Mining microsatellites in the peach genome: development of new long-core SSR markers for genetic analyses in five Prunus species

Dettori et al. SpringerPlus (2015) 4:337 DOI 10.1186/s40064-015-1098-0 Open Access RESEARCH Mining microsatellites in the peach genome: development of new long‑core SSR markers for genetic analyses in five Prunus species Maria Teresa Dettori1*, Sabrina Micali1, Jessica Giovinazzi1, Simone Scalabrin2, Ignazio Verde1 and Guido Cipriani3 Abstract A wide inventory of molecular markers is nowadays available for individual fingerprinting. Microsatellites, or simple sequence repeats (SSRs), play a relevant role due to their relatively ease of use, their abundance in the plant genomes, and their co-dominant nature, together with the availability of primer sequences in many important agricultural crops. Microsatellites with long-core motifs are more easily scored and were adopted long ago in human genetics but they were developed only in few crops, and Prunus species are not among them. In the present work the peach whole-genome sequence was used to select 216 SSRs containing long-core motifs with tri-, tetra- and penta-nucleotide repeats. Microsatellite primer pairs were designed and tested for polymorphism in the five diploid Prunus species of economic relevance (almond, apricot, Japanese plum, peach and sweet cherry). A set of 26 microsatellite markers covering all the eight chromosomes, was also selected and used in the molecular characterization, population genetics and structure analyses of a representative sample of the five diploid Prunus species, assessing their transportability and effectiveness. The combined probability of identity between two random individuals for the whole set of 26 SSRs was quite low, ranging from 2.30 × 10−7 in peach to 9.48 × 10−10 in almond, confirming the usefulness of the proposed set for fingerprinting analyses in Prunus species. Keywords: P. armeniaca, P avium, P. persica, P. salicina, P. dulcis, Fingerprinting Background The Prunus genus includes several diploid species of economic relevance. Comparative mapping studies showed that the genomes of the diploid Prunus species are essentially colinear and syntenic (Dettori et al. 2001; Dirlewanger et al. 2004; Verde et al. 2005; Dondini et al. 2007; Jung et al. 2009) and DNA fingerprinting of accessions belonging to these species consistently revealed a high transportability of molecular markers (Cipriani et al. 1999; Dirlewanger et al. 2002; Vendramin et al. 2007). Fingerprinting based on molecular markers is a popular *Correspondence: 1 Consiglio per la Ricerca in Agricoltura e l’analisi dell’economia agraria, Centro di Ricerca per la Frutticoltura, Rome, Italy Full list of author information is available at the end of the article tool for studies of population genetics and diversity, including the resolution of synonymy/homonymy controversies, the protection of plant breeders’ rights, paternity and kinship analyses. SSR markers (simple sequence repeats), or microsatellites, consist of tandemly repeated DNA sequences with a core unit of 1–6 base pairs (bp). They offer a number of positive features for the genetic profiling of individuals including wide distribution in plant genomes, prevalent single-locus tagging in diploid species, multi-allelic co-dominant patterns, simple use and availability of several primer sequences in many important agricultural crops (Schlötterer 2004). The high variability of microsatellites is mainly due to a different number of repeats in the region of the repeated motif but also to short insertion/deletion events (Decroocq et al. 2003). © 2015 Dettori et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Dettori et al. SpringerPlus (2015) 4:337 In humans and animals long nucleotide repeats, namely tetra- and penta- motifs, were adopted because neighbor alleles are more easily separated from each other (Hammond et al. 1994; Ruitberg et al. 2001; Butler et al. 2004; Butler 2006; Hellmann et al. 2006). Moreover, di-nucleotide SSRs, even though frequent in eukaryotic genomes, suffer from the presence of ghost bands (stuttering), which make the interpretation of electropherograms and the allele call less reliable. The first SSRs developed by plant scientists were mainly di-nucleotide repeats, which are the most abundant in plant genomes. The isolation procedure was costly, microsatellites were isolated from SSR-enriched libraries with the aim of producing a high number of potentially useful markers for mapping purposes. The availability of whole-genome sequences offers the opportunity to mine the genomes and retrieve thousands of different kind of markers including single nucleotide polymorphisms (SNPs), structural variants and microsatellites. SNPs are widely used for the generation of saturated genetic maps due to the availability of high-throughput automated genotyping platforms (Gunderson 2009). High-throughput SNP tools have been recently developed in Prunus species using an Illumina platform (Peace et al. 2012; Verde et al. 2012) and have been used to genotype cultivars and accessions to perform large scale genetic analyses (Micheletti et al. 2012). However, mapping technologies using SNP markers are still rather expensive and not applicable in every laboratory. Due to their relative abundance in the genome and simple relatively low cost detection, microsatellites are still preferable in population genetics and fingerprinting studies with a low or moderate number of markers. As the regions flanking the repeated motif are in many cases highly conserved, microsatellite markers are easily amplified by PCR in many different accessions and close species. Long-core repeats microsatellites have been developed in a few tree species: grape (Cipriani et al. 2008, 2010), Eucalyptus (Faria et al. 2011) and olive (De la Rosa et al. 2013). The availability of the peach genome sequence (Verde et al. 2013) has allowed the scanning of the whole genome with the aim of retrieving microsatellites to be used for molecular analyses in peach and in its closely related species belonging to the Prunus genus. The aim of this study was to find a universal set of polymorphic tri-, tetra- or penta-nucleotide SSRs distributed in the eight chromosomes for the following diploid Prunus species: peach (P. persica), almond (P. dulcis), apricot (P. armeniaca), Japanese plum (P. salicina), sweet cherry (P. avium). These SSRs were also required to preferably be single locus and to have common amplification parameters. Page 2 of 18 Methods Retrieving microsatellites from the peach genome sequence Penta-, tetra- tri- and di-nucleotide core simple sequence repeats with a minimum length of 12 bp were retrieved from the peach whole-genome sequence ( (...truncated)