Principles of Plant Genetics and Breeding

ands for identification may minimize its shortcomings.
Further, one may include parental genomes where available
to help determine bands of genetic origin.
DNA amplification fingerprinting (DAF)
DAF is a variation of the RAPD methodology. It produces
more variation than RAPD because it uses very
short (5–8 bases) random primers. Because of the great
capacity for producing polymorphisms, DAF is best
used where plants are genetically closely related (e.g.,
used to distinguish among GM cultivars that differ only
in transgenes). It is less effective for distinguishing
among species of plants at a higher taxonomic level
where genetic variation is already pronounced. The procedure
can be made more effective and efficient by
digesting the template DNA with restriction enzymes
prior to conducting the PCR technique and optimizing
the PCR environment for reproducible results.
Simple sequence repeats (SSRs)
Repetitive DNA sequences are common in the eukaryotic
genome. These short repetitive sequences are called
microsatellites (or variable nucleotide tandem repeats
– VNTRs – with tandem repeats of about 9–100). SSRs
are random tandem repeats of 2–5 nucleotides (e.g.,
GT, GACA) that occur in microsatellites. The copy
number of these repeats varies among individuals and is
a source of polymorphism in plants. The SSR technique
is also PCR based. Because the DNA sequences that flank
microsatellite regions are usually conserved, primers
specific for these regions are designed for use in the PCR
reaction.
The SSR and RFLP techniques are more tedious to
conduct, but they are more reliable than the RAPD and
DAF techniques. These procedures require nucleotide
information for primer design for the polymerase chain
reaction, and sophisticated electrophoresis systems and
computer software for accurate separation and scoring
of bands.
Amplified fragment length polymorphisms (AFLPs)
AFLPs are simply RFLPs visualized by selective PCR
amplification of DNA restriction fragments. The technique
uses primers that are 17–21 nucleotides in length
and are capable of annealing perfectly to their target
sequences (the adapter and restriction sites) as well as a
small number of nucleotides adjacent to the restriction
sites. This property of the AFLP technology makes it
BIOTECHNOLOGY IN PLANT BREEDING 249
very reliable, robust, and immune to small variations
in PCR amplification parameters (e.g., thermal cyclers,
template concentration). The technique also produces
a high marker density. A typical AFLP fingerprint (the
restriction fragment patterns generated by the technique)
contains 50–100 amplified fragments, of which
up to 80% may serve as genetic markers. Another
advantage of the technology is that it does not require
sequence information or probe collections prior to
generating the fingerprints. This is particularly useful
when DNA markers are scarce. The markers generated
are unique DNA fragments (usually exhibit Mendelian
inheritance) and are mostly monoallelic (the corresponding
allele is not detected).
Some of the applications of AFLP markers include
biodiversity studies, analysis of germplasm collections,
genotyping of individuals, identification of closely linked
DNA markers, construction of genetic DNA marker
maps, construction of physical maps, gene mapping, and
transcript profiling
Sequence characterized amplified regions (SCARs)
and sequence tagged sites (STSs)
SCAR and STS markers are derived from PCR-based
markers by sequencing the ends of fragments to develop
primers. SCAR markers are obtained by sequencing the
ends of RAPD fragments, whereas STS markers are
obtained by sequencing the ends of RFLP markers.
Single nucleotide polymorphisms (SNPs)
An SNP is a single base pair site in the genome that is
different from one individual to another. The more
common the marker, the more likely it would be for
scientists to discover difference among individuals in
the population. SNPs are often linked to genes, making
them very attractive subjects to study by scientists
interested in locating, for example, disease genes.
Sometimes, SNPs have no detectable phenotypic effect.
However, in other cases, SNPs are responsible for dramatic
changes.
Introduction to genetic mapping:
RFLP mapping
RFLP makers are inherited in a Mendelian fashion.
Gene mapping simply entails obtaining a set of crossover
frequencies between genes (see Chapter 3). Recombination
between homologous chromosomes is the basis of