Molecular characterization of the citrate transporter gene TaMATE1 and expression analysis of upstream genes involved in organic acid transport under Al stress in bread wheat (Triticum aestivum).

In bread wheat, besides malate, the importance of citrate efflux for Al tolerance has also been reported. For better understanding the Al tolerance mechanism in bread wheat, here, we performed both a molecular characterization of the citrate transporter gene TaMATE1 and an investigation on the upstream variations in citrate and malate transporter genes. TaMATE1 belong to multidrug transporter protein family, which are located on the long arm of homoeologous group 4 chromosomes (TaMATE1-4A, TaMATE1-4B TaMATE1-4D). TaMATE1 homoeologues transcript expression study exhibited the preponderance of homoeologue TaMATE1-4B followed by TaMATE1-4D whereas homoeologue TaMATE1-4A seemed to be silenced. TaMATE1, particularly homoeologue TaMATE1-4B and TaALMT1 transcripts were much more expressed in the root apices than in shoots of Al tolerant genotype Barbela 7/72/92 under both control and Al stress conditions. In addition, in both tissues of Barbela 7/72/92, higher basal levels of these gene transcripts were observed than in Anahuac (Al sensitive). Noticeably, the presence of a transposon in the upstream of TaMATE1-4B in Barbela 7/72/92 seems to be responsible for its higher transcript expression where it may confer citrate efflux. Thus, promoter variations (transposon in TaMATE1-4B upstream and type VI promoter in TaALMT1) associated with higher basal transcript expression of TaMATE1-4B and TaALMT1 clearly show how different mechanisms for Al tolerance operate simultaneously in a single genotype. In conclusion, our results demonstrate that Barbela 7/72/92 has favorable alleles for these organic acids transporter genes which could be utilized through genomic assisted selection to develop improved cultivars for acidic soils.

Molecular characterization of TaSTOP1 homoeologues and their response to aluminium and proton (H(+)) toxicity in bread wheat (Triticum aestivum L.).

BACKGROUND: Aluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat. RESULTS: TaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H(+)) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat. CONCLUSIONS: Physical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H(+)) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H(+)) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat.

Differential rRNA genes expression in bread wheat and its inheritance.

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.

Interleukin-6 gene -174G>C and -636G>C promoter polymorphisms and prostate cancer risk.

Prostate cancer (PCa) is one of the most commonly diagnosed internal malignancies affecting men. Due to the important roles of IL-6 in different physiological and pathophysiological processes, IL-6 polymorphisms may modulate PCa risk. IL-6 -174 G>C (rs 1800795, also designated -236 G>C) and -636 G>C (rs 1800796, also designated -572 G>C) promoter polymorphisms have been implicated in PCa susceptibility, albeit still controversial. A literature search using PubMed and Highwire databases was conducted, resulting in eight case-control studies concerning the IL-6 -174 G>C polymorphism (11,613 PCa cases and 13,992 controls) and four case-control publications regarding the IL-6 -636 G>C polymorphism (1,941 PCa cases and 3,357 controls). In order to derive a more precise estimation, a meta-analysis based upon these selected case-control studies was performed. There was no significant association between IL-6 -174 G>C polymorphism and PCa increased risk. Nevertheless, the presence of allele C and the CC genotype were statistically significantly associated with decreased PCa risk in the overall analysis for IL-6 -636 G>C polymorphism. Additional studies in larger samples and analyses of functional repercussions of these SNPs in prostate tumor cells are necessary to validate these findings.

Analysis of new lactotransferrin gene variants in a case-control study related to periodontal disease in dog.

The molecular and genetic research has contributed to a better understanding of the periodontal disease (PD) in humans and has shown that many genes play a role in the predisposition and progression of this complex disease. Variations in human lactotransferrin (LTF) gene appear to affect anti-microbial functions of this molecule, influencing the PD susceptibility. PD is also a major health problem in small animal practice, being the most common inflammatory disease found in dogs. Nevertheless, the research in genetic predisposition to PD is an unexplored subject in this species. This work aims to contribute to the characterization of the genetic basis of canine PD. In order to identify genetic variations and verify its association with PD, was performed a molecular analysis of LTF gene in a case-control approach, including 40 dogs in the PD cases group and 50 dogs in the control group. In this study were detected and characterized eight new single nucleotide variations in the dog LTF gene. Genotype and allele frequencies of these variations showed no statistically significant differences between the control and PD cases groups. Our data do not give evidence for the contribution of these LTF variations to the genetic background of canine PD. Nevertheless, the sequence variant L/15_g.411C > T leads to an aminoacid change (Proline to Leucine) and was predicted to be possibly damaging to the LTF protein. Further investigations would be of extreme value to clarify the biological importance of these new findings.

Sequence variants and haplotype analysis of cat ERBB2 gene: a survey on spontaneous cat mammary neoplastic and non-neoplastic lesions.

The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine.

An efficient method for genomic DNA extraction from different molluscs species.

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.

Polymerase chain reaction-single strand conformation polymorphism applied to sex identification of Accipiter cooperii.

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.

Genetic diversity and variation among botanical varieties of old Portuguese wheat cultivars revealed by ISSR assays.

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.

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues.

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.

Development of Colletotrichum acutatum on tolerant and susceptible Olea europaea L. cultivars: a microscopic analysis.

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192 h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.

Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae).

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

Suiformes orthologous satellite DNAs as a hallmark of Pecari tajacu and Tayassu pecari (Tayassuidae) evolutionary rearrangements.

In a broad general way, eukaryotic satellite DNA sequences are characterized by a highly dynamic molecular behavior due to concerted evolution that leads to rapid change between repeat sequences of different species, achieved by amplification of new variants during speciation or by gradual sequence evolution due to the accumulation of nucleotide substitutions. There are, although exceptions for this almost universal rule. We isolated variants from both the Mc1 and Ac2 pig (Sus scrofa, Suidae) satellite DNA families from the genomes of two Tayassuidae members: Pecari tajacu and Tayassu pecari, which have highly derived karyotypes. The presence of these sequences in both families' genomes (Suidae and Tayassuidae) implies their existence in a common ancestor, what confers to the variants the status of orthology and the approximate age of, at least 40 million years. While at the molecular composition level these orthologous sequences are highly homologous, cross-species physical mapping revealed a completely different chromosomal location in Suidae versus Tayassuidae families, most probably, reflecting the high level of divergence and chromosomes evolution pathways after radiation of each family. Detailed comparative analysis of the satellites assignment on the peccary's chromosomes revealed its co-localization with homologous evolutionary breakpoints in both species, suggesting their involvement in the rearrangement events. The complex behavior of the repeats evolution in the pig/peccaries genomes is here clearly illustrated. These sequences are molecularly preserved for a considerable period of time and display slow rates of sequence change, but show a dynamic motion behavior throughout the peccary's genomes that accompanied the great architectonic reorganization of Tayassuidae chromosomes during evolution.

The karyotype and sex chromosomes of Praomys tullbergi (Muridae, Rodentia): a detailed characterization.

Here we present the first detailed characterization of Praomys tullbergi karyotype, enlightening several chromosome features such as constitutive heterochromatin, telomeric and LINE-1 sequences. The combination of these approaches provided some interesting insights about the genome organization of this African species, which is one of the tullbergi complex elements, a group of species belonging to Murinae (Rodentia, Muridae). Evolutionary considerations on Praomys chromosomes were also achieved, namely, the autosomal complement and the X chromosome from P. tullbergi seem to be derivative chromosomes, most probably resulting from extensive reshufflings during the course of evolution. This conclusion came from the fact that the majority of the chromosomes telomeric sequences are located interstitially, seeming footprints of evolutionary chromosome rearrangements. The detailed analysis of Praomys tullbergi X chromosome suggests that chromosome rearrangements and/or centromere transpositions and addition/elimination of heterochromatin must have been the main evolutionary events that shaped this chromosome.

Different evolutionary trails in the related genomes Cricetus cricetus and Peromyscus eremicus (Rodentia, Cricetidae) uncovered by orthologous satellite DNA repositioning.

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.

Identification and characterization of four splicing variants of ovine POU1F1 gene.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly (p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.

Phylogenetic relationships and the primitive X chromosome inferred from chromosomal and satellite DNA analysis in Bovidae.

The early phylogeny of the 137 species in the Bovidae family is difficult to resolve; knowledge of the evolution and relationships of the tribes would facilitate comparative mapping, understanding chromosomal evolution patterns and perhaps assist breeding and domestication strategies. We found that the study of the presence and organization of two repetitive DNA satellite sequences (the clone pOaKB9 from sheep, a member of the 1.714 satellite I family and the pBtKB5, a 1.715 satellite I clone from cattle) on the X and autosomal chromosomes by in situ hybridization to chromosomes from 15 species of seven tribes, was informative. The results support a consistent phylogeny, suggesting that the primitive form of the X chromosome is acrocentric, and has satellite I sequences at its centromere. Because of the distribution of the ancient satellite I sequence, the X chromosome from the extant Tragelaphini (e.g. oryx), rather than Caprini (sheep), line is most primitive. The Bovini (cow) and Tragelaphini tribes lack the 1.714 satellite present in the other tribes, and this satellite is evolutionarily younger than the 1.715 sequence, with absence of the 1.714 sequence being a marker for the Bovini and Tragelaphini tribes (the Bovinae subfamily). In the other tribes, three (Reduncini, Hippotragini and Aepycerotini) have both 1.714 and 1.715 satellite sequences present on both autosomes and the X chromosome. We suggest a parallel event in two lineages, leading to X chromosomes with the loss of 1.715 satellite from the Bovini, and the loss of both 1.714 and 1.715 satellites in a monophyletic Caprini and Alcelaphini lineage. The presence and X chromosome distribution of these satellite sequences allow the seven tribes to be distributed to four groups, which are consistent with current diversity estimates, and support one model to resolve points of separation of the tribes.

Chromosome restriction enzyme digestion in domestic pig (Sus scrofa) constitutive heterochromatin arrangement.

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.

A case-control study between interleukin-10 gene variants and periodontal disease in dogs.

Periodontal disease (PD) refers to a group of inflammatory diseases that affect the periodontium, the organ which surrounds and supports the teeth. PD is a highly prevalent disease with a multifactorial etiology and, in humans the individual susceptibility is known to be strongly determined by genetic factors. Several candidate genes have been studied, namely genes related with molecules involved in the inflammatory response. Interleukin-10 (IL-10) is a cytokine with important anti-inflammatory and immunomodulatory roles, and several studies indicate an association between IL10 polymorphisms and PD. In dogs, an important animal model in periodontology, PD is also a highly prevalent naturally occurring disease, and only now are emerging the first studies evaluating the genetic predisposition. In this case-control study, a population of 90 dogs (40 dogs with PD and 50 healthy dogs) was used to study the IL10 gene, and seven new genetic variations in this gene were identified. No statistically significant differences were detected in genotype and allele frequencies of these variations between the PD cases and control groups. Nevertheless, one of the variations (IL10/2_g.285G>A) leads to an amino acid change (glycine to arginine) in the putative signal peptide, being predicted a potential influence on IL-10 protein functionality. Further investigations are important to clarify the biological importance of these new findings. The knowledge of these genetic determinants can help to understand properly the complex causal pathways of PD, with important clinical implications.

Analysis of new Matrilin-1 gene variants in a case-control study related to dental malocclusions in Equus asinus.

Prognathism and brachygnathism are craniofacial deformities that severely affect the health of human and vertebrates, such as donkeys. The multifactorial etiology of this disease makes the genetic analysis a powerful tool for its understanding and prevention of spreading these deformities. This study aims to contribute to the characterization of the genetic basis of prognathism and brachygnathism in donkeys, using the Zamorano-Leonés donkey, an endangered Spanish breed, as a model. Matrilin-1 (MATN1) polymorphisms have been previously described as markers for mandibular prognathism in Korean and Japanese human populations. Genetic variations in MATN1 gene were sought, in order to verify its association in a case-control study, including 30 donkeys presenting brachygnathism, 30 donkeys presenting prognathism and 30 donkeys with normal occlusion phenotypes. One genetic variation (g503G > A) located in an intronic region of MATN1 gene was identified and characterized. Statistically significant differences were detected between the control group and prognathism cases, but no statistical significant results were found between the control group and the brachygnathism cases. These results support evidence for an important role of MATN1 on prognathism in the analyzed population with MATN1 genetic variation - 503G>A - having a protective effect. Further studies should be developed in order to understand the whole role of MATN1 and the mechanisms affected by its genetic variations.