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59
1983,
with
some
modifications.
DNA
was
dissolved
in
200
µl
of
1×
TE
buffer
and
stored
at
–20°C,
and
thereafter
diluted
to
25
ng/µl
using
double-distilled
water
to
become
the
working
stock
.
The
DNA
harvested
and
quality
was
determined
using
1%
agarose
gel
at
150
V
for
45
minutes
in
0.5×
TBE
buffer
and
spectrophotometer.
4.4 Polymorphism
and
Parental Genome Coverage
using
SNPs
The
SNP
genotyping
and
analysis
was
done
in
collaboration with
IRRI
in Los Baños, The Philippines.
A
customized
chip
comprising
of
384 SNP
IRRI
panel
for
indica×indica
covering
all
the
12
chromosomes
of
rice
genome
was
used
for
parental
polymorphic
survey
between
the
2
parents
Hasawi
and
IR29,
For
each OPA Oligo
Pool All,
reagent,
run,
a
plate
of
96
samples
with
5µl
of
unamplified
genomic
DNA
normalized
to
50
ng/µl
concentration
was
genotyped
using
the
‘GoldenGate
Genotyping
Assay
for
VeraCode Manual
Protocol’
Illumina
Part
#
11275211
from
Illumina,
San
Diago,
CA
USA),
following
the
manufacturer’s
instructions.
Scoring
of
SNP
genotyping
data
was
done
using
the
BeadStudio
genotyping
software
(Teo
et
al.,
2007),
SNPs with
the
same
genotype
as
Hasawi
were
scored
as
‘‘1’’,
and
those
with
the
same
genotype
as
IR29
were
scored
as
‘‘2’’,
heterozygous
SNPs
were
scored
as
“3”,
and
missing
SNPs
were
scored
“0”.
Since
the
SNP
map
obtained
from
the
BeadStudio
software
gave
marker
position
in
bp
base
pair,
physical
map),
the
position
was
converted
to
genetic
map
in
cM,
using
the
equation:
1
cM=250
kb
(Tanksley
et
al.,
1989),
Markers
with
missing
call
or
showing
same
alleles
for
both
parents
were
discarded.
4.5 Marker
Analysis
and
QTL
identification
QTL
were
identified
by Windows
QTL
Cartographer
version
2.5
(Wang
et
al.,
2011),
The
analysis
was
conducted
by
regressing
the
F
5:6
RIL
screening
performance
on
the
F
5
genotypes
based
on
SNPs
anchored
in
a
linkage
map
previously
developed
by
Temnykh
et
al.
(2001),
and McCouch
et
al.
(2002),
The
chromosomal
locations
of QTL were
determined
by
composite
interval
mapping
CIM,
A
QTL
was
declared
if
the
phenotype
was
associated
with
two
adjacent
marker
loci
at
a
probability
P
,
below
0.05.
Significance
thresholds
for
CIM
were
determined
using
1,000
permutations
for
each
trait
with
4
iterations.
The
experiment-wise
significance
level
of
P
<0.01
and
P
<0.05
corresponds
to
an
average
LOD
of
the
traits. When
two
LOD
peaks
fell
in
a
common
support
interval,
only
one QTL was
considered
to
be
present,
and
its
approximate
position was
taken
as
the
highest
peak.
The
proportion
of
observed
phenotypic
variation
attributed
to
a
particular QTL was
estimated
by
the
coefficient
of
determination
R
2
, QTL
found
in
our
study
were
compared
with
previously
detected
rice QTL
using
RICE QTL
in
TRAITS
option
on
the
Gramene
Web
site
www.gramene.org/,
The
linkage
map
was
constructed
using
142
RILs
from
the
cross
IR29×Hasawi
4.6 QTL
Nomenclature
We
used
a
2
-
or
3
-
letter
abbreviation
in
which
the
number
of
chromosome where
the QTL
is
found
and
a
terminal
suffix,
separated
by
a
period;
provide
a
unique
identifier
to
distinguish
multiple
QTLs
on
a
single
chromosome
(McCouch
et
al.,
1997),
The
distribution,
number
of
the
polymorphic markers
and
allele’s
distribution
across
each
chromosome
was
done
using
the
computer
software
Flapjack
(Milne
et
al.,
2010).
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