Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina Japonica

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing. The total length of 45S rDNA repeat unit of S. japonica was 8995 bp, including 5420 bp of 18s-5.8s-25s rDNA and 3575 bp of IGS (Intergenic Spacer), with the GC content of 51.4%. IGS was composed of 465 bp 3’-outer transcribed spacer (ETS), 874 bp 5’-ETS, and 2236 bp non transcribed spacer (NTS), with the GC content of 50.1%.Fiber-FISH (fiber-fluorescence in situ hybridization, fiber-FISH) analysis of 45S rDNA on the BAC molecule of female gametophytes of S. japonica illustrated that each fiber had at least five continuous moniliform hybridization signal points, indicating the distribution of 45S rDNA repeat unit on the bacterial artificial chromosome. This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S. japonica, and the successful fiber-FISH analysis of 45S rDNA on BAC molecule would contribute to the construction of the physical map and Map-based cloning of this kelp.

Ribosomal DNA intergenic spacer of indoor wood-decay fungi

Indoor wood-decay fungi are economically very important. Approximately half of the total damage caused by indoor fungi in Germany is caused by species of the Coniophoraceae. The sequence of the intergenic spacer (IGS) region of the ribosomal DNA was elucidated with the following fungi of this family:Serpula lacrymans(true dry rot fungus),S. himantioides(wild merulius),Meruliporia incrassata(North American dry rot fungus),Leuco-gyrophana pinastri(mine dry rot fungus),Coniophora puteana(brown cellar fungus), andC. marmorata(marmoreus cellar fungus). The IGS length ranges between 2584 and 3785 bp and consists of the short IGS 1 (253–440 bp), the 5S rDNA (118 bp) and the long IGS 2 (2193–3310 bp). IGS 1 is phylogenetically less informative for the investigated Coniophoraceae species. 5S rDNA is transcribed in the reverse direction. IGS 2 contains extended repeat blocks of copies of different length. Intraspecific length polymorphism as a result of different copy number occurs inM. incrassataandL. pinastri. In combination with previous results, the full-length sequence of the rDNA repeat unit is available for important indoor wood-decay fungi. The various rDNA regions can now be used for future identification of unknown sequences by BLAST and also for phylogenetic studies.

Identification of the Dry Rot Fungus, Serpula lacrymans, and the Wild Merulius, S. himantioides, by Amplified Ribosomal DNA Restriction Analysis (ARDRA)

SummaryIsolates of the dry rot fungus,Serpula lacrymans, and of the morphologically similar wild merulius,S. himantioides, were investigated by amplified ribosomal DNA restriction analysis (ARDRA) of the internal transcribed spacer (ITS) region to prove this method as diagnosis tool for the economically important indoor rot fungi. The technique uses the polymerase chain reaction (PCR) to amplify the relatively variable sequences of the ITS region arranged between the highly conserved portions of the 18S and 28S RNA genes of the nuclear ribosomal DNA (rDNA) repeat unit. Subsequent digestion of the amplicon with restriction endonucleases may exhibit differences at species and subspecies level. Using the universal ITS 1/ITS 4 primer combination, the ITS region of all isolates ofS. lacrymansandS. himantioideswas amplified. The size of the amplified products was about 630bp in both species, as estimated from agarose gel electrophoresis. Digestion of the amplicon with the endonuclease pairsAluI/HhaI andAvaII/MboII, respectively, revealed identical rDNA-ITS fragments for the isolates of both species, indicating their genetic relationship. On the other hand, digestion withBglI/HinfI andHaeIII/TaqI, respectively, separated the fungi by means of different fragment patterns. Thus, ARDRA-ITS proved to be suited for the identification of both fungi.

Sequence and PCR–RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts

The Cryptosporidium ITS1, 5·8S and ITS2 rDNA regions from a number of Cryptosporidium isolates from different hosts and geographical areas were cloned and sequenced in order to investigate the extent of sequence heterogeneity between human and cattle-derived isolates from different geographical locations and also between isolates of Cryptosporidium from different hosts such as cats, pigs, mice and a koala. Calf-derived isolates from different continents were virtually identical as were human-derived isolates from the UK and Australia. Genetic differences between Cryptosporidium isolates were extensive and were in fact greater than the level of nucleotide divergence between Toxoplasma gondii and Neospora caninum rDNA sequences. Based on the sequence information derived from this study, PCR–RFLP of the ITS1 region was undertaken in order to directly amplify and genotype Cryptosporidium isolates from different hosts. This PCR–RFLP approach can now be used for molecular epidemiology studies, circumventing the need for costly sequencing and allowing a wider range of genetically different isolates to be examined.

A rapid means of identifying wild rice species DNA using dot blots and genome-specific rDNA probes

Intergenic spacer fragments from the rDNA repeat unit were isolated from a single accession of each of 9 species that cover the range of genomes found in the Oryza genus (A-F). Seven of the 9 species contained 1 size class of rDNA repeat unit only, while Oryza sativa and Oryza latifolia contained 3 and 2 size classes, respectively, of which fragments were cloned for the major size class only. Oryza australiensis contained an additional BamHI site in the intergenic spacer. Dot blots were prepared and hybridised with a repeat unit from each species. Under high stringency conditions, all probes were specific to species possessing the same genome or genomes.Key words: rDNA, rice, genome-specific, dot blots.

Evolutionary conservation of the transcribed spacer sequences of the rDNA repeat unit in three species of the genus Aspergillus.

We have cloned and sequenced the two intervening transcribed spacers in the rDNA repeat unit of three Aspergillus species--A. nidulans, A. awamori and A. wentii. The A. wentii and A. awamori spacers are almost identical and share a high degree of homology with the A. nidulans spacers. All spacers have a high G-C content (66%-76%) and the potential of forming complex secondary structures, which may indicate that they play a role in the maturation of pre-rRNA molecules.

Ribosomal DNA, peroxidase and extensin variation in genomic DNA of Medicago laciniata (L.) Miller from Australia and Africa

Fifty-four Medicago laciniata (L.) Miller accessions from diverse locations in eastern Australia, Africa and Israel were characterized for genotypic differences by restriction endonuclease cleavage of genomic DNA and probing with radio-labelled DNA sequences to detect restriction fragment length polymorphisms (RFLP). Digestion of the genomic DNA with the restriction endonuclease, Bam HI, and probing with the complete ribosomal DNA (rDNA) repeat unit of soybean (pGmrl) and its subclones revealed that the variable region in M. laciniata spans the Bam H1 sites of the intergenic spacer region between the 18s and 26s DNA. The length variation in the rDNA repeat unit clearly distinguished South African from Australian accessions. The rDNA variants from SA1847 (Morocco) and SA7750 (Tunisia) were the same as the Australian accessions and slightly larger than the rDNA spacer variant unique to SA3428 (Israel). When Eco R1 digested M. laciniata genomic DNA was probed with peroxidase and extensin cDNA clones, all Australian accessions were again characterized by the same RFLP genotype with further differentiation between African and Israeli accessions. An accession of unknown origin, SA3412, possessed the same RFLP genotype as the Australian accessions for all endonuclease and probe combinations. For the African accessions, differences in RFLP genotype were more frequent than differences in phenotype (leaflet laciniation and colour). However, of three South African accessions with the same RFLP genotype, SA26844, SA26847, and SA26848, SA26847 had more pronounced leaflet laciniation.