Nuclear genome characterization and carrageenan analysis ofGymnogongrus griffithsiae (Rhodophyta) from North Carolina

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inGymnogongrus griffithsiae (Gigartinales, Rhodophyta). Results indicate the presence of three second order components corresponding to fast (3%), intermediate (8%) and slow (89%) fractions. Thus the genome consists mainly of unique sequences. Thermal denaturation (T _m) indicated a nuclear DNA base pair composition of 40 mol% G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporoblastic phases. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.32 pg/2 C genome forGymnogongrus griffithsiae. Karyological studies using aceto-orcein revealed the presence of ca. 23 bivalents during diakinesis of tetrasporangial mother cells. Total carrageenan content in water extraction was 30% dry weight. Infrared spectroscopy confirmed the isolated carrageenan to be the iota-fraction.     

Karyology,nuclear genome quantification and characterization of the carrageenophytesEucheuma andKappaphycus (Gigartinales)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteKappaphycus alvarezii. Results indicate the presence of three second order components corresponding to fast (12%), intermediate (38%) and slow (50%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Comparison of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in pg/2C genome estimates:Eucheuma denticulatum=0.35,E. isiforme=0.44,Kappaphycus alvarezii=0.32 andK. striatum=0.42. Karyological studies of tetraspore mother cells during diakinesis using aceto-orcein revealed a chromosome complement of 10 forEucheuma denticulatum andKappaphycus alvarezii.     

Nuclear genome characterization of the carrageenophyteHypnea musciformis (Rhodophyta)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in the carrageenophyteHypnea musciformis (Gigartinales, Rhodophyta). Results indicate the presence of three second order components corresponding to fast (2%), intermediate (33%) and slow (65%) fractions. Microspectrophotometry with the DNA-localizing fluorochrome DAPI confirmed ploidy level differences in the gametophytic and tetrasporophytic phases. Elevated (endopolyploid) nuclear DNA levels were observed in both gametophytic and cystocarpic tissue. Comparison of mean nuclear DNA (If) values to chicken erthrocytes (RBC) resulted in an estimate of 0.22 pg/2 C genome forHypnea musciformis. Karyological studies using aceto-orcein revealed a chromosome complement of five bivalents during diakinesis of tetraspore mother cells.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity.Author for correspondence     

Agar Analysis,nuclear genome quantification and characterization of four agarophytes (Gracilaria) from the Mexican Gulf Coast

DNA reassociation kinetics were used to determine nuclear genome organization and complexity in four species of Gracilaria (Gracilariales, Rhodophyta). In Gracilaria tikvahiae, G. caudata, G. cervicornis and G. divaricata, results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–46% and unique DNA ranged from 45–78%, Thermal denaturation (T _m) indicated guanine + cytosine (G + C) levels of 41.9–46.0 mol % G + C. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA content. Comparisons of mean nuclear DNA (I _f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.37–0.40 pg/2C genomes for the four Gracilaria species. Total agar content following alkaline pretreatment ranged from 7–15% dry weight. Gel strengths were generally below commercial levels, ranging from 40–260 g cm⁻² Nuclear genome profiles developed from information for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity as previously speculated.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

Chloroplast DNA insertions into the nuclear genome of rice: the genes,sites and ages of insertion involved

Rice (Oryza sativa) is one of three predominant grain crops, and its nuclear and organelle genomes have been sequenced. Following genome analysis revealed many exchanges of DNA sequences between the nuclear and organelle genomes. In this study, a total of 45 chloroplast DNA insertions more than 2 kb in length were detected in rice nuclear genome. A homologous recombination mechanism is expected for those chloroplast insertions with high similarity between their flanking sequences. Only five chloroplast insertions with high sequence similarity between two flanking sequences from an insertion were found in the 45 insertions, suggesting that rice might follow the non-homologous end-joining (NHEJ) repair of double-stranded breaks mechanism, which is suggested to be common to all eukaryotes. Our studies indicate that the most chloroplast insertions occurred at a nuclear region characterized by a sharp change of repetitive sequence density. One potential explanation is that regions such as this might be susceptible target sites or “hotspots” of DNA damage. Our results also suggest that the insertion of retrotransposon elements or non-chloroplast DNA into chloroplast DNA insertions may contribute significantly to their fragmentation process. Moreover, based on chloroplast insertions in nuclear genomes of two subspecies (indica and japonica) of cultivated rice, our results strongly suggest that they diverged during 0.06–0.22 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Ligation-mediated suppression-PCR as a powerful tool to analyse nuclear gene sequences in the green alga Chlamydomonas reinhardtii

To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches. The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3′ rapid amplification of cDNA ends (3′ RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of Chlamydomonas reinhardtii. This revised version was published online in June 2006 with corrections to the Cover Date.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Nuclear DNA content of Vitis species,cultivars, and other genera of the Vitaceae

The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.     

DNA base composition heterogeneity in some agarophytes (Gracilariales,Rhodophyta) from Mexico and the Philippines

Estimates of nuclear DNA base composition by determination of thermal denaturation temperatures (T_m) indicate guanine + cytosine (G + C) levels of 35.4–46.8% for ten species of the Gracilariaceae, representing the generaGracilaria andHydropuntia. T_m values were found to be reproducible with variation among most samples and replicates of less than 1 °C and 2 mol%. Interspecific variation in G + C values was less than 11.4% amongGracilaria species. Calculation of intragenomic base pair composition distribution based on mid-resolution thermal denaturation (A 1 °C/min with 4s interval H and dT logging) indicated an inverse relationship between maximum similarity values and taxonomic rank. Intraspecific (population level) maximum similarity (homology) values were estimated to range from 79–90% inGracilaria tikvahiae (4 isolates). Interspecific values of 46–69% were found in 13 species ofGracilaria. Nucleotide distribution similarity values for the Gracilariaceae are compared with previous information for genome organization and complexity, genome size and karyotype patterns.Author for correspondence     

Interspecific hybridization inBrassica: Application of flow cytometry for analysis of ploidy and genome composition in hybrid plants

Interspecific hybrids from the crosses betweenBrassica campestris, B. carinata, B. juncea andB. napus were obtained throughin vitro ovary and ovule culture. F₁ hybrids were studied morphologically and flow cytometry was used to estimate 2C nuclear DNA content both in parentalBrassica species and their hybrids. It was found that in comparison with the A genome, the B and the C genomes ofBrassica contained 26.9 % and 43.9 % more DNA, respectively. This finding may be used to distinguish interspecific hybrids containing various genome combinations. It was concluded that flow cytometric analysis of nuclear DNA content might be useful tool inBrassica breeding.     

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.     

cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (-subunit) of F₁-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species

The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58%±0.56 (SE) in S. oleracea leaves and 4.97%±0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r₍₁₉₎=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication.Abbreviations ctDNA chloroplast DNA - nDNA nuclear DNA - RuBPCase ribulose-1,5-bisphosphate carboxylase - DAPI 4,6-diamidine-2-phenylindole     

Nuclear DNA changes within Helianthus annuus L.: cytophotometric,karyological and biochemical analyses

Summary Cytophotometric measurement of the root meristems of seedlings after Feulgen-staining reveals that large differences (up to 58.16%) in nuclear DNA content may occur in the thirty-one cultivated varieties or lines of Helianthus annuus tested. Significant variations (not exceeding 25%) in the amount of DNA, which does not differ between the root and the shoot meristems of a single seedling, are also found to exist within cultivars or lines; even seedlings obtained from seeds collected from different portions of single heads of plants belonging to a selfed line may vary one from the other in this respect. Variations in the number of chromosomes or alterations in the chromosome structure do not account for the differences observed in nuclear DNA content. Karyometric analyses demonstrate that the surface area of squashed interphase nuclei and metaphase chromosomes and the total length of the latter increase with the increase in Feulgen/DNA absorption. DNA thermal denaturation and reassociation kinetics indicate that a frequency variation in repeated DNA sequences goes hand in hand with changes in the size of the genome. These results, supporting the concept that a plant genome is highly flexible, are discussed in relation to other data to be found in the literature on the intraspecific variation in the nuclear DNA content and in relation to the way in which it is produced in H. annuus.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.