Characterization of Four Members of the Alpha-Tubulin Gene Family in Ceratopteris richardii

2007·12.8 MB·English

by  Rodney J Scott

American
Fern
Journal 97(2):47-65
(2007)
Members
Four
Characterization of
of the
Alpha-Tubulin Gene Family
Ceratopteris
in richardii
RODNKY
SC0
rIT
J.
Department Wheaton
of Biology, College, Wheaton, IL 60187
Gerald Gas tony
}.
Department
of Biology, Indiana University, Blooniington, IN 47405-700J
5
W.
Jeremy Weatiierford
MO
Department of Computer Science and Engineering, Washington University, Louis, 63108
St.
Takuya Nakazato
Department
of Biology, Indiana University, Blooniington, IN 47405-7005
—
Abstract. Four members of the alpha-tubulin gene family were examined
in Ceratopteris
mapping
richardii. Genetic linkage based on a population of nearly 500 Doubled Haploid Lines was
members
able to position three or four of this gene family on linkage groups 17, 24, and 28,
respectively (two of the four observed polymorphic restriction fragments containing alpha-tubulin
map
genes are either identical or too close to each other on linkage group 17 to be distinguishable
map
Non-mapable monomorphic
in distance). bands observed on probed Southern blots suggest
that the alpha-tubulin gene family in this species large. Four alpha-tubulin genes from
is C.
richardii were sequenced and found to be fairly similar to each other in terms of their amino acid
BLAST
sequences, with
their greatest diversity the carboxy-terminal ends. comparisons found
at
each of these four amino acid sequences more similar an alpha-tubulin from
to a dicot,
gymnosperm, or alga species than was to any other alpha-tubulin sequence presently known
it
from Ceratopteris or from the fern Anemia phyllitidis or the moss Physcomitrella patens. Bayesian
phylogenetic analysis of nucleotide sequences placed three of the four Ceratopteris alpha-tubulin
gene copies in a clade with copies from Pseudotsuga and Anemia, consistent with a history of two
gene duplication events, one following and one preceding the divergence of ferns and seed plants.
The copy
fourth is robustly separated from the preceding three and placed in a clade of algal alpha-
^
tubulin genes, suggesting its d-"ivewirgence from the ancestor of the other three before the divergence
•"{-> it
- * e>
and As
of algae land plants. characterized thus far, the alpha-tubulin gene family of C. richardii
is
compared known
relatively large as to the six copies from fully sequenced Arabidopsis thaliana,
may
a condition that be correlated with the large genome size and diverse history constraints of
life
homosporous new
These
this fern species. findings suggest several opportunities for research into
and
the evolution, function, regulation of the alpha-tubulin gene familv in Ceratopteris.
DNA
This report describes the application of sequencing and genetic linkage
mapping how
to the alpha-tubulin genes of Ceratopteris richardii and shows
such can enhance model
studies further the utility of this system.
Initial
successes with Ceratopteris were in the areas of cytogenetics Hickok,
(e.g.,
1976, 1977a, 1977b, 1978, 1979a, 1979b; Hickok and Klekowski,
1973,
1974),
physiology and development, and Mendelian
genetics (reviewed Hickok,
in
Hickok More
1987; et 1987, 1995). recent studies have focused on the
a/.,
molecular genetics of Ceratopteris (Minister 1997; Hasebe 1998;
et et
a/., al.,
Aso
et al, 1999; Stout et a/., 2003; Rutherford et al, 2004; Salmi et 2005).
al.,
These employ
recent studies that robust molecular methods are especially
encouraging, since the lack of a technique to induce stable genetic trans-
VOLUME NUMBER
AMERICAN FERN JOURNAL:
97 (2007)
48 2
impeded
formation Ceratopteris has undoubtedly function as a unifying
in
its
from
genes of richardii are described, and the phylogenetic relationships of these
C.
Molec
used
polymorphic alpha-tubulin of richardii are to position
three loci C.
for
new map and
these on the genetic linkage of this species, several potential
loci
new
using information molecular studies are outlined.
strategies for this in
These should provide encouragement those seeking
perspectives further for to
model
utilize Ceratopteris as a system.
The alpha-tubulin gene family was selected these studies because of the
for
gametophyte
biological significance and tractability of tubulin in the
new may
and because be gained
generation of ferns of the insights that for
among
genomics by studying them. Inferring evolutionary relationships
members of gene families generally problematic because distinguishing
is
known
paralogues and orthologues difficult without appropriate outgroups
is
polarize the gene phylogeny. Tubulin genes are ideal for studying gone
to
known
family evolution because alpha-, beta-, and gamma-tubulin genes are
1)
and
have diverged well before the divergence of plant lineages, the highl\
2)
to
These
conserved nature of the genes allows their sequences to be aligned.
gene
features allow inference of gene phylogenies within a given tubulin
group, using sequences from other tubulin groups as outgroups.
compo-
Tubulins and the microtubules they form are obviously essential
nents of eukaryotic cells. In fern gametophytes, however, their roles are
all
directly observable in various stages of development that can be easily studied.
For example, one of the events that signals preparation for spore
first
germination the migration of the nucleus within the cytoplasm (Banks,
is
1999). This event, which critical to the continued development of the
is
Onoclea by microtubule
gametophyte, inhibited in sensibilis several
is
An
(Vogelmann important
including colchicine ah, 1981). role
inhibitors, et
microtubules development has been suggested by the
for at a later stage of
Murata ai on blue
studies of (1997)
et
1
dark-grown Ceratopteris gametophytes. These investigators found that
in
same
cortical microtubules reorient in response to blue light at the time that
inhibition of elongation occurs. Microtubules also serve a key role in the
cell
and sperm (Raghavan, and Ceratopteris
organization function of fern 1989),
sperm has been used extensively in studies characterizing these roles
(Hoffman and Vaughn, 1995a, 1995b, 1996; Hoffman 1994; Renzaglia
et
a/.,
ai, 2004).
et
Because homosporous ferns compose most of the sister group to seed plants
knowledge
(including flowering plants; Pryer et 2001), increased of
a/.,
genome structure and organization in the genomes of homosporous ferns will
genome
significantly broaden our knowledge of structure and evolution in
gene
vascular Furthermore, characterization of the alpha-tubulin
plants.
answer
family in Ceratopteris will ultimately help to questions specificall}
i
fu
Ml)
SCOTT ET ALPHA-TUBULIN GENE FAMILY CERATOPTERIS
AL.: IN RICHARDII
49
genome
of C. richardii is ca. 110 times the size of Arabidopsis thaliana, 24
times that of rice, 12 times that of tomato, 4 times that of maize, and times
2
Some
Do
that of barley. relevant questions include the following. organisms
with larger genomes have larger gene families? Do such organisms (with
their
DNA)
apparent excess amounts have more pseudogenes among members
of
the
How
gene members
of their families? are the various of gene families regulated
development
during organisms with genomes? The
in large question
latter
is
an especially interesting one as relates to the ferns, whose alternation of
it
generations features fully independent sporophyte and gametophyte genera-
tions.
em
genes from The approach involved and
C. richardii. first identifying, isolating,
sequencing alpha-tubulin genes were expressed gametophyte
that in the
was
generation. This accomplished by using an antibody-based screening
me cDNA
hod
with
a library derived from the gametophyte generation.
Bayesian Inference analyses determined the phylogenetic relationships of
newly
these obtained sequences The
to other plant alpha-tubulin genes.
second approach mapping
developed
utilized a strategy the recently
for
completed Nakazato
project of et al. (2006) to generate a high-resolution
map
mapping
linkage This used polymorph-
for C. richardii. project genetic
isms present in a population of nearly 500 Doubled Haploid Lines (DHLs)
generated from an between
cross diploid inbred highly diverged
initial lines of
ON
geographic races of C. richardii (Hickok et ah, 1995): 8 (derived from
Nicaraguan and Ha-PQ45, mutant Hn-n Cuban
a collection) a of (derived from a
collection). Together these two approaches provide detailed insights regarding
new
a previously uncharacterized gene family These
in C. richardii. insights
fam
ome
Materials and Methods
—
cDNA cDNA
library screening. The used study was
library in this a
gift
Ann
from Banks (Purdue was made from
University). 12-dav-old cultures
Jo
It
of C. richardii gametophytes of the Hn-n strain containing both males and
cDNA
The was
hermaphrodites. cloned EcoRl lambda
into the of the
site
ZipLox bacteriophage vector (Life Technologies). The aliquot used to screen
was
for alpha-tubulin genes from a sample thai had been amplified from the
original library. The library was screened using standard methods detecting
for
specific proteins via antibody labeling (Young and Davis, 1991). In the
brief,
bacteriophage was grown on lawn
vector a of bacteria and, plaques
first after
were produced, gene expression was induced with IPTG. Proteins from the
library were adhered nylon membranes and detected by hybridization an
to
to
monoclonal number
anti alpha-tubulin antibody (Sigma, catalogue T5168).
The presence of this antibody was detected with a labeled secondary antibody.
and
After detection plaques
initial isolation of that tested positive
for
expression of alpha-tubulin, several rounds of re-screening were conducted
VOLUME NUMBER
\MKRICAN FERN JOURNAL:
97 (2007)
50 2
i
eliminate contaminating vectors that were not positive for alpha-tubulin.
to
cDNAs
were converted
Bacteriophage vectors containing alpha-tubnlin to
plasmids via an in vivo excision process as described in technical reference
supplied by Technologies.
materials Lite
—
DNA cDNA
sequencing. Plasmids with the inserts were used as the
two sequencing
sequencing For each plasmid, reactions
templates. least
at
where
were conducted using the two primer sites that flank the EcoRl site the
cDNA When
from two sequencing reactions indicated
inserted. the results
is
that the insert was longer than 1220 base pairs, two additional pairs of primers
new new
were constructed based on the sequence data. These internal
more
sequencing primers provided reliable results for the central regions of
these longer inserts. The sequence reads from either two or four reactions were
Ambigu-
assembled using the software program ContigExpress (NTI Vector).
were
from discrepancies between overlapping sequences
resulted
that
ities
resolved by relying on the sequence(s) with the clearest chromatogram.
—
BLAST
comparisons. The sequences were compared using the standard
BLAST BLAST) NGBI
program proteins (protein-protein provided on the
for
web compare sequences with sequences contained
individual other
in
site to
GenBank The Composition-
the database as described in the Results section.
based option and the options were not enabled so that the results
statistic filter
BLAST
would on and/or
of the comparisons be based solely similarities
GenBank
differences of individual amino acids. Since the database
is
BLAST
constantly being updated, should be noted that the reported
it
comparisons were confirmed on 2005.
July 29,
last
—
A
gene Bayesian
Phylogenetic analysis alpha-tubulin relationships.
of
was
sequences used phylogenetic
Inference analysis of nucleotide to infer the
alpha-tubulin genes with regard other
relationships of the four C. richardii to
GenBank
plant alpha-tubulin gene nucleotide sequences available in for
Viridiplantae. Sequences used were from abroad selection of plants, including
BLAST
amino
those corresponding the results of the acid search above,
to
amino
except nucleotide sequences precisely corresponding the acid
that to
Q6VAG0
Chlamydomonas
sequences Gossypium hirsutum and reinhardtii
of
P09204 could not be located in GenBank. Prior to analysis, nucleotide
The which were
sequences were aligned three nucleotides,
visually.
first
invariable, and the 45 nucleotides, which caused ambiguous alignments,
last
were removed. MrModeltest 2.2, a modified version of Modeltest 3.6 (Posada
and Crandall, 1998) determined that the suitable nucleotide substitution
model analyzed sequences GTR+I+G. Bayesian analysis was
for the
is
conducted using MrBayes (Huelsenbeck and Ronquist, 2001; Ronquist
3.1
and sample
and Huelsenbeck, 2003) with three million generations
a
frequency of 1000. The 300 "burn-in" trees were discarded after the
first
analysis. Five independent runs using the same setting converged to identical
Two
one node was one
except resolved only run. beta-tubulin
trees, that in
genes were used an outgroup root the resulting The species used and
as to tree.
GenBank numbers
their accession are identified in the Results section, as are
the Bayesian posterior probability confidence values for the tree's clades.
SCOTT ET ALPHA-TUBULIN GENE FAMILY CERATOPTERIS RICHARDII
AL.: IN 51
—
A
mapping map
Linkage the alpha-tubulin genes. genetic linkage of the
of
genome was developed independently
Ceratopteris richardii of this alpha-
—500
The mapping
tubulin study (Nakazato 2006). population of
et
al.,
Doubled Haploid Lines (DHLs) was generated by intragametophytic
selfing of
gametophytes derived from spores of an cross between diploid inbred
initial
lines of highly diverged geographic races of C. richardii (Hickok et 1995):
al.,
from Nicaraguan Nichols GH) and Ha-PQ45,
<£>N8 (derived a collection, 1719,
map
a mutant of Hn-n (derived from a Cuban collection, Killip 44595, GH). The
based on analysis of Restriction Fragment Length Polymorphism (RFLP),
is
Amplified Fragment Length Polymorphism (AFLP), and allozyme markers.
DNA
RFLPs used in the general mapping project were generated from genomic
DHL
of parental and sporophytes digested with EcoRl and Hindlll, separated
on 0.8% IX TAE, and Southern nylon membranes.
agarose gels in blotted to
Further details of the methods used in development of mapping materials and
map
construction are in Nakazato and Gastony (2006) and Nakazato et
al.
map
Alpha-tubulin gene copies were located on the linkage in the
(2006).
following way. RFLPs containing the alpha-tubulin genes were detected by
made
probing the Southern blots with an alpha-tubulin probe cheiluminescent
by digoxigenin (DlG)-labeling according protocol optimized the
to a for C.
genome mapping The alpha-tubulin probe used
richardii project. C.
is
cDNA E18SP6
CrilO (GenBank sequence num-
richardii clone
cDNA
BQ086953), from gametophytic
ber the library of C. richardii tissue
Ann
provided by Banks (Purdue University). This clone sequence
Jo
GenBank AY231146 TuaCRl
corresponds sequence Table according
of in
to
1,
BLAST
an NCBI search using blastn, which found sequence identity 651/
to at
mapping
652 (99.85%) of the bases compared. Probing of the project's
Southern blots with the alpha-tubulin probe was carried out toward the end
when
mapping Southern were beginning wear
of the project the blots to out.
Thus
the quality of the autorads presented here are suboptimal but neverthe-
DHL
RFLPs mapping
scorable. Parental alpha-tubulin segregating in the
less
MAPMAKER/EXP
maps
population were scored and placed on the linkage by
UNIX
3.0 (Lander et 1987) at a workstation at Indiana University,
al.,
mapping
Department used
of Biology, settings for the general project.
at
Results
—
A
Sequencing comparison amino sequences
results. cursory of the acid for
the four Ceratopteris alpha-tubulin genes suggests immediately that
(Fig.
1)
they are relatively similar to one another. Although only one of the four
sequences complete, possible to note at least two trends directly from
is it is
where
the comparison presented (Fig. First, for the portion sequences are
1).
TuaCR3
available four genes residues 195 332 of TuaCRl), and
for to
all
(i.e.,
TuaCR4 TuaCR3
most unique
are the distinct. In this region, contains five
amino and TuaCR4 while two sequences
acid residues contains four, the other
each possess only a single unique amino acid. The second observation that can
made
be directly from these data relates only to the three sequences that
AMERICAN VOLUME NUMBER
FERN JOURNAL:
52 97
(2007)
2
A
T.\m.i; comparison of the ten Ceratopteris alpha-tubulin sequences identified in this study to
1.
othur alpha-tubulin sequences recorded GenBank.
in
Number
GiMiBcink of bases Size of Accession number, description, and
Names
for Accession sequenced in Predicted identity for amino acid sequence(s) most
DNA
new number ORF new
gene 1 similar sequences :{
to
TuaCRl AY23114H Q6VAG0,
1,676 450 Tubulin alpha-2 chain
1)
AY8625B1
1,159 306 Gossypium hirsutum, 436/450 (96.2%)
AY862563
450
,564
1
TuaCR2 AY862565
1,015 266 AAK81858.1, alpha tubulin subunit
1)
AY862566
1,016 267 Rosa Hybrid cultivar, 260/267 (97.4%)
AY862567
1,018 267 AAV92379.1, alpha tubulin
2)
1
AY86256U
1,016 267 Pseudotsuga 260/267
menziesii, (97.4%)
AY862562
938 257 BAA03955, alpha-tubulin
TuaCK.'i
1) 1
AY862f)(i4 044 257 Chlorella vulgaris 245/257 (95.3%)
1
P09204, Tubulin alpha-1 chain
2)
Chlamydomonas
245/257
reinhardtii,
(95.3%)
TuaCR4 AY862568
9 7 5 325 AAV92379.1, alpha tubulin
1) 1
Pseudotsuga menziesii, 316/325 (97.2%)
DNA
This includes reliable sequences before and after the open reading frame present, but not
if
polyA
the predicted
tail.
2
If two or more versions of the same gene occurred in GenBank. only the most recent one listed
is
here. If several identical sequences were; submitted by the same author(s) and are listed with the
same submission date, only the one with the last accession number in the numerical sequence
is
listed here. The descriptions are given in the form in which they were submitted to GenBank.
BLAST
Sequence similarities were determined using the standard program protein sequences
for
GenBank The
to search the database. Composition-based statistic option and the options were
filter
BLAST
The
not enabled. searches were performed on 2065.
fuly 29,
include complete carboxy-terminal ends TuaCRl, and
In this region,
(i.e., 2, 3).
TuaCR3
these three sequences are highly diverse, with once again exhibiting
number
unique
the greatest of residues.
An
sequence
additional identified an alpha-tubulin sequence from
as
GenBank
Ceratopteris has been deposited in by a separate research group
(Salmi 2005). This 825 base-long sequence (GenBank accession
et
a/.,
BE642799)
is a single pass sequence from a collection of expressed sequence
The NCBI web
tags. bl2seq tool on the site was used to compare this sequence
A
with the four alpha-tubulin sequences described here (data not shown). large
78%
region of this sequence (ranging from 574 to 640 bases) 77 or identical
is
to the beginning portions of TuaCRl and TuaCR4 respectively. has no
It
TuaCRl. The (88%
significant similarity to highest identity for a region that
is
195 bases long) occurs between this sequence and TuaCR2. This region of
TuaCR2
similarity occurs the beginning of and the end of the sequence
at at
described by Salmi et al. (2005). Hecause this sequence was derived from
may
a single sequencing experiment, be expected contain
to a relatively large
it
number
of incorrect bases. Furthermore, the authors did not provide deduced
a
SCOTT ET ALPHA-TUBULIN GENE FAMILY CERATOPTERIS RICHARDII
AL.: IN 53
TuaCRl MRECISIHIGQAGIQVGNACWELYCLEHGIQPDGQMPSDKTVGGGDDAFNT
TuaCR2
TuaCR3
_************************"LJ*******************
TuaCR4 H
TuaCRl FFSETGAGKHVPRAIFVDLEPTVIDEVRTGTYRQLFHPEQLISGKEDAANN
TuaCR2
TuaCR3
**************T7'**************** ********
**********fcT
TuaCR4 V N
FE
, ,
TuaCRl FARGHYTIGKEIVDLCLDRIRKLADNCTGLQGFLVFNAVGGGTGSGLGSLL
TuaCR2
TuaCR3
**************^*****^***************TJ*******
TuaCR4 H A
TuaCRl LERLSVDYGKKSKLGFTVYPSPQVSTSWEPYNSVLSTHSLLEHTDVAVLL
****************C!***
TuaCR2
s
********
TuaCR3 M *
***************************************************
TuaCR4
TuaCRl DNEAIYDICRRSLDIDRPTYTNLNRLVSQVISSLTASLRFDGALNVDVTEF
***************T^***********************************
TuaCR2
*******************************T***
***************TI
TuaCR3
E
I
1
***************!?**** *****T********"*"*'*r*************
TuaCR4
N
E XT
I
TuaCRl QTNLVPYPRIHFMLSSYAPVISAEKAYHEQLSVAEITNSAFEPSSMMAKCD
************************
TuaCR2
*******************************************
*****
TuaCR3 * X *
***********************X*************C*************
TuaCR4 A
S
TuaCRl PRHGKYMACCLMYRGDWPKDVNAAVATIKTKRTIQFVDWCPTGFKCGINY
***************************************************
TuaCR2
**********M*************0**************************
TuaCR3 M
S
**************************
TuaCR4
TuaCRl QPPTWPGGDLAKVQRAICMISNSTSVAEVFSRIDFKFDLMYCKRAFVHWY
***************** *****************v******»********
TuaCR2 V Y A
•*7,
******X********
*****************Tr********T****"^*
TuaCR3 V L*H A
I
TuaCR4
TuaCRl VGEGMEEGEFSEAREDLAALEKDYEEVGAEGQDDDEPGD DEY
*******************************
*n*
TuaCR2 DEG**GE*G
* *
***********************jp*****
TuaCR3 DSTEG*GEDEGE
* *
TuaCR4
Alignment of the predicted amino acid sequences of four alpha-tubulin proteins from
Fig. 1.
Ceratopteris richardii; the standard symbols for amino acids are used. Only the sequence for
unknown
TuaCRl complete, and other proteins are compared to Dashes represent amino
is all
it.
acids, asterisks represent known amino acids that are identical to those shown for TuaCRl, bold
known amino between TuaCRl and one TuaCR
letters represent acids that differ at least other
copy, and blank space represents an apparent gap the alignment.
a in
AMERICAN VOLUME NUMBER
FKRN JOURNAL:
54
(2007)
2
\)7
may
protein sequence gene. For these reasons, and because represent
for this
it
an otherwise uncharacterized portion of one of the genes described here, was
it
not included any of the comparisons described below.
in
A
BLAST
comparison sequences
of the four protein the Ceratopteris
for
GenBank
genes with other alpha-tubulin sequences recorded in reveals
somewhat column
a diverse pattern (see the last of Table First, each of
1).
was more
the lour sequences similar to an alpha-tubulin from a different plant
species than was any other Ceratopteris alpha-tubulin sequence. Second,
to
it
the sequences from other plants that are most similar to the Ceratopteris
sequences are quite diverse. The two Ceratopteris amino acid sequences
first
(those of TuaCRl and TuaCR2) are most similar sequences derived from
to
dicotyledonous plants and/or gymnosperm, the third most similar two
a to
is
different algal sequences, and the fourth most similar sequence from
to a
is
a gymnosperm. These observations are perhaps more noteworthy in light of the
Anemia
fact that two distinct sequences for alpha-tubulin genes from the fern
phyllitidis (one a partial sequence) and two from the moss Physcomitrella
is
When
patens are deposited GenBank. compared the Ceratopteris genes
in to
some many
(data not shown), of the four lower plant genes have nearly as
amino
identical acid sequences as do the sequences from the higher plants and
shown when
algae in Table However, these high levels of identity exist, the
1.
lower sequences have one amino
plant also at least additional acid or at least
amino
one less acid than the Ceratopteris sequences, resulting in gaps in the
The
sequence homologies. homologies indicated Table do have such
in not
1
gaps and therefore the sequences they refer to are considered to be more
similar the Ceratopteris sequences.
to
BLAST
For comparative purposes, searches were conducted each
of the
for
known
alpha-tubulin genes Arabidopsis determine what sequences from
of to
would show
Arabidopsis or other organisms the highest similarities to each of
The genome
these genes (data not shown). entire of Arabidopsis has been
sequenced, and six separate alpha-tubulin genes (TUAl-TtJAtt) have been
genome. The
located in patterns of similarity for these genes are
its
dramatically different from those of Ceratopteris. general, the
In six
show much
Arabidopsis alpha-tubulin genes more one another
similarity
to
Among
than do the four Ceratopteris genes. these six genes are two pairs
TUA2 TUA4
whose protein sequences are identical. The proteins of and are
TUA3 TUA5
and those and Furthermore, TUAfi
identical, of are identical.
is
almost identical to TUA2/4, sharing 448 out of 450 amino acids (99.6%
The amino
sequences most
identity). acid that are the next similar to these five
Arabidopsis genes (after comparing them to other Arabidopsis genes) are
all
The TUA2/4
sequences from angiosperms. sequence 98.7%
of identical
to
is
TUA6
sequence from Brassica napns, sequence 99.1%
a the of identical
is to
%
same sequence from napus, and sequence TUA3/5
the B. the of 97.1
is
sequence from Oryza Only sequence remaining
identical to a sativa. the of the
TUAl,
Arabidopsis gene product, that of more similar to the sequence of
is
TUAl
The
a different plant than to another Arabidopsis gene. protein
is
it
shares 414 amino acids out of 450 (92% identity) with the grass Miscanthus.
5
/
SCOTT
ET ALPHA-TUBULIN GENE FAMILY
CERATOPTER1S
AL.: IN RICHARDII
55
Carboxy
termini of the four protein sequences
of
alpha- tubulins from Arabidopsis
thaliana:
TUA1 AREDLAALEKDYEEVGGEGAEDDDEEGDEY
TUA2 AREDLAALEKDYEEVGAEGGDDEDDEGEEY
4
TUA3 AREDLAALEKDYEEVGAEGGDDEEDEGEDY
/
TUA6 AREDLAALEKDYEEVGAEGGDDEDDEGEEY
Carboxy termini of three inferred protein sequences
alpha-tubulins
of from Ceratopteris
richardii:
TuaCRl AREDLAALEKDYEEVGAEGQDDDEPGD
DEY
TuaCR2 AREDLAALEKDYEEVGAEGDEGDEGEDGDEY
TuaCR3 AREDLAALEKDFEEVGADSTEGDGEDEGEEY
Comparison
Fig. 2. of the carboxy terminus regions of Arabidopsis and Ceratopteris alpha-tubulin
genes. Bold letters represent regions when; the sequences within each set are not identical: a blank
space represents an apparent gap the alignment.
in
To compare
further the relative diversity of the Ceratopteris and Arabidopsis
alpha-tubulin proteins, the sequences from the carboxy terminus
region,
which
considered be highly
is to variable in alpha-tubulin proteins see
(e.g.,
and
Sullivan, 1988; Fosket Morejohn, 1992), were aligned with one another
(Fig. Since only three of the four Ceratopteris sequences contain the coding
2).
region for the carboxy terminus portion of the protein, TuaCR4 was omitted
from
this analysis. For both species the amino acid sequence
relatively
is
conserved
until the last 14 residues at the carboxy terminus end, or in the case
TuaCRl, which
of appears have amino
to a single acid deletion in this region,
amino
the last 13 acids. In this region the Arabidopsis sequences share 6
amino
identical acids out of 14, while the Ceratopteris sequences share only
3
amino
identical
acids.
—
Gene phylogeny The
Bayesian
results. tree infers phylogenetic
(Fig. 3)
relationships of nucleotide sequences of the four sequenced richardii alpha-
C.
om
and Meth
Materials
resolving one of the nodes, the resolved subnodes being toward
the top of the
tree with probabilities of 0.80, 0.50, and 0.81. The central clade from Zea mays
22149 Oryza
to sativa japonica cultivargroup 1136121 was not resolved any
in
Bayesian show many
of the runs. Posterior probability values
that clades of
ome
branches had weak The
support. unresolved and weakly supported
clades,
however, are inconsequential to this paper, which focuses instead on the four
copies of C. richardii genes and their placement strongly supported
in clades.
nodes
All separating the four Ceratopteris genes are strongly supported. Three
Ceratopteris copies (TuaCRl, TuaCR2, and TuaCR4) toward
the top of Fig. 3
are relatively closely related to each other in a strongly supported subclade
that is part of the maior clade stretching from C. richardii TuaCRl 2942381 2 tn
5 2 7
1
VOLUME NUMBER
AMERICAN FERN JOURNAL:
97 (2007)
2
56
TuaCRl
29423812
Ceratopteris richardii
.00
TuaCR2
Ceratopteris richardii
1.00 r
57867862
Pseudotsuga menziesii var. menziesii 56481496
1.00
Anemia
296493
phyllitidis
TuaCR4
-
57867864
Ceratopteris richardii
0.99
TUA6
r—
Arabidopsis thaliana 30683073
0.80
0.9J3
TUA4
[L
Arabidopsis thaliana 166915
1.00
TUA2
Arabidopsis thaliana 166913
-
0.50
Brassica napus 34733238
Pisum sativum 525331
Daucus carota 9965990
tC
0.81
Prunus 20412
duicis
0.96
Betula pendula 6723477
1.00
Populus tremuloides 29124982
0.95 —
tabacum
Nicotiana 1967905
1
Rosa hybrid cultivar 15029367
o/w
Zea mays 22149
00
C-
Setaria viridis 51988171
Zea mays
22 46
1
|
1—
Setaria viridis 51988173
i
0.99
0.69
Eleusine indica 3163945
Hordeum
vulgare 1556445
.ool
I
l~L
Triticum aestivum 4098271
0.79
1.00
T
Hordeum
vulgare subsp. vulgare 4165487
0.91
Oryza sativajaponica cultivargroup 136121
1
ANT3
Chloromonas 2625153
sp.
ANT1
Chloromonas 2625155
sp.
Volvox carteri nagariensis 22000
f.
Euglena gracilis 39791
Chiorel vulgaris 391652
la
0.97
0.72
TuaCR3
Ceratopteris richardii 57867856
TU
A5
Arabidopsis thaliana 6691
.00 1
1
TU
A3
l-Arabidopsis thaliana 30687472
0.99
Anemia
296495
phyllitidis
0.84
0.99
TUA1
Arabidopsis thaliana 30697090
Eleusine indica 251 1532
Mi
scan thus jloridulus 793 62
0.98 3
1
.
95
i.ooL Miscanthus 37936209
sinensis
Zea mavs
22155
Avena
1279205
sativa
0.98
o
Hordeum
vulgare vulgare 1477427
Sllbsp.
Oryza sativa japonica cultivargroup 51979001
Physcomitrella patens 25396544
Euglena tubulin 66 70
OUTGROUF gr< icilis bet a 13 2
//
Zea mavs beta tubulin 416146
Bayesian inference gene based on aligned nucleotide sequences of alpha-tubulin genes
tree
Fig. 3.
of Viridiplantae in Gen Bunk (excluding the invariant first three nucleotides and the non-alignable
showing sequenced alpha-tubulin gene
45 nucleotides), relationships of the four C. rich ardii
last
copies discussed. Taxon sources and GenBank numbers are given for each gene copy. Bayesian
gi
posterior probability values indicate support for olades.