In order to facilitate the high throughput transformation required to use Fragaria vesca (wild strawberry) as a tool in genomic research, functional genomics, and gene discovery not only for strawberry but for fruit crops in general, we need to increase its regeneration frequency and transformation efficiency using Agrobacterium. Ten accessions of F. vesca representing a range of germplasm with worldwide distribution were obtained from the USDA National Germplasm Repository, Corvallis, Ore. for use in shoot regeneration experiments. Seed germination with or without vernalization ranged from 0% to 90%. In vitro growth varied for the accessions with five accessions eliminated from further experiments due to poor growth. In preliminary experiments with 125 leaf explants and 40 petiole explants combined representing PI 551573, PI 602923, and F. vesca `Alpine'; 100% of the uncontaminated explants regenerated at least one shoot after 8 weeks on medium supplemented with 1 mg·L-1 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ) and 0.2 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D). In a replicated study of `Alpine' comparing regeneration on the above TDZ/2,4-D medium with control medium [0.25 mg·L-1 indole-3-butyric acid (IBA)/3 mg·L-1 benzyladenine (BA)], regeneration frequency at 6 weeks for leaf or petiole explants on control medium was 8% (n = 180) compared to 27% (n = 210) on the TDZ/2,4-D medium. This optimized shoot regeneration protocol for F. vesca `Alpine' is currently under investigation in transformation experiments with several other accessions and Agrobacterium constructs.
Phillip A. Wadl* and Richard E. Veilleux
Matthew A. Cutulle, Howard F. Harrison Jr., Chandresakar S. Kousik, Phillip A. Wadl and Amnon Levi
A greenhouse trial was used to evaluate 159 accessions of bottle gourd [Lagenaria siceraria (Mol.) Standl.] obtained from the U.S. National Plant Germplasm for tolerance to clomazone herbicide. Most accessions tested were moderately or severely injured by clomazone at 3.0 mg·kg−1 incorporated into greenhouse potting medium; however, several exhibited lower injury. Seeds were produced from tolerant and susceptible plants for use in a greenhouse concentration–response experiment. About three to four times higher clomazone concentrations were required to cause moderate injury to tolerant bottle genotypes in comparison with susceptible genotypes. The differences in tolerance among genotypes were observed with injury ratings, chlorophyll measurements, and shoot weights. Clomazone may be used safely on tolerant bottle gourd genotypes, but the herbicide may not be safe for susceptible genotypes. Also, tolerant genotypes such as Grif 11942 may be desirable for use as rootstocks in grafted watermelon production.
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.
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.
Thomas J. Molnar, Megan Muehlbauer, Phillip A. Wadl and John M. Capik
Robert N. Trigiano, Alan S. Windham, Mark T. Windham and Phillip A. Wadl
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, 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.
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.