White mold, (Sclerotinia sclerotiorum), is an aggressive pathogen of beans and is capable of inflicting devastating damage on yield. Finding resistance is a major concern to bean breeders. The scarlet runner bean (Phaseolus coccineus) is generally known to have greater resistance to white mold than does the common bean, (P. vulgaris). Since it is possible to cross these two species, we have started to examine the NPGS core collection of P. coccineus for resistance to this pathogen. A straw test was used to measure physiological resistance of bean stems to white mold. A rating of one equates to a small lesion, resulting from contact with inoculum, and a rating of nine describes total plant collapse. Controls that were used were two common beans, 91G, a commercially produced, blue lake type snap bean and ExRico, a small, white dry bean. The bean 91G received a straw test rating of 8.3, which correlates to a field test rating of 8.5. ExRico rated 7.4 with the straw test and had a field test score of 6.5. Within the P. coccineus collection we found very strong resistance, with straw test values of 1 and 2 in several individual plants and in some accessions. Accessions that had individuals that displayed the strongest resistance of all the plants tested were: PI201299, PI361302, PI406938, and PI535278. These accessions appeared to be segregating for white mold resistance. Accessions showing the best average resistance were: PI313221, PI361372, PI361539, and PI583553. Because P. coccineus is outcrossed, we expected to find variation within accessions for white mold resistance. Some accessions had uniformly high levels of resistance, while other accessions showed variability.
Barbara Gilmore and James R. Myers
Barbara Gilmore, Nahla Bassil, April Nyberg, Brian Knaus, Don Smith, Danny L. Barney, and Kim Hummer
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 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.
Michael Dossett, Jill M. Bushakra, Barbara Gilmore, Carol A. Koch, Chaim Kempler, Chad E. Finn, and Nahla V. Bassil
The advent of next-generation, or massively parallel sequencing technologies has been a boon to the cost-effective development of molecular markers, particularly in nonmodel species. Here, we demonstrate the efficiency of microsatellite or simple sequence repeat (SSR) marker development from short-read sequences in black and red raspberry (Rubus occidentalis L. and R. idaeus L., respectively), compare transferability of markers across species, and test whether the rate of polymorphism in the recovered markers can be improved upon by how marker sequences are chosen. From 28,536,412 black raspberry reads and 27,430,159 reads in red raspberry, we identified more than 6000 SSR sequences in each species and selected 288 of these (144 from each species), for testing in black and red raspberry. A total of 166 SSR primer pairs were identified with informative polymorphism in one or both species. SSRs selected based on different percentages (90% to 97% as compared with ≥98%) of read cluster similarity did not differ in polymorphism rates from each other or from those originating from singletons. Efficiency of polymorphic SSR recovery was nearly twice as high in black raspberry from black raspberry-derived sequences as from red raspberry-derived sequences, while efficiency of polymorphic SSR recovery in red raspberry was unaffected by the source of the primer sequences. Development of SSR markers that are transferable between red and black raspberry for marker-assisted selection, evaluation of genome collinearity and to facilitate comparative studies in Rubus L. will be more efficient using SSR markers developed from black raspberry sequences.