Botanical Studies (2009) 50: 137-147.
Present address: Department of Plant Pathology and
Microbiology, Texas A & M University, College Station, TX
77840, USA.
Corresponding author: E-mail: Peter.Ueng@ars.usda.
gov;; Tel: +1-301-504-6308; Fax:
Length polymorphisms in the ribosomal RNA (rRNA)
genes are due to DNA fragment insertions, deletions,
duplications and the presence of group I introns. Group
I introns are one of the major class introns widespread
in mitochondria, chloroplasts and nuclear rDNA of
eukaryotes including fungi, algae, slime molds and plants,
and in eubacteria and protist (Turmel et al., 1991; Haugen
et al., 2005). Group I introns are also infrequently reported
in mitochondrial genomes of lower sea animals, viruses
and phages, and absent in prokaryotes including archaea
and bacteria (Lonergan and Gray, 1994; Beagley et al.,
Group I introns in small subunit ribosomal DNA (SSU-
rDNA) of cereal Phaeosphaeria species
Chih-Li WANG
, Pi-Fang Linda CHANG
, Ying-Hong LIN
, Arkadiusz MALKUS
, Ling-Yan
and Peter P. UENG
Department of Plant Protection, Fengshan Tropical Horticultural Experiment Station, Agricultural Research Institute,
Kaohsiung 830, Taiwan
Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan
Department of Plant Pathology, Plant Breeding and Acclimatization Institute, Radzikow, Poland
Inner Mongolia Agriculture University, College of Ecology and Environmental Science, Inner Mongolia
Molecular Plant Pathology Laboratory, Plant Science Institute, U.S. Department of Agriculture, ARS, Beltsville, MD
20705, USA
(Received May 14, 2008; Accepted November 20, 2008)
In a study of small subunit ribosomal RNA (SSU-rRNA) gene sequences in cereal and a grass
Phaeosphaeria species, group I introns were found in 9 of 10 P. avenaria f. sp. avenaria (Paa) isolates from
oat (Avena sativa L.), 1 of 2 Phaeosphaeria sp. (P-rye) isolates (Sn48-1) from Polish rye (Secale cereale L.), 1
Phaeosphaeria sp. (P-dg) isolate (S-93-48) from dallis grass (Paspalum dilatatum Poir.) and both heterothallic
P. a. f. sp. triticea (Pat2) isolates (ATCC26370 and ATCC26377) from foxtail barley (Hordeum jubatum L.).
There were no group I introns in wheat- and barley-biotype P. nodorum (PN-w and PN-b), homothallic P.
a. f. sp. triticea (Pat1) and P. a . f. sp. triticea (Pat3) from the state of Washington. Based on the reference
16S rDNA nucleotide sequence of Escherichia coli (accession number J01695), the intron-inserted positions
of Pav.nS943, Pse.nS943, Ppa.nS1199 and Pho.nS1533 were determined to be at nt943, nt943, nt1199 and
nt1533, respectively. The sizes of the introns were 362 bp for Pav.nS943 (from Paa), 363 bp for Pse.nS943 (from
P-rye), 460 bp for Pho.nS1533 (from Pat2) and 383 bp for Ppa.nS1199 (P-dg). The intron-inserted position at
nt1533 found in SSU-rRNA of Pat2 pathogen was newly discovered. The phylogenetic relationships based on
aligned conserved secondary structure component sequences of group I introns showed that three introns from
cereal Phaeosphaeria species (Pav.nS943, Pse.nS943 and Pho.nS1533) were likely affiliated with subgroup
IC1 introns while Ppa.nS1199 intron from the dallis grass pathogen belonged to subgroup IE3.
Keywords: Introns; Phaeosphaeria; Phylogenetic relationships; Ribosomal RNA gene; Small subunit; Wheat.
1998; Nishida et al., 1998; Riipinen and Alatossava,
2004; Sandegren and Sjoberg, 2004; Fukami et al.,
2007; Stankovic et al., 2007). Some group I introns are
experimentally proven to act as mobile genetic elements
by reverse splicing and site-specific endonuclease
restriction (Lambowitz and Belfort, 1993; Saldanha et al.,
1993; Belfort and Perlman, 1995; Roman and Woodson,
1998; Haugen et al, 2005).
The insertion positions of group I introns in nuclear
SSU-rRNA gene were standardized by using 16S rDNA
nucleotide sequence of Escherichia coli (Accession
number J01695) as reference. Group I introns with the
same insertion positions are suggested to have evolved by
vertical transmission, along with the occasional loss and
horizontal transfer (Nikoh and Fukatsu, 2001; Feau et al.,
2007). In vertical transfer, the phylogenetic relationship
of introns inserted at the same position is expected to be
significantly congruent with the phylogeny of their hosts
(Nikoh and Fukatsu, 2001). When the phylogeny of group
I introns differ significantly from the host genomes, it is
Botanical Studies, Vol. 50, 2009
a strong evidence of horizontal transfer (Goddard and
Burt, 1999; Holst-Jensen et al., 1999; Simon et al., 2005).
The presence of group I introns in rRNA in some fungal
species is useful in developing molecular markers for PCR
amplification to separate them from other species which
had no introns (Neuveglise et al., 1997; Chen et al., 1998).
In a preliminary survey, length polymorphisms
were observed in the small subunit ribosomal RNA
(SSU-rRNA) genes of several Phaeosphaeria species
(Anamorph: Stagonospora). The SSU-rRNA gene
sequence of P. avenaria f. sp. avenaria (Paa) AFTOL-ID
280 isolate containing an intron was deposited previously
(Accession number AY544725). Characteristics of these
length polymorphisms in SSU-rRNA of three cereal
and one grass Phaeosphaeria species were studied here.
This is the first report of a new group I intron inserted at
position nt1533 corresponding to the Escherichia coli 16S
rDNA found in P. avenaria f. sp. triticea (Pat2) pathogens
isolated from foxtail barley (Hordeum jubatum L.).
PCR amplification and sequencing
Procedures for fungal culture in a liquid medium and
for genomic DNA (gDNA) isolation were described
previously (Ueng et al., 1992). The gDNA was purified
by CsCl gradient ultracentrifugation. The primer
(TCCGCAGGTGCACCTACGGA) designed from the
rDNA sequence (nt10138-nt10120 / nt8368-nt8387) of
Saccharomyces cerevisiae (Accession number Z73326)
were used for amplifying partial SSU fragments in strains
of Phaeosphaeria. Phaeosphaeria species including 5
wheat-biotype P. nodorum (PN-w), 6 barley-biotype P.
nodorum (PN-b), 10 P. avenaria f. sp. avenaria (Paa), 5
homothallic P. avenaria f. sp. triticea (P. a. f. sp. triticea,
Pat1), 2 heterothallic P. a. f. sp. triticea from foxtail barley
(Pat2), 1 P. a. f. sp. triticea from the state of Washington
(Pat3), 2 Phaeosphaeria sp. from Polish rye (P-rye)
(Reszka et al., 2006) and 1 from dallis grass (Paspalum
dilatatum Poir.) (P-dg) were used (Table 1). For examining
intron insertion in the large subunit ribosomal RNA
(LSU-rRNA) genes of those Phaeosphaeria species,
amplifications. PCR amplification was performed in 50 £gl
reaction mixtures containing reaction buffer (50 mM KCl,
10 mM Tris-HCl, pH 9.0 at 25¢XC, 0.1 % Triton X-100), 1.5
mM MgCl
, 0.2 mM dNTPs, 1.5 £gM of each primer, 80 ng
gDNA, and 1.0 unit of Ta q DNA polymerase (Promega,
Madison, WI). Reaction parameters were: denaturation (94
¢XC, 3 min) followed by 40 cycles of 94¢XC (20 s), 55¢XC (30
s), and 72¢XC (1 min), and a final incubation step at 72¢XC
(10 min). Isolation and direct sequencing of PCR products
were conducted as described previously (Ueng et al.,
Intron splicing in SSU rRNA
To determine the intron splicing in rRNA, the total RNA
digested with DNase I was used. Both gDNA and total
RNA were isolated from Phaeosphaeria cultures grown
in YMS (0.5% malt extract, 0.5% yeast extract and 2.0%
sucrose) liquid medium with shaking at 125 rpm for 7-14
days at 27¢XC (Ueng et al., 1992). The gDNA was partially
purified, ribonuclease A treated and without CsCl gradient
ultracentrifugation (Ueng et al., 1992). Extraction of total
RNA mainly followed the protocols previously described
(Wang et al., 2007). Lack of residual gDNA in total RNA
was evidenced by not being able to amplify the partial
histidinol dehydrogenase (Hdh1) gene fragment by the
primer set 15A/12-1 (ATGCCGGCAGGACCCAGTGA/
polymerase (Unpublished data, Wang et al., 2007).
The first strand (1¡Ñ) cDNA was synthesized with
random primers p(dN)
and the First Strand cDNA
Synthesis Kit (Roche Diagnostics Corporation,
Indianapolis, IN). PCR amplified fragments amplified
from gDNA and 1¡Ñ cDNA were isolated, sequenced
and compared (Ueng et al., 2003). Primers, NS1 and
NS8, and other specifically designed ones, such as Fun5
and SR6 (TGTTACGACTTTTACTT), were used for
amplifying and sequencing partial SSU-rDNA fragments
from gDNA and the first strand cDNA.
Secondary structure modeling of group I
introns in pre-rRNA
The secondary structures of group I introns were
predicted and constrained according to the expected
conserved sequences and structures for subgroup IC
(Cech, 1988) and subgroup IE (Suh et al., 1999; Li and
Zhang, 2005) by utilizing the ¡¥RNA structure, version 4.4¡¦
program (Mathews et al., 1999, 2004) and exported in ¡¥.ct¡¦
format for further refinement with the ¡¥RnaViz 2¡¦ program
(De Rijk et al., 2003). The intron nomenclatures followed
Johansen and Haugen¡¦s proposal (2001) and the structural
conventions for group I introns followed Burke et al.
(1987) and Gutell (1993).
Subgroup introns by sequence analysis
Regardless of the exon sequences, the insertion
positions, and their host phylogenetic relationships, group
I introns were divided into 5 major groups (IA-IE), which
included 14 subgroups (IA1-AI3, IB1-IB4, IC1-IC3, ID,
and IE1-IE3). Most of group I introns in the nuclear SSU-
rDNA have been grouped in either IC1 or IE, whilst in
the organelle (mitochondria and chloroplast), SSU-rDNA
grouped in IA3 and IC2 except for 1 in IB1 (www.rna., Cannone et al., 2002). To identify sub
groupings of the introns found in the nuclear SSU-rDNA
WANG et al. ¡X Group I intron in rDNA
Botanical Studies, Vol. 50, 2009
of Phaeosphaeria species, 12 intron nucleotide sequences
representative of subgroup IC, IE, IB2 and IA3 were
randomly chosen from the alignments made by Michel &
Westhof (1990) and Li & Zhang (2005), manually aligned
with 4 Phaeosphaeria intron nucleotide sequences based
on the core secondary structure components P1 to P10,
and refined by removing the components P2, P2.1, P7.1,
P7.2, and P9.1 to P9.3, which are not presented in all
groups (Table 2). The final alignment data were analyzed
using Mega Version 4.0 (
index.html, Tamura et al., 2007). One thousand data sets
analyzed from 167 informative nucleotides sites were
generated by bootstrap re-sampling and evaluated by
Neighbor-Joint (NJ) method with Jukes-Cantor model.
Identification of the intron sequences in SSU-
Partial SSU-rDNA fragments amplified from gDNA
with the primer set NS1/NS8 showed length variations in
several cereal Phaeosphaeria species (Figure 1A). There
were two DNA fragments produced in P-rye Sn48-1
isolate (Figure 1A, lane 6). Based on the nucleotide
sequences, group I introns were identified in the following
isolates: 9 of 10 Paa, 1 of 2 P-rye, both Pat2 and 1 P-dg
(Table 1). The analysis also showed that group I introns in
SSU-rDNA in Phaeosphaeria will be removed from the
pre-rRNA during rRNA maturation (Figure 1B). Without
group I introns, the length of partial SSU fragments
amplified by the primer set NS1/NS8 are expected to be
1,769 bp in Phaeosphaeria species. Pat1 and Pat3 had the
same nucleotide sequences for the partial SSU-rDNA as
that in PN-w. Compared to partial SSU-rDNA sequence
of PN-w, there is l nucleotide substitution in PN-b, 2 in
Paa and Pat2, 3 in P-dg and 4 in P-rye isolates (Data not
shown). The sizes of group I intron in SSU-rDNA were
as followed: 362 bp in Paa, 363 bp in P-rye, 460 bp in
Pat2 and 383 bp in P-dg isolates (Table 1). On the other
hand, partial nucleotide sequences of large subunit (LSU-)
rRNA gene amplifying with three primers sets LR0R/LR7,
LR7R/LR10 and LR10R/LR13 found no intron insertions
in those Phaeosphaeria species (Accession numbers
EF590318-EF590322 and EU223256-EU223257, Table 1).
Intron positions and naming
Based on the 1,542 bp-long reference 16S rDNA
sequence of E. coli (accession number J01695), the intron
positions were determined to be at nt943 for Paa and
P-rye, nt1199 for P-dg and nt1533 for Pat2 (Figure 2).
Since Paa (P. avenaria f.sp. avenaria) was first identified
as a pathogen from oat [Av ena sativa L.], the group I
intron found in this pathogen was named as Pav.nS943
following Johansen and Haugen¡¦s proposal (2001).
However, the scientific names and classification of P-rye,
Pat2 and P-dg pathogens have not yet been recognized.
In this paper, the first two letters of their respective host
WANG et al. ¡X Group I intron in rDNA
Figure 1. Amplification of partial small subunits of ribosomal DNA (SSU-rDNA) in Phaeosphaeria species. A, Partial SSU-
rDNA was amplified from the genom ic DNA (gDNA) with NS1/NS8 primer set. M = Molecular markers of £f DNA cut with
HindIII and EcoRI restriction enzymes. 1-2 = Phaeosphaeria avenaria f. sp. avenaria (Paa) (ATCC12277, Saa001NY-85); 3
= P. avenaria f. sp. triticea (P. a . f. sp. triticea, Pat3) (S-81-W10); 4-5 = Heterothallic P. a . f. sp. triticea (Pat2) (ATCC26370,
AT CC26377); 6 = Phaeosphaeria sp. from Polish r ye (P-rye) (Sn48-1); 7 = P. nodorum, whe at-biot ype (PN-w) (Sn27-1);
8-9 = P. nodor um, barley-biotype (PN-b) (S-84-2, S-83-2); 10-11 = Homothallic P. a. f.s p. triticea (Pat1) (Sat24-1, 12889);
12 = Phaeosphaeria sp. from dallis grass (P-dg) (S-93-48); B, Pa rtial SSU-rDNA was amplified from the gDNA (a) and the
first strand (1¡Ñ) cDNA (b) with NS1/NS8 primer set. Solid arrows indicated either no introns in gDNA and cDNA or spliced
cDNA. Open arrows indicated the presence of introns in SSU-rDNA of gDNA. Isolates used were: PN-b = S-80-611; Paa =
ATCC12277; Pat2 = ATCC26370; P-dg = S-93-48; P-rye = Sn48-1.
Figure 2. Group I insertions in the small subunit of ribo-
som al DNA (SSU-rDNA) of Phaeosphaeria sp ecie s. T he
numbers below each t riangle corresponds to the intron posi-
tions relative to the SSU-rDNA sequence of Escherichia coli
(accession number J01695). Paa = Phaeosphaeria avenaria
f. sp. avenaria; P-rye = Phaeosphaeria sp. from Polish
rye; P-dg = Phaeosphaeria sp. from dallis grass; Pat2 =
Heterothallic P. avenaria f.sp. triticea.
plant genus names were temporarily used as the 2
letters in the naming of the introns. Therefore, group
I introns found in P-rye (from rye [Secale cereale L.]),
Pat2 (from foxtail barley [Hordeum jubatum L.]) and P-dg
(from dallis grass [Paspalum dilatatum L.]) are called Pse.
nS943, Pho.nS1533 and Ppa.nS1199, respectively.
Predicted secondary structures of group I
The secondary structures of 4 group I introns found in
the SSU-rDNA in Phaeosphaeria species were composed
of nine base-paired segments, characterized by a conserved
active structure that was formed by the assembly of the
¡¥J¡¦ (including P4-P6) and the ¡¥P¡¦ (including P3, P7 - P9)
Botanical Studies, Vol. 50, 2009
structural elements, and other peripheral elements needed
for splicing (Figure 3) (Burke et al., 1987; Cech, 1988;
Michel and Westhof, 1990; Golden et al., 1998; Adams et
al., 2004). Internal guide sequences (IGS) were recognized
which base pair tightly with the 5¡¦ exon to form a P1 helix
before docking into the catalytic core and that permits to
stable tertiary interactions during the course of the self-
splicing (Been and Cech, 1986; Pyle and Cech, 1991)
(Figure 3). Also as expected, a 3¡¦ end exon base ¡¥U¡¦
immediately upstream of the 5¡¦ intron splicing site and a
3¡¦ end intron base ¡¥G¡¦ preceding the 3¡¦ intron splicing site
were located (Michel and Westhof, 1990). However, there
was an exception in Pho.nS1533 intron, which had ¡¥GU¡¦
preceding the 3¡¦ intron-splicing site (Figure 3C).
Based on 4 consensus sequences (P, Q, R and S
elements), Pav.nS943, Pse.nS943 and Pho.nS1533
complied with the expected consensus for groups IA-ID
introns, and Ppa.nS1199 with group IE introns (Table 3)
Figure 3. Predicted secondary structure models of Phaeosphaeria group I introns. Intron representatives of Pse.nS943 (A), Pav.
nS943 (B), Pho.nS1533 (C) and Ppa.nS1199 (D) were shown. The intron sequences were numbered from 5¡¦ end to 3¡¦ end and in
uppercase letters whilst the 5¡¦ and 3¡¦ exons in lowercase letters. The bolded and circled letters were the conserved sequence ele-
ments, P, Q, R and S. The characteristics of helixes were termed from P1 to P10 following the struct ural conventions for Group
I introns (Burke et al., 1987). T he P13 stem formed by base pairing of two remote peripheral elements P2.1 and P9.1 in (D) was
indicated. The recognition and binding of a 5¡¦ end internal guide sequences (IGS) with the 3¡¦ exon sequences in splicing were
boxed. Three shaded letters in (B) were base substitutions occurred in respective isolates, 1919WRS (at nt100, C ¡÷ T), Sa37-2
(at nt128, A ¡÷ G) and ATCC12277 (at nt336, A ¡÷ G).
WANG et al. ¡X Group I intron in rDNA
(Cech, 1988; Michel and Westhof, 1990; Suh et al., 1999;
Gibb and Hausner, 2003; Li and Zhang, 2005). Three
introns from cereal Phaeosphaeria pathogens (Pav.nS943,
Pse.nS943 and Pho.nS1533) had typical long extended P5
domains (P5a-P5d) in their predicted secondary structures
(Figure 3A-C). Presence of long extended P5 domains
in these introns might suggest that they were ancestral
type and able to self-splice (Haugen et al., 2004). Like
other group IE introns, there was only a short peripheral
P5 region (without P5a-P5d) in the secondary structure
of the Ppa.nS1199 intron (Li and Zhang, 2005) (Figure
3D). In the secondary structure of the Ppa.nS1199 intron,
formation of a P13 stem by base-pairing of two remote
peripheral elements, P2.1 and P9.1, was reported to be an
important feature, and play a role in the functional folding
of the active structure in subgroup IE intron (Figure 3D)
(Li and Zhang, 2005; Xiao et al., 2005).
Subgrouping group I introns
Based on nucleotide sequences of the secondary
structure components, phylogenetic relationships of
introns from 4 Phaeosphaeria with other 11 known group
I introns were compared. It appeared that Pav.nS943, Pse.
nS943 and Pho.nS1533 introns were closely related to
IC1 subgroup, and Ppa.nS1199 belonged to subgroup IE3
(Figure 4).
Table 3. Alignments of 4 consensus sequence elements in the group I introns of ribosomal DNA in Phaeosphaeria species.
Species Intron size (bp)
Consensus conserved sequences in the elements
Groups IA - ID
a uncnngaAn
Guu ga
cA GacUana
aauugcggggaa aauccgcagc guucagagacuaaa aagauauagucc
P-rye 363
aauugcggggaa aauccgcagc guucagagacuaaa aagauauagucc
aauugcggggaa aauccgcagc
Group E
ua g g
GG cn gG An
Au u a
a c u
AUc ng Gg
g a a
uc ga ca
uc A GCncG
gu ac gu
a g c
c u u
Group E
g ua g gg
G cn g An
u au u aa
a c u
AUc ng Gg
g a a
uc ga c ca
uc A G ncG
gu ac u gu
a g g c
AGG Acgu C
c u a u
383 gguacagggaac gauccugugg ucgcaacgcgcgga aagguacgugcu
Consensus conserved sequences were after
Cech, 1988,
Suh et al., 1999 and
Gibb and Hausner, 2003.
Underlined nucleotides are proposed to be base-paired in the secondary structure. Bulged nucleotides within base-paired
segments are found in #7 nucleotide of the ¡¥R¡¦ element. An uppercase letter in consensus conserved sequences designates >90%
conservation of the particular nucleotide; a lowercase letter designates 70-90% conservation. A pair of lowercase letters indicates
that two nucleotides frequently occupy the position and together account for >90% of the sequences; "n" in a position indicates
that no nucleotide is conserved at the level of these criteria. Shaded nucleotides in the group I introns are not identical to consensus
Fi gure 4 . Phylogenetic relationships of group I introns
in the small su bu n it r ib oso mal DNA (SSU -rDNA) of
Phaeosphaeria species. Data of aligned secondary structure
components from 4 Phaeosphaeria int rons (see Table 2) and
6 introns re presenting groups IA-IB and 6 of the group IE
aligne d respectively by Michel and Westhof (1990) and Li
and Zhang (2005) were used for analysis. The numbers in
pa rentheses are GenBank acce ssion numbers. Except for 4
underlined introns from Phaeosphaeria species which puta-
tively follows the nomination of Johansen and Haugen¡¦s pro-
posal (2001), int ron names in square brackets are abbreviated
as by Cech (1988). Bootst rap values (with 1000 replications)
of the internal branches larger than 50% are indicated.
Botanical Studies, Vol. 50, 2009
Group I introns are found in both the SSU and LSU
rRNA genes in numerous parasitic, lichen-forming and
mycorrhizal fungi (De Wachter et al., 1992; Lin et al.,
1992; Liu and Leibowitz, 1993; Mercure et al., 1993;
Egger et al., 1995; Chen et al., 1996, 1998; Neuveglise et
al., 1997; Tan, 1997; Ito and Hirano, 1999; Myllys et al.,
1999; Mavridou et al., 2000; Perotto et al., 2000; Suga et
al., 2000; Gibb and Hausner, 2003; Lickey et al., 2003;
Wang et al., 2003; Cote et al., 2004). Group I introns were
found in the LSU-rDNA of the soil-borne wheat take-all
disease pathogen, Gaeumannomyces graminis (Tan, 1997).
Group I introns were also found in the SSU-rDNA of
numerous Septoria and other anamorphic species related
to the teleomorphic genus Mycosphaerella from banana
and woody and ornamental plants (Feau et al., 2007).
However, the present of group I introns in the rDNA of
M. graminicola (anamorph: Septoria tritici) and Septoria
passerinii, two important leaf blotch pathogens in cereals,
have not been studied.
Allelic heterogeneity of group I introns in rDNA has
been reported in numerous fungi. In some organisms,
group I introns only inserted in partial repeats of ribosomal
DNA repeats tandem located in genome of the same strain.
(Lin et al., 1992; Wetzel et al., 1999; Lickey et al., 2003).
In strain P-rye Sn48-1, ribosomal repeat regions inserted
with intron were amplified by PCR at less frequent than
intron-less regions (Figure 1A, lane 6 and Figure 1B,
P-rye). The presence of the intron was often variable with
strains of a species or among strains of different species.
For example, only some strains of Candida albicans and
Monilinia fructicola are reported to have the same group
I introns in the rDNA (Mercure et al., 1993; Cote et al.,
2004). Also, in the ericoid mycorrhizal fungi, no group
I intron was present in all SSU rDNA repeats in 1 of 11
Oidiodendron maius isolates (Perotto et al., 2000). The
variation in the presence of the introns also found in both
Paa and P-rye (Table 1).
Group I introns are widely distributed in nuclear SSU-
and LSU-rDNA. With 2,179 introns so far reported in
SSU-rDNA, there are 117 intron positions identified in
the 1542 bp-long reference Escherichia coli 16S rDNA
(Accession number J01695) (
Of which 16.5% and 6.2% introns are inserted at the
nt943 and nt1199 positions, respectively. In this study,
three group I introns from Phaeosphaeria (Pav.nS943,
Pse.nS943, and Ppa.nS1199) were located in these two
common positions (Figure 2). Nevertheless, here we report
a new group I intron-inserted position at nt1533 of E.
coli 16S rDNA, which is identified in Pat2 (Pho.nS1533)
(Figure 2).
It had been showed that nucleotide sequences of
secondary structure components were conserved in the
same subgroups of group I introns which inserted in
either nucleus or organelles and even in distantly relative
organisms (Michel and Westhof, 1990; Suh et al., 1999).
For instance, Pav.nS943, Pse.nS943 and Pho.nS1533
introns were grouped in IC1 with an intron inserted in
the nuclear LSU-rDNA of Tetrahymena thermophila (Tt.
LSU), a ciliated protozoan, rather than other subgroup
such as the IE2 in LSU-rDNA of Candida albicans
(Ca. LSU, X74272), which was more closely related
t o Phaeosphaeria (Figure 4). Furthermore, types of
subgroups seem to be associated with the exons in which
group I introns inserted. In a collective survey of 747 group
I introns from fungal nuclear SSU-rRNA genes, almost
all of them belong to either group IC introns (65.6%) or
group IE (34.3%) (, Cannone
et al., 2002). Four group I introns from Phaeosphaeria
spp. are also grouped into these two subgroups (Figure
4). Group I introns are commonly inserted in highly
conserved regions of the SSU-rRNA genes. It had been
shown that the same subgroup group I introns that occupy
the same position in SSU-rDNA but in distantly related
hosts, tend to share a number of structure features as well
as high levels of primary sequence similarities compared
to introns at different insertion positions, thus allowing to
explore their phylogenetic relationships (Suh et al., 1999;
Nikoh and Fukatsu, 2001). It is suggested that the intron
insertion positions are related to the nature of the intron
subgroups to which they belong. In a comparison with
numerous group I introns found in the SSU-rRNA genes
from several fungi and green algae, the position at nt1199
tended to be a common position for subgroup IE3, and the
position at nt943 for subgroup IC1 (Michel and Westhof,
1990; Takashima and Nakase, 1997). Like the Ppa.nS1199
intron from P-dg, many group I introns inserted at position
nt1199 are reported to have group IE secondary structures
(Gibb and Hausner, 2003; Li and Zhang, 2005; Feau et al.,
Group I introns were suggested to be either transferred
horizontally to the distinct insertion sites or inherited and
diverged vertically (Shinohara et al., 1996; Tan, 1997;
Mavridou et al., 2000; Gibb and Hausner, 2003, Martin et
al., 2003; Wang et al., 2003). Horizontal transfer events
were suggested to occur to group I introns of SSU-rDNA
in Septoria and other anamorphic species related to the
teleomorphic genus Mycosphaerella (Feau et al., 2007).
Based on the Phylogenetic relationships inferred from the
£]-tubulin (tubA), £]-glucosidase (bgl1), and the second
largest subunit of RNA polymerases II (RPB2) genes, two
sister clades, P-rye-PN-w and Paa-Pat3, were consistently
recovered with well supports (Malkus et al., 2005; Reszka
et al., 2005; Malkus et al., 2006). An additional intron
found in P. eustoma isolate AFTOL-ID 1570 (DQ678011),
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