A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60 000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

Involvement of polyamines in the post-anthesis development of inferior and superior spikelets in rice

Early-flowered superior spikelets usually exhibit a faster grain filling rate and heavier grain weight than late-flowered inferior spikelets in rice (Oryza sativa L.). But the intrinsic factors responsible for the variations between the two types of spikelets are unclear. This study investigated whether and how polyamines (PAs) are involved in regulating post-anthesis development of rice spikelets. Six rice genotypes differing in grain filling rate were field grown, and PA levels and activities of the enzymes involved in PA biosynthesis were measured in both superior and inferior spikelets. The results showed that superior spikelets exhibited higher levels of free spermidine (Spd) and free spermine (Spm) and higher activities of arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and Spd synthase (EC 2.5.1.16) than inferior spikelets at the early endosperm cell division and grain filling stage. The maximum concentrations of free Spd and free Spm and the maximum activities of ADC, SAMDC and Spd synthase were significantly correlated with the maximum cell division and grain filling rates, maximum cell number and grain weight. Application of Spd and Spm to panicles resulted in significantly higher rates of endosperm cell division and grain filling in inferior spikelets along with the activities of sucrose synthase (EC 2.4.1.13), ADP glucose pyrophosphorylase (EC 2.7.7.27) and soluble starch synthase (EC 2.4.1.21), suggesting that these PAs are involved in the sucrose-starch metabolic pathway. The results indicate that the poor development of inferior spikelets is attributed, at least partly, to the low PA level and its low biosynthetic activity.     

Identification of gibberellins in the rice plant and quantitative changes of gibberellin A19 throughout its life cycle

The major endogenous gibberellin (GA) in shoots, roots and ears of the rice plant, Oryza sativa L. japonica cv. Nihonbare, was identified as GA₁₉ by combined gas liquid chromatography-mass spectrometry (GC-MS) and GC-selected ion current monitoring (GC-SICM). Another GA present in these tissues in small quantity was tentatively identified as GA₁ by GC-SICM, and GA₄ may be present in the seeds (kernels) of 3rd-leaf-stage seedlings. Using GC-SICM, the GA₁₉ content was quantified throughout the life cycle of rice plants. It was found to reach high levels (ca. 10–15 g/kg fresh weight) in 3rd-leaf seedlings, at panicle initiation (shoots), and during heading and anthesis (ears). The levels of GA₁₉ in Oryza sativa indica cv. T-136 underwent changes closely similar to those found in Nihonbare. The growth-promoting activity in rice of exogenous GA₁₉ is generally considerably less than that of GA₁. It therefore seems possible that GA₁₉ functions as a pool GA. The level of active GAs such as GA₁ may be regulated by the rate of biosynthesis of GA₁₉ or its metabolic conversions.Abbreviations GA(s) gibberellin(s) - GA_n gibberellin A_n - GA_n-MeTMS trimethylsilyl ether of GA_n methyl ester - GC-MS combined gas liquid chromatography-mass spectrometry - GC-SICM combined gas liquid chromatography-selected ion current monitoring - TLC thin-layer chromatography     

Identification of repeat structure in large genomes using repeat probability clouds

The identification of repeat structure in eukaryotic genomes can be time-consuming and difficult because of the large amount of information (3 × 10⁹ bp) that needs to be processed and compared. We introduce a new approach based on exact word counts to evaluate, de novo, the repeat structure present within large eukaryotic genomes. This approach avoids sequence alignment and similarity search, two of the most time-consuming components of traditional methods for repeat identification. Algorithms were implemented to efficiently calculate exact counts for any length oligonucleotide in large genomes. Based on these oligonucleotide counts, oligonucleotide excess probability clouds, or “P-clouds,” were constructed. P-clouds are composed of clusters of related oligonucleotides that occur, as a group, more often than expected by chance. After construction, P-clouds were mapped back onto the genome, and regions of high P-cloud density were identified as repetitive regions based on a sliding window approach. This efficient method is capable of analyzing the repeat content of the entire human genome on a single desktop computer in less than half a day, at least 10-fold faster than current approaches. The predicted repetitive regions strongly overlap with known repeat elements as well as other repetitive regions such as gene families, pseudogenes, and segmental duplicons. This method should be extremely useful as a tool for use in de novo identification of repeat structure in large newly sequenced genomes.     

Polysomes and intracisternal accumulations in enucleate sieve elements of rice (Oryza sativa L.)

Polysomes in sieve elements of rice (Oryza sativa L.) were studied with the electron microscope. The polysomes were found on the rough endoplasmic reticulum (ER) present in immature sieve elements and also on the cisternae of aggregated ER in the parietal layer of mature, enucleate sieve elements. In the immature sieve elements the ER cisternae existed as narrow profiles while in the mature sieve elements the ER cisternae were considerably dilated and contained a fibrillar material and, occasionally, electron-opaque inclusions. In addition to the aggregated ER, single profiles of ER were found applied to the lateral walls and also the sieve plates. These cisternae also bore ribosomes and were separated from the plasmalemma by a narrow, dense space. In the mature sieve elements much of the surface of the ER membranes was covered with polysomes. The dimensions of the polysomes are described and the possibility that they contribute to the formation of the fibrillar material in the intracisternal space is discussed.Abbreviations ER endoplasmic reticulum     

Organization of DNA sequences highly repeated in tandem in rice genomes

Digestion of the total genomic DNA from rice Oryza sativa L. cv. C5924 with EcoRI generated an intense band of a DNA fragment of about 0.36 kb long. The DNA fragment cloned into pUC19 was used to hybridize with the total rice genomic DNA partially digested with EcoRI. A ladder of bands of DNA fragments with multiplied length of 0.36 kb was observed, demonstrating that this sequence occurs in tandem in the genome. The copy number of the sequence estimated by dot blot hybridization analysis was 2000-3000 copies per haploid genome from callus or seedling of C5924. This sequence was present in other O. sativa cultivars, such as Sasanishiki in 700-900 copies, Koshihikari in 3400-4300, and Nipponbare in 4600-6000 copies. Another rice species, O. glaberrima, also had this sequence in 540-680 copies, but four lines of foxtail millet had none. The DNA fragments containing the repeated sequences in Nipponbare were then cloned into lambda EMBL3, and sequences of nine units consecutively repeated and an AT-rich sequence connected with them in a phage clone could be determined. Each repeating unit showed sequence divergency mostly by substitution of bases in a range from 3% to 7%, when compared with a 355-bp consensus sequence. Analyses of the substituted bases indicate that these are due to spontaneous mutations which occurred at random, after reiteration of a unit sequence by unequal crossing over events. Gene conversion within the repeated sequences might have further diversified their sequences.     

Inter-simple-sequence repeat (ISSR)-PCR for the identification of saprophytic strains of Leptospira

The inter-simple-sequence repeat (ISSR) primers that anneal to a simple repeat of various length and at non-repetitive motifs at 3 and 5 end were attempted for PCR amplification of Leptospira genome. Of the six ISSR primers tested, namely, (AG)₈T, (AG)₈C, (AG)₈G, (CA)₈A, (TG)₈C and (TG)₈G, only primer (AG)₈T produced amplification of 1000 bp in the two non-pathogenic Leptospira species tested, viz; Leptospira biflexa serovar patoc and L. meyeri serovar ranarum, with no amplification in any of the 16 standard pathogenic serovars tested. The remaining five ISSR primers did not exhibit any amplification of the Leptospira genome in either pathogenic or non-pathogenic species. From among 35 Leptospira isolates recovered from hospitalized patients with pyrexia of unknown origin and/or febrile jaundice (12 in number) and from different environmental water sources (23 in number), (AG)₈T ISSR-PCR correctly identified all the 22 isolates from water sources that were confirmed to be non-pathogenic by conventional tests. The results therefore, confirmed the ability of a primer, based on simple-sequence repeat motif, to produce a fragment that is useful as a group genetic marker in Leptospira species. The single nucleotide anchor, T, at the 3 end of the primer appeared to play an important role in differentiation of pathogenic and non-pathogenic species of Leptospira. Multiplex PCR, using ISSR primer, (AG)₈T and the reported 16S rRNA gene primers, specific for pathogenic Leptospira species, or the 23S rRNA Leptospira genus specific primers, provided clear identification of serovars and isolates into pathogenic or non-pathogenic groups.     

Amplification and dispersion of repeated DNA sequences in theTriticeae

Four representatives of a family of dispersed repetitive sequences which were prominent and dispersed in the E genome ofThinopyrum elongatum but poorly represented in wheat, were studied in detail. The 1.4kb sequences were present both as part of tandem and more complex arrays and appeared to have resulted from repeated amplification of the sequence and their dispersion throughout the genome. Subcloning of sections of the 1.4 kb sequences resulted in probes which improved the resolution of the E genome from the genomes in wheat and enabled identification of single E genome chromosomes introduced into wheat. The generality of these types of sequences in the tribeTriticeae was confirmed by isolating analogous sequences from the R (rye,Secale cereale), V (Dasypyrum villosum), and N (Psathyrostachys juncea) genomes. — The cloned repetitive sequences from the R, V, and N genomes each showed characteristic fluctuations in amount within the grasses examined in addition to being virtually absent from wheat. It is thus possible that these sequences may provide useful taxonomic indicators for establishing relationships within theTriticeae, as well as valuable probes for tracing alien chromatin introduced into wheat.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Submergence tolerance in relation to variable floodwater conditions in rice

Flash floods adversely affect rice productivity in vast areas of rainfed lowlands in South and Southeast Asia and tropical Africa. Tolerant landraces that withstand submergence for 1–2 weeks were identified; however, incorporation of tolerance into modern high-yielding varieties through conventional breeding methods has been slow because of the complexity of both the tolerance phenotype and floodwater conditions, and the ensuing discrepancies encountered upon phenotyping in different environments. Designing an effective phenotyping strategy requires a thorough understanding of the specific floodwater characteristics that most likely affect survival during flooding. We investigated the implications of floodwater temperature and light penetration, caused by artificial shading, seasonal variation, or water turbidity, for seedling survival after submergence. Three field experiments were conducted using rice genotypes contrasting in their tolerance of submergence: FR13A and Kusuma (tolerant); Gangasiuli (intermediate); Sabita, CRK-2-6 and Raghukunwar (elongating/avoiding types); and IR42 (sensitive). We tested the hypotheses that warmer floodwater decreases plant survival and that turbid water augment plant mortality by causing effects similar to those caused by shading, by reducing light penetration. Plants survive better when water is cooler, and survival decreased at about 8% per unit increase in water temperature above 26 °C. Lower intensity of light and warmer temperatures seem to reduce biomass and increase mortality under flooding. An increase in the concentrations of O₂ and CO₂ and a decrease in water pH did not improve survival in clear unshaded water. Turbid floodwater was more damaging to rice as plant mortality increased as the percentage of silt increased, and the effects of water turbidity cannot be explained by the reduction in light penetration alone. Even the most tolerant rice cultivar, FR13A, experienced higher mortality when flooded with turbid floodwater. Correlation studies revealed that cultivars with the capacity to maintain higher biomass, higher chlorophyll, and non-structural carbohydrate concentrations after submergence had higher survival. These findings help to understand the variation observed in submergence tolerance when screening is done under different environments. The study could have implications for designing proper screening strategies and assessing the damage submergence causes across different rice-growing regions.     

Growth responses of rice seedlings to triacontanol in light and dark

Triacontanol, a 30-carbon primary alcohol, applied in nutrient culture solutions to rice (Oryza sativa L.) seedlings at 2.3×10^-8 M (10 g/l), caused an increase in dry weight and leaf area of the whole plants. The response could be observed as early as 3 h of treatment. It was observed at relatively high and low light intensities as well as in the dark where control plants lost but triacontanol-treated plants gained in dry weight. The dry weight gain in the dark was, however, eliminated by removing CO₂ from the atmosphere. Triacontanol-treated plants also increased their content of Kjeldahl-N and contained 30% more total N per plant than controls after 6 h in the dark.     

The occurrence of Ds-like sequences in cereal genomes

Summary The occurrence of DNA sequences similar to the Ds-element of sh-m5933 maize (Ds-like sequences) was studied in other representatives of the Gramineae. The approximate number of copies of such sequences found under gentle and stringent conditions of washing was determined by dot-hybridization. It was shown that in the maize genome the number of copies of Ds-like sequences exceeds about ten-fold the content of such sequences found in wheat, rye and barley genomes. Quantitative differences in Ds-like sequences between wheat species with various genomes and ploidies (when estimated per genome) as well as between different H. vulgare varieties was not determined. The various melting points (T_m) of DNA-duplexes formed when the Ds-element is hybridized with wheat, rye and barley DNA respectively do not show significant differences and are essentially lower than the T_m of the Ds-element (by 8°–9°C). Thus, these duplexes have 9–11% of nucleotide substitutions in comparison to Ds sh-m5933. The data obtained permit one to suppose the presence of a series of Ds-like sequences heterogenous for the length and degree of homology to the Ds-element isolated from the shrunken locus (sh-m5933) of maize DNA.     

Influence of low irradiance on chloroplast proteins and photosystem activities in rice cultivars

In six rice cultivars the relative amounts of 55, 26–28 and 15 kDa polypeptides were mostly lower in plants grown for 15 d under low irradiance than in plants grown under saturating irradiance. This decline in the polypeptides, especially in those related to light-harvesting chlorophyll proteins, was accompanied with a decrease in the whole chain electron transport and the photosystem 2 activity.