De novo transcriptome assembly and SSR marker development in apricot (Prunus armeniaca)

Turkish Journal of Agriculture and Forestry http://journals.tubitak.gov.tr/agriculture/ Research Article Turk J Agric For (2017) 41: 305-315 © TÜBİTAK doi:10.3906/tar-1705-105 De novo transcriptome assembly and SSR marker development in apricot (Prunus armeniaca) 1,2 1,2 1,2, Muhammed Ali KÖSE , Necati ÇETİNSAĞ , Kahraman GÜRCAN * Genome and Stem Cell Research Center, Erciyes University, Kayseri, Turkey 2 Department of Agricultural Biotechnology, Erciyes University, Kayseri, Turkey 1 Received: 25.05.2017 Accepted/Published Online: 10.07.2017 Final Version: 25.08.2017 Abstract: Apricot (Prunus armeniaca) is an important fruit crop worldwide. We have performed a de novo transcriptome assembly for 7 apricot accessions (‘Stark Early Orange’ (SEO), ‘Hacıhaliloğlu’ (HH), ‘Perfection’, ‘Iğdır’, ‘Roxana’, ‘Esen1’, and ‘Esen2’), which yielded a total number of transcripts ranging from 30,363 for ‘SEO’ to 59,751 for ‘Iğdır’. The pool of the reads produced from 7 accessions were assembled into 85,766 transcripts, with an average of 1165.69 nt. Functional annotation (Gene Ontology- GO and Kyoto Encyclopedia of Genes and Genomes- KEGG) was performed successfully for the transcripts. Simple sequence repeats (SSRs) were searched in the transcript pool and 14,722 di-, tri- tetra-, penta-, and hexanucleotide motif loci with a minimum of 5 repetitions for all motifs were identified. Primers were designed for 206 loci, and 72 of them were found to be polymorphic by amplifying diverse 24 apricot accessions, including 7 Plum Pox Virus (PPV)-resistant and 17 PPV-susceptible accessions. In order to test the amplification success of publicly available genomic SSRs (gSSRs) for diverse apricot accessions, an additional 88 published Prunus gSSRs were characterized amplifying the same 24 apricots and only 54 (62%) produced polymorphic bands. The new EST-SSRs could be a reliable source of primers for characterization and mapping studies of apricots, especially because they mostly flank easily scorable tri- and tetranucleotide repeats. Key words: Microsatellite, molecular markers, functional annotation 1. Introduction The common apricot (Prunus armeniaca L.) is diploid, with eight pairs of chromosomes (2n = 16) and an estimated genome size of 240 million nucleotides (nt) according to the Genome Database for Rosaceae (https://www.rosaceae. org/). Apricot belongs to the family Rosaceae, subfamily Prunoideae, and it is native to China and Central Asia, which are two primary genetic diversity centers for the species. The near-eastern group, including Turkey, Iran, and the Caucasus, is considered as a secondary center of diversity (Vavilov, 1951; Ercisli, 2009; Halasz et al., 2010; Hegedus et al., 2010). High-throughput next-generation sequencing (NGS) technologies produce large amounts of data and are thus widely used for transcriptome analysis, allowing quantification of RNA transcripts, discovery of new genes, and a vast amount of polymorphic loci. The potential of NGS in apricot science was reviewed by MartínezGómez et al. (2011). NGS technologies have been applied in apricot species to facilitate the transcriptome analysis of several biological and agronomical aspects: seasonal bud dormancy (Zhong et al., 2013) and self- and cross* Correspondence: pollinated pistils (Habu et al., 2014) in Japanese apricot, global gene profiling and the search for potential SSR markers (Dong et al., 2014), oil dynamic accumulation in developing seed kernels for the development of woody biodiesel (Niu et al., 2015) in Siberian apricot (Prunus sibirica L.), Plum Pox Virus (PPV) (Sharka) susceptibility/ resistance (Rubio et al., 2015), single nucleotide polymorphism (SNP) discovery (Salazar et al., 2015), the study of the development of embryos (Bai et al., 2016), and SNP discovery and genetic characterization via genotyping by sequencing in common apricot (Gürcan et al., 2016). Simple sequence repeats (SSRs) have been highly preferred due to their high variability, codominant inheritance, suitability for sharing among laboratories, and cross-species transferability (İpek et al., 2016; Sakar and Ünver, 2016; Sorkheh and Khaleghi, 2016). A variety of SSR markers have been developed for Prunus. Examples include almond (Prunus dulcis) (Testolin et al., 2004; Messina et al., 2004), apricot and Japanese apricot (Prunus mume) (Lopes et al., 2002; Decroocq et al., 2003; Vilanova et al., 2006; Li et al., 2010; Wang et al., 2014), cherries (Clarke and Tobutt, 2003; Sorkheh et al., 2016), 305 KÖSE et al. / Turk J Agric For and peach (Aranzana et al., 2002; Yamamoto et al., 2002; Howad et al., 2005; Chen et al., 2014; Dettori et al., 2015). SSR markers along with other marker systems have been used in the construction of apricot genetic maps, genetic diversity assessments, and characterization of apricot germplasm collections (Lambert et al., 2007; Lalli et al., 2008; Dondini et al., 2011; Soriano et al., 2012; Rubio et al., 2014, Decroocq et al., 2014, 2016; Gürcan et al., 2015). Although a variety of SSRs have been identified in Prunus species including apricot, more reliable SSR markers are needed for apricot molecular breeding, particularly for anchoring the parental maps and construction of saturated maps of apricot. Available primers occasionally fail during polymerase chain reaction (PCR), probably due to their having not been tested on a wide set of apricots representing overall apricot genetic diversity. Furthermore, the useful number of SSRs lowers drastically while mapping them to biparental segregation populations since SSRs do not always produce polymorphic alleles for parental accessions. Additionally, the majority of available primers were usually developed from SSR-enriched genomic libraries, derived primarily from intergenic DNA regions. In contrast, ESTSSRs are specifically developed from transcribed regions of the genome and present high potential for linkage to loci of interest. Thus, polymorphic EST-SSRs are valuable in constructing linkage maps, presenting considerable utility for MAS. Here, we report the NGS sequencing and transcriptome profiling of 7 apricot accessions and the development of 72 polymorphic EST-SSR loci obtaining allele sizes of 24 diverse accessions, including 7 PPVresistant and 16 PPV-susceptible accessions. Additionally, we have studied 88 previously published gSSRs amplifying the same diverse 24 apricots in order to exhibit how many of the published Prunus primers are useable in apricot breeding programs. 2. Materials and methods 2.1. Plant material We used 7 accessions including international, national, and local accessions for the transcriptome analysis: ‘SEO’, ‘Hacıhaliloğlu’ (HH), ‘Perfection’, ‘Iğdır’, ‘Roxana’, ‘Esen1’, and ‘Esen2’. ‘SEO’ is PPV-resistant and the most commonly used donor for PPV resistance breeding programs in Europe. ‘HH’ is susceptible to PPV and accounts for most dried apricots (about 70%) in Turkey, and thereby is also the main cultivar for ap (...truncated)