Probes pTa71 Research Articles

Identification and comparison of individual chromosomes of three accessions of Hordeum chilense, Hordeum vulgare, and Triticum aestivum by FISH.

Karyotypes of three accessions of Hordeum chilense (H1, H16, and H7), Hordeum vulgare, and Triticum aestivum were characterized by physical mapping of several repetitive sequences. A total of 14 repetitive sequences were used as probes for fluorescence in situ hybridization (FISH) with the aim of identifying inter- and intraspecies polymorphisms. The (AG)12 and 4P6 probes only produced hybridization signals in wheat, the BAC7 probe only hybridized to the centromeric region of H. vulgare, and the pSc119.2 probe hybridized to both wheat and H. chilense, but not to H. vulgare. The remaining repetitive sequences used in this study produced a hybridization signal in all the genotypes. Probes pAs1, pTa-535, pTa71, CCS1, and CRW were much conserved, showing no significant polymorphism among the genotypes studied. Probes GAA, (AAC)5, (CTA)5, HvT01, and pTa794 produced the most different hybridization pattern. We identified large polymorphisms in the three accessions of H. chilense studied, supporting the proposal of the existence of different groups inside species of H. chilense. The set of probes described in this work allowed the identification of every single chromosome in all three species, providing a complete cytogenetic karyotype of H. chilense, H. vulgare, and T. aestivum chromosomes, which could be useful in wheat and tritordeum breeding programs.

Nucleolar activity and physical location of ribosomal DNA loci in Vitis vinifera L. by silver staining and sequential FISH

Abstract Vitis vinifera L. is one of the most important fruit crops in the world, having thousands of different varieties. This diploid species has 38 small chromosomes that have been characterised with Giemsa staining, fluorochrome banding, silver staining, and fluorescence in situ hybridisation (FISH). Previous works reported that the number of ribosomal DNA (rDNA) loci in V. vinifera is four. In this work, we intend to study the nucleolar activity and to physically map the 45S rDNA loci in seven varieties of V. vinifera using an optimized protocol of silver nitrate staining and sequential FISH, respectively. Nucleoli and mitotic chromosomes of root-tips and leaves of the seven V. vinifera varieties were stained with silver nitrate. The number of nucleoli per interphase cell was scored and statistically analysed. Most of the varieties showed a maximum of three nucleoli per nucleus and three Ag-NORs per metaphase. The variety Bastardo showed a maximum of two nucleoli per nucleus in leaf tissue whereas Pinot Noir presented a maximum of four nucleoli per interphase in roots. However, in both tissues of all varieties, 1 nucleolus per nucleus was most frequent. High-quality chromosome spreads were selected for sequential FISH with the 45S rDNA probe pTa71 which allowed the physical location of four 45S rDNA loci in all varieties.

Molecular Cytogenetic Analysis of Deschampsia antarctica Desv. (Poaceae), Maritime Antarctic.

Deschampsia antarctica Desv. (Poaceae) (2n = 26) is one of the two vascular plants adapted to the harshest environment of the Antarctic. Although the species is a valuable model for study of environmental stress tolerance in plants, its karyotype is still poorly investigated. We firstly conducted a comprehensive molecular cytogenetic analysis of D. antarctica collected on four islands of the Maritime Antarctic. D. antarctica karyotypes were studied by Giemsa C- and DAPI/C-banding, Ag-NOR staining, multicolour fluorescence in situ hybridization with repeated DNA probes (pTa71, pTa794, telomere repeats, pSc119.2, pAs1) and the GAA simple sequence repeat probe. We also performed sequential rapid in situ hybridization with genomic DNA of D. caespitosa. Two chromosome pairs bearing transcriptionally active 45S rDNA loci and five pairs with 5S rDNA sites were detected. A weak intercalary site of telomere repeats was revealed on the largest chromosome in addition to telomere hybridization signals at terminal positions. This fact confirms indirectly the hypothesis that chromosome fusion might have been the cause of the unusual for cereals chromosome number in this species. Based on patterns of distribution of the examined molecular cytogenetic markers, all chromosomes in karyotypes were identified, and chromosome idiograms of D. antarctica were constructed. B chromosomes were found in most karyotypes of plants from Darboux Island. A mixoploid plant with mainly triploid cells bearing a Robertsonian rearrangement was detected among typical diploid specimens from Great Jalour Island. The karyotype variability found in D. antarctica is probably an expression of genome instability induced by environmental stress factors. The differences in C-banding patterns and in chromosome distribution of rDNA loci as well as homologous highly repeated DNA sequences detected between genomes of D. antarctica and its related species D. caespitosa indicate that genome reorganization involving coding and noncoding repeated DNA sequences had occurred during the divergence of these species.

Analysis of chromosomal polymorphism in barley (Hordeum vulgare L. ssp. vulgare) and between H. vulgare and H. chilense using three-color fluorescence in situ hybridization (FISH)

The aim of the present work was to study chromosomal polymorphism within cultivated barley (Hordeum vulgare ssp. vulgare) using three-color fluorescence in situ hybridization (FISH). The physical distribution of the most frequently used, highly repetitive DNA sequences (GAA)7 specific for pericentromeric heterochromatic regions, the ribosomal DNA clone pTa71, specific for the 45S rDNA, and the barley-specific telomere-associated sequence HvT01, was investigated to reveal genetic diversity in metaphase spreads of ten barley genotypes with diverse geographical origin, growth habit and row number. A wild relative of barley, Hordeum chilense was also studied in order to compare the polymorphism between and within Hordeum species. Significant differences in the hybridization patterns of all three DNA probes could be detected between the two related species, but only probes pTa71 and HvT01 showed variation in the intensity and/or position of hybridization sites among genotypes of H. vulgare ssp. vulgare. The extent of polymorphism was less than that earlier reported for molecular markers and was restricted to the long chromosome arms, with differences between the chromosomes. 1H and 3H proved to be the most variable chromosomes and 4H and 6H the most conserved.

Characterization of a 5HS-7DS.7DL wheat-barley translocation line and physical mapping of the 7D chromosome using SSR markers

A spontaneous wheat-barley translocation line was previously detected in the progenies of the Mv9kr1 × 'Igri' wheat-barley hybrid and the translocation was identified as 5HS-7DS.7DL. Multicolor genomic in situ hybridization (mcGISH) with D and H genomic DNA probes and three-color fluorescence in situ hybridization (FISH) with repetitive DNA probes (Afa-family, pSc119.2, and pTa71) were performed to characterize the rearranged chromosome. The effect of 5HS and the deleted 7DS fragment on the morphological traits (plant height, fertility, yield, and spike characteristics) of wheat was assessed. Despite the non-compensating nature of the translocation, the plants showed good viability. The aim of the study was to physically localize SSR markers to the telomeric and subtelomeric regions of the 7DS chromosome arm. Of the 45 microsatellite markers analyzed, ten (Xbarc0184, Xwmc0506, Xgdm0130, Xgwm0735, Xgwm1258, Xgwm1123, Xgwm1250, Xgwm1055, Xgwm1220, and Xgwm0635) failed to amplify any 7DS-specific fragments, signaling the elimination of a short chromosome segment in the telomeric region. The breakpoint of the 5HS-7DS.7DL translocation appeared to be more distal than that of reported deletion lines, which provides a new physical landmark for future deletion mapping studies.

Chromosomal characterization in native populations of Elymus scabrifolius from Argentina through classical and molecular cytogenetics (FISH–GISH)

The karyotype of Elymus scabrifolius (Döll) J.H. Hunz. (2n = 4x = 28) was investigated by DAPI staining and in situ hybridization. All the accessions studied presented a symmetric and uniform karyotype constituted by 9m+2m-sm+3sm. DAPI stain showed 1-7 conspicuous bands in all the chromosomes and polymorphisms between accessions. FISH experiments carried out with 45S rDNA as probe (pTa71) showed strong hybridization signals on the metacentric SAT-chromosome pair 8; the submetacentric SAT-chromosome pair 13 presented weaker hybridization. FISH using pSc119.2 clone as probe identified five chromosome pairs. Then, the combination of chromosome morphology, DAPI-staining, and FISH enabled the accurate identification of each chromosome pair in E. scabrifolius. Genomic in situ hybridization (GISH) experiments using Hordeum DNA as probe on mitotic metaphases confirmed unequivocally the presence of the H genome in E.scabrifolius, allowing us to observe six uniformly labeled chromosome pairs and two chromosome pairs with only one arm labeled. The remaining six chromosome pairs were weakly labeled. The rehybridization of FISH slides with Hordeum DNA as probe allow us to assign the genomic provenance of most of the chromosomes in the studied accessions. Moreover, intergenomic rearrangement was detected between genome H and the still unknown progenitor genome.

Connection between rod bivalents and incomplete meiotic association at NORs in Hordeum marinum Huds.

The hypothesis of a connection between rod bivalent formation and incomplete meiotic association at NORs of SAT-chromosomes of H. marinum is supported. PMCs of H. marinum ssp. gussoneanum (2x), two diploid ssp. marinum × ssp. gussoneanum (2x) hybrids and two ssp. gussoneanum (4x) × Secale cereale hybrids at metaphase I (M-I) were analyzed by in situ hybridization. The probe pTa71 labelled rDNA sites at NORs of a single pair of homologous or near-homologous SAT-chromosomes of H. marinum in each material. In the three diploids, M-I was regular with ring bivalents and one or a few rods (av.13.52 bound arms cell(-1)). More rod bivalents than the expected one out of seven, i.e. 30, 67 and 89% included rDNA-carrying chromosomes. Corresponding bound short arm frequencies were 0.89, 0.72 and 0.52, while long arms and arms of other chromosomes presented complete or near- complete association. The two heterogenomic hybrids had a less regular M-I (av. 8.04 bound arms cell(-1)) including 20% rDNA-carrying rods with bound arm frequencies of 0.29 in short and 0.87 in long arms. Positions of chromosome associations were established in all 150 rDNA-carrying bivalents. In 77 bivalents with short arm associations, 4% of these occurred proximally to, none at, and 96% distally to rDNA sites, i.e. in satellites. In 143 bivalents with long arm associations, 83% occurred at interstitial and 17% at terminal positions. The observations combine increased frequency of rDNA-carrying rods with decreased frequency of association at NOR regions of SAT-chromosomes. The basis for the relationship is discussed.

Karyotypic analysis of Triticum monococcum using standard repetitive DNA probes and simple sequence repeats

Triticum monococcumrepresents an important source of useful genes and alleles that it would be desirable to use in wheat breeding programmes. The well-defined landmarks on the Amchromosomes could accelerate the targeted introgression ofT. monococcumchromatin into the wheat genome.Fluorescencein situhybridization (FISH) using the repetitive DNA probes pSc119.2, Afa family and pTa71 showed that the pSc119.2 probe was not suitable for the identification of Amchromosomes. In contrast, the whole set of Amchromosomes (especially chromosomes 1, 4, 5 and 7) could be discriminated based on the hybridization pattern of pTa71 and Afa family.In situhybridization with microsatellite motifs (GAA, CAG, AAC and AGG) proved that SSRs represent additional landmarks for the identification of Amchromosomes. The most promising SSR probes were the GAA and CAG motifs, which clearly discriminated the 6Amchromosome and, when used in combination with the Afa family and pTa71 probes, allowed the whole set of Amchromosomes to be reliably identified.In conclusion, fluorescencein situhybridization using the repetitive DNA probes Afa family and pTa71, combined with SSR probes, makes it possible to identify the Amchromosomes ofT. monococcumand to discriminate them from Auchromosomes in the polyploid wheat background.

Molecular cytogenetic characterization of two high protein wheat-Thinopyrum intermedium partial amphiploids

Fluorescence and genomic in situ hybridization (FISH and GISH) were used to establish the cytogenetic constitution of two wheat × Thinopyrum intermedium partial amphiploids H95 and 55(1-57). Both partial amphiploids are high-protein lines having resistance to leaf rust, yellow rust and powdery mildew and have in total 56 chromosomes per cell. Repetitive DNA probes (pTa71, Afa family and pSc119.2) were used to identify the individual wheat chromosomes and to reveal the distribution of these probes within the alien chromosomes. FISH detected 6B tetrasomy in H95 and a null (1D)-tetrasomy (1B) in 55(1-57). GISH was carried out using biotin labeled Th. intermedium DNA and digoxigenin labeled Pseudoroegneria spicata DNA as probes, subsequently. GISH results revealed 44 wheat chromosomes and four Thinopyrum chromosome pairs, including three S and one J chromosome pairs in line H95. Line 55(1-57), contained 42 wheat chromosomes and six Th. intermedium pairs, including two S and one J(S) pairs. Additionally, two identical translocated chromosome pairs with diminished affinity to the alien chromatin were detected in both amphiploids. Another two translocations were found in 55(1-57), with satellite sections from the Thinopyrum J genome.

A new chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C. Koch) based on FISH analysis of monosomic lines

Fluorescent in situ hybridization (FISH) with multiple probes was used to analyze mitotic and meiotic chromosome spreads of Avena sativa cv 'Sun II' monosomic lines, and of A. byzantina cv 'Kanota' monosomic lines from spontaneous haploids. The probes used were A. strigosa pAs120a (a repetitive sequence abundant in A-genome chromatin), A. murphyi pAm1 (a repetitive sequence abundant in C-genome chromatin), A. strigosa pITS (internal transcribed spacer of rDNA) and the wheat rDNA probes pTa71 (nucleolus organizer region or NOR) and pTa794 (5S). Simultaneous and sequential FISH employing pairs of these probes allowed the identification and genome assignation of all chromosomes. FISH mapping using mitotic and meiotic metaphases facilitated the genomic and chromosomal identification of the monosome in each line. Of the 17 'Sun II' lines analyzed, 13 distinct monosomic lines were found, corresponding to four monosomes of the A-genome, five of the C-genome and four of the D-genome. In addition, 12 distinct monosomic lines were detected among the 20 'Kanota' lines examined, corresponding to six monosomes of the A-genome, three of the C-genome and three of the D-genome. The results show that 19 chromosomes out of 21 of the complement are represented by monosomes between the two genetic backgrounds. The identity of the remaining chromosomes can be deduced either from one intergenomic translocation detected on both 'Sun II' and 'Kanota' lines, or from the single reciprocal, intergenomic translocation detected among the 'Sun II' lines. These results permit a new system to be proposed for numbering the 21 chromosome pairs of the hexaploid oat complement. Accordingly, the A-genome contains chromosomes 8A, 11A, 13A, 15A, 16A, 17A and 19A; the C-genome contains chromosomes 1C, 2C, 3C, 4C, 5C, 6C and 7C; and the D-genome consists of chromosomes 9D, 10D, 12D, 14D, 18D, 20D and 21D. Moreover, the FISH patterns of 16 chromosomes in 'Sun II' and 15 in 'Kanota' suggest that these chromosomes could be involved in intergenomic translocations. By comparing the identities of individually translocated chromosomes in the two hexaploid species with those of other hexaploids, we detected different types of intergenomic translocations.

Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora

Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.

Comparative analysis of diploid species of Avena L. Using cytogenetic and biochemical markers: Avena pilosa M. B. and A. clauda Dur.

The diploid oat species containing the Cp genome - Avena pilosa and A. clauda - were studied using C-banding, fluorescence in situ hybridization with probes pTa71 and pTa794, and electrophoresis of grain storage proteins (avenins). Species with the C genome differed considerably from the species of the A genome group in the karyotype structure, heterochromatin type and distribution, relative positions of the 45S and 5S rRNA gene loci, and avenin patterns. These facts confirmed that the C genome had diverged from the ancestral genome before the radiation of the various A genome. Presumably, further evolution of the A -and C genome species occurred separately.

Molecular systematics ofBrassicaand allied genera in subtribes Brassicinae, Raphaninae, Moricandiinae, and Cakilinae (Brassicaceae, tribe Brassiceae); the organization and evolution of ribosomal gene families

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of ribosomal DNA (rDNA) of Brassica and allied genera. The total genomic DNA of 95 accessions of 52 species representing 16 genera was restricted with six enzymes, and the restriction fragments were probed with three ribosomal clones (pTA71, Ver 18-6, and Ver 6-5). Eleven repeat unit length classes were recognized. The repeat unit size classes of 8.9 kb and 9.5 kb were observed most commonly, being represented in 17 and 14 species, respectively. The restriction enzyme SacI produced three to six (generally three) bands with detectable hybridization to the probe pTA71. This probe–enzyme combination indicated a remarkable uniformity amongst Brassica and allied genera in the coding region of repeat units. By contrast, an extensive size variation in the restriction fragments could be localized in the intergenic spacer (IGS) region. Eleven IGS-containing length variants were detected. Complex hybridization patterns, resulting from extensive repeat unit heterogeneity and taxon-specific methylation of one or more cleavage sites, were obtained with the EcoRI + pTA71 combination. The relative homologies between the coding regions were evident from the presence of 1.5 kb in all the taxa, and 0.4-, 1.3-, and 1.7-kb fragments in 33, 27, and 24 species, respectively. The SacI + pTA71 and EcoRI + pTA71 combinations were generally able to distinguish taxa both within and between genera. Three restriction endonuclease digests probed with three ribosomal clones yielded essentially identical fragmentation patterns across all the accessions within the cultivated species Brassica campestris, B. oleracea, and B. juncea. In B. napus, three and seven accessions exhibited restriction profiles similar to one and both diploid progenitor species, respectively. Overall, rDNA repeat unit length polymorphism showed good correlation with the cytodeme-based classification of Brassica and allied genera. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 545–557.

Comparative analysis of diploid species of Avena L. using cytogenetic and biochemical markers: Avena canariensis Baum et Fedak and A. longiglumis 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 3A1 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.

Gene Expression and Nucleolar Dominance in Hexaploid Triticale and T. Aestivum

ABSTRACTThe expression of rRNA genes located in the nucleolar organizing region (NOR) present in the short arm of chromosome 1R from rye (Secale cereale L.) was examined in several hexaploid genotypes of T. aestivum cv. Chinese Spring (AABBDD), Triticale 171 (AABBRR), sphaerococcum mutant form MT47–2D(2R) and Secale cereale (RR). By silver staining and in situ (FISH) hybridization with the DNA probe pTa71, was established a corresponance between the expression and organization patterns of rDNA domains in metaphase and interphase cells. We show here that the substitution of the rye chromosome 2R by a chromosome from hexaploide wheat 2D (triticale–2D(2R) in MT47sph, prevents suppression of the rye-origin rDNA and leads to activity of all six major rDNA loci. In spite of that in triticale 171, containing 28 chromosomes of wheat origin and 14 from rye, rDNA of rye origin is not normally expressed, while the 1B- and 6B- origin rDNA from wheat shows strong expression.

A comparative cytogenetic study of the tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi

Differential C-banding of chromosomes and in situ hybridization with the probes pTa71 and pTa794 were used for a comparative cytogenetic study of the three tetraploid oat species with the A and C genomes: Avena insularis, A. magna, and A. murphyi. These species were similar in the structure and patterns of C-banding of several chromosomes as well as in the location of the loci 5S rRNA genes and main NOR sites; however, they differed in the number and localization of minor 45S rDNA loci as well as in the morphology and distribution of heterochromatin in some chromosomes. According to the data obtained, A. insularis is closer to A. magna, whereas A. murphyi is somewhat separated from these two species. Presumably, all the three studied species originated from the same tetraploid ancestor, and their divergence is connected with various species-specific chromosome rearrangements. The evolution of A. murphyi is likely to have occurred independently of the other two species.

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.

Comparison of somatic chromosomes in some species of <i>Chrysanthemum sensu lato</i> in Russia

The karyotypes of seven species of Chrysanthemum sensu lato collected in Russia were documented for the first time: Chrysanthemum maximowiczii V. Komarov, 2n=54=36m+16sm+2st; C. sinuatum Ledeb., 2n=18=14m+2sm+2st; C. zawadskii Hebrich, 2n=54=34m+16sm+4st; Tanacetum achilleifolium (M. Bieb.) Schuttz-Bip., 2n=18=14m+4sm; T. boreale Fisch. ex DC., 2n=18=16m+2sm; T. kittaryanum (C. A. Mey.) Tzvel., 2n=36=26m+10sm; T. uralense (H. Kraschen.) Tzvel., 2n=18=13m+5sm. This result supported the assumption that C. sinuatum could hybridize naturally with Brachanthemum krylovii (2n=18, diploid) to produce the hybrid as Brachanthemum baranovii (Krasch. et Poljak) Krasch. The karyotypes of T. vulgare and T. boreale were different from each other. Fluorescence in situ hybridization (FISH) experiment using 45S rDNA probe pTa71 was applied to nine species of Chrysanthemum sensu lato. The numbers of 45S rDNA signals in C. crassum, C. ornatum, C. sinuatum, C. zawadskii, T. kittaryanum and T. uralense were reported here for the first time. Fourteen 45S rDNA signals were found in C. crassum (2n=90, decaploid), C. zawadskii (2n=54, hexaploid) and T. uralense (2n=18, diploid). Twelve signals were found in C. ornatum (2n=54, hexaploid). Ten signals were found in T. kittaryanum (2n=36, tetraploid). Eight signals were observed in T. parthenium. Six signals were observed in C. sinuatum (2n=18, diploid) and T. vulgare (2n=18, diploid). The number of hybridization signals obtained for T. vulgare verified Honda et al. (1997). Frequency of signal number/chromosome complement was calculated. Tanacetum uralense show the highest frequency of 77.7%, T. vulgare and C. sinuatum showed the frequency of 33.3%, T. kittaryanum showed the frequency of 27.8%, D. zawadskii showed the frequency of 25.9%, T. parthenium and C. ornatum showed the frequency of 22.2%, and C. crassum showed the frequency of 15.5%.

Karyotypes of Elymus scabrifolius (Poaceae: Triticeae) from South America studied by banding techniques and in situ hybridization.

Karyotypes of 4 accessions of Elymus scabrifolius (2n = 4x = 28) were investigated by Giemsa C- and N-banding, GAA-banding (one accession), AgNO3-staining and in situ hybridization with the rDNA probe pTa71. Two additional accessions were studied in less detail. The chromosomes were large (9-14 microns). The complements included 11 pairs of metacentrics, one with conspicuous satellites on the short arms, and 3 pairs of submetacentrics. Two of 4 accessions from Eastern Argentina and Uruguay had minute or small satellites on a submetacentric pair. No such satellites were observed in the other two accessions. In two accessions from the Cordoba province, a non-homologous submetacentric pair had very long satellites. AgNO3-staining established the presence of 4 nucleoli, two larger and two small ones, in 5 accessions. The C-banding patterns comprised from one to 12 conspicuous bands per chromosome at no preferential positions. The amount of constitutive heterochromatin (19-21%) was the highest hitherto established in the Triticeae. Similarities in banding patterns and chromosome morphology identified homologous and discriminated between non-homologous chromosomes within and, except for two chromosomes, between plants. Heteromorphic chromosome pairs were identified in satellite-carrying chromosomes only. N-banding produced conspicuous bands overall at the same positions as C-banding. GAA-banding patterns were similar to N-banding patterns. The rDNA probe hybridized to chromosome segments at nucleolar constrictions only. The production of C- and N-banding patterns in both genomes of E. scabrifolius suggests the presence of two H genomes and the absence of the pivotal St genome of Elymus. On account of the uncertain identity of one genome, and the overall similar gross morphology of E. scabrifolius and other tetraploid South American species referred to Elymus, E. scabrifolius is retained in Elymus.

Heterochromatin and rDNA sites inCoffeaL. chromosomes revealed by FISH and CMA/DAPI II:C canephoracv. Apoatã,C. salvatrixandC. sessiliflora

Abstract Cytogenetic studies in coffee species present a lack of information caused by some cytological characteristics, such as small chromosomes, symmetric karyotypes and the remarkable genetic homogeneity presented by the genus Coffea. Cytomolecular techniques based on in situ hybridization brought up new possibilities to improve the knowledge of cytogenetic characters and cytological processes involved in speciation of taxa with these cytological features. In the present work we analyzed one cultivar (C. canephora cv. Apoatã) and two wild diploid species (C. sal-vatrix and C. sessiliflora) of the Coffee Germplasm Bank of IAC attempting to cytogenetic characterization. Chromosome banding with fluorochromes DAPI and CMA3 and in situ hybridization with specific probes for rDNA sites 18S-5.8S-26S (probe pTa71) and 5S (probe pScT7) were carried out. For C. salvatrix and C. sessiliflora the CMA3 positive bands agreed with the in situ hybridized rDNA sites in number, size and position. The little variation in number and length of the rDNA sites between species of coffee is discussed.