J. AMER.SOC.HORT.SCI. 138(1):64–74. 2013. Microsatellite Marker Development in Peony using Next Generation Sequencing Barbara Gilmore Formerly at USDA ARS Arctic and Subarctic Plant Gene Bank, 1509 South Georgeson Road, Palmer, AK 99645 Nahla Bassil and April Nyberg USDA ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333 Brian Knaus USDA Forest Service, 3200 SW Jefferson Way, Corvallis, OR 97331 Don Smith Yellow Peonies and More, West Newton, MA 02465 Danny L. Barney USDA ARS North Central Regional Plant Introduction Station, G212 Agronomy Building, Ames, IA 50011 Kim Hummer1 USDA ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333 ADDITIONAL INDEX WORDS. Paeonia lactiflora, Paeonia ·suffruticosa, intersectional, simple sequence repeat primers, Illumina libraries 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 (Harding, 1917). Peonies are heritage perennial flowers that hold a medicinal plant in Asia several thousand years ago (Hsu special cultural value in the United States. They are a traditional et al., 1986). In the late 1700s, peonies were imported from Asia Memorial Day cut flower and have become a popular wedding and Europe into North America for use as a garden flower 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. Received for publication 26 July 2012. Accepted for publication 27 Sept. 2012. This work was supported by USDA-ARS CRIS 5341-21000-004-00D and These flowers have been widely sold in European markets for 5358-21000-038-00D. centuries and were first sold in Chicago in 1884 (Rogers, 1995). We appreciate funding from ARS CRIS 5341-21000-004-00D and the generous Commercial production is found on every continent except donation of plant materials from Adelman Peony Gardens, Brooks, OR, and Jim Antarctica. Production areas in North America range from Alaska Oliphant, Corvallis, OR. The Illumina sequencing was performed at the Center for Genome Research and Biocomputing at Oregon State University, Corvallis, OR. and Canada in the North through northern California through the We are indebted to Caprice Rosato, Mark Dasenko, and Chris Sullivan for their center of the continent to North Carolina in the South. In 2009, technical advice on the bioanalyzer, sequencing, and core facility computing sup- world peony sales through Dutch auctions resulted in nearly 63 port. We greatly appreciate the support of the Alaska Peony Growers Association for million stems sold, valued at almost V24 million [$30 million their support of this project. We appreciate Pat Holloway and Barbara Reed for (Vakblad voor de Bloemisterji, 2012)]. critical review of this manuscript. We are grateful to Charlotte Boches, Jeremy Jones, and Estefania Elorriaga for their assistance with field and laboratory work. Jakubowski et al. (2007) listed the names and descriptions of Mention of a trademark, proprietary product, or vendor does not constitute 7995 peony cultivars worldwide. Many more cultivars have been a guarantee or warranty of the product by the U.S. Dept. of Agriculture and named each year since then. Peonies are generally recognized as does not imply its approval to the exclusion of other products or vendors that three distinct types. Herbaceous peonies are perennial plants that also may be suitable. Dedicated to the memory of Charlotte (Charlie) Boches, 1987–2011, for all she have soft, succulent, green stems that die back to the ground will never do. every fall. The crowns of the plants are below the surface of the 1Corresponding author. E-mail: [email protected]. ground and can survive extremely cold winter temperatures and 64 J. AMER.SOC.HORT.SCI. 138(1):64–74. 2013. resume growth in spring. These new shoots grow, flower, set the relationships between three tree species, P. yananensis, seed, and die at the end of the season. The tree peonies are P. jishanensis,andP. rockii (Yuan et al., 2010). Paeonia perennial plants that have woody stems above the ground at all yananensis was found to be a hybrid of P. jishanensis and times of the year. Their stem buds break in the spring and the P. rockii (Yuan et al., 2010). Eight additional SSRs were stems elongate over the years to form a bush 1 to 1.5 m in reported to cross-amplify in six Peonia species (Homolka height. Because of the exposed stems, tree peonies are unable to et al., 2010). In 2011, researchers used seven of 21 SSRs, survive temperatures as low as the herbaceous types. Intersec- developed from peony expressed sequence tags, in cultivar tional hybrids are crosses between the two groups. Intersec- identification of tree peonies (Zhang et al., 2012). Fewer tionals (Itohs) have a similar growth pattern to herbaceous SSRs have been developed for P. lactiflora where only 20 peonies, so they are able to withstand temperatures that would polymorphic SSRs are reported (Li et al., 2011; Sun et al., kill tree peonies, but their foliage and the flowers have the tree 2011). peony appearance (La Pivoinerie D#Aoust Peony Nursery, The objectives of this study were to develop new SSR 2012). Itohs are named for Toichi Itoh, who made the first markers using barcoded multiplexed libraries of multiple peony successful cross in 1948 between an herbaceous peony and tree species, and to evaluate these markers for fingerprinting peony (Rogers, 2004). herbaceous peony (P. lactiflora and hybrids), tree peony Currently, the standard way to identify cultivars requires (moutan), and intersectional (Itohs) individuals. 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 Methods and Materials incorrect sales. Adding to the complexity of identity determi- nation, growers attest that some cultivars produce variant PLANT MATERIALS. In early Spring 2010, leaf material was flower colors when grown in different regions or countries obtained from seven peony individuals from multiple sources. (D. Hollingsworth, personal communication). Growers may Leaves of P. lactiflora ‘Bowl of Beauty’, P. lutea · P. often wait two to 10 years for bloom appearance to confirm the ·suffruticosa hybrid ‘Souvenir de Maxime Cornu’, P. identity of planted stock. ·suffruticosa ‘Feng deng bai’, and P. delavayi were collected Identity determination of other horticultural crops such as from the U.S. Department of Agriculture (USDA), Agricultural blueberry [Vaccinium corymbosum (Boches et al., 2005)], Research Service (ARS), National Clonal Germplasm Repos- peach [Prunus persica (Rojas et al., 2008)], and mango itory (NCGR), Corvallis, OR. Samples from P. tenuifolia [Mangifera indica (Wahdan et al., 2011)] benefitted from using ‘Rubra Flora Plena’ and P. peregrina were obtained from SSRs as molecular markers for identity verification. SSRs are plants donated by Adelman Peony Gardens, Brooks, OR, easy to use, codominant, multiple allelic, abundant, and highly whereas leaves of P. rockii were obtained from J. Oliphant, reproducible across laboratories for genotype identification Corvallis, OR (Table 1). Leaf samples were collected, bagged, (Powell et al., 1996). Application of this technique to peony kept cool, and transported to the laboratory. Each leaf sample cultivars could simplify the identification process for growers was placed in a ceramic mortar and ground with liquid nitrogen. and allow identification of rhizomes or leaves at an early stage The ground leaf material was stored at –80 °C until extraction. of production. The DNA extraction protocol for the library preparation was To develop SSRs, many laboratories use the chain termina- performed using Qiagen (Valencia, CA) reagents (Gilmore tion method of DNA sequencing that was developed by Sanger in et al., 2011). 1975 (Sanger et al., 1977). This protocol entails construction of DNA SEQUENCING. The Illumina library preparation of the genomic libraries using enriched recombinant DNA (Boccacci DNA for sequencing included fragmentation of purified geno- et al., 2005; Boches et al., 2005; Castillo et al., 2010), resulting mic DNA. This was accomplished by sonication [Bioruptor in a procedure that is time- and labor-intensive and ultimately XL(BR_XL); Diagenode, Denville, NJ]. An aliquot of the DNA yields low numbers