Contents - Faperta

Physico-Chemical and Molecular Markers for Resistance to Insect Pests 169
1996; Burns et al., 2001; Sharopova et al., 2002; Zhang et al., 2002). The SSRs provide highquality
and consistent results, but the markers are expensive to develop as they require
extensive sequence data from the species of interest. The sequence data can be generated
from libraries of cloned DNA (with or without enrichment for a specifi c SSR motif). Existing
EST resources for a specifi c crop can also be used. EST-derived SSRs are less polymorphic
than those derived from genomic libraries, but both are useful as they are PCR-compatible
and typically inherited in a codominant manner. Polymorphic SSRs have been used to
map resistance to shoot fl y, A. soccata, in sorghum (Folkertsma et al., 2003; Hash et al., 2003);
greenbug, Schizaphis graminum (Rondani), resistance in wheat (Weng and Lazar, 2002); and
for covering the soybean genome (Narvel et al., 2000).
Sequence-tagged microsatellite markers (STMs) primers are complementary to the fl anking
regions of SSR loci, and yield highly polymorphic amplifi cation products. Polymorphism
appears because of variation in the number of tandem repeats (VNTR loci) of a given
repeat motif. Tri- and tetranucleotide microsatellites are more popular for STMS analysis
(based on PCR product separation on PAGE gels) because they present a clear banding pattern
after PCR and gel electrophoresis. However, dinucleotides are abundant in genomes
and have been used as markers. The di- and tetranucleotide repeats are present mostly in
the noncoding regions of the genome, while 57% of trinucleotide repeats reside in or around
the genes. A good relationship has been observed between the number of alleles detected
and the total number of SSRs within the targeted microsatellite DNA of a reference genotype.
Thus, the larger the repeat number in the VNTR locus, the greater the number of
alleles that are expected to be detected in a large population of diverse accessions.
Randomly Amplified Polymorphic DNA
The randomly amplifi ed polymorphic DNAs (RAPDs) are based on PCR, and use arbitrary
primers for initiating amplifi cation of random pieces of the sampled plant DNA, and
requires no knowledge of the genome to be screened. PCR-based markers involve in vitro
amplifi cation of particular DNA sequences or loci with the help of specifi cally or arbitrarily
chosen oligonucleotide sequences (primers) and a thermostable DNA polymerase enzyme.
The amplifi ed fragments are separated electrophoretically and banding patterns detected
by staining or autoradiography. PCR is a versatile technique invented during the mid-
1980s (Saiki et al., 1985). However, the results are inconsistent across populations and laboratories
(Powell et al., 1996; Staub, Serquen, and Gupta, 1996). The PCR primers used for
RAPD analysis are short random DNA sequences approximately 10 nucleotides long that
amplify homologous genomic DNA sequences during the PCR process. Differences in the
DNA sequences of resistant and susceptible plants result in different primer binding sites,
which result in differences in PCR products that allow the visualization of polymorphisms
between the DNA in the resistant and susceptible plants. Insect resistance genes in apple,
rice, and wheat have been mapped using RAPD primers (Dweikat et al., 1994; Nair et al.,
1995, 1996; Roche et al., 1997; Botha and Venter, 2000; Venter and Botha, 2000; Selvi et al.,
2002; Jena et a1., 2003). Reproducibility of RAPD markers has been quite diffi cult in many
studies. In addition, DNA banding patterns based on RAPD primer amplifi cation do not
reveal heterozygotes as they are inherited in a dominant manner.
Inter Simple Sequence Repeat
In this technique, primers based on microsatellites are utilized to amplify inter-simple
sequence repeat (SSR) DNA sequences (Staub, Serquen, and Gupta, 1996, Srivastava and
Narula, 2004). Here, various microsatellites anchored at the 3′ end are used for amplifying