Variation in the ribosomal DNA intergenic spacer of a maize population mass-selected for high grain yield

Summary Variation in the intergenic spacer of ribosomal DNA (rDNA) was detected among individual plants of the open-pollinated maize variety Hays Golden and populations derived from this variety. rDNA intergenic spacer-length variants were detected at approximately 200 bp intervals, consistent with the number of 200 bp subrepeats as the basis for this variation. Inheritance data revealed that more than one spacer-length class may be present on an individual chromosome. Fourteen different predominant rDNA intergenic spacer hybridization fragment patterns were detected. C-29, a population developed by 29 cycles of mass-selecting Hay Golden for high grain yield, exhibited a significant change in rDNA intergenic spacer hybridization fragment pattern composition in comparison to Hays Golden. This change included a reduction in frequency of the shortest predominant space-length variant (3.4 kb) and an increase in a 5.2 -kb hybridization fragment. I-31, a population developed through thermal neutron irradiation of Hays Golden and 31 generations of mass selection for high grain yield, did not exhibit a significant change in overall rDNA intergenic spacer composition. I-31 did exhibit an increase in frequency of the 5.2-kb hybridization fragment and a significant change in two specific hybridization fragment patterns that had also changed in C-29. These data, particularly for the C-29 population, suggest that rDNA intergenic spacer-length variants and/or associated loci were influenced by selection.Paper No. 8701, Journal Series, Nebraska Agricultural Research Division     

Genetic variability of rumen Selenomonads

Molecular diversity of rumen bacteria belonging to the species Selenomonas ruminantium was evaluated by biochemical and PCR analyses targeted at the 16S rRNA operon and lactate dehydrogenase gene. While extremely variable in metabolic characteristics, two different RISA (ribosomal intergenic spacer analysis), and five lactate dehydrogenase gene RFLP profiles were observed among the twelve strains studied. The strains showed very limited variability ARDRA ( amplified ribosomal DNA restriction analysis) when two different profiles were observed only. 16S rDNA sequence comparisons indicate complex genetic structure within S.ruminantium population.     

Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Heritable somaclonal variation in wild barley (Hordeum spontaneum)

Summary Progenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.     

Distribution of gymnostoma spp. microsymbiotic frankia strains in new caledonia is related to soil type and to host-plant species

The diversity of the Frankia strains that are naturally in symbiosis with plants belonging to the Gymnostoma genus in New Caledonia was investigated. A direct molecular characterization of DNA extracted from nodules was performed, followed by characterization by restriction fragment length polymorphism (RFLP) of the ribosomal rrs-rrl (16S-23S) intergenic spacer (IGS) polymerase chain reaction (PCR)-amplified region. Seventeen different patterns were identified among the 358 microsymbiotic strains studied in the eight species of host plant present in New Caledonia. This genotypical approach permitted us to show that a large diversity existed among the patterns and that these did not exhibit a strict specificity to any host-plant species comparable with that previously found in the Casuarina and Allocasuarina symbioses in Australia. Despite this lack of specificity, a correspondence analysis nevertheless showed that the distribution of these patterns was related to soil type and to host-plant species. Furthermore, several Frankia strains were exclusively associated with the ultramafic soils.     

中国大麦叶绿体DNA和核糖体RNA基因限制性片段长度多型性

本文报道了我们对我国不同大麦区80份大麦品种叶绿体DNA和核糖体RNA基因限制性片段长度多型性的研究。结果表明:rDNA间隔序列长度存在丰富的多样性,80份材料中出现了8种长度变异炎型共组成8种表现型。长度变异类型及其表现型在地理分布上存在着明显的区域性。推测这种分布上的地区性与植物对环境的适应性有关。所用的两个叶绿体DNA克隆片段未检测到限制性片段长度多型性,说明栽培大麦叶绿体DNA变异程度低。     

Pigment and isozyme variation in aspen shoots regenerated from callus culture

Pigment as well as isozyme variations were observed among aspen (Populus tremuloides Michx.) plants regenerated from callus cultures. Out of more than 600 plantlets, two chimeric plants (one with green base and two albino shoots and the other with an albino shoot) were produced. Callus derived from albino shoots produced albino as well as chimeric plants when transferred to shoot inducing medium. Isozyme patterns of 119 plants were examined by starch gel electrophoresis. Thirty plants showed variation in shikimic dehydrogenase isozyme and 41 in isocitric dehydrogenase. Variation was also observed in malate dehydrogenase and phosphoglucose isomerase. No variation was seen in 6-phosphogluconate dehydrogenase. Pigment variation was not associated with any isozyme changes.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - GD Gresshoff & Doy medium - WPM woody plant medium - SKD shikimic dehydrogenase - IDH isocitric dehydrogenase - MDH malate dehydrogenase - PGI phosphoglucose isomerase - 6-PGD 6-phosphogluconate dehydrogenase     

Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants

Sequences of the chloroplast trnC-trnD region and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were obtained for all species of Panax L. (the ginseng plant genus, Araliaceae) to reconstruct phylogenetic relationships. The trnC-trnD phylogeny is congruent with the ITS phylogeny for the diploid taxa of Panax. This study is the first use of the trnC-trnD sequence data for phylogenetic analysis at the interspecific level. We evaluated this DNA region for its phylogenetic utility at the lower taxonomic level for flowering plants. The trnC-trnD region includes the trnC-petN intergenic spacer, the petN gene, the petN-psbM intergenic spacer, the psbM gene, and the psbM-trnD intergenic spacer. The petN and psbM genes are small, 90 and 104-114 bp across angiosperms, respectively, and have conserved sequences. We have designed universal amplification and sequencing primers within these two genes. Using these primers, we have successfully amplified the entire trnC-trnD region for a diversity of flowering plant groups, including Aralia L. (Araliaceae), Calycanthus L. (Calycanthaceae), Corylus L. (Betulaceae), Hamamelis L. (Hamamelidaceae), Hydrocotyle L. (Apiaceae), Illigera Blume (Hernandiaceae), Nelumbo Adans. (Nelumbonaceae), Nolana L. ex L.f. (Solanaceae), Prunus L. (Rosaceae), and Staphylea L. (Staphyleaceae). In Panax, the trnC-trnD region provides a similar number of informative phylogenetic characters as the ITS regions and a slightly higher number of informative characters than the chloroplast ndhF gene. We thus demonstrate the utility of the trnC-trnD region for lower-level phylogenetic studies in flowering plants.     

Fine structure and evolution of the rDNA intergenic spacer in rice and other cereals

Summary The intergenic spacer of a rice ribosomal RNA gene repeating unit has been completely sequenced. The spacer contains three imperfect, direct repeated regions of 264–253 bp, followed by a related but more highly divergent region. Detailed analysis of the sequence allows the presentation of an evolutionary scenario in which the 264–253-bp repeats are derived from an ancestral 150-bp sequence by deletion and amplification. Comparison of the rice sequence with those of maize, wheat, and rye shows that, despite considerable divergence from the ancestral sequence, several regions have been highly conserved, suggesting that they may play an important role in the structure and/or expression of the ribosomal genes.Abbreviations IGS ribosomal gene intergenic spacer - rDNA ribosomal DNA - rRNA ribosomal RNA Offprint requests to: M. Delseny     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

ARISA方法研究产甲烷菌共存及去除条件下瘤胃真菌多样性变化

摘要：【目的】建立厌氧真菌多样性分析方法,并研究厌氧真菌与产甲烷菌共培养液在传代过程中厌氧真菌的区系变化及共培养液中去除产甲烷菌条件下厌氧真菌多样性的变化。【方法】根据厌氧真菌ITS1序列长度多态性,设计厌氧真菌特异性引物,然后PCR扩增样品中厌氧真菌ITS1序列,在基因分析仪中分析PCR产物序列长度多态性,分析共培养液在传代过程中及共培养液中去除产甲烷菌后厌氧真菌多样性的变化。【结果】对瘤胃厌氧真菌Caecomyces属YC301菌株、Neocallimastix属菌株（YC501与YC502）的ARI     

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Structure and evolution of the 4.5-5S ribosomal RNA intergenic region from Glycine max (soya bean).

The nucleotide sequence for the 4.5-5S ribosomal DNA region from the chloroplastids of soya beans was determined as the basis of further comparative studies on the structure and evolution of this intergenic region. Comparisons with other plant sequences as well as equivalent sequences in eubacteria suggest that the longer internal transcribed spacer regions of plants have evolved, at least in part, by DNA sequence duplications and that the presence of the 4.5S rRNA in chloroplast may result from the accidental acquisition of a RNA maturation site during the evolution of longer internal transcribed spacer regions. Estimates of the secondary structures also indicate only a very limited retention of structural features and suggest that the primary role of the intergenic sequences may be to bring processed sites into close proximity.     

Change in ribosomal DNA spacer-length composition in maize recurrent selection populations. 2. Analysis of BS10, BS11, RBS10, and RSSSC

Four maize (Zea mays L.) populations selected for grain yield (BS10, Iowa Two-ear Synthetic; BS11, formerly Pioneer Two-ear Composite; RBS10, Illinois strain of BS10; and RSSSC, Illinois strain of Iowa Stiff Stalk Synthetic) were assayed for molecular variation in the ribosomal DNA (rDNA) intergenic spacer (IGS) at initial and advanced cycles of selection. RSSSC and RBS10 underwent reciprocal recurrent selection with an inbred tester in a high-yield environment, whereas BS10 and BS11 were subjected to full-sib reciprocal recurrent selection. Maize rDNA, which encodes the ribosomal RNA genes, is highly repetitive and shows IGS length variation within and among individuals. Five different ribosomal spacer-length variants (rslvs) and a polymorphic SstI restriction site in the IGS were detected in the four populations. The five rslvs and the polymorphic restriction fragment were observed in 20 different combinations or hybridization fragment patterns (HP). RSSSC, RBS10, and BS11 showed significant changes in the overall rslv and HP frequencies between cycle 0 and the advanced cycle of selection, whereas BS10 did not. In general, two specific HPs were more frequent in the majority of the advanced cycles of the four populations. The frequency changes between initial and advanced cycles were more dramatic for HPs than rslvs. These results are consistent with earlier findings and further support the hypothesis that certain rDNA HPs and/or linked loci may be responding to selection for grain yield and may be associated with a selective advantage in US Corn Belt environments.     

Bacterial communities associated with the rhizosphere of transgenic Bt 176 maize (Zea mays) and its non transgenic counterpart

The effect of transgenic Bt 176 maize on the rhizosphere bacterial community has been studied with a polyphasic approach by comparing the rhizosphere of Bt maize cultivated in greenhouse with that of its non transgenic counterpart grown in the same conditions. In the two plants the bacterial counts of the copiotrophic, oligotrophic and sporeforming bacteria, and the community level catabolic profiling, showed no significant differences; differences between the rhizosphere and bulk soil bacterial communities were evidenced. Automated ribosomal intergenic spacer analysis (ARISA) showed differences also in the rhizosphere communities at different plant ages, as well as between the two plant types. ARISA fingerprinting patterns of soil bacterial communities exposed to root growth solutions, collected from transgenic and non transgenic plants grown in hydroponic conditions, were grouped separately by principal component analysis suggesting that root exudates could determine the selection of different bacterial communities.