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
Giovanni A. Caputo, Phillip A. Wadl, Lambert McCarty, Jeff Adelberg, Katherine M. Jennings, and Matthew Cutulle
Weed competition is a main factor limiting sweetpotato [Ipomoea batatas (L.) Lam] production. Yellow nutsedge (Cyperus esculentus L.) is a problematic weed to control due to its ability to quickly infest a field and generate high numbers of tubes and shoots. Compounding this is the lack of a registered herbicide for selective postemergence control of yellow nutsedge. Research was conducted to evaluate the bentazon dose response of two sweetpotato cultivars and one advanced clone and to evaluate the plant hormone melatonin to determine its ability to safen bentazon post emergence. Bioassays using Murashige and Skoog (MS) media supplemented with melatonin (0.232 g a.i./L and 0.023 g a.i./L) and bentazon (0.24 g a.i./L) were conducted to evaluate the effect of bentazon on sweetpotato and to determine the interactive response of the Beauregard cultivar to bentazon and exogenous applications of melatonin. Beauregard swas the most tolerant cultivar and required dosages of bentazon that were two-times higher to cause the same injury compared with other cultivars. MS media containing melatonin and bentazon showed fewer injuries and higher plant mass than plants treated with bentazon alone. These results indicate that sweetpotato injury caused by bentazon may be reduced by melatonin.
Matthew A. Cutulle, H. Tyler Campbell, Monica Farfan, and Phillip A. Wadl
Weed management is an important component of sweetpotato production. Currently, S-metolachlor is the only herbicide registered in sweetpotato that has some suppressive effect on nutsedge species (Cyperus spp.). It is integral that the release of any new germplasm from sweetpotato breeding programs be tolerant to S-metolachlor. Screening for thousands of experimental clones for S-metolachlor in a field trial would be cumbersome. Therefore, screening for tolerant lines might be streamlined in an hydroponics system. Research was conducted to determine whether a hydroponics assay could detect differences in S-metolachlor response between a known sensitive sweetpotato cultivar (Centennial) and a tolerant sweetpotato cultivar (Beauregard) in 10 days. Results of the study show that ‘Beauregard’ was ≈50 times more tolerant to S-metolachlor than ‘Centennial’ when accessing injury at the 25% threshold. No differences were detected in S-metolachlor response between cultivars in the soil-based assay. This assay could be used for screening for S-metolachlor tolerance in a sweetpotato breeding program.
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
D. Michael Jackson, Howard F. Harrison, Robert L. Jarret, and Phillip A. Wadl
During 2012–14, 737 sweetpotato, Ipomoea batatas (L.) Lam. (Convolvulaceae), plant introduction (PI) accessions from the U.S. Department of Agriculture, Agricultural Research Service (USDA, ARS) sweetpotato germplasm collection were evaluated for several phenotypic leaf and plant characteristics, and a photographic record of each accession was made. Data were prepared for placement in the USDA, ARS Germplasm Resources Information Network (GRIN) database and the sweetpotato ontology. The parameters recorded for each genotype were canopy coverage, vine length, general leaf outline, leaf lobing, shape of the central leaf lobe, number of leaf points, leaf petiole length, leaf width, leaf length, leaf width × length, and leaf width/length (aspect ratio). The data indicate that there is wide genetic diversity for vegetative phenotypic characteristics within the USDA, ARS sweetpotato germplasm collection. This study provides important phenotype information for the USDA, ARS sweetpotato collection that has been lacking and can be used for curation of the collection and by researchers and breeders working with this important global food crop.
Robert N. Trigiano, Alan S. Windham, Mark T. Windham, and Phillip A. Wadl
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.