The Chloroplast Genome Structure of the Vascular Plant Isoetes Is Similar to That of the Liverwort Marchantia

9 Pages·2000·5 MB·English

by  R Joel Duff

American Fern
Journal 90(2):51-59 (2000)
The Genome
Chloroplast VascuK^X\\C''^'^
Structure of the
Plant Isoetes Similar That
to
is o^\ffi9 ^a^^
^
Liverwort Marchantia ^
a
^^^^
Duff
R. Joel
ar)0^^^^
OH
Department
of Biology, University of Akron, Akron,
44315-39fil0k
Edward
Schilling
E.
TN
Department
of Botany, University of Tennessee, Knoxville, 37996-1100
—
Abstract. Restriction site mapping was used to characterize the chloroplast genome of the ly-
cophyte, Isoetes melonopodo. The Isoetes chloroplast genome approximately 139-145 kb
is in size
with an inverted repeat of 12-13 kb. The gene content and consensus gene order are similar
to
A
that of Marchantia. distinctive feature of the Isoetes genome an increased size of the small
is
DNA
single copy (SSC) region possibly due to the insertion of a piece of (3-8 kb) of unknown
The
composition. inferred insertion, along with a slightly larger inverted repeat, are responsible
genome
for the apparent size difference in the total relative to Marchantia. Patterns of restriction
fragments were also consistent with the presence of a small inversion (2-3 kb) in the large single
copy
(LSC) region.
Structural differences in chloroplast genomes have proven be
to povirerful
among
characters in understanding relationships land plants because of their
and corresponding apparent low homoplasy
rarity levels of (Palmer, 1985a,
b,
Palmer and Palmer Manhart and
1987, 1991; Stein, 1986; 1988; Palmer,
et
al.,
Raubeson and Downie and Lew
1990; Jansen, 1992; Palmer, 1992; Doyle, 1992;
and Manhart, Raubeson and The genome
1993; Stein, 1995). chloroplast ex-
remarkably and gene and among
hibits a consistent structure order content
a
wide members kingdom
range of of the plant (reviewed in: Stein et 1986;
al.,
Among
Palmer Olmstead and
1988; Palmer, 1994). photosynthetic plants
et
al.,
120—160
this circular molecule is usually kilobases (kb) in length and has a
capacity to code for approximately 120 genes.
Our genome comes
understanding of the chloroplast primarily from angio-
been
sperms, but little has reported about the size, overall structure, or vari-
genomes
ation of the chloroplast of lower vascular plants (Lycophyta, Psilo-
Among
toph)^a, and Sphenopsida) or most of the bryophytes. the lower vas-
cular plants only the presence or absence of a 30 kb inversion (Raubeson and
Among
been
Jansen, 1992) has demonstrated. the pteridophytes Stein
et
al.
shown
and Conant, have multiple
(1992) et (1994) that structural rearrange-
al.
among
ments groups and
exist the diverse of ferns that these structural features
may
be useful phylogenetic markers the familial and higher More
at levels.
information about the genomes of the primitive land plants will be needed
to
comprehensive understanding of the chloroplast genome.
attain a
we maps
complete
In this study provide the restriction site of the chlo-
first
We
genome melanopoda.
roplast of a lycophyte, the quillwort Isoetes find that
genome with two
the Isoetes chloroplast shares significant features those of
AMERICAN FERN VOLUME
52 JOURNAL:
non-vascular plants, the bryophytes Marchantia (liverwort) and Physcomitrel-
The genome
la (moss). Isoetes chloroplast has the following
features:
its
(1)
inverted repeat (IR) is several kb larger than the IR of either of the bryophytes
but is significantly smaller than the IR of most ferns and seed plants, the
(2)
small copy
single region (SSC) has
a large (3-8 kb) region of extra genetic
unknown
and
origin,
(2-4
Materials and Methods
Leaves melanopoda Gay &
oi Isoetes Durieu were from
(Isoetaceae) collected
a single population in Alabama and voucher specimen
a {Duff 9201) v^as de-
DNA
posited at the University of Tennessee Herbarium. was
Total isolated
using the procedure of Doyle and Doyle
(1987). Single digests of sixteen
re-
striction endonucleases [BamHI,
Banl, Banll, Dral, EcoRl, EcoRV, Haell,
Hindlll, Neil, Ncol, Pstl, Pvull, Sad, SaR, Stul, and Xhol) and selected double
digests [Banl/Banll, EcoRl/EcoRW,
Pstl/SaR, SaclfPvull, and StuVXhol] were
made
and
the fragments separated on 0.9% agarose run out 15 cms and
gels
transferred by dry Amersham (Hybond N+)
blotting to nylon membranes.
cpDNA
Cloned
fragments from and and
lettuce (Jansen Palmer, 1987) tobacco
(Olmstead and
Palmer, 1992; Shinozaki 1986) were used probes
et al., as for
mapping
physical and DNAs
gene Membrane-bound
localization. were hy-
bridized 32P-dCTP random
to labeled probes using primer
oligolabeling (Fein-
and
berg BRL
Vogelstein, 1983, 1984; Gibco labeling 24 hr 55°
kit) for at C.
Hybridization and
buffers conditions were used as described by the manufac-
DNA
turer (Gibco BRL) except that hybridizations were done 55° Tobacco
at C.
fy
from Hin
dill
Mappin
Palmer
and
(1982, 1986) Jansen and Palmer
(1987).
Results
Hybridization both and
to lettuce tobacco probes generally gave good
results
mapping
limited
As
was
probes. a result possible generate complete maps only
it to for restric-
enzymes
The
problematic
regions are those covered by tobacco probes
20a, 21, 29,
3,
and
March
Adiantum
also reported a lack of hybridization with tobacco probe which
3,
March
(ORFs)
that
March
map
STRUCTURE * 4
53
double melanopoda
digests of Isoetes were successfully generated
(Fig. Par-
1).
maps
seven enzymes were
tial for additional obtained for a great majority of
genome
the including the inverted repeat and small copy and
single region,
were
utilized in determining portions genome Completed
of the
structure.
re-
maps
gave genome
striction site total chloroplast size estimates for Isoetes of
139-145 kb with an average 141 The minimum
of kb. estimates the
for size of
+
the inverted repeat varied from 11.8 [EcoRl EcoRV] kb The
to 13.2 [Nsn).
copy was
size of the large single region (LSC) approximately 85 kb and
the
for
small single copy region (SSC) was 24-29 kb. Consistent with the results of
Raubeson and genome
Jansen presumed
[1992), the Isoetes exhibits the ances-
gene order exhibited bryophytes and Evidence genome
tral in lycophtes. of this
came
from two
architecture the fact that non-adjacent pairs of tobacco probes,
31-11 and
2-12,
respectively, consistently hybridized overlapping
to
frag-
A
ments
(Fig. large insertion in the SSC, relative both Marchantia and
1). to
tobacco, was inferred from mapping studies and appears be unique
to a feature
An
genome was
to Isoetes. additional feature of the Isoetes the apparent pres-
ence of a small inversion (1.5-3.0 kb) found in the large single copy region
may
(LSC) as well as several other smaller inversions which be postulated from
maps
individual restriction but cannot be characterized more completely
site
due
to the resolution of the current data
set.
—
Inverted Repeat. The
size of the inverted repeat in Isoetes (11.8-13.2 kb)
is
Marchantia
IR
homologous
of regions to those of tobacco fragment 32 (rps' 12, Tps7) and a
which
very small portion of probe 31, are found in the IR of tobacco but are
copy
restricted to the large single region adjacent to IR^ in Marchantia (Ohya-
ma
cpDNA
et 1986). Just as in Marchantia, regions of homologous
al., to to-
bacco probes 36 and which
28, 29, 30, all of are part of the IR of tobacco,
1,
mapped
were copy
to the large single region of the Isoetes chloroplast genome.
—
Small Single Copy Region. The single copy region was estimated be 24-
to
29 kb The was due
in size. variation in this estimate to lack of hybridization
and
presence
of genetic material in Isoetes not represented in the tobacco
probes resulting in difficulties in resolving the boundaries of the region. The
combined
lack of hybridization to several probes with the size of spanning
made
SSC
fragments precise estimations of the size of the and the entire ge-
DNA
nome
shows amount
Figure
difficult. 1 that the of in the area adjacent
was more
to the edge of IR^ than could be accounted for from the sizes of the
tobacco probes used and than expected content compared with Mar-
the
its
DNA
genome (Ohyama
chantia For example, 17 kb
et 1986). a chloroplast
al.,
by
fragment, the result of digestion PvuII, only hybridized probes
to 36, 37,
maximum DNA
and very weakly to 35 which account for a of 10 kb of in
The SSC came map
tobacco. best estimate of total size of the from the of SacL
This enzyme yielded only two fragments that span the SSC; a 23-27 kb
frag-
ment and a 1.8 kb fragment each of which hybridized to probe 35. For the
same enzyme two 5.4 kb fragments hybridized strongly to probe 35 and very
AMERICAN FERN VOLUME NUMBER
JOURNAL:
54 90
2 (2000)
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DUFF & SCHILLING: CHLOROPLAST GENOME STRUCTURE OF ISOETES
55
weakly to probe 34 indicating that the 5.4 kb fragment accounts for the ma-
jority of the 4.7 kb tobacco probe 35. The approximately 27 kb and kb
1.8
fragments that weakly hybridize probe 35 must primarily within SSC,
to the
lie
This gives an upper estimate of the SSC of approximately 29 kb. Considering
maps of more than 10 enzymes completed for the SSC, an estimate of 24-29
kb can be made. Compared with Marchantia kb SSC), PhyscomitreJJa
(20
(21
kb SSC, Calie and Hughes, 1986], and Adiantum (20-22 kb SSC, Hasebe and
DNA
Iwatsuki, 1990), this would place the amount of extra in the Isoetes SSC
3-8
at kb.
—
KB
Absence of 30 Based on
Inversion, the presence of fragments from
BamHl
and show
Hindlll digests that overlap tobacco probes 31 and
to 10,
and and Raubeson and
12 2, Jansen (1992) report the absence of a 30 kb in-
found and Our
version in ferns seed plants. data supported the absence of this
inversion in these higher plants. Figure 1 shows that genetic material homol-
ogous to tobacco probes 29, 30 and a portion of probe 1 were detected between
fragments homologous to probes 2 and 12. Tobacco probe hybridized
1 to frag-
ments in two areas: between probes 2 and 29 and between probes 28 and 32
the edge of This what would be expected were genome
at IRg. the oi Isoetes
is
to have the same arrangement as Marchantia. In Marchantia the transfer RNA,
H(GUG), found in probe 1 of tobacco, is present next to probe 2 but the re-
maining portion of probe frnl(CAU), rp723, rp/2, can be found in the LSC
1; at
The weak
the edge of in Marchantia, presence of very hybridization
IR^^ to
probe 1 adjacent to probe 2 suggested that the arrangement of genes in Isoetes,
in the region of this 30 kb inversion in ferns and seed plants, identical
to
is
Marchantia,
that in
—
A
Additional was LSC
Inversions. small (1-3 kb) inversion detected in the
but precise size was difficult to determine. The restriction enzymes used
its
DNA
produced fragments that were too large to allow localization of the end-
The was
points of the inversion. presence of the inversion supported by the
detection of multiple fragments that hybridized identically both tobacco
to
maybe
and
fragments 14 Additional, smaller inversions hypothesized from
15.
several individual restriction maps. These inversions could not be accurately
characterized because they are located in a portion of the genome which
for
was
there poor hybridization signal. This region corresponds to the position
=
of a large open reading frame (ORF2136 tobacco fragments 29,30). Only
maps genome
Fig. 1. Linearized restriction site of the chloroplast of Isoetes melanopodo. En-
map genome
zymes used in single or double digestions to the are indicated each end of the
at
map. The top line represents the tobacco probe set (Olmstead and Palmer, 1992) in positions
cpDNA.
relative to their hybridization with Isoetes Large solid bars indicate relative position and
boundaries of the inverted repeat in Isoetes. Asterisks denote tobacco probes for which very week
hybridization was observed to Isoetes fragments. Fragments less than 0.5 kb were not consistently
observed and therefore are postulated based on data from double digests. Fragment sizes larger
than 18 kb could not be accurately calculated and so reflect estimations based on additive sizes
of fragments detected by double digests.
AMERICAN FERN VOLUME NUMBER
56 JOURNAL:
90
2 (2000)
fragments spanning were mapping
the entire region utilized in the data pre-
Raubeson and
sented here. Jansen (1992) also report the probable occurrence
up two
of to small inversions in this region as well, but did not characterize
further
Discussion
Although
Isoetes is a highly distinctive genus, whose evolutionary lineage
is separated from the majority of vascular plants by up 300 million years
to
and from
the non-vascular
plants long
for as or longer, exhibits a chloroplast
it
genome
remarkably
similar to the liverwort Morchantia. Most vascular plants
appear have
to significantly larger chloroplast genomes than the bryophytes
because cpDNA
the inverted repeat The genome
is larger. Isoetes also larger
is
than bryophyte genome
the
in size but the difference is the result of an in-
SSC
creased cpDNAs
size of the relative to other characterized and to a lesser
an
extent increase The
in the size of the IR. overall size, including the IR, and
genome
structure of the chloroplast
of Selaginella unpublished
(Duff, data)
March
Sim
Marchantia
of the 30 kb inversion some
confers
structural integrity to these molecules.
The
kb
30 inversion that took place in the ancestor of the ferns and seed plants
appears be
to correlated with a relaxation of physical constraints on the
size
of the inverted Hence
repeat region. the IR subsequently underwent
rapidly
expand
LSC
to incorporate genetic material from the in the vast majority of
ferns and seed plants (Palmer and Thompson 1982; Jansen and Palmer 1987;
Howe
et 1988]. Stein has proposed around
al. et (1992] that the region the
al.
Marchantia
mlike
those
the
genome
integrity of the not These by
to affect the IR. taxa are accounts
all
highly divergent and
yet the sizes of their respective inverted repeats are not
significantly different nor have they been demonstrated have undergone
to
inversion events to the extent observed in plants which contain the 30 kb
March
March
and
Physcomitrella,
Isoetes is directly related to their gene order.
the was
29 unex-
kb) in Isoetes
fficulty
homol
example, has been shown
that the chIL gene has been from angiosperm
it lost
cpDNAs
although
it is present in Isoetes as well as all bryoph5^es. lycophytes,
ferns except Psilotum, and gymnosperms
except Welwitschia (Burke
et
al.,
This gene SSC
1993]. is typically located in the adjacent to the IR^ though the
map
restriction generated does
for Isoetes not clearly define this region due to
the present of
several large fragments spanning
this region. Furthermore, the
DUFF & SCHILLING: CHLOROPLAST GENOME STRUCTURE OF ISOETES
57
map
tobacco probes used in constructing the lack this gene region and thus
it
possible that several small fragments generated by Sad, and Pvull
is Nsil,
could have escaped between and Even
detection in this region probes 40
35.
accounting SSC
for these sources of uncertainty the total size of the appears
to be increased over that of all other previously characterized land plant chlo-
DNA
genomes. Furthermore most
roplast this increase in content apparent
is
in the fragments that hybridized to tobacco probe 35, found in the IR of both
and and The
tobacco tobacco probe ma-
Isoetes, 36. latter represents genetic
found whose homologous
terial in the IR of tobacco but content can be found
DNA
SSC
Marchantia had
in the of adjacent This
to IRg. extra insufficient
sequence similarity with any portion of the tobacco or lettuce genomes
to
produce hybridization signals and presence was only determined by the
its
presence of fragments that spanned the entire region (see Stul, SaR, Pstl in
DNA
SSC may
This found be an
Fig. in the Isoetes the result of either
1).
may
increased size of spacer regions between the genes or be due to an inser-
DNA
DNA.
Very few non-homologous
tion of foreign definitive cases of extra,
found genome known. Conant
in the chloroplast are et (1994) report the
al.
DNA
presence of about 0.9 kb of in the IR of Cyathea furfuracea (Cyatheaceae)
which no Adiantum genome and
for there counterpart in the thus cannot
is
A
be explained by duplication or inversion events. more dramatic example
genome
can be seen in the unusual choloroplast of Trachelium (Campanula-
DNA
many
which
seven have been
ceae) in as as insertions of foreign postu-
lated (Cosner et 1997). In any case, more detailed analyses will need to be
al.
undertaken to confirm the existence and to determine the nature of any ad-
sequences genome.
ditional in the Isoetes chloroplast
we genome
In summary, have established that the of Isoetes has the overall
Marchantia and The
structure of the bryophytes, Physcomitrella. only
signif-
between bryophyte genome and
icant difference the that of Isoetes the pos-
is
DNA
SSC genome and
sible increase in content of the region of the Isoetes
work and
increased IR content. Further to characterize the insertion in Isoetes
map
genomes Lycopodium and
Equisetum, Psilotum
to the of sens, will
lat.,
most
likely result in a better understanding of the evolution of the chloroplast
genome and possibly provide clues regarding the relationships of the lower
vascular
plants.
Acknowledgments
This paper represents a portion of a Ph.D. dissertation submitted the University of Tennessee.
to
We
thank Diana Stein and Linda Raubeson for their advice and help on the mapping of the genome
and the comments of one anonymous reviewer. This research was supported by National Science
Foundation Doctoral Dissertation Improvement Grant DEB-9321767, and grants from the A.
J.
Sharp Fund, Department of Botany, University of Tennessee.
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