Agriculture Reference
In-Depth Information
Most RAPD studies are conducted on tissue from growing plants. McDonald et al. (1994) developed a
DNA extraction procedure directly from dry seeds for ive agronomic crops, reducing the time of analysis.
Other studies with soybean seed revealed that seed deterioration, contamination by fungi, and differing
seed maturation environments did not affect the stability of RAPD markers (McDonald, 1995). Studies
with corn seed showed that RAPD markers were dominant and that hybrid seed generally possessed mark-
ers from both inbred parents (Zhang et al., 1996a). Interestingly, RAPD markers obtained from the corn
embryo were characteristic of the parents while those from the seed coat and endosperm were not; this was
attributed to the desiccation of these seed tissues and subsequent degradation of DNA during seed matura-
tion (McDonald, 1995). RAPD markers have been used to determine genetic purity of a wide range of crops
such as barley (Selbach and Cavalli-Molina, 2000), coffee (Diniz et al., 2005), Kentucky bluegrass (Curley
and Jung, 2004), pepper (Ilbi, 2003), perennial ryegrass (Sweeney and Danneberger, 1994), petunia and
cyclamen (Zhang et al., 1997), rice (Masataka et al., 2003) and soybean (Zhang et al., 1996b).
The use of RAPD markers is relatively inexpensive, simple, fast, avoids the use of growing plant
tissue, is applicable to a number of crops and can be successfully used to achieve greater sensitivity in the
determination of genetic purity in seeds. Other advantages of RAPDs include the following (McDonald
1995): (1) RAPDs can provide greater potential discrimination of varieties since the nucleotide composi-
tion of a gene is being determined instead of the product of a gene such as an enzyme, (2) RAPDs are more
versatile than protein electrophoresis; over 700 primers are available for screening in the former compared
with 20 enzyme systems in the latter, (3) RAPDs do not pose the potential human health and environmental
disposal issues associated with radioisotopes used in other DNA technologies; (4) RAPDs require the same
general equipment and technical expertise as protein electrophoresis with the exception of a DNA ther-
mocycler; and (5) the cost of a RAPD analysis and the time taken to complete it are equivalent to those of
current protein electrophoresis protocols. Since no knowledge of the DNA sequence for the targeted gene is
necessary, RAPD analysis is particularly useful for comparing the DNA of lesser known or studied species,
or in situations where a relatively few DNA sequences are compared. The major continuing concern about
the use of RAPDs is the inability to obtain reproducible results among different laboratories (Riedy et al.,
1992; Heun and Helentjaris, 1993) although satisfactory repeatability can usually be obtained for samples
ampliied within the same laboratory provided care is taken and check samples are used to evaluate varia-
tions in ampliication that do not have a genetic basis. Further research into the standardization of the pro-
tocol is still necessary. Other limitations of RAPD analysis include its lower resolving power compared to
species-speciic DNA methods, and the requirement for large intact DNA templates. Mismatches between
the DNA template and primers can result in either the absence or decrease of PCR products, making valid
interpretations of analysis results more dificult.
Numerous variations of the RAPD technique exist once a marker linked to the genotype is discov-
ered to improve speciicity. These include allele-speciic PCR (Wu et al., 1989), allele- speciic ligation
(Nickerson et al., 1990) and sequence-characterized ampliied region (SCAR) (Paran and Michelmore,
1993) assays. While the advantages of RAPDs are obvious, continuing advancements in molecular biology
techniques provide even greater promise for enhancing the sensitivity of genetic purity determinations.
Simple Sequence repeats
(SSr)
Simple sequence repeats (also known as microsatellites, microsatellite repeat polymorphisms or short
tandem repeats) have become one of the most important molecular markers for plant genome analysis
as well as marker-assisted breeding. SSRs are genetic loci consisting of 1 to 6 bp (base pairs) repeated in
tandem and ubiquitously distributed throughout the eukaryotic genomes, where the whole repetitive region
does not exceed 150 bp due to high mutation rates affecting the number of repeat units. SSRs show exten-
sive length polymorphism (Morgante and Olivieri, 1993) and are therefore ideal for DNA ingerprinting and
diversity studies. Since SSRs can be readily assayed by PCR, they are also considered ideal genetic markers
for the construction of high-density linkage maps.
