To determine the optimal conditions for mutagenesis of Hydrangea macrophylla (Thunb.) Ser. and Hydrangea paniculata Siebold, cold-treated and untreated seed from representative cultivars were exposed to 0.5%, 2.5%, and 5.0% ethyl methane sulfonate (EMS). Most untreated H. macrophylla seed exposed to 2.5% and 5.0% EMS had substantially lower germination percentages. H. paniculata seed germination percentages did not differ from controls until EMS concentrations reached 5.0%. Cold treatment of H. macrophylla and H. paniculata seed made germination more tolerant to all concentrations of EMS tested and increased germination of most H. macrophylla cultivars when compared with lower dosages. Germination of cold-treated seed from H. paniculata cultivars rose substantially above control levels even at the highest dosages. For the most part, we observed more nondormant seed, or seed available for germination, but less viability with increasing EMS dosage in untreated Hydrangea L. seed versus controls. In contrast, cold-treated seed displayed higher levels of mutagen tolerance and nondormancy versus controls when exposed to these same doses of EMS.
Stephen Patrick Greer and Timothy A. Rinehart
Sandra M. Reed and Timothy A. Rinehart
Genetic diversity studies using 39 simple-sequence repeat (SSR) markers were carried out with 114 taxa of Hydrangea macrophylla (Thunb.) Ser., including 87 H. macrophylla ssp. macrophylla cultivars and 20 members of H. macrophylla ssp. serrata (Thunb.) Makino. The SSR loci were highly variable among the taxa, producing a mean of 8.26 alleles per locus. Overall allelic richness was relatively high at 5.12 alleles per locus. H. macrophylla ssp. serrata contained nearly twice the allelic diversity of H. macrophylla ssp. macrophylla. The majority of genetic diversity was found to reside within the subspecies, with only 12% of the total genetic diversity observed occurring between subspecies. Although the elevation of H. macrophylla ssp. serrata to species level has recently been recommended by several hydrangea authorities, these data support the subspecies designation. Four cultivars (Preziosa, Pink Beauty, Tokyo Delight, and Blue Deckle) appeared to be hybrids between the two subspecies. Genetic similarities were found among five remontant cultivars (Bailmer, Oak Hill, David Ramsey, Decatur Blue, and Penny Mac) and several nonremontant cultivars, including General Vicomtesse de Vibraye, Nikko Blue, All Summer Beauty, and La France. No close genetic relationship was found between the remontant cultivar Early Sensation and other remontant cultivars. Genetic similarities were found among variegated and double-flower cultivars. Within H. macrophylla ssp. macrophylla, cultivars with mophead inflorescences clustered separately from most lacecap cultivars. This indicates the cultivars with lacecap inflorescences that were among some of the earliest introductions to Europe were not widely used in the breeding of mophead forms. Some presumed synonyms were found to be valid (‘Preziosa’ and ‘Pink Beauty’, ‘Rosalba’ and ‘Benigaku’, ‘Geoffrey Chadbund’ and ‘Mowe’), whereas others were not (‘Harlequin’ and ‘Monrey’, ‘Nigra’ and ‘Mandschurica’). This study identified potentially unexploited sources of germplasm within H. macrophylla and relationships between existing cultivars of this popular shrub. This information should be of value when selecting parents for breeding programs.
Sandra M. Reed and Timothy A. Rinehart
Genetic diversity studies using 26 simple-sequence repeat (SSR) markers were conducted with 36 cultivars, breeding lines, and wild-collected accessions of Hydrangea paniculata Sieb. The SSR markers were highly variable among the genotypes, producing a mean of 5.8 alleles per marker. Three cultivars (Boskoop, Compact Grandiflora, and Webb) were either identical to or sports of the popular cultivar Grandiflora. The name ‘Pee Wee’ appears to have been applied to two phenotypically different compact forms of H. paniculata, one of which seems to be a sport of ‘Tardiva’, whereas the other is likely derived from ‘Grandiflora’. No close genetic similarity was observed among several cultivars from a long-term Belgium breeding program, although many had one parent in common. Early-flowering genotypes clustered separately from genotypes that flower in midsummer, but close genetic relationships were not observed among early-flowering cultivars. Two genotypes from Taiwan were genetically similar but were distinctly different from the Japanese genotypes. These, along with the early-flowering genotypes and a new collection from Japan, may represent unexploited sources of germplasm for improvement of H. paniculata.
Timothy A. Rinehart, Brian E. Scheffler, and Sandra M. Reed
Using 14 codominant microsatellite markers that amplify loci across 14 different Hydrangea L. species, we analyzed gene diversity and genetic similarity within Hydrangea. Samples also included Dichroa Lour., Platycrater Sieb. and Zucc., and Schizophragma Sieb. and Zucc. genera to establish their relatedness to Hydrangea species since previous work suggests they may be closely related. Our results support the close affiliation between Macrophyllae E.M. McClint. and Petalanthe (Maxim.) Rehder subsections and their separation from the other Hydrangea species. Most of the Hydrangea species analyzed cluster within their designated sections and subsections; however, genetic distance between species within each subsection varied considerably. Our data suggest that morphological analyses which labeled H. serrata (Thunb.) Ser. as a subspecies of H. macrophylla (Thunb. Ex J.A. Murr.) Ser. are probably more accurate than recent genome size data suggesting H. macrophylla ssp. macrophylla (Thunb.) Ser. and H. macrophylla ssp. serrata (Thunb.) Makino are separate species. Gene diversity estimates indicate that 64.7% of the total diversity is due to differences between species and 49.7% of the overall variation is due to differences between subsections. Low diversity suggests a lack of gene flow between species and subsections and underscores the difficulty in making wide hybrids. Since only 35.3% of the genetic variation is common to all species, unique alleles were used to develop a molecular key for unambiguous species identification and interspecific hybrid verification. Genetic similarity estimates for all 85 samples suggests a roadmap for introgressing horticulturally important traits from different Hydrangea species.
Cecil Pounders, Brian E. Scheffler, and Timothy A. Rinehart
Timothy A. Rinehart, Brian E. Scheffler, and Sandra M. Reed
Recent evidence suggests a close genetic relationship between Hydrangea macrophylla (Thunb.) Ser. and D. febrifuga Lour., which supports previous morphological and DNA sequence data. This relationship was confirmed by the production of fertile intergeneric hybrids. We characterize the genetic diversity of available D. febrifuga plants, both cultivars and wild-collected taxa, as breeding material to improve H. macrophylla. Relatively high genetic diversity is seen among D. febrifuga, which splits into two main clusters. We also document considerable differences in genome size when compared with previously characterized D. febrifuga. Dichroa versicolor (Fortune) D.R. Hunt plants were also included and data suggest that D. versicolor could be a hybrid between H. macrophylla and D. febrifuga, similar to the intergeneric hybrids produced by recent breeding efforts. Because native H. macrophylla plants do not overlap extensively with D. febrifuga populations, we tested Hydrangea indochinensis Merr. as a possible parent because endemic H. indochinensis populations overlap regions where D. febrifuga and D. versicolor have been collected. However, results suggest that H. indochinensis does not share a genetic background with D. versicolor. Taxonomic revision of Dichroa is warranted, especially because we document several more intergeneric hybrids from self-sown, open-pollinated sources.
Timothy A. Rinehart, Ned C. Edwards, and James M. Spiers
Sandra M. Reed, Keri D. Jones, and Timothy A. Rinehart
The potential of producing an intergeneric hybrid between Dichroa febrifuga Lour. and Hydrangea macrophylla (Thunb.) Ser. was investigated. Reciprocal hybridizations were made between a D. febrifuga selection (GUIZ 48) and diploid (‘Veitchii’) and triploid (‘Kardinal’ and ‘Taube’) cultivars of H. macrophylla. Embryo rescue was employed for about one-third of the crosses that produced fruit, and the rest were allowed to mature on the plant and seed-collected and germinated. Reciprocal hybrids, which were verified with simple sequence repeat markers, were produced from both embryo rescue and seed germination and with both diploid and triploid H. macrophylla cultivars. Hybrids were intermediate in appearance between parents, but variability in leaf, inflorescence, and flower size and flower color existed among the hybrids. A somatic chromosome number of 2n = 6x = 108 was tentatively proposed for D. febrifuga GUIZ 48. Chromosome counts and flow-cytometric measurements of nuclear DNA content indicated that some of the hybrids may be aneuploids, but neither analysis was definitive. Although hybrids with H. macrophylla as the pistillate parent did not form pollen-producing anthers, D. febrifuga × H. macrophylla hybrids had normal-appearing anthers that produced abundant pollen. F2 and BC1 progeny were obtained using D. febrifuga × ‘Veitchii’ hybrids. This work documents the first step in an effort to combine desirable horticultural features from D. febrifuga and H. macrophylla.
Keri D. Jones, Sandra M. Reed, and Timothy A. Rinehart
Ploidy level was estimated in Hydrangea macrophylla (Thunb) Ser. using flow cytometry. For H. macrophylla ssp. macrophylla, 42 diploid and 19 triploid cultivars were identified. All 14 H. macrophylla ssp. serrata (Thunb.) Makino cultivars tested were diploids. Somatic chromosome counts confirmed the ploidy of three diploid (2n = 2x = 36) and three triploid (2n = 3x = 54) cultivars. Stomatal guard cell length and pollen diameter of H. macrophylla ssp. macrophylla diploid cultivars were smaller than those of triploid cultivars. However, because the range of measurements for the diploids overlapped that of the triploids, neither guard cell nor pollen measurements are recommended for determining ploidy of H. macrophylla cultivars. Fertility was estimated using pollen staining and controlled pollinations. Stainable pollen for triploid cultivars averaged 63% and ranged from 25% in ‘Masja’ to 85% in ‘Marechal Foch’. Viable seed was obtained when four triploid cultivars were used as pistillate or staminate parents in controlled pollinations to diploid H. macrophylla ssp. macrophylla cultivars. A bimodal distribution of pollen sizes, which is suggestive of unreduced gamete production, was observed in one cultivar; however, more detailed genetic and cytologic studies are needed to elucidate the mechanism behind triploid formation in H. macrophylla taxa.
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.