I. THE EXPRESSION OF INSTABILITY IN N. TABACUM × N. PLUMBAGINIFOLIA

Moav , Rom (Hebrew U., Jerusalem), and D. R. Cameron . Genetic instability in Nicotiana hybrids. I. The expression of instability in N. tabacum × N. plumbaginifolia. Amer. Jour. Bot. 47(2): 87—93. 1960.—N. tabacum (n = 24) and N. plumbaginifolia (n=10) are distantly related species both from morphological and cytological points of view. Hybrids of these species with various genome dosages have exhibited somatic variegation when plumbaginifolia dominant characters were superimposed on an appropriate tabacum genetic background. Five loci were studied in this respect: Wh and Tg—for flower coloration; Ws—for chlorophyll production; Kl—for pollen abortion and Bs—for black shank resistance. All 5 were found to be unstable. Backcross progenies of the sesquidiploid hybrid (tbc-tbc-pbg) to tabacum showed a marked increase in intensity of variegation. This has been attributed to the breaking up of the plumbaginifolia genome into individual chromosomes. The evidence indicates that variegation was due to somatic chromosomal aberrations which probably characterized all the plumbaginifolia chromosomes. An hypothesis regarding the heterogeneity of F₁ hybrids of distantly related homozygous species is outlined and the occurrence of instability due to hybridization in other Nicotiana hybrids is discussed.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin. Received: 1 July 1998 / Accepted: 7 September 1998     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

Chromosomal structure is altered by mutations that suppress or enhance position effect variegation

We examined the genetic, morphological, and molecular effects of position effect variegation inDrosophila, and the effects of mutations that either suppress [Su(var)] or enhance [E(var)] this phenomenon. All eightSu(var) mutations examined strongly suppress the inactivation of variegating alleles of the genes white [In(l) w ^m4 ], brown [In (2R)bw ^VDe2 ] and Stubble [T(2;3)Sb ^V ]. TheE(var) mutation enhances variegation of these loci. The chromosomal region 3C-E (26 bands) which includes the white locus is usually packaged as heterochromatin in salivary glands of the variegating strainw ^m4 . Addition of any of theSu(var) mutations restores a more euchromatic morphology to this region. In situ hybridization to polytene chromosomes and DNA blot analyses of gene copy number demonstrate that the DNA of thew ⁺ gene is less accessible to its probe in the variegatingw ^m4 strain than it is in the wildtype or variegation-suppressed strains. Blot analysis of larval salivary gland DNA indicates that the white gene copy number does not vary among the strains. Hence, the differences in binding of thew ⁺ gene probe in the variegating and variegation-suppressed strains reflect differences in chromosomal packaging rather than alterations in gene number. The effects of variegation and theSu(var) mutations on chromatin structure were analyzed further by DNAse I digestion and DNA blot hybridization. In contrast to their dramatic effects on chromosomal morphology and gene expression, theSu(var) mutations had negligible effects on nuclease sensitivity of the white gene chromatin. We suggest that the changes in gene expression resulting from position effect variegation and the action of theSu(var) mutations involve alterations in chromosomal packaging.     

Phyletic and evolutionary relationships ofBrachyscome lineariloba (Compositae)

A comparison of karyotypes ofBrachyscome breviscapis (2n = 8),B. lineariloba cytodemes E (2n = 10), B (2n = 12) and C (2n = 16) suggests that these species have a homoelogous basic set of four chromosome pairs, two large pairs and two small, and that theB. lineariloba cytodemes E, B and C are related toB. breviscapis by successive additions of small chromosomes. A pronounced asynchrony of chromosome condensation between these large and small chromosomes has been observed. In the artificial hybrids betweenB. dichromosomatica (2n = 4) ×B. breviscapis, and theB. lineariloba cytodemes, theB. dichromosomatica chromosomes are similar in size and condensation behaviour to the small chromosomes ofB. breviscapis and ofB. lineariloba cytodemes E, B and C. Meiotic pairing in these hybrids also demonstrates the strong affinities between these chromosomes. It is suggested thatB. breviscapis may be of amphidiploid origin between a species with two large early condensing chromosome pairs and another,B. dichromosomatica-like species with two small late condensing pairs. It seems most likely that the additional small and late condensing chromosomes inB. lineariloba cytodemes E, B and C are derived from theB. dichromosomatica-like parent, and that each addition increases vigour, fecundity and drought tolerance, allowing these cytodemes to colonize more open and arid environments. Transmission of the univalents in the quasidiploidB. lineariloba cytodeme E was verified as being via the pollen, and not via the embryo sacs.The cytology ofBrachyscome lineariloba (Compositae, Asteroidae), 10.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture. Received: 17 November 1999; in revised form: 3 February 2000 / Accepted: 3 February 2000     

Comparison of the Giemsa C-banded and N-banded karyotypes of twoElymus species,E. dentatus andE. glaucescens (Poaceae: Triticeae)

The karyotypes ofElymus dentatus from Kashmir andE. glaucescens from Tierra del Fuego, both carrying genomesS andH, were investigated by C- and N-banding. Both taxa had 2n = 4x = 28. The karyotype ofE. dentatus was symmetrical with large chromosomes. It had 18 metacentric, four submetacentric and six satellited chromosomes. The karyotype ofE. glaucescens resembled that ofE. dentatus, but a satellited chromosome pair was replaced by a morphologically similar, non-satellited pair. The C-banding patterns of both species had from one to five conspicuous and a few inconspicuous bands per chromosome. N-banding differentiated the chromosomes of the constituent genomes by producing bands in theH genome only. TheS genomes of both species were similar with five metacentric and two satellited chromosomes having most conspicuous C-bands at telomeric and distal positions. They resembled theS genome of the genusPseudoroegneria. TheH genomes had four similar metacentric and two submetacentric chromosomes. The seventhH genome chromosome ofE. dentatus was satellited, that ofE. glaucescens nonsatellited, but otherwise morphologically similar. The C-bands were distributed at no preferential positions. TheH genome ofE. dentatus resembles theH genomes of some diploidHordeum taxa.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

The karyotype and meiosis of the 12-ploid plants—one of the offspring of the natural F₁ hybrid (Aster ageratoides subsp.ovatus (2n=36) ×Kalimeris incisa (2n=72), 2n=72)—were examined. The 2n=108 chromosomes of the 12-ploids were found to consist of 18 large chromosomes and 90 small chromosomes. In meiosis of the PMCs of the 12-ploid, chromosome configurations of 3_III+46_II+7_I, 2_III+48_II+6_I and 3_III+47_II+5_I were observed. All the univalents and trivalents were small, and among the 46–48 bivalents nine were large and the remaining 37–39 were comparatively small. The large bivalents were found to represent autosyndetic pairings, and the small bivalents and trivalents were probably formed by autosyndetic pairings. The large chromosomes of the 12-ploids were found to coincide with the large chromosomes ofovatus, and the 90 small chromosomes to correspond to small chromosomes ofovatus andK. incisa. The 12-ploids were concluded to have been produced by a fusion of an unreduced gamete of the F₁ plant and a reduced gamete ofK. incisa which was growing in proximity to the F₁s. Thus the 12-ploids were regarded to be an amphidiploid having 36 chromosomes ofovatus and 72 chromosomes ofK. incisa.     

Characterization of aLycopersicon esculentum xSolanum lycopersicoides somatic hybrid lacking a glutamate oxaloacetate transaminase isozyme

Morphological, cytological, isozyme and chloroplast DNA analyses were used to determine possible mechanism(s) for the loss of glutamate oxaloacetate transaminase-4 (GOT-4) isozyme activity in a somatic hybrid. Plant 204-1, derived by cell fusion between tomato (Lycopersicon esculentum) andSolanum lycopersicoides, was characterized for bothGot-4 and acid phosphatase-2 (Aps-2), two isozyme loci which are closely linked (recombination 2.5 cM). This hybrid was determined to be chimeric for bothGot-4 andAps-2. TheS. lycopersicoides plant used to provide cells for the fusion was determined to be heterozygous for bothGot-4 andAps-2. Only oneS. lycopersicoides allelic form ofAps-2 andGot-4 was found in plant 204-1. This observation indicated that either the alternative copy of theS. lycopersicoides chromosome region encodingGot-4 andAps-2 is deleted or the entire chromosome is absent. Plant 204-1 was cytologically determined to be aneuploid with approximately 62 chromosomes. Sixty-two somatic hybrids of separate callus origin were analysed for GOT-4 and a high proportion (27%) lacked theS. lycopersicoides form ofGot-4. The loss of this allele and the linkedAps allele most likely occurred in the suspension culture ofS. lycopersicoides used to provide cells for fusion.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

Natural intergeneric hybrids betweenAster ageratoides subsp.ovatus (2n=36) andKalimeris incisa (2n=72) were found. All of the hybrids studied were found to have 2n=72, 18 more chromosomes than a regular F₁ hybrid. The hybrids were found to be of two types: one having 18 large chromosomes ofovatus, and the other having 9 large chromosomes of the same subspecies. In meiosis of the PMCs of the hybrid with 18 large chromosomes, a regular chromosome configuration, 36_II, was observed. In PMCs of the hybrid with 9 large chromosomes an irregularity of chromosome pairings was observed, showing varied chromosome configurations: 35_II+2_I, 34_II+4_I, 33_II+6_I, I_III+33_II+3_I, 1_IV+32_II+4_I, 32_II+8_I, 31_II+10_I, 29_II+14_I, 3_III+29_II+5_I. Most univalents were large, but a few were small. The hybrids with 18 large chromosomes were found to be partial amphidiploid and possessing double chromosome complements ofovatus. The hybrids with 9 large chromosomes were found to be the first backcrossed generation between the hybrid with 18 large chromosomes andK. incisa.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

Intergeneric hybrids ofAster ageratoides subsp.ovatus (2n=36)×Kalimeris pinnatifida (2n=18) were produced artificially. The chromosomes of the hybrid were found to be 2n=27, and to consist of 9 large chromosomes and 18 small chromosomes. In meiosis of the PMCs of the hybrid, a chromosome configuration of 9_II+9_I was regularly observed. While all the univalents were large, and all the bivalents were comparatively small. The large and small chromosomes ofA. ageratoides subsp.ovatus were found to be rather distant in homology, and the small chromosomes of the subspecies and the chromosomes ofK. pinnatifida were found to have a high degree of homology. The tetraploidovatus was concluded to be an amphidiploid, composed of the large chromosomes ofAster and the small chromosomes ofKalimeris.     

Hybridization of chromosome-polymorphic populations of the inquiline ant,Doronomyrmex kutteri (Hym., Formicidae)

Summary The workerless, inquiline ant,Doronomyrmex kutteri has isolated populations with a haploid chromosome number ofn=23 both in the Alps (Swiss and South Tyrolean Alps) and in Sweden, and a population withn=25 in southern Germany. Crossbreeding of sexuals from all populations proved successful. Backcrosses of F1-females with males from the parental populations produced F2-females, and hybrid males withn=23, 24, or 25 chromosomes. The chromosome polymorphism is not due to B-chromosomes. Probably then=25 karyotype originated from then=23 karyotype by two Robertsonian fissions (2 ¯M 4 ¯A), since then=25 karyotype was found in only one of the populations. Diploid males occurred frequently in colonies from four out of five sites investigated.     

Isolation and chromosomal distribution of a novel Ty1-copia-like sequence fromSecale, which enables identification of wheat—Secale africanum introgression lines

A repetitive sequence of 411 bp, named pSaO5₄₁₁, was identified in theSecale africanum genome (R^a) by random amplified polymorphic DNA (RAPD) analysis of wheat and wheat—S. africanum amphiploids. GenBank BLAST search revealed that the sequence of pSaO5₄₁₁ was highly homologous to a part of a Ty1-copia retrotransposon. Fluorescence in situ hybridization (FISH) analyses indicated that pSaO5₄₁₁ was significantly hybridized toS. africanum chromosomes of a wheat—S. africanum amphiploid, and it was dispersed along theSecale chromosome arms except the terminal regions. Basing on the sequence of pSaO5₄₁₁, a pair of sequence-characterized amplified region (SCAR) primers were designed, and the resultant SCAR marker was able to target both cultivated rye and the wildSecale species, which also enabled to identify effectively theS. africanum chromatin introduced into the wheat genome.     

Elements causing hybrid dysgenesis on the second chromosome ofDrosophila melanogaster

Summary Hybrid dysgenesis inDrosophila melanogaster is a syndrome of germline abnormalities including temperature-dependent gonadal dysgenesis (GD sterility), high rates of mutation and male recombination. In theP-M system, hybrid dysgenesis results from interaction between chromosomally-linked factors (P factors) and a particular extrachromosomal state refered to as theM cytotype. TheT007/Cy strain, shown by other authors to induce a high level of mutation and male recombination, is presently studied with respect to gonadal dysgenesis. TheP activity appears mainly linked with theT007 second chromosome and has been essentially mapped to a 0.6 centimorgan long interval, i.e. betweenhk andpr. On the other hand, 14 strains balanced for deficiencies on the left arm of the second chromosome are studied for their relative level ofM cytotype activity.In F1 females, inheriting the same maternal cytotype and the same paternalT007 chromosome, significant differences inGD sterility are found between flies receiving the maternal deficiency and those receiving the alternate non-deleted chromosome. This effect appears only when the chromosomes are deleted for a common region (37F5-38A7), suggesting the presence of elements intervening in the determinism ofGD sterility in this zone. As this region is included in the correspondinghk-pr interval (37C1-38B6), these results state the problem of the nature of the elements located in this interval and two hypotheses are discussed.     

Aluminum effects on embryo suspensor polytene chromosomes of Phaseolus coccineus L

Aluminum (Al) represents a widespread environmental pollutant, with severe toxic impacts on plants. In this study, we documented for the first time the structural and functional responses induced by two concentrations of AlCl₃ (10^?2 M and 10^?1 M) in the polytene chromosomes that characterize the chromatin organization in the embryo suspensor cells of Phaseolus coccineus. Polytene chromosomes showed signs of dose-dependent genotoxicity following AlCl₃ treatments with a significant increase in both chromatin stickiness and chromatin fragmentation. Polytene chromosomes specifically reacted to AlCl₃ also in terms of DNA and RNA puffing activity: with respect to the control, the treatments promoted ex-novo and/or inhibited puff formation along chromosome arms, suggesting a fine modulation of the differential genome activity in response to the treatments. The nuclei of suspensors from control and treated seeds showed nucleoli mainly arranged by more than one NOR-bearing chromosome. In addition, AlCl₃ treatments affected the frequency of nucleoli organized by singular organizer chromosomes, with an increase in the frequencies of nucleoli organized by chromosome II and a reduction in the frequencies of those organized by chromosomes I or V. These results confirm that, also in our system, nucleolus may react as stress response organelle.     

The karyotype ofFestucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

The karyotypes of two populations ofFestucopsis serpentini (2n = 2x = 14) endemic to Albania were investigated in detail by Giemsa C- and N-banding, AgNO₃ staining, and in situ hybridization with an rDNA probe. The complements consisted of 14 large chromosomes, 10 metacentric and 4 SAT-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming activity was established through AgNO₃ staining; however, the rDNA probe additionally hybridized to intercalary positions in the short arms of two metacentric chromosomes revealing two inactive rDNA sites. C-banding patterns comprised from zero and up to four very small to larger, generally telomeric bands per chromosome giving low levels of constitutive heterochromatin. Similarities in chromosome morphology and C-banding patterns identified the homologous relationships of all chromosomes in one population, but of three pairs only in the other. Reliable identification of homologous chromosomes between plants was only possible for the SAT-chromosomes. A comparison between the C-banded karyotypes ofF. serpentini andPeridictyon sanctum supports their position in two genera.     

Chromosome banding and pairing behaviour inFestuca andVulpia (Poaceae,Pooideae)

Giemsa C-banding patterns of the grassesFestuca rubra (2n = 6x = 42),Vulpia fasciculata (2n = 4x = 28) and their wild F1 hybrid ×Festulpia hubbardii (2n = 5x = 35) show marked differences between the chromosomes of the two parents that enable unequivocal identification ofFestuca andVulpia chromosomes in the hybrid. Moreover, meiotic banding patterns reveal that both homogenetic (Festuca-Festuca andVulpia-Vulpia) and heterogenetic (Festuca-Vulpia) pairing occurs in the F1. The significance of this in relation to the formation of backcrosses to theFestuca parent and to the evolution of theFestuca polyploid complex in general is discussed.