Principles of Plant Genetics and Breeding

248 CHAPTER 14
Isozymes
Enzymes are macromolecular compounds that catalyze
specific biochemical reactions. Most enzymes are proteins.
Scientists have developed methods that allow the
coupling of certain chemical reactions to the biochemical
processes for colorimetric detection and location of
specific enzymes. Isozymes are multiple forms of an
enzyme that differ from each other by the substrate
they act on, their maximum activity, or their regulatory
properties. The term refers to enzyme polymorphisms
that result from different loci. Another term, allozyme,
is reserved for allelic enzymes.
As previously discussed, proteins can exist at one of
four levels of structural complexity, of which the primary
structure is the simplest. The most complex form,
the quaternary structure, is attained through the folding
and aggregation of polypeptide units. When an
enzyme comprises one polypeptide chain, it is called
a monomer. An enzyme comprising aggregates of
polypeptide chains is called a multimer (or polymer). If
an allozyme is multimeric, both homomers and heteromers
will be produced in heterozygous individuals.
Isozyme technology has certain limitations, the major
ones being the paucity of isozymes in plants and their
tendency to be limited to certain chromosomes (not
evenly distributed in the genome). Also, isozymes are
sensitive to tissue type and age. However, the technology
is inexpensive and relatively easy to apply. Some of
the earlier successful applications were made in tomato
(e.g., tagging of the Aps-1 locus for acid phosphatase,
and the exploitation of its linkage to nematode resistance).
In spite of advances in molecular marker technology,
isozymes are still used for certain purposes, such
as the authentication of hybridity in hybrid development.
Restriction fragment length polymorphisms (RFLPs)
RFLP markers are the first generation of DNA markers
and one of the best for plant genome mapping. The
RFLP variations are codominantly inherited. Mutation
events (e.g., insertion, deletion) cause natural variations
to occur in the genome. These variations may cause
alterations (abolish) in the recognition sites for restriction
enzymes. As a consequence, when homologous
chromosomes are subjected to restriction enzyme digestion,
different restriction products are produced upon
electrophoresis (hence the term restriction fragment
length polymorphisms). RFLPs are randomly distributed
throughout the genome of an organism and may
occur in both exons and introns. The DNA profiles or
fingerprints produced are specific to the combination of
the restriction enzyme and probe (used to detect the
polymorphism, using Southern blotting). Probes may
be derived from random genomic DNA libraries, cDNA
libraries, or minisatellites from other organisms.
One of the advantages of RFLPs is that the sequence
used as a probe need not be known. All that a researcher
needs is a genomic clone that can be used to detect the
polymorphism. Very few RFLPs have been sequenced
to know what sequence variation is responsible for the
polymorphism. In the absence of sequence information,
interpreting complex RFLP allelic systems may be
problematic.
There are different types of RFLP polymorphisms,
the simplest being the two-allele system involving the
presence or absence of a recognition site for a single
restriction enzyme. Screening reveals three different
types of banding patterns: a large band (homozygous),
two smaller bands (restriction site occurs on both
homologues), and all three bands (heterozygous). It is
assumed that a single base pair change within the recognition
site will result in a chromosome that would either
have the restriction site or not. In another allele system,
one band corresponds to one allele. This system is also
easy to score. One variable band corresponds to a
homozygote. An individual inherits only two of the
variant types of fragment sizes. Tomato was one of the
first plant species to be characterized by RFLPs. The disadvantage
of this marker system is that it is expensive
and has low throughput.
Random amplified polymorphic DNA (RAPD)
PCR is a technology discovered in 1986 for directly
amplifying a specific short segment of DNA without the
use of a cloning method. This eliminates the tedious
process of repeated cloning to obtain ample quantities
of DNA for a study. An attractive feature of a PCRbased
marker system is that only a minute amount of
DNA is needed for a project. Also, it has a higher
throughput than RFLP. Because of the sensitivity of
PCR technology to contamination, it is common to
observe a variety of bands that are not associated with
the target genome but are artifacts of the PCR condition.
Consequently, certain bands may not be reproducible.
RAPD is a PCR-based marker system. In RAPD, the
total genomic DNA is amplified using a single, short
(about 10 bases), random primer. The PCR product is
electrophoresed. This method yields high levels of polymorphism
and is simple and quick to conduct. When
using RAPD markers, using only the reproducible major