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.
Barbara Gilmore, Nahla Bassil, April Nyberg, Brian Knaus, Don Smith, Danny L. Barney, and Kim Hummer
Barbara S. Gilmore, Nahla V. Bassil, Danny L. Barney, Brian J. Knaus, and Kim E. Hummer
Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.