Agriculture Reference
In-Depth Information
RFLP is a technique in which varieties (or, more generally, organisms) are differentiated on the basis
of patterns derived from cleavage of their DNA at speciic sites. Restriction enzymes (restriction endonucle-
ases) cut DNA at speciic points (restriction sites) producing DNA fragments of precisely deined length.
Differences such as polymorphisms in homologous DNA sequences between two individuals are detected
by the presence of different length fragments upon separation by electrophoresis. The DNA fragments can
be visualized using a radioactive or luorescent probe. A probe is typically a sequence of short, single-
stranded genomic or cDNA that hybridizes with one or more fragments having a complementary sequence
of nucleotides, after those fragments have been separated by electrophoresis. Most RFLP markers are co-
dominant (both alleles in heterozygous sample will be detected) and provide complete genetic information
at a single locus.
One advancement that helped expand the use of RFLP markers in routine genetic purity analysis has
been the substitution of radioactive probes by sensitive non-radioactive marker systems. However, the
use of RFLP for routine variety identiication has been limited by several factors. One factor is the high
amount of DNA required for RFLP analysis, typically ranging from 5 to 10 μg making the isolation of sufi-
cient DNA amounts time-consuming and labor intensive. The relative low level of polymorphism observed
among crops like wheat or tomato (Bryan et al., 1997; Manifesto et al., 2001; Smith, 1995) is another
factor limiting the utility of RFLP analysis. Finally, the cost and complexity of the method, especially when
compared to other DNA marker methods, has limited the use of RFLP for varietal identiication. A second
generation of simple, low-cost tests, made possible by PCR techniques, now accounts for most variety test-
ing using molecular markers. These tests include RAPD, SSR and RFLP.
random Amplification of Polymorphic dnA (rAPd)
One advance in genetic purity tests has been the polymerase chain reaction (PCR) technology called random
ampliication of polymorphic DNA or RAPD (Williams et al., 1990). RAPD reactions amplify unknown
(random) DNA segments. Therefore, one of the principal advantages of RAPD analysis is that it does not
require extensive or costly marker development since no knowledge of target DNA sequences is necessary,
making it the least expensive PCR-based method to initiate in a genetic purity testing program. This tech-
nique uses several arbitrary short primers (8-12 nucleotides), all or some of which will hybridize to a large
genomic DNA template of individual seeds at two different sites, one on each strand of the complementary
DNA. Under appropriate temperature alternations, a thermostable DNA polymerase synthesizes discrete
DNA products (usually 200 to 2,000 base-pairs long). Each primer can consistently amplify several unique
DNA fragments that are then separated on an electrophoretic gel. Some of these fragments are characteristic
of a genotype and useful in genetic purity tests (Figure 9.1).
Figure 9.1. RAPD fragment polymorphisms for different soybean genotypes. M=molecular weight marker
(Weian et al., 2009).
