Marker-Assisted Backcrossing Selection for High O/L Ratio in Cultivated Peanut - Translational Genomics for Crop Breeding: Abiotic Stress, Yield and Quality - page 182

Fig. 11.2. Structures of ahFAD2A and ahFAD2B genes and mutational polymorphic sites between 'Nakateyutaka' and

'YI0311.'

was performed according to the protocol of
the TaqMan Genotyping Master Mix (Applied
Biosystems, USA).
The transposon insertional polymorphism on
ahFAD2B was identified on a 2% agarose gel as a
mobility difference of the DNAs amplified with
primers, bF19: 5 -CAGAACCATTAGCTTTG-
3 and R1: 5 -CTCTGACTATGCATCAG-3
(Patel et al. 2004). PCR reactions were per-
formed in a 5 μl reaction volume using 0.5 ng
of genomic DNA in 1X PCR buffer (Bioline,
UK), 3 mM MgCl 2 ,0.04UofBIOTAQDNA
polymerase (Bioline, UK), 0.2 mM dNTPs, and
0.8 μM of each primer. The thermal cycling con-
ditions were as follows: 1 min denaturation at
94 ◦ C; 35 cycles of 30 s denaturation at 94 ◦ C,
30 s annealing at 60 ◦ C, and 1 min extension at
72 ◦ C; and a final 3 min extension at 72 ◦ C.
The genotypes of 'YI-0311' identified on
ahFAD2A and ahFAD2b were the same as the
previously reported mutational alleles found
on high O/L peanut lines (Jung et al. 2000;
Lopez et al. 2000; Patel et al. 2004, Figure

11.2), and the loci identified on the two candi-
date genes were used as selection markers for
a high O/L ratio. However, because the Taq-
Man assay required expensive chemicals and
facilities, the TaqMan SNP marker was con-
verted to a PCR-based SNP marker that was
able to identify the SNP on a 3% agarose gel
for practical use (Hayashi et al. 2004). A mix-
ture of the following four primers was used for
PCR; FAD2AF: ATCCAAGGCTGCATTCTC-
CCT, FAD2AR4: CCTAGTGTGAGTGTGAT-
GCAG, HachiAF: ACACCGGTTCCCTCGAC-
CTCA and NakaR: GGTTTTGGGACAAA-
CACTTCTTC. PCR reactions were performed
ina15μl reaction volume using 1.5 ng of
genomic DNA in 1X PCR buffer (Bioline,
UK), 1.5 mM MgSO 4 , 0.375 U of Takara Taq
HS (Takara Bio Inc.), 0.2 mM dNTPs, and
0.25 μM of each primer. The thermal cycling
conditions were as follows: 5 min denaturation
at 94 ◦ C; 35 cycles of 30 s denaturation at 94 ◦ C,
30 s annealing at 55 ◦ C, and 1 min extension at
72 ◦

C.