Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated. The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h.The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol.It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.