Optimisation of the randomly amplified polymorphic DNA (RAPD) technique for the characterisation of selected South African maize (Zea mays L.) breeding material.
Loading...
Date
2000
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an
important component of the South African economy. Considerable effort has been directed
towards the genetic improvement of maize through both conventional breeding and
biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity
in plant breeding. A widely used molecular based and relatively inexpensive method for DNA
fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD
technique was tested in this study for its potential use in maize breeding programmes. Initial
results using the technique showed a low degree of reproducibility, therefore both the DNA
isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice
of Taq polymerase buffer were three of the variables found to be influential in ensuring
reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was
evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a
total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible
profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA
fragments. RAPD markers were able to distinguish between all seven genotypes with five primers
producing specific fragments for four genotypes. Genetic similarity matrices were calculated
using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic
Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to
generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only
minor differences were observed between the Genstat or PAUP method of analysis). Genetic
diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with
the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential
for genome analysis of maize. The applicability of the technique for marker assisted selection was
also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened
for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between
the NILs. Four primers produced five polymorphic fragments present in the resistant inbred
K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals
was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested
seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3')
showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the
HtN gene for leaf blight resistance. This study shows that the RAPD technique does have
application in maize breeding programmes.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.
Keywords
Maize--Breeding., Maize--Genetics--Technique., Random amplified polymorphic DNA., Genetic markers., Genetic polymorphisms., DNA fingerprinting of plants., Theses--Genetics.