SSR Assays
Primers were designed to sequences flanking SSRs using the computer program PRIMER. 10l PCRs
consisting of 20ng genomic DNA, 1x PCR buffer, 0.3U Taq polymerase (Boehringer-Mannheim),
0.3Mols of forward and reverse primers, 200M dNTPs and 0.5 Ci of alpha32P-dCTP. The cycling
conditions for PCR on a PE 9600 were optimised to one of the following PCR conditions:
A, B, C were PCR conditions 1, 2 and 3 of Liu et al. (1996) respectively.
NB ‘I’ and ‘J’ are the ‘55’ and ‘60’ of the GMS*** primers from IPK
SSR Assays
A:
18 cycles: 1 min @ 94C, 30 secs @ 64C (decrease 1C per 2 cycles until 55C), 1 min @ 72C,
30 cycles: 1 min @ 94C, 1 min @ 55C, 1 min @ 72C,
1 cycle: 5 mins @ 72C.
B:
18 cycles: 1 min @ 94C, 30 secs @ 69C (decrease 1C per 2 cycles until 60C), 1 min @ 72C,
20 cycles: 1 min @ 94C, 1 min @ 55C, 1 min @ 72C,
1 cycle: 5 mins @ 72C.
C:
1 cycle: 3 min @ 94C, 2 min @ 55C , 1.5 min @ 72C,
30 cycles: 1 min @ 94C, 1 min @ 55C, 1.5 min @ 72C,
D:
1 cycle: 3 mins @ 94C, 1 min @ 66C, 1 min @ 72C,
5 cycles: 30 secs @ 94C, 30 secs @ 65C (decrease 1C per cycle until 60C is reached), 30 secs @ 72C,
24 cycles: 30 secs @ 94C, 30 secs @ 60C, 30 secs @ 72C,
1 cycle: 5 mins @ 72C.
E:
1 cycle: 3 min @ 94C, 1 min @ 55C , 1 min @ 72C,
30 cycles: 1 min @ 94C, 1 min @ 55C, 1 min @ 72C,
1 cycle: 5 mins @ 72C.
F:
1 cycle : 3 min @ 94C, 1 min @ 58C, 1 min @ 72C,
30 cycles: 30 secs @ 94C, 30 secs @ 58C, 30 secs @ 72C,
1 cycle: 5 mins @ 72C.
G:
1 cycle: 3 min @ 94C, 1 min @ 69C, 1 min @ 72C,
5 cycles: 30 secs @ 94C, 30 secs @ 68C (decrease 1C per cycle until 63C is reached), 30 secs @ 72C,
24 cycles 30 secs @ 94C, 30 secs @ 63C, 30 secs @ 72C,
1 cycle 5 mins @ 72.
H:
1 cycle: 3 min @ 94C, 1 min @ 53C, 1 min @ 72C
30 cycles: 30 secs @ 94C, 30 secs @ 53C, 30 secs @ 72C,
1 cycle: 5 mins @ 72C.
I:
35-45 cycles: 1 min @ 94C, 1 min @ 55C, 2 min @ 72C,
1 cycle: 60 mins @ 72C.
J:
35-45 cycles: 1 min @ 94C, 1 min @ 60C, 2 min @ 72C,
1 cycle: 60 mins @ 72C.
The conditions that amplified the strongest product of the expected size on 1.5% agarose gel were
utilised in our mapping studies which were carried out as described by Morgante et al., (1994). Equal
volumes of 95% formamide electrophoresis loading buffer were added to the samples, which were then
denatured, snap cooled on ice, and electrophoresed in 6% Easigel (Scotlab) according to standard
procedures. An M13 sequencing marker was run to estimate product sizes. Gels were double loaded.
Visualisation of results was acheived by exposure of fixed, dried gels to x-ray film.