Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Different behaviour of Xbb+ and Xbb chromosomes in D. melanogaster with only one nucleolus organizer.

Summary We have examined the rDNA content of male and female adult flies having only one nucleolus organizer (NO), using X chromosomes carrying wild or partially deleted bobbed loci (Xbb ⁺/O, Xbb ⁺/X_NO-and Xbb/O, Xbb/X_NO ^-).The results show that in Xbb ⁺/O and Xbb ⁺/X_NO ^-flies, where only somatic gene compensation is supposed to occur, the rDNA increase, although less pronounced than previously reported, is directly proportional to the number of rRNA genes initially present in the nucleolus organizer. In Xbb/O and in Xbb/X_NO ^-flies the rDNA increase is relatively much higher than that observed in flies carrying bb ⁺ instead of bb. It is suggested that this may be due to rDNA premagnification and somatic gene compensation occurring simultaneously in the former flies.On leave of absence from International Institute of Genetics and Biophysics, Naples, Italy     

Regulation of rRNA gene number in Drosophila melanogaster: new aspects resulting from the use of free duplications

Summary We have isolated a bobbed (bb) mutant on the free duplication Dp(1; f)122bb ⁺ and we have measured the rDNA content of the bb ⁺ and the bb loci in genetic combinations in which none of the phenomena involved in the change of the rDNA redundancy occurs. We have also measured the rDNA content of the two bb loci carried by the free duplications in two different genetic combinations: (1) and females in which there are two attached X chromosomes completely deleted for the nucleolus organizer (NO) regions and there-fore the only rDNA is contributed by the free duplication; (2) X/Dp122bb ⁺ and X/Dp122bb males, in which there are two bb loci, one on the X chromosome and the other on the X free duplication.The bb ⁺ and the bb duplications produced an overall increase of the rDNA content in the two genetic conditions tested.These results are not in favour of both a cis and trans effect of the regulator locus (cr ⁺ locus) hypothesised as being involved in the disproportionate replication of rRNA genes.     

Comparative cytomolecular analyses reveal karyotype variability related to biogeographic and species richness patterns in Bombacoideae (Malvaceae)

Bombacoideae is one out of nine subfamilies of Malvaceae and encompasses 160 tree species. The subfamily is karyotypically characterized by small and numerous chromosomes and is traditionally known by a remarkable inter- and intraspecific chromosome number variation. We conducted a comparative cytogenetic analysis to investigate karyotype diversity and chromosome evolution within Bombacoideae. To achieve this, we performed new chromosome counts, CMA/DAPI double staining, genome size estimations, and localization of 5S and 45S rDNA by fluorescence in situ hybridization for 21 species distributed across the Bombacoideae phylogeny. We performed ancestral states reconstruction analyses to elucidate chromosome evolution and provide insights into the systematics and evolution of Bombacoideae in comparison with other Malvaceae species. Newly generated data on chromosome number on Bombacoideae revealed diploids (Ochroma (2n = 84), Cavanillesia, Pochota, Pseudobombax (2n = 88), and Pachira (2n = 92)) and polyploids (Adansonia digitata (2n = 160) and Eriotheca species (2n = ca. 194 and 2n = 276)). For most species, in situ hybridization revealed karyotype, with two pairs of 45S rDNA sites co-located with CMA⁺ bands, and 5S rDNA sites in only one chromosome pair. Taken together, our results provide support to the hypothesis of karyotypic stability in Bombacoideae. Only the Pachira s.l. clade displayed some variability in ploidy level, number of CMA⁺ bands and 45S rDNA sites, and genome size compared to other Bombacoideae clades. The Striated bark clade was characterized by comparatively small genomes and low cytomolecular variability. Karyotypic data were related to biogeographic and species richness patterns of Bombacoideae.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

Comparative analysis of diploid species of <Emphasis Type="Italic">Avena</Emphasis> L. using cytogenetic and biochemical markers: <Emphasis Type="Italic">Avena canariensis</Emphasis> Baum et Fedak and <Emphasis Type="Italic">A. longiglumis</Emphasis> Dur.

The diploid oat species containing the A genome of two types (Al and Ac) were studied by electrophoresis of grain storage proteins (avenins), chromosome C-banding, and in situ hybridization with probes pTa71 and pTa794. The karyotypes of the studied species displayed similar C-banding patterns but differed in size and morphology of several chromosomes, presumably, resulting from structural rearrangements that took place during the divergence of A genomes from a common ancestor. In situ hybridization demonstrated an identical location of the 45S and 5S rRNA gene loci in Avena canariensis and A. longiglumis similar to that in the A. strigosa genome. However, the 5S rDNA locus in A. longiglumis (5S rDNA1) was considerably decreased in the chromosome 3Al long arm. The analysis demonstrated that these oat species were similar in the avenin component composition, although individual accessions differed in the electrophoretic mobilities of certain components. A considerable similarity of A. canariensis and A. longiglumis to the Avena diploid species carrying the As genome variant was demonstrated.     

Banding and FISH in three species of <Emphasis Type="Italic">Vernonia</Emphasis>, subsection <Emphasis Type="Italic">Macrocephalae</Emphasis> (Asteraceae,Vernonieae)

To complement our knowledge about the karyotypes of the genus Vernonia Schreb., different techniques of chromosome banding, including AgNOR, triple staining with fluorochromes CMA/DA/DAPI (CDD), and fluorescence in situ hybridization (FISH) for the 45S rDNA probe, were applied to three species of subsection Macrocephalae. Vernonia bardanoides was collected from an area of cerrado (savanna) vegetation in Itirapina, São Paulo State, Brazil, and V. linearifolia and V. tomentella were collected from areas of rocky, open altitudinal vegetation in Joaquim Felicio and Diamantina, respectively, in Minas Gerais State. All species showed two terminal CMA⁺ and NOR bands. FISH indicated two terminal 45S rDNA sites in V. linearifolia and V. tomentella, and six in V. bardanoides.     

X-Linkage of Human α-Galactosidase

A deficiency in α-galactosidase (α-Gal) activity–as measured aspecifically with the use of artificial substrates–is a regular feature in leucocytes and fibroblasts of patients affected by angiokeratoma corporis diffusum or Fabry's disease, a well-known X-linked trait in man^1–4. Fibroblast clones derived from mothers of affected males exhibit either normal or deficient activity of α-galactosidase. This demonstrates that the deficiency of α-galactosidase is caused by an X-linked mutation, but does not necessarily prove that the structural locus for this enzyme is itself located on the X-chromosome.     

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

Summary D. melanogaster males of bb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb ⁺/O females, yielding bb/O male progeny, and to X_NO-w sn bb ⁺ fameles, yielding bb/X_NO- females. The male and female offspring were scored for the bb ⁺ phenotype.Results show that there is a higher percentage of bb ⁺ flies in the bb/O male progeny than in bb/X_NO- females progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters.These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.     

<Emphasis Type="Italic">X</Emphasis> Chromosome Inactivation in Cells from an Individual heterozygous for Two <Emphasis Type="Italic">X</Emphasis>-Linked Genes

THE Lyon hypothesis of X chromosome inactivation predicts that in mammalian females, somatic cells are mosaic with respect to whether the active X chromosome is of maternal or paternal origin and that this chromosomal mosaicism is heritable somatically¹. Studies of cell clones derived from females who were heterozygous for genes at one of several X-linked loci^2–6 have provided good evidence for such mosaicism. Proof that only one of the two X chromosomes, however, is active in any given cell rests on the demonstration that the cell or its clone expresses all of the X-linked genes from one parent and none from the other parent. For this purpose it is useful to examine cloned cells from female subjects for genetic markers representing allelic genes at two or more of the parental loci. This study was undertaken to determine whether genes at the X-linked loci for glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK) are consistently expressed in the eis position in cloned cells as would be expected from a single parental contribution.     

Study of the oxidation of alkanes in their mixtures with oxygen and air under the action of a pulsed volume nanosecond discharge

The slow oxidation of alkanes (from methane to hexane) in their stoichiometric mixtures with oxygen or air under the action of nanosecond pulsed discharges was investigated. The discharges were excited in a tube of diameter 5 cm and length of 20 cm by 25-ns voltage pulses with an amplitude of 10 kV and a repetition rate of 40 Hz. The initial pressure in the mixture was varied in the range 0.76–10.1 torr. The current, the electric field strength, and the power deposited in a discharge were measured with a nanosecond time resolution. In time-resolved and time-integrated measurements, the intensities of the following bands were determined: CO ₂ ⁺ (B²Σ → X²Π, δv=0), CH(A²Δ, v′=0 → X²Π, v″=0), OH(A²Σ, v′=0 → X²Π, v″=0), CO(B¹Σ, v′=0 → A¹Π, v″=2), NO(A²Σ → X²Π, δv=3), N₂(C³Π, v′=1 → B³Π, v″=7), N₂(B³Π, v′=6 → A³Σ, v″=3), and N ₂ ⁺ (B²Σ, v′=0 → X²Σ, v″=2). The methane concentration was measured from the absorption of He-Ne laser radiation. Based on the results of optical measurements, the times of the complete oxidation of hydrocarbons were determined.     

Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse

Background

Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F₂ female mice: F₂A ^y mice (F₂ mice with the A ^y allele) and F₂ non-A ^y mice (F₂ mice without the A ^y allele). These were produced by crossing B6 females and DDD.Cg-A ^y males. DDD.Cg-A ^y is a congenic mouse strain for the A ^y allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The A ^y allele is dominant and homozygous lethal; therefore, living A ^y mice are invariably heterozygotes.

Results

Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F₂ non-A ^y mice, and on chromosomes 2, 6, and 12 for F₂A ^y mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8.

Conclusions

The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone. Identifying the genes responsible for the QTLs will be essential for understanding the molecular basis of X-zone function, which is currently unclear.

     

Microwave electrode discharge in nitrogen: Structure and characteristics of the electrode region

The parameters of the electrode region of an electrode microwave discharge in nitrogen are studied by emission spectroscopy. The radial and axial distributions of the intensities of the bands of the second (N₂(C ³Π _u → B ³Π _g )) and first (N₂(B ³Π _g → A ³Σ _u ⁺ )) positive systems of molecular nitrogen and the first negative system of nitrogen ions (N ₂ ⁺ (B ²Σ _u ⁺ → X ²Σ _g ⁺ )), the radial profiles of the electric field E and the electron density N _e , and the absolute populations of the vibrational levels v _C = 0–4 of the C ³Π _u excited state of N₂ and the vibrational level v _Bi = 0 of the B ²Σ _u ⁺ excited state of a molecular nitrogen ion are determined. The population temperature of the first vibrational level T _V of the ground electronic state X ¹Σ _g ⁺ of N₂ and the excitation temperature T _C of the C ³Π _u state in the electrode region of the discharge are measured. The radius of the spherical region and the spatially integrated plasma emission spectra are studied as functions of the incident microwave power and gas pressure. A method for determining the electron density and the microwave field strength from the plasma emission characteristics is described in detail.     

Haploid yeast cells undergo a reversible phenotypic switch associated with chromosome II copy number

Background

SUP35 and SUP45 are essential genes encoding polypeptide chain release factors. However, mutants for these genes may be viable but display pleiotropic phenotypes which include, but are not limited to, nonsense suppressor phenotype due to translation termination defect. [PSI ⁺] prion formation is another Sup35p-associated mechanism leading to nonsense suppression through decreased availability of functional Sup35p. [PSI ⁺] differs from genuine sup35 mutations by the possibility of its elimination and subsequent re-induction. Some suppressor sup35 mutants had also been shown to undergo a reversible phenotypic switch in the opposite direction. This reversible switching had been attributed to a prion termed [ISP ⁺]. However, even though many phenotypic and molecular level features of [ISP ⁺] were revealed, the mechanism behind this phenomenon has not been clearly explained and might be more complex than suggested initially.

Results

Here we took a genomic approach to look into the molecular basis of the difference between the suppressor (Isp^?) and non-suppressor (Isp⁺) phenotypes. We report that the reason for the difference between the Isp⁺ and the Isp^? phenotypes is chromosome II copy number changes and support our finding with showing that these changes are indeed reversible by reproducing the phenotypic switch and tracking karyotypic changes. Finally, we suggest mechanisms that mediate elevation in nonsense suppression efficiency upon amplification of chromosome II and facilitate switching between these states.

Conclusions

(i) In our experimental system, amplification of chromosome II confers nonsense suppressor phenotype and guanidine hydrochloride resistance at the cost of overall decreased viability in rich medium. (ii) SFP1 might represent a novel regulator of chromosome stability, as SFP1 overexpression elevates frequency of the additional chromosome loss in our system. (iii) Prolonged treatment with guanidine hydrochloride leads to selection of resistant isolates, some of which are disomic for chromosome II.

     

Nitrogen Metabolism Genes from Temperate Marine Sediments

In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO₃ ^? was present in the top 5 cm below the sediment-water interface at both sites. NH₄ ⁺ increased with depth below 5 cm where it overlapped with the NO₃ ^? zone. Steady-state modelling of NO₃ ^? and NH₄ ⁺ porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dissimilatory NO₃ ^? reduction to NH₄ ⁺ (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO₃ ^? reduction to NO₂ ^? or NH₄ ⁺ as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.     

Effect of cumulative polymery of tomato keeping life genes

Results from works involving the study of hetero-and homozygous interaction of the keeping quality genes alc, nor, and rin in tomato plants are presented. It is shown that cumulative polymery leading to the formation of a new “long ripening” phenotype is observed in the double heterozygotes alc/alc ⁺//nor/nor ⁺, nor/nor ⁺//rin/rin ⁺, and alc/alc ⁺//rin/rin ⁺. Strong inhibition of processes of ripeness and synthesis of carotenoids occurs with nonallelic interaction in the double homozygotes alc/alc//rin/rin, nor/nor//rin/rin, and alc/alc//nor/nor. This promotes the formation of a new “non-ripening” phenotype with the absence of visual signs of ripeness, i.e., a whitish-green coloring of the fruit.     

Microsatellite based linkage disequilibrium analyses reveal <Emphasis Type="Italic">Saltol</Emphasis> haplotype fragmentation and identify novel QTLs for seedling stage salinity tolerance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A set of 84 diverse rice genotypes were assessed for seedling stage salt tolerance and their genetic diversity using 41 polymorphic SSR markers comprising of 19 Saltol QTL linked and 22 random markers. Phenotypic screening under hydroponics identified three indica landraces (Badami, Shah Pasand and Pechi Badam), two Oryza rufipogon accessions (NKSWR2 and NKSWR17) and one each of Basmati rice (Seond Basmati) and japonica cultivars (Tompha Khau) as salt tolerant, having similar tolerance as of Pokkali and FL478. Among the salt tolerant genotypes, biomass showed positive correlation with shoot fresh weight and negative association with root and shoot Na⁺ content. The results indicated repression of Na⁺ loading within the tolerant plants. Linkage disequilibrium (LD) of the Saltol linked markers was weak, suggestive of high fragmentation of Pokkali haplotype, a result of evolutionary active recombination events. Poor haplotype structure of the Saltol region, may reduce its usefulness in marker assisted breeding programmes, if the target foreground markers chosen are wide apart. LD mapping identified eight robust marker-trait associations (QTLs), of which RM10927 was found linked to root and shoot Na⁺ content and RM10871 with shoot Na⁺/K⁺ ratio. RM271 on chromosome 10, an extra Saltol marker, was found associated to root Na⁺/K⁺ ratio. This marker showed a distinct allele among O. rufipogon accessions. There were also other novel loci detected on chromosomes 2, 5 and 10 influencing salt tolerance in the tested germplasm. Although Saltol remained as the key locus, the role of other genomic regions cannot be neglected in tailoring seedling stage salt tolerance in rice.     

New insights into sex chromosome evolution in anole lizards (Reptilia,Dactyloidae)

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X₁X₁X₂X₂/X₁X₂Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n?=?36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.     

Mapping of novel salt tolerance QTL in an Excalibur × Kukri doubled haploid wheat population

Key message

Novel QTL for salinity tolerance traits have been detected using non-destructive and destructive phenotyping in bread wheat and were shown to be linked to improvements in yield in saline fields.

Abstract

Soil salinity is a major limitation to cereal production. Breeding new salt-tolerant cultivars has the potential to improve cereal crop yields. In this study, a doubled haploid bread wheat mapping population, derived from the bi-parental cross of Excalibur?×?Kukri, was grown in a glasshouse under control and salinity treatments and evaluated using high-throughput non-destructive imaging technology. Quantitative trait locus (QTL) analysis of this population detected multiple QTL under salt and control treatments. Of these, six QTL were detected in the salt treatment including one for maintenance of shoot growth under salinity (QG_(1–5).asl-7A), one for leaf Na⁺ exclusion (QNa.asl-7A) and four for leaf K⁺ accumulation (QK.asl-2B.1, QK.asl-2B.2, QK.asl-5A and QK:Na.asl-6A). The beneficial allele for QG_(1–5).asl-7A (the maintenance of shoot growth under salinity) was present in six out of 44 mainly Australian bread and durum wheat cultivars. The effect of each QTL allele on grain yield was tested in a range of salinity concentrations at three field sites across 2 years. In six out of nine field trials with different levels of salinity stress, lines with alleles for Na⁺ exclusion and/or K⁺ maintenance at three QTL (QNa.asl-7A, QK.asl-2B.2 and QK:Na.asl-6A) excluded more Na⁺ or accumulated more K⁺ compared to lines without these alleles. Importantly, the QK.asl-2B.2 allele for higher K⁺ accumulation was found to be associated with higher grain yield at all field sites. Several alleles at other QTL were associated with higher grain yields at selected field sites.