Agriculture Reference
In-Depth Information
dwarfism, leaf shape, absence of petals, etc. It is pos-
sible, of course, to choose ones that are easily scored
but the difficulties with morphological markers are
that they: cannot always be scored early in develop-
ment (e.g. flower colour); are often associated with
deleterious effects (e.g. albinism); are often relatively
rare; their expression is not always independent of
the environment in which they grow; often show
dominance/recessiveness.
of polymorphism; have no discernible effects on the
phenotype; only pieces of tissue are required from
any stage of development; and some systems show
codominance.
Given the above characteristics of molecular markers,
particularly their relatively unlimited numbers, it is no
surprise that the advent of the possibilities of molecular
markers in the 1990s was greeted with some excitement
and is seen as providing a major change in the potential
to exploit the ideas for using markers advocated some
70 years earlier.
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Biochemical markers - such as isozyme markers.
Isozymes (an abbreviation for isoenzyme) are variant
forms of an enzyme, which are functionally identical,
but which can be distinguished by electrophoresis -
in other words when placed in an electric field. Under
these circumstances the different forms of the enzyme
will migrate to different points in the electric field
depending on their charge, size and shape. Isozymes
have been used very successfully in certain aspects
of plant breeding and genetics since they: gener-
ally appear to be nearly neutral in their effects on
fitness: are rarely associated with undesirable phe-
notypic effects on other traits: are usually free of
environmental influence; and can often be extracted
from tissue early in development. So they have a
number of inherent properties that allows them to
be used effectively for characterizing, and selection
for, qualitative and quantitative characters. Unfortu-
nately, the number of genetic markers provided by
isozyme assays is not over-abundant, and they can
be either co-dominant or dominant in expression. As
a result, the use of isozymes as genetic markers did
not allow the full potential of genetic mapping to be
realized.
Molecular markers
Non-PCR methods - DNA/DNA hybridization
The first and most widely known of these is Restriction
Fragment Length Polymorphism (RFLP). Other non-
PCR methods do exist, for example the use of tandemly
repeated regions of DNA, known as mini-satellites or
micro-satellites, but these will not be described here.
RFLP analysis involves digesting the DNA (cutting
it at sites with specific sequences - there are a number
of different enzymes, called restriction enzymes that cut
different patterns of sequences) into fragments, which
can then be separated out by gel electrophoresis (as for
isozymes separating them by their differing mobilities
in an electric field). To visualize their positions, they are
'blotted' onto a filter, where they are hybridized with
a labelled (usually radioactive) 'probe'. The probe is a
short fragment of DNA, which may be from a known
gene, an expressed sequence or an unknown fragment
of the genome. When the 'blotted DNA', having first
denatured it to reduce it to single strands (rather the
usually double-stranded state of DNA), and the probe
(also denatured) are brought together, where there is
an exact match in the complementary sequences they
will hybridize (by hydrogen bonding) or bond. The
filter is then washed to remove all the excess probe and
leave only that which is now bonded with our sample
DNA. If we expose the filter to X-ray film, when it
is developed it will show where the probe still remains,
hence where the probe has hybridized and so where there
was a piece of the DNA we were investigating which
had a complementary sequence. The pattern of bands
obtained in this way is called the restriction fragment
pattern. Using a varied combination of enzymes and
Molecular markers - there are basically two systems
by which molecular markers are generated and these
need to be described briefly to allow an understanding
of their application. The two systems can conve-
niently be classified as non-PCR based methods and
PCR based methods. Before briefly describing each
it is worth pointing out that molecular markers are
simply differences in the DNA between individuals,
groups, species taxa etc. Clearly the type and level of
variation in DNA that we would want to examine is
different depending on what level of distinction we
are interested in and what questions we are answer-
ing. But the main characteristics of molecular markers
are that they: are a ubiquitous form of variation; are
free from environmental influence; show high levels
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