tools in plant biotechnology, which includes genomics and proteomics, are gel electrophoresis, polymerase chain reaction (PCR), and DNA fingerprinting. To incorporate these techniques into middle and high school and college curricula, content instruction
R.N. Trigiano, B.H. Ownley, A.N. Trigiano, J. Coley, K.D. Gwinn, and J.K. Moulton
Ashok K. Ghosh, Lewis N. Lukens, David M. Hunter, and Judith N. Strommer
The genus Pyrus (pear) includes species and cultivars of great diversity. We have tested the feasibility of a polyacrylamide gel eletrophoresis (PAGE)-based +/– simple sequence repeat (SSR) screen as a means of defining relationships amongst pears of commercial importance in North America. The screen included 28 pear accessions, including economically important cultivars, numbered selections from breeding programs and interspecific hybrids. It relied on 18 SSR primer pairs, each of which produced polymorphic banding patterns in all the genotypes examined. Fragments were scored for presence or absence within genotypes. The results show that amplification and analysis of a small number of SSR loci enable identification of cultivars and reasonable definition of genetic relationships in North American pears. Seven primer pairs were sufficient to distinguish the 28 pear cultivars. Analyses using both distance and parsimony criteria grouped cultivars in a manner consistent with known pedigrees and sites of origin.
Robert D. Marquard, Larry J. Grauke, Tommy E. Thompson, and Ruth S. Janos
More than 170 pecan [Carya illinoensis (Wangenh.) K. Koch] cultivars were evaluated formalate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase, leucine aminopeptidase (LAP), and diaphorase (DIA). Isozymes of LAP were observed in two regions after starch gel electrophoresis. The faster region of activity (Lap-1) was polymorphic and consistently expressed in leaves, wood, and roots. Controlled crosses suggest that Lap-1 is simply inherited and controlled by at least two alleles. DIA was well resolved and storable only from leaf material and produced a complex banding pattern. The ability to differentiate among cultivars by isozymes was good. The 177 cultivars sorted into 72 classes. Forty of the cultivars (23%) possessed a unique series of isozyme patterns. Most cultivars (124 of 177) shared common banding patterns with less than four other cultivars. From the inheritance models of four isozymes, some historical pedigrees can be questioned. Most notably,' Western Schley' could not have been parented by `San Saba' based on the inheritance of Mdh-1 and Lap-1.
R.N. Trigiano and G. Caetano-Anollés
The primary objectives of these laboratory exercises are to familiarize advanced undergraduate and graduate students (and instructors) with the general concepts, techniques, and uses of DNA fingerprinting and to remove some of the perceived mystique underlying molecular genetics. The technique of DNA amplification fingerprinting (DAF) is partitioned into four independent laboratory exercises that include DNA isolation, DNA amplification, gel electrophoresis and silver staining, and data collection and analysis. Although the DNA amplification and gel electrophoresis exercises are emphasized, very detailed and easy-to-follow instructions and protocols are provided for all aspects of the DNA fingerprinting process. These exercises, or similar ones, have been successfully completed on the first attempt by several classes of novice graduate students and other researchers.
Stephen P. Lee, Paul M. Chen, Tony H.H. Chen, Diane M. Varga, and Eugene A. Mielke
A proportion of `d'Anjou' pear fruit (Pyrus communis L.) developed a disorder, “black speck” or “skin speckling”, after prolonged controlled atmosphere (CA) storage (1% O2, - 0.5 C). A comparative study of biochemical components revealed that there was no significant difference in succinic, citric, fumaric, and pyruvic acids between the speckled' and normal skin tissues. The content of malic acid in the affected tissue was almost three times lower than that in the normal tissue. The specific activity of NADP-malic enzyme (EC 220.127.116.11) in the affected tissue was also lower, but the total activities were similar. The affected tissue contained higher percentages of dry matter and soluble proteins than the normal tissue. Two-dimensional gel electrophoresis of proteins showed that two groups of novel polypeptides appeared only in the affected skin tissue. This study indicated that a certain proportion of `d'Anjou' pear fruit might have been exposed to unfavorable preharvest environmental stresses, and, therefore, could no longer tolerate the subsequent semi-anaerobic and chilling stresses during prolonged CA storage.
Priscila L. Gratão, Carolina C. Monteiro, Lázaro E.P. Peres, and Ricardo Antunes Azevedo
polyacrylamide gel electrophoresis (PAGE) were carried out as described by Gomes-Junior et al. (2007) . Electrophoresis buffers and gels were prepared as described by Vitória et al. (2001) except that sodium dodecyl sulphate was excluded. The experimental
Clíssia Barboza da Silva, Julio Marcos-Filho, Pablo Jourdan, and Mark A. Bennett
staining for peroxidase in bell pepper seeds of two cultivars, AF-6 and AF-7, following nondenaturing polyacrylamide gel electrophoresis. Each cultivar was represented by three and four seed lots, respectively. (a) Unprimed seeds; (b) drum-primed seeds
Lyn A. Gettys and Dennis J. Werner
Stokes aster is a herbaceous perennial native to the southeastern United States. Stokesia is a monotypic genus belonging to the tribe Vernonieae Cass. (family Asteraceae Dumont). The level of genetic diversity within the genus is unknown. The goal of this study was to determine the level of genetic diversity and relatedness among cultivars of stokes aster. The genetic relatedness among 10 cultivars of stokes aster, one accession of Vernonia crinita Raf. (syn. V. arkansana DC.), and one accession of Rudbeckia fulgida Ait. var. sullivantii (Beadle et Boynton) Cronq. `Goldsturm' was estimated using 74 randomly amplified polymorphic DNA (RAPD) primers. Similarity indices suggest that cultivars of stokes aster are very closely related, with values for all pairwise comparisons of cultivars of stokes aster ranging from 0.92 to 0.68. One cultivar, `Omega Skyrocket', had markedly lower similarity indices from the other cultivars, ranging from 0.72 to 0.68. Similarity indices between stokes aster and Vernonia and between stokes aster and Rudbeckia were 0.44 and 0.50, respectively.
Ben-Hong Wu, Ning Niu, Ji-Hu Li, and Shao-Hua Li
may be regulated by this ratio. The aim of the present study was to assess the effect of different LF treatments on protein variation in grape berry skin using two-dimensional gel electrophoresis (2-DE). Materials and Methods Plant materials and
Franco Famiani and Robert P. Walker
western blotting of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with specific antisera, an approach that has proved successful for a number of soft fruit ( Famiani et al., 2005 , 2009 ). To use this approach, it is