Identification and validation of novel EST-SSR markers in olives

Matjaž Hladnik,Jernej Jakše,Dunja Bandelj,Alenka Baruca Arbeiter

doi:10.1590/1678-992x-2016-0111

Abstract

The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.

Highlights

Olive (Olea europaea L.) production is the most important agricultural branch in the Mediterranean basin, and olive oil is the main source of fats in the wealthy Mediterranean diet
We report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project
Sequences containing SSRs longer than 20 nucleotides were first reviewed with the use of Tablet (Milne et al, 2010) in order to exclude all SSR-including sequences that were inappropriate for primer designing according to the following criteria: (a) very short DNA sequence flanking the microsatellite or (b) microsatellite sequence repeat was used by assembler as an overlapping part for the adjacent reads, there is a probability that this contig is a chimeric one

Summary

Introduction

Olive (Olea europaea L.) production is the most important agricultural branch in the Mediterranean basin, and olive oil is the main source of fats in the wealthy Mediterranean diet. Breeders and the olive industry are currently focused on identifying high-performance genotypes, while the increasing need for diversity of varieties that are well-adopted to changes in the environment, cultivation conditions and consumer requirements dictate the development of new olive varieties (Lavee et al, 2014). For these reasons, the use of new technologies, including new molecular markers, are essential for breeding success and validation of authenticity and traceability of primary products entering the agro-food chain (Corrado, 2016; Pasqualone et al, 2016). The validation and putative functional annotation of a new set of EST-SSRs and their applicability in olive diversity study are reported in this paper

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