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Recently, members of the MATE family have been implicated in aluminium (Al) tolerance by facilitating citrate efflux in plants. The aim of the present work was to perform a molecular characterisation of the MATE2 gene in bread wheat. Here, we cloned a member of the MATE gene family in bread wheat and named it TaMATE2, which showed the typical secondary structure of MATE-type transporters maintaining all the 12 transmembrane domains. Amplification in Chinese Spring nulli-tetrasomic and ditelosomic lines revealed that TaMATE2 is located on the long arm of homoeologous group 1 chromosomes. The transcript expression of TaMATE2 homoeologues in two diverse bread wheat genotypes, Barbela 7/72/92 (Al-tolerant) and Anahuac (Al-sensitive), suggested that TaMATE2 is expressed in both root and shoot tissues of bread wheat, but mainly confined to root rather than shoot tissues. A time-course analysis of TaMATE2 homoeologue transcript expression revealed the Al responsive expression of TaMATE2 in root apices of the Al-tolerant genotype, Barbela 7/72/92. Considering the high similarity of TaMATE2 together with similar Al responsive expression pattern as of ScFRDL2 from rye and OsFRDL2 from rice, it is likely that TaMATE2 also encodes a citrate transporter. Furthermore, the TaMATE2-D homoeologue appears to be near the previously reported locus (wPt0077) on chromosome 1D for Al tolerance. In conclusion, molecular cloning of TaMATE2 homoeologues, particularly TaMATE2-D, provides a plausible candidate for Al tolerance in bread wheat that can be used for the development of more Al-tolerant cultivars in this staple crop.

Human leukocyte antigen (HLA)-B27 is the mostly known major histocompatibility complex (MHC) gene associated with ankylosing spondylitis (AS). Nonetheless, there is substantial evidence that other MHC genes appear to be associated with the disease, although it has not yet been established whether these associations are driven by direct associations or by linkage disequilibrium (LD) mechanisms. We aimed to investigate the contributions of HLA class I and II alleles and B27-haplotypes for AS in a case-control study. A total of 188 HLA-B27 AS cases and 189 HLA-B27 healthy controls were selected and typed for HLA class I and II by the Luminex polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. Allelic and haplotypic distributions were estimated by maximum likelihood method using Arlequin v3.11 and statistical analysis were performed by Stata10.1. No associations were found between non-HLA-B27 loci and AS susceptibility, but several associations were observed for phenotypic features of the disease. DRB1*08 was identified as a risk factor for uveitis and DQB1*04 seems to provide protection for AS severity (functional, metrological and radiological indexes). A*02/B27/C*02/DRB1*01/DQB1*05 [P

The expression of the ribosomal RNA (rRNA) genes from rye, located within the nucleolus organizer regions (NORs), is repressed by cytosine methylation in wheat x rye hybrids and in triticale, as consequence of nucleolar dominance. Our previous study revealed that bread wheat cultivars with a maximum number of four Ag-NORs presented high level of rDNA cytosine methylation when compared to others with a maximum of six Ag-NORs. In order to evaluate the inheritance of the Ag-NORs number and NOR methylation patterns, we produced F1 hybrids between bread wheat cultivars with four Ag-NORs and bread wheat cultivars with six Ag-NORs (in the direct and reciprocal senses). The F2 progenies of these F1 hybrids were also evaluated for the NOR number and methylation patterns. Parent bread wheat cultivars with a maximum of four Ag-NORs after treated with 5-azacytidine evidenced a maximum of six Ag-NORs per metaphase cell and a maximum of six nucleoli per interphase nucleus, confirming that the expression of the rRNA genes in bread wheat is related to cytosine methylation. Most of the F1 hybrids showed a maximum number of four or six Ag-NORs, similarly to that of the female parent suggesting a non-mendelian inheritance, while other hybrids presented four or six Ag-NORs in both senses of the cross. The F1 NOR methylation patterns showed some fragments common to their parents but also novel fragments suggesting genomic and/or chromosome rearrangements after hybridization. Despite the different NOR patterns among the parents, an invariable NOR pattern was found among the F1 plants suggesting a tendency to stability, which was also transmitted to the F2. The F2 progenies showed plants with a maximum of four, five and/or six Ag-NORs. The ratio of plants with four, five and/or six Ag-NORs per F2 progeny was variable and did not follow any specific mendelian proportion. These results allowed us to suggest that the inheritance of the number of Ag-NORs by the F1 and F2 plants did not follow any mendelian inheritance and were not correlated to NOR methylation patterns in contrast to what was verified for their parents.

Lack of suitable biological material for genetic studies in wildlife species can be a bottleneck for studies of conservation, phylogeny, evolution and sustainable management of specific wildlife species. This is due to animal stress during immobilization for sample collection, restricted rules in Zoos and Natural Parks or even physical difficulties in getting samples from living animals. Noninvasive techniques are more often used to obtain samples like hair, faeces and old museum collections, among others. However, the amount and quality of DNA obtained is a major concern and comparison of suitable methodologies must be done for optimization of methodologies for specific purposes. Suiformes are a large group that represents an important repository of biodiversity and constitutes an excellent group for clarification of the phylogenetic relationships based on the results of comparative genomic approaches. They include several species that constitute an important group for meat production and hunting. Pigs and babirusa, peccaries and hippopotamuses belong to the mammalian order Artiodactyla, the even-toed ungulates, of which there are three major lineages or clades — the Suiformes, Tylopoda and Pecora — each ranked as a suborder. Of these, the Suiformes are the only nonruminants (Groves and Grubb 1993). Among them Suiformes species possess several million years of distinct evolutionary history and show several morphological size variations, changes in several external and internal structures, different environmental adaptations, a characteristic geographical distribution (Macdonald 1988), and also different karyotype (Adega et al. 2006). These

Olea europaea L. presents a huge number of cultivars that differ in terms of end-use, and consequently in their chemical composition. The use of different phenotypic (fruit dimension, shape, and color) and chemical parameters (phenolic compounds, flavonoids, and antioxidant activity) during olive fruit maturation in three olive cultivars (Blanqueta, Cobrancosa, and Galega), grown in the Protected Denomination Region of Tras-os-Montes, revealed clear differences among the cultivars, indicating that they behave differently during the maturation process, Galega being the first to reach full maturity. All colorimetric parameters (L*, b*, C*, and H*) decreased with maturity. Galega cultivar presented the highest phenolic content in all the maturation stages analyzed (128.25–175.46 mg GAE g−1). Also, this cultivar presented the highest antioxidant activity in semi-green stage fruits (231.72 μmol Trolox g−1). The flavonoid content increased in all cultivars from the green drupe to black stage, with differential ...

L1 distribution in mammal's genomes is yet a huge riddle. However, these repetitive sequences were already found in all chromosomic regions, and in general, they seem to be nonrandomly distributed in the genome. It also seems that after insertion and when they are not deleterious, they are always involved in dynamic processes occurring on that particular chromosomic region. Furthermore, it seems that large-scale genome rearrangements and L1 activity and accumulation are somehow interconnected. In the present study, we analysed L1 genomic distribution in Tatera gambiana (Muridae, Gerbillinae), Acomys sp. (Muridae, Deomyinae), Cricetomys sp. (Nesomyidae, Cricetomyinae), Microtus arvalis (Cricetidae, Arvicolinae), Phodopus roborovskii and P. sungorus (Cricetidae, Cricetinae). All the species studied here seems to exhibit a species-specific pattern.Possible mechanisms, and processes involved in L1 distribution and preferential accumulation in certain regions are di scussed.

The olive tree disease, named in Portugal as “gafa”, is caused by fungi of Colletotrichum genus. Two species of this genus (C. gloesporioides and C. acutatum) were identified as causal agents of the disease. However, our research has pointed out that C. acutatum is the prevalent pathogen on the southern region of the country. A comparative study among four olive tree cultivars, two from Portugal (‘Galega’ and ‘Cobrancosa’) and the other two from Spain (‘Picual’ and ‘Arbequina’) was conducted to evaluate the variability of reaction types to C. acutatum infections. The analysis of phenotypic reactions revealed significant differences between ‘Galega’ and ‘Picual’, with the most susceptibility for ‘Galega’ versus characteristics of resistance shown by ‘Picual’. The cultivar ‘Cobrancosa’ and the cultivar ‘Arbequina’ had performances classified as intermediates, which could be connected with differences on the timing of maturation. Several factors involved in the response of the four olive tree cultivars face to the attack of C. acutatum are discussed in this work, as a contribution to the knowledge of the resistance to “gafa” and, consequently, to the disease management.

In humans, a germline mutation (c.309C>G) in the TWIST1 oncogene may predispose to breast cancer and its expression has been associated with tumour progression and metastasis. In this study, the feline TWIST1 gene was screened for sequence variations in 37 neoplastic and eight hyperplastic mammary gland lesions from cats. In addition, mRNA levels were examined in 15 mammary tumours and three cases of mammary hyperplasia by quantitative real-time reverse-transcriptase PCR. Feline mammary carcinomas had significantly lower levels of expression of TWIST1 mRNA than benign mammary tumours. No variations were identified in the TWIST1 coding region in feline mammary tumours and the mutation described in humans was not detected. However, two germline variants in the TWIST1 gene intron were identified in four and three carcinomas, respectively: GQ167299:g.535delG and GQ167299:g.460C>T. There was no association between these sequence alterations and TWIST1 mRNA levels.

Sex identification in birds through molecular methods is an important tool for the management and preservation of species. Advances in real-time PCR-based techniques overcome some limitations of the more classical molecular analysis methodologies. Here, we describe a new approach, based on high-resolution melting (HRM) curve analysis of the CHD1 gene, for avian gender identification. This method was successfully applied to carry out sexual differentiation based on melting curve patterns in common quail (Coturnix c. coturnix) and Japanese quail (Coturnix c. japonica). We clearly demonstrate the efficacy of a simple HRM assay for a rapid and efficient gender differentiation of these subspecies and propose this methodology as a valuable addition to expand the applicability of real-time PCR-based technology in avian molecular sexing.

Objective.Unconfirmed reports describe association of ankylosing spondylitis (AS) with several candidate genes includingANKH. Cellular export of inorganic pyrophosphate is regulated by the ANK protein, and mutant mice (ank/ank), which have a premature stop codon in the 3’ end of theankgene, develop severe ankylosis. We tested the association between single-nucleotide polymorphisms (SNP) in these genes and susceptibility to AS in a population of patients with AS. We investigated the role of these genes in terms of functional (BASFI) and metrological (BASMI) measures, and the association with radiological severity (mSASSS).Methods.Our study was conducted on 355 patients with AS and 95 ethnically matched healthy controls. AS was defined according to the modified New York criteria. Four SNP inANKH(rs27356, rs26307, rs25957, and rs28006) were genotyped. Association analysis was performed using Cochrane-Armitage and linear regression tests for dichotomous and quantitative variables. Analyses of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), BASFI, and mSASSS were controlled for sex and disease duration.Results.None of the 4 markers showed significant single-locus disease associations (p > 0.05), suggesting thatANKHwas not a major determinant of AS susceptibility in our population. No association was observed between these SNP and age at symptom onset, BASDAI, BASFI, BASMI, or mSASSS.Conclusion.These results confirm data in white Europeans thatANKHis probably not a major determinant of susceptibility to AS.ANKHpolymorphisms do not markedly influence AS disease severity, as measured by BASMI and mSASSS.

Retrotransposon-based markers, such as the inter-retrotransposon-amplified polymorphism (IRAP) and the retrotransposon microsatellite-amplified polymorphism (REMAP) are highly informative, multilocus, and reveal insertional polymorphisms among plant individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, genetic mapping linkage, and for the detection of genetic rearrangements induced by polyploidization. In this study, we used IRAP and REMAP markers to assess the genetic diversity among 51 old Portuguese durum wheat cultivars belonging to 27 botanical varieties and to define their genetic relationships. Five IRAP and four REMAP primer combinations were used. IRAP markers revealed 66.3% of polymorphism and an average of 18.4 bands per primer combination which ranged in size from 450 to 3,100 bp. The REMAP technique allowed the detection of 86.36% of inter-cultivar polymorphism and an average of 11 bands per primer combination. The molecular weight of the REMAP bands ranged from 250 to 2,750 bp. The durum wheat cultivars analyzed here belong to 27 botanical varieties of the subspecies Triticum turgidum subsp. turgidum L. [syn. T. turgidum] and Triticum turgidum L. subsp. durum [syn. T. durum] (Desf.) Husn.. Our results showed that the genetic variability assessed by both the IRAP and REMAP markers did not allow the clustering of the durum wheat cultivars according to their taxonomical criteria (subspecies or botanical variety) or homonymy. Nonetheless, these markers were useful for the assessment of genetic diversity at the individual level, for the definition of genetic relationships among cultivars, and for estimation of the genetic structure of the Old collection under analysis. The achieved data could be valuable for future experiments of DNA fingerprinting, genetic improvement, and germplasm conservation in wheat.

“Barbela” (Triticum aestivum L. em. Thell) constitutes an aggregate of Old Portuguese landraces highly genetically diverse. We characterized 30 “Barbela” populations from four regions at the cytogenetic, molecular, morphological, and yield levels. The hexaploid wheat “Chinese Spring” was included in all analyses as a control. No polymorphisms at the nucleolar organizer regions were detected. Instead, he intergenic spacer (IGS) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) markers identified four IGS length variant phenotypes within “Barbela” and one in “Chinese Spring”. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering based in these markers reflected the IGS length variant phenotypes identified. Inter-Simple Sequence Repeat (ISSR) markers revealed high genetic variability within “Barbela” and between “Barbela” and “Chinese Spring”. Consequently, “Chinese Spring” was clustered apart from “Barbela” in the UPGMA tree constructed based on the po...

Regardless of the high economic value and large geographical distribution of oysters, the current knowledge of oyster taxonomic relationships and systematics is still limited, particularly for flat oysters. In this study, the molecular cytogenetic characterization of mitotic chromosomes of the Provence flat oyster or dwarf oyster, Ostrea stentina, was performed through Giemsa staining, chromosome measurements, in situ restriction endonuclease banding, C-banding, fluorescence in situ hybridization with major ribosomal RNA genes (ITS1), and telomeric sequence (TTAGGG)n. The karyotype (2n = 20) consisted of 6 metacentric (1, 3, 4, 6, 8, and 10) and 4 submetacentric (2, 5, 7, and 9) chromosome pairs. Chromosome treatment with HaeIII produced specific banding patterns for all chromosomal pairs, confirming the efficiency of this restriction enzyme for chromosome banding in oysters. Results for C-banding revealed the presence of heterochromatin in the telomeric regions of the short arms: on a large meta...

TWIST1 is thought to be a novel oncogene. Understanding the molecular mechanisms regulating the TWIST1 gene expression profiles in tumor cells may give new insights regarding prognostic factors and novel therapeutic targets in veterinary oncology. In the present study we partially isolated the TWIST1 gene in Felis catus and performed comparative studies. Several primer combinations were used based on the alignments of homologous DNA sequences. After PCR amplification, three bands were obtained, purified and sequenced. Several bioinformatic tools were utilized to carry out the comparative studies. Higher similarity was found between the isolated TWIST1 gene in Felis catus and Homo sapiens (86%) than between Homo sapiens and Rattus norvegicus or Mus musculus (75%). Partial amino acid sequence showed no change in the four species analyzed. This confirmed that coding sequences presented high similarity (~96%) between man and cat. These results give the first insights regarding the TWIST1 gene in cat but further studies are required in order to establish, or not, its role in tumor formation and progression in veterinary oncology.

Ribosomal RNA genes expression was analysed in 18 Portuguese bread wheat accessions (Triticum aestivum L. em Thell.), and the number of argyrophilic-nucleolar organiser regions (Ag-NORs) per cultivar was scored. Ten accessions presented six Ag-NORs per metaphase and six nucleoli per interphase, and eight accessions presented four Ag-NORs per metaphase and four nucleoli per interphase. Fluorescent in situ hybridisation with the 45S rDNA sequence pTa71 and genomic DNA from Aegilops tauschii (2n = 2x= 14, DD) confirmed the Ag-NOR location and identified the six satellited chromosomes as being the chromosome pairs 1B, 6B and 5D. The methylation pattern of the NOR region was studied by Southern blot using pTa71 as probe, which represented a complete rDNA unit of bread wheat. DNA digestions performed by MspI and HpaII resulted in different patterns revealing the high level of cytosine methylation at their recognition sequences. The total percentage values of NOR methylation indicated that wheat accessions with a maximum of four Ag-NORs were more heavily methylated at the NOR region than accessions with a maximum of six Ag-NORs.

The pullet carpet shell Venerupis senegalensis (=V. pullastra) is a commercially important species in Portugal, Spain, France, and Italy. In Portugal, this species was once abundant in the Ria Formosa (southern Portugal). However, in the early 1980s, its abundance declined dramatically due to overfishing. In order to reverse this negative trend, the genetic sustainable management of the wild stocks of V. senegalensis should be performed by promoting successful restocking actions and the development of an aquaculture commercial production program of this species. In order to find the best broodstock for aquaculture purposes and therefore minimize the deleterious effects of hatchery practices, we analyzed the genetic diversity of the natural population to be restocked (Ria Formosa) but also of another potential genetically close population (Ria de Aveiro) by RAPD. Similar and substantive percentage of polymorphic loci, effective number of alleles, Nei’s gene diversity, and Shannon’s diversity index was found within both populations. This high genetic variability within populations suggests that they might have a gene pool with sufficient genetic plasticity to support changes in the environmental conditions. Analyses of population genetic structure also revealed a small genetic differentiation between the two populations. The high genetic variability of the natural population to be restocked makes it the preferential broodstock for aquaculture purposes. However, the Ria de Aveiro population could also be a viable alternative, due to its genetic plasticity and the genetic similarity of both populations. The results of this study can be useful to the sustainable management of wild stocks as well as in promoting successful restocking actions based on aquaculture production.

Determination of sex in birds is valuable for studying population dynamics and structure, habitat use, behavior and mating systems. The purpose of the present study was to optimize a DNA-based methodology to allow the sex identification in Accipiter cooperii nestlings. Chromo-helicase-DNA-binding (CHD1) gene was used in this work as a marker for sex identification. CHD-W and CHD-Z sequences should present length and/or sequence differences providing a way to identify gender. We used a non-invasive method for DNA extraction from feathers and performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. The length difference between CHD-W and CHD-Z amplified fragments observed by electrophoresis in conventional agarose gel was not enough to provide a clear differentiation between males and females. However, patterns obtained by PCR-SSCP differentiated undoubtedly males and females in A. cooperii. This tool provides a precise gender identification assay and will be applied to confirm and refine morphometrically based sexing techniques used in the field.

A collection of 18 landraces of common bean (Phaseolus vulgarisL.) from northern Portugal was evaluated for both phaseolin pattern and a set of qualitative and quantitative traits. The phaseolin Contender and Tendergreen types were the most commonly occurring, with Boyaca and Huevo de Huanchaco present, but rare. The colour of the flower standard and wing, and seed shape, colour and size were all highly variable. On the basis of morphological traits, the landraces and testers clustered into two main groups. No relationship was found between phaseolin profile and any of the quantitative traits measured. Based on phaseolin patterns, the origin of Portuguese common bean populations was probably the Andean region of South America.

The Fl AABBRH ch hybrids studied here were produced by crosses between the Portuguese triticale cultivar 'Douro' (AABBRR) and the tritordeum line HT9 (AABBH ch H ch ). Fluorescent in situ hybridization performed with genomic DNA probes genomic in situ hybridization (GISH) from rye and Hordeum chilense allowed the unequivocal parental genomes discrimination in all hybrids. Among 55 plants, one presented a spontaneous wheat-rye translocation which was successfully detected after GISH. Recombinant chromosomes identification was made after reprobe with pTa71 and pScll9.2. Nine rDNA loci were detected by pTa71 and pSc119.2 identified the chromosome arms involved in the translocation, after comparing the observed hybridization patterns with those described by several authors. We identified the spontaneous wheat-rye translocation as being the 7BS/7RL. Many wheat-rye translocations have been found (e.g. 1BL.1RS and 1AL.1RS), but as far as we know, this is the first time that this translocation is reported. We considered it helpful for wheat breeding programmes as it could provide the transference of interesting agronomic characteristics from rye (e.g. leaf rust resistance) to wheat.

Inter-simple sequence repeats (ISSRs) were used for genetic diversity analyses of an Old Portuguese wheat collection. Eighteen primers produced 96.3 and 98.5% of ISSR polymorphism in bread and durum wheat cultivars, respectively. The unweighted pair group method with arithmetical averages (UPGMA) phenogram clearly split all cultivars based on their species/ploidy, reflecting a defined genetic structure. ISSRs revealed high genetic diversity at interspecific, intraspecific, and intercultivar levels. Thirty-three exclusive ISSR markers were found. Cultivars were clustered according to their botanical varieties and, in a few cases, with their homonym(s). No statistically significant differences were found between genetic diversity parameters of durum and bread wheat, probably due to high intraspecific diversity. Similar analyses were performed among botanical varieties, and their relationships were defined. Cladograms resembled UPGMA clustering. This highly genetically diverse Old wheat collection will be conserved and maintained, and it could be further used in breeding programs to widen the narrow genetic basis of modern wheat varieties and to avoid the loss of rare and unique alleles.

The Quercus genus includes several species and presents a huge genetic variability. In the last decades, studies regarding genetic diversity and molecular characterisation in Quercus emerged. In this work, we intend to characterise nine Quercus species at cytogenetic and molecular levels. Cytogenetically, we aim to evaluate the ribosomal RNA genes expression, and to determine the physical mapping of the rDNA loci using both salt‐nylon silver staining and double‐target fluorescent in situ hybridisation (FISH) techniques. Molecularly, our goals were to characterise and determine the genetic variability using Inter‐Simple Sequence Repeats (ISSRs). All individuals were euploid (2n = 2x =24), and presented a maximum number of four Ag‐NORs and four nucleoli per mitotic cell. FISH confirmed the Ag‐NORs localisation, and evidenced that all 45S rDNA loci are being actively expressed. Two additional 5S rDNA loci were also detected. Cytogenetic analyses did not allow us to distinguish the oak species. Inver...

Eukaryotic genomes contain far more DNA than needed for coding proteins. Some of these additional DNA sequences comprise non-coding repetitive DNA sequences, mostly satellite DNAs and also transposable elements usually located at the heterochromatic regions of chromosomes. Satellite DNAs consist of tandemly repeated DNA sequences inhabiting the mammalian genome, typically organized in long arrays of hundreds or thousands of copies. Different important functions have been ascribed to satellite DNA, from the imperative centromeric function in mitosis and meiosis to the recent discovery of its involvement in regulatory functions via satellite transcripts. Moreover, satellite DNAs, among other repetitive sequences, are believed to be the ‘engine’ triggering mammalian genome evolution. Repetitive DNAs are, most likely, the genetic factors responsible for promoting genomic plasticity and therefore higher rates of chromosome mutation. Furthermore, constitutive heterochromatin regions are thought to be ‘hotspots’ for structural chromosome rearrangements. A considerable collection of evidences places these sequences in the landscape of mammalian evolution. However, the mechanisms that could explain how this alliance between chromosome evolution and satellite DNA is made are still enigmatic and subject of debate. Throughout the mammalian taxa, different patterns of chromosome evolution have been widely registered from heterochromatin additions/eliminations, Robertsonian translocations, whole-arm reciprocal translocations to tandem translocations; the fact is genome’s repetitive fraction is playing a central role in mammalian genome structuring. Throughout this review we will focus on the evidences that associate satellite DNAs and constitutive heterochromatin to the process of chromosome evolution and consequently to domestic species genome’s remodeling.

Constitutive heterochromatin comprises a substantial fraction of the eukaryotic genomes and is mainly composed of tandemly arrayed satellite DNAs (satDNA). These repetitive sequences represent a very dynamic and fast evolving component of genomes. In the present work we report the isolation of Cricetus cricetus (CCR, Cricetidae, Rodentia) centromeric repetitive sequences from chromosome 4 (CCR4/10sat), using the laser microdissection and laser pressure catapulting procedure, followed by DOP-PCR amplification and labelling. Physical mapping by fluorescent in situ hybridisation of these sequences onto C. cricetus and another member of Cricetidae, Peromyscus eremicus, displayed quite interesting patterns. Namely, the centromeric sequences showed to be present in another C. cricetus chromosome (CCR10) besides CCR4. Moreover, these almost chromosome-specific sequences revealed to be present in the P. eremicus genome, and most interestingly, displaying a ubiquitous scattered distribution throughout this karyotype. Finally and in both species, a co-localisation of CCR4/10sat with constitutive heterochromatin was found, either by classical C-banding or C-banding sequential to in situ endonuclease restriction. The presence of these orthologous sequences in both genomes is suggestive of a phylogenetic proximity. Furthermore, the existence of common repetitive DNA sequences with a different chromosomal location foresees the occurrence of an extensive process of karyotype restructuring somehow related with intragenomic movements of these repetitive sequences during the evolutionary process of C. cricetus and P. eremicus species.

The certification of olive oil has led to the definition of Protected Denomination of Origin (PDO) producing regions in European countries. PDO products should be protected, and a solution could be by using DNA fingerprinting. In this work we evaluate the efficiency of RAPD, ISSR, and SSR molecular markers for olive oil varietal identification and their possible use in certification purposes. Twenty-three Portuguese olive oil samples (11 obtained monovarietal and 12 purchased commercial oils) were screened by means of two RAPD, four ISSR, and four SSR markers. The quality of amplified products was used to evaluate the reproducibility and the level of polymorphism. Principal component analysis was performed with DCENTER using unweighted pair group mathematical average (UPGMA) that allowed group formation according to olive oil varietal geographic origin.

Chromosome identification is essential in oyster genomic research. Fluorescence in situ hybridization (FISH) offers new opportunities for the identification of oyster chromosomes. It has been used to locate satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been conducted with simple sequence repeats (SSRs). FISH was used to probe the physical organization of three particular SSRs, (GGAT)(4), (GT)(7) and (TA)(10) onto metaphase chromosomes of the Pacific oyster, Crassostrea gigas. Hybridization signals were observed in all the SSR probes, but the distribution and intensity of signals varied according to the oligonucleotide repeat. The intercalary, centromeric and telomeric bands were observed along the chromosomes, and for each particular repeat every chromosome pair presented a similar pattern, allowing karyotypic analysis with all the SSRs tested. Our study is the first in mollusks to show the application of SSR in situ hybridization for chromosome identification and karyotyping. This technique can be a useful tool for oyster comparative studies and to understand genome organization in different oyster taxa.

The DNA hypomethylation effect of 5-azacytine (5-AC; a cytosine analog) is widely known. This agent has been used for rRNA gene expression studies of Triticeae amphiploids and hybrids regarding rye rRNA genes suppression caused by the wheat nucleolar dominance phenomenon. However, this situation is reverted by 5-AC treatment which activates rye rRNA gene expression as it has been intensively observed in triticale. For nucleolar dominance studies, we produced F1 multigeneric hybrids (AABBRHch; 2n = 6x = 42) from crosses between the triticale cultivar ‘Corgo’ (AABBRR; 2n = 6x = 42) and the tritordeum cultivars HT9 and HT31 (AABBHchHch; 2n = 6x = 42). The hybrid seeds were germinated in a low concentration of 5-AC (treatment) and in distilled water (nontreated control plants). Silver nitrate staining performed in one 5-AC-treated F1 hybrid revealed a reduced number of interphase cells with seven nucleoli, metaphases with eight Ag-NORs, and neocentromeres in the long arm of three wheat chromosomes. Nontreated hybrids presented six Ag-NORs per mitotic metaphase cell and a maximum of six nucleoli per interphase because of the 1R Ag-NOR suppression. No neocentromere was found in the control F1 hybrid plants. Both treated and nontreated seedlings were subsequently evaluated by fluorescent in situ hybridization performed with genomic and repetitive DNA probes to identify Hch and rye genomes, to confirm Ag-NORs location, and to detect inactive rDNA loci. DAPI counterstaining was also helpful for the detection of neocentromeres in the long arm of three wheat chromosomes. This study allowed us to suggest that 5-AC treatment specifically induced wheat neocentromeres in the F1 multigeneric triticale × tritordeum hybrids.

Clinical animal cytogenetics development began in the 1960’s, almost at the same time as human cytogenetics. However, the development of the two disciplines has been very different during the last four decades. Clinical animal cytogenetics reached its ‘Golden Age’ at the end of the 1980’s. The majority of the laboratories, as well as the main screening programs in farm animal species, presented in this review, were implemented during that period, under the guidance of some historical leaders, the first of whom was Ingemar Gustavsson. Over the past 40 years, hundreds of scientific publications reporting original chromosomal abnormalities generally associated with clinical disorders (mainly fertility impairment) have been published. Since the 1980’s, the number of scientists involved in clinical animal cytogenetics has drastically decreased for different reasons and the activities in that field are now concentrated in only a few laboratories (10 to 15, mainly in Europe), some of which have become highly specialized. Currently between 8,000 and 10,000 chromosomal analyses are carried out each year worldwide, mainly in cattle, pigs, and horses. About half of these analyses are performed in one French laboratory. Accurate estimates of the prevalence of chromosomal abnormalities in some populations are now available. For instance, one phenotypically normal pig in 200 controlled in France carries a structural chromosomal rearrangement. The frequency of the widespread 1;29 Robertsonian translocation in cattle has greatly decreased in most countries, but remains rather high in certain breeds (up to 20–25% in large beef cattle populations, even higher in some local breeds). The continuation, and in some instances the development of the chromosomal screening programs in farm animal populations allowed the implementation of new and original scientific projects, aimed at exploring some basic questions in the fields of chromosome and/or cell biology, thanks to easier access to interesting biological materials (germ cells, gametes, embryos ...).

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe. (c) 2007 Elsevier B.V. All rights reserved.

The mammalian family Tayassuidae (peccaries) is confined to the New World and comprises three recognized extant species, white-lipped (Tayassu pecari), collared (Pecari tajacu) and chacoan (Catagonus wagneri) peccaries, which exhibit distinct morphological and chromosomal features. The phylogenetic relationships among the tayassuids are unclear and have instigated debate over the palaeontological, cytogenetic and molecular aspects. Constitutive heterochromatin analysis can be used in understanding the phylogenetic relationships between related species. Here we describe, for the first time, the constitutive heterochromatin (C-positive heterochromatin) of two tayassuid species, Tayassu pecari and Pecari tajacu. We demonstrate that in situ restriction endonuclease digestion with sequential C-banding could be a complementary tool in the study of constitutive heterochromatin heterogeneity in chromosomes of the Tayassuidae. Our characterization of peccary chromosomes suggests that the Pecari tajacu autosomal karyotype is more primitive and has accumulated great diversity in its constitutive heterochromatin. This idea is supported by several other studies that analysed nuclear and mitochondrial sequences of the living peccary species. Finally, the tayassuid X chromosome primitive form seems to be the one of Tayassu pecari.

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria.In humans,polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case-control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A>C and IL1A /1_g.521T>A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03-0.76),P=0.022; 5.76 (1.03-32.1),P=0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G>T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.

To examine DNA abnormalities in bladder papillary tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female rats, using image cytometric DNA analysis and cytogenetics. Thirty female rats were exposed to BBN in their drinking water for 20 weeks. One group of 10 animals served as controls. The animals exposed to BBN were killed at a rate of two per week, with the bladder being collected under aseptic conditions and those tumours with exophytic growth removed. The nuclear DNA content of the tumours was evaluated using image cytometric analysis. In two rats part of the tumour pieces was stipulated for culturing. Cytogenetic analysis was performed on at least 30 cells from each cell population and on both tumours. Papillary carcinomas were classified as low grade and high grade. DNA ploidy studies were carried out on 28 low-grade and 21 high-grade papillary carcinomas. Histograms obtained by image analysis showed that a normal urothelium was diploid; 28.6% and 100% of low-and high-grade papillary carcinomas were aneuploid respectively. Both tumours used for cell culture showed multiple numerical and structural chromosome alterations and several marker chromosomes. Image cytometric DNA analysis proved to be a good and reliable method for examining DNA alterations in papillary bladder carcinomas. The present findings establish that the DNA content is statistically different between low-grade and high-grade papillary carcinomas and that deviation from the diploid number is markedly higher in the high-grade ones. In addition, the occurrence of marker chromosomes seems to be related to the aggressiveness of the tumour.

Reliable banding techniques are a major necessity for the genetic research in marine bivalves. Restriction enzyme banding (HaeIII) was performed, in this study, on chromosomes of two commercially important species of veneroid bivalves: the clam Ruditapes decussatus (Adams and Reeve) and the cockle Cerastoderma edule. Identification of the nineteen individual chromosome pairs was obtained for both species. The cytogenetic studies made in marine molluscs have recently experienced a very fast development caused by the introduction of new molecular techniques mainly fluorescence in situ hybridization (FISH). Recently it has been shown in mammalian chromosomes that restriction enzyme banding is compatible with FISH, allowing simultaneous banding, and consequent accurate identification of the localization of the probes and unambiguously identification of the chromosome(s) carrier(s). As far as we know this is the first RE-banding obtained in karyotypes of veneroid species. The application of restrictio...

We used chromosome painting with chromosome-specific probes derived from domestic sheep and pig for a high-resolution cytogenetic comparison with the karyotype of collared peccary (Pecari tajacu sonoriensis). A reorganization of the karyotype involving at least 62-66 conserved segments were observed between the sheep and collared peccary. This is an extremely high number compared with other members of the same mammalian order (Cetartiodactyla). The comparison between pig and collared peccary, both belonging to the Suiformes, however, revealed various changes in the gross organization of both karyotypes that may have already occurred in a common ancestor of both species suggesting a monophyletic origin of Suidae/Tayassuidae. The sheep probes, however, also revealed several rearrangements between the two Suidae/Tayassuidae, indicating that these probes represent a useful tool for a more detailed analysis of the evolutionary history of Suiformes. Our sample of the collared peccary from North America (Arizona, USA) showed distinct differences to those already described from South America. The chromosome painting results defined a complex translocation that involves chromosomes including about one-quarter of the entire collared peccary karyotype. This considerable rearrangement indicates subspecies or even species status of both peccary populations, as it should present a significant barrier for their hybridization.

Most mammalian chromosomes have satellite DNA sequences located at or near the centromeres, organized in arrays of variable size and higher order structure. The implications of these specific repetitive DNA sequences and their organization for centromere function are still quite cloudy. In contrast to most mammalian species, the domestic cat seems to have the major satellite DNA family (FA-SAT) localized primarily at the telomeres and secondarily at the centromeres of the chromosomes. In the present work, we analyzed chromosome preparations from a fibrosarcoma, in comparison with nontumor cells (epithelial tissue) from the same individual, by in situ hybridization of the FA-SAT cat satellite DNA family. This repetitive sequence was found to be amplified in the cat tumor chromosomes analyzed. The amplification of these satellite DNA sequences in the cat chromosomes with variable number and appearance (marker chromosomes) is discussed and might be related to mitotic instability, which could explain the exhibition of complex patterns of chromosome aberrations detected in the fibrosarcoma analyzed.

Tritordeum (X Tritordeum Ascherson et Graebner) is a synthetic amphiploid belonging to the Triticeae tribe, which resulted from crosses between Hordeum chilense and wheat. It presents useful agronomic traits that could be transferred to wheat, widening its genetic basis.

Inter-simple-sequence repeat (ISSR) were used to fingerprint three kinds of F1 interspecific hybrids: the Portuguese durum wheat cultivar ‘Candial’ × tritordeum, the Portuguese bread wheat cultivar ‘Barbela’ × tritordeum and the Portuguese triticale cultivar ‘Douro’ × tritordeum. Among the 30 primers tested, the number of discriminative primers varies from 13 to 20 in each hybrid combination, the total number of ISSR loci analysed ranged from 101 to 183, and the number of discriminative ISSR loci varied from 50 to 71. ISSR-PCR technique revealed to be a valuable fingerprint system for all F1 hybrid combinations and respective parents analysed here. The complementary use of molecular cytogenetic techniques and molecular markers could allow a more accurately evaluation and characterisation of different plant species.

The bimodal karyotype of pig appears to contain two types of constitutive heterochromatin, reflecting different satellite DNA families: GC-rich heterochromatin located mainly in the centromeric regions of the biarmed chromosomes, and less-GC-rich heterochromatin in the centromeric regions of the one-armed chromosomes. In order to better discriminate this constitutive heterochromatin, we treated pig chromosome preparations with eight different restriction endonucleases, followed by C-banding. This technique allowed an expedited characterization of the constitutive heterochromatin and demonstrated its great heterogeneity in pig chromosomes. Our work allowed the detection and identification of twenty-two heterochromatin subclasses (twelve centromeric, four interstitial, five telomeric, and the Yq band). Moreover, several cryptic interstitial and telomeric bands were revealed. The work presented here is useful not only for fundamental studies of chromosome banding and constitutive heterochromatin, but also offers a new approach for pig clinical cytogenetics.

Cattle chromosomes were digested with two restriction endonucleases, MspI and HaeIII. The banding pattern induced by each enzyme allowed the identification and paring of all individual chromosomes and consequently the elaboration of the karyotype. This method is rapid and technically easy, and proved to be of great utility in cattle cytogenetic studies.

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.

We have analysed and mapped physically the satellite I, III (subunits pvu and sau) and IV DNA sequences in cattle using in-situ hybridization. Four breeds were analysed including individuals with a chromosome number of 2n=60 and individuals with the widespread t(1;29) in the homozygous (2n=58) and heterozygous state (2n=59). All three satellite DNA families were present at the centromeres of the many but not all of the autosomal acrocentric chromosomes, and essentially absent from the sex chromosomes. In the translocated t(1;29) chromosome, the satellite DNA families showed a different pattern from that simply derived by fusion of the acrocentric autosomes and loss of satellite sequences, with no variation between breeds. A model of centromeric evolution is presented involving two independent events. Knowledge of mechanisms of translocation formation within cattle is important for a functional understanding of centromere and satellites, investigation of chromosomal abnormalities, and for understanding chromosomal fusion during evolution of other bovids and genome evolution in general.

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.

Limited genetic knowledge is available regarding crossability between hexaploid triticale (2n= 6x= 42, 21″, AABBRR, amphiploid Triticum turgidum L.-Secale cereale L.) and rye (2n= 14, 7″, RR). Our objectives were to determine (1) the crossability between triticales and rye and (2) the inheritance of crossability between F2 progeny from intertriticale crosses and rye. First, ‘8F/Corgo’, a hexaploid triticale, was crossed as a female with two landrace ryes, ‘Gimonde’ and, ‘Vila Pouca’ and two derived north European cultivars, ‘Pluto’ and ‘Breno’. These crosses produced 21.7, 20.9, 5.9, and 5.6%, seed-set or crossability, respectively, showing that the landrace ryes produced higher seed-set than the cultivars. Second, ‘Gimonde’ rye was crossed as a male with four triticales for 3 years. The control cross, ‘Chinese Spring’ wheat × rye, produced 80-90% seed-set. Of the four triticales, ‘Beagle’ produced 35.7-56.8% seed-set. The other three triticales produced less than 20% seed-set, showing that the triticales differ in crossability with ‘Gimonde’ rye. Third, six FiS from intertriticale crosses (‘8F/Corgo’בBeagle’, ‘Beagle’בCachirulo’, ‘Lasko’בBeagle’, ‘8F/Corgo’בCachirulo’, ‘Lasko’בCachirulo’, ‘Lasko’ב8F/Corgo’) were crossed to ‘Gimonde’ rye. Results indicated that lower crossability trait was partially dominant in the two F1S from crosses involving ‘Beagle’(high crossability) with‘8F/Corgo’ and ‘Cachirulo’(low crossability) and completely dominant in the ‘Beagle’בLasko’ cross, as it happens in wheat. Fourth, segregants in four F2 populations (‘Lasko’בBeagle’, ‘8F/Corgo’בBeagle’, ‘Lasko’ב8F/Corgo’, and‘8F/Corgo’בCachirulo’) were crossed with rye. Segregation for crossability was observed, although distinct segregation classes were blurred by environmental and perhaps other factors, such as self-incompatibility alleles in rye. Segregation patterns showed that ‘Beagle’, with high crossability to rye, carries either Kr1 or Kr2. The three triticales with low crossability with rye were most likely homozygous for Kr1 and Kr2. Therefore, it is likely that the Kr loci from A and B genomes acting in wheat also play a role in triticale × rye crosses.

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize.