Genetic aspects of the organization of legumin genes in pea

We have compared physical and genetic maps of the region around the legJ gene in pea. In this vicinity there are four B-type legumin genes, arranged as two close pairs. The detection of a recombination event within this gene cluster allows the orientation of this group of genes within the surrounding linkage group to be determined. The relationship between physical and genetic distances in this region is discussed, as are the implications of this for relating physical and genetic maps elsewhere in the pea genome.     

Foreign DNA introgression caused heritable cytosine demethylation in ribosomal RNA genes of rice

Significant cytosine demethylation in ribosomal RNA genes (18S or 25S) were detected in all four studied rice lines containing introgressed DNA from wild rice, Zizania latifolia Griseb. In each line, the changed RFLP (restriction fragment length polymorphism) patterns produced with the methylation-sensitive enzyme (HpaII) were identical between two randomly selected individual plants both within and between generations. This indicates that the methylation changes are non-random and stably inherited. Cytosine demethylation in ribosomal RNA genes could be a major cause for the drastically altered phenotypic variations observed in the introgression lines.     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

Molecular characterization and identification ofSaprolegnia by restriction analysis of genes coding for ribosomal RNA

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species ofSaprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except forS. hypogyna, S. delica, S. lapponica, andS. mixta. The rDNA data indicate thatS. lapponica andS. mixta are conspecific withS. ferax, whereas there is no support for the proposed synonymies ofS. diclina withS. delica and ofS. mixta withS. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in theS. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains ofS. parasitica originating from the U.K. and Japan.HhaI andBsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonucleaseBstUI was diagnostic forS. parasitica, generating identical fingerprints for all strains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved inS. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed thatS. diclina andS. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification ofSaprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.     

Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis

The time course of replication of the 9 kb ribosomal DNA repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3 end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.     

Ribosomal RNA genes structure in somePopulus spp. (Salicaceae) and their hybrids

The tandemly repeated multigene families encoding 18S and 25S rRNAs were studied at the restriction enzyme level inPopulus alba L.,Populus deltoides Bartr. exMarsh.,Populus trichocarpa Torr. & Gray and in the hybrids between the last two mentioned species. The analysis of single and double digestion with EcoRI, BamHI, XbaI, and SstI endonucleases showed the presence of single repetitive unit types of 12.25 and 11.75kb inP. alba andP. trichocarpa, respectively.P. deltoides showed two rDNA gene types having the same length (12.25Kb) but different nucleotide sequence in the IGS. The rDNAs genes ofP. deltoides andP. triochocarpa are inherited codominantly in their hybrids.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

5S ribosomal gene variation in the soybean and its progenitor

Summary The soybean, Glycine max and its wild progenitor, Glycine soja, have been surveyed for repeat length variation for the nuclearly encoded 5S ribosomal RNA genes. There is little variation among the 33 accessions assayed, with a common repeat length of 345 bases being typical of both taxa. A 334 base size variant was encountered in individuals from two populations of G. soja from China. The low level of variability is in marked contrast to the variation observed within and between the species of the perennial subgenus Glycine.     

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2)

Summary Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S-23S-5S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16S-23S-5S.     

Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas

A given plant species is able to resist most of the potentially pathogenic microorganisms with which it comes in contact. This phenomenon, known as non-host resistance, can be overcome only by a very small number of true pathogens which can use that plant as a host. In some cases, plants have developed mechanisms for overcoming infection by specific races or strains of a true pathogen. This race-specific resistance can be easily manipulated into agronomically important cultivars by plant breeders. We have previously described nine cDNA clones which represent pea genes active during non-host resistance against the fungus Fusarium solani f. sp. phaseoli. In the present work, we have used these cDNAs as probes to compare non-host resistance with race-specific responses of peas against three races of Pseudomonas syringae pv. pisi. Five of the genes most active during non-host resistance were also active in direct correlation with the phenotypic expression of resistance in race-specific reactions of five differential pea cultivars against three races of Pseudomonas syringae pv. pisi.     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads

Summary In cryptomonads, unicellular phototrophic flagellates, the plastid(s) is (are) located in a special narrow compartment which is bordered by two membranes; it harbours neither mitochondria nor Golgi dictyosomes but comprises eukaryotic ribosomes and starch grains together with a small organelle called the nucleomorph. The nucleomorph contains DNA and is surrounded by a double membrane with pores. It is thought to be the vestigial nucleus of a phototrophic eukaryotic endosymbiont. Cryptomonads are therefore supposed to represent an intermediate state in the evolution of complex plastids from endosymbionts. We have succeeded in isolating pure nucleomorph fractions, and can thus provide, using pulsed field gel electrophoresis, polymerase chain reaction and sequence analysis, definitive proof for the eukaryotic nature of the symbiont and its phylogenetic origin.     

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.)

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F₂ progeny of a cross between two cultivated types (Primo and OSU442-15) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (Slow) and a P. sativum ssp. humile accession (JI1794). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the Primo x OSU442-15 cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the JI1794 x Slow cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the Primo x OSU442-15 and JI1794 x Slow crosses.     

Fasciation in pea: Basic principles of morphogenesis

A study of fasciated pea Pisum sativum L. (Fabaceae) mutant Shtambovy in comparison with the wild type (Nemchinovsky cultivar) has shown that fasciation is a result of abnormal cohesion of axial or other structures which arise in a superfluous amount due to uncontrolled meristic processes. In some cases, the organs with the same number and position as in the wild type can be fascinated. Subsequent defasciation and some features of tissue differentiation suggest that the meristem of a fasciated shoot retains a certain degree of discreteness which reflects its complex structure. The number and position of leaves in a node is a function of the diameter of the leaf primordium inhibitory zone, size of the shoot apical meristem, and number of bundles in a shoot. In the absence of the apex proliferative activity combined with the reduction of phyllomes in the upper nodes, abnormal cohesion of the second order axes, racemes, can take place. As a result, inflorescences of special type develop.