Molecular cloning of TaMATE2 homoeologues potentially related to aluminium tolerance in bread wheat (Triticum aestivum L.).

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.

LINE-1 distribution in six rodent genomes follow a species-specific pattern.

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.

Variants in the interleukin-1 alpha and beta genes, and the risk for periodontal disease in dogs.

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.

HLA alleles and HLA-B27 haplotypes associated with susceptibility and severity of ankylosing spondylitis in a Portuguese population.

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<0.0001; odds ratio (OR) = 39.06; 95% confidence interval (CI) (2.34-651)] is the only haplotype that seems to confer susceptibility to AS. Moreover, the haplotype A*02/B27/C*01/DRB1*08/DQB1*04 seems to provide protection for disease functional and radiological repercussions. Our findings are compatible with the hypothesis that other genes within the HLA region besides HLA-B27 might play some role in AS susceptibility and severity.

Genetic differences between wild and hatchery populations of Diplodus sargus and D. vulgaris inferred from RAPD markers: implications for production and restocking programs design.

Restocking and stock enhancement programs are now recognized as an important tool for the management of fishery resources. It is important, however, to have an adequate knowledge on the genetic population structure of both the released stock and the wild population before carrying out such programs. In this study, random amplified polymorphic DNA (RAPD) markers were applied to assess genetic diversity and population structure of wild and hatchery populations of the white seabream Diplodus sargus and the common two-banded seabream D. vulgaris (Sparidae). The estimated values for intrapopulation genetic variation, measured using the percentage of polymorphic loci (%P), Shannon index (H'), and Nei's gene diversity (h), showed high values for all populations. The percentage of genetic variation within D. sargus and D. vulgaris populations, based on coefficient of gene differentiation, reached 82.5% and 90% of the total genetic variation, respectively. An undeniable decrease in genetic variation was found in both hatchery populations, particularly in D. sargus, compared to the wild ones. However, the high values of variation within all populations and the low levels of genetic variation among populations did not indicate inbreeding or depression effects, thus indicating a fairly proper hatchery management. Nevertheless, the results of this study highlight the importance of monitoring the genetic variation of hatchery populations, particularly those to be used in restocking programs. The creation of a genetic baseline database will contribute to a more efficient conservation management and to the design of genetically sustainable restocking programs.

Satellite DNA in the karyotype evolution of domestic animals--clinical considerations.

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.

Association of IL23R and ERAP1 genes with ankylosing spondylitis in a Portuguese population.

OBJECTIVE: Association between ankylosing spondylitis (AS) and two genes, ERAP1 and IL23R, has recently been reported in North American and British populations. The population attributable risk fraction for ERAP1 in this study was 25%, and for IL23R, 9%. Confirmation of these findings to ERAP1 in other ethnic groups has not yet been demonstrated. We sought to test the association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to AS among a Portuguese population. We also investigated the role of these genes in clinical manifestations of AS, including age of symptom onset, the Bath Ankylosing Spondylitis Disease Activity, Metrology and Functional Indices, and the modified Stoke Ankylosing Spondylitis Spinal Score. METHODS: The study was conducted on 358 AS cases and 285 ethnically matched Portuguese healthy controls. AS was defined according to the modified New York Criteria. Genotyping of IL23R and ERAP1 allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests of genotypes as implemented in PLINK for dichotomous and quantitative variables respectively. A meta-analysis for Portuguese and previously published Spanish IL23R data was performed using the StatsDirect Statistical tools, by fixed and random effects models. RESULTS: A total of 14 nsSNPs markers (8 for IL23R, 5 for ERAP1, 1 for LN-PEP) were analysed. Three markers (2 for IL23R and 1 for ERAP1) showed significant single-locus disease associations, confirming that the association of these genes with AS in the Portuguese population. The strongest associated SNP in IL23R was rs1004819 (OR=1.4, p=0.0049), and in ERAP1 was rs30187 (OR=1.26, p=0.035). The population attributable risk fractions in the Portuguese population for these SNPs are 11% and 9.7% respectively. No association was seen with any SNP in LN-PEP, which flanks ERAP1 and was associated with AS in the British population. No association was seen with clinical manifestations of AS. CONCLUSION: These results show that IL23R and ERAP1 genes are also associated with susceptibility to AS in the Portuguese population, and that they contribute a significant proportion of the population risk for this disease.

Chromosomal organization of simple sequence repeats in the Pacific oyster (Crassostrea gigas): (GGAT)(4), (GT)(7) and (TA)(10) chromosome patterns.

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.

Cytogenetic screening of livestock populations in Europe: an overview.

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 ...).

A complex intersex condition in a Holstein calf.

A case of disrupted embryonic development of the genital tract in a newborn Holstein calf is described. The physical examination of the calf evidenced several abnormalities, like atresia ani, rudimentary external genitalia and caudal vertebral agenesis. On necropsy, the excised genitalia consisted of bilateral streak gonads, apparently normal uterine tubes, a fluid-filled uterus, a long vagina and a very narrow clitoris-like structure covered with a discrete skin-fold. The urinary tract seemed normal and the urethra's opening was at the vestibule-vaginal junction. A cytogenetic analysis was requested. Karyotype revealed the existence of Y chromosome material in the two X chromosomes. However, the search for the sex-determining region Y (SRY) showed that this was an apparently absent gene. The histological examination of the gonads revealed the existence of ovarian dysplasia. Uterine sections evidenced the absence of the uterine epithelium, with only sporadic caruncles. Under microscopic examination, the uterine tubes and vagina structure was normal. The external genitalia sections revealed the existence of a skin-fold covering an erectile structure surrounding the urethra, a structure more similar to a penis than to a clitoris. This is an unusual situation of gonadal dysplasia combined with genital tract anomalies in cattle, probably associated to a genetic defect.

[Genetic basis of ankylosing spondylitis]. / Espondilite Anquilosante e sua base genética.

Ankylosing spondylitis (AS) is a common rheumatic condition, highly heritable. Much of the genetic contribution to the disease lies in the major histocompatibility complex (MHC). The association with the allele group HLA-B*27 has been described worldwide for 30 years. On the other hand, genome wide scans have provided some interesting results showing that other MHC and non-MHC genes could be implicated either in disease susceptibility and phenotypic manifestations. Different hypothesis for disease pathophysiology have been investigated which contribute for a better understanding of the genetic basis of AS. This review aims to summarize the status of the knowledge in this exciting area. New data may, in a near future, change the screening of patients and generate new insights for the emergence of novel therapies.

Chromosomal evolution and phylogenetic analyses in Tayassu pecari and Pecari tajacu (Tayassuidae): tales from constitutive heterochromatin.

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.

Morphological, yield, cytological and molecular characterization of a bread wheat x tritordeum F1 hybrid.

The morphological, yield, cytological and molecular characteristics of bread wheat x tritordeum F(1) hybrids (2n = 6x = 42; AABBDH(ch)) 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 H(ch) and A, B or D chromosomes. The average number of univalents was higher in the wheat genome (6.8) than in the H(ch) 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 F(1) 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.

Cattle rob(1;29) originating from complex chromosome rearrangements as revealed by both banding and FISH-mapping techniques.

Sixteen carriers of rob(1;29) (one of which was homozygous) from six different breeds (four Italian and two Portuguese), two heterozygous carriers of rob(26;29), three river buffaloes and two sheep were cytogenetically investigated in this study by using banding and FISH-mapping techniques (the latter only in cattle and river buffalo). Single- and dual- colour FISH were used with bovine probes containing both INRA143 (mapping proximally to BTA29) and bovine satellite (SAT) DNA SAT I, SAT III and SAT IV (mapping at the centromeric regions of cattle chromosomes). The combined use of these probes, the comparison of rob(1;29) with the dicentric rob(26;29) and with both river buffalo and sheep chromosomes (biarmed pairs) allowed us to hypothezise that rob(1;29) originated from complex chromosomal rearrangements through at least three sequential events: (a) centric fusion with the formation of a dicentric chromosome; (b) formation of a monocentric chromosome with loss of SAT I from both BTA1 and BTA29, most of SAT IV from BTA29 and, probably, some repeats of SAT III from BTA1; (c) double pericentric inversion or, more probably, a chromosome transposition of a small chromosome segment containing INRA143 from proximal p-arms to proximal q-arm of the translocated chromosome.

Physical organization of the 1.709 satellite IV DNA family in Bovini and Tragelaphini tribes of the Bovidae: sequence and chromosomal evolution.

Repetitive DNA in the mammalian genome is a valuable record and marker for evolution, providing information about the order and driving forces related to evolutionary events. The evolutionarily young 1.709 satellite IV DNA family is present near the centromeres of many chromosomes in the Bovidae. Here, we isolated 1.709 satellite DNA sequences from five Bovidae species belonging to Bovini: Bos taurus (BTA, cattle), Bos indicus (BIN, zebu), Bubalus bubalis (BBU, water buffalo) and Tragelaphini tribes: Taurotragus oryx (TOR, eland) and Tragelaphus euryceros (TEU, bongo). Its presence in both tribes shows the sequence predates the evolutionary separation of the two tribes (more than 10 million years ago), and primary sequence shows increasing divergence with evolutionary distance. Genome organization (Southern hybridization) and physical distribution (in situ hybridization) revealed differences in the molecular organization of these satellite DNA sequences. The data suggest that the sequences on the sex chromosomes and the autosomes evolve as relatively independent groups, with the repetitive sequences suggesting that Bovini autosomes and the Tragelaphini sex chromosomes represent the more primitive chromosome forms.

Restriction enzyme digestion chromosome banding in Crassostrea and Ostrea species: comparative karyological analysis within Ostreidae.

Reliable banding techniques are a major necessity for genetic research in oysters. In this study, we carried out the cytogenetic characterization of four oyster species (family Ostreidae) using restriction endonuclease treatments. Chromosomes were treated with three different restriction enzymes, stained with Giemsa, and examined for banding patterns. The following species were studied: Crassostrea gigas (2n = 20; total number of bands with ApaI, 74; HaeIII, 61; PstI, 76), Crassostrea angulata (2n = 20; ApaI, 62; HaeIII, 61; PstI, 55) (subfamily Crassostreinae), Ostrea edulis (2n = 20; ApaI, 82; HaeIII, 59; PstI, 66), and Ostrea conchaphila (2n = 20; ApaI, 68; HaeIII, 62; PstI, 69) (subfamily Ostreinae). Treatment of samples with ApaI, HaeIII, and PstI produced specific banding patterns, which demonstrates the potential of these enzymes for chromosome banding in oysters. This is of special interest, since it has been recently shown in mammalian chromosomes that restriction enzyme banding is compatible with fluorescence in situ hybridization. This study therefore provides a fundamental step in genome mapping of oysters, since chromosome banding with restriction enzymes facilitates physical gene mapping in these important aquaculture species. The analysis of the banded karyotypes revealed a greater similarity within the genera of Crassostrea and Ostrea than between them.

High levels of genetic diversity throughout the range of the Portuguese wheat landrace 'Barbela'.

BACKGROUND AND AIMS: Landrace populations represent an important intra-crop reservoir of biodiversity and source of novel gene alleles for use in breeding programmes. Here the aim was to measure the diversity of a wheat landrace, 'Barbela', from the north of Portugal. METHODS: DNA was extracted from 59 accessions of Barbela collected across its geographical range. Diversity was measured by microsatellite length polymorphisms using 27 primer pairs amplifying 34 polymorphic microsatellite loci. KEY RESULTS: High levels of polymorphism were found, with an average polymorphism information content of 0.52; an average of 4.77 alleles (range 2-11) were present at each locus, and half of these loci showed an additional allele in the reference variety 'Chinese Spring'. CONCLUSIONS: 'Barbela' is maintained from seeds collected by farmers, but it maintains high allelic variation, and no groupings of accessions were detected when analysed by geographical region, farm or climate, indicating that the wheat landrace is a homogeneous entity. The diversity within the farmer-maintained landrace demonstrates the importance of characterization and maintenance of landrace collections before valuable genetic combinations are lost as uniform commercial crops are introduced.

Chromosomal localization of the major satellite DNA family (FA-SAT) in the domestic cat.

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.