Application Studies Of Microsatellite DNA And ISSR Techniques In Protozoa

Microsatellite DNA and ISSR (inter simple sequence repeat) techniques are first applied in the free-living protozoa:I. The specificity of the microsatellite DNA loci and applicability of microsatellite DNA primers were first studied in protozoa in details. Eight microsatellite loci primers developed from Trypanosoma cruzi were employed to amplify microsatellite DNA sequences in four free-living protozoa. Eight 300-500bp amplification fragments were cloned and sequenced. Nevertheless, all sequences did not contain corresponding microsatellite sequences. Thus, the microsatellite DNA primers can not be applied among order or more far taxa and the specificity of microsatellite DNA is very high in protozoa.II.Microsatellite sequences in the genome of free-living protozoa were identificated and cloned based on Southern blotting. The partial genome libraries for Euglena gracilis FACHB-848 and Tetrahymena thermophila BF1 were constructed using RAPD (the random amplified polymorphic DNA) method. Subsequently, positive RAPD fragments in Southern blotting were cloned and sequenced. From a total of 70 RAPD primer profiles, eight microsatellite loci were detected and characterized and their abundances were calculated. Results show that: 1) similar to animal genomes, the GT repeats were much more abundant than the CT repeats; 2) the CT, GT microsatellites of T. thermophila BF1 were much less than E. gracilis FACHB-848, maybe this is due to the special characters of macronucleus in ciliates. 3) there were large numbers of the mononucleotide repeats in the sequences, maybe they originated in past retrotranspositional events.Furthermore, ISSR analysis was conducted to investigate the genetic relationships and genetic diversity of some ciliates.I. The genetic relationships of five ciliate strains were analyzed. Out of 13 ISSR primers, 6 primers generated highly reproducible and polymorphic DNA fragments. The genetic trees were constructed using both unweighted pair-group method for arithmetic averages analysis (UPGMA) and neighbor-joining (NJ) methods. Two T. thermophila...