Exploiting the Transcriptome of Euphrates Poplar, Populus euphratica (Salicaceae) to Develop and Characterize New EST-SSR Markers and Construct an EST-SSR Database

Populus euphratica (Salicaceae) to Develop and Characterize New EST-SSR Markers and Construct an EST-SSR Database. PLoS ONE 8(4): e61337. doi:10.1371/journal.pone.0061337 Exploiting the Transcriptome of Euphrates Poplar, Populus euphratica (Salicaceae) to Develop and Characterize New EST-SSR Markers and Construct an EST- SSR Database Fang K. Du 0 Fang Xu 0 Hong Qu 0 Sisi Feng 0 Jijun Tang 0 Rongling Wu 0 Jean-Marc Lacape, CIRAD, France 0 1 Center for Computational Biology, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University , Beijing, China, 2 Center for Bioinformatics , National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University , Beijing , China , 3 Department of Computer Science and Engineering, University of South Carolina , Columbia, South Carolina , United States of America Background: Microsatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/ conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries. Methodology/Principal Findings: A total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/ poplar/resources/static_page/index.html). Conclusions: The large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database. - Funding: This work is financially supported by Fundamental Research Funds for the Central Universities (TD 2012-01), the Research Fund for the Doctoral Programme of Higher Education of China to FKD; Changjiang Scholars Award and Thousand-person Plan Award to RLW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author Rongling Wu is a PLOS ONE Editorial Board member; this does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Populus euphratica Oliv. (Salicaceae) inhabits semi-arid areas, with a natural distribution ranging from Western China and the Middle East to Spain and Western Morocco. As a desert tree, it is considered to be salinity and drought tolerant [1]. Moreover, it plays an important role in maintaining the ecological equilibrium of desert environments by counteracting the effects of dust storms, stabilizing sand, and retaining water [2]. Changes in its habitats and distribution have caused natural stands of P. euphratica to diminish over the last few decades, and some populations have disappeared [3]. P. euphratica is a useful model organism for studying salt and drought resistance mechanisms in trees because it can tolerate NaCl concentrations of up to 450 mM [4,5] and mannitol concentrations up to 400 mM [6] in controlled experiments. Much of the work conducted on this species to date has focused on the physiological mechanisms responsible for its resistance to abiotic factors (e.g. [7] and references therein). More recent studies using Amplified Fragment Length Polymorphism (AFLP) or microsatellite analysis based on polyacrylamide gel electrophoresis (PAGE) with silver staining have provided useful information on its genetics and evolutionary history [80]. However, partly because of the scarcity of suitable molecular markers, much remains to be learned about the genetic factors responsible for the ability of P. euphratica to cope with various adverse environmental conditions. SSRs (simple sequence repeats) [11] are tandem repeated DNA sequences with a repeating motif of one to six base pairs. They are also known as STRs (short tandem repeats) [12], SSLPs (simple sequence length polymorphisms) [13], and microsatellites [14]. SSRs are common and widely distributed in prokaryotic and eukaryotic genomes [15]. Because of their high rates of mutation, they tend to be highly polymorphic [16]. This makes it possible to distinguish between alleles based on their length using either classical PAGE, or by DNA sequencing when working on a larger scale or when higher accuracy is required [17]. Because of these useful properties, SSRs are the most popular markers in population genetics [17] and have been used in many applications such as gene tagging, Quantitative Trait Locus (QTL) mapping, marker assisted selection (MAS), parentage analysis, fingerprinting, and phylogenetic and taxonomic studies [18]. Two categories of SSRs can be defined based on their locations in the genome: Expressed Sequence Tag-SSRs (eSSRs) are embedded in transcribed sequences [19], while genomic SSRs (gSSRs) are embedded in both transcribed and non-transcribed sequences. Until recently, the lack of the detailed genomic information for many plants has meant that SSR marker development was primarily conducted using selective hybridization strategies, which require fragmented genomic DNA that is bound to either a nylon membrane using repeat-containing probes or to biotinylated probes that are subsequently captured on streptavidin-coated beads [2023]. This is complicated by the difficulty of preparing uniformly digested genome DNA, the cumbersome nature of the molecular cloning, the limited scope for obtaining sufficiently long sequences, and the high cost of the overall process. However, thousands of ESTs can be obtained from a single cDNA library comprising fragments with lengths of 300500 bp, from which plenty of eSSRs can be designed. Because ESTs are conserved across phylogenetically related species, eSSRs developed for one species are often highly transferable and thi (...truncated)