Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason w...Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason we tested this region for phylogenetic studies. This report focuses on the study of IGS sequences of isolates belonging to Trichoderma section (T. viride, T. koningii, T. hamatum, T. erinaceus, T. asperellum) and Pachybasium section (T. harzianum, T. crassum, T. fasciculatum, T. oblongisporum, T. virens). Using the primer pair 28STD and CNS1, the Fast Start Taq DNA Polymerase (Roche), and a three temperature PCR protocol, products ranging from ca 1900 to 2400 bp were obtained from all tested isolates. The PCR product of 16 Trichoderma spp. isolates was cloned into a pGEM-TEeasy Vector (Promega) and sequenced. Based on a BLAST search we can conclude that the PCR product represents the whole IGS region. Multiple alignments of IGS sequences revealed two portions with different homology level. Portion A (ca 1660 bp) is the portion that contains 3’ end of 28S gene and is the more variable, while portion B (ca 830 bp), that contains the 3’ end of IGS region and the 5’ end of 18S gene, is the less variable. Comparing all sequences in region A 705 identical pairs occur out of 1704 total nucleotides (41.4%), while in region B identical pairs were 723 out of 832 total nucleotides (86.9%) . Sequence comparison of the two regions at intraspecific level (where it was possible) showed higher variability in region A (0.17%-6.8%) than in region B (0.0%-1.0%) . At interspecific level, performing all possible comparisons, the variability of region A (19.5%-52.7%) and B (0.8%- 16.9%). were significantly higher. Comparing sequences of species belonging to Trichoderma section variability of the two regions appears reduced if compared with that obtained from comparisons of species belonging to Pachybasium section. On the basis of sequence alignment, phylogenetic trees were obtained either with entire IGS, with region A, and with region B. Results of this analysis revealed that all isolates belonging to Trichoderma section grouped separately from isolates belonging to Pachybasium section. IGS region allowed us to group species according to their taxonomic position. The topology of the tree did not change substantially, varying in genetic distance only. Performing a GenBank search sequences representing the final portion of the IGS region of other fungal species were found, and we carried out a multiple alignment using also our sequences of Trichoderma spp. and Diaporthe helianthi. The phylogeny inferred from sequence alignment matched the generally accepted morphology-based classification and was identical to other molecular schemes at high taxonomic level. Data analysis was useful in establishing a broad-scale phylogeny of Ascomycota and was also useful in sorting them into statistically-supported clades. The tree showed that Trichoderma occurred in a well-supported terminal subclade of a larger clade that also contained other genera belonging to Hypocreales order. Sequence analysis of the Trichoderma spp. IGS region allowed us to design a specific PCR primer that was successfully used to amplify region A. The new reverse primer LCR2, that recognize all Trichoderma isolates, was identified in region B and confirmed for its specificity on the DNA of fungi belonging to other Ascomycota genera. Results obtained showed that IGS region seems to be an interesting and versatile tool for phylogenetic analysis, for resolving some taxonomic problems and for constructing specific primer useful for different purposes.展开更多
The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced wi...The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .展开更多
文摘Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason we tested this region for phylogenetic studies. This report focuses on the study of IGS sequences of isolates belonging to Trichoderma section (T. viride, T. koningii, T. hamatum, T. erinaceus, T. asperellum) and Pachybasium section (T. harzianum, T. crassum, T. fasciculatum, T. oblongisporum, T. virens). Using the primer pair 28STD and CNS1, the Fast Start Taq DNA Polymerase (Roche), and a three temperature PCR protocol, products ranging from ca 1900 to 2400 bp were obtained from all tested isolates. The PCR product of 16 Trichoderma spp. isolates was cloned into a pGEM-TEeasy Vector (Promega) and sequenced. Based on a BLAST search we can conclude that the PCR product represents the whole IGS region. Multiple alignments of IGS sequences revealed two portions with different homology level. Portion A (ca 1660 bp) is the portion that contains 3’ end of 28S gene and is the more variable, while portion B (ca 830 bp), that contains the 3’ end of IGS region and the 5’ end of 18S gene, is the less variable. Comparing all sequences in region A 705 identical pairs occur out of 1704 total nucleotides (41.4%), while in region B identical pairs were 723 out of 832 total nucleotides (86.9%) . Sequence comparison of the two regions at intraspecific level (where it was possible) showed higher variability in region A (0.17%-6.8%) than in region B (0.0%-1.0%) . At interspecific level, performing all possible comparisons, the variability of region A (19.5%-52.7%) and B (0.8%- 16.9%). were significantly higher. Comparing sequences of species belonging to Trichoderma section variability of the two regions appears reduced if compared with that obtained from comparisons of species belonging to Pachybasium section. On the basis of sequence alignment, phylogenetic trees were obtained either with entire IGS, with region A, and with region B. Results of this analysis revealed that all isolates belonging to Trichoderma section grouped separately from isolates belonging to Pachybasium section. IGS region allowed us to group species according to their taxonomic position. The topology of the tree did not change substantially, varying in genetic distance only. Performing a GenBank search sequences representing the final portion of the IGS region of other fungal species were found, and we carried out a multiple alignment using also our sequences of Trichoderma spp. and Diaporthe helianthi. The phylogeny inferred from sequence alignment matched the generally accepted morphology-based classification and was identical to other molecular schemes at high taxonomic level. Data analysis was useful in establishing a broad-scale phylogeny of Ascomycota and was also useful in sorting them into statistically-supported clades. The tree showed that Trichoderma occurred in a well-supported terminal subclade of a larger clade that also contained other genera belonging to Hypocreales order. Sequence analysis of the Trichoderma spp. IGS region allowed us to design a specific PCR primer that was successfully used to amplify region A. The new reverse primer LCR2, that recognize all Trichoderma isolates, was identified in region B and confirmed for its specificity on the DNA of fungi belonging to other Ascomycota genera. Results obtained showed that IGS region seems to be an interesting and versatile tool for phylogenetic analysis, for resolving some taxonomic problems and for constructing specific primer useful for different purposes.
基金The project supported by the Grant from Presidentof Chinese Academy of Sciences
文摘The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .
