Contents - Faperta

Physico-Chemical and Molecular Markers for Resistance to Insect Pests 167
origin or sequence. Ever since thermostable DNA polymerase was introduced, the use of
PCR in genomic research has increased tremendously (Saiki et al., 1988; Mullis, 1990). The
primer sequences are chosen to allow base-specifi c binding to the template in reverse
orientation. PCR is extremely sensitive and operates at a very high speed. Several types of
molecular markers have been used for developing genetic linkage maps of different crops,
and to identify quantitative trait loci (QTLs) associated with resistance to insects (Terry
et al., 2000; Yencho, Cohen, and Byrne, 2000; Smith, 2005). Different types of markers used
to detect QTLs associated with insect resistance are discussed below.
Restriction Fragment Length Polymorphisms
The restriction fragment length polymorphisms (RFLPs) markers detect the differences
between genotypic DNA of different genotypes when restriction enzymes from bacteria
cut genomic DNA at specifi c nucleotide binding sites and yield variable sizes of DNA fragments
(Karp et al., 1997; Primrose, 1998; Srivastava and Narula, 2004). Restriction enzymes
are also called restriction endonucleases because they cut the DNA molecule at a particular
nucleotide sequence, and different restriction enzymes cut DNA into pieces of different
sizes. The digested DNA is separated by electrophoresis according to size, normally on
agarose or polyacrylamide gel. The separated DNA fragments are then transferred to a
nylon membrane and exposed to a particular DNA probe in a procedure referred to as
Southern blotting. Complementary binding between the probe DNA and membrane DNA
provides information about the possible location of the gene for insect resistance. The differences
in DNA banding patterns on an autoradiogram of a Southern blot indicate the
presence of one or more restriction sites in a sequence. The sequence containing a restriction
site is one allele, while the corresponding sequence missing the restriction site is the other
allele. When restriction sites are compared between two genotypes, one genotype may have
the site, while the other does not. If differences exist, they are referred to as polymorphisms
between the two genotypes. RFLP probes allow mapping of loci linked to resistance genes,
and hundreds of RFLP loci have been mapped in several crop genomes. RFLP markers usually
behave in a codominant manner and thus can detect heterozygotes and more allelic
variation in natural plant populations. RFLPs, being codominant markers, can detect the
coupling phase of DNA molecules, as DNA fragments from all homologous chromosomes
are detected. They can easily determine if a linked trait is present in a homozygous or
heterozygous state in an individual. Such information is highly desirable for recessive traits.
Their inability to detect single base changes restricts their use in detecting point mutations
occurring within the regions at which they are detecting polymorphism. Use of RFLP
markers needs more time to complete the analysis (7 to 10 days), and involves the use of
radioactive isotopes. RFLP markers have been used effectively to map insect resistance
genes in several crops (Yencho, Cohen, and Byrne, 2000; Smith 2005). The RFLP probes from
one species can often be used to detect homologous regions in the genomes of related species
and, thus, are widely used to identifi y syntenic genomic regions (Devos and Gale, 2000).
Sequence-Tagged Sites
RFLP probes specifi cally linked to a desired trait can be converted into PCR-based sequencetagged
sites (STS) markers based on nucleotide sequence of the probe giving polymorphic
band pattern to obtain specifi c amplicon (Karp et al., 1997; Srivastava and Narula, 2004).
Using this technique, tedious hybridization procedures involved in RFLP analysis can be
overcome. However, in many cases, the power to detect polymorphism is reduced compared