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The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present an advanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to pre-fixation and fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182bp.

Evaluation of the genetic diversity for six genes in forty animals of the Portuguese indigenous sheep breed (Ovis aries) ""Churra da Terra Quente"" was done. A non-radioactive method to allow single-strand conformation polymorphism (SSCP) detection was optimised, starting from genomic DNA and PCR amplification of seven fragments: exon 1 of the alpha-lactalbumin gene; exons 10 and 11 of the alpha s1-casein gene; exon 7 of the beta-casein gene; exon 4 of the kappa-casein gene; exons 4 and 5 of the growth hormone gene and exon 6 of the growth hormone receptor gene. Polymorphisms were detected in five of the seven PCR products. Only kappa-casein and growth hormone receptor were monomorphic. Alpha-lactalbumin and alpha s1-casein exons showed three conformational patterns, beta-casein and growth hormone exon 4 showed two electrophoretic patterns and growth hormone exon 5 showed five conformational patterns. These data provide evidence that ""Churra da Terra Quente"" has a high genetic variability, which opens interesting prospects for future selection programs and also for preservation strategies. Also, our data show that PCR-SSCP is an appropriate tool for evaluating genetic variability.

The morphological, yield, cytological and molecular characteristics of bread wheat x tritordeum F1 hybrids (2n = 6x = 42; AABBDHch) and their parents were analysed. Morphologically, these hybrids resembled the wheat parent. They were slightly bigger than both parents, had more spikelets per spike, and tillered more profusely. The hybrids are self-fertile but a reduction of average values of yield parameters was observed. For the cytological approach we used a double-target fluorescence in situ hybridization performed with total genomic DNA from Hordeum chilense L. and the ribosomal sequence pTa71. This technique allowed us to confirm the hybrid nature and to analyse chromosome pairing in this material. Our results showed that the expected complete homologous pairing (14 bivalents plus 14 univalents) was only observed in 9.59% of the pollen mother cells (PMCs) analysed. Some PMCs presented autosyndetic pairing of Hch and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the Hch genome (5.4). The maximum number of univalents per PMC was 20. We only observed wheat multivalents (one per PMC) but the frequency of trivalents (0.08) was higher than that of quadrivalents (0.058). We amplified 50 RAPD bands polymorphic between the F1 hybrid and one of its parents, and 31 ISSR polymorphic bands. Both sets of markers proved to be reliable for DNA fingerprinting. The complementary use of morphological and yield analysis, molecular cytogenetic techniques and molecular markers allowed a more accurate evaluation and characterization of the hybrids analysed here. [ABSTRACT FROM AUTHOR]

A major satellite DNA sequence was isolated from the cat genome and its sequencing data revealed homology to the FA-SAT family. In situ hybridization of the cat satellite DNA and telomeric sequences to cat chromosomes, together with staining of constitutive heterochromatin, allowed the physical mapping of the FA-SAT sequences, and also an overall constitutive heterochromatin study in cat chromosomes. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The old Portuguese wheat landrace aggregate known as 'Barbela' shows good productivity under the low-fertility conditions often associated with acid soils. The use of genomic rye DNA, in combination with 45S rDNA and the repetitive sequences dpTa1 and pSc119.2 as probes, in two sequential in situ hybridization steps enabled the identification of all chromosomes in the 'Barbela' wheat lines and the detection of the introgression of rye-origin chromatin onto wheat chromosome arm 2DL in two of the lines. Amplification of microsatellite loci using published primer pairs showed that the distal segment of wheat chromosome 2DL, which was involved in the rye translocation, was deleted. The identification and characterization of small recombinant chromosome segments in wheat–rye lines may allow their use in plant breeding programmes. Their presence in farmer-maintained material demonstrates the importance of maintaining, characterizing, and collecting landrace material before valuable genetic combinations are lost as uniform commercial crops are introduced.Key words: biodiversity, in situ hybridization, microsatellites, plant breeding, recombination, alien chromosomes, marker selection.La vieille variété de pays amalgamée d'origine portugaise connue sous le nom de 'Barbela' montre une bonne productivité en conditions de faible fertilité, lesquelles sont souvent rencontrées dans des sols acides. L'emploi comme sondes de l'ADN génomique du seigle en combinaison avec l'ADNr 45S et les séquences répétitives dpTa1 ainsi que pSc119.2, lors d'hybridations in situ séquentielles, a permis d'identifier tous les chromosomes au sein des lignées du blé 'Barbela' et la détection d'une introgression de chromatine provenant du seigle au sein du bras chromosomique 2DL chez deux des lignées. L'amplification de microsatellites à l'aide d'amorces déjà caractérisées a montré que le segment distal du chromosome 2DL, celui impliqué dans la translocation avec le seigle, a été délété. L'identification et la caractérisation de courts segments chromosomiques recombinants dans des lignées blé–seigle pourraient ouvrir la voie à leur utilisation dans des programmes de sélection. Leur présence au sein de lignées retenues par les agriculteurs démontre l'importance du maintien, de la caractérisation et de la collection de variétés de pays avant que ne soient perdues des combinaisons génétiques de valeur avec l'introduction de cultures commerciales uniformes.Mots clés : biodiversité, hybridation in situ, microsatellite, amélioration génétique, recombinaison, chromosomes étrangers, sélection de marqueurs[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]