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

5.7
260 nm. To check for DNA degradation, the samples were separated
eledrophoretically on ethidium bromide stained 0.75% agarose gels in 0.5 x TA€ buffer
(trislsodiumlaœtatelEDTA pH 8.0). lsolated DNA was stored in TE buffer at -20" C.
Polymerase Chain Readon (PCR)
A set of 30 primers (oligonucleotides), obtahed from the University of British
Columbia (UBC), was tested to detect polyrnorphism between the donor populations of
DCSJ and Reward. PCR was pe~omed using 50 ngîpl of genomic DNA in 25 pl
volumes using a PTC-1 OON (MJ Research, Inc.), as reported by Mailer et al. (1 994).
RAPD products were separated by electrophMesis on ethidium bromide stained 1 -4 Oh
agarose gels in lx TAE buffer. Genomic DNA from 8. napus and a negative control
were exposed to the PCR conditions with the analysis of each new primer. Lambda
DNA diçested with Hindll (Phamiacia Biotech) was included as the sire marker.
RA PD Analysis
Primers that expressed polyrnorphism between DSC-3 and Reward donor
populations were selected from the initial primer set. These primers, shown in Table
5.1, were tested against each of the DH lines produced from the two donor populations.
UBC primers 329 and 338 were selected for RAPD analysis because they
resulted in the highest amount of informative bands being expressed among al1 of the
primers used to analyze the DH lines (Table 5.2). This was detennined by calculating