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.
Karen Harris-Shultz, Melanie Harrison, Phillip A. Wadl, Robert N. Trigiano, and Timothy Rinehart
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.
Ying Chen, Xinlu Chen, Fei Hu, Hua Yang, Li Yue, Robert N. Trigiano, and Zong-Ming (Max) Cheng
Agave species are economically important plants in tropical and subtropical desert ecosystems as ornamentals as well as potential bioenergy crops. However, their relatively long life cycles and the current lack of biotechnology tools hinder their breeding. In this study, an efficient system for micropropagation was developed for Agave americana L. by using basal stems as explants and grown on a modified Murashige and Skoog medium (MSI) or a 1/2 MSI medium supplemented with various concentrations of 6-benzylaminopurine (BA) for shoot proliferation. The highest number of shoots (18.5 shoots/explant) from basal stems was obtained on MSI supplemented with 13.32 μM BA. An efficient shoot regeneration system was also developed from leaf tissues. Combinations of auxin with cytokinin, basal media, and leaf regions were optimized for shoot induction. Adventitious shoot formation from leaf segments was induced and proliferated with combination ranging of 0.54 to 2.68 μM [α-naphthaleneacetic acid (NAA)] with 8.88 to 13.32 μM (BA), and the maximum frequency (≈69%) was obtained with 2.68 μM NAA plus 13.32 μM BA. MSI medium and the basal segment of leaf affected shoot induction. The highest rooting frequency and mean number of shoots occurred in 1/2 MSI containing with 4.92 μM indole-3-butyric acid (IBA) alone (90%, 3.4) or 1.48 μM IBA plus 1.61 μM NAA (92%, 5.2). Survival of in vitro plantlets after transfer and acclimatization to ex vitro conditions was 87%. This is the first complete protocol for micropropagation of A. americana.
Yonghao Li, Mark T. Windham, Robert N. Trigiano, Donna C. Fare, James M. Spiers, and Warren E. Copes
Infection process of Puccinia hemerocallidis, the causal agent of daylily rust, and resistance responses in eight daylily cultivars, were studied macroscopically and microscopically. After germination of urediniospores, appressoria were formed at the tip of germ tubes and penetrated through stomatal openings. Intercellular hyphae aggregated and formed uredia under the infection sites, and released urediniospores after rupturing the epidermis. In highly resistant cultivars `Prairie Blue Eyes' and `Bertie Ferris', intercellular hyphal growth was restricted and uredia were not formed. No macroscopic symptoms of the disease were present on the leaf surface, although a few collapsed cells were observed microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed on the moderately resistant cultivar `Chicago Apache', but not on resistant cultivars, `Buttered Popcorn' and `Stella De Oro'. Sporulation was observed on both moderately susceptible and susceptible cultivars, but latent periods were delayed and the amount of urediniospore production was reduced on moderately susceptible cultivars, `Mary Todd' and `Chorus Line', compared to the susceptible cultivar `Pardon Me'. The results indicate that the hypersensitive cell death is one of the resistance responses to daylily rust, but necrotic lesions on leaf surfaces are associated with the amount of collapsed host cells. The delayed latent periods and reduced sporulation that resulted from restricted intercellular hyphal growth could represent another resistance mechanism in the daylily rust pathosystem.
Xinwang Wang, Robert N. Trigiano, Mark T. Windham, Renae DeVries, Timothy A. Rinehart, James M. Spiers, and Brain Scheffler
The genus Cornus consists of many species, of which C. florida, C. kousa, C. mas, and C. stolonifera are four main ornamental species in North America, Asia, and Europe. For example, over 200 cultivars of C. florida alone have been developed for the nursery industry. Microsatellite loci, or SSR, are useful markers for studying genetic diversity and for creating linkage maps of the various species. The objective of this study was to investigate the genetic diversity between these four Cornus species and eight hybrids. Evaulation of the diversity will be useful in assessing the selection pressure of breeders and/or genetic drift of these dogwood cultivars/lines. Fifteen SSR primer pairs were selected to examine 56 Cornus cultivars and/or lines of the four species and hybrids. The study included 28 C. florida cultivars and lines, 15 C. kousa cultivars and lines, four C. stolonifera cultivars, one cultivar of C. mass and eight hybrids between various Cornus species. An exceptionally high level of diversity was detected among the 56 entries in both the number and size range of SSR alleles. A total of 95 alleles with an average of 7.8 alleles per loci were detected among these 56 genotypes. These selected Cornus cultivars and/or lines could be clustered into four to six subgroups. Some Cornus species were integrated into other species groups, suggesting gene flow between species via the breeding or evolution. SSR markers can contribute to the exploitation of genetic diversity for existing Cornus germplasm. For further study, examination of more SSR loci could explain more completely the diversity among these Cornus cultivars and lines.
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.
Naomi R. Smith, Robert N. Trigiano, Mark T. Windham, Kurt H. Lamour, Ledare S. Finley, Xinwang Wang, and Timothy A. Rinehart
Flowering dogwood (Cornus florida L.) is an important tree of forests and urban landscapes in the eastern United States. Amplified fragment length polymorphism (AFLP) markers were generated from genomic DNA of 17 cultivars and lines, and four duplicate samples of selective cultivars. Specific markers were identified for all except the following two lines and cultivar: MW94-67, MW95-12, and ‘Plena’. A dichotomous cultivar identification key was constructed based on AFLP data, and specific peaks or combinations of peaks were identified for all cultivars and lines. The key was assessed with seven anonymous (unlabeled) dogwood samples, and all unknowns except one were identified using the dichotomous key. Two of the unknown samples, ‘Cherokee Chief’ and ‘Cherokee Brave’, were difficult to distinguish using the AFLP markers. Intracultivar variation, up to 36% dissimilarity, was observed between duplicate samples of the same cultivar from different trees, suggesting that some mislabeling of trees had occurred at the nursery. The cultivar-specific AFLP markers can be used in breeding applications, patent protection, and in future projects, such as mapping the C. florida genome.
Nicolas C. Strange, John K. Moulton, Ernest C. Bernard, William E. Klingeman III, Blair J. Sampson, and Robert N. Trigiano
Helianthus verticillatus Small (whorled sunflower) is a federally endangered plant species found only in the southeastern United States that has potential horticultural value. Evidence suggests that H. verticillatus is self-incompatible and reliant on insect pollination for seed production. However, the identity of probable pollinators is unknown. Floral visitors were collected and identified during Sept. 2017 and Sept. 2018. Thirty-six species of visitors, including 25 hymenopterans, 7 dipterans, 2 lepidopterans, and 2 other insect species, were captured during 7 collection days at a site in Georgia (1 day) and 2 locations in Tennessee (6 days). Within a collection day (0745–1815 hr), there were either five or six discrete half-hour collection periods when insects were captured. Insect visitor activity peaked during the 1145–1215 and 1345–1415 hr periods, and activity was least during the 0745–0845 and 0945–1015 hr periods at all three locations. Visitors were identified by genus and/or species with morphological keys and sequences of the cox-1 mitochondrial gene. The most frequent visitors at all sites were Bombus spp. (bumblebees); Ceratina calcarata (a small carpenter bee species) and members of the halictid bee tribe Augochlorini were the second and third most common visitors at the two Tennessee locations. Helianthus pollen on visitors was identified by microscopic observations and via direct polymerase chain reaction of DNA using Helianthus-specific microsatellites primers. Pollen grains were collected from the most frequent visitors and Apis mellifera (honeybee) and counted using a hemocytometer. Based on the frequency of the insects collected across the three sites and on the mean number of pollen grains carried on the body of the insects, Bombus spp., Halictus ligatus (sweat bee), Agapostemon spp., and Lasioglossum/Dialictus spp., collectively, are the most probable primary pollinators of H. verticillatus.
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.
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.