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

BackgroundThe 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 FindingsNext-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/SignificanceThis 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.

Single nucleotide polymorphism discovery through Illumina-based transcriptome sequencing and mapping in lentil

“…Combining complex trait analysis with transcriptome analysis is an important step in molecular genetic studies (Li et al, 2005). Transcriptome SNPs are associated with genes or functional regions of the genome (Xu et al, 2012;Kaya et al, 2013), which is why a number of researchers have focused on combining high-throughput transcriptome data and quantitative trait loci (QTL) detection to understand biological pathways related to complex traits (Mootha et al, 2003;Kirst et al, 2004;Schadt et al, 2005). This new approach is referred to as 'genetical genomics' or 'integrative genomics' and involves the use of gene and genetic marker expression levels to define genomic regions referred to as expression quantitative trait loci (eQTL) (Mignon et al, 2009).…”

“…In another study, 6 genotypes were examined to discover SSRs in lentil and 1.38 × 10 6 reads were generated (Kaur et al, 2011). In olive, Kaya et al (2013) performed transcriptome sequencing of cDNA from 5 distinct olive genotypes. The resulting 126,542,413 sequencing reads in this study were assembled into 22,052 contigs and identified 2987 SNP markers.…”

“…Similar to our study, transcriptome sequencing using the Illumina platform in olive was successfully performed and the authors obtained high-quality reads for SNP discovery. Different numbers of reads have been obtained through transcriptome sequencing in peanut (Wu et al, 2013), coconut (Fan et al, 2013), black pepper (Joy et al, 2013), sweet potato (Wang et al, 2010), ramie , and olive (Kaya et al, 2013). This finding could be explained by the different expression levels present in the examined tissues collected from different growing stages (Wu et al, 2013).…”

“…There are 2 potential reasons why the SNP primers might have shown monomorphism: either false positive predictions were generated, or the SNPs were not present in the tested parent genotypes (Hubert et al, 2010;Helyar et al, 2012). Different percentages of polymorphisms have been obtained among the SNPs discovered in many studies (Hyten et al, 2010a(Hyten et al, , 2010bShirasawa et al, 2010;Helyar et al, 2012;Kaya et al, 2013;Loridon et al, 2013;Sharpe et al, 2013). Hyten et al (2010a) reported that, among 3072 SNPs used in genotyping, 9% failed to produce a good assay on RIL mapping populations.…”

Association of SSR markers with contents of fatty acids in olive oil and genetic diversity analysis of an olive core collection

“…Olive has been considered to be one of the most diverse tree species with about 1200 olive cultivars grown worldwide (Bartolini et al, 1998). The characterization of olive germplasm has been mainly carried out by using morphological and agronomic characters (Cantini et al, 1999), but most of them used for the characterization of olive genotypes can be affected by environmental factors which could lead to misidentification and incorrect naming of olive genotypes (Ipek et al, 2012;Kaya et al, 2013). Therefore, DNA molecular markers, which only reflect nucleotide diversity in the genome of genotypes, have been utilized to estimate genetic relationships between olive genotypes and cultivars in most of the recent genetic diversity studies (reviewed by Bracci et al, 2011).…”