The Application of Ooubled Haploid Plants to Population ... - MSpace

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analysis (Edwards et al., 1991 ). The extradion procedure uses ionic detegents. such
as SDS or CTAB. which should be removed by phenol extraction because they inhibit
Taq polymerase activity. Residual traces of phenol, which also inhibits PCR. should be
removed by chlorofom : isoamyl alcohol(24:l) extraction or by ethanol prdpitation of
the DNA (Newton and Graham. 1994).
Concems have been expressed regarding the reliability of RAPD markers.
These concems are based on the fact that random primers do not bind 100 % to target
DNA. as is the case with primers based on specific sequence information in other PCR
assays. The accuracy of random primers in RAPD analysis compared to RFLP
analysis was tested in the detedion of geneüc relationships within and among
cruciferous species (Thomann et al., 1994). They found a discrepancy for interspecific
but not intraspecific relationships between RFLP and WPD data due to the false
scoring of non-homologous amplified sequences as homologous.
DNA extraction procedures can affect repeatability of RAPD markers as well. It
is important to use consistent methods to optimize the template concentration relative
to the primer to minimize cornpetition for primer sites. Themocyclers have been tested
for reliability and different machines have produced repeatable RAPD products (Smith
and Chin, 1992; Weeden et al., 1992; Mailer et al.. 1994). Vanous concentrations of
Taq polymerase and numbers of cycles of PCR also produce repeatable RAPD
products (Smith and Chin. 1992). Methods of scoring RAPD products may also result
in non-repeatable results. Faint bands tend to be less repeatable and therefore should
not be scored as RAPD pmducts unless tested repeatedly. The optimization and