1. Are
different
alleles
of
the
APETALA1
gene
responsible
for
differences
in
development
among
varie6es
of
Brassica
oleracea?
Daniel
Dorado
and
Marilyn
Cruz
Alvarez
Department
of
Biological
Sciences,
Florida
Gulf
Coast
University,
10501
FGCU
Blvd.
South,
Fort
Myers,
Florida
Abstract
Agriculture
is
one
of
the
most
important
developments
in
human
history
and
understanding
the
influences
genes
have
over
crops
is
crucial.
Brassica
oleracea
is
an
important
agricultural
plant
species
which
shows
many
differences
in
its
varie6es.
The
two
primary
varie6es
we
are
focusing
on
are
cauliflower
and
Rbo.
Rbo
is
a
rapid
cycling
variety
and
flowers
normally,
while
cauliflower
(B.
oleracea
var.
botry5s)
shows
an
arrest
in
flowering
development
and
forms
an
edible
curd.
Currently
we
are
aNemp6ng
to
see
which
genes
are
responsible
for
the
developmental
differences
between
cauliflower
and
Rbo.
Ini6ally
we
had
crossed
cauliflower
and
Rbo
to
obtain
F1
and
F2
genera6ons.
The
F2
genera6on
resulted
in
segrega6ng
phenotypes
for
traits
such
as
flowering
6me
and
number
of
flowers
produced.
The
APETALA1
(AP1)
gene
has
been
shown
to
be
involved
in
flowering
in
many
species.
Our
hypothesis
is
that
differences
in
alleles
of
the
AP1
gene
contribute
to
the
differences
in
phenotype
in
B.
oleracea.
AP1
has
three
copies
in
this
species,
two
of
which
have
been
shown
to
be
expressed:
AP1a
and
AP1c.
To
test
our
hypothesis
we
are
determining
the
genotype
for
the
AP1a
and
AP1c
alleles
of
the
F2
plants
and
studying
any
correla6on
between
genotypic
and
phenotypic
differences.
To
carry
out
this
analysis
we
first
need
to
determine
the
AP1
alleles
present
in
the
cauliflower
and
Rbo
parents.
We
are
cloning
AP1a
and
AP1c
from
both
varie6es
by
using
PCR
and
primers
corresponding
to
sequences
that
are
highly
conserved
in
the
AP1
gene.
Contrary
to
previously
published
results,
we
found
that
the
deduced
amino
acid
sequence
corresponding
to
the
first
exon
of
the
AP1c
gene
from
cauliflower
is
iden6cal
to
that
deduced
from
the
broccoli
and
kale
genes,
sugges6ng
that
AP1
may
not
be
responsible
for
the
phenotypic
differences
as
hypothesized.
Introduc.on
Brassica
oleracea
is
a
species
in
the
Brassica
genus.
Brassica
varie6es
include
Brussels
sprouts,
cabbage,
cauliflower,
broccoli,
kale,
kohlrabi,
and
Rbo
(a
non
agricultural
rapid
cycling
variety).
In
cauliflower
and
broccoli
there
is
prolifera6on
of
cells
at
the
6p
of
the
shoot
and
developmental
arrest
that
produces
a
head.
In
cauliflower
these
cells
are
arrested
during
flower
development,
while
in
broccoli
the
arrest
takes
place
aYer
flower
forma6on.
There
is
much
specula6on
as
to
what
molecular
differences
contribute
to
the
arrest
in
flower
development
and
curd
forma6on
in
cauliflower.
Studying
Arabidopsis
thaliana,
which
is
a
model
system
and
belongs
to
the
same
family
as
B.
oleracea,
has
provided
some
clues
as
to
possible
genes
that
could
influence
flowering
phenotype.
When
studying
this
species
it
was
found
that
muta6ons
in
both
the
CAULIFLOWER
(CAL)
and
APETALA1
(AP1)
genes
result
in
a
phenotype
similar
to
that
of
cauliflower
in
B.
oleracea.
Smith
and
King
(1)
studied
the
effect
of
muta6ons
in
CAL
and
AP1
on
the
phenotype
of
B.
oleracea.
They
crossed
a
Calabrese
variety
which
displays
a
broccoli
phenotype
with
cauliflower,
resul6ng
in
an
F1
genera6on
with
Calabrese
phenotype
(figure
1).
AYer
self-­‐
crossing
the
F1
genera6on,
the
resul6ng
F2
genera6on
consisted
of
plants
with
Calabrese
phenotype,
intermediate
phenotype,
and
cauliflower
phenotype.
When
the
F2
plants
were
genotyped
a
correla6on
was
found
between
their
alleles
for
the
AP1
and
CAL
genes
and
their
phenotypes.
If
both
AP1
and
CAL
were
wild
type
(AACC)
the
resul6ng
phenotype
was
like
that
of
broccoli,
if
plants
were
homozygous
for
the
mutant
alleles
of
either
gene
(aaCC/AAcc)
they
had
intermediate
phenotype,
and
homozygosity
for
the
mutant
alleles
for
both
AP1
and
CAL
(aacc)
resulted
in
a
cauliflower
phenotype.
Smith
and
King
(1)
used
a
polymorphism
upstream
of
the
start
codon
to
dis6nguish
between
the
AP1
alleles
from
broccoli
and
cauliflower
but
did
not
show
differences
in
the
coding
region
sequence
between
these
alleles.
Further
research
showed
that
there
are
three
copies
of
AP1
in
B.
oleracea:
AP1a,
AP1b
and
AP1c
(2,
3).
AP1b
has
a
muta6on
that
makes
it
non-­‐func6onal
(3).
AP1a
and
AP1c
are
expressed
during
curd
development
in
cauliflower.
Therefore
one
or
both
copies
may
contribute
to
the
differences
in
flower
development
seen
in
this
variety.
Sequencing
has
revealed
several
differences
in
AP1a
between
cauliflower
and
kale
or
broccoli
(table
1)
(2,
4).
Results
v No
differences
in
the
amino
acid
sequences
in
exon
1
can
be
observed
between
cauliflower
AP1a
and
broccoli
or
kale
AP1a.
v No
differences
in
the
amino
acid
sequences
in
exon
1
can
be
observed
between
cauliflower
AP1c
and
broccoli
or
kale
AP1c.
v Since
the
cauliflower
cul6var
used
is
a
hybrid,
it
is
possible
that
only
one
of
the
two
alleles
were
cloned.
The
other
allele
may
s6ll
show
a
sequence
difference
with
respect
to
broccoli
and
kale.
However,
several
clones
from
cauliflower
showed
the
same
sequence
sugges6ng
this
is
not
the
case.
v Our
results
suggest
that
differences
in
AP1
between
cauliflower
and
broccoli
are
not
responsible
for
their
phenotypic
differences.
v Future
research
includes
cloning
and
sequence
analysis
of
exons
7
and
8,
where
addi6onal
differences
were
previously
observed.
Conclusions
References
Acknowledgements
Methods
Primers
were
designed
that
would
allow
for
amplifica6on
of
exon
1
of
AP1a,
AP1b
and
AP1c
(table
2).
These
primers
were
used
to
amplify
the
DNA
extracted
from
cauliflower
leaves
using
the
following
thermo-­‐cycler
program:
94°
10
minutes,
30
cycles
of
94°
1
minute,
57°
1
minute,
72°
1
minute,
and
72°
for
10
minutes.
Table
2.
Primers
designed
to
amplify
Exon
1
of
AP1a,
AP1b
and
AP1c.
The
products
of
amplifica6on
were
separated
by
agarose
gel
electrophoresis
and
amplified
fragments
extracted
from
the
gel
using
QIAEXII
(Qiagen).
PCR
products
were
cloned
into
pCR
II
TOPO
and
bacteria
transformed
with
these
plasmids.
White
colonies
were
selected
and
bacteria
grown
overnight.
Plasmids
were
prepared
from
the
cultures
and
digested
with
EcoRI
to
check
that
they
contained
a
PCR
insert
of
the
expected
size.
Plasmids
were
sent
to
be
sequenced
at
Cornell
University
Life
Sciences
Core
Laboratories
Center.
Figure
1.
According
to
Smith
and
King,
muta6ons
in
the
BoCAL
and
BoAP1a
loci
explain
the
curding
phenotype
of
Brassica
oleracea
var.
botry5s.
A=
BoAP1a,
C=
BoCAL.
Lower
case
leNers
represent
mutant
alleles
(1).
Table
1.
Four
amino
acid
subs6tu6ons
seen
in
the
AP1a
protein
from
cauliflower
(4).
Changes
in
the
sequences
of
the
AP1a
and
AP1c
genes
in
cauliflower
may
result
in
lack
of
func6on
of
the
encoded
proteins
and
contribute
to
the
arrest
in
flowering
in
cauliflower.
Cloning
AP1a
and
AP1c
from
Rbo
and
cauliflower
will
allow
us
to
see
if
the
sequence
differences
previously
reported
are
always
present
between
these
varie6es.
We
will
later
analyze
if
there
is
a
correla6on
between
sequence
differences
for
AP1
and
differences
in
phenotype
in
the
F2
popula6on
from
a
cross
between
Rbo
and
cauliflower.
Intron
4
AP1a
Reverse
Intron
4
AP1b
Reverse
Intron
4
AP1c
Reverse
ataaacgtacca=aca=gactaatcata
aaagacacatcacatgatc=aa=ataca
gatcagtaaaatgaatc=ataacaacaca
Exon
1
AP1
forward
atggggaggggtaggg=c
atggggaggggtaggg=c
atggggaggggtaggg=c
Figure
2.
Panel
A
shows
the
comparison
of
the
deduced
amino
acid
sequences
corresponding
to
exon
1
from
the
cloned
cauliflower
AP1
a
fragment
and
broccoli/kale
AP1a.
Panel
B
shows
the
comparison
of
the
deduced
amino
acid
sequences
corresponding
to
exon
1
from
the
cloned
cauliflower
AP1c
fragment
and
broccoli/kale
AP1c.
The
highlighted
green
regions
show
an
amino
acid
difference
between
AP1a
and
AP1c.
The
highlighted
yellow
region
shows
sequence
iden6ty
where
a
difference
was
expected
between
AP1a
in
broccoli
(N)
and
cauliflower
(S)
(2).
1. Smith,
L.
B.
and
King.
G.
J.
(2000).
The
distribu6on
of
BoCal-­‐a
alleles
in
Brassica
oleracea
is
consistent
with
a
gene6c
model
for
curd
development
and
domes6ca6on
of
the
cauliflower.
Molecular
Breeding.
6,
603-­‐613
2. Smith,
L.B.
1999.
The
molecular
gene6cs
of
curd
morphology
and
domes6ca6on
of
cauliflower
(Brassica
oleracea
L.
var.
botry5s
L.).
Ph.D.
thesis,
University
of
Warwick,
Coventry,
United
Kingdom.
3. Carr,
S.M.
and
Irish.
V.F.
1997.
Floral
homeo6c
gene
expression
defines
developmental
arrest
stages
in
Brassica
oleracea
L.
vars.
botry5s
and
italica.
Planta.
201,
179-­‐188.
4. Lowman,
A.
C.
and
Purugganan.
M.D.
1999.
Duplica6on
of
the
Brassica
oleracea
APETALA1
floral
homeo6c
gene
and
the
evolu6on
of
the
domes6cated
cauliflower.
J.
Heredity.
90,
514-­‐520.
I
would
like
to
thank
Dr.
Cruz-­‐Alvarez
for
her
guidance
and
support
presen6ng
this
research.
Also
a
special
thanks
to
Nina
Infantado
for
always
being
available
to
lend
a
helping
hand.
Exon
Posi.on
Kale
Cauliflower
Exon
1
16
Asparagine
Serine
Exon
7
170
Lysine
Asparagine
Exon
7
199
Serine
Proline
Exon
8
251
Leucine
Phenylalanine
A.
B.