Microsatellite Marker Development in Peony using Next Generation Sequencing

in Journal of the American Society for Horticultural Science

Peonies (Paeonia), the grand garden perennial of spring and early summer, are economically important to the international cut flower market. Herbaceous peonies (Paeonia section Paeonia), tree peonies (Paeonia section Moutan), and intersectional crosses between the two types (Itoh Paeonia hybrids) are of interest to gardeners, growers, and nursery producers. Thousands of peony cultivars exist and identity is traditionally determined by experienced horticulturists knowledgeable in plant and bloom characteristics. With DNA extraction possible during any time of the year, molecular markers can provide genotype identity confirmation for dormant roots or mature post-bloom plants. The primary objective of our research was to rapidly and inexpensively develop microsatellite markers in a range of Paeonia species using barcoded Illumina libraries. A secondary objective was to apply these simple sequence repeat (SSR) markers to fingerprint 93 accessions that include tree, intersectional, and herbaceous peonies. We used 21 primers to distinguish cultivars and their close relatives. Also from our sequence information, greater than 9000 primers were designed and are made available.

Abstract

Peonies (Paeonia), the grand garden perennial of spring and early summer, are economically important to the international cut flower market. Herbaceous peonies (Paeonia section Paeonia), tree peonies (Paeonia section Moutan), and intersectional crosses between the two types (Itoh Paeonia hybrids) are of interest to gardeners, growers, and nursery producers. Thousands of peony cultivars exist and identity is traditionally determined by experienced horticulturists knowledgeable in plant and bloom characteristics. With DNA extraction possible during any time of the year, molecular markers can provide genotype identity confirmation for dormant roots or mature post-bloom plants. The primary objective of our research was to rapidly and inexpensively develop microsatellite markers in a range of Paeonia species using barcoded Illumina libraries. A secondary objective was to apply these simple sequence repeat (SSR) markers to fingerprint 93 accessions that include tree, intersectional, and herbaceous peonies. We used 21 primers to distinguish cultivars and their close relatives. Also from our sequence information, greater than 9000 primers were designed and are made available.

Peonies, family Paeoniaceae, were first recognized as a medicinal plant in Asia several thousand years ago (Hsu et al., 1986). In the late 1700s, peonies were imported from Asia and Europe into North America for use as a garden flower (Harding, 1917). Peonies are heritage perennial flowers that hold special cultural value in the United States. They are a traditional Memorial Day cut flower and have become a popular wedding flower (D. Hollingsworth, personal communication). These plants are produced as a commercial nursery crop, and the blooms are a significant component of the cut flower industry. These flowers have been widely sold in European markets for centuries and were first sold in Chicago in 1884 (Rogers, 1995). Commercial production is found on every continent except Antarctica. Production areas in North America range from Alaska and Canada in the North through northern California through the center of the continent to North Carolina in the South. In 2009, world peony sales through Dutch auctions resulted in nearly 63 million stems sold, valued at almost €24 million [≈$30 million (Vakblad voor de Bloemisterji, 2012)].

Jakubowski et al. (2007) listed the names and descriptions of 7995 peony cultivars worldwide. Many more cultivars have been named each year since then. Peonies are generally recognized as three distinct types. Herbaceous peonies are perennial plants that have soft, succulent, green stems that die back to the ground every fall. The crowns of the plants are below the surface of the ground and can survive extremely cold winter temperatures and resume growth in spring. These new shoots grow, flower, set seed, and die at the end of the season. The tree peonies are perennial plants that have woody stems above the ground at all times of the year. Their stem buds break in the spring and the stems elongate over the years to form a bush 1 to 1.5 m in height. Because of the exposed stems, tree peonies are unable to survive temperatures as low as the herbaceous types. Intersectional hybrids are crosses between the two groups. Intersectionals (Itohs) have a similar growth pattern to herbaceous peonies, so they are able to withstand temperatures that would kill tree peonies, but their foliage and the flowers have the tree peony appearance (La Pivoinerie D′Aoust Peony Nursery, 2012). Itohs are named for Toichi Itoh, who made the first successful cross in 1948 between an herbaceous peony and tree peony (Rogers, 2004).

Currently, the standard way to identify cultivars requires knowledge and experience in recognizing the morphological characteristics of the flower and plant. Misidentification of cultivars can sometimes cost thousands of dollars as a result of incorrect sales. Adding to the complexity of identity determination, growers attest that some cultivars produce variant flower colors when grown in different regions or countries (D. Hollingsworth, personal communication). Growers may often wait two to 10 years for bloom appearance to confirm the identity of planted stock.

Identity determination of other horticultural crops such as blueberry [Vaccinium corymbosum (Boches et al., 2005)], peach [Prunus persica (Rojas et al., 2008)], and mango [Mangifera indica (Wahdan et al., 2011)] benefitted from using SSRs as molecular markers for identity verification. SSRs are easy to use, codominant, multiple allelic, abundant, and highly reproducible across laboratories for genotype identification (Powell et al., 1996). Application of this technique to peony cultivars could simplify the identification process for growers and allow identification of rhizomes or leaves at an early stage of production.

To develop SSRs, many laboratories use the chain termination method of DNA sequencing that was developed by Sanger in 1975 (Sanger et al., 1977). This protocol entails construction of genomic libraries using enriched recombinant DNA (Boccacci et al., 2005; Boches et al., 2005; Castillo et al., 2010), resulting in a procedure that is time- and labor-intensive and ultimately yields low numbers of SSRs. Next generation sequencing (NGS) platforms termed “next generation” or “massively parallel” were recently developed. These platforms are changing genomic discovery in plants, delivering large amounts of sequence data, but require specialized and devoted computer infrastructure and bioinformatics (Cronn et al., 2008). The resulting sequence data can be applied to the development of SSR markers in species that lack or have few available SSRs (Jennings et al., 2011). For example, Illumina, Inc. (San Diego, CA) sequencing has been used to develop SSR markers for port-orford cedar (Chamaecyparis lawsoniana) and alaska yellow cedar (Callitropsis nootkatensis) (Jennings et al., 2011), and for mile-a-minute weed [Mikania micrantha (Yan et al., 2011)].

In peony, less than 90 SSRs are available from the tree peony, P. ×suffruticosa, (Homolka et al., 2010; Wang et al., 2009; Yuan et al., 2010; Zhang et al., 2012). Wang et al. (2009) identified 59 SSRs from P. ×suffruticosa and designed corresponding primer pairs. Fourteen of these SSRs were polymorphic in P. ×suffruticosa and were used to examine the relationships between three tree species, P. yananensis, P. jishanensis, and P. rockii (Yuan et al., 2010). Paeonia yananensis was found to be a hybrid of P. jishanensis and P. rockii (Yuan et al., 2010). Eight additional SSRs were reported to cross-amplify in six Peonia species (Homolka et al., 2010). In 2011, researchers used seven of 21 SSRs, developed from peony expressed sequence tags, in cultivar identification of tree peonies (Zhang et al., 2012). Fewer SSRs have been developed for P. lactiflora where only 20 polymorphic SSRs are reported (Li et al., 2011; Sun et al., 2011).

The objectives of this study were to develop new SSR markers using barcoded multiplexed libraries of multiple peony species, and to evaluate these markers for fingerprinting herbaceous peony (P. lactiflora and hybrids), tree peony (moutan), and intersectional (Itohs) individuals.

Methods and Materials

Plant materials.

In early Spring 2010, leaf material was obtained from seven peony individuals from multiple sources. Leaves of P. lactiflora ‘Bowl of Beauty’, P. lutea × P. ×suffruticosa hybrid ‘Souvenir de Maxime Cornu’, P. ×suffruticosa ‘Feng deng bai’, and P. delavayi were collected from the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository (NCGR), Corvallis, OR. Samples from P. tenuifolia ‘Rubra Flora Plena’ and P. peregrina were obtained from plants donated by Adelman Peony Gardens, Brooks, OR, whereas leaves of P. rockii were obtained from J. Oliphant, Corvallis, OR (Table 1). Leaf samples were collected, bagged, kept cool, and transported to the laboratory. Each leaf sample was placed in a ceramic mortar and ground with liquid nitrogen. The ground leaf material was stored at –80 °C until extraction. The DNA extraction protocol for the library preparation was performed using Qiagen (Valencia, CA) reagents (Gilmore et al., 2011).

Table 1.

Peony (Paeonia) species used for DNA sequence determination and for designing simple sequence repeat (SSR) primers.z

Table 1.

DNA sequencing.

The Illumina library preparation of the DNA for sequencing included fragmentation of purified genomic DNA. This was accomplished by sonication [Bioruptor XL(BR_XL); Diagenode, Denville, NJ]. An aliquot of the DNA subjected to agarose gel electrophoresis to ensure that shearing was successful and that DNA fragments were within the expected size ranges, 200 to 1500 bp. The DNA was then cleaned using the QIAquick PCR purification Kit (Qiagen). DNA ends were repaired and purified using the Agencourt AMPure Kit (Beckman Coulter, Brea, CA).

DNA samples were loaded onto a gel along with a low-molecular-weight DNA ladder (New England BioLabs, Ipswich, MA). The sample was viewed with the gel doc and a 350-bp band was excised from the sample with a 5-Prime SafeXtractor-25 (Fisher, Waltham, MA) and stored at –20 °C. Samples were selectively enriched for those DNA fragments that had adapter molecules on both ends using the polymerase chain reaction (PCR) with Phusion DNA Polymerase Mix (Thermo Scientific, Wilmington, DE). The PCR product was cleaned with a QIAquick PCR Purification Kit and run on a 1.5% agarose gel to verify library size and to visually estimate the concentration. The sample was then quantified and the 260/280 absorbance ratio obtained with a spectrophotometer (NanoDrop ND-1000 ultraviolet-Vis; Thermo Scientific). Samples were then submitted to the Oregon State University Center for Genome Research and Biocomputing (CGRB) to determine band size and for sequencing. An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) was used to determine band size. After diluting and pooling, barcoded Peonia samples were submitted for paired-end 80-bp sequencing with the Illumina Genome Analyzer II. Illumina Version 3.0 reagents were used for cluster generation, sequencing, and image acquisition. Illumina pipeline Version 1.5 was used for base calling and resulting microreads were sorted by bar codes using a custom perl script bcsort (Knaus, 2011). Sorted reads were searched only for dinucleotide motifs. The dinucleotide motif guidelines were: microreads containing at least four perfect repeats and each nucleotide represented at least four times and with fewer than eight ambiguous bases. Paired-end microsatellite-containing reads were joined into a single sequence by concatenating Read 1 and the reverse complement of Read 2 and separated by 50 Ns. This was to identify the break between microreads. The output was filtered for redundant sequences (identity, 95%) to a single unique microread using the program cd-hit-454 (Niu et al., 2010). A stringent filter was then applied to identify microreads with microsatellites located near the center of the sequence; this yielded the largest possible flanking sequences for subsequent primer design (Jennings et al., 2011). The filtered SSR-containing singleton cluster and contig sequences were then evaluated with BatchPrimer3 (You et al., 2008) to identify PCR primer sequences. Default settings were used except for product size, which was increased to a maximum of 300 bp.

Initial primer screening.

SSR primers (384 of 1504) designed from P. lactiflora sequences were ordered from Integrated DNA Technologies (IDT, San Diego, CA). These primers were screened on 3% agarose gel using DNA from four Chinese cultivars, Yin Long Han Zhu, Zhu Guang, Fen Yu Nu, and Zi Hong Kui, from the field collection at NCGR for amplification and PCR product size. Seventy-two primer pairs that appeared polymorphic by 3% agarose gel electrophoresis were screened further using these four cultivars with the Beckman Coulter CEQ 8000 capillary electrophoresis. The M13 sequence TGTAAAACGACGGCCAGT was added to the 5′ end of each of the forward primers. Then the M13 tagged forward primer, a universal fluorescent-labeled M13(-21) forward primer (WellRed D2, D3, or D4), and a reverse primer were ordered from IDT to allow economic fluorescent labeling of PCR products following the procedure outlined by Schuelke (2000). The 28 primers that were developed in P. ×suffruticosa were also tested after the addition of the M13 tag to the 5′ end of the forward primer (Homolka et al., 2010; Wang et al., 2009; Yuan et al., 2010).

Fingerprinting.

Leaf samples from 93 unique peony cultivars from Adelman Peony Gardens, Salem, OR, were sampled (Table 2). Samples consisted of 15 tree peony cultivars (moutan types) with one tree cultivar duplicated; 61 herbaceous peony cultivars with two herbaceous cultivars duplicated; 15 intersectional hybrids (Itohs) samples; and two species. Each leaf sample weighed between 33 mg and 50 mg and was placed in a cluster tube (Corning, Tewsbary, MA). The cluster tubes were frozen in liquid nitrogen and stored at a –80 °C until extraction. The DNA extraction protocols for SSR fingerprinting and cultivar identification were performed using an Omega Plant DNA extraction kit 96 well format (Gilmore et al., 2011).

Table 2.

Pedigrees, types, and breeders for 93 peony (Paeonia) genotypes.z

Table 2.Table 2.

Thermocycler amplification of all the M13 tagged SSRs was performed with a touchdown program (PMTD52) using an initial denaturing step of 94 °C for 3 min, then 10 cycles of 94 °C for 40 s, 62 °C for 45 s (lowering the annealing temperature –1.0 °C per cycle), and 72 °C for 45 s followed by 20 cycles of 94 °C for 40 s, 52 °C for 45 s, and 72 °C for 45 s; eight cycles of 94 °C for 40 s, 53 °C for 45 s, 72 °C for 45 s; and a final extension of 72 °C for 30 min. The 15-μL PCR reaction mix contained: 3 μL of GoTaq DNA Polymerase Buffer (Promega, Madison, WI), 5×; 1.2 μL of 2.5 mm dNTPs; 1.2 μL of 25 mm MgCl2; 0.075 μL of 5 U/μL GoTaq DNA polymerase; 0.18 μL of 10 μM forward primer; 0.75 μL of 10 μM reverse primer; 0.75 μL of 10 μM M13 fluorescent tag, WellRed D2, D3, or D4; and 1.5 μL of 3 ng·μL−1 template DNA.

The resultant PCR products were separated by 3% agarose gel electrophoresis at 90 V for 150 min and then examined for product size and amplification. Primers that had bands in the 90- to 500-bp range were screened with capillary electrophoresis (CEQ 8000; Beckman Coulter) using the first four cultivars on the DNA plate, Cherry Ruffles, Do Tell, Brightness, and Sunny Girl. The Beckman Coulter CEQ software was used for estimating fragment size, ease of scoring, and polymorphism. PCR products generated from 21 polymorphic SSRs (Table 3) were pooled into nine multiplexes to fingerprint 93 peony samples.

Table 3.

List of 21 simple sequence repeat primer pairs (SSRs) evaluated in 93 peony individuals.z

Table 3.

PowerMarker Version 3.25 (Liu and Muse, 2005) was used for cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) algorithm. The bootstrap function was used at 1000 reiterations. Shannon’s-Wiener’s index (H) (Spellerberg and Fedor, 2003) was calculated using Excel 2007 (Microsoft, Redmond, WA).

Results and Discussion

Simple sequence repeat primers.

As many as 9657 SSR primers were designed from the seven peony libraries (Table 1). The total number of reads generated was 48,457,692; which was comprised of 48,157,663 barcoded reads and 300,029 non-barcoded reads. The singleton sequence length was equal to 244 bp and the average contig length was 295 bp. This result supported previous studies that report a large number of SSR primers when using NGS platforms (Zalapa et al., 2012). Up to 368,303 SSRs were documented in 22 NGS publications compared with 8,332 SSRs reported in 71 publications that used Sanger sequencing (Zalapa et al., 2012). In this study, we generated 1504 SSR primer pairs from P. lactiflora, one of seven Paeonia Illumina libraries sequenced (Table 1). We tested 384 of these for amplification in four cultivars of herbaceous peonies. The rest of the dinucleotide primer pairs from P. lactiflora and other species sequenced in this study (almost 9400) are available for download at the USDA, ARS web site (USDA, 2012). These primers were generated at the Genome Database for Rosaceae (Sook et al., 2008). Sequence data will also be available for further mining of tri-, tetra-, or pentanucleotide SSRs or single nucleotide polymorphisms (National Center for Biotechnology Information, 2012).

We estimated that the Illumina library laboratory preparation time for these eight samples was ≈20 h and at a cost of $51.00 per sample using the TruSeq DNA sample reagents (Illumina), whereas short read sequence processing through primer design required 8 h. The cost of sequencing the libraries at CRGB, Corvallis, OR, was $1700 per lane. The approximate cost of primer development was $0.22 per primer pair, number of primer pairs divided by our costs, but not including the cost of labor for library preparation.

We first tested 384 primer pairs by agarose gel electrophoresis, and 230 produced polymorphic DNA fragments that ranged from 72 to 500 bp in size; 17 were questionable; and 137 failed to produce a product or generated a product that exceeded 500 bp in size and so were discarded. We then selected 12 SSRs (Table 3) that were polymorphic by capillary electrophoresis in addition to nine previously reported SSRs (Homolka et al., 2010; Wang et al., 2009) to evaluate in 93 herbaceous, tree, and intersectional peonies.

Diversity parameters were calculated in the 93 accessions including number of alleles per primer pair (A), H index per locus (Spellerberg and Fedor, 2003), and group-specific alleles. These parameters were also calculated in each of the three groups, herbaceous, intersectional (Itohs), and tree peonies. The average number of alleles, A, in the 93 individuals was 26.9. It ranged from 12.2 per primer pair in Itohs and 13.2 alleles in tree peonies to 21.6 in herbaceous individuals. The number of alleles in the 93 samples ranged from five at Pae28 to 65 alleles at Pdel06. The average number of alleles varied between the sections. The herbaceous peonies always had more alleles than did section moutan or the hybrids, except for primer pair AT8051, which had 17 alleles for the intersectional group, 13 alleles for the tree group, but only 11 for the herbaceous group. Pae28 had the same amount of alleles, four, for both the tree group and the herbaceous group but generated only two alleles in the Itoh group. The excessive number of alleles in the herbaceous peonies (61) may be explained by the large number of herbaceous peonies that we sampled as compared with only a few moutan (15) or Itoh (15) specimens in this study. Furthermore, a high amount of genetic variation in the herbaceous peonies illustrated by a large number of alleles is expected given that many of these peonies are species hybrids. As more moutan and intersectional peonies genotypes are examined, the estimates of allelic frequency will likely increase for these two groups. Using a Student’s paired t test, the average number of alleles (A) in the herbaceous group was significantly higher (P < 0.001) than that found in Itohs or tree peonies (Table 4). No statistical significance in A was observed between intersectional and tree peonies. The H usually is used as a measure of species diversity (Bay et al., 2009; Wu et al., 2010), but we used it as a measure of primer diversity because the ploidy level was unknown for many of the peonies. The H index in the 93 samples ranged from 0.8 at Pae28 to 3.4 at Pdel06 and Pae100 with an average of 2.5. The average H was also higher in herbaceous peonies than that found in the other two groups but no statistical significance was found among the three groups (Table 4). H ranged from 0.8 at Pae28 to 3.3 at Pae100 with an average of 2.3 in the herbaceous group; 0.7 at Pae28 to 3.6 at Pdel06 with an average of 2.2 in the Itoh group; and 0.8 at Pae28 to 3.4 at Pdel06 and Pae100 with an average of 2.1. In this study, all but one primer pair, Pae28, had high levels of H (H 1.7 or greater). In Wedelia tribobata, H for 10 primer pairs that generated between two and five alleles in four populations ranged from 0.7 to 1.4 (Wu et al., 2010), whereas in coral endosymbiotic dinoflagellates, H in seven primer pairs evaluated in five populations of dinoflagellates ranged from 0.7 to 2.8 (Bay et al., 2009).

Table 4.

Peony primer pairs, alleles per peony type (A), Shannon-Weiner index (H) for the three types and the total peony population, and group-specific alleles.

Table 4.

The group-specific alleles varied from one allele (at Pae28 and Pae102) to 28 alleles (at ATG9706) with an average of 10.4 per primer pair for the herbaceous group. The intersectionals had the lowest number of group-specific alleles, which ranged from zero to two alleles with an average of 0.7 alleles per primer pair. The tree peonies had zero to 12 group-specific alleles with an average of 2.0 alleles per primer pair (Table 4).

All SSRs except for Pae28 were highly polymorphic and can be used to distinguish among unique peony cultivars. Pae28 had five alleles, but most cultivars had the same two alleles. In contrast, Pae100 had 58 alleles, which were more evenly distributed throughout the population. There was little genetic variability in the intersectional group as a result of the limited parent pool used in breeding. There was large variability in the herbaceous peonies, possibly as a result of the high number of cultivars used in our studies and the different species involved. The tree peony population was small but still had many unique alleles possibly resulting from the many species involved in hybridizing of the garden cultivars that were used in this study.

Analytic factors.

As expected for dinucleotide-containing SSRs, stutter was observed for most of the primers (Table 4). Another PCR artifact, split peaks, caused by incomplete non-templated addition of adenosine by Taq polymerase, was less common and found only at P05, Pae06, Pae28, and Pae110. These PCR artifacts render automated allele scoring challenging and raise the cost of genotyping by decreasing the number of PCR products that can be pooled for capillary electrophoresis separation and increasing the amount of time needed to score these alleles. Products from 10 SSRs were easy to score and 11 were mildly more challenging to score, but no primer kept for this fingerprinting set was rated as difficult. To further improve the primer products to assist ease of scoring, one could easily determine the optimal annealing temperature by gradient PCR or the reverse primers could be pigtailed by adding bases GTTT (Brownstein et al., 1996). We suspected some of the primer pairs to amplify multiple loci (ATG9706, P05, and Pae100) based on the large number of alleles generated even in diploid species. All observed alleles were scored in the peony samples because ploidy status was unknown in many of the cultivars. The intersectional hybrids are reported sterile and unable to set viable seed, which could be the result of differences in ploidy and triploid plants have been found in garden hybrids (Halda and Waddick, 2004). Cytometric analysis was beyond the scope of the present research but is needed to confirm ploidy levels for these samples and will facilitate allele calling for single-loci SSRs. Mapping these markers will allow identification of single and multiple-loci SSRs. An alternative to optimization of dinucleotide-containing markers is the development of SSRs that have longer core repeats and do not generate these PCR artifacts. We recommend using the sequences generated in this project to identify SSRs that contain larger core repeats.

Although the SSR markers can begin to determine relatedness of cultivars, this analysis needs to be examined in concordance with pedigree and by including founding clones. Forty-eight of the 93 peonies evaluated in this study had unknown or unspecified pedigrees and most parental types were not included.

Cluster analysis.

UPGMA cluster analysis separated the peonies into two groups: 1) the herbaceous; and 2) the moutan/Itoh. The herbaceous dendrogram subdivided into three major cultivar subgroups: P. officinalis, P. lactiflora, and P. lobata (Fig. 1). These groups were labeled by available pedigree information. Six cultivars did not group with any of these three herbaceous subgroups and they included: Sunny Girl, Campagna, Honored Guest, and Nosegay as well as May Lilac and Picotee, which were grouped together with high bootstrap support. These six genotypes were bred by A.P. Saunders or were the progeny of a Saunders-bred cultivar. Saunders experimented with unusual interspecific crosses, and this is likely the reason his peonies were separated from the other herbaceous peonies. These peonies have either P. daurica ssp. macrophylla or P. daurica ssp. mlokosewitschii in their backgrounds (Burkhardt, 2012).

Fig. 1.
Fig. 1.

Unweighted pair group method with arithmetic mean dendrogram of herbaceous “h” peonies included in this study. Bootstrap support of 50 or greater is indicated.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 1; 10.21273/JASHS.138.1.64

Subgroup P. officinalis consists of ‘Salmon Beauty’, replicated, P. officinalis, ‘Chocolate Soldier’, ‘Red Satin’, ‘Red Charm’, ‘Rose Heart’, and ‘Pink Teacup’. The bootstrap value for the ‘Salmon Beauty’/P. officinalis was 100. Although ‘Salmon Beauty’, ‘Chocolate Soldier’, ‘Red Charm’, and ‘Pink Teacup’ had P. officinalis registered in their pedigree; ‘Red Satin’ and ‘Rose Heart’ did not. ‘Rose Heart’ was listed as a P. lactiflora and ‘Red Satin’ had an unknown pedigree; it is likely that both have P. officinalis in their linage. Bootstrap support was found for ‘Chocolate Soldier’ and ‘Red Satin’ (69).

The largest subgroup of the dendrogram, P. lactiflora, contained 39 of the 61 herbaceous samples. It was composed of 25 peonies with no pedigree information (Burkhardt, 2012), five with one known parent and nine in which both of the parents were registered. The only patterns that appear are that four cultivars have ‘Monsieur Jules Elie’ as one parent and two cultivars have ‘Charle’s White’ as a parent. Further sampling of additional potential founding species might better resolve relationships in this group.

The third herbaceous subgroup, P. lobata, contained ‘Pastelegance’, ‘Salmon Dream’, ‘Coral Sunset’, ‘Coral Tide’, ‘Mary Jo Legare’, ‘Brightness’, ‘Prairie Moon’, ‘Ann Berry Cousins’, ‘Blaze’, and ‘Cherry Ruffles’ replicated. Six of these cultivars have P. lobata in their pedigree, ‘Pastelegance’, ‘Salmon Dream’, ‘Mary Jo Legare’, ‘Prairie Moon’, ‘Ann Berry Cousins’, and ‘Blaze’, but this name is a nomen nudum and may refer to P. officinalis, P. broteri, or P. peregrina (Hong, 2010). Because of the ambiguity of P. lobata, commonality among the parents made relationships difficult to identify, P. lobata may or may not refer to the same founding species in each case. Seven of the cultivars in this group have P. officinalis listed in their pedigree (Burkhardt, 2012) explaining why these peonies grouped together and includes ‘Pastelegance’, ‘Salmon Dream’, ‘Coral Sunset’, ‘Mary Jo Legare’, ‘Brightness’, ‘Ann Berry Cousins’, and ‘Cherry Ruffles’. Bootstrap support was found for ‘Blaze’ and ‘Cherry Ruffles’ (67).

The tree and intersectional types grouped together in the moutan/intersectional group (Fig. 2). This group separated into four subgroups and several stand-alone peonies. Subgroups were labeled with the name(s) of the breeder(s) of the majority of cultivars in that subgroup. Paeonia section Moutan has eight species and one hybrid species. In the past, many tree peonies were referred to as P. lutea hybrids, but the taxon P. lutea was been submerged into P. delavayi (Hong, 2010). This complicates the relationships, because a P. lutea hybrid might imply a cross between P. lutea and P. delavayi or P. lutea and P. ×suffruticosa or any other tree species. If more pedigree information was available on the hybrid reference, deductions could more easily be made.

Fig. 2.
Fig. 2.

Unweighted pair group method with arithmetic mean dendrogram of the moutan “t” and Itoh “I” peonies analyzed. Bootstrap support of 50 or greater is indicated.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 138, 1; 10.21273/JASHS.138.1.64

The Saunders/Daphnis subgoup had only four tree peonies, ‘Renoun’, ‘Vesuvian’, ‘Boreas’, and ‘Hephestos’. Little pedigree information is available (Burkhardt, 2012), but this relationship might indicate that Daphnis used Saunders’ peonies as parents or possibly both used the same species for their crosses.

Subgroup Anderson is comprised only of Itohs: ‘Copper Kettle’, ‘Canary Brilliants’, ‘First Arrival’, ‘Ballarena de Saval’ (bred by Tolomeo), ‘Bartzella’, ‘Julia Rose’, ‘Pastel Splendor’, ‘Hillary’, and Don Smith’s two unnamed hybrids. Six of these peonies have ‘Martha W.’ as the P. lactiflora parent (Table 2). ‘Martha W.’ may be a chance seedling of ‘Monsieur Jules Elie’ (Burkhardt, 2012). The information available for the pollen parents is limited because none of the pollen parents were included in our study. We do know that most of these hybrids were either bred by Reath or Daphnis or are the progeny of Daphnis’ or Saunders’ cultivars. We also observed that many of these intersectionals display red or reddish flares, a trait associated with P. rockii.

Subgroup Hollingsworth contained one tree peony, ‘Alice Harding’, and three intersectionals, ‘Border Charm’, ‘Garden Treasure’, and ‘Love Affair’. All three of these intersectionals have the same pollen parent, ‘Alice Harding’; ‘Border Charm’ and ‘Garden Treasure’ are pod siblings. Strong bootstrap support was found for this subgroup (Fig. 2).

The Saunders subgroup was composed of four tree peonies: ‘Spring Carnival’, ‘Banquet’, ‘Hesperus’, and ‘Pluto’ (bred by Daphnis). This section was comprised of yellow- and red-colored peonies with some having red and purple flares at the petal attachment point. Once again Saunders’ material was separated from peonies developed by other breeders.

The study objectives of developing SSRs using new technology platforms and distinguishing cultivars were accomplished. The sequence data generated in the study was successfully used to develop SSR markers. Sequences of the herbaceous peony ‘Bowl of Beauty’ produced 1504 potential SSR markers and more SSRs could easily be designed. The nine SSRs obtained from previous studies (Homolka et al., 2010; Wang et al., 2009; Yuan et al., 2010) were also polymorphic in our herbaceous peonies. Using these SSRs, we were able to distinguish among each of the unique cultivars evaluated in this study.

Published pedigree information for peonies is obscure because some breeding information is often confidential and in other cases information may be lost or breeding records not kept (Burkhardt, 2012). This lack of information hampered our relationship determination. Additional molecular studies should contrast pure species and derived genotypes. The raw data from this study will be available at the sequence read archive (http://www.ncbi.nlm.nih.gov/sra) at the National Center for Biotechnology Information and the accession number is: SRA054037. These resources can be used to develop additional microsatellite or single nucleotide polymorphic markers for further molecular studies.

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  • GilmoreB.HummerK.BassilN.2011DNA Extraction protocols from dormant buds of twelve woody plant generaJ. Amer. Pomol. Soc.65201207

  • HaldaJ.WaddickJ.2004The genus Paeonia. Timber Press Portland OR/Heartland Peony Soc. Gladstone MO

  • HardingA.1917The book of the peony. Lippincott Philadelphia PA/London UK

  • HomolkaA.BerenyiM.BurgK.KopeckyD.FluchS.2010Microsatellite markers in the tree peony, Paeonia ×suffruticosa (Paeoniaceae)Amer. J. Bot.97e42e44

    • Search Google Scholar
    • Export Citation
  • HongD.2010Peonies of the world taxonomy and phytogeography. Kew Publishing Richmond UK

  • HsuH.ChenY.ShenS.HsuS.ChenC.ChangH.1986Oriental material medica: A concise guide. Oriental Healing Arts. Inst. Keelung Taiwan

  • JakubowskiR.(compiler)2012The Canadian Peony Society, peony parentage data1 June 2012. <http://www.peony.ca/assets/pdf/peonyparentsweb.pdf>

    • Export Citation
  • JakubowskiR.HollingsworthD.NordickJ.BuchiteH.SchroerC.2007Peonies 1997–2007Amer. Peony Soc. Gladston MO

  • JenningsT.KnausB.MullinsT.HaigS.CronnR.2011Multiplexed microsatellite recovery using massively parallel sequencingMol. Ecol. Notes1110601067

    • Search Google Scholar
    • Export Citation
  • KnausB.2011Short read toolbox1 Oct. 2012. <http://brianknaus.com/software/srtoolbox/shortread.html>

    • Export Citation
  • La Pivoinerie D′Aoust Peony Nursery2012Learn about peonies1 May 2012. <http://www.paeonia.com/html/peonies/about.htm#5.QCJ0P1J0>

    • Export Citation
  • LiL.ChengF.ZhangQ.2011Microsatellite markers for the chinese herbaceous peony Paeonia lactiflora (Paeoniaceae)Amer. J. Bot.98e16e18

  • LiuK.MuseS.2005PowerMarker: An integrated analysis environment for genetic marker analysisBioinformatics2121282129

  • National Center for Biotechnology Information2012Accession number: SRA05403729 June 2012. <http://www.ncbi.nlm.nih.gov/sra>

    • Export Citation
  • NiuB.FuL.SunS.LiW.2010Artificial and natural duplicates in pyrosequencing reads of metagenomic dataBMC Bioinformatics11187

  • PowellW.MachrayG.ProvanJ.1996Polymorphism revealed by simple sequence repeatsTrends Plant Sci.1215222

  • RogersA.1995Peonies. Timber Press Portland OR

  • RogersA.2004Peonies. Timber Press Portland OR

  • RojasG.MéndezM.MunbozC.LemusG.HinrichsenP.2008Identification of a minimal microsatellite marker panel for the fingerprinting of peach and nectarine cultivarsElectron. J. Biotechnol.11112

    • Search Google Scholar
    • Export Citation
  • SangerF.NicklenS.CoulsonA.1977DNA sequencing with chain-terminating inhibitorsProc. Natl. Acad. Sci. USA7454635467

  • SchuelkeM.2000An economic method for the fluorescent labeling of PCR fragmentsNat. Biotechnol.18233234

  • SmithD.2000Producing high quality intersectional hybridsPaeonia3014

  • SookJ.StatonM.LeeT.BlendaA.SvancaraR.AbbottA.MainD.2008GDR (Genome Database for Rosaceae): Integrated web-database for Rosaceae genomics and genetics dataNucleic Acids Res.36D1034D1040

    • Search Google Scholar
    • Export Citation
  • SpellerbergI.FedorP.2003A tribute to Claude Shannon (1916–2001) and a plea for more rigorous use of species richness, species diversity and the ‘Shannon-Wiener’ indexGlob. Ecol. Biogeogr.12177179

    • Search Google Scholar
    • Export Citation
  • SunJ.YuanJ.WangB.PanJ.ZhangD.2011Development and characterization of 10 microsatellite loci in Paeonia lactiflora Pall. (Paeoniaceae)Amer. J. Bot.98e242e243

    • Search Google Scholar
    • Export Citation
  • U.S. Department of Agriculture2012SSR summary report15 June 2012. <http://www.ars.usda.gov/sp2UserFiles/Place/53581500/Paeonia.SSRs.files.xlsx>

    • Export Citation
  • Vakblad voor de Bloemisterji2012De meest complete international vakbeurs voor de snijbloemen en bioeiende pot planten! 1 Feb. 2012. <http://www.vakbladvoordebloemisterij.nl/home/artikelen/6428/aanvullingen-bij-nummer-23-2010>

  • WahdanM.AbdelsalamA.El-NaggarA.HusseinM.2011Preliminary horticultural studies to describe and identify of two new egyptian mango strains using DNA fingerprintJ. Amer. Sci.7641650

    • Search Google Scholar
    • Export Citation
  • WangJ.XisT.ZhangJ.ZhouS.2009Isolation and characterization of fourteen microsatellites from a tree peony (Paeonia ×suffruticosa)Conserv. Genet.1010291031

    • Search Google Scholar
    • Export Citation
  • WuW.ZhouR.HuangH.GeX.2010Development of microsatellite for the invasive weed Wedelia trilobata (Asteraceae)Amer. J. Bot.97e114e116

  • YanY.HuangY.FangX.LuL.ZhouR.GeX.ShiS.2011Development and characterization EST-SSR markers in the invasive weed Mikania micrantha (Asteracea)Amer. J. Bot.98e1e3

    • Search Google Scholar
    • Export Citation
  • YouF.HuoN.GuY.LuoM.MaY.HaneD.LazoG.DvorakJ.AndersonO.2008Batchprimer3: A high throughput web application for PCR and sequencing primer designBMC Bioinformatics9253

    • Search Google Scholar
    • Export Citation
  • YuanJ.ChengF.ZhouS.2010Hybrid origin of Paeonia yananensis revealed by microsatellite markers, chloroplast gene sequences, and morphological characteristicsIntl. J. Plant Sci.171409420

    • Search Google Scholar
    • Export Citation
  • ZalapaJ.CuevasH.ZhuH.SteffanS.SenalikD.ZeldinE.McCownB.HarbutR.SimonP.2012Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciencesAmer. J. Bot.99193208

    • Search Google Scholar
    • Export Citation
  • ZhangJ.ShuQ.LuiZ.RenH.WangL.De KeyserE.2012Two EST-derived marker systems for cultivar identification in tree peonyPlant Cell Rptr.31299310

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This work was supported by USDA-ARS CRIS 5341-21000-004-00D and 5358-21000-038-00D.We appreciate funding from ARS CRIS 5341-21000-004-00D and the generous donation of plant materials from Adelman Peony Gardens, Brooks, OR, and Jim Oliphant, Corvallis, OR. The Illumina sequencing was performed at the Center for Genome Research and Biocomputing at Oregon State University, Corvallis, OR. We are indebted to Caprice Rosato, Mark Dasenko, and Chris Sullivan for their technical advice on the bioanalyzer, sequencing, and core facility computing support. We greatly appreciate the support of the Alaska Peony Growers Association for their support of this project. We appreciate Pat Holloway and Barbara Reed for critical review of this manuscript. We are grateful to Charlotte Boches, Jeremy Jones, and Estefania Elorriaga for their assistance with field and laboratory work.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.Dedicated to the memory of Charlotte (Charlie) Boches, 1987–2011, for all she will never do.

Corresponding author. E-mail: kim.hummer@ars.usda.gov.

  • View in gallery

    Unweighted pair group method with arithmetic mean dendrogram of herbaceous “h” peonies included in this study. Bootstrap support of 50 or greater is indicated.

  • View in gallery

    Unweighted pair group method with arithmetic mean dendrogram of the moutan “t” and Itoh “I” peonies analyzed. Bootstrap support of 50 or greater is indicated.

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  • GilmoreB.HummerK.BassilN.2011DNA Extraction protocols from dormant buds of twelve woody plant generaJ. Amer. Pomol. Soc.65201207

  • HaldaJ.WaddickJ.2004The genus Paeonia. Timber Press Portland OR/Heartland Peony Soc. Gladstone MO

  • HardingA.1917The book of the peony. Lippincott Philadelphia PA/London UK

  • HomolkaA.BerenyiM.BurgK.KopeckyD.FluchS.2010Microsatellite markers in the tree peony, Paeonia ×suffruticosa (Paeoniaceae)Amer. J. Bot.97e42e44

    • Search Google Scholar
    • Export Citation
  • HongD.2010Peonies of the world taxonomy and phytogeography. Kew Publishing Richmond UK

  • HsuH.ChenY.ShenS.HsuS.ChenC.ChangH.1986Oriental material medica: A concise guide. Oriental Healing Arts. Inst. Keelung Taiwan

  • JakubowskiR.(compiler)2012The Canadian Peony Society, peony parentage data1 June 2012. <http://www.peony.ca/assets/pdf/peonyparentsweb.pdf>

    • Export Citation
  • JakubowskiR.HollingsworthD.NordickJ.BuchiteH.SchroerC.2007Peonies 1997–2007Amer. Peony Soc. Gladston MO

  • JenningsT.KnausB.MullinsT.HaigS.CronnR.2011Multiplexed microsatellite recovery using massively parallel sequencingMol. Ecol. Notes1110601067

    • Search Google Scholar
    • Export Citation
  • KnausB.2011Short read toolbox1 Oct. 2012. <http://brianknaus.com/software/srtoolbox/shortread.html>

    • Export Citation
  • La Pivoinerie D′Aoust Peony Nursery2012Learn about peonies1 May 2012. <http://www.paeonia.com/html/peonies/about.htm#5.QCJ0P1J0>

    • Export Citation
  • LiL.ChengF.ZhangQ.2011Microsatellite markers for the chinese herbaceous peony Paeonia lactiflora (Paeoniaceae)Amer. J. Bot.98e16e18

  • LiuK.MuseS.2005PowerMarker: An integrated analysis environment for genetic marker analysisBioinformatics2121282129

  • National Center for Biotechnology Information2012Accession number: SRA05403729 June 2012. <http://www.ncbi.nlm.nih.gov/sra>

    • Export Citation
  • NiuB.FuL.SunS.LiW.2010Artificial and natural duplicates in pyrosequencing reads of metagenomic dataBMC Bioinformatics11187

  • PowellW.MachrayG.ProvanJ.1996Polymorphism revealed by simple sequence repeatsTrends Plant Sci.1215222

  • RogersA.1995Peonies. Timber Press Portland OR

  • RogersA.2004Peonies. Timber Press Portland OR

  • RojasG.MéndezM.MunbozC.LemusG.HinrichsenP.2008Identification of a minimal microsatellite marker panel for the fingerprinting of peach and nectarine cultivarsElectron. J. Biotechnol.11112

    • Search Google Scholar
    • Export Citation
  • SangerF.NicklenS.CoulsonA.1977DNA sequencing with chain-terminating inhibitorsProc. Natl. Acad. Sci. USA7454635467

  • SchuelkeM.2000An economic method for the fluorescent labeling of PCR fragmentsNat. Biotechnol.18233234

  • SmithD.2000Producing high quality intersectional hybridsPaeonia3014

  • SookJ.StatonM.LeeT.BlendaA.SvancaraR.AbbottA.MainD.2008GDR (Genome Database for Rosaceae): Integrated web-database for Rosaceae genomics and genetics dataNucleic Acids Res.36D1034D1040

    • Search Google Scholar
    • Export Citation
  • SpellerbergI.FedorP.2003A tribute to Claude Shannon (1916–2001) and a plea for more rigorous use of species richness, species diversity and the ‘Shannon-Wiener’ indexGlob. Ecol. Biogeogr.12177179

    • Search Google Scholar
    • Export Citation
  • SunJ.YuanJ.WangB.PanJ.ZhangD.2011Development and characterization of 10 microsatellite loci in Paeonia lactiflora Pall. (Paeoniaceae)Amer. J. Bot.98e242e243

    • Search Google Scholar
    • Export Citation
  • U.S. Department of Agriculture2012SSR summary report15 June 2012. <http://www.ars.usda.gov/sp2UserFiles/Place/53581500/Paeonia.SSRs.files.xlsx>

    • Export Citation
  • Vakblad voor de Bloemisterji2012De meest complete international vakbeurs voor de snijbloemen en bioeiende pot planten! 1 Feb. 2012. <http://www.vakbladvoordebloemisterij.nl/home/artikelen/6428/aanvullingen-bij-nummer-23-2010>

  • WahdanM.AbdelsalamA.El-NaggarA.HusseinM.2011Preliminary horticultural studies to describe and identify of two new egyptian mango strains using DNA fingerprintJ. Amer. Sci.7641650

    • Search Google Scholar
    • Export Citation
  • WangJ.XisT.ZhangJ.ZhouS.2009Isolation and characterization of fourteen microsatellites from a tree peony (Paeonia ×suffruticosa)Conserv. Genet.1010291031

    • Search Google Scholar
    • Export Citation
  • WuW.ZhouR.HuangH.GeX.2010Development of microsatellite for the invasive weed Wedelia trilobata (Asteraceae)Amer. J. Bot.97e114e116

  • YanY.HuangY.FangX.LuL.ZhouR.GeX.ShiS.2011Development and characterization EST-SSR markers in the invasive weed Mikania micrantha (Asteracea)Amer. J. Bot.98e1e3

    • Search Google Scholar
    • Export Citation
  • YouF.HuoN.GuY.LuoM.MaY.HaneD.LazoG.DvorakJ.AndersonO.2008Batchprimer3: A high throughput web application for PCR and sequencing primer designBMC Bioinformatics9253

    • Search Google Scholar
    • Export Citation
  • YuanJ.ChengF.ZhouS.2010Hybrid origin of Paeonia yananensis revealed by microsatellite markers, chloroplast gene sequences, and morphological characteristicsIntl. J. Plant Sci.171409420

    • Search Google Scholar
    • Export Citation
  • ZalapaJ.CuevasH.ZhuH.SteffanS.SenalikD.ZeldinE.McCownB.HarbutR.SimonP.2012Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciencesAmer. J. Bot.99193208

    • Search Google Scholar
    • Export Citation
  • ZhangJ.ShuQ.LuiZ.RenH.WangL.De KeyserE.2012Two EST-derived marker systems for cultivar identification in tree peonyPlant Cell Rptr.31299310

    • Search Google Scholar
    • Export Citation
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