AJPS  Vol.6 No.19 , December 2015
Exploitation of Concatenated Olive Plastome DNA Markers for Reliable Varietal Identification for On-Farm Genetic Resource Conservation
Abstract: Rapid and reliable identification of olive plants using DNA markers has been attempted in the past but the selection of polymorphic regions for discrimination at varietal level remained obscure. Recent sequencing of plastid genome of the olive flaunts high resolution Cp markers for olive DNA fingerprinting. Using this information, we designed a combination of chloroplast markers to amplify genes recruited in photosynthesis, ribosomal and NADH energy metabolism for varietal identification of olive plants. Concatenated DNA sequences of more than 100 unknown and 10 reference plants samples were analyzed using various bioinformatics and phylogenetic tools. Conserved blocks of nucleotide sequences were detected in multiple alignments. Phylogenetic reconstruction differentiated the unknown plants into various clusters with known varieties. Further narrowing down of the samples through UPGMA tree clearly separated the plants into Arbosana, Frantoio and Koroneiki as the major varieties. Multiple alignments of these clusters revealed important variety specific SNPs including G and T nucleotides at specific positions. Sequence identifying at intra cultivar level was more than 98.79% while it dropped to 97%, and even to 96% at inter varietal level. Furthermore, a neighbor net network analysis separated these three clusters, thus validating the results of UPGMA tree. Over all, out of 100 plants samples, 49 plants were identified that fall into 10 varieties including Arbosana, Carolea, Chetoui, Coratina, Domat, Frantoio, Gemlik, Koroneiki,Leccino and Moraiolo. The maximum number of known plants belongs to Frantoio and Gemlik (8 each). The least number of samples was identified from Carolea, Domat and Moraiolo with 2 samples each. However, 51 plants could not be identified, as plants were not clustered with any of reference control. Our results have implications in on-farm conservation of olive germplasm and provision of genuine material for multiplication of authentic varieties. This strategy can be extended to varietal identification of other plant species.
Cite this paper: Noman, M. , Ajmal, W. , Khan, M. , Shahzad, A. , Ali, G. (2015) Exploitation of Concatenated Olive Plastome DNA Markers for Reliable Varietal Identification for On-Farm Genetic Resource Conservation. American Journal of Plant Sciences, 6, 3045-3074. doi: 10.4236/ajps.2015.619299.

[1]   Angiolillo, A., Mencuccini, M. and Baldoni, L. (1999) Olive Genetic Diversity Assessed Using Amplified Fragment Length Polymorphisms. Theoretical and Applied Genetics, 98, 411-421.

[2]   Doveri, S. and Baldoni, L. (2007) Olive. Fruits and Nuts, Springer, 253-264.

[3]   Golding, B., Reale, S., Doveri, S., Díaz, A., Angiolillo, A., Lucentini, L., Pilla, F., Martín, A., Donini, P. and Lee, D. (2006) SNP-Based Markers for Discriminating Olive (Olea europaea L.) Cultivars. Genome, 49, 1193-1205.

[4]   Green, P. (2002) A Revision of Olea L.(Oleaceae). Kew Bulletin, 91-140.

[5]   Carriero, F., Fontanazza, G., Cellini, F. and Giorio, G. (2002) Identification of Simple Sequence Repeats (SSRs) in Olive (Olea europaea L.). Theoretical and Applied Genetics, 104, 301-307.

[6]   Lavee, S. (1985) Olea europaea. Handbook of Flowering, Vol. 6, CRC Press, Boca Raton, 423-434.

[7]   Rugini, E. and Lavee, S. (1992) Olive. Biotechnology of Perennial Fruit Crops, CAB International, Wallingford, 371-382.

[8]   Colmogro, S., Collins, G. and Sedgley, M. (2010) Processing Technology of the Table Olive. Horticultural Reviews, 25, 235.

[9]   Connels, J.H. (2005) History and Scope of the Olive Industry. Olive Production Manual, Vol. 3353, UCANR Publications, Oakland, 1-10.

[10]   Sanz-Cortés, F., Martinez-Calvo, J., Badenes, M., Bleiholder, H., Hack, H., Llacer, G. and Meier, U. (2002) Phenological Growth Stages of Olive Trees (Olea europaea). Annals of Applied Biology, 140, 151-157.

[11]   Besnard, G., Breton, C., Baradat, P., Khadari, B. and Bervillé, A. (2001) Cultivar Identification in Olive Based on RAPD Markers. Journal of the American Society for Horticultural Science, 126, 668-675.

[12]   Lanza, B., Marsilio, V. and Martinelli, N. (1996) Olive Pollen Ultrastructure: Characterization of Exine Pattern through Image Analysis-Scanning Electron Microscopy (IA-SEM). Scientia Horticulturae, 65, 283-294.

[13]   Barranco, D., Cimato, A., Fiorino, P., Rallo, L., Touzani, A., Castaneda, C., Serafin, F. and Truijillo, I. (2000) World Catalogue of Olive Varieties. International Olive Oil Council, Madrid.

[14]   Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985) Individual-Specific “Fingerprints” of Human DNA. Nature, 316, 76-79.

[15]   Giannoulia, K., Gazis, F., Nikoloudakis, N., Milioni, D. and Haralampidis, K. (2002) Breeding, Molecular Markers and Molecular Biology of the Olive Tree. European Journal of Lipid Science and Technology, 104, 574-586.<574::AID-EJLT574>3.0.CO;2-1

[16]   Belaj, A., Caballero, J.M., Barranco, D., Rallo. L. and Trujillo, I. (2003) Genetic Characterization and Identification of New Accessions from Syria in an Olive Germplasm Bank by Means of RAPD Markers. Euphytica, 134, 261-268.

[17]   Besnard, G., Khadari, B., Villemur, P. and Bervillé, A. (2000) Cytoplasmic Male Sterility in the Olive (Olea europaea L.). Theoretical and Applied Genetics, 100, 1018-1024.

[18]   Besnard, G., Khadari, B., Baradat, P. and Bervillé, A. (2002) Combination of Chloroplast and Mitochondrial DNA Polymorphisms to Study Cytoplasm Genetic Differentiation in the Olive Complex (Olea europaea L.). Theoretical and Applied Genetics, 105, 139-144.

[19]   Dumolin-Lapegue, S., Pemonge, M. and Petit, R. (1997) An Enlarged Set of Consensus Primers for the Study of Organelle DNA in Plants. Molecular Ecology, 6, 393-397.

[20]   Besnard, G., Casas, D., Rubio, R. and Vargas, P. (2003) A Set of Primers for Length and Nucleotide-Substitution Polymorphism in Chloroplastic DNA of Olea europaea L. (Oleaceae). Molecular Ecology Notes, 3, 651-653.

[21]   Mariotti, R., Cultrera, N.G., Díez, C.M., Baldoni, L. and Rubini, A. (2010) Identification of New Polymorphic Regions and Differentiation of Cultivated Olives (Olea europaea L.) through Plastome Sequence Comparison. BMC Plant Biology, 10, 211.

[22]   Olson, S.A. (1994) MacVector: An Integrated Sequence Analysis Program for the Macintosh. Computer Analysis of Sequence Data, Springer, 195-201.

[23]   Doyle, J.J. (1987) A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin, 19, 11-15.

[24]   Hall, T.A. (1999) BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.

[25]   Tamura, K, Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725-2729.

[26]   Huson, D.H. and Bryant, D. (2006) Application of Phylogenetic Networks in Evolutionary Studies. Molecular Biology Evolution, 23, 254-267.

[27]   Tanksley, S.D. and Orton, T.J. (1983) Isozymes in Plant Breeding and Genetics. Elsevier.

[28]   Busconi, M., Foroni, C., Corradi, M., Bongiorni, C., Cattapan, F. and Fogher, C. (2003) DNA Extraction from Olive Oil and Its Use in the Identification of the Production Cultivar. Food Chemistry, 83, 127-134.

[29]   Shaw, J., Lickey, E.B., Schilling, E.E. and Small, R.L. (2007) Comparison of Whole Chloroplast Genome Sequences to Choose Noncoding Regions for Phylogenetic Studies in Angiosperms: The Tortoise and the Hare III. American Journal of Botany, 94, 275-288.

[30]   Besnard, G., de Casas, R.R., Christin, P.A. and Vargas, P. (2009) Phylogenetics of Olea (Oleaceae) Based on Plastid and Nuclear Ribosomal DNA Sequences: Tertiary Climatic Shifts and Lineage Differentiation Times. Annals of Botany, 105.

[31]   Wallander, E. and Albert, V.A. (2000) Phylogeny and Classification of Oleaceae Based on rps16 and trnL-F Sequence Data. American Journal of Botany, 87, 1827-1841.