Agriculture Reference
In-Depth Information
SSRs are believed to be the result of unequal crossing-over or DNA replication errors, leading to the
formation of such DNA secondary structures as hairpins or slipped strands (Pearson and Sinden, 1998).
These loci then mutate by either insertions or deletions of one or more repeat units resulting in length poly-
morphisms. Polymorphisms can be easily detected by gel separations of PCR-ampliied fragments obtained
using a unique pair of primers lanking the repeat region (Weber and May, 1989). Allelic proiles can be
generated by deining the allelic constitution of varieties at multi-allelic loci. The number of targeted loci,
although relatively few, should be the minimum needed to produce a unique proile for deinitive variety
identiication.
SSRs offer several advantages over other molecular markers. SSR analysis can be semi-automated
with little DNA required and no radioactivity. Microsatellites are ubiquitously distributed throughout the
genome, allowing for the identiication of multiple alleles at a single locus and are co-dominant. SSR analy-
sis has been used for genotype identiication of a wide range of crops such as alfalfa (Mengoni et al., 2000),
barley (Fernandez et al., 2002; Ramsay et al., 2000), melon and cucumber (Danin-Poleg, 2001), peanut
(Raina et al., 2001), peas (Burstin et al., 2001), rape (Tommasini, 2003), rice (Blair et al., 1999; Joshi et al.,
2000), soybean (Rongwen, 1995), sunlower (Paniego et al., 2002), tomato (He et al., 2003; Areshehenkova
and Ganal, 2002), and wheat (Lima et al., 2003; Roder et al., 2002).
Amplified fragment Length Polymorphism (AfLP)
Ampliied fragment length polymorphism, irst described by Zabeau and Vos (1993), is a powerful DNA
ingerprinting technique. Similar to RAPD, and contrary to RFLP and SSR, AFLP eliminates the need for
DNA-sequence information. Extremely small amounts (-50 ng) of either intact or partially degraded DNA
can be used. Vos et al. (1995) described the AFLP method as one which combines the reliability of RFLP
with the power of PCR ampliication. The method exploits the genetic variations between closely related
varieties generating DNA restriction fragments which, after PCR ampliication and gel electrophoresis,
results in unique “ingerprints.” Although AFLP technology has been used predominantly for identifying
and assessing the degree of variability among plant varieties, it is also used in phylogeny analysis and
population genetics.
The procedure starts with using two restriction endonucleases (EcoRI and Msel) to digest cellular
DNA and generate restriction fragments. Adaptors (linkers) are then ligated to the ends of the DNA frag-
ments, with the combined adaptor and restriction site sequences serving as the primary sites for PCR selec-
tive ampliication. Although thousands of fragments can be generated by DNA digestion, depending on the
size of the genome, only a subset of those fragments is ampliied. This is achieved by two ampliication
steps, a “pre-selective” and a “selective” ampliication. The irst primers that extend one base into the
unknown part of the fragments are used, reducing the number of PCR ampliied fragments by a factor of 4.
This is followed by a more selective primer with a 3 bp extension into the fragments. Because of the high
selectivity, using combinations of primers differing by just one single base in the extension will amplify dif-
ferent subsets of fragments. Polyacrylamide gel electrophoresis is then used to generate handling patterns
with the ability to differentiate between fragments with a single nucleotide length difference.
The main advantage of this method is the large number of polymorphisms that it can generate, making
it ideal for plant variety registration (Law et al., 1998). Several studies have shown that the number of
polymorphic bands generated by AFLP is several folds higher than that generated by RFLP, SSR or RAPD
(Maughan et al., 1996; Nakajima et al., 1998; Tyrka, 2004). Other advantages to using this method include
the small amount of DNA needed, and no requirement for prior knowledge of the DNA sequence. One fre-
quently cited disadvantage of AFLP is that the choice of primers could have a signiicant inluence on the
magnitude of observed variation. Therefore, a preliminary screening of primer combinations, especially for
relatively unstudied species, is necessary to verify that the primers used will produce suficient and mean-
ingful results.
