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were selected, genome regions derived from the
donor parent were not excluded. The reason for
the higher ARy values on LG1.2 and 5.2 is
not clear.
tively. In order to disturb the amplification of
the
FA D 2 B
locus, terminal sequences of the con-
trol primer pairs were used as
FA D 2 A
specific
bases. In addition, the third bases from the ter-
minals of each of the primers were designed as
mismatch bases for obtaining a high specificity
of PCR (Hayashi et al. 2004). It is well known
that the result of a PCR often changes depend-
ing on the enzymes used and the thermal cycler.
Because the two-step PCR proposed by Chen
and colleagues (2010) used more simple primer
combinations than are used in the one-step PCR,
it is expected that the two-step PCR will deliver
a more stable result under diverse lab conditions.
Meanwhile, the advantage of the one-step PCR
over the two-step PCR is the former's lower cost
and less intensive labor.
Comparison with the Other Agarose Gel
Base Genotyping Technique
for FAD2Loci
In this study, we first used the TaqMan assay
for SNP identification on the
FA D 2 A
locus for
genotyping of F
2
individuals, then the agarose
gel base SNP identification assay (Hayashi et al.
2004) was employed for MAS, since our breed-
ing program preferred SNP genotyping with sim-
ple and inexpensive equipment. As with our
study, Chen and colleagues (2010) also reported
an agarose gel base allele-specific PCR assay
for identification of SNPs on
FA D 2
loci, for
simplifying the techniques and decreasing the
cost of SNP genotyping. In the study by Chen
and colleagues (2010), five primer sequences
(one forward primer and four reverse primers)
were developed. One of the four reverse primers
was used for amplification of control DNA frag-
ments, while the other three reverse primers were
designed to amplify allele-specific amplicons.
Wild and mutation-specific amplicons were
obtained from individual PCR, then the both wild
and the mutation-specific amplicons were mixed
and loaded in agarose gel for electrophoresis.
While Chen and colleagues (2011) proposed
a two-step PCR for identifying mutation alle-
les on the
FA D 2
loci, in our study a one-
step PCR was employed for identification of
the
FA D 2 A
mutation. PCR was performed with
a mixture of four primers, that is, a control
forward primer (FAD2AF), a control reverse
primer (FAD2AR), a wild type-specific reverse
primer (NakaR), and a mutant-specific forward
primer (HachiAF). A primer pair made up of
FAD2AF and FAD2AR was used for obtain-
ing control amplicons (293bp), whereas primer
pairs FAD2AF and NakaR, and HachiAF and
FAD2AR were used for obtaining wild (127bp)
and mutant-specific (209bp) amplicons, respec-
Comparison with the Other Breeding
Program for High O/L with MAS
Chu and colleagues (2011) reported MAS for
high O/L and nematode resistance in peanut.
There are many parameters in our study that are
similar to and others that contrast with the work
carried out by Chu and colleagues (2011). In the
study of Chu and colleagues (2011), two lines,
'Georgia-02C' and 'Florida-07,' were used as
donors of the high O/L alleles and crossed with
'Tifguard.' 'Tifguard' was a donor of nematode
resistance traits and used as maternal and recur-
rent parent. The mutation allele of 'Georgia-02C'
and 'Florida-07' in
FA D 2 A
locus was same as
'YI-0811,' while that in the
FA D 2 B
locus was
different from 'YI-0811,' that is, 441_442insA
mutation. In the study of Chu and colleagues
(2011), CAPS markers were first used for SNP
identification, then the HybProbe assay (Bernard
et al. 1998) was employed for polymorphic anal-
ysis of the 441_442insA mutation, in order to
increase through-put and accuracy of genotyp-
ing. Both studies changed the genotyping assay
to fit the breeding program, but the directions of
modification were opposite. The two contrast-
ing results indicate that there is no single correct
answer for a genotyping system that uses MAS,
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