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Hongmei Ma and Margaret Pooler

Ornamental flowering cherry trees (Prunus species) are popular landscape plants that are used in residential and commercial landscapes throughout most temperate regions of the world. Most of the flowering cherry trees planted in the United States represent relatively few species. The U.S. National Arboretum has an ongoing breeding program aimed at broadening this base by developing new cultivars of ornamental cherry with disease and pest resistance, tolerance to environmental stresses, and superior ornamental characteristics. Knowledge of the genetic relationships among species would be useful in breeding and germplasm conservation efforts. However, the taxonomy of flowering cherry species and cultivars is complicated by differences in ploidy levels and intercrossing among species. We have used simple sequence repeat (SSR) markers developed for other Prunus species to screen a diverse collection of over 200 ornamental cherry genotypes representing 70 taxa in order to determine the genetic relationships among species, cultivars, and accessions. Data were generated from 9–12 primer pairs using an automated DNA genetic analyzer (ABI3770), and subjected to UPGMA cluster analysis. Extremely high levels of polymorphism were exhibited among the materials studied, thus indicating that ornamental flowering cherry germplasm has substantial inherent genetic diversity. This information, combined with traditional morphological characteristics, will be useful in determining genetic relationships among accessions in our collection and for predicting crossability of taxa.

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Margaret Pooler and Hongmei Ma

Flowering cherries belong to the genus Prunus, consisting primarily of species native to Asia. Despite the popularity of ornamental cherry trees in the landscape, most ornamental Prunus planted in the United States are derived from a limited genetic base of Japanese flowering cherry taxa. Controlled crosses among flowering cherry species carried out over the past 30 years at the U.S. National Arboretum have resulted in the creation of interspecific hybrids among many of these diverse taxa. We used simple sequence repeat (SSR) markers to verify 73 of 84 putative hybrids created from 43 crosses representing 20 parental taxa. All verified hybrids were within the same section (Cerasus or Laurocerasus in the subgenus Cerasus) with no verified hybrids between sections.

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Hongmei Ma, Margaret Pooler, and Robert Griesbach

Many studies have examined anthocyanin gene expression in colorless tissues by introducing anthocyanin regulatory genes of the MYC/R and MYB/C1 families. Expression of the two regulatory genes under the control of a strong promoter generally results in high anthocyanin accumulation. However, such approaches usually have a negative effect on growth and development of the recovered plants. In this study the author used two promoters of different strengths—a weak (Solanum tuberosum L. polyubiquitin Ubi3) and a strong (double 35S) promoter—and generated two sets of expression constructs with the Zea mays L. anthocyanin regulatory genes MycLc and MybC1. A transient expression system was developed using biolistic bombardment of white Phalaenopsis amabilis (L.) Blume flowers, which the authors confirmed to be anthocyanin regulatory gene mutants. Transient expression of different combinations of the four constructs would generate three different MycLc-to-MybC1 ratios (>1, 1, <1). The enhanced green florescent protein gene (EGFP) was cotransformed as an internal control with the two anthocyanin regulatory gene constructs. These results demonstrate that the ratio of the two transcription factors had a significant influence on the amount of anthocyanin produced. Anthocyanin accumulation occurred only when MybC1 was under the control of the 35S promoter, regardless of whether MycLC was driven by the 35S or Ubi3 promoter.

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Hongmei Ma, Margaret Pooler, and Robert Griesbach

Anthocyanin biosynthesis requires the coordinated expression of Myc, Wd, Chs, Dfr, and Myb. Chs and Dfr are structural genes, while Myc, Myb, and Wd are regulatory genes. Reverse transcription polymerase chain reaction was used to measure the expression of these genes in Phalaenopsis amabilis and Phalaenopsis schilleriana. P. amabilis expresses an albescent phenotype with petals and sepals that are anthocyanin free, while P. schilleriana has a wild-type phenotype with anthocyanin-containing petals and sepals. As expected, the petals and sepals of P. schilleriana expressed high levels of Chs and Dfr. The petals and sepals of P. amabilis expressed high levels of Chs and very low levels of Dfr. In P. amabilis and P. schilleriana, anthocyanin-specific Myc and Wd were expressed; however, Myb specific for anthocyanin biosynthesis were undetectable in P. amabilis. This suggests that the absence of Myb expression was responsible for the lack of dihydroflavonol 4-reductase and results in the absence of anthocyanin pigmentation in P. amabilis petals and sepals. This was confirmed by particle bombardment of P. amabilis petals with functional Mybs isolated from P. schilleriana. Comparisons of anthocyanin-related Myb gene expression between P. schilleriana and P. amabilis are between genetically different species. Phalaenopsis ‘Everspring Fairy’ expresses a harlequin phenotype with white petals and sepals containing large anthocyanin sectors. Harlequin flowers are ideal to evaluate anthocyanin-related Myb gene expression within genetically identical but differently pigmented tissue. High levels of anthocyanin-specific Myb and Dfr transcripts were present in the purple, but not in the white, sectors of Phalaenopsis ‘Everspring Fairy’ petals and sepals. There was no differential expression of Chs, Wd, and Myc between the purple and white sectors. These results are in agreement with the results from P. amabilis and P. schilleriana.

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Hongmei Ma, Richard Olsen, Margaret Pooler, and Matthew Kramer

Flowering cherries belong to the genus Prunus, consisting primarily of species native to Asia. Despite the popularity of ornamental cherry trees in the landscape, most ornamental Prunus planted in the United States are derived from a limited genetic base of Japanese flowering cherry taxa. A diverse collection of ornamental Prunus germplasm is maintained at the U.S. National Arboretum as part of an ongoing flowering cherry improvement program, but the genetic backgrounds of many trees are unclear. We characterized this germplasm using five simple sequence repeat (SSR) primer pairs, including one chloroplast primer pair. These primers generated 140 unique alleles that were used to assess genetic relationships among species, hybrids, and cultivars in this collection. We found that these markers followed expected Mendelian inheritance from parents to progeny in controlled hybridizations. In general, species clustered according to published taxonomic groupings, including a distinct separation of the ornamental cherries (Prunus subgenus Cerasus section Pseudocerasus) from other subgenera. Individual accessions of several taxa did not cluster with other samples of the species, indicating possible misidentification or interspecific combinations. The resulting information will be useful in guiding decisions on breeding methodology and germplasm preservation.