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.
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