Abstract
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.