Comparative chromosome painting in Spizaetus tyrannus and Gallus gallus with the use of macro- and microchromosome probes

Although most birds show karyotypes with diploid number (2n) around 80, with few macrochromosomes and many microchromosomes pairs, some groups, such as the Accipitriformes, are characterized by a large karyotypic reorganization, which resulted in complements with low diploid numbers, and a smaller number of microchromosomal pairs when compared to other birds. Among Accipitriformes, the Accipitridae family is the most diverse and includes, among other subfamilies, the subfamily Aquilinae, composed of medium to large sized species. The Black-Hawk-Eagle (Spizaetus tyrannus-STY), found in South America, is a member of this subfamily. Available chromosome data for this species includes only conventional staining. Hence, in order to provide additional information on karyotype evolution process within this group, we performed comparative chromosome painting between S. tyrannus and Gallus gallus (GGA). Our results revealed that at least 29 fission-fusion events occurred in the STY karyotype, based on homology with GGA. Fissions occurred mainly in syntenic groups homologous to GGA1-GGA5. On the other hand, the majority of the microchromosomes were found fused to other chromosomal elements in STY, indicating these rearrangements played an important role in the reduction of the 2n to 68. Comparison with hybridization pattern of the Japanese-Mountain-Eagle (Nisaetus nipalensis orientalis), the only Aquilinae analyzed by comparative chromosome painting previously, did not reveal any synapomorphy that could represent a chromosome signature to this subfamily. Therefore, conclusions about karyotype evolution in Aquilinae require additional painting studies.

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.

Identification and comparison of individual chromosomes of three Hordeum chilense accessions, 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 fourteen repetitive sequences were used as probes for fluorescence in situ hybridization (FISH) with the aim of identifying inter‐ and intra-species 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, pTa535, 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 H. chilense species. 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, useful in wheat and tritordeum breeding programs.

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

Triticum monococcum represents 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 Am chromosomes could accelerate the targeted introgression of T. monococcum chromatin into the wheat genome.Fluorescence in situ hybridization (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 Am chromosomes. In contrast, the whole set of Am chromosomes (especially chromosomes 1, 4, 5 and 7) could be discriminated based on the hybridization pattern of pTa71 and Afa family. In situ hybridization with microsatellite motifs (GAA, CAG, AAC and AGG) proved that SSRs represent additional landmarks for the identification of Am chromosomes. The most promising SSR probes were the GAA and CAG motifs, which clearly discriminated the 6Am chromosome and, when used in combination with the Afa family and pTa71 probes, allowed the whole set of Am chromosomes to be reliably identified.In conclusion, fluorescence in situ hybridization using the repetitive DNA probes Afa family and pTa71, combined with SSR probes, makes it possible to identify the Am chromosomes of T. monococcum and to discriminate them from Au chromosomes in the polyploid wheat background.

Primary intracranial dural-based synovial sarcoma with an unusual SYT fluorescence in situ hybridization pattern

The authors present the case of an elderly man with a primary dural-based intracranial synovial sarcoma. Histological and immunohistochemical profiles of the lesion were diagnostic for a synovial sarcoma, and molecular studies using fluorescence in situ hybridization were compatible with a synovial sarcoma. A wide array of spindle cell neoplasms has been described as originating in the dura. To the authors' knowledge, however, this is only the second primary durabased intracranial synovial sarcoma ever reported, emphasizing the importance of a broad differential diagnosis when encountering spindle cell lesions of the meninges.

First Report on the Occurrence of Grapevine leafroll-associated virus 7 and 9 in Chilean Grapevines

Grapevine is one of the oldest horticultural crops and represents a highly valuable agricultural commodity. So far, nine distinct Grapevine leafroll-associated viruses (GLRaVs) within the Closteroviridae family have been found to be associated with grapevine leafroll disease (3). Previous studies have demonstrated a high incidence of GLRaV-1, -2, and -3 in Chile (2). To determine if other GLRaVs were present, 21 dormant cane samples were screened with a comprehensive 70-mer oligonucleotide microarray designed to simultaneously detect all grapevine viruses with total or partial genomic sequence available. The array contained 570 unique probes designed against specific regions of more than 40 viral genomes (E. Engel et al., 15th ICVG [Abstr.], 2006). One sample (cv. Black Seedless) showing a microarray hybridization pattern compatible with a mixed infection of GLRaV-7 and GLRaV-1 was analyzed by ELISA using GLRaV-7 specific antibodies (Agritest, Valenzano, Italy) and reverse transcription (RT)-PCR using virus-specific primers LR7-F: 5′- TAT ATC CCA ACG GAG ATG GC -3′ and LR7-R: 5′- ATG TTC CTC CAC CAA AAT CG -3′ (based on GenBank Accession No. Y15987). The serological analysis confirmed the presence of GLRaV-7 with further confirmation by the RT-PCR product of 502 bp corresponding to a fragment of the HSP70h gene that was cloned and sequenced. The Chilean GLRaV-7 sequence (GenBank Accession No. EU334662) showed 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-7 isolate from Albania (GenBank Accession No. Y15987). GLRaV-1 infection was confirmed by ELISA (Bioreba AG, Reinach, Switzerland) and RT-PCR. A second sample (cv. Tintorera) showing microarray hybridization pattern compatible with a mixed infection of GLRaV-9 and Grapevine virus A (GVA) was analyzed by RT-PCR using virus-specific primers LR9-F: 5′- CGG CAT AAG AAA AGA TGG CAC -3′ and LR9-R: 5′- TCA TTC ACC ACT GCT TGA AC -3′ (1). The RT-PCR product of 393 bp corresponding to a fragment of the HSP70h gene was cloned and sequenced (GenBank Accession No. EU334663), showing 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate from the United States (GenBank Accession No. AY297819). Since there are no commercial antibodies available for GLRaV-9 detection, a second pair of primers, LR9-F1: 5′- AAA GGT TTC TGC TGG TTA CC -3′ and LR9-R1: 5′- CTT TCA GAA CAG TCC TCC TC -3′ that amplified a fragment of ORF1a was also used. The 301-bp product was cloned and sequenced (GenBank Accession No. EU588989) showing 93.7% nucleotide and 98% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate (GenBank Accession No. AY297819). GVA infection was confirmed by ELISA (Bioreba AG) and RT-PCR. To our knowledge, this is the first report of GLRaV-7 and GLRaV-9 in Chile. Further studies will help determine the effect and incidence of these viruses in Chilean grapevines. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) N. Fiore et al. J. Plant Pathol. 90:125, 2008. (3) G. P. Martelli and E. Boudon-Padieu. Options Méditerr. B55, 2006.