Ten cultivars of Cornus florida (Barton, Cherokee Princess, Cloud 9, First Lady, Fragrant Cloud, Plena, Purple Glory, Rubra, Springtime, and Welch's Jr. Miss) were evaluated for horticultural characters of canker incidence, susceptibility to spot anthracnose and freeze damage, bloom number, bract length, and phenology. Of the ten cultivars, only `Barton' and `Cloud 9' were similar for all characteristics studied. DNA amplification fingerprinting (12 standard primers) and Arbitrary Signatures from Amplification Profiles (four minihairpin primers) were used to test our hypothesis that `Barton' and `Cloud 9' were genetically distinct, but phenotypically similar. Polymorphisms were not observed; therefore, we could not reject the null hypothesis that `Barton' and `Cloud 9' are the same genotype.
Mark T. Windham and Robert N. Trigiano
Naomi R. Smith and Robert N. Trigiano
Flowering dogwood (Cornus florida L.) is an important tree of forests and urban landscapes in the eastern United States. Currently, there are over 100 cultivars of flowering dogwood commercially available. An identification process based on genotype would be of use to researchers, breeders, and nurserymen, as many cultivars are similar phenotypically. Molecular markers offer a promising way of definitively identifying flowering dogwood cultivars. Amplified fragment length polymorphism (AFLP) is a technique that can be used to generate DNA fingerprints. DNA was isolated from leaves of 17 common cultivars of dogwood and AFLP fingerprints were generated by a Beckman Coulter CEQ™ 8000. Fingerprints were converted to binary data and verified manually. Two drafts of a cultivar identification key were generated based on the corrected, verified binary data and cultivar-specific peaks. Six primer combinations were used to construct all keys and were tested with seven unknown dogwood cultivar samples. Six unknown samples were correctly identified using the keys. Only one unknown, `Cherokee Brave', was unidentifiable with any key. In all cases, some intracultivar variation was observed. A similarity index was calculated and visualized with a tree of genetic relatedness using NTSYSpc. Intracultivar variation was observed in the similarity index as well. This database for cultivar-specific molecular markers will serve as a starting point to which other cultivars can be added and also can be used in breeding applications, patent application and other projects, such as mapping the C. florida genome.
Denita Hadziabdic, Robert N. Trigiano, Stephen Garton and Mark T. Windham
Axillary and apical buds from five Cornus kousa cultivars (`Little Beauty', `Samaritan', `Heart Throb', `Rosabella', and `Christian Prince') were initially established on two basal media, woody plant medium (WPM) and woody plant medium/broad leaved tree medium (BW), amended with the following concentrations of 6–benzylaminopurine (BA): 0, 2, 4, and 8 μm. After explants were transferred at 4-week intervals for 28 weeks beginning in April, only microshoots of `Samaritan', `HeartThrob' and `Rosabella', were harvested from proliferating cultures and placed on rooting media. `Little Beauty' and `Christian Prince' did not perform well in multiplication phase of tissue culture and were excluded from further studies. Rooting media contained WPM or BW supplemented with either 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) at the following concentrations: 0, 0.5, 1.5, 4.5, and 13.5 μm. Six weeks following rooting experiment, preliminary data were collected and results showed that total of nine plants rooted on both WPM and BW media supplemented with IBA, 17 plants rooted on media supplemented with NAA, and 14 plants rooted on media supplemented with IAA. These results indicated that NAA and IAA appeared to be better for root production on C. kousa cultivar microshoots than IBA. Additionally, WPM supported more root production, when compared to BW, even though both media resulted in rooted microshoots. Proliferating masses were placed on fresh medium with 2μm BA and were used again for the rooting projects.
Robert N. Trigiano, Alan S. Windham, Mark T. Windham and Phillip A. Wadl
Denita Hadziabdic, Robert N. Trigiano, Stephen Garton, Mark T. Windham and William E. Klingeman
Axillary buds from a single Cladrastis kentukea tree were initially cultured on two media, woody plant medium (WPM) and Murashige and Skoog (MS) containing 0, 1, 2, or 4 μm 6–benzylaminopurine (BA). Cultures were transferred to fresh media every 4 weeks. Elongated shoots were harvested after 39 weeks and transferred to half-strength MS medium supplemented with the following concentrations of IBA: 0, 3, 30, 100, and 300 μm for 3 d, then returned to half-strength MS without growth regulators. Explants exposed to 300 μm of IBA produced significantly more roots (75%) compared to explants exposed to other treatments. Fifty-four and 45% of the microshoots rooted when exposed to 100 and 30 μm IBA, respectively. Only 4% of the microshoots rooted when exposed to 3 μm IBA and none of the control microshoots rooted. Although the 300 μm treatment yielded the most rooted plantlets, there was significantly higher terminal meristem abortion compared to other treatments. There were no statistical differences between the numbers of roots and total root length among all treatments. Additionally, all microshoots that rooted had lenticels, suggesting that presence of lenticel cambial activity can possibly improve rooting abilities of selected microshoots. Rooted microshoots were gradually acclimatized to nonsterile environment.
Justin A. Porter, Hazel Y. Wetzstein, David Berle, Phillip A. Wadl and Robert N. Trigiano
Georgia plume, Elliottia racemosa (Ericaceae), is a small tree endemic only to the state of Georgia, where it is listed as a threatened species. Information about genetic relatedness is critical for establishing approaches for safeguarding, reintroduction, and conservation of this rare species. The genetic relationships among and within selected georgia plume populations were evaluated using random amplified polymorphic DNA (RAPD) in conjunction with site visits at which time a census and GPS survey were conducted. Populations ranged from those containing eight to over 1000 individuals with most populations containing few plants (less than 50 individuals). With one exception, small populations with less than 50 individuals had more genetic similarity than populations with greater numbers of plants. Two protected populations containing large numbers of individuals were sampled extensively. Genetic similarity of individuals was not associated with plant proximity within a population. The small number of individuals and geographic isolation characteristic of many populations were associated with high within-population genetic similarity. Conservation priorities should be given to preserving as many different populations as possible to retain the genetic diversity of the species. Whether the narrow genetic variation found in some populations may be contributing to lack of sexual reproduction in the wild is an area for further study.
Anjana R. Sharma, Robert N. Trigiano, Willard T. Witte and Otto J. Schwarz
Cultivars of flowering dogwood (Cornus florida L.) are commercially propagated by vegetative methods such as rooting cuttings or grafting. The results of these methods can be unpredictable. A reliable method of producing dogwoods through tissue culture would be very useful to rapidly produce many copies of important genotypes with horticulturally important characters such as resistance to diseases. One of the primary difficulties of propagating dogwoods (seedlings only) by axillary bud multiplication has been the low rooting efficiency of the microshoots. Various treatments were tried in order to enhance rooting. Eighty-three percent of microshoots harvested between 5 and 7 weeks and treated continuously with 4.9 micromolar IBA rooted after 4 weeks, whereas <20% of microshoots harvested before 5 weeks and after 7 weeks rooted after 4 weeks of continuous exposure to IBA. Differences were also observed in rooting potentials of microshoots that had reddish brown stems rooting at a higher frequency compared to those that had green stems. We hope to translate this method to the propagation of cultivars and potential new releases.
Phillip A. Wadl, Mark T. Windham, Richard Evans and Robert N. Trigiano
Karen Harris-Shultz, Melanie Harrison, Phillip A. Wadl, Robert N. Trigiano and Timothy Rinehart
Little bluestem (Schizachyrium scoparium) is a perennial bunchgrass that is native to North American prairies and woodlands from southern Canada to northern Mexico. Originally used as a forage grass, little bluestem is now listed as a major U.S. native, ornamental grass. With the widespread planting of only a few cultivars, we aimed to assess the ploidy level and genetic diversity among some popular cultivars and accessions in the U.S. Department of Agriculture National Plant Germplasm System collection. Ten microsatellite markers, with successful amplification, were developed by using sequences available in Genbank and additional simple sequence repeat (SSR) markers were generated by using ion torrent sequencing of a genomic library created from the cultivar The Blues. A total of 2812 primer sets was designed from high-throughput sequencing, 100 primer pairs were selected, and 82 of these primers successfully amplified DNA from the Schizachyrium accessions. Only 35 primer pairs, generating 102 scored fragments, were polymorphic among S. scoparium accessions. Twenty-two primer pairs generated more than four fragments per accession. The use of a repetitive sequence identifier found that of 117 examined sequences, only nine sequences did not have similarity to DNA transposons, retrotransposons, viruses, or satellite sequences. The most frequently identified fragments were the long terminal repeat retrotransposons Gypsy (177 fragments) and Copia (98 fragments) and the DNA transposon EnSpm (60 fragments). Using the software program Structure, cluster analysis of the SSR data for S. scoparium revealed four groups. The lowest genetic similarity between little bluestem samples was 86%, which was surprising as a high degree of morphological variation is seen in this species. Furthermore, no variation in ploidy level was seen among little bluestem samples. These microsatellite markers are the first sequence-specific markers designed for little bluestem and can serve as a resource for future genetic studies.
Phillip A. Wadl, Robert N. Trigiano, Dennis J. Werner, Margaret R. Pooler and Timothy A. Rinehart
There are 11 recognized Cercis L. species, but identification is problematic using morphological characters, which are largely quantitative and continuous. Previous studies have combined morphological and molecular data to resolve taxonomic questions about geographic distribution of Cercis species, identifying botanical varieties, and associations between morphological variation and the environment. Three species have been used in ornamental plant breeding in the United States, including three botanical varieties of C. canadensis L. from North America and two Asian species, C. chingii Chun and C. chinensis Bunge. In this article, 51 taxa were sampled comprising eight species of Cercis and a closely related species, Bauhinia faberi Oliv. Sixty-eight polymorphic simple sequence repeat markers were used to assess genetic relationships between species and cultivars. For all samples the number of alleles detected ranged from two to 20 and 10 or more alleles were detected at 22 loci. Average polymorphic information content was 0.57 and values ranged from 0.06 to 0.91 with 44 loci 0.50 or greater. Cross-species transfer within Cercis was extremely high with 55 loci that amplified at 100%. Results support previously reported phylogenetic relationships of the North American and western Eurasian species and indicate suitability of these markers for mapping studies involving C. canadensis and C. chinensis. Results also support known pedigrees from ornamental tree breeding programs for the widely cultivated C. canadensis and C. chinensis species, which comprised the majority of the samples analyzed.