The literature is full of different techniques and approaches to the isolation, purification and quantitative analysis of plant hormones. From this body of literature it is possible to deduce that 1) a lot of investigators are interested in how much of these compounds are in plants and 2) that the techniques for phytohormone analysis are still largely “under development”. This talk will discuss different approaches to hormone analysis, suitability of each approach, and criteria for the evaluation of techniques and results. The goal will be to highlight points that are important to obtaining reliable analytical information and knowing what to do when problems occur. Nevertheless, having reliable numbers is frequently only the first step in understanding hormonal systems involved in plant development, It is often the case that the expected results are not what is found in experiments involving plant hormone quantitation. We will consider experimental design, tissue localization, developmental stages, sampling and extraction procedures, and the limits of what to expect when “dogma confronts reality”. Work reported was supported by grants from the National Science Foundation DCB-8917378, USDA-CRGO 89-3721-4734, US-Israel BARD US-1362-87, and by funds from the USDA Argicultural Research Service,
Jerry D. Cohen and Janet P. Slovin
Anik L. Dhanaraj*, Janet P. Slovin, and Lisa J. Rowland
To gain a better understanding of changes in gene expression associated with cold stress in the woody perennial blueberry (Vaccinium spp.), a genomics approach based on the analysis of expressed sequence tags (ESTs) was undertaken. Two cDNA libraries were constructed using RNA from cold acclimated (mid winter conditions when the plants are cold stressed) and non-acclimated (before they received any chilling) floral buds of the blueberry cultivar Bluecrop. About 600 5'-end ESTs were generated from each of the libraries. Putative functions were assigned to 57% of the cDNAs that yielded high quality sequences based on homology to other genes/ESTs from Genbank, and these were classified into 14 functional categories. From a contig analysis, which clustered sequences derived from the same or very similar genes, 430 and 483 unique transcripts were identified from the cold acclimated and non-acclimated libraries, respectively. Of the total unique transcripts, only 4.3% were shared between the libraries, suggesting marked differences in the genes expressed under the two conditions. The most highly abundant cDNAs that were picked many more times from one library than from the other were identified as representing potentially differentially expressed transcripts. Northern analyses were performed to examine expression of eight selected transcripts and seven of these were confirmed to be preferentially expressed under either cold acclimating or non-acclimating conditions. Only one of the seven transcripts, encoding a dehydrin, had been found previously to be up-regulated during cold stress of blueberry. This study demonstrates that analysis of ESTs is an effective strategy to identify candidate cold-responsive transcripts in blueberry.
Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin, and Mark K. Ehlenfeldt
Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.