Rapid microsatellite development for tree peony and its implications
© Gao et al.; licensee BioMed Central Ltd. 2013
Received: 3 June 2013
Accepted: 4 December 2013
Published: 16 December 2013
Microsatellites are ubiquitous in genomes of various organisms. With the realization that they play roles in developmental and physiological processes, rather than exist as ‘junk’ DNA, microsatellites are receiving increasing attention. Next-generation sequencing allows acquisition of large-scale microsatellite information, and is especially useful for plants without reference genome sequences.
In this study, enriched DNA libraries of tree peony, a well-known ornamental woody shrub, were used for high-throughput microsatellite development by 454 GS-FLX Titanium pyrosequencing. We obtained 675,221 reads with an average length of 356 bp. The total size of examined sequences was 240,672,018 bp, from which 237,134 SSRs were identified. Of these sequences, 164,043 contained SSRs, with 27% featuring more than one SSR. Interestingly, a high proportion of SSRs (43%) were present in compound formation. SSRs with repeat motifs of 1–4 bp (mono-, di-, tri-, and tetra-nucleotide repeats) accounted for 99.8% of SSRs. Di-nucleotide repeats were the most abundant. As in most plants, the predominant motif in tree peony was (A/T)n, with (G/C)n less common. The lengths of SSRs were classified into 11 groups. The shortest SSRs (10 bp) represented 1% of the total number, whereas SSRs 21–30 and 101–110 bp long accounted for 26% and 29%, respectively, of all SSRs. Many sequences (42,111) were mapped to CDS (coding domain sequence) regions using Arabidopsis as a reference. GO annotation analysis predicted that CDSs with SSRs performed various functions associated with cellular components, molecular functions, and biological processes. Of 100 validated primer pairs, 24 were selected for polymorphism analysis among 23 genotypes; cluster analysis of the resulting data grouped genotypes according to known relationships, confirming the usefulness of the developed SSR markers.
The results of our large-scale SSR marker development using tree peony are valuable for investigating plant genomic structural evolution and elucidating phenotypic variation in this species during its evolution and artificial selection. The newly identified SSRs should be useful for genetic linkage map construction, QTL mapping, gene location and cloning, and molecular marker-assisted breeding. In addition, the genome-wide marker resources generated in this study should aid genomic studies of tree peony and related species.
KeywordszzzMicrosatellite Next-generation sequencing Tree peozzzzny Ornamental SSR marker
Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated 1–6-bp DNA regions ubiquitous in prokaryotes and eukaryotes. As components of genomes, they can be found both in protein-coding and non-coding regions. SSRs have been universally utilized as genetic markers because of their abundance and inherent potential for variation . The functions of SSRs were previously unclear, and until recently they were regarded as ‘junk’ (i.e., having no significant genomic role). At present, much progress has been achieved in regard to elucidation of SSR function. SSR locations appear to determine the types of functional roles that SSRs play, and alterations in SSR lengths at different locations can lead to changes in organismal phenotypes [2, 3]. SSRs in different gene positions (i.e., promoter regions, 5′ untranslated regions (UTRs), 3′ UTRs, exons, and introns) may play important roles in determining protein function, genetic development, and regulation of gene expression. For example, expansion of CAG repeats in the HD gene coding region can lead to Huntington’s disease in humans, possibly through activation of some so-called ‘toxic’ proteins . With expanding knowledge of SSR functions in terms of development, gene regulation, and evolution, SSRs are receiving increasing attention. Because genomic information is lacking for most species, however, it is difficult to study microsatellite origin, distribution, and evolution, or even to develop new SSR-based molecular markers.
Traditional SSR development is time-consuming, and involves laborious iterations of genomic DNA library screening with SSR probes required to isolate microsatellite-containing sequences . Next-generation sequencing technologies are remarkably well-developed, and are widely used for genome sequencing, transcriptome sequencing, and genome deep-sequencing in plants [5, 6]. It has been successfully used for identifying molecular markers, including SSRs and simple nucleotide polymorphisms (SNPs), in organisms such as the water strider , copperhead snake , blue duck , pine pathogen fungus , and scuttle fly . Because of the complicated structure of plant genomes, however, molecular marker development using next-generation sequencing has had limited application, especially in non-model plants lacking genomic information.
Among next generation-sequencing approaches, Roche 454 pyrosequencing (R454) holds great promise with respect to the long reads obtained as well as acquisition of sufficient genetic information of interest within single reads. The large amount of generated data facilitates sequence assembly without genomic information , and increases the likelihood that a single read contains microsatellite repeats along with suitable flanking regions of unique sequences. Another approach to mining molecular markers, involving in silico methods, has also been successful; examples include the derivation of markers from a draft genome  and the mining of existing expressed sequence tag (EST) libraries . Compared with traditional library-based and in silico methods, R454 offers great advantages, being faster, less costly, and less dependent on existing genetic resources . Another advantage is the huge amount of genetic information produced, with the possibility of future use. This is greatly beneficial for studies of plants without genomic information, especially woody plants; in such species, no established systems exist for in vitro culture or transformation for genetic manipulation, hampering new cultivar breeding. Molecular marker-assisted breeding is efficient for such organisms. DNA markers developed via next-generation sequencing are also increasingly being used for genetic diagnostics, drug discovery, gene cloning, genome analysis, comparative genomics, and molecular evolution studies.
The purpose of this study was to apply next-generation sequencing, such as R454, to SSR development in tree peony (Paeonia suffruticosa Andrews). Such an approach was expected to drastically shorten the time required for effective marker development and utilization. Tree peony belongs to sect. Moutan DC. of the genus Paeonia L. (Paeoniaceae). It is a well-known ornamental plant enjoying worldwide popularity on account of its large, showy, colorful and fragrant flowers. Little genomic information is currently available for this species. In a previous study, we constructed a cDNA library from flower buds and obtained 2,241 ESTs, from which 167 SSRs were derived and a dataset of 185 putative SNPs obtained for breeding based on their high availability and stability . Although more than 200 SSRs have been submitted to public databases , the number is inadequate with respect to the 1,500 cultivars of tree peony. Compared with crop plants such as maize, wheat, and soybean, or ornamental plants such as rose, molecular markers, especially SSRs, are still needed for future breeding of tree peony. This is especially true taking into consideration its importance, barely transformed nature, and woody characteristics, and the lack of genomic information. Construction of a dense genetic linkage map and development of genome-wide molecular markers are also essential for marker-assisted selection of new tree peony hybrids. Because relationships among wild species of tree peony and their taxonomic position within Paeoniaceae are still unclear, developed polymorphic SSRs would also aid studies of Paeonia evolution, comparative genetics, and population structure.
Sequencing and characterization of reads
Identification of SSR loci
Occurrence of microsatellites in the surveyed tree peony genome
Total number of sequences examined
Total size of examined sequences (bp)
Total number of identified SSRs
Number of SSR-containing sequences
Number of sequences containing more than 1 SSR
Number of SSRs present in compound formation
Microsatellite motif length distribution
Number of motif repeats
Relative frequency of different SSR repeat motifs
Frequency of mono-, di-, tri-, and tetra-nucleotide repeat motifs in the tree peony genome
Tree peony genome
Relative frequencies of different SSR repeat lengths
Compound SSR analysis
Microsatellite distribution in different genomic regions of tree peony using Arabidopsis, poplar, and grape reference sequences
Microsatellite distribution in different genomic regions of tree peony using the Arabidopsis genome as a reference
Functional annotation of SSR-containing coding sequences
Validation of SSR assays
SSR loci amplified from 23 accessions of tree peony
Forward primer (5′-3′)
Reverse primer (5′-3′)
The number of SSRs obtained in this study from tree peony was higher than that generated from other plants, including Arabidopsis, Medicago truncatula, Oryza sativa (rice), and Sorghum bicolor (sorghum) [1, 19]. The frequency of A/T repeats present in tree peony was between dicots and monocots . The percentage of tetra-, penta-, and hexa-nucleotide repeats observed in tree peony (8%) was higher than in Sorghum (5.4%), Populus (1.66%), Medicago (0.94%), rice (2.54%), Brachypodium (2.45%), and Arabidopsis (0.53%) .
The frequency of di-nucleotide repeats in tree peony was not consistent with that observed in Brachypodium by Sonah et al. . Similar to rice, AG/CT repeats were well represented. AG/CT and AT/AT repeats were abundant in tree peony, accounting for 41.9% and 41.0%, respectively, of identified SSRs, while AT/AT repeats were more frequent in Populus (60.5%) and Medicago (59.9%) . CG/CG repeats were relatively uncommon in tree peony, however, similar to Populus, Medicago, and Arabidopsis, suggesting that CG-rich motifs are the least preferred in dicot genomes. In human, Caenorhabditis, and Arabidopsis genomes, the most common di-nucleotide repeats are (AC)n, (AG)n, and (AT)n, respectively, demonstrating that different species have different motif frequency distributions.
With respect to tri-nucleotide repeats, AGC/CGT, AGG/CCT, and CCG/CGG have been observed more frequently in monocots than in dicots. A/T-rich repeats were the dominant tri-nucleotide SSRs in tree peony, similar to the results of Sonah et al. . In tree peony, the sparseness or absence of CCG and ACG repeats may be due to highly mutable CpG di-nucleotide repeats, as evidenced in rice by the tendency of tri-nucleotide repeats, with few exceptions, to consist of various combinations of C and G. Transcriptional repression by DNA methylation depends on CpG density; CCG repeats may also be selected against by the requirements of the splicing machinery, with maintenance or absence of CCG possibly an active process . The total absence of a particular repeat motif may indicate that the sequence is not preferred by the mechanism generating repeats or that strong selective pressure exists against repeated occurrence of particular sequences .
The characteristically short lengths of SSRs may have functional implications with respect to their evolution or the genes involved in plant physiology and development. In a previous study , rice SSRs were divided into two groups based on the length of SSR tracts and their potential as informative genetic markers: Class I microsatellites contained perfect SSRs ≥ 20 bp long and Class II microsatellites contained perfect SSRs 12–20 bp long. Class II microsatellites tended to be less variable because of less possibility of slipped-strand mispairing over the shorter SSR template. In tree peony, 85% of SSRs were categorized as Class I microsatellites and 1% as Class II microsatellites. Longer perfect repeats (Class I) have been determined to be highly polymorphic . In future studies of tree peony SSRs, attention should focus on Class I microsatellites, with an emphasis on evaluation of polymorphism and its implications.
Length variation of repeated units may be due to differences in generation and fixation mechanisms of simple repetitive DNA. The inherent ability of a sequence to form alternative DNA conformations may be important for SSR generation, but does not explain differences observed among taxa. Enzymes or other proteins responsible for various aspects of DNA processing, such as replication and repair, and for chromatin remodeling, may be involved in the taxon specificity of microsatellite characteristics. It should be emphasized that not only do genomes differ in degree of repetitiveness , but also in preferred microsatellite types. In plant genomes, the frequent occurrence of repeat motifs of a particular sequence and length is the result of selection pressure applied on the specific motif during evolution . The molecular mechanism responsible for the origin of microsatellites is still a subject of controversy, with many theories—such as replication slippage and unequal crossing-over—proposed to explain their occurrence [19, 23, 24]. The essential basis for species-specific accumulation of particular motif repeats, repeat lengths, and G/C content, which may influence unique microsatellite distribution patterns and evolution, is also still unclear. Variations in repetition purity and motif length enable site-specific adjustment of mutation rate and mutation effect, evidence indicating that common SSR alleles may offer potential selective advantages . The increasing number of species with sequenced genomes should provide a foundation for the study of microsatellite evolution and even lead to discovery of the genetic/genomic role of microsatellites.
SSR frequency in monocot CDS regions is twice that of dicots . It has been suggested that SSRs in different gene positions may perform varied functions. In animals, including mammals and other vertebrates, introns contain more poly (A/T) than poly (C/G) repeats. In Caenorhabditis elegans, however, intergenic regions show an interesting preference for poly (C/G) over poly (A/T) repeats , indicating that preferences may vary among organisms. In tree peony, the abundance of tri-nucleotide repeats mapping onto CDS regions was consistent with results found for the six species studied by Sonah et al. . Tang et al.  examined SSRs in the Arabidopsis genome, and found that SSRs generally were preferentially located in upstream gene regions, especially 5′ UTRs; as in tree peony, tri-nucleotide repeats were the most common repeats found in coding regions. The accumulation of tri-nucleotide repeats in coding regions is primarily due to the triplet-repeat nature of codons . The various numbers of repeats in coding regions are a potential source of quantitative and qualitative phenotypic variation . SSRs in 5′ UTRs and CDSs may modify the expression or function of genes with which they are associated .
In rice, 80% of GC-rich tri-nucleotide repeats occur in predicted exons, while AT-rich tri-nucleotide repeats are distributed evenly across all genomic components. Di-nucleotide and tetra-nucleotide repeats are predominantly situated in noncoding—mainly intergenic—regions. (GA)n repeats usually occur in regions with a balanced (close to 50%) GC content, favoring robust PCR amplification, whereas (CA)n and (AT)n are rare in gene-rich regions . Tri- and hexa-nucleotide repeats have been shown to be the most common repeats in eukaryotic coding regions [20, 27]. In our study, SSR-containing genes encoding for binding, catalytic, and structural molecules were abundant in the GO molecular function category, similar to results found in Brachypodium. While such SSR-containing genes may perform multiple functions in tree peony, the importance of SSRs within genes remains to be further explored.
The SSR markers identified in this study should be useful for population genetic studies, and are potentially amplifiable across the genus. Plant genomes are complex, and contain large amounts of repetitive DNA, including microsatellites, which has immediate practical implications for the success of SSR marker development. Observed differential patterns of SSR marker distribution may be helpful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study have potential application to genomic research, marker-assisted breeding, DNA fingerprinting of genetic resources, molecular mapping of tree peony and related species, and map-based cloning of candidate genes. Hypervariable microsatellites are a useful source of polymorphic DNA markers for linking genetic maps with genomic sequences, and ultimately with phenotypic variation. They provide an opportunity to use SSR markers to investigate the wide range of genetic diversity that exists in wild relatives outside of the tree peony gene pool. Because SSRs are associated with vital functions and characteristics, such as transcription factor binding, RNA shape, DNA structure and packaging, and DNA length and orientation , the SSRs obtained in this study may be important for investigating plant genomic structural evolution and for providing insights into phenotypic variation in species during their evolution.
This study represents the first application of next-generation sequencing for high-throughput microsatellite development in tree peony. The large size of the tree peony genome, approximately 16 G (data from private correspondence), hampers its sequencing, and the species is not highly amenable to transformation because of its woody characteristics. Consequently, the 237,134 microsatellites obtained in this study should be useful for marker-assisted breeding and functional characterization of genes related to trait formation. In addition, because the phylogenetic position of Paeoniaceae is still unresolved, the uncovered microsatellites may serve as a data resource for evolutionary studies in the family.
Leaves of tree peony (Paeonia suffruticosa Andrews) were collected from the Peony Germplasm Garden, Institute of Botany, Chinese Academy of Sciences (Beijing, China). Three cultivars—‘Liu li guan zhu’ , ‘Fu gui hong’ , and ‘Wu cai die’—were used for primer validation. Twenty-three accessions of tree peony were used for marker validation (Additional file 2: Table S2).
Genomic DNA isolation, library preparation, and R454 sequencing
Total genomic DNA was extracted using the CTAB method . Genomic DNA (500 μl; 600 μg) was fragmented with nitrogen at 45 psi for 2 min; 500–750-bp fragments were used for further study. Both fragment ends were polished and ligated to adaptors using T4 ligase. After PCR amplification of fragments with adaptor primers, selective hybridization was performed using eight biotin-labeled probes—pGA, pAC, pAAT, pAAC, pAAG, pATGT, pGATA, and pAAAT—and streptavidin-coated beads (Dynabeads; Invitrogen, Grand Island, NY 14072, USA) [28–31]. Library quality inspection and sequencing of clones was carried out as described by Yang et al. .
DNA (5 μg per plate) was sequenced on a Roche 454 GS FLX sequencer using Titanium reagents. Processing and analysis of sequencing data was performed with GS-FLX Software v2.0.01 (454 Life Sciences/Roche, Werk Penzberg82372, Penzberg, Germany). Raw sequences in SFF files were base-called using the python script sff_extract.py developed by COMAV (http://bioinf.comav.upv.es), and then processed to remove low-quality and adaptor sequences using the programs tagdust , LUCY , and SeqClean  with default parameters.
SSR locus search and mapping
The program MISA (Microsatellite identification; http://pgrc.ipk-gatersleben.de/misa/) was used to identify reads and contigs containing SSRs. Criteria used for selection were a minimum of five repeats for simple motifs and three repeats for complex or imperfect repeats, a motif length of 2–10 bp, and, for compound SSRs, a maximum interruption distance of 100 bp between different SSRs. To facilitate SSR detection, only 1- to 6-nucleotide motifs were considered, and the minimum repeat unit was defined as 10 for mono-, 6 for di-, and 5 for tri-, tetra-, penta-, and hexa-nucleotides. SSR position, number of different repeat types, and length (motif bp × number of motifs) were analyzed using the ‘bespoke’ function in MISA  and plotted using Open Office Calc.
To map coding regions, all reads containing SSRs were compared against Arabidopsis (ftp://ftp.arabidopsis.org/home/tair), grape (http://www.genoscope.cns.fr) and poplar (ftp://ftp.jgi-psf.org) public databases using the program BWA-SW [36, 37]. Map position was categorized as follows: 3′/5′ UTR, CDS, intergenic, intron, non-mapped, or multi-mapped. The repeat unit type (1–6, compound, or compound with interruption) was then determined.
Primer acquisition and validation
Primer pairs for flanking sequences of each unique SSR were designed automatically using Primer3 , with target microsatellites containing at least five repeats and yielding PCR products of 80–500 bp. One hundred primer pairs were synthesized and used for validation (Additional file 1: Table S1). Screened primer pairs giving good amplification were subsequently used to characterize genetic diversity among 23 accessions of tree peony (Additional file 2: Table S2). PCR protocols and components were as described in , with modifications to annealing temperatures.
Number of alleles and expected and observed heterozygosities were calculated using POPGEN1.32 . A dendrogram was constructed based on Nei’s unbiased genetic distances  using the unweighted pair-group method with arithmetic averages (UPGMA) as implemented in NTSYSpc-2.02 .
Simple sequence repeat
Quantitative trait locus
Expressed sequence tag
We appreciated for the technical help by Dr. Dahai Wang from Autolab. LTD. Co. (Beijing, China) and Miss Jing Sun from HeBei United University for the marker development. This study was supported by the National Natural Science Foundation of China (Grant No. 31272201) and the National High Technology Research and Development Program of China (863 Program, Grant No. 2011AA100207).
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