Genomic studies

Plants possess three different genomes most commonly used in molecular studies, namely: chloroplast, mitochondrial and nuclear genomes. The chloroplast genome is highly conserved in structure, gene content and order and is widely used in diversity studies at taxonomic levels. The ribosomal RNA gene family of the nuclear genome are located at specific chromosomal loci and are organised in tandem repeats. They possess highly conserved and variable regions that are extensively used in polymorphism studies. Universal primer pairs have been designed to enable amplification of different regions of these genomes in a wide range of organisms for taxonomic studies at all levels.  Polymerase Chain Reaction (PCR) sequencing is considered the most comprehensive method of molecular analysis. This approach is applicable to extremely small DNA samples and the technique is fully automated. Sequence data can provide hypotheses on the relationships between different classes of individuals, in addition to providing groupings for individuals. Various non-coding regions of the chloroplast and nuclear DNA of Stangeria sp. have been amplified by PCR and subjected to sequencing. The trnL intron region of the chloroplast DNA yielded sequences that were analysed by computer software (CLUSTALX and BIOEDIT). The nuclear DNA has not yielded any satisfactory sequences and experiments are ongoing to optimise the reaction.  A marker system called Inter-Simple Sequence Repeats (ISSRs) recently developed as an anonymous approach, can access variability in the microsatellite regions (simple sequence repeats that are highly mutable) dispersed throughout various genomes (particularly the nuclear genome). They can be detected by Polymerase Chain Reaction and visualised by electrophoresis on agarose or polyacrylamide sequencing gels. They are generally used for the multi-allelic study of a single locus, in below-species level population studies, and for assessing diversity among genotypes within a species. A preliminary study done by 4 ISSR primers on Stangeria DNA have produced specific fingerprints (markers) for individuals. Various aspects of the PCR reactions are being optimised, after which a genetic map will be created for each plant included the study.

Choroplast trnL intron region primed PCR products of Stangeria DNA as visualised on agarose gel. ISSR1and ISSR4 primed PCR of 3-nucloetide long microsatellite regions of Stangeria DNA.    PCR products of the intergenic transcribed spacer region of nuclear DNA of Stangeria as visualised on agarose gel.

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