Cantini et al.,999). In the case of ‘Picual’ progeny, fruit traits appear toe also associated to oil content. However, contrary to previ-us in situ evaluations of wild olive populations (Belaj et al.,011), no association between oil content and fruit traits wasound for the wild o.p. progeny. It would be interesting tourther explore the relations between fruit size and oil con-ent and their interaction with the cultivated/wild nature ofhe genitors, as this seems to be the main difference amonghem.
The six microsatellites markers revealed high levels of polymor-hism and genetic diversity values, in agreement with qualitativend quantitative morphological description and as expected inffsprings coming from open pollination (Lavee and Avidan, 2011).imilar levels of polymorphism were documented in previous stud-es carried out on wild (Belaj et al., 2010, 2011; Munoz-Díez et al.,011) and cultivated olives (Corrado et al., 2009). At the same time,iversity parameters were higher than those reported by Belaj et al.2011) and Corrado et al. (2009) in wild, and cultivated olive respec-ively, but lower than the ones observed by Belaj et al. (2010), Erret al. (2010) and Munoz-Díez et al. (2011) in both wild and culti-ated materials. The differences in the number of genotypes andicrosatellites used in these studies may be the cause of these
iscrepancies. In addition, HO was superior to HE, in both prog-nies, indicating an excess of heterozygosity and thus a possibleeterozygote preference during the selection processes, as previ-usly reported for cultivated (Erre et al., 2010) and wild materialLumaret et al., 2004).
The analysis of molecular variance, in agreement with the qual-tative and quantitative data analyses, located most of the geneticariability between genotypes within progenies. The high geneticistance among the two female genitors may explain the high andignificant molecular differentiation found between these proge-ies also observed in their respective PCA and FCA. Furthermore, asreviously shown in wild olive populations (Belaj et al., 2011), thesefulness and complementariness of morphological, agronomicalnd molecular tools for a more complete knowledge of the diver-ity available in wild and cultivated o.p. progenies have also beenonfirmed.
In conclusion, this first report on the segregation of a wild olive.p. progeny has shown high variability at morphologic, agronomicnd molecular levels. This variability was found to be significantlyifferent from the one observed in a ‘Picual’ o.p. progeny. Wild oliveermplasm could represent a future source of genes for interesting
haracters such as short juvenile period, although several breedingycles could be necessary to obtain new cultivars with improvedgronomic performance. These results underline the need for con-ervation and evaluation of these genetic resources.
turae 152 (2013) 92–101
Acknowledgements
The present study was partly financed by the INIA projectsRF2006-00017-C02 and RF2009-00005-00-00. National Institute ofAgricultural Research (INIA), Ministry of Education and Culture,Spain, partially funded by European Regional Development Fund(ERDF). We are in debt with Gema Gonzalez Becerra for her helpin laboratory and field work. A. Belaj has got a post doctoral INIAcontract (Subprograma DOC-INIA) National Institute of AgriculturalResearch (INIA), Ministry of Education and Culture, Spain. T. Klepois grateful to the International Olive Oil Council for the MSc grant.
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