M.Sc. / Mnandi and Astrid are two commercially important potato cultivars in South Africa. These two cultivars are closely related and are morphological virtually identical. It is, however, necessary to be able to distinguish between these two cultivars, because each of these cultivars has certain desirable characteristics. It was decided to use DNA markers, since DNA markers are not influenced by the environment and the polymerase chain reaction (PCR) based DNA markers are relatively easy, cheap and fast. It was decided to develop a sequenced characterized amplified region (SCAR) due to the problems with the reproducibility of random amplified polymorphic DNA (RAPDs). SCARs are derived from RAPD fragments by using the sequence of a RAPD derived fragment to design a set of new longer primers (usually 20-24mer) which are less sensitive to PCR conditions. Ten commercial potato cultivars (Astrid, Mnandi. BP,, Buffelspoort, VanderPlank, Up-to-Date, Hoevelder, Hertha, Pimpernel and Agria) were used in this study. Commercially available RAPD primers (102) were evaluated to seek a polymorphism unique to either Mnandi or Astrid. Thirtyseven polymorphisms between Astrid and Mnandi were identified but only three were unique. The polymorphism obtained with OPH-15 was however, not reproducible. The polymorphisms obtained with UBC 509 and 582, corresponding to the presence in Mnandi of a 300 and 900 by fragment respectively, were reproducible. These two fragments, UBC 509 3" and UBC 582900, were cloned into the pMosBlue TA cloning vector and sequenced. The identity if the inserts in the recombinant plasmids were verified with PCR and Southern blotting. The sequences were used to develop two sets of SCAR primers, SCAR UBC 509 3" and SCAR UBC 582900 . The two SCAR primer pairs were then used in PCR reactions. The SCAR UBC 509 300 primer pair amplified a fragment of 230 by in both Astrid and Mnandi and a fragment of 260 by in Mnandi. The polymorphism is thus retained and SCAR UBC 509 3" can be used to distinguish between Astrid and Mnandi. The SCAR UBC 582' primer pair amplify a fragment of 500 by in both Astrid and Mnandi as well as some other longer fragments. It was not possible to regain a polymorphism by either elevating the annealing temperature or by digesting the amplification products with restriction enzymes. SCAR UBC 582' could thus not be used to distinguish between Astrid and Mnandi.

The ultimate goal of the· breeding scheme, of which the present study is a part, is to introduce exotic germplasm into existing cultivars of Solanum tuberosum through· 4x X 2x crosses using the South American diploid potato species Solanum phureja as the pollen parent. The first phase of this program includes the 'reconstruction' of a highly heterozygous diploid, pollen parent by .first : reducing the chromosome number of the S. phureja clones to the monoploid level and subsequently fusing genomes of two unrelated monoploid plants either by somatic hybridization or by cross-pollination between fertile doubled monoploids. Within this framework, the objectives of this research were to analyze variation among anther-derived plants of Solanum phureja regarding their: 1) ploidy level and morphology, 2) net photosynthesis and its biochemical components, and 3) nuclear genomic structure, particularly with regard ·to the amplification of rRNA genes as influenced by the anther-culture process. Based on the analysis of several morphological characters of the anther-derived plants by canonical discriminant function, four characters (anther length, number of chloroplasts/pair of guard cells, leaf width, corolla width) were selected for most effective assignment of plants to their ploidy groups by clustering procedures. Clustering of the anther-derived plants proved to be an efficient means of separating monoploids from higher ploidy levels. To assess the impact of the process of anther-culture on the physiology of the resulting plants and to evaluate the possibility of selecting anther-derived genotypes for further breeding efforts, monoploid, diploid and tetraploid anther-derived plants were studied regarding their net photosynthesis and its component characteristics. Leaf area, net photosynthesis and chlorophyll content increased with increasing ploidy' Among .the. monoploids I. Rubisco activity and concentration displayed a. significant genotypic effect; whereas in the diploid group variation among genotypes was significant for total protein content and maximum specific activity of ribulose bisphosphate carboxylase. Among the tetraploid genotypes, significant differenc.es were found with respect to net photosynthesis and specific leaf weight. Two exceptional genotypes were identified: a monoploid with an increase of 28% fcfr maximum activity of ribulose bisphosphate carboxylase and a tetraploid with an increase of 30% for net photosynthesis over the anther-donor plant. To evaluate DNA variation among the anther-derived plants, the nuclear genomes of anther-derived monoploid and diploid plant were studied by DNA reassociation kinetics. It was found that the nuclear genome of the monoploid has undergone differential replication resulting in an increase of sequences consisting of highly· repetitive DNA. Free solution RNA-DNA hybridization showed that the monoploid DNA contained 30% more rDNA sequences than the diploid. Southern blot analysis using rRNA as the probe revealed variation for copy number of certain restriction fragments and for restriction enzyme cleavage sites. / Ph. D.