Sequence analysis of the internal transcribed spacer of ribosomal DNA in the genus rhizopus.

The internal transcribed spacer (ITS) regions including the 3'-end of 18S rRNA gene, 5.8S rRNA gene and the 5'-end of the 28S rRNA gene of Rhizopus spp. were amplified by PCR and analyzed by DNASIS program. Length polymorphism of these region ranged from 564 bp in R. oryzae to 789bp in R. stolonifer. The length and sequence of 5.8S was very conserved with 154~155 bp. The sequence of ITS2 was more variable than that of ITS1. The base substitution rates were ranged from 0 to 0.6069 per site, and higher rate was found in R. stolonifer. In general, transition was usually more frequent than transversion. On the basis of sequencing results, four groups were clustered with value of 61.9% similarity; R. oryzae, R. microspores, R. homothallicus, and R. stolonifer groups.

Rhizopus is a small genus of Zygomycetes (Mucorales). Rhizopus spp. produce a variety of enzymes, proteins, and by-products. About 82 strains have been investigated with very divergent views on the recognition of most taxa by different authors (Shipper, 1984). There are very few molecular techniques currently in use for the Rhizopus, and there is still very little experimental data available.

Molecular techniques are being employed more extensively for determining taxonomic assignments. The use of DNA complementarity studies (Ellis, 1985) and PCR or other molecular techniques are providing the basis for the assignment of fungi into their appropriate class, family, genus, species, and variety (Sutton, 1998).

ITS sequence comparisons are becoming an increasingly popular tool for phylogenetic analysis and for the differentiation of populations. DNA sequencing is generally accepted to be the more reliable method for revealing genetic relationship and could be used to evaluate relationships of organisms at any taxonomic rank. The internal transcribed spacer of rDNA is considered to be a variable region among genera and even among species (Paul, 2002). The degree of variability can be used in the classification of species or varieties (Su et al., 1999). As a preliminary report of this work, PCR-RFLP analysis of twenty strains in Rhizopus spp. showed the polymorphism in length and restriction site of ITS (Park et al., 2003) and a total of five ITS haplotypes were identified among these strains. To compare the result obtained from RFLP loci for the same strains, one or two strains representing the each haplotype were sequencing. For the purpose of clarifying the taxonomic status and genetic relationships which has been issued on the morphological classification, we compared the levels of DNA sequence divergence among the ITS regions of Rhizopus spp.

Materials and Methods

Fungal strains and culture condition

The information for strains of the Rhizopus spp. used this study is given at Table 1. All species were maintained at 4℃ on potato dextrose agar (PDA). Mycelium for DNA extraction was grown in 250 ml of PDB (potato dextrose broth) in a rotary shaker on 180 rpm for 24 h at 28℃. After vacuum filtration, the mycelia were lyophilized, ground with sea sand and stored at -20℃. Genomic DNA for PCR was extracted according to the method of Lee et al. (2000).

Table 1

List of Rhizopus spp. examined in this study

aATCC : American Type Culture Collection, IFO : Institute for Fermentation, Osaka, Japan.

bPark et al., 2003.

Sequencing of entire ITS region

For sequencing, ITS region of the rDNA was amplified using primer ITS1 (5'-TCCGTTGGTGAACCAGCGG-3') and primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al., 1990). Amplification was performed in 100 µl of reaction mixture containing 10 µl of 10 × buffer [500 mM KCl, 100 mM Tris-HCl (pH 9.0), 1% Triton X-100], 2.5 units Ex Taq DNA polymerase (Takara, Japan), 200 µM dNTPs, 2 mM MgCl2, and 10 pmol of both primers, ITS 1 and ITS4. The mixture was subjected to PCR in an MWG-Biotech (Germany). Using a TOPO TA Cloning kit (Invitrogen, USA), the amplified PCR products and the pCR2.1 vector were ligated and the resulting plasmids were introduced into Escherichia coli. Transformed cells were chosen at random from an LB agar plate (to which 50 µg ampicillin had been added), and extracted plasmid by Qiagen mini-prep columns. For sequencing [ABI PRISM® 3100 Genetic Analyzer (Applied Biosystem)], M13 forward and M13 reverse primers were used to amplify the template, and sequencing was carried out using plasmid-specific and insert-specific primers.

Sequences analysis

Sequences analyses were carried out using programmes within DNASIS (version 2.1). Phylogenetic tree was obtained from multiple align plot of ITS sequences among Rhizopus species studied. The sequence identity (%) between species was showed on the tree and the G+C content (%) were calculated. Pair-wise comparisons were calculated from the data matrix using maximum-matching method to generate a similarity matrix among strains. Kimura's two-parameter method was used to calculate the total number of nucleotide substitutions per site (K): K = -0.5 ln [(1 - 2P - Q) √1 - 2Q], where P is the proportion of transitions, and Q is the proportion of transversion (Kimura, 1980). The sequences for all species examined have been deposited in GenBank and taken accession numbers.

Results and Discussion

The ITS sequence of the different strains were PCR amplified using primers with ITS1 and ITS4 primer. The PCR fragments were cloned, and several indepent clones of each strains were sequenced. These revealed a length polymorphism at the sequence level ranged from 564 to 789 bp.

The sizes of th ITS1 and ITS2 regions varied among strains from 199 bp to 292 bp and 190 bp to 343 bp, respectively (Table 2). The length of ITS regions including 5.8S for R. stolonifer was found to be considerably longer (789 bp) than those of other strains.

Table 2

The size (bp) and G+C content (%) of ITS 1, 5.8S and ITS2, respectively. The 3'-end of the 18S gene and 5'-end of the 28S gene was not calculated

The total G+C contents of ITS1 and ITS2 varied between 32.87 to 42.72% in the ITS1 and 23.61 to 41.25% in the ITS2. The spaces of R. stolonifer had a very low G+C content with 32.87% in ITS and 23.61% in ITS2. When the non-coding regions of Zygomycete Mucor miehei were analyzed (Maicas et al., 2000), ITS (198 bp) and ITS2 (255 bp) are A-T rich (66% and 77%, respectively) and ITS1 is longer than the corresponding sequence of other related zygomycota (Maicas et al., 2000). But, in this study, ITS1 and ITS2 of M. miehei is still shorter than those of R. stolonifer. The length and G+C content of 5.8S in M. miehei and genus Rhizopus were very similar; 158 bp long, 40.5 G+C for M. miehei and 154~155 bp long, 39.99~40.89 G+C for the genus Rhizopus. In contrast, G+C contents of ITS region in Fusarium solani showed relatively high G+C contents; 46.70~51.30% in ITS1 region and 52.01~68.96% in ITS2 (Lee et al., 2000). Takamatsu et al. (1998) reported that the secondary structure shown on the ITS region of powdery mildew fungi was supported by high G+C contents. It has been reported that ITS region form secondary structures, which are actively involved in the processing of the primary transcript in yeast (Rau and Planta., 1995). ITS1 is required for the processing of the 3' end of the 18S and the 5' end of the 5.8S molecules, while ITS2 is required for the processing of the 3' end of the 5.8S and the 5' end of the 28S molecules. The yeast results furthermore that the secondary-structure of these spaces is important for the processing reaction (van der Sande et al., 1992). We could detect the restriction sites of restriction enzymes and coincided with ITS-RFLP analysis (Park et al. 2003). We have identified some more highly conserved regions in ITS1 than ITS2 sequence. The 3'-termini of the 18S rRNA sequence from 8 strains showed complete homology. No information about ITS region involving 5.8S sequence from any other Rhizopus species was found in the databases.

The nucleotide sequence data have been lodged in the EMBL nucleotide sequence database under the accession numbers shown at Table 2.

The range of sequence variation was from single and multi base pair change to multiple changes including deletions and insertions. Most variation of sequences in the 5.8S rRNA gene, was shown in R. stolonifer and R. homothallicus. The single base pair changes in the position 346 (T-C), 431 (G-A), 448 and 452 (T-C), 448, 453 (A-G), 464 (C-T) in R. stolonifer and in the position 405 (deletion), 448 (T-C), 452 (T-C), 473 (T-C) in R. homothallicus were observed. In general, transition was usually more frequent than transversion.

We have used the alignment to calculate the average number of nucleotide substitutions per site for the whole spacer sequences. This was done in pair wise sequence comparisons by using the method of Kimura (1980). The ITS is useful for examining the rates of base substitution among closly related taxa and for studying fixation by molecular drive. The average number of substitutions is apparently correlated with the evolutionary distance of the species within the Drosophila melanogaster species group. In Drosophila, average base substitution number per site for seven species was ranging from 0.014 to 0.433 (Scholtterer et al., 1994). In Rhizopus, the base substitution number per site was ranged from 0 to 0.6069 (Table 3). Substitution number between R. microsporous and R. oryzae 4772 and those of between R. sexualis 42542 and R. oryzae 24794 showed zero. The highst was found in R. stolonifer which is biggest species in the genus Rhizopus. Unfortunately, we could not compare with related data about other fungi.

Table 3

Matrices for the average numbers of substitutions per site

Number: 1. R. microsporus var. oligosporus 22959, 2. R. oryzae 4772, 3. R. oryzae 22580, 4. R. oryzae 11145, 5. R. sexualis 42542, 6. R. oryzae 24794, 7. R. homothallicus 42221, 8. R. stolonifer 6227b.

For getting the similarity among these strains, the maximum-matching method on the DNASIS program was used (Table 4). The Similarity of 5.8S rDNA was 95~100%, whereas those of ITS1 and ITS2 were 50~99% and 39~100% respectively. It was proved that the 5.8S rRNA gene was highly conserved among strains, whereas ITS region was variable.

Table 4

Nucleotide sequence identities among each strains using Maximum matching program

Number : 1. R. oryzae 22580, 2. R. oryzae 11145, 3. R. oryzae 24794, 4. R. sexualis 42542, 5. R. homothallicus, 6. R. oryzae 4772, 7. R. microsporus var. oligosporus 22959, 8. R. stolonifer 6227b.

The phylogenetic tree obtained from multi-alignment plot was shown in Fig. 1. The cluster analysis separated the 8 strains into four major groups based on above 61.9% similarity; R. oryzae, R. microspores, R. homothallicus and R. stolonifer groups. The results of cluster analysis were coincided with those of PCR-RFLP analysis. The strains belonging to haplotypes I and II showed 99.8% similarity. R. stolonifer 42221 showed lowest similarity 47.9%, with other strains on tree. The similarity between R. micro var. oligo and R. homothallicus was 61.9%.

Fig. 1

The phylogenetic tree based on the nucleotide sequence multialignment of the ITS region of the Rhizopus spp. Numbers on the tree nodes indicate percentage of the similarity.

When taxonomical study of the genus Rhizopus was made by examining the morphological, cultural and physiological characteristics of 449 culture strains, Inui et al. (1965) divided the genus Rhizopus into 4 section, 13 species and one new variety; section Sexualis, section Nigricans (referred to stolonifer), section Oryzae and section Chinensis.

Schipper (1984) did not accept the taxonomic decisions in the monograph of the genus by Inui et al. (1965). Instead, Schipper divided the genus Rhizopus into R. stolonifer, R. oryzae and R. microspous groups. According to Schipper (1984), the homothallic species R. sexualis belonging to R. stolonifer group resembles R. stolonifer in the shape and size of its sporangiospores, but differs in having relatively small sporangia and small sporangiophores. In our study, R. sexuals ATCC 42542 was distantly related to R. stolonfer and showed above 99.8% similarity with R. oryzae. R. microsporus revised by Schipper and Stalpers (1984) was recognized in two species, R. homothallicus and R. microsporus, the latter with three additional varieties : var. chinenesis, var. oligosporus and var. rhizopodiformis. And, judging from their general morphology and maximum growth temperature, R. homothallicus and R. microsporus are closely related. But, R. homothallicus and R. microsporus var. oligosorus were separated in cluster analysis in our results.

As a results of this study, R. sexualis ATCC 42542 and R. oryzae IFO 4772 would be revised. And the genus Rhizopus might be divided into four groups; R. oryzae, R. microsporous, R. homothallicus and R. stolonifers groups at the level of ITS sequence. Due to the lack of molecular taxonomic information about the genus Rhizopus, the information provided by these studies will be useful in evaluating the existing taxonomy of Rhizopus.