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.