Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune

The common split-gilled mushroom Schizophyllum commune is found throughout the world on woody substrates. This study addresses the dispersal and population structure of this fungal species by studying the phylogeny and evolutionary dynamics of ribosomal DNA (rDNA) spacer regions. Extensive sampling (n = 195) of sequences of the intergenic spacer region (IGS1) revealed a large number of unique haplotypes (n = 143). The phylogeny of these IGS1 sequences revealed strong geographic patterns and supported three evolutionarily distinct lineages within the global population. The same three geographic lineages were found in phylogenetic analysis of both other rDNA spacer regions (IGS2 and ITS). However, nested clade analysis of the IGS1 phylogeny suggested the population structure of S. commune has undergone recent changes, such as a long distance colonization of western North America from Europe as well as a recent range expansion in the Caribbean. Among all spacer regions, variation in length and nucleotide sequence was observed between but not within the tandem rDNA repeats (arrays). This pattern is consistent with strong within-array and weak among-array homogenizing forces. We present evidence for the suppression of recombination between rDNA arrays on homologous chromosomes that may account for this pattern of concerted evolution.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Ribosomal DNA, species structure, and biogeography of the cactophilic yeast Clavispora opuntiae

The ribosomal DNA of the cactophilic yeast species Clavispora opuntiae was studied in order to clarify the global distribution of the yeast. Over 500 strains, including isolates from several new localities worldwide, were characterized by rDNA restriction mapping. An unusual restriction pattern previously encountered only in one strain, from Conception Island in the Bahamas, was found in several Brazilian isolates. Sequences of the D1/D2 and D7/D8 divergent domains of the large subunit (LSU) and of the intergenic spacers (IGS) confirmed that these strains represent a genetically distinct variety of Clavispora opuntiae. This divergence had previously been hypothesized on the basis of reduced genetic recombination in inter-varietal crosses and the presence of a polymorphic ApaI restriction site located in the LSU. The exact position of the ApaI site in the D8 divergent domain and the nature of the variation that it reveals were determined. The complete sequences of 12 intergenic spacers clarified the significance of the species-wide variation uncovered by restriction mapping. Most of the polymorphic sites occur in the IGS1 and IGS2 regions, on either side of the 5S gene, and the variation is largely due to differences in the numbers and the sequences of internal repeats. Two other polymorphic sites are located in the external transcribed spacer (ETS) region. The reliability of various sites as indicators of overall spacer sequence divergence differed from one case to another. Variety-specific probes were devised and used to screen 120 strains for the presence of recombinant rDNA spacers. Three strains gave ambiguous results, but these did not constitute evidence that inter-varietal recombination has taken place in nature. The hypothesis that the global movement of Clavispora opuntiae has been influenced by the worldwide biological control of prickly pear with Cactoblastis cactorum, a moth of Argentinian origin, has received additional support from the demonstration that Argentinian strains have rDNAs similar to those found where the moth has been introduced. A dramatic founder effect was identified in a yeast population collected in cacti (Maui, Hawaii) in a site where the moth had been recently introduced.     

Plant rDNA: organisation,evolution, and using

Ribosomal DNA comprises a considerable part of a plant genome and is organized in tandemly arranged repeats composed of conservative coding sequences for ribosomal RNA and rapidly evolving spacer elements. We determined the nucleotide sequences of intergenic spacer regions (IGS) for five species from Solanacaea family: Solanum tuberosum, Atropa belladonna, Nicotiana tabacum, N. tomentosiformis, and N. sylvestris. The detailed comparative analysis of these and some other rDNA sequences allowed us to reveal the general regularities of evolution and functional organization of the rDNA spacer region and to clarify better phylogenetic relationships between the species within Solanacea family. A large body of experimental data on the application of rDNA in plant breeding, taxonomical studies and biotechnology are provided and discussed.     

Assessment of rDNA IGS as a molecular marker in the Simulium damnosum complex

For five cytospecies of the Simulium damnosum Theobald complex of blackflies (Diptera: Simuliidae) from West Africa, both ends of the intergenic spacer region (IGS) of the rDNA have been sequenced with the aim of developing specific molecular markers. No specific differences in these two regions were detected between Simulium sanctipauli V. & D., Simulium sirbanum V. & D., Simulium soubrense V. & D., Simulium squamosum Enderlein and Simulium yahense V. & D., except in the number of A subrepeats at the 5' end of the IGS (two in S. squamosum and four or five in the others) and in position 310 of the 3' end (a C in S. squamosum and a G in the others). However, genetic distances within and between species overlapped. These DNA sequences had no strong phylogenetic signal, and the trees obtained were mostly unresolved. Although most sequences from S. squamosum clustered together, a few of them were more similar to those in other cytospecies. These results could be explained either by hybridization with genetic introgression or by ancestral polymorphism and recent speciation.     

Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba

Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.by D. Schweizer     

A comparative study of vicilin genes in Lens: negative evidence of concerted evolution

Genes for vicilin, a component of legume seed storage proteins, have been identified in the cultivated lentil (Lens culinaris ssp. culinaris) and in wild species of the genus Lens. Five different types of vicilin sequences (designated A-E) have been identified in each lentil individual. The different types of sequences, and some possible variants of them (also present in each individual) are part of the vicilin family of genes. Type D sequences have the characteristics of nonprocessed pseudogenes. Comparison of nucleotide sequences indicates that lentil vicilin sequences are similar to vicilin sequences of other legume species, in particular to those of the tribe Vicieae, in which the genes Lens is included. Sequence comparison and distance and parsimony trees indicated that two groups or subfamilies of sequences, including, respectively, types A, B, and E (47 kDa vicilins) and types C and D (50 kDa), can be distinguished in lentil and other Vicieae species, and that in the Vicieae species there is no evidence of concerted evolution among the vicilin sequences of different gene subfamilies or sequences groups, as has been suggested for other legume species.      

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.     

阴道分离因肯毛孢子菌的分子鉴定和种内分型

分别采用rRNA基因内转录间隔区(ITS)和基因间隔区(IGS1)测序,ITS和IGS1区PCR限制性片段长度多态性分析(PCR-RFLP)和基因组DNA的随机扩增多态性DNA(RAPD)等方法,对三株因肯毛孢子菌Trichosporon inkin进行分子特征及种内分型研究。结果显示,不同菌株的rRNA基因ITS区和IGS1区的序列相似性均高达100%,RFLP酶切图谱具有较理想的种内一致性,而不同菌株的RAPD图谱不尽相同。研究表明:rRNA基因IGS1区测序及RFLP酶切可考虑用于因肯毛孢子菌的菌种分子鉴定,而基因组DNA的RAPD则较适合于菌种的种内分型。     

Asterotremella gen. nov. albida, an anamorphic tremelloid yeast isolated from the agarics Asterophora lycoperdoides and Asterophora parasitica

Using a genotypic approach (PCR-fingerprinting, DNA/DNA reassociation, partial sequences of the 26S rDNA gene, complete sequences of the 18S rDNA gene, and sequences of the internal transcribed spacers) five tremelloid yeast isolates from the agarics Asterophora lycoperdoides and A. parasitica were shown to be conspecific with Cryptococcus ramirezgomezianus. It was not possible to distinguish the yeast strains from A. lycoperdoides and A. parasitica using sequences from the intergenic spacer (IGS1). Phylogeny based on the 26S (D1/D2-domain), ITS1-5.8S-ITS2 and complete 18S rDNA demonstrated that C. ramirezgomezianus is closely related to several additional Cryptococcus species (C. humicola, C. longus, C. musci, C. pseudolongus) within the Trichosporonales. A new genus, Asterotremella, and a new family, Asterotremellaceae were introduced for Cryptococcus species clustering within the Trichosporonales having a ubiquinone Q-9. Cryptococcus ramirezgomezianus is a synonym of Asterotremella albida.