Eric L. Zeldin

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

Nodule Culture: A Developmental Pathway with High Potential for Regeneration, Automated Micropropagation, and Plant Metabolite Production from Woody Plants

Nodules are independent, spherical, dense cell clusters which form a cohesive unit and display a consistent internal cell/tissue differentiation. At a minimum, three cell types (meristematic cells, plastid-dense parenchyma, and vascular elements) and two cell layers (epidermal and internal cortex/vascular) can be distinguished in nodules of poplar. Although nodules have been randomly observed by many researchers working with a myriad of plant species, they most commonly are seen in cultures of woody plant species being differentiated from dedifferentiated cells. We have developed liquid culture systems in which nodules are the predominant structures. Such cultures grow via nodule enlargement (a three-stage process) and nodule multiplication (via two general pathways). In general, nodules display a high capacity for plant/organ regeneration via organogenesis. The nodular developmental pathway parallels that of the embryogenic developmental pathway; a theoretical comparison of the two pathways as bridges between totipotency and competence is discussed. Although the research is still very preliminary, nodule cultures have apparent applications in regeneration strategies, automated micropropagation, and in vitro phytochemical production.

Detectableβ-glucuronidase activity in transgenic cranberry is affected by endogenous inhibitors and plant development

Extracts of cranberry, rich in flavonols and proanthocyanins, inactivatedβ-glucuronidase (GUS) in assays using either purified bacterial GUS or preparations of transgenic tobacco (Nicotiana tabacum L.) or transgenic cranberry (Vaccinium macrocarpon Ait.) expressing thegusA gene. Histochemical GUS assays produced random and generally unpredictable staining. The addition of poly-vinylpolypyrrolidone (PVPP) during the preparation of transgenic cranberry leaf extracts increased the detectable GUS activity in fluorogenic assays more than 200-fold. Detectable GUS activity varied among transclones and also within a transclone depending on the developmental and physiological state of the tissue, as well as the growth environment. Use of PVPP altered the relative ranking of plants based on their total transgenic enzyme activity and resulted in different conclusions as to the effects of genotype or growth environment on transgene expression.

Immunocytochemical localization of taxol in Taxus cuspidata

The supply of taxol, a valuable anticancer compound, depends completely on the extraction of taxol from Taxus brevifolia (Pacific yew) plants. Although taxol is found in virtually all parts of the plant, nothing is known about the localization of taxol within the cells or tissues of the plant. Portions of T. cuspidata stems were chemically fixed, dehydrated in ethanol, and then embedded in standard resins. In these tissues, immunolocalization using polyclonal antitaxol antiserum indicates that taxol is associated with vacuolar tannin inclusions in axial phloem parenchyma cells. Little or no label is bound over the cell walls of any cell type. However, chemical preparation causes diffusion of taxol, which results in an erroneous localization of taxol. In contrast, using cryotechniques and a water-soluble melamine resin (Nanoplast), taxol is localized almost exclusively in the cell walls of phloem, vascular cambium, and xylem. Our method yields insight into the problems associated with the intra- and extracellular localization of lipophilic plant secondary compounds. It also offers an alternative tissue-preparation protocol that could be useful for the localization of other plant metabolites.

Resistance to the birch leafminer Fenusa pusilla (Hymenoptera: Tenthredinidae) within the genus Betula.

Abstract Thirteen Betula species were tested for resistanceto the birch leafminer, Fenusa pusilla (Lepeletier), usingno-choice assays. Birch leafminers were able to oviposit into expandingleaves of all Betula individuals tested. Larvae did notsurvive within any of the tested individuals of three species, B.alleghaniensis (Britt.), B. grossa (S. & Z.), andB. lenta (L.). Leafminer eggs deposited into the leaves ofthese species hatched, and larvae fed for a short period before dying.These three species were classified as highly resistant to birchleafminer, based on very low percent of mines (0.6-3.1%) with adiameter >3 mm. Eight species, B. papyrifera (Marsh),B. pendula (Roth), B. turkestanica (Litvin),B. glandulifera (Regal), B. ermanii (Cham.),B. platyphylla variety japonica [(Miq.) Hara],B. populifolia (Marsh) and B. maximowicziana(Regal) were classified as susceptible, with percent of mines >3 mmdiameter of 87-94%. Two species, B. costata (Trautv.) andB. davurica (Pall.), displayed intermediate and variableresistance. B. davurica exhibited a mechanism of resistancenot observed in the other species. Eggs oviposited into the leaves ofresistant B. davurica individuals became surrounded by anarea of discolored and necrotic tissue, and died. This responseresembles the programmed cell death associated with a hypersensitiveresponse.

Selection of American hazelnut as a potential oilseed crop

Development of perennial oilseed crops for the Lakes States region of the United States would provide another crop opportunity for farmers, while improving ecosystem services from agricultural lands. In order to effectively select hazelnut (Corylus americana Marsh.) genotypes from existing wild populations, we needed to know at least three parameters: (1) measures for indirect assessment of yield, (2) between and within population genetic variability, (3) fatty acid characteristics of the nuts. American hazelnut populations at 21 sites were screened for high-yielding plants. Yield component analysis was used to determine the relationship of specific component of yield (nut clusters/m2, nuts/cluster, mass in-shell/nut, mass of nutmeat/mass in-shell) to yield of nutmeat/m2. Nut clusters/m2 explained the majority of the variation in yield of nutmeat/m2. Ten microsatellite loci were used to determine the within and between population variation. The majority of the variance was between individuals within populations. Fatty acid profiles were determined for a subset of high-producing plants. Hazelnut oil is predominantly oleic acid. The fatty acid profiles do not vary greatly between individual plants, although linoleic acid is the most open to selection.