International Journal of Agronomy and Agricultural Research (IJAAR) ISSN: 2223-7054 (Print) 2225-3610 (Online) http://www.innspub.net Vol. 5, No. 2, p. 87-93, 2014 OPEN ACCESS RESEARCH PAPER Morphological and molecular characterization of the main olive varieties cultivated in the region of Hbebsa (North West of Tunisia) Mnasri Rahmani Sameh1*, Saddoud Debbabi Olfa1, Ben Saleh3 and Ferchichi Ali2 1 National Gene Bank of Tunisia, Street Yesser Arafet, Tunis, Tunisia 2 National Institute of Agronomy of Tunisia,Charles Nicolle Tunis, Mahrajène Tunisia 3 Institute of Arid Regions of Gabes, Nahal Gabes Tunisia Article published on August 12, 2014 Key words: Morphology, SSR markers, olive varieties, Hbebsa. Abstract A group of seven olive varieties, commonly cultivated in the region of ‘Hbebsa’ localized in the North West of Tunisia were described using both morphological and molecular approaches. The morphological characters of each cultivar were collected according to the International olive council descriptor (1997). Biometric indexes of leaf, fruit, and endocarp were compared to the molecular data obtained on the same set of cultivars using ten SSR markers. We have noted a significant fluctuation of the flush percentage (70.68 to 84.82%), the fruit weight (from 0.58 to 4.48 g) and the endocarp weight (from 0.17 to 0.68g). The morphological study permitted a specific description of the characteristics for the tested varieties and their repartition into three groups according to their fruit and endocarp weight. Whereas, the molecular analyses based on SSR markers didn’t present any clear segregation of the seven olive varieties relative to their fruit weight and their end-use. These result proved the insufficiency of the morphological parameters to discriminate the olive varieties and the importance of the SSR markers for studying variation between olive cultivars and for future breeding and olive germplasm management efforts. * Corresponding Author: Mnasri Rahmani Sameh  mnasrisameh@yahoo.fr Introduction Sameh et al. Page 87 Tunisia is the fourth largest producer of olive oil Therefore, the objective of this analysis was to study country in the world and oil exports represent 40 % of for the first time in Tunisia the morphological and the overall value of agronomic exports and 5.5 % of molecular aggregate exports, making it the fifth largest source of (Toufehhi, Besbessi, Meski, El Hor and Neb Jmel) foreign currency earnings for the country (IOC, 1997). and two major cultivars (Chetoui and Chemlali) The distribution of Olea varieties in Tunisia gave rise cultivated in the region of Hbebsa. The molecular to a very complex and highly articulated structure of analysis was based on microsatellite markers which olive culture which was marked by the existence of a are considerable number of different olive cultivars. The heterozygosity because of their codominant character main variety cultivated is ‘Chemlali’ in the south and (Carriero et al., 2002). The major goal is to the centre of the country and “Chetoui” in the north. differentiate a number of Tunisian minor olive These two varieties account for 95 % of the total olive cultivars and to explore the genetic relationships tree orchards and contribute more than 90 % of the among these genotypes, specially the autochthones national production of olive oil (Trigui and Msallem varieties “Neb Jmel”, “Besbssi”, “Toufehhi” and “El 2002). Conversely, several minor varieties are Hor” which are characterized by a small geographic maintained in restricted areas. The number is dispersion in the North West of Tunisia. more parameters effective of than five minor others in cultivars estimating probably underestimated because of the scarce information on minor local varieties widespread in Materials and methods the different Tunisian olive growing areas. Thus, Plant Material there is an urgent need to study and to inventory The study was carried out during the growing season these traditional varieties before their lost (Abaza et 2012-2013 localized in the region of Hbebsa (North al., 2005; Baccouri et al., 2007). West of Tunisia). Morphological and genotype description of the seven cultivated olive cultivars The region of Hbebsa localized in the North West of (Meski, Neb Jmel, El Hor, Chetoui, Chemlai, Touffehi Tunisia is a rural area, which provides optimal and Tounsi) was carried out on three olive cultivars to growing conditions for most tree fruit crops, specially a total of 21trees. The olive grove under study is not the olive trees. Our previous research on the irrigated, pruned each 2 years and subject to the morphological variability of the olive patrimony in traditionally cultural practices in the area. This olive this region (Mnasri et al., 2013 a) was proved an grove was selected due to the regularity of the important phenotypic variability of the analyzed olive productions of the last years and because all the cultivars for all the studied traits, especially for the accessions fruit and endocarp parameters. However, these homogeneity of the pedologic and climate conditions. are presented, guaranteeing the morphological markers have the disadvantage of the small number of polymorphism detected and of being Morphological characterization environmentally dependent ( Kamoun 1999 ; Trigui The morphological analysis was carried out by using and Msallem, 2002) to overcome these problems the methodology for primary characterization of olive several Tunisian research teams have used PCR-based varieties, proposed by the International Olive oil markers for basic and applied research to assess the Council (IOC, 1997). This investigation include the genetic diversity of Tunisian olive cultivars. These analysis of 29 distinct characters: four related to the markers types include RAPD (Zitoun et al., 2008), leaf (length “V1”, width “V2”, shape “V3” and AFLP (Kamoun et al., 2006; Taamalli et al., 2006), Longitudinal curvature of the blade “V12” ) , 12 SSR (Taamalli et al., 2010; Rekik et al., 2008) and related with the fruit (length “V4”, maximum SNP (Rekik et al., 2010). diameter “V5”, shape “V6”, weight “V7”, symmetry in position (A) “V13”, position of maximum transversal diameter “V14”, apex “V15”, base “V16”, nipple Sameh et al. Page 88 presence “V17”, presence of small lens “V18”, SSR data were analyzed using several genetic dimension of small lens “V19” and the localization of parameters such as: number of alleles per locus; initial turning “V20” ), and 13 related to the endocarp observed heterozygosity (Ho, calculated as the (length “V8”, maximum diameter “V9”, shape “V10”, number of heterozygotes per locus divided by the weight “V11”, symmetry in position (A) “V21”, number symmetry in position (B) “V22”, position of maximum heterozygosity (He) or gene diversity (Nei, 1987), and transversal diameter “V23”, apex “V24”, base “V25”, the surface “V26”, number of grooves “V27”, distribution calculated for each locus (Botstein et al., 1980). Pair of grooves “V28” and the mucro presence “V29”). wise genetic similarities were calculated using Dice of individuals polymorphism typed); information expected content (PIC) similarity coefficient (Dice, 1945; Neil and Li, 1979). A Molecular Characterization dendrogram was constructed from the resultant DNA extraction matrix via the unweighted pair group method with Total genomic DNA was extracted from young leaf the tissue following the method described by (Angiolillo methods. All calculations were performed with the et al., 1999) using a CTAB buffer with a concentration use of NTSYS-pc version 2.1 (Rohlf, 1998). arithmetic averages algorithm (UPGMA) measured on agarose gel by lambda ladder. Results and discussion SSR markers Morphological polymorphism Ten microsatellite (SSR) markers were used in this The morphological characteristic of the seven olive study. Four markers (GAPU59, GAPU71A, GAPU71B, cultivars, including mean value, variability range, GAPU103A) from the primer set designed by Carriero variation et al. (2002), four markers (UDO03, UDO12, UDO28, difference among accessions are shown on table 1. UDO39) from Cipriani et al. (2002) and two markers The (DCA9, DCA18) from Sefc et al. (2000) were selected variability among the seven cultivars, especially the for their high polymorphism among olive cultivars, fruit parameters V4, V5 and V7, as well those that their easily scored patterns and their small-scale were measured in the endocarp V8, V9 and V11. The stuttering (Table 3). The 20-μl reactions contained 50 fruit weight varied from 0.58 to 4.48 g, the endocarp ng template DNA, 1.5 mM MgCl2, 0.3 mM dNTP, 10 weight ranged from 0.17 to 0.68g and the flush pmol of each primer, and 1.5 U Taq DNA polymerase percentage from 70.68 to 84.82%. The variety (Gibco-BRL) in 1X PCR buffer. The cycling regime ‘’Chemlali’’ which is classified as oil olive cultivar consisted of 94°C for 4 min, followed by 34 rounds of present the lowest fruit and endocarp weight, while 94°C for 30 s; 50-60°C (primer pair dependent; Sefc the table olive variety “Meski” present the highest et al., 2000; Cipriani et al., 2002) for 45 s and 72°C values. for 60 s, with a final step of 72°C for 10 min. description of the morphological characteristics is the coefficient, morphological Previous and traits studies minimum showed explained significant considerable that the usual methodology accepted from a legal point of Data analysis view for patenting and registration of varieties An average value for each trait and accession was (Badanes, 1998), especially the importance of fruit calculated. The value of the quantitative and and endocarp parameters to discriminate between the qualitative morphological traits was standardized and olive varieties (Zaher et al., 2011; Paula et al., 2005, subject to a Principal Component Analysis (PCA). Mnasri et al., 2013 a and Mnasri et al., 2014). Each trait was also subject of one-way analysis of variance (ANOVA) at a significant level of P\0.05. All calculations were done by the using of XLSTAT software (2010). Sameh et al. Page 89 Table 1. Descriptive statistic analysis of the morphorphological parameters. and Trait Minimum Maximum Average V1 47,33 64,76 56,64 V2 9,04 15,05 11,84 V3 4,04 6,79 4,97 V4 13,32 24,11 19,83 V5 8,38 18,97 14,78 V6 1,18 1,58 1,36 V7 0,58 4,48 2,81 V8 10,41 16,63 14,55 V9 5,16 9,37 7,15 V10 1,70 2,49 2,06 V11 0,17 0,68 0,43 CV% 11*** 16,27*** 19,83*** 21,72*** 26,41*** 10,63*** 55,3*** 18,42*** 22,72*** 15,52*** 47,81*** P-value: ** significant (P < 0.05); *** Highly significant (p < 0.01). CV% Variation coefficient expressed in percentage. The principal component analysis performed on the morphological descriptors of the fruit, endocarp, and leaf (ACP) is presented in Fig 1. The eigenvalues of the first, second and third axis of the principal components, accounted the 62.14%, 69.15%, 5.84% of the total variance, respectively. The relative magnitude of the first PC eigenvectors showed that weight, length, and maximum diameter of fruit and endocarp, as well the qualitative parameters of the fruit (symmetry in position A and nipple presence) and the endocarp (number and distribution of grooves, surface in position B and base in position A) were important attributes for the classification of cultivars in cluster. dimension of small lens) , as well with the endocarp (shape, symmetry in position A, symmetry in position B and apex in position A). The leaf traits (Length, shape and the longitudinal curvature of the blade) had relatively high eigenvectors in the third PC. The projection of individuals in the plane generated by the axis 1, 2 and 3 showed the distribution of the seven varieties in three main groups. The cluster 1 grouped the varieties (‘Meski’, ‘Touffehi’ and ‘Besbessi’) characterized by the highest fruit and endocarp weight. These cultivars were classified in the olive categories of high to very high weight fruit and they can be used for canning (Barranco et al., 2000). In turn, the cluster 2 which grouped the cultivars (‘Neb Jmel’ and ‘Chetoui’) is characterized by medium weight fruit and a sharp-pointed apex, as well by elliptic and mean weight endocarp, these cultivars can be used with a double aptitude. The oil varieties (‘Chemlali’ and ‘El Hor’) were grouped in cluster 3 and characterized by low weight fruit with an around apex in position A and an oval and low weight endocarp with rounding apex and base. Molecular polymorphism Microsatellites were successfully amplified in the seven analyzed varieties with the ten primer pairs. A total of 41 alleles were observed across the used 4 El hor 3 PC 2(29,16 %) maximum transversal diameter, apex in position A markers, the number of alleles per locus ranging from 2 5 (GAPU103A) to 3 (DCA09 and DCA18) with a mean Besbessi 1 value of 4.1 alleles per locus (Table 2). Allele sizes Chemlali 0 vary among the ten loci, differences between the Meski Touffehi -1 longest and shortest allele ranged from 121 to 228 bp. Chetoui Neb Jmel -2 The observed heterozygosity ranged between 1.00 at -3 -5 -4 -3 -2 -1 0 1 2 3 4 5 PC 1(62,15 %) locus (GAPU71B, UDO12) and 0.42 at DCA18, with a mean value of 0.75 which proved the important variability of the analyzed cultivars. The mean PIC Fig. 1. Projection of the twenty two accessions in the values were high (0.65) and ranging from 0.78 at plane generated by the first two principal components locus (UDO28) to 0.57 at locus (DCA18). In fact, this based on leaf, fruit and endocarp traits. diversity may be associated with the variation in the loci. An important number of reports have indicated The inertia accounted for the second PC was due to the high variability in the average number of alleles the contribution of the fruit (shape, position of per locus in olive cultivars (Carriero et al., 2002; De Sameh et al. Page 90 La Rosa et al., 2002; Diaz et al., 2006; Sarri et al., Tunisian olive cultivars based on SSR markers (Rekik, 2006 and Abdelhamid et al., 2012). Moreover, these 2008 and Tamalli et al., 2006). findings are in good agreement with those of other authors working on the molecular variability of Table 2. SSR locus, allelic number, Ho, He, PIC and product size range of the 10 SSR loci studied. SSR locus N° alleles GAPU59 GAPU71A GAPU71B GAPU103A UDO03 UDO12 UDO28 UDO39 DCA09 DCA18 Total Mean 4 4 4 5 4 4 5 5 3 3 41 4.1 Observed Heterozygosity 0,71 0,57 1 0,85 0,85 1 0,71 0,71 0,71 0,42 Expected heterozygosity 0,62 0,65 0,69 0,82 0,64 0,69 0,84 0,81 0,69 0,61 0,58 0,61 0,64 0,76 0,6 0,64 0,78 0,75 0,64 0,57 Range size (pb) 208-218 210-228 121-144 136-184 135-202 166-193 143-210 108-220 182-206 174-190 0,75 0,7 0,65 0,75 The diversity of the studied sample was approached PIC 0.4 by calculating a dendrogram of genetic similarity (fig 2) based on Jaccard index (1901) with NTSYS-PC 0.5 (Rohlf, 1998). Two main groups were revealed by cutting the dendrogram at a GS value of 0.7. The first 0.6 cluster grouped the cultivars (Besbessi, El Hor, Chetoui and Chemlali) characterized by oval fruit 0.7 symmetric in position A and presented an around base. The second cluster grouped the cultivars 0.8 (Touffehi, Meski and Neb Jmel) characterized by elliptic fruit and endocarp asymmetric in position A. 0.9 In fact, there is no clear structuration of the seven Neb Jmel Meski Touffehi Chemlali and endocarps and proved our previous analyses of Chetoui according to the qualitative parameters of their fruits El hor use. Nevertheless, the seven cultivars clustered 1 Besbessi varieties relative to their fruit weight and their end- the molecular biodiversity of the autochthon Tunisian Fig. 2. Dendrogram of the seven olive cultivars based on AFLP markers (Mnasri et al., based on SSR data using Jaccard’s GS matrix and the 2013b and Mnasri et al., 2014). Further, Kamoun et UPGMA clustering method. olive cultivars al., (2006), Taamaalli et al., (2006) and Abdelhamid et al., (2012), in their analysis of Tunisian olive Conclusion cultivars by AFLP and SSR obtained a comparable The morphological and molecular analyses of the seven clustering of cultivars based on the qualitative predominant olive varieties in the region of Hbebsa morphological and proved the importance of this germoplasm. The studied endocarps. These similar results emerge from analysis cultivars featured phenotypic variability for all the of different olive cultivars using different approaches analyzed traits, especially for the fruit and endocarp would seem to indicate the efficiently of the parameters. The principal components analysis based on qualitative morphological marker to discriminate morphological markers revealed the distribution of the olive germplasm. seven varieties in three main groups according to their parameters of their fruits Sameh et al. Page 91 fruit and endocarp weight. Whereas, the molecular 2000. World catalogue of olive varieties. Madrid, analyses based on SSR markers didn’t present any clear Spain: International Olive Council Publishing, 17–21. segregation of the seven olive varieties relative to their fruit weight and their end-use. These results proved the Botstein D, White RL, Skolnick M, Davis RW. insufficiency of the morphological markers, especially 1980. Construction of a genetic linkage map in man the quantitative traits to discriminate the olive varieties. using restriction fragment length polymorphisms. For that reason, the use of SSR markers is essential to American Journal in Human Genetics 32, 314-331. verify the denomination of each cultivar and increase the knowledge about the olive germoplasm in the region of Carriero F, Fontanazza G, Cellini F, Giorio G. Hbebsa which is in despite of its arid climate 2002. Identification of simple sequence repeats characterized by an important olive biodiversity. These (SSRs) in olive (Olea europaea L.). Theoretical and biotechnological tools can provide significant insights for Applied Genetics 104, 301–307. research in crop breeding and germplasm conservation. Cipriani G, Marrazzo MT, Marconi R, Cimato References A, Testolin R. 2002. Microsatellite markers isolated Abaza L, Taamalli M, Ben Temime S, Daoud D, in olive (Olea europaea L.) are suitable for individual Gutierrez fingerprinting and reveal F, Zarrouk M. 2005. Natural polymorphism within antioxidant composition as correlated to stability of ancient cultivars. Theoretical and Applied Genetics some Tunisian virgin olive oils. Rivista Italiana delle 104, 223–228. Sostanze Grasse 82, 12-18. De La Rosa R, James C, Tobutt KR. 2002. Abdelhamid S, Kamoun G, Francesco M, Isolation Tiziano C. 2012. Genetic similarity among Tunisian microsatellite in olive (Olea europaea L.) and their cultivated olive estimated through SSR markers. transferability to other genera in the Oleacea. Scientia Agricola 70 (1), 33-38. Molecular Ecology 2, 265-267. Angiolillo A, Mencuccini M, Baldoni L. 1999. Diaz A, De La Rosa R, Martin A, Rallo P. 2006. Olive genetic diversity assessed using amplified Development, characterization and inheritance of fragment length polymorphism. Theoretical and new microsatellites in olive (Olea europaea L.) and Applied Genetics 98, 411–421. evaluation of their usefulness in cultivar identification and characterization of polymorphic and genetic relationship studies. Tree Genetic Baccouri O, Ceerretani L, Bendini A , Caboni Genomes 2, 165-175. MF, Zarrouk M, Pirrone L, Daoud D. 2007. Preliminary chemical characterization of Tunisian Dice LR. 1945. Measures of the amount of ecologic mono-varietal virgin olive oils and comparison with association between species. Ecology 26, 297-302. Sicilian ones. European Journal of Lipid Science and Kamoun GN, Khlif M, Hamdi MT. 1999. Technology 109, 1208-1217. Evolution of oils characteristics with maturity of olives in Sfax: Chemlali variety. Acta Horticulturea Badanes ML, Martinez-Calvo J, Llacer G. 1998. 474, 701–704. Analysis of apricot germplasm from the European ecogeographical group. Euphytica 102, 93-99. Kamoun GN, Lamy FM, Rebaî A, Gargouri A, Panaud O, Saar A. 2006. Genetic diversity of Barranco D, Ciamato A, Fiorino P, Rallo L, Tunisian olive tree (Olea europaea L.) cultivars Touazini A, Ccastaneda C, Serafin F, Trujillo I. assessed by AFLP markers. Genetic Resources and Crop Evolution 53, 265–275. Sameh et al. Page 92 Mnasri Rahmani S, Saddoud Dabbebi O, Sarri V, Baldoni L, Porceddu A, Cultrera NG. Ferchichi A. 2013 a. The study of olive oil quality 2006. Microsatellite markers are powerful tools for and morphological biodiversity of Olea europaea L. in discriminating among olive cultivars and assigning the region of “Hbebsa. Journal of Biodiversity and them to geographically defined populations. Genome. Environmental Sciences 3 (4), 59-66. 49, 1606-1615. Mnasri Rahmani S, Saddoud Dabbebi O, Ferchichi Sefc K M, Lopes M S, Mendoncxa D, Rodrigues A. 2013 b. AFLP marker-based identification and genetic dos Santos M, Laimer da Camara Machado M, relationships of olive cultivars in the region of Hbebsa Da Camara Machado A. 2000. Identification of “North West of Tunisia”. Journal of Biodiversity and microsatellite loci in olive (Olea europea L.) and their Environmental Sciences 3 (8), 36-41. characterization in Italian and Iberian olive trees. Molecular Ecology 9, 1171–1173. Mnasri Rahmani S, Saddoud Dabbebi O, Ben Saleh M, Ferchichi A. 2014. DNA fingerprinting of Taamalli W, Geuna F, Banfi R, BassiD, Daoud millennium olive varieties in Tunisia by AFLP D, Zarrouk M. 2006. Agronomic and molecular markers. Journal of Biodiversity and Environmental analyses for the characterisation of accessions in Sciences 4 (4), 310-317. Tunisian olive germplasm collections. Electronic Journal of Biotechnology 9, 467–481. Nei M, Li WH. 1979. Mathematical model for studying genetic variation in terms of endonucleases. Taamalli Proceedings of the National Academy Sciences of the Carretero AS, Gutierrez, AF. 2010. Determination United States of America 76, 5269–5273. of a polar and minor polar compounds and other A, Goemez CAM, Zarrouk M, chemical parameters for the discrimination of six Nei M. 1987. Estimation of average heterozygosity different varieties of Tunisian extra-virgin olive oil and genetic distance from a small number of cultivated in their traditional growing area. European individuals. Genetics 89,583–590. Food Research and Technology 231, 965-975. Paula BM, Pinheiro PBM, Joaquim CG, Esteves Trigui A, Msallem M. 2002. Olive of Tunisia, Da Silva JCG. 2005. Chemometric classification of Tunisian Catalogue of Olive Varieties. Tunis, Tunisia, olives from three Portuguese cultivars of Olea europaea IRESA Publishing,1-159. L. Analytica Chimica Acta 544, 229–235. Zaher H, Boulouha B, Baaziz M, Sikaoui L, Rekik I, Salimonti A, Innocenzo M, Oliver L, Gaboun F, Sripada MU. 2011. Morphological and Sophie G. 2008. Characterization and Identification genetic diversity in olive (Olea europaea subsp. of Tunisian Olive Tree Varieties by Microsatellite europaea L.) clones and varieties. Plant Omics Markers. Horticulture Sciences 43(5), 1371–1376. Journal 4(7), 370-376. Rekik hakim I, kammoun GN, Makhloufi E, Zitoun B, Bronzini de Caraffa V, Giannettini J, Rebaï A. 2010. Discovery and potential of SNP Breton CMM, Trigui A, Maury J, Gambotti C, markers Marzouk B, Berti L. 2008. Genetic diversity in in characterization of Tunisian olive germplasm. Diversity 2, 17–27. Tunisian olive accessions and their relatedness with other Rohlf, M. 1998. NTSYS-pc. Numerical Mediterranean olive genotypes. Scientia Horticulturae 115, 416–419. Taxonomy and Multivariate Analysis System: Version 2.02. Exeter Software, Setauket, NY, USA. Sameh et al. Page 93