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Phillip A. Wadl, Mark T. Windham, Richard Evans, and Robert N. Trigiano

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Phillip A. Wadl, Timothy A. Rinehart, Adam J. Dattilo, Mark Pistrang, Lisa M. Vito, Ryan Milstead, and Robert N. Trigiano

Pityopsis ruthii is an endangered species endemic to the Hiwassee and Ocoee Rivers in Tennessee. As part of a recovery effort focused on P. ruthii, vegetative propagation and in vitro multiplication and seed germination techniques were developed. Plants were vegetatively propagated using greenhouse stock plants and wild-collected stems. Rooting occurred with and without auxin treatments but was greatest when 0.1% indole-3-butyric acid (IBA) talc was applied to the vegetative cuttings; rooting was lowest when flowering stems were used. Pro-Mix BX substrate provided the most consistent rooting. In vitro multiplication was accomplished by the removal of lateral shoots from in vitro-grown plants that were rooted on Murashige and Skoog (MS0) basal medium with 270 clones produced from a single individual after 4 months. Nineteen clones were transplanted and secured with bonded fiber matrix into their natural habitat and 14 survived for 1 year. To avoid genetic swamping of native populations with the introduction of large numbers of genetically identical individuals through clonal propagation, seed-based propagation efforts were explored. Open-pollinated seeds were collected, disinfested and germinated, and seedlings established on MS medium. Seeds were submersed in 70% ethanol for 1 minute and briefly flamed. Seeds were surface-sterilized in a range [10% to 50% (v/v)] Clorox® bleach solutions with vigorous shaking for 20 minutes, rinsed three times in sterile water, and germinated on MS0. Removal of pappus from seeds was required for successful disinfestations, but the bleach concentration was not critical. Successful propagation is a step toward the conservation and recovery of P. ruthii and should allow future reintroduction projects.

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Phillip A. Wadl, Xinwang Wang, John K. Moulton, Stan C. Hokanson, John A. Skinner, Timothy A. Rinehart, Sandra M. Reed, Vincent R. Pantalone, and Robert N. Trigiano

Cross-species transferability of simple sequence repeats (SSRs) is common and allows SSRs isolated from one species to be applied to closely related species, increasing the use of previously isolated SSRs. The genus Cornus consists of 58 species that are ecologically and economically important. SSRs have previously been isolated from C. florida and C. kousa. In this study, 36 SSRs were tested on taxa from 18 Cornus species and hybrids for cross-species transferability and genetic diversity was calculated for each locus using polymorphism information content (PIC). Cross-species transferability of SSR loci was higher in more closely related species and PIC values were high. Evidence was found for conserved primer sites as determined by the amplification of SSR loci in the taxa examined. Polymerase chain reaction products were cloned and sequenced for three SSR loci (CF48, CF59, and CF124) and all individuals sequenced contained the appropriate repeat. Phylogenetic relationships of 14 Cornus species were inferred using nucleotide sequences of SSR locus CF48. The most parsimonious tree resulting from this analysis was in concordance with phylogenies based on matK and internal transcribed spacer sequences. The SSR loci tested in this study will be useful in future breeding, population, and genetic studies within Cornus.

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Phillip A. Wadl, John A. Skinner, John R. Dunlap, Sandra M. Reed, Timothy A. Rinehart, Vincent R. Pantalone, and Robert N. Trigiano

Flowering (Cornus florida L.) and kousa (C. kousa Hance) dogwoods are ornamental trees valued for their four-season appeal, but also for their importance to retail and wholesale nurseries. The popularity of kousa dogwood has increased in recent years as a result of its resistance to dogwood anthracnose and powdery mildew as compared with flowering dogwood, which is typically susceptible to those diseases. This range of resistance allows the development of intra- and interspecific cultivars with multiple disease resistance or a combination of disease resistance and specific ornamental traits. Breeding requires controlled crosses that are usually done manually, which is a labor-intensive process. Cornus florida and C. kousa have generally been found to be self-incompatible allowing for the breeding process to be made more efficient by not having to emasculate flowers. We have capitalized on the natural ability of honeybees and the self-incompatible nature of dogwood to perform self- and crosspollinations of flowering and kousa dogwood. Self-pollinations were conducted in 2006 and 2007 with C. florida ‘Appalachian Spring’ and ‘Cherokee Brave’ and with C. kousa ‘Blue Shadow’ and Galilean®. The flowering dogwood self-pollinations resulted in no seed production, whereas the kousa dogwood self-pollinations resulted in low seed production, indicating self-incompatibility. Intra- and interspecific crosses of flowering and kousa dogwood cultivars and breeding lines were conducted in 2006 to 2008. Honeybees were effective in facilitating seed production for all intraspecific crosses conducted. Seedling phenotypes of putative intra- and interspecific hybrids are similar and practically indistinguishable, so dogwood-specific simple sequence repeats were used to verify a sample of the putative hybrids. The results demonstrated that honeybees were effective in performing controlled pollinations and that honeybee-mediated pollinations provide an alternative to time-consuming hand pollinations for flowering and kousa dogwood.

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Phillip A. Wadl, Xinwang Wang, Andrew N. Trigiano, John A. Skinner, Mark T. Windham, Robert N. Trigiano, Timothy A. Rinehart, Sandra M. Reed, and Vincent R. Pantalone

Flowering dogwood (Cornus florida) and kousa dogwood (C. kousa) are popular ornamental species commonly used in the horticultural industry. Both trees are valued for their beautiful floral display and four-season appeal. Species-specific simple sequence repeat (SSR) loci were used to genotype and assess genetic diversity of 24 flowering dogwood cultivars and breeding lines and 22 kousa dogwood cultivars. Genetic diversity was determined by allele sharing distances and principal coordinate analysis and was high in both species. Molecular identification keys were developed for cultivars and breeding lines of each species using a few polymorphic SSRs loci (four in C. florida and five in C. kousa). Most (18 of 24) of the flowering dogwood and all (22 of 22) kousa dogwood accessions could be distinguished from each other using these SSRs; those that could not were resolved using DNA amplification fingerprinting. The reliability of both keys was assessed using five anonymous cultivars for each dogwood species, which were correctly identified using the molecular keys. The genetic information presented here will be useful for identification and verification of cultivars for nurseries and as molecular markers for breeders and researchers.

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Xinwang Wang, Phillip A. Wadl, Cecil Pounders, Robert N. Trigiano, Raul I. Cabrera, Brian E. Scheffler, Margaret Pooler, and Timothy A. Rinehart

Genetic diversity was estimated for 51 Lagerstroemia indica L. cultivars, five Lagerstroemia fauriei Koehne cultivars, and 37 interspecific hybrids using 78 simple sequence repeat (SSR) markers. SSR loci were highly variable among the cultivars, detecting an average of 6.6 alleles (amplicons) per locus. Each locus detected 13.6 genotypes on average. Cluster analysis identified three main groups that consisted of individual cultivars from L. indica, L. fauriei, and their interspecific hybrids. However, only 18.1% of the overall variation was the result of differences between these groups, which may be attributable to pedigree-based breeding strategies that use current cultivars as parents for future selections. Clustering within each group generally reflected breeding pedigrees but was not supported by bootstrap replicates. Low statistical support was likely the result of low genetic diversity estimates, which indicated that only 25.5% of the total allele size variation was attributable to differences between the species L. indica and L. fauriei. Most allele size variation, or 74.5%, was common to L. indica and L. fauriei. Thus, introgression of other Lagestroemia species such as Lagestroemia limii Merr. (L. chekiangensis Cheng), Lagestroemia speciosa (L.) Pers., and Lagestroemia subcostata Koehne may significantly expand crapemyrtle breeding programs. This study verified relationships between existing cultivars and identified potentially untapped sources of germplasm.

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Deborah Dean, Phillip A. Wadl, Denita Hadziabdic, William E. Klingeman, Bonnie H. Ownley, Timothy A. Rinehart, Adam J. Dattilo, Brian Scheffler, and Robert N. Trigiano

Viburnum rufidulum is a deciduous tree native to North America that has four-season appeal, which provides commercial horticultural value. In addition, the plant has unique and attractive red pubescence on leaf buds and petioles, common to no other Viburnum species. As habitat undergoes development and subsequent fragmentation of native plant populations, it is important to have baseline genetic information for this species. Little is known about the genetic diversity within populations of V. rufidulum. In this study, seven microsatellite loci were used to measure genetic diversity, population structure, and gene flow of 235 V. rufidulum trees collected from 17 locations in Kentucky and Tennessee. The genotype data were used to infer population genetic structure using the program InStruct and to construct an unweighted pair group method with arithmetic mean dendrogram. A single population was indicated by the program InStruct and the dendrogram clustered the locations into two groups; however, little bootstrap support was evident. Observed and expected heterozygosity were 0.49 and 0.78, respectively. Low-to-moderate genetic differentiation (F ST = 0.06) with evidence of gene flow (Nm = 4.82) was observed among 17 populations of V. rufidulum. A significant level of genetic diversity was evident among V. rufidulum populations with most of the genetic variations among individual trees (86.37%) rather than among populations (13.63%), and a Mantel test revealed significant correlation between genetic and geographical distance (r = 0.091, P = 0.001). The microsatellites developed herein provide an initial assessment or a baseline of genetic diversity for V. rufidulum in a limited area of the southeastern region of the United States. The markers are a genetic resource and can be of assistance in breeding programs, germplasm assessment, and future studies of V. rufidulum populations, as this is the first study to provide genetic diversity data for this native species.

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Deborah Dean, Phillip A. Wadl, Xinwang Wang, William E. Klingeman, Bonnie H. Ownley, Timothy A. Rinehart, Brian E. Scheffler, and Robert N. Trigiano

Viburnum dilatatum is a popular and economically important ornamental shrub. The wide range of desirable horticultural traits, paired with a propensity for seedlings to become invasive, has created interest in the genetics and breeding of this species. To investigate the genetic diversity of V. dilatatum, microsatellite loci were identified from a GT-enriched genomic library constructed from V. dilatatum ‘Asian Beauty’. Eleven microsatellite loci have been characterized on a group of 16 different related V. dilatatum cultivars and hybrids. Two to 12 alleles were identified per locus, and the polymorphism information content (PIC) values ranged from 0.36 to 0.87. Expected heterozygosity (He) ranged from 0.48 to 0.88 and observed heterozygosity (Ho) ranged from 0 to 0.73. This set of molecular markers also exhibited expected transferability between various V. dilatatum cultivars and two hybrids with V. japonicum. As a consequence, these markers will aid in breeding for new cultivar development, assist with early detection and screening of plants that have escaped cultivation, and are expected to help in refining the phylogenetic relationship of V. dilatatum to other species and genera within the Adoxaceae.