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recipient, and subsequently restores the genetic
background of the respective local variety by
repeated backcrossing and marker selection.
Generally, three types of molecular markers are
needed for this approach: (1) QTL-specific fore-
ground markers, (2) flanking markers to select
for plants with a crossover at the 5
and 3
bor-
der of the QTL, and (3) background markers to
select for plants with maximum recovery of the
recipient parent genome (Collard and Mackill
2008). The outcome of this selection is a mod-
ified rice variety that is indistinguishable from
the original recipient parent with respect to plant
type and grain quality, except that it is toler-
ant. The MABC approach has the advantage that
crop duration and desirable traits, such as supe-
rior grain quality and disease resistance, remain
unchanged and farmers therefore do not need to
adjust crop management practices.
Applying this technology for the breeding
of P-deficiency-tolerant rice, molecular mark-
ers for the
Pup1
QTL were developed based
on a comparative sequence analysis between the
Pup1
locus in Kasalath and Nipponbare (Fig-
ure 5.3b, c). For regions within the
Pup1
locus
that are partially conserved between the two loci,
codominant markers were developed based on
sequence polymorphisms mainly targeting small
(
2011). However, since dominant markers do not
distinguish between homozygous and heterozy-
gous plants, additional genotyping with codomi-
nant markers is required. Based on these data and
a genotype x marker association analysis, three
Pup1
markers (two codominant and one domi-
nant; Figure 5.3c) have been identified that are
ideal foreground markers to select for
Pup1
intro-
gression in MABC-derived progenies (Chin et al.
2011). These three markers also provide suffi-
cient information on the
Pup1
haplotype across
diverse rice genotypes (Chin et al., 2011). The
primer sequences and additional information on
the markers are provided in Table 5.2.
Evaluation of
Pup1
in Different
Genetic Backgrounds and
Environments
As mentioned above, only QTLs that show a sig-
nificant effect in different genetic backgrounds
and environments are of interest to breeders. For
the evaluation of
Pup1
, it was necessary to trans-
fer
Pup1
into
indica
-type rice varieties, since
the initially developed Nipponbare-
Pup1
NILs
flower early under tropical short-day conditions
and therefore were not suitable for field exper-
iments in most of the targeted Asian countries.
For this purpose, two
indica
-type irrigated rice
varieties (IR64 and IR74) and three Indonesian
upland varieties (Situ Bagendit, Dodokan, Batur)
were initially selected as
Pup1
recipient parents.
For the development of IR64-
Pup1
and IR74-
Pup1
, two continuous generations of segregat-
ing populations were genotyped to select plants
with a homozygous
Pup1
locus (Figure 5.4). The
use of markers located within (K46-2) and out-
side (K20-2, K29-1) the INDEL (Figure 5.3b, c)
ensured that no recombination occurred within
the
Pup1
region. In addition to foreground selec-
tion, background markers were used to select
for plants with minimum remaining donor seg-
ments in combination with visual selection of
plants most similar to the recipient parent (Fig-
ure 5.4). For background selection, a set of SNP
(single nucleotide polymorphism) markers was
50 bp) INDELs and single nucleotide poly-
morphism (SNPs). In contrast, markers target-
ing the Kas-
Pup1-
specific INDEL region are
naturally dominant since this region is absent
from Nip-
Pup1
(Chin et al. 2010; Chin et al.
2011). The developed codominant and domi-
nant markers were subsequently tested in a wide
range of diverse rice accessions, which revealed
that some markers that are polymorphic between
Nip-
Pup1
and Kas-
Pup1
were monomorphic in
other genotypes (data not shown). Such mark-
ers are not generally suitable for breeding appli-
cations, but may be used in specific crosses
if the parental lines are polymorphic. Among
the various markers tested, dominant markers
targeting the INDEL (Figure 5.3a, b) were gen-
erally the most robust and best associated with
Pup1
across diverse rice genotypes (Chin et al.
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