Materials
and
Methods
Plant
Material
:
The
five
S
.
chacoense
families
utilized
for
map
construction
have
been
previously
described
(Ronning
et
al
.
,
1998,
1999)
.
Lines
55
-
1
and
55
-
3
are
sibling
selections
from
PI
320387
and
produce
high
and
nil
leptine
as
a
percentage
of
total
foliar
glycoalkaloids,
respectively
.
Line
8380
-
1
is
a
sibling
selection
from
PI
458310
and
produces
high
levels
of
leptine
(Sinden
et
al
.
,
1986;
Sanford
et
al
.
,
1997)
.
Reciprocal
crosses
were
made
between
55
-
1
and
55
-
3
to
produce
two
segregating
F
1
families,
9501
(55
-
3
x
55
-
1,
94
individuals)
and
9502
(55
-
1
x
55
-
3,
65
individuals)
.
Reciprocal
crosses
were
also
made
between
55
-
3
and
8380
-
1
(family
55
-
3
x
8380
-
1,
29
individuals;
family
8380
-
1
x
55
-
3,
31
individuals)
.
The
fifth
mapping
population
was
derived
from
a
cross
between
the
two
high
leptine
-
producing
genotypes
55
-
1
and
8380
-
1
(family
55
-
1
x
8380
-
1,
20
individuals)
.
PCR
analysis
:
AFLP
reactions
were
carried
out
with
the
AFLP
Analysis
System
I
kit
(Gibco
BRL)
using
the
manufacturer's
protocol,
but
omitting
the
labeling
reactions
.
Twelve
primer
pair
combinations
that
produced
numer
ous
polymorphic
markers
were
selected
for
screen
rs
.
Markers
that
deviated
strongly
from
expected
chi
-
square
ratios
(p<0
.
001)
were
Haanstra
et
al
.
1999)
.
Maps
generated
from
the
55
-
1x8380
-
1
cross
and
the
two
reciprocal
crosses,
9501
/
9502
and
55
-
3
x
8380
-
1
/
8380
-
1
x
55
-
3,
were
subsequently
combined
using
JoinMap
2
.
0
.
A
recombination
threshold
of
0
.
49
and
Kosambi’s
mapping
function
were
utilized
for
calculating
map
distances
.
The
modified
LOD
threshold
value
for
the
integrated
map
was
set
at
0
.
1
(Haanstra
et
al
.
,
1999)
.
Results
and
Discussion
ing
of
progeny
.
Markers
amplified
by
respective
primer
combinations
were
designated
as
follows:
M1E6,
M
-
CAA/E
-
ACT;
M1E7,
M
-
CAA/E
-
AGC;
M2E3,
M
-
CAC/E
-
ACA;
M2E4,
M
-
CAC/E
-
ACC;
M3E3,
M
-
CAG/E
-
ACA;
M3E4,
M
-
CAG/E
-
ACC;
M3E8,
M
-
CAG/E
-
AGG;
M4E1,
M
-
CAT/E
-
AAC;
M5E4,
M
-
CTA/E
-
ACC;
M6E4,
M
-
CTC/E
-
ACC;
M6E5,
M
-
CTC/E
-
ACG;
and
M6E7,
M
-
CTC/E
-
AGC
(M,
Mse
-
I
+3
primer;
E,
Eco
-
RI
+3
primer)
.
Following
selective
amplification,
AFLP
products
were
separated
via
electrophoresis
using
5%
denaturing
polyacrylamide
gels
and
visualized
using
the
Silver
Stain
System
(Promega,
Madison
WI)
.
RAPD
reactions
were
performed
as
previously
described
(Ronning
et
al
.
1999)
.
Individual
loci
were
named
according
to
the
primer(s)
used
to
generate
the
fragment
followed
by
its
size
in
base
pai
omitted
from
subsequent
mapping
analysis
.
Map
analysis
:
Map
construction
was
conducted
using
JoinMap
version
2
.
0
(Stam
and
Van
Ooijen,
1996)
.
Since
the
parents
are
cross
-
pollinating
and
heterozygous,
segregation
types
and
ratios
varied
between
loci
within
each
F
1
population
.
Therefore,
the
JoinMap
population
code
"CP"
(Cross
Pollinator),
which
allows
for
the
simultaneous
analysis
of
different
segregation
types,
was
used
.
Marker
linkage
groups
were
determined
for
each
family,
analyzing
only
those
markers
that
were
nonsignificant
by
chi
-
square
test
for
fit
to
expected
ratios
(p>0
.
5)
.
Modified
LOD
thresholds
of
7
.
0,
7
.
0,
3
.
0,
3
.
5,
and
3
.
0
based
on
the
chi
-
square
test
for
independence
of
segregation
were
utilized
for
grouping
markers
into
linkage
groups
for
families
9501,
9502,
55
-
1x8380
-
1,
8380
-
1
x
55
-
3,
and
55
-
3
x
8380
-
1,
respectively
.
Estimates
of
recombination
frequencies
were
calculated
and
the
pairwise
recombination
estimates,
together
with
their
modified
LOD
scores,
were
used
to
order
markers
within
each
linkage
group
.
The
LOD
threshold
for
map
construction
was
0
.
1
and
the
recombination
threshold
0
.
49
.
Markers
were
included
in
a
linkage
group
if
they
exhibited
linkage
to
other
markers
with
less
than
5,
10
or
20%
recombination
with
corresponding
LOD
values
for
linkage
greater
than
10,
5,
and
1,
respectively
(
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