DNA Sequence-The lournal of Sequencing and Mapping, Vol. 5 , pp.
381-383 0 1995 Harwood Academic Publishers CmbH
Reprints available directly from the publisher
Photocopying permitted by license only
SHORT COMMUNICATION
Complete nucleotide sequence of Beauveria
bassiana 5.8s rRNA coding gene and flanking
internal transcribed spacers
Mitochondrial DNA Downloaded from informahealthcare.com by AMS-Laguna-Nautica-Es Mar
H. L. SHIH, C. P. LIN, R. F. LlOU and S. S. TZEAN*
Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, 106 17, R.O.C.
Database A'ccession No. EMBL 247354, 247355
The nucleotide sequence of two clones of Beauveria bassiana in
5.8s rRNA coding gene and ITS regions were completely se-
For personal use only.
quenced. The overall sequence similarity of these two clones is
96%. The identities of internal transcribed spacer (ITS) regions
are 91% (ITSI) and 100% (ITSII), respectively. Both of 5.8s
rRNA sequences have 98% homology.
KEY WORDS: 5.8s rDNA, internal transcribed spacer, PCR, se-
quencing
Beauveria bassiana is an entomopathogenic fungus
and is a member of Deuteromycotina which lacks a
sexual stage (Paccolla-Meirelles and Azevedo,
1991). Though both heterokaryon incompatibility
and parasexuality in B. bassiana had been reported
i n laboratory conditions (Paccolla-Meirelles and
Azevedo, 19911, the parasexual cycle has never
been demonstrated in nature (St Leger et a/., 1992).
Thus, mutation was the way responsible for most of
the genetic variations in 5. bassiana population and
was maintained from generation to generation by
asexual reproduction. Conventional, taxonomic
study is primarily based on morphological (Mugnai
et a/., 1989) and physiological (Mugnai et a/., 1989,
St Leger et a/., 1992) characters. The great hetero-
geneity demonstrated within this species complex
has permitted few conclusions to be drawn about
population structures and taxonomy (Mugnai et a/.,
1989). Both morphological and biological charac-
Address for correspondence: Dr. S. S. Tzean, Department of Plant
Pathology and Entomology, National Taiwan University, Taipei,
Taiwan, 10617, R.O.C.
381
Printed in Malaysia
ters (Gerbi, 1985) may reflect the environmental
conditions and may thus lead to controversial
species reassignments (Curtis eta/.,1994).
Darwinian evolution requires selection operating
on the phenotype to favor changes in the genotype
which are to be maintained and perpetuated (Gerbi,
1985). The ribosomal DNA (rDNA) repeated units
contain highly conserved DNA sequence as well as
more variable D N A sequence regions and have
been used to detect genetic variation in population
(White et a/., 1990). The products of rDNA are
RNAs, this is one case where evolutionary selection
may act at the RNA level (Gerbi, 1985). In this re-
port, the 5.8s rRNA gene and the noncoding adja-
cent regions were amplified by PCR using the ITS?
and ITS4 primers (White et a/., 1990) and were se-
quenced in both orientation, with ITS1, ITS2, ITS3
and ITS4 primers (White eta/., 1990). Both ITS1 and
ITS4 primers were sequenced completely. We have
sequenced a total of tweleve isolates of B. bassiam
from different geographic areas of the world. The
ITS regions and 5.8s rDNA sequences of B. bassiana
isolate LE73 (from Taiwan) and ARSl51 (from
France) are shown in Figure 1. The overall similarity
of ITS regions and 5.8s rDNA of these two isolates i s
98%. The length of ITS1 region is 167 bp (LE73) and
162 bp (ARSlSl), respectively. The length of ITS2
region is 191 bp in both isolates. The identity of ITS
regions is 91% (ITS1 region) and 100°/~(ITS2 re-
gion). The sequence length of both isolates of 5.8s
rDNA of 8.bassiana is 156 bp and has 98% iden-
tity. We found that 5.8s rDNA of B. bassiana i s
 3 82 H. L.SHIH ETAL.

50
A151 CCTGCCCACCCATCATTACCCACTTTTCAACTCCC. .CA. ACCCTTCTCT
~ l ! i i l ~l l ~ l ~ l l I l i I l l ~ l l l l l ~ I ~ ~l l ll l l : l ' ~l I i l
LE7J CCTCCI;CAGCCATCATTACCCAGTTTTCAACTCCCGTTACACCCTTCTGT
100
4151 (;A ACCTACCTATCCTTCCTTCC(;CCGA(;CTCCCCCCAGCCC(;CACGCCCA
l ~ l ~ ~ ~ ~ ~ ! l l ~~ ~~ ~l l~ l l l i i l i ~! t~ ~ l i ~ l l~ ! ~ I ~ i ~~ ~ ~ ~ l l
LE73 CAACCTACCTATCCTTCCTTCCGCGCA .CTCCCCCCAGCCCCC ACCCCCA
I50
A151 CT(;(;ACCAGC(;CCCC(;CCT(;GCGAC. CTCAAAC. . .TCCTCTATTCCACC
l l l l l l l l l l l l i l l l l ~1 1 1 I I 1 1 1 1'l11~I/l11
LE73 CTI;GACCACC(;CCCCGCCCC(;CACCTCAAACTCTTCTA~CTATTCCACC
200
Mitochondrial DNA Downloaded from informahealthcare.com by AMS-Laguna-Nautica-Es Mar

A151 hTL'TT~T(;AA'TACCCC(;CAACCCAAAAACAAAT(;AAT~AAAA~TTTCAAC
l l l ~ i ' l l l l ' l~ l i l ~ ~ l I l l l l l l l l l l l l l l l l l l l ~ / I l l ~ I
LE7.3 ATCTTCTGAATACCCCGCAACGC.ACAACAAAT(;AATCAAAACTTTCAAC
250
A151 AACCGATCGTCTTCCCTCTGGCATCCATCAACAACGCAGCCAAATCCGAT
!
i , i 1 : I 1 I I I 1 I I I I I I I I I I I I I I I I I I 1.1 I 1 1 I I 1 i I 1 I 1 I 1 I
~

LE73 AACCCATC .TCTT(;CCTCTCCCATCCATCA ACACCGCACC(;AAATCCCAT

300
A151 AA(;TAATGTGAATTCCACAATCCACTGAATCATCCAATCTTTGAACGCAC
l l I I l i l l l ~ l l l l l ~ ~ l l l l l ~ l l l l l l l l ~ l ~ l l ~ l l ~ l l l l l ~ l ~ l
LE73 AACTAATGTCAATTGCACAATCCACTCAATCATCGAATCT~CAAC~CAC
For personal use only.

350
A151 .4.TTGCCCCCCCCACCA~CT(;(;C(;CGCATCCCTGTTCATTTC
~ ll~lllllllllllllll/I:~llllll~l~lllllll~~lill~~ll
LE7G ATTTGCCCCCCCCAGCATTCTCCCGCGCATGCCTCTTCCA~CCT~ATTTC
400
A151 AACCCTC(:ACCTCCCCTTCCCGACCTCCCCCTTCC(;CACC(;(:CA(;CACAC
l " / I ~ l / l / / / l ~ l ~ l ~ I l I I ~ l l l l l i l ~ l l ~ l l l l l i ~ l ~ ~ ~ ~ l ' l I
LEY3 AACCCTC(;ACCTCCCCTTC(;GGA~(;TCCGCCTT(;(;(;GACC(;CCACCACAC
450
A151 CCCCCGCCCTGAAATCCACTCCCGCCCCCTCCCCGGCGACCTCTCCCTAC
l l I I l l l l l l l l l l ~ l ~ ~ l l l ~ ~ ~ l ~ l l l i l l l l l ~ l ~ l l ~ l i ' l l I ! ~ l
LE73 C(;CCCGCCCTGAAATGGAGT(;CC(;GCCCC~CCCCCCCCACCTCTCC~TA(;
500
A151 TAATACACCTCCCACCCC;AACCCC(;ACGC(~CCCAC(
l l , i ' i l l l l l l l l l l ~ l l l l ~ l ~ l l l l l l l l ~ l l l l l ~ l i l l l ' l l l l l
LE73 TAATACACCTCCCACCGCAACCCCGACCCCCCCACCCCCTAAAACACCCA
550
Aljl ACTTCT(;AACCTT(;ACCTCCAATCAGCTACCCTACCCCCTCAACTTAACC
I I I1.i I I I I I i I i I I 1 1 I I I I I I I 1 i i I i I I I I I I I I 1 I I I I I 1 1 I I I I
~

LE7R ACTTCTCAACCTTCACCTCGAATCACCTA(;GCTACCC(;CT(;AACTTAAGC
508
A151 ATATCAAT
liIll!i'
LE73 ATATCAAT

Figure 1 The complete sequence of two clpnes of 19.bassiana (A151, LE73) ITS regions and 5.8s rRNA gene. "." indicates deletion of the
base. Positions 19-187 and 346-537 are ITS1 and ITSII, respectively. Bases 188-305 are 5.8s rDNA gene. Fungal universal primers ITS1
(5'-TCCCTAGCTGAACCTGCGG-3')and ITS4 (5'-TCCTCCGCTTATTGATATCC-3') have been used to amplify ITS regions and 5.8s rDNA
from fungal genomic DNA (200ng) using Polymerase Chain Reaction (PCR). The contained 1OmM Tris-CI, PH 8.3, 50mM KCI, 1.5mM
MgC12, 0.1 % Triton X-100, lOOnM each of dATP, dCTP, dCTP and dTTP, 0.2uM primers, and 1 unit of Super Taq DNA polymerase (HT).
Amplification was performed with hot start at, 94"C, 3 mim, and then 40 cycles of 1 min at 9 4 T , 1 min at 50"C, 2 rnin at 72°C. Amplified
products were sequenced directly on both strands using the dideoxynucleotide chain termination method with primers ITSl, ITS2 (5'-CCT-
CCGTTCT-TCATCCATGC-3'), ITS3 (5'-CCATCCATG AACAACCCACC-3') and ITS4. 35S-dATP was incorporated during sequencing reac-
tion. The sequencing products were separated by 6% polyacrylamide-urea gel electrophoresis and visualized by autoradiography (Kodak
X-OMAT film).
 BEAUVERIA BASSIANA 5.8s RRNA
closely related to Penicillium purpruogenum
(L14506) (92% homology), and Neurospora c r a m
(M13906) (94% homology). The results are not un-
expected, since 5.8s rDNA i s convinced to be
highly conserved (Gerbi, 1985; White eta/., 1990).
(Received 13th January 1995)
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the tomato leaf mould fungus Cladosporium fulvurn syn. Fulvia
Mitochondrial DNA Downloaded from informahealthcare.com by AMS-Laguna-Nautica-Es Mar
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3 83
fulva by analysis of r D N A sequences Curr. Genet. 2 5 ,
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Gerbi, S.A. (1985). Chap. 7, In Molecular Evolutionary Genetics.
p. 419-517, ed. R.J. Maclntype, Plenum, New York.
Mugnai, L., Bridge, P.D. and Evans, H.C. (1989). A chemotaxo-
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Paccola-Meirelles, L.D. and Azevedo, J.L. (1991). Parasexuality
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