Plant Science 203–204 (2013) 115–123 Contents lists available at SciVerse ScienceDirect Plant Science j our na l ho me p a ge: www.elsevier.com/locate/plantsci Single nucleotide polymorphism isolated from a novel EST dataset in garden asparagus (Asparagus officinalis L.) Francesco Mercati a , Paolo Riccardi b , Jim Leebens-Mack c , Maria Rosa Abenavoli a , Agostino Falavigna b , Francesco Sunseri a,∗ a Dipartimento di AGRARIA, Università “Mediterranea” di Reggio Calabria, Salita Melissari, 89124 Reggio Calabria (RC), Italy b CRA-ORL, Unità di Ricerca per l’Orticoltura, via Paullese 28, 26836 Montanaso L. (LO), Italy c Department of Plant Biology, University of Georgia, Athens, GA 30602, USA a r t i c l e i n f o Article history: Received 18 October 2012 Received in revised form 7 January 2013 Accepted 9 January 2013 Available online 16 January 2013 Keywords: Molecular markers cDNA library 454 pyrosequencing Genetic map a b s t r a c t Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSR) are abundant and evenly distributed co-dominant molecular markers in plant genomes. SSRs are valuable for marker assisted breeding and positional cloning of genes associated traits of interest. Although several high throughput platforms have been developed to identify SNP and SSR markers for analysis of segregant plant popula- tions, breeding in garden asparagus (Asparagus officinalis L.) has been limited by a low content of such markers. In this study massively parallel GS-FLX pyro-sequencing technology (454 Life Sciences) has been used to sequence and compare transcriptome from two genotypes: a rust tolerant male (1770) and a sus- ceptible female (G190). A total of 122,963 and 99,368 sequence reads, with an average length of 245.7 bp, have been recovered from accessions 1770 and 190 respectively. A computational pipeline has been used to predict and visually inspect putative SNPs and SSR sequences. Analysis of Gene Ontology (GO) slim annotation assignments for all assembled uniscripts indicated that the 24,403 assemblies represent genes from a broad array of functions. Further, over 1800 putative SNPs and 1000 SSRs were detected. One hundred forty-four SNPs together with 60 selected SSRs were validated and used to develop a pre- liminary genetic map by using a large BC 1 population, derived from 1770 and G190. The abundance of SNPs and SSRs provides a foundation for the development of saturated genetic maps and their utilization in assisted asparagus breeding programs. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Molecular markers are useful for identifying and select- ing genotypes with favorable traits. Molecular markers linked to traits of interest can be used to select for a desirable trait in a segregant population and facilitate crop improve- ment through marker-assisted selection (MAS) [1,2]. Expressed sequence tag (EST) or transcriptome databases are being used to develop gene-anchored molecular markers in several crops, including corn [3], soybean [4], barley [5,6], tomato [7] and wheat [8,9]. The EST sequences available in the NCBI EST database (http://www.ncbi.nlm.nih.gov/dbEST) range from less than 10,000 for little investigated crops to over a million ESTs in major crop plants and 1.5 million ESTs for the model plant Arabidopsis. With the advent of Next-Generation Sequencing (NGS) tech- nologies much greater volumes of transcriptome and genome data are being deposited into the NCBI’s Sequence Read Archive (SRA; ∗ Corresponding author. Tel.: +39 0965 1870659; fax: +39 0965 1870459. E-mail address: francesco.sunseri@unirc.it (F. Sunseri). http://www.ncbi.nlm.nih.gov/sra). The pyrosequencing-based 454 NGS technology provides massive volumes of sequence infor- mation at low cost and relative low error frequency [10]. This technology is widely utilized for genome re-sequencing [11], de novo sequencing of small genomes [12], SNP detection [13,14], or transcriptome sequencing [15]. Several software packages are available for assembling 454 sequence reads and develop- ing genetic markers, including single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs). SNP and SSR-based markers are abundant and evenly distributed throughout most plant genomes [16,17]. SNPs are easily developed from sequence data, highly reproducible, amenable to high throughput assays [17,18] and useful for the construction of high-density genetic maps [19]. Further, transcript-associated SNPs can be applied to develop allele-specific assays for the discovery of intraspe- cific cis-regulatory variation [20]. SSRs based on short (1–6 bp) tandem repeats and can be identified through straightforward anal- yses of ESTs (EST–SSR) and developed into gene-anchored marker loci [21,22]. To date, however, few SSRs and no SNP markers have been reported for the high value vegetable crop Asparagus officinalis L. 0168-9452/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2013.01.002