This document summarizes research characterizing the CesA6 gene, which encodes a catalytic subunit of cellulose synthase, in oat. Key findings include: - CesA6 was cloned and sequenced from multiple Avena species, allowing determination of its genomic origin in hexaploid oat. - The oat CesA6 gene is approximately 5kb with 12 introns and a 3.2kb coding sequence, similar to the CesA6 gene in barley. - Sequencing of hexaploid oat revealed three distinct CesA6 alleles presumed to originate from the A, C, and D genomes. - Comparison to sequences from diploid oat species is being used

1. Abstract:
Cellulose
synthase
cataly.c
subunit
genes
(CesAs)
are
involved
in
the
biosynthesis
of
cellulose,
an
insoluble
fiber
that
is
the
main
component
of
the
plant
cell
wall.
In
all
land
plants,
cellulose
microfibrils
are
synthesized
from
a
roseAe
complex
known
as
the
cellulose
synthesis
complex,
embedded
in
the
plasma
membrane.
Each
complex
consists
of
six
globules
that
all
contain
mul.ple
cellulose
synthase
cataly.c
subunits
(CesAs).
CesA6
is
a
member
of
the
cellulose
synthase
cataly.c
subunit
family
and
research
shows
it
is
specialized
for
cellulose
synthesis
in
the
primary
cell
wall.
In
this
study,
EST
sequence
data
from
several
oat
germplasms
were
u.lized
to
amplify
and
clone
the
CesA6
cataly.c
subunit
gene
from
mul.ple
Avena
species,
including
the
diploid
species
A.
strigosa
(AA),
A.
canariensis
(DD),
and
A.
ventricosa
(CC).
Comparison
of
the
A.
sa2va
sequences
to
the
sequences
of
the
diploids
allowed
for
the
determina.on
of
genomic
origin.
The
CesA6
gene
in
Oat
is
~
5kb
with
12
introns
and
a
coding
sequence
of
3.2
kb.
Materials
and
Methods:
Germplasm
was
acquired
from
Na.onal
Small
Grains
Collec.on
at
the
USDA-­‐ARS
in
Aberdeen,
ID,
and
from
the
Laren
Robinson
Memorial
Genbank
at
Brigham
Young
University
Provo,
UT.
Assembly
of
CesA6
Expressed
Sequence
Tags
(ESTs)
from
a
database
created
by
the
Collabora.ve
Oat
Research
Enterprise
(CORE)
was
done
using
the
barley
CesA6
coding
sequence
as
a
reference.
Primers
were
designed
from
this
assembly
and
used
to
amplify
the
CesA6
gene.
Clones
of
two
different
lengths
were
created
for
each
line.
The
first
clone
(f3-­‐r7)
began
at
the
5’
end
and
proceeded
into
the
tenth
exon,
while
the
second
(f7-­‐r2)
shorter
clone
contains
sequence
through
the
next
three
exons
un.l
the
end.
Sequencing
was
performed
at
the
BYU
DNA
Sequencing
Center
using
Applied
Biosystems
3730xl
DNA
Analyzer
and
BigDye
v3.1
chemistry.
Sequences
were
assembled
and
aligned
using
Geneious
Pro
assembly
so]ware
by
BiomaAers
LTD.
available
at
www.geneious.com.
Results
and
Discussion:
Gene
Ontology:
The
CesA6
gene
in
Oat
is
approximately
5
kb
with
12
introns
and
a
coding
sequence
of
approximately
3.2
kb.
The
CesA6
gene
of
Oat
is
similar
to
the
CesA6
gene
of
Barley.
The
size
and
loca.on
of
the
exons
are
very
similar.
The
size
of
the
introns
are
more
variable,
as
would
be
expected.
An
alignment
showed
a
89.4%
homology
between
Oat
and
Barley.
Allele
varia:on:
Sequencing
of
A.
sa2va
revealed
three
dis.nct
alleles
of
the
CesA6
gene
(Fig2).
Different
alleles
are
presumed
to
be
from
each
of
the
three
genomes
A,
C
and
D.
Consistently,
two
of
the
alleles
were
closely
related
while
the
third
allele
was
more
divergent
from
the
other
two.
There
is
a
98.6%
iden.ty
between
the
three
Hifi
homeologs.
As
expected
the
intron
regions
had
the
greatest
sequence
diversity
including
frequent
small
dele.ons
and
inser.ons.
Differences
in
the
coding
regions
between
alleles
consisted
almost
exclusively
of
single
nucleo.de
polymorphisms.
Separa.on
of
A.
sa2va
homeologs
is
being
done
by
comparison
of
sequence
data
from
diploid
strains
such
as
A.
canariensis,
which
is
a
D
genome
diploid,
allowing
for
genome
allele
assignment
(Fig
3).
A
92.6%
iden.ty
was
found
between
Hifi
and
A.
canariensis.
Sequencing
is
currently
being
conducted
on
mapping
parents
Provena,
Mn-­‐84,
Hi-­‐Fi,
Sol-­‐fi,
GS-­‐7
and
Assiniboia
which
feeds
into
the
physically-­‐anchored
hexaploid
oat
linkage
map2.
With
homeolog
specific
SNPs
it
is
now
possible
to
conduct
allele
specific
expression
analysis
with
RNAseq
reads.
References:
1. Ding
SY,
Himmel
ME
(2006)
The
maize
primary
cell
wall
microfibril:
a
new
model
derived
from
direct
visualiza.on.
J.
Agric.
Food
Chem.
54:597-­‐606.
2. Oliver
RE,
Tinker
NA,
Lazo
GR,
Chao
S,
Jellen
EN,
et
al.
(2013)
SNP
Discovery
and
Chromosome
Anchoring
Provide
the
First
Physically-­‐Anchored
Hexaploid
Oat
Map
and
Reveal
Synteny
with
Model
Species.
PLoS
ONE
8:
e58068.
doi:
10.1371/journal.pone.
0058068
3. Mutwil
M,
Debolt
S,
Persson
S
(2008).
Cellulose
synthesis:
a
complex
complex.
Curr.
Opin.
Plant
Biol.
11:
252-­‐257.
Characteriza.on
of
CesA6
in
Oat
Cassandra
Anne
Dohse,
Melissa
C.
Fogarty,
Eric
N.
Jellen
Brigham
Young
University,
Department
of
Plant
and
Wildlife
Sciences,
Provo,
Utah
Figure
1:
Cellulose
synthases,
such
as
CesA6,
form
hexameric
roseAes
known
as
cellulose
synthesis
complexes.
1-­‐C
shows
a
roseAe
array
in
the
plasma
membrane1.
Figure
3:
Alignment
of
Hi-­‐fi
(high
fiber
A.
sa2va
cul.var)
homeologs
and
A.
canariensis
sequence.
Mul.ple
SNPs
can
be
seen.
Base
pair
345
is
of
par.cular
interest
as
it
infers
the
first
Hi-­‐fi
homeolog
is
of
the
D
genome
like
A.
canariensis.
Figure
2:
Alignment
of
Hi-­‐fi
(high
fiber
A.
sa2va
cul.var)
homeologs
represen.ng
various
polymorphisms.
Unique
polymorphisms
can
be
seen
at
bp
709,
713,
759
and
796.