SNP discovery by illumina-based transcriptome sequencing of the olive and the genetic characterization of Turkish olive genotypes revealed by AFLP, SSR and SNP markers

Abstract

Background: The olive tree (Olea europaea L.) is a diploid (2n = 2x = 46) outcrossing species mainly grown in the Mediterranean area, where it is the most important oil-producing crop. Because of its economic, cultural and ecological importance, various DNA markers have been used in the olive to characterize and elucidate homonyms, synonyms and unknown accessions. However, a comprehensive characterization and a full sequence of its transcriptome are unavailable, leading to the importance of an efficient large-scale single nucleotide polymorphism (SNP) discovery in olive. The objectives of this study were (1) to discover olive SNPs using next-generation sequencing and to identify SNP primers for cultivar identification and (2) to characterize 96 olive genotypes originating from different regions of Turkey.

Methodology/principal findings: Next-generation sequencing technology was used with five distinct olive genotypes and generated cDNA, producing 126,542,413 reads using an Illumina Genome Analyzer IIx. Following quality and size trimming, the high-quality reads were assembled into 22,052 contigs with an average length of 1,321 bases and 45 singletons. The SNPs were filtered and 2,987 high-quality putative SNP primers were identified. The assembled sequences and singletons were subjected to BLAST similarity searches and annotated with a Gene Ontology identifier. To identify the 96 olive genotypes, these SNP primers were applied to the genotypes in combination with amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers.

Conclusions/significance: This study marks the highest number of SNP markers discovered to date from olive genotypes using transcriptome sequencing. The developed SNP markers will provide a useful source for molecular genetic studies, such as genetic diversity and characterization, high density quantitative trait locus (QTL) analysis, association mapping and map-based gene cloning in the olive. High levels of genetic variation among Turkish olive genotypes revealed by SNPs, AFLPs and SSRs allowed us to characterize the Turkish olive genotype.

Figure 1. Map of Turkey indicating the location of the olive tree genotypes used in the study.

See Table 1 for the code numbers.

Figure 2. Overview of high-throughput RNA-seq library preparation.

mRNA is isolated from total RNA and fragmented. The mRNA is used to make first and second strands of cDNA and this double stranded cDNA molecules are subsequently synthesized, end-repaired and adenylated. Illumina adaptors are ligated to the processed double-stranded DNA and size selected. Finally, the ligated samples are then enriched by amplification using adapter specific primers and purified for sequencing.

Figure 3. Workflow for De novo transcriptome assembly and analysis.

Figure 4. Length distribution of assembled Olive transcript contigs.

Figure 5. GO Classification.

Olive transcriptome GO terms from level 2 of the biological process (A), molecular function (B) and cellular component (C) categories.

Figure 6. UPGMA dendrogram based on Jaccard’s coefficient illustrating the genetic similarities and distance among olive genotypes.

Figure 7. Principal coordinate analysis based on combined marker data showing distribution of 96 olive genotypes.

Figure 8. Bar plot diagrams for Structure.

Codes are defined in Table 1. Each cultivar is represented by a vertical column, which is partitioned into K colored segments that represent the cultivar’s estimated common fractions in the K clusters.

Figure 9. ΔK values over 10 runs for increasing K-values, from 2 to 9.