**2.3 SSR markers**

As discussed before, SSR efficiency compared with other DNA-based markers in olive was demonstrated and nowadays they are preferred by several authors for biodiversity studies, germplasm characterization and varietal fingerprinting. Moreover, the recent introduction of fluorescent labeled primers that avoid radioactive labeled ones for PCR-based markers let the use of automatic sequencers, less time consuming and with minor health implications for operators. Besides, automatic sequencers are more effective, because capillary

Olive (*Olea Europaea* L.): Southern-Italian Biodiversity

when resolved on agarose gel (data not shown).

fraud.

Assessment and Traceability of Processed Products by Means of Molecular Markers 61

(Official Journal of the Italian Republic. 1998). In Apulia region at least 3 different type of Ogliarola, namely barese, salentina, and garganica, derive their qualifications from the three different geographical districts of diffusion. The SSR GAPU 89 confirmed the assumption, due to geographical aspects, of the identity of the generic Ogliarola with Ogliarola salentina, since its intense cultivation with respect to the other two in the production area of the "Collina di Brindisi" PDO oil. Infact, Ogliarola garganica e Ogliarola barese showed different electrophoretic pattern between them and with respect to Ogliarola salentina, which showed monomorphic pattern when compared to Ogliarola profile (Tab. 6). Beside this, the allelic profile of "Ogliarola" was detectable also in the binary blends, providing a qualitative result of a product. Its prevailing in Collina di Brindisi PDO oil in amounts higher than 70% could be used as a positive control, since the absence of the Ogliarola salentina pattern in a commercial Collina di Brindisi PDO oil could be revealed to be a

In Apulia region, Southern Italy, five extra-virgin olive oil bear the PDO mark of quality (Tab. 5). Besides the "Collina di Brindisi", "Terra di Bari" represents one of the most produced PDO oils in Apulia and can be subdivided into three distinct sub-denominations: "Bitonto", "Castel del Monte" and "Murgia dei Trulli e delle Grotte". In particular, the subdenomination "Bitonto" was focused by the DiBCA for traceability purposes by means of SSR (Alba et al., 2008a). The disciplinary of production of "Terra di Bari", sub-denomination "Bitonto" provides that this oil is obtained from the following varieties, present for at least 80%, alone or in combination: Cima di Bitonto, Ogliarola barese or Coratina. These 3 cultivars were screened for allele biodiversity at 4 SSR loci together with other 6 cultivars diffused in Southern Italy and a dendrogram of genetic similarity based on Jaccard index was calculated (Fig. 2). Despite the low number of SSRs tested, all the cultivars were clearly distinguished, revealing a Jaccard value of at least 0.50 when comparing all the cultivars each other, except Cima di Mola e Ogliarola barese (Jaccard = 0.68). More, table 7 shows the allelic profile at 3 SSR loci investigated on the cultivars comprised in the disciplinary of production of "Terra di Bari" and it is evident that all the SSR were singularly able to discriminate cultivars. The automation of the electrophoresis, conducted on a sequencer ABI PRISM 3100 Avant Genetic Analyzer, instead of agarose or PAGE gels, allowed a more precise read of the amplicons, revealing minimal differences between alleles differing by 2 bp. Fig. 3 provides a comparison between SSR amplification resolved on agarose gel and on automatic sequencer. As an example, samples amplified with DCA03 and run on sequencer revealed an allele at 237 bp in Cima di Bitonto and at 239 bp in Coratina, undistinguishable

Once established the efficiency of SSR in olive biodiversity assessment and in olive oil traceability, they have been largely used in the last years supplanting other more laborious and time-consuming molecular marker classes. Pasqualone et al. (2007b) presented a paper at the 3rd Commission Internationale du Genie Rural (CIGR) International Symposium held in Naples (September 24th - 26th) in which a set of 75 olive cultivars native from the Mediterranean basin and coming from different germplasm collections was subjected to SSR analysis with a DCA04 SSR locus, in order to provide a DNA-bank database. The choice of the SSR DCA04 (Sefc et al., 2000) employed in this paper was based on the simple repeat motif [(GA)16]. Genotyping with di-nucleotide SSRs is difficult because separation from neighbourhood alleles requires very precise and reliable protocols for allele separation and identification to avoid allele misidentification (Baldoni et al., 2009). However, despite its dinucleotide nature, the SSR DCA04 revealed to be reliable in our previous SSR screening, in

electrophoresis allows to distinguish alleles with very small differences in molecular weight, and has a higher resolutive power than classical methods. The sensibility of the method allows to evidence also weak signals, such as those that could come from olive oil DNA, known to be partially degraded (Alba et al., 2009b).

Baside AFLP analysis, Montemurro et al. (2005) started to screen 65 SSR on a sub-set ot an entire collection of 112 olive accessions. The goal was to select those SSR most efficiently employable in further studies of biodiversity and traceability in olive. Based on simplicity and reproducibility of electrophoretic patterns, 27 were chosen and analysed further on the entire set of olive accessions. Dendrograms of genetic similairity based on Jaccard index (1901) were calculated by means of NTSYS-PC (Rohlf, 1992) using AFLP and SSR data separately and combined. The analysis revealed the ability of AFLP to distinguish accessions belonging to the same cultivars. Conversly, SSR were not able to distinguish all the accessions, making necessary the combination with AFLP data for a complete distinction of all the accessions.

As described, the first attempts to amplify DNA from oil at the DiBCA by ISSR failed, suggesting a different molecular approach to the problem. The analysis of SSR at the DiBCA started in 2004 by Pasqualone et al.. DNA from leaves, drupes and virgin oil of 10 olive cultivars usually grown in Southern Italy were screened with 7 SSR. Virgin olive oil was obtained mechanically as reported before and the commercial Gene Elute Plant kit (Sigma) was used to extract DNA from all matrices. As expected, all the SSRs employed, resolved on agarose gel and ethidium bromide staining, yielded clear and reproducible electrophoretic patterns for leaves, drupes and virgin oil. Full correspondence of the alleles amplified was verified when comparing the electrophoretic profiles from the three different matrices of each olive cultivar. Three SSRs were used as a minimum number of molecular markers able to distinguish among the 10 olive cultivars.

These encouraging results and the standardization of DNA extraction and SSR amplification conditions led the DiBCA to verify the suitability of SSR for the traceability of a PDO extravirgin olive oil like "Collina di Brindisi" (Pasqualone et al., 2007a). Collina di Brindisi PDO is a typical Apulian olive oil (Tab. 5) prepared from Ogliarola (minimum 70%), and other olive cultivars diffused in the production area alone or together, accounting for a maximum of 30%, with no other indications of their single ratios (Official Journal Euopean. Community, 1996; Official Journal of the Italian Republic. 1998). The research consisted on the screening of different virgin oils, filtered and unfiltered, obtained by different oil mills and derived from the olive cultivars included in the disciplinary protocol. The screening was conducted both on monovarietal and binary blends. DNA was extracted by Gene Elute Plant kit (Sigma) and 7 SSR were employed to amplify DNA from different matrices. The suitability of the SSR technique in olive oil traceability was confirmed by the good amplification levels obtained both from cloudy unfiltered and from clear filtered oils. Sufficient cellular sediments for DNA extraction was obtained from filtered oils by augmenting starting oil volumes, centrifuging at higher speeds and for longer times respect to unfiltered oils, and improving the efficiency of the commercial kit used for DNA extraction by using higher amounts of the extracting reagents. The interesting aspect of this paper was represented not only by the fact that SSR confirmed the composition of a certain olive oil, but GAPU89 discovered a wrong designation attributed by one of the mills that provided the experimental material. In particular, an oil supposed to be composed by Leccino was verified to be composed by Ogliarola. Moreover, the paper tried to shed light on the cultivar "Ogliarola", registered in the national list of O. europaea Italian cultivars

electrophoresis allows to distinguish alleles with very small differences in molecular weight, and has a higher resolutive power than classical methods. The sensibility of the method allows to evidence also weak signals, such as those that could come from olive oil DNA,

Baside AFLP analysis, Montemurro et al. (2005) started to screen 65 SSR on a sub-set ot an entire collection of 112 olive accessions. The goal was to select those SSR most efficiently employable in further studies of biodiversity and traceability in olive. Based on simplicity and reproducibility of electrophoretic patterns, 27 were chosen and analysed further on the entire set of olive accessions. Dendrograms of genetic similairity based on Jaccard index (1901) were calculated by means of NTSYS-PC (Rohlf, 1992) using AFLP and SSR data separately and combined. The analysis revealed the ability of AFLP to distinguish accessions belonging to the same cultivars. Conversly, SSR were not able to distinguish all the accessions, making necessary the combination with AFLP data for a complete distinction

As described, the first attempts to amplify DNA from oil at the DiBCA by ISSR failed, suggesting a different molecular approach to the problem. The analysis of SSR at the DiBCA started in 2004 by Pasqualone et al.. DNA from leaves, drupes and virgin oil of 10 olive cultivars usually grown in Southern Italy were screened with 7 SSR. Virgin olive oil was obtained mechanically as reported before and the commercial Gene Elute Plant kit (Sigma) was used to extract DNA from all matrices. As expected, all the SSRs employed, resolved on agarose gel and ethidium bromide staining, yielded clear and reproducible electrophoretic patterns for leaves, drupes and virgin oil. Full correspondence of the alleles amplified was verified when comparing the electrophoretic profiles from the three different matrices of each olive cultivar. Three SSRs were used as a minimum number of molecular markers able

These encouraging results and the standardization of DNA extraction and SSR amplification conditions led the DiBCA to verify the suitability of SSR for the traceability of a PDO extravirgin olive oil like "Collina di Brindisi" (Pasqualone et al., 2007a). Collina di Brindisi PDO is a typical Apulian olive oil (Tab. 5) prepared from Ogliarola (minimum 70%), and other olive cultivars diffused in the production area alone or together, accounting for a maximum of 30%, with no other indications of their single ratios (Official Journal Euopean. Community, 1996; Official Journal of the Italian Republic. 1998). The research consisted on the screening of different virgin oils, filtered and unfiltered, obtained by different oil mills and derived from the olive cultivars included in the disciplinary protocol. The screening was conducted both on monovarietal and binary blends. DNA was extracted by Gene Elute Plant kit (Sigma) and 7 SSR were employed to amplify DNA from different matrices. The suitability of the SSR technique in olive oil traceability was confirmed by the good amplification levels obtained both from cloudy unfiltered and from clear filtered oils. Sufficient cellular sediments for DNA extraction was obtained from filtered oils by augmenting starting oil volumes, centrifuging at higher speeds and for longer times respect to unfiltered oils, and improving the efficiency of the commercial kit used for DNA extraction by using higher amounts of the extracting reagents. The interesting aspect of this paper was represented not only by the fact that SSR confirmed the composition of a certain olive oil, but GAPU89 discovered a wrong designation attributed by one of the mills that provided the experimental material. In particular, an oil supposed to be composed by Leccino was verified to be composed by Ogliarola. Moreover, the paper tried to shed light on the cultivar "Ogliarola", registered in the national list of O. europaea Italian cultivars

known to be partially degraded (Alba et al., 2009b).

to distinguish among the 10 olive cultivars.

of all the accessions.

(Official Journal of the Italian Republic. 1998). In Apulia region at least 3 different type of Ogliarola, namely barese, salentina, and garganica, derive their qualifications from the three different geographical districts of diffusion. The SSR GAPU 89 confirmed the assumption, due to geographical aspects, of the identity of the generic Ogliarola with Ogliarola salentina, since its intense cultivation with respect to the other two in the production area of the "Collina di Brindisi" PDO oil. Infact, Ogliarola garganica e Ogliarola barese showed different electrophoretic pattern between them and with respect to Ogliarola salentina, which showed monomorphic pattern when compared to Ogliarola profile (Tab. 6). Beside this, the allelic profile of "Ogliarola" was detectable also in the binary blends, providing a qualitative result of a product. Its prevailing in Collina di Brindisi PDO oil in amounts higher than 70% could be used as a positive control, since the absence of the Ogliarola salentina pattern in a commercial Collina di Brindisi PDO oil could be revealed to be a fraud.

In Apulia region, Southern Italy, five extra-virgin olive oil bear the PDO mark of quality (Tab. 5). Besides the "Collina di Brindisi", "Terra di Bari" represents one of the most produced PDO oils in Apulia and can be subdivided into three distinct sub-denominations: "Bitonto", "Castel del Monte" and "Murgia dei Trulli e delle Grotte". In particular, the subdenomination "Bitonto" was focused by the DiBCA for traceability purposes by means of SSR (Alba et al., 2008a). The disciplinary of production of "Terra di Bari", sub-denomination "Bitonto" provides that this oil is obtained from the following varieties, present for at least 80%, alone or in combination: Cima di Bitonto, Ogliarola barese or Coratina. These 3 cultivars were screened for allele biodiversity at 4 SSR loci together with other 6 cultivars diffused in Southern Italy and a dendrogram of genetic similarity based on Jaccard index was calculated (Fig. 2). Despite the low number of SSRs tested, all the cultivars were clearly distinguished, revealing a Jaccard value of at least 0.50 when comparing all the cultivars each other, except Cima di Mola e Ogliarola barese (Jaccard = 0.68). More, table 7 shows the allelic profile at 3 SSR loci investigated on the cultivars comprised in the disciplinary of production of "Terra di Bari" and it is evident that all the SSR were singularly able to discriminate cultivars. The automation of the electrophoresis, conducted on a sequencer ABI PRISM 3100 Avant Genetic Analyzer, instead of agarose or PAGE gels, allowed a more precise read of the amplicons, revealing minimal differences between alleles differing by 2 bp. Fig. 3 provides a comparison between SSR amplification resolved on agarose gel and on automatic sequencer. As an example, samples amplified with DCA03 and run on sequencer revealed an allele at 237 bp in Cima di Bitonto and at 239 bp in Coratina, undistinguishable when resolved on agarose gel (data not shown).

Once established the efficiency of SSR in olive biodiversity assessment and in olive oil traceability, they have been largely used in the last years supplanting other more laborious and time-consuming molecular marker classes. Pasqualone et al. (2007b) presented a paper at the 3rd Commission Internationale du Genie Rural (CIGR) International Symposium held in Naples (September 24th - 26th) in which a set of 75 olive cultivars native from the Mediterranean basin and coming from different germplasm collections was subjected to SSR analysis with a DCA04 SSR locus, in order to provide a DNA-bank database. The choice of the SSR DCA04 (Sefc et al., 2000) employed in this paper was based on the simple repeat motif [(GA)16]. Genotyping with di-nucleotide SSRs is difficult because separation from neighbourhood alleles requires very precise and reliable protocols for allele separation and identification to avoid allele misidentification (Baldoni et al., 2009). However, despite its dinucleotide nature, the SSR DCA04 revealed to be reliable in our previous SSR screening, in

Olive (*Olea Europaea* L.): Southern-Italian Biodiversity

areas of other olive cultivars genetically compatible.

Assessment and Traceability of Processed Products by Means of Molecular Markers 63

Ogliarola Barese 180 – 200 Ogliarola Garganica 180 – 190 Ogliarola Salentina 180 - 210 *Ogliarola* **180 - 210** 

SSR marker Cima di Bitonto Ogliarola barese Coratina DCA03 237 243 243 256 239 243 DCA04 130 132 140 190 130 165 DCA18 177 179 177 177 177 181

line with Baldoni et al. (2009) which reported the SSR DCA04 to have low peak stuttering and strong peak intensity during electrophoresis. The main drawbacks for DCA04 were represented by the n° of loci amplified (2) and by a high frequency of null alleles (> 0.05). DCA4 was used to amplify DNA from both the leaves and the oils of 5 cultivars: Cellina di Nardò, Coratina, Frantoio, Leccino, Ogliarola barese. Materials and methods on how to extract monovarietal virgin olive oil is reported in this and other here revised papers produced by the DiBCA. In this research, in particular, the authors begun to question the possibility that the full DNA match between the leaves and oil can be undermined by the emergence of different alleles of paternal origin. In fact, if leaf DNA is practically identical to oil DNA since oils derived mainly from olive flesh, that is derived from the evolution of the ovary (e.i. from mother plant), on the other side the contribution of the pollen from the pollinator plant resides in the seed whose oily fraction represents only a minor percentage of the final oil, and whose solid phase is almost totally eliminated during oil processing. It is evident that this problem occurs for self-incompatible cultivars. Thus, before treating as a fraud the incoming of different alleles when a DNA from oil is amplified with SSR and compared to DNA from leaves, one should ask the pollinating nature of the cultivar employed to produce oil, the percentage of allogamy, the presence in the same cultivations

Therefore, given the efficiency and speed with which SSR analyses can be conducted on DNA extracted from both leaves and oil, a study of correspondence between genetic profiles obtained from leaves and oil was undertaken at the DiBCA by considering 7 olive cultivars, and 10 SSR (Alba et al., 2009b). The cultivars chosen are all used in different disciplinaries of production of PDO oils. Three kg of drupes from certified clones were collected and milled to obtain monovarietal oils. DNA was extracted as previously reported, yielding as expected a mean concentration of 100 ng/μl for DNA from leaves and 5 ng/μl from DNA from oil. Six out of 10 SSR gave clear and unequivocal electrophoretic patterns, while 4 SSRs failed to amplify on at least 2 of the 7 cultivars tested, being the Coratina the most difficult to amplify. The research revealed identical patterns between leaves and oil DNA for 90% of the experiments, even in cases where SSR revealed a multilocus nature. Fig. 4 shows a comparison between oil and leaves electrophoretic patterns of cultivar Leccino obtained with SSR DCA03 and it is evident how peaks for alleles amplified from oil were different in

Table 7. Allelic profile for 3 SSR markers of the 3 olive cultivars comprised in the disciplinary of production of "Terra di Bari", sub-denomination "Bitonto",

Table 6. Allelic profile for the SSR GAPU89 in the "Ogliarola" cultivars

Allele size (bp)


Table 5. Main Italian PDO and IGP extra-virgin olive oils from different regions and legislative references at Italian and European level. (\*Official Journal of Italian Republic; \*\*Official Journal of European Community) Source: http://www.frantoionline.it/dop-eigp/olio-dop-zone-di-produzione.html

Italian Region O.J.E.C.\*\*

Romagna n° 1263/96

Romagna n° 149/03

legislative reference

O.J.I.R.\*

Brisighella O.J. 11.11.1998, n° 264 Emilia

Colline di Romagna O.J. 11.9.2003, n° 211 Emilia

igp/olio-dop-zone-di-produzione.html

legislative reference

Colline Teatine O.J. 7.10.1998, n° 234 Abruzzo n° 1065/97 Aputino Pescarese O.J. 20.8.1998, n° 193 Abruzzo n° 1263/96

Tramane Abruzzo n° 1491/03 Dauno O.J. 20.8.1998, n° 193 Apulia n°2325/97 Terra d'Otranto O.J. 20.8.1998, n° 193 Apulia n° 1065/97 Terra di Bari O.J. 29.9.1998, n° 227 Apulia n° 2325/97 Collina di Brindisi O.J. 21.12.1998, n° 297 Apulia n° 1263/96 Terre Tarentine Apulia n° 189/04 Bruzio O.J. 28.10.1998, n° 252 Calabria n° 1065/97 Lametia O.J. 11.11.1999, n° 265 Calabria n° 2107/99 Alto Crotonese O.J. 21.8.2003, n° 193 Calabria n° 1257/03 Irpinia - Colline dell'Ufita Campania n° 203/2010 Cilento O.J. 20.8.1998, n° 193 Campania n° 1065/97 Penisola Sorrentina O.J. 20.8.1998, n° 193 Campania n° 1065/97 Colline Salernitane G.U 11.9.2003, n° 211 Campania n° 1065/97

Canino O.J. 11.11.1998, n° 264 Lazio n° 1263/96 Sabina O.J. 20.6.1995, n° 142 Lazio n° 1263/96 Riviera Ligure O.J. 20.8.1998, n° 193 Liguria n° 123/97 Molise G.U 21.8.2003, n° 193 Molise n° 1257/03 Cartoceto Marche n° 1897/04 Laghi Lombardi O.J. 7.10.1998, n° 234 Lombardy n° 2325/97 Garda O.J. 7.10.1998, n° 234 Lombardy n° 2325/97 Terra di Siena O.J. 15.1.2001, n° 11 Tuscany n° 3446/00 Chianti Classico O.J. 17.1.2001, n° 13 Tuscany n° 3446/00 Lucca Tuscany n° 1845/04 Toscano Tuscany n° 644/98 Tuscia Tuscany n° 1623/05 Umbria Umbria n° 2325/97 Veneto O.J. 15.11.2001, n° 266 Veneto n° 2036/01 Val di Mazara O.J. 28.3.2001, n° 73 Sicily n° 138/01 Valli Trapanesi O.J. 26.10.1998, n° 250 Sicily n° 2325/97 Monti Iblei O.J. 24.10.1998, n° 249 Sicily n° 2325/97 Valdemone Sicily n° 205/05 Monte Etna O.J. 21.8.2003, n° 193 Sicily n° 1491/03 Sardegna Sardinia n° 148/07 Table 5. Main Italian PDO and IGP extra-virgin olive oils from different regions and legislative references at Italian and European level. (\*Official Journal of Italian Republic; \*\*Official Journal of European Community) Source: http://www.frantoionline.it/dop-e-

Italian PDO and IGP extra-virgin olive oils

Pretuziano delle Colline


Table 6. Allelic profile for the SSR GAPU89 in the "Ogliarola" cultivars


Table 7. Allelic profile for 3 SSR markers of the 3 olive cultivars comprised in the disciplinary of production of "Terra di Bari", sub-denomination "Bitonto",

line with Baldoni et al. (2009) which reported the SSR DCA04 to have low peak stuttering and strong peak intensity during electrophoresis. The main drawbacks for DCA04 were represented by the n° of loci amplified (2) and by a high frequency of null alleles (> 0.05). DCA4 was used to amplify DNA from both the leaves and the oils of 5 cultivars: Cellina di Nardò, Coratina, Frantoio, Leccino, Ogliarola barese. Materials and methods on how to extract monovarietal virgin olive oil is reported in this and other here revised papers produced by the DiBCA. In this research, in particular, the authors begun to question the possibility that the full DNA match between the leaves and oil can be undermined by the emergence of different alleles of paternal origin. In fact, if leaf DNA is practically identical to oil DNA since oils derived mainly from olive flesh, that is derived from the evolution of the ovary (e.i. from mother plant), on the other side the contribution of the pollen from the pollinator plant resides in the seed whose oily fraction represents only a minor percentage of the final oil, and whose solid phase is almost totally eliminated during oil processing. It is evident that this problem occurs for self-incompatible cultivars. Thus, before treating as a fraud the incoming of different alleles when a DNA from oil is amplified with SSR and compared to DNA from leaves, one should ask the pollinating nature of the cultivar employed to produce oil, the percentage of allogamy, the presence in the same cultivations areas of other olive cultivars genetically compatible.

Therefore, given the efficiency and speed with which SSR analyses can be conducted on DNA extracted from both leaves and oil, a study of correspondence between genetic profiles obtained from leaves and oil was undertaken at the DiBCA by considering 7 olive cultivars, and 10 SSR (Alba et al., 2009b). The cultivars chosen are all used in different disciplinaries of production of PDO oils. Three kg of drupes from certified clones were collected and milled to obtain monovarietal oils. DNA was extracted as previously reported, yielding as expected a mean concentration of 100 ng/μl for DNA from leaves and 5 ng/μl from DNA from oil. Six out of 10 SSR gave clear and unequivocal electrophoretic patterns, while 4 SSRs failed to amplify on at least 2 of the 7 cultivars tested, being the Coratina the most difficult to amplify. The research revealed identical patterns between leaves and oil DNA for 90% of the experiments, even in cases where SSR revealed a multilocus nature. Fig. 4 shows a comparison between oil and leaves electrophoretic patterns of cultivar Leccino obtained with SSR DCA03 and it is evident how peaks for alleles amplified from oil were different in

Olive (*Olea Europaea* L.): Southern-Italian Biodiversity

same field.

Assessment and Traceability of Processed Products by Means of Molecular Markers 65

to a low signal of the amplicons, or to troublesome working conditions for the polymerase. On the other hand, a wrong denominations of the cultivars or the occurrence of somaclonal mutations as causes of the gain of additional alleles in oil profiles with respect to leaf DNA could be excluded since the certified origin of cultivars and the belonging of leaves and drupes to the same clone. An accredited hypothesis is represented by a paternal contribution of embryos in oil samples extracted from entire drupes, in line with Doveri et al. (2006) that recorded diverse profiles in oil and plant tissues of the cultivar ''Leccino'', suggesting a nonmaternal origin of additional alleles in oil profiles arisen from out-crossing. A confirmation of the hypothesis of a paternal contribution is offered by the presence of all the scored additional alleles in the electrophoretic profiles of the other cultivars of the research, suggesting, in fact, that out-crosses could be occurred between clones grown in the

Fig. 4. Electropherograms of Leccino cultivar oil (up) and leaves (down) obtained with SSR

Next, the screening of olive cultivar diversity continued when a total of 30 cultivars was analyzed with 16 SSR (Alba et al., 2009a). A molecular identification key with the minimum number of markers revealed that 2 SSR were able to distinguish all the cultivars. In order to identify the SSR that better respond to this purposes we used two measures of the ability of primers to distinguish between genotypes by calculating Resolving Power (RP) (Prevost & Wilkinson, 1999) and Power of Discrimination (Kloosterman et al., 1993). RP consists in the use of a function strongly correlated to the proportion of genotypes identified by a certain molecular marker, independently of the number of genotypes composing the collection. PD is calculated by considering in the formula the genotype frequency instead of the allele frequency. A correlation between the number of distinguished cultivars (NDC) and the values of PD and RP of each SSR was estimated. The comparison between the two possible indices of diversity revealed a higher suitability of PD, with respect to RP, to predict the

DCA03. The reduced height of oil peaks was determined by the lower quality and

concentration of template with respect to leaves.

Fig. 2. Dendrogram of genetic similarity based on Jaccard Index of 3 Italian olive cultivars included in the disciplinary of production of "Terra di Bari", sub-denomination "Bitonto", indicated with \*, compared to other 6 Italian olive cultivars.

Fig. 3. Comparison of SSR electrophoretic pattern on agarose gel 2% (left) and on ABI PRISM 3100 Avant Genetic Analyzer (right). Arrows indicate polymorphisms between olive cultivars. The polymorphism revealed by the sequencer is more evident than that revealed by agarose gel.

shape and height but not in molecular weight with respect to alleles from leaves. The reduced height of oil peaks was determined by the lower quality and concentration of template with respect to leaves. In our previous researches cases of a non-complete concordances between leaves and oil DNA did not emerged, probably due to the low resolution of the methods employed to resolve amplifications (agarose, PAGE). In this research the use of a sequencer let to identify some miss-matches between leaves and oil DNA due to the loss or gain of alleles in the oil DNA. The loss of some peaks in oil profiles was hypothesized to be related to the degraded DNA for the process of olive oil extraction,

**Corati**

**Cima di** 

**Maiati**

**Toscanina** 

**Cima di** 

**Ogliarola barese\*** 

**Cellina di** 

**Simon**

**Nociar**

Fig. 2. Dendrogram of genetic similarity based on Jaccard Index of 3 Italian olive cultivars included in the disciplinary of production of "Terra di Bari", sub-denomination "Bitonto",

Fig. 3. Comparison of SSR electrophoretic pattern on agarose gel 2% (left) and on ABI PRISM 3100 Avant Genetic Analyzer (right). Arrows indicate polymorphisms between olive cultivars. The polymorphism revealed by the sequencer is more evident than that revealed

shape and height but not in molecular weight with respect to alleles from leaves. The reduced height of oil peaks was determined by the lower quality and concentration of template with respect to leaves. In our previous researches cases of a non-complete concordances between leaves and oil DNA did not emerged, probably due to the low resolution of the methods employed to resolve amplifications (agarose, PAGE). In this research the use of a sequencer let to identify some miss-matches between leaves and oil DNA due to the loss or gain of alleles in the oil DNA. The loss of some peaks in oil profiles was hypothesized to be related to the degraded DNA for the process of olive oil extraction,

indicated with \*, compared to other 6 Italian olive cultivars.

**0. 0. 0. 0. 1. Jaccard** 

by agarose gel.

to a low signal of the amplicons, or to troublesome working conditions for the polymerase. On the other hand, a wrong denominations of the cultivars or the occurrence of somaclonal mutations as causes of the gain of additional alleles in oil profiles with respect to leaf DNA could be excluded since the certified origin of cultivars and the belonging of leaves and drupes to the same clone. An accredited hypothesis is represented by a paternal contribution of embryos in oil samples extracted from entire drupes, in line with Doveri et al. (2006) that recorded diverse profiles in oil and plant tissues of the cultivar ''Leccino'', suggesting a nonmaternal origin of additional alleles in oil profiles arisen from out-crossing. A confirmation of the hypothesis of a paternal contribution is offered by the presence of all the scored additional alleles in the electrophoretic profiles of the other cultivars of the research, suggesting, in fact, that out-crosses could be occurred between clones grown in the same field.

Fig. 4. Electropherograms of Leccino cultivar oil (up) and leaves (down) obtained with SSR DCA03. The reduced height of oil peaks was determined by the lower quality and concentration of template with respect to leaves.

Next, the screening of olive cultivar diversity continued when a total of 30 cultivars was analyzed with 16 SSR (Alba et al., 2009a). A molecular identification key with the minimum number of markers revealed that 2 SSR were able to distinguish all the cultivars. In order to identify the SSR that better respond to this purposes we used two measures of the ability of primers to distinguish between genotypes by calculating Resolving Power (RP) (Prevost & Wilkinson, 1999) and Power of Discrimination (Kloosterman et al., 1993). RP consists in the use of a function strongly correlated to the proportion of genotypes identified by a certain molecular marker, independently of the number of genotypes composing the collection. PD is calculated by considering in the formula the genotype frequency instead of the allele frequency. A correlation between the number of distinguished cultivars (NDC) and the values of PD and RP of each SSR was estimated. The comparison between the two possible indices of diversity revealed a higher suitability of PD, with respect to RP, to predict the

Olive (*Olea Europaea* L.): Southern-Italian Biodiversity

molecular marker, only monovarietal oils can be traced.

oil profile rather than to another.

**4. Aknowledgments** 

**5. References** 

Assessment and Traceability of Processed Products by Means of Molecular Markers 67

traditional lab protocols laborious, in some cases hazardous and subjected to many environmental variables, and arriving to the use of health-safe reagents and standardized procedures. In this sense, the efficacy and the advantages showed by SSR with respect to ISSR and AFLP is well known. In particular, in olive oil traceability they revealed their ability to amplify highly degraded DNA extracted from oil. SSR generally amplify fragments of 100-300 bp, while ISSR generate amplicons of higher molecular weight (500- 2000bp) and it can be argued that a degraded DNA from oil works better with short fragments. Furthermore, the step of enzymatic digestion in the AFLP can represent a severe bottleneck when processing degraded oil DNA. For all these reasons, SSR are now widely used for cultivar identification, while more needs to be done for their use in traceability. As an example, most of the PDO oils are multivarietal, so the traceability of a single cultivar becomes hard to be verified. Thus, approaches based on quantitative Real Time PCR can help to correctly define which cultivars are present in a certain oil and in which percentages. Indeed, if the approach is based on qualitative PCR, independently from the class of

Genomes of several crops and woody species have been completely sequenced and characterized, while few is known about the olive genome. However, the increasing availability of Expressed Sequence Tags (EST) deduced from mRNA extracted from olive and annotated on the web (www.Oleadb.it) represent an interesting starting point for "candidate gene" approach, with the goal of a cultivar characterization based on gene functionality. It cannot be ignored that functional genomics offer the possibility not only to distinguish cultivars merely on restricted genomic areas often harbouring intronic regions, but it let the knowledge of how gene biodiversity in olive can lead to a certain organoleptic

Actually, the DiBCA is attempting researches on SSR alleles specific for some Southern Italian olive cultivars, while the approach of the "candidate gene" has been enterprised to isolate and characterize genes involved in the Kennedy pathway that leads to the syntesis or desaturation of long chain fatty acids in olive oil. In the future, the complete genome sequencing of olive will offer new opportunities to better understand the evolution, the existing biodiversity and the physiological aspects of a species that fused its hystory with

This work was conducted within the framework of ''Progetto interregionale OLVIVA'' funded by Programma Interregionale - ''Sviluppo rurale - Innovazione e Ricerca'' D.M. n. 25279 of 23/12/03'' and of "Progetto Strategico PS59" funded by Regione Puglia, Italy .

Alba, V., Montemurro, C. & Blanco A. (2008) Valorizzazione dell'olio "Terra di Bari"

Alba, V., Montemurro, C., Sabetta, W., Pasqualone, A. & Blanco, A. (2009a). SSR-based

mediante l'applicazione dei marcatori molecolari microsatellite. *Proceedings of the VIII National Congress on Biodiversity – La Biodiversità: Risorsa per Sistemi* 

identification key of cultivars of Olea europaea L. diffused in Southern-Italy.

that of mankind all over the Mediterranean basin in the last 5000 years.

*Multifunzionali*, Lecce, Italy, April 21 – 23, pp. 15 – 17.

*Scientia Horticulturae*, No. 123, pp. 11–16.

ability of SSR marker in distinguishing among genotypes, suggesting that indices based on genotypic frequency are more suitable than those based on allelic frequency. A Principal Component Analysis (PCA) was performed for this review by using SSR profiles (Fig. 5) and computing data to construct a three-dimensional array of eigenvectors by the DCENTER module of the NTSYS-PC program (Rohlf, 1992).

The first Component accounted for 9.8% of the total allelic variation, while the second for 9.2% and the third for 6.0%, for a total SSR diversity of 25.0%. This low value suggested that more Components should be considered when representing the PCA and indicated that 16 SSR allowed the screening of a minimal part of the total biodiversity contained by the 30 olive cultivars investigated.

Fig. 5. Principal Component Analysis of 30 Italian olive cultivars analysed with 16 SSRs. Lines represent vectors.
