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Ralstonia solanacearum is a β-proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain-specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex. [ABSTRACT FROM AUTHOR]

The photocatalytic degradation of carbaryl, present in aqueous solutions containing suspended TiO2 particles, has been studied in a continuous flow system, using a polytetrafluoroethylene photochemical reactor, equipped with an UV lamp. In order to optimize the process the effect of pH, catalyst and pesticide concentration, and irradiation time have been evaluated, working in the best conditions, the measured half‐life of carbaryl was 26 seconds. More than 99% of carbaryl present in solutions containing 0.2 mgL‐1 of pesticide decomposes after less than 1 minute irradiation. [ABSTRACT FROM PUBLISHER]

14 children, aged between 15 months and 14 years, with brucellosis were treated with oral rifampicin only (20 mg/kg per day) for 3 weeks. Laboratory diagnosis depended on blood culture (positive for Brucella melitensis in 11 of the cases), serum agglutination, complement-fixation test, and Coomb's test. Response was good in each child, with fever clearing between the 2nd and 8th day. 2 children relapsed, but one relapse was probably a reinfection from contaminated cheese. Both children were given a further course of treatment (rifampicin and co-trimoxazole) which was successful. Despite the reasonably good results with rifampicin alone, it is advisable to combine the drug with co-trimoxazole when treating brucellosis. [ABSTRACT FROM AUTHOR]

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients., (© 2024. The Author(s).)

Background: The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion., Results: Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples., Conclusions: The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them., (© 2022. The Author(s).)

Background: Interspecific hybridizations and admixtures were key in Citrus domestication, but very little is known about their impact at the transcriptomic level. To determine the effects of genome introgressions on gene expression, the transcriptomes of the pulp and flavedo of three pure species (citron, pure mandarin and pummelo) and four derived domesticated genetic admixtures (sour orange, sweet orange, lemon and domesticated mandarin) have been analyzed at color break., Results: Many genes involved in relevant physiological processes for domestication, such sugar/acid metabolism and carotenoid/flavonoid synthesis, were differentially expressed among samples. In the low-sugar, highly acidic species lemon and citron, many genes involved in sugar metabolism, the TCA cycle and GABA shunt displayed a reduced expression, while the P-type ATPase CitPH5 and most subunits of the vacuolar ATPase were overexpressed. The red-colored species and admixtures were generally characterized by the overexpression in the flavedo of specific pivotal genes involved in the carotenoid biosynthesis, including phytoene synthase, ζ-carotene desaturase, β-lycopene cyclase and CCD4b, a carotenoid cleavage dioxygenase. The expression patterns of many genes involved in flavonoid modifications, especially the flavonoid and phenylpropanoid O-methyltransferases showed extreme diversity. However, the most noticeable differential expression was shown by a chalcone synthase gene, which catalyzes a key step in the biosynthesis of flavonoids. This chalcone synthase was exclusively expressed in mandarins and their admixed species, which only expressed the mandarin allele. In addition, comparisons between wild and domesticated mandarins revealed that the major differences between their transcriptomes concentrate in the admixed regions., Conclusion: In this work we present a first study providing broad evidence that the genome introgressions that took place during citrus domestication largely shaped gene expression in their fruits., (© 2022. The Author(s).)

We performed genomic analyses on wild species of the genus Citrus to identify major determinants of evolution. The most notable effect occurred on the pathogen-defense genes, as observed in many other plant genera. The gene space was also characterized by changes in gene families intimately related to relevant biochemical properties of citrus fruit, such as pectin modifying enzymes, HDR (4-hydroxy-3-methylbut-2-enyl diphosphate reductase) genes, and O-methyltransferases. Citrus fruits are highly abundant on pectins and secondary metabolites such as terpenoids and flavonoids, the targets of these families. Other gene types under positive selection, expanded through tandem duplications and retained as triplets from whole genome duplications, codified for purple acid phosphatases and MATE-efflux proteins. Although speciation has not been especially rapid in the genus, analyses of selective pressure at the codon level revealed that the extant species evolved from the ancestral citrus radiation show signatures of pervasive adaptive evolution and is therefore potentially responsible for the vast phenotypic differences observed among current species., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

We performed genomic analyses on species and varieties of the genus Citrus to identify several determinants of domestication, based on the pattern of pummelo [Citrus maxima (Burr. f) Merr] and mandarin (Citrus reticulata Blanco) admixture into the ancestral genome, as well as population genetic tests at smaller scales. Domestication impacted gene families regulating pivotal components of citrus flavor (such as acidity) because in edible mandarin varieties, chromosome areas with negative Tajimas values were enriched with genes associated with the regulation of citric acid. Detection of sweeps in edible mandarins that diverged from wild relatives indicated that domestication reduced chemical defenses involving cyanogenesis and alkaloid synthesis, thus increasing palatability. Also, a cluster of SAUR genes in domesticated mandarins derived from the pummelo genome appears to contain candidate genes controlling fruit size. Similarly, conserved stretches of pure mandarin areas were likely important as well for domestication, as, for example, a fragment in chromosome 1 that is involved in the apomictic reproduction of most edible mandarins. Interestingly, our results also support the hypothesis that various genes subject to selective pressure during evolution or derived from whole genome duplication events later became potential targets of domestication., (© 2021 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Background: Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species., Results: The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides., Conclusion: Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.

Copper(II) sulfate-loaded chitosan microparticles were herein prepared using ionic cross-linking with sodium tripolyphosphate (STPP) followed by spray drying. The microencapsulation process was optimal using an inlet temperature of 180 °C, a liquid flow-rate of 290 mL/h, an aspiration rate of 90%, and an atomizing gas flow-rate of 667 nL/h. Chitosan particles containing copper(II) sulfate of approximately 4 µm with a shrunken-type morphology were efficiently attained and, thereafter, fixated on a paper substrate either via cross-linking with STPP or using a chitosan hydrogel. The latter method led to the most promising system since it was performed at milder conditions and the original paper quality was preserved. The developed cellulose substrates were reduced and then exposed to different humidity conditions and characterized using colorimetric measurements in order to ascertain their potential as irreversible indicators for moisture detection. The results showed that the papers coated with the copper(II) sulfate-containing chitosan microparticles were successfully able to detect ambient moisture shown by the color changes of the coatings from dark brown to blue, which can be easily seen with the naked eye. Furthermore, the chitosan microparticles yielded no cytotoxicity in an in vitro cell culture experiment. Therefore, the cellulose substrates herein developed hold great promise in paper packaging as on-package colorimetric indicators for monitoring moisture in real time.

Citrus is an extremely important genus in terms of world fruit production. Despite its economic importance and the small genome sizes of its species (2 n = 18, 1C = 430 ± 68 Mbp), entire genomic assemblies have only recently become available for some of its representatives. Together with the previous CMA/DAPI banding and fluorescence in situ hybridization (FISH) in the group, these data are important for understanding the complex relationships between its species and for assisting breeding programs. To anchor genomic data with the cytogenetic map of mandarin ( Citrus reticulata ), the parental species of several economically important hybrids such as sweet orange and clementine, 18 BAC (bacterial artificial chromosome) clones were used. Eleven clementine BACs were positioned by BAC-FISH, doubling the number of chromosome markers so far available for BAC-FISH in citrus. Additionally, six previously mapped BACs were end-sequenced, allowing, together with one BAC previously sequenced, their assignment to scaffolds and the subsequent integration of chromosomes and the genome assembly. This study therefore established correlations between mandarin scaffolds and chromosomes, allowing further structural genomic and comparative study with the sweet orange genome, as well as insights into the chromosomal evolution of the group.

Speciation of the genus Citrus from a common ancestor has recently been established to begin ∼8 Ma during the late Miocene, a period of major climatic alterations. Here, we report the changes in activity of Citrus LTR retrotransposons during the process of diversification that gave rise to the current Citrus species. To reach this goal, we analyzed four pure species that diverged early during Citrus speciation, three recent admixtures derived from those species and an outgroup of the Citrus clade. More than 30,000 retrotransposons were grouped in ten linages. Estimations of LTR insertion times revealed that retrotransposon activity followed a species-specific pattern of change that could be ascribed to one of three different models. In some genomes, the expected pattern of gradual transposon accumulation was suddenly arrested during the radiation of the ancestor that gave birth to the current Citrus species. The individualized analyses of retrotransposon lineages showed that in each and every species studied, not all lineages follow the general pattern of the species itself. For instance, in most of the genomes, the retrotransposon activity of elements from the SIRE lineage reached its highest level just before Citrus speciation, while for Retrofit elements, it has been steadily growing. Based on these observations, we propose that Citrus retrotransposons may respond to stressful conditions driving speciation as a part of the genetic response involved in adaptation. This proposal implies that the evolving conditions of each species interact with the internal regulatory mechanisms of the genome controlling the proliferation of mobile elements., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Harvest time is a relevant economic trait in citrus, and selection of cultivars with different fruit maturity periods has a remarkable impact in the market share. Generation of early- and late-maturing cultivars is an important target for citrus breeders, therefore, generation of knowledge regarding the genetic mechanisms controlling the ripening process and causing the early and late phenotypes is crucial. In this work we analyze the evolution of the transcriptome during fruit ripening in 3 sport mutations derived from the Fina clementine (Citrus clementina) mandarin: Clemenules (CLE), Arrufatina (ARR) and Hernandina (HER) that differ in their harvesting periods. CLE is considered a mid-season cultivar while ARR and HER are early- and late-ripening mutants, respectively., Results: We used RNA-Seq technology to carry out a time course analysis of the transcriptome of the 3 mutations along the ripening period. The results indicated that in these mutants, earliness and lateness during fruit ripening correlated with the advancement or delay in the expression of a set of genes that may be implicated in the maturation process. A detailed analysis of the transcription factors known to be involved in the regulation of fruit ripening identified a member of the MADS box family whose expression was lower in ARR, the early-ripening mutant, and higher in HER, the late-ripening mutant. The pattern of expression of this gene during the maturation period was basically contrary to those of the ethylene biosynthetic genes, SAM and ACC synthases and ACC oxidase. The gene was present in hemizygous dose in the early-ripening mutant., Conclusions: Our analysis provides new clues about the genetic control of fruit ripening in citrus and allowed the identification of a transcription factor that could be involved in the early phenotype.

Fruits have been traditionally classified into two categories based on their capacity to produce and respond to ethylene during ripening. Fruits whose ripening is associated to a peak of ethylene production and a respiration burst are referred to as climacteric, while those that are not are referred to as non-climacteric. However, an increasing body of literature supports an important role for ethylene in the ripening of both climacteric and non-climacteric fruits. Genome and transcriptomic data have become available across a variety of fruits and we leverage these data to compare the structure and transcriptional regulation of the ethylene receptors and related proteins. Through the analysis of four economically important fruits, two climacteric (tomato and apple), and two non-climacteric (grape and citrus), this review compares the structure and transcriptional regulation of the ethylene receptors and related proteins in both types of fruit, establishing a basis for the annotation of ethylene-related genes. This analysis reveals two interesting differences between climacteric and non-climacteric fruit: i) a higher number of ETR genes are found in climacteric fruits, and ii) non-climacteric fruits are characterized by an earlier ETR expression peak relative to sugar accumulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The genus Citrus, comprising some of the most widely cultivated fruit crops worldwide, includes an uncertain number of species. Here we describe ten natural citrus species, using genomic, phylogenetic and biogeographic analyses of 60 accessions representing diverse citrus germ plasms, and propose that citrus diversified during the late Miocene epoch through a rapid southeast Asian radiation that correlates with a marked weakening of the monsoons. A second radiation enabled by migration across the Wallace line gave rise to the Australian limes in the early Pliocene epoch. Further identification and analyses of hybrids and admixed genomes provides insights into the genealogy of major commercial cultivars of citrus. Among mandarins and sweet orange, we find an extensive network of relatedness that illuminates the domestication of these groups. Widespread pummelo admixture among these mandarins and its correlation with fruit size and acidity suggests a plausible role of pummelo introgression in the selection of palatable mandarins. This work provides a new evolutionary framework for the genus Citrus.

Background: After its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes., Results: A collection of 217 rice varieties mainly cultivated in temperate regions was generated. The collection encompasses modern elite and old cultivars, as well as traditional landraces covering a wide genetic diversity available for rice breeders. Whole Genome Sequencing was performed on 14 cultivars representative of the collection and the genomic profiles of all cultivars were constructed using a panel of 2697 SNPs with wide coverage throughout the rice genome, obtained from the sequencing data. The population structure and genetic relationship analyses showed a strong substructure in the temperate rice population, predominantly based on grain type and the origin of the cultivars. Dendrogram also agrees population structure results., Conclusions: Based on SNP markers, we have elucidated the genetic relationship and the degree of genetic diversity among a collection of 217 temperate rice varieties possessing an enormous variety of agromorphological and physiological characters. Taken together, the data indicated the occurrence of relatively high gene flow and elevated rates of admixture between cultivars grown in remote regions, probably favoured by local breeding activities. The results of this study significantly expand the current genetic resources available for temperate varieties of rice, providing a valuable tool for future association mapping studies.

Previous RNA-Seq studies in citrus have been focused on physiological processes relevant to fruit quality and productivity of the major species, especially sweet orange. Less attention has been paid to vegetative or reproductive tissues, while most Citrus species have never been analysed. In this work, we characterized the transcriptome of vegetative and reproductive tissues from 12 Citrus species from all main phylogenetic groups. Our aims were to acquire a complete view of the citrus transcriptome landscape, to improve previous functional annotations and to obtain genetic markers associated with genes of agronomic interest. 28 samples were used for RNA-Seq analysis, obtained from 12 Citrus species: C. medica, C. aurantifolia, C. limon, C. bergamia, C. clementina, C. deliciosa, C. reshni, C. maxima, C. paradisi, C. aurantium, C. sinensis and Poncirus trifoliata. Four different organs were analysed: root, phloem, leaf and flower. A total of 3421 million Illumina reads were produced and mapped against the reference C. clementina genome sequence. Transcript discovery pipeline revealed 3326 new genes, the number of genes with alternative splicing was increased to 19,739, and a total of 73,797 transcripts were identified. Differential expression studies between the four tissues showed that gene expression is overall related to the physiological function of the specific organs above any other variable. Variants discovery analysis revealed the presence of indels and SNPs in genes associated with fruit quality and productivity. Pivotal pathways in citrus such as those of flavonoids, flavonols, ethylene and auxin were also analysed in detail., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Citrus genus includes some of the most important cultivated fruit trees worldwide. Despite being extensively studied because of its commercial relevance, the origin of cultivated citrus species and the history of its domestication still remain an open question. Here, we present a phylogenetic analysis of the chloroplast genomes of 34 citrus genotypes which constitutes the most comprehensive and detailed study to date on the evolution and variability of the genus Citrus. A statistical model was used to estimate divergence times between the major citrus groups. Additionally, a complete map of the variability across the genome of different citrus species was produced, including single nucleotide variants, heteroplasmic positions, indels (insertions and deletions), and large structural variants. The distribution of all these variants provided further independent support to the phylogeny obtained. An unexpected finding was the high level of heteroplasmy found in several of the analyzed genomes. The use of the complete chloroplast DNA not only paves the way for a better understanding of the phylogenetic relationships within the Citrus genus but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance, heteroplasmy, and gene selection., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored., Results: Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion. The Arrufatina Mule displayed "dissimilar" 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome., Conclusions: Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes--a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes--and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.

Background: Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a 'Mediterranean' mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine., Results: Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome., Conclusions: A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the 'Mediterranean' mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Premise of the Study: Indel markers were developed from BAC-end sequences of Citrus clementina cv. Nules. Transferability and polymorphism were tested in the Citrus genus to estimate the potential of indel markers mined from a single genotype for use in genetic studies., Methods and Results: Using polyacrylamide gel electrophoresis and DNA silver staining, 89 indel markers were tested for their transferability and polymorphism. Thirty-eight markers were selected. Heterozygosity in C. clementina cv. Nules was confirmed for 33 of these indel pairs. A preliminary diversity study using a capillary electrophoresis fragment analyzer was conducted with 21 indels using 45 accessions representing Citrus genus diversity. Intraspecific and interspecific polymorphisms were observed., Conclusions: These results indicate the utility of indel markers developed from sequence data of a single genotype of interspecific origin. In Citrus, these markers will be useful for genetic mapping, germplasm characterization, and phylogenetic assignment of DNA fragments.

Background: With the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species., Results: Fifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping., Conclusions: A set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.

Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified., (© 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.)

Premise of the Study: Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. •, Methods and Results: Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata, C. maxima, C. medica, C. sinensis, C. aurantium, C. paradisi, C. lemon, C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra- and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. •, Conclusions: These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps.

Background: Research on citrus fruit ripening has received considerable attention because of the importance of citrus fruits for the human diet. Organic acids are among the main determinants of taste and organoleptic quality of fruits and hence the control of fruit acidity loss has a strong economical relevance. In citrus, organic acids accumulate in the juice sac cells of developing fruits and are catabolized thereafter during ripening. Aconitase, that transforms citrate to isocitrate, is the first step of citric acid catabolism and a major component of the citrate utilization machinery. In this work, the citrus aconitase gene family was first characterized and a phylogenetic analysis was then carried out in order to understand the evolutionary history of this family in plants. Gene expression analyses of the citrus aconitase family were subsequently performed in several acidic and acidless genotypes to elucidate their involvement in acid homeostasis., Results: Analysis of 460,000 citrus ESTs, followed by sequencing of complete cDNA clones, identified in citrus 3 transcription units coding for putatively active aconitate hydratase proteins, named as CcAco1, CcAco2 and CcAco3. A phylogenetic study carried on the Aco family in 14 plant species, shows the presence of 5 Aco subfamilies, and that the ancestor of monocot and dicot species shared at least one Aco gene. Real-time RT-PCR expression analyses of the three aconitase citrus genes were performed in pulp tissues along fruit development in acidic and acidless citrus varieties such as mandarins, oranges and lemons. While CcAco3 expression was always low, CcAco1 and CcAco2 genes were generally induced during the rapid phase of fruit growth along with the maximum in acidity and the beginning of the acid reduction. Two exceptions to this general pattern were found: 1) Clemenules mandarin failed inducing CcAco2 although acid levels were rapidly reduced; and 2) the acidless "Sucreña" orange showed unusually high levels of expression of both aconitases, an observation correlating with the acidless phenotype. However, in the acidless "Dulce" lemon aconitase expression was normal suggesting that the acidless trait in this variety is not dependent upon aconitases., Conclusions: Phylogenetic studies showed the occurrence of five different subfamilies of aconitate hydratase in plants and sequence analyses identified three active genes in citrus. The pattern of expression of two of these genes, CcAco1 and CcAco2, was normally associated with the timing of acid content reduction in most genotypes. Two exceptions to this general observation suggest the occurrence of additional regulatory steps of citrate homeostasis in citrus.

To better understand the molecular and physiological mechanisms underlying maintenance and release of seasonal bud dormancy in perennial trees, we identified differentially expressed genes during dormancy progression in reproductive buds from peach (Prunus persica [L.] Batsch) by suppression subtractive hybridization (SSH) and microarray hybridization. Four SSH libraries were constructed, which were respectively enriched in cDNA highly expressed in dormant buds (named DR), in dormancy-released buds (RD) and in the cultivars with different chilling requirement, 'Zincal 5' (ZS) and 'Springlady' (SZ), sampled after dormancy release. About 2500 clones picked from the four libraries were loaded on a glass microarray. Hybridization of microarrays with the final products of SSH procedure was performed in order to validate the selected clones that were effectively enriched in their respective sample. Nearly 400 positive clones were sequenced, which corresponded to 101 different unigenes with diverse functional annotation. We obtained DAM4, 5 and 6 genes coding for MADS-box transcription factors previously related to growth cessation and terminal bud formation in the evergrowing mutant of peach. Several other cDNAs are similar to dormancy factors described in other species, and others have been related to bud dormancy for the first time in this study. Quantitative reverse transcription polymerase chain reaction analysis confirmed differential expression of cDNAs coding for a Zn-finger transcription factor, a GRAS-like regulator, a DNA-binding protein and proteins similar to forisome subunits involved in the reversible occlusion of sieve elements in Fabaceae, among others.

The Andalusian Kidney Transplant Registry is a Public Health Service Regional Registry of Andalusia, Spain. We have analyzed the causes of death among 5599 kidney transplantations performed between January 1, 1984 and December 31, 2007. The total number of patients who died after renal transplantation was 1106. Of these, 656 patients died with functioning renal grafts, which constituted the group analyzed in this study. No significant differences in the causes of death were observed as a function of age, sex, retransplant status, cause of end-stage renal disease, diabetes, and duration of the previous renal replacement therapy. Infections were the most frequent cause of death in the first year posttransplantation (early deaths). A significant difference was observed when these early deaths were compared with those that occurred after the first year posttransplantation (late deaths); there were fewer deaths due to infections (40.4% vs 15.9%, early vs late) and more deaths from cancer (4.9% vs 23.7%). The causes of death in the period 1984-1995 were compared with those in 1996-2007, excluding the deaths that occurred more than 12 years posttransplantation (not possible in the 1996-2007 periods n = 583). The causes of early death did not change. A significant difference was observed among late deaths with an increase in infections (14% vs 17%) and cancers (20% vs 29.7%). Thus, malignancies became the most frequent cause of late death in the 1996-2007 period. In conclusion, in our region, a long-term change in renal transplant patient mortality is taking place, with a significant increase in deaths due to cancer.

Graft failure with return to dialysis is associated with higher mortality, but the determining factors have not been completely studied. We determined predictive factors for survival after renal transplant failure by analyzing demographic and clinical data on kidney recipients in a Spanish regional registry (Andalusia). Among 5547 single-organ cadaveric grafts in Andalusia between 1984 and 2007, 1534 patients returned to dialysis and were followed to death (n = 450), retransplantation (n = 574), or the end of the study period. The most frequent cause of death was cardiovascular disease (36.9%). Actuarial survival rates were 89%, 72%, and 51% at 1, 5, and 10 years, respectively. Upon univariate (Kaplan-Meier) analysis, survival was significantly related only to age at return to dialysis and diabetes. Cox forward step wise multivariate regression analysis showed that predictive factors for lower survival (relative risk -95% confidence interval) were: age (per year), 1.05 (1.05-1.06); male sex, 1.33 (1.1-1.6); and duration of pre-graft renal replacement therapy (per year), 1.02 (1.00-1.05). According to Cox backward stepwise regression analysis, a more saturated model, the predictive factors were: age; male sex; duration of pre-graft renal replacement therapy (per year), 1.03 (1.01-1.06); later study period (1996-2007), 0.81 (0.6-1.0); and primary etiology of chronic kidney disease (CKD). Survival after renal transplant failure was lower among male recipients of advanced age or with a long period of prior renal replacement therapy. Time of transplantation and primary etiology of CKD may also play roles.

Many studies have shown a trend to improved long-term survival of renal transplant recipients. We analyzed the survival of recipients in Andalusia, Spain, from 1984-2007. The study included all the deceased donor, non-multiorgan grafts (n = 5599), grouped over successive 6-year periods, compared for corrected recipient survival. Changes were noted in the recipient characteristics: increased age, diabetes, vascular nephropathy, retransplantation, duration of prior replacement therapy, and reduction in positive hepatitis C virus (HCV+) serology. The univariate analysis showed a significantly worse survival associated with increased age (P < .001), diabetes (P < .001), HCV+ serology (P < .01; 1996-2007), and longer times on replacement therapy, but not with sex or retransplantation. The respective survivals at 1, 5, and 10 years in 1984-1989 were 93%, 86%, and 75%; in 1990-1995, 97%, 92%, and 84%; in 1996-2001, 96%, 91%, and 84%; and in 2002-2007, 96% and 92%, respectively. There was a significant improvement between the first and second periods (P < .001), but no change thereafter. The multivariate analysis (Cox) showed, a significant influence of age >40 years, female gender (relative risk [RR] 0.8; 95% confidence interval [CI] 0.7-0.9), diabetes (RR 2.5; 95% CI 1.8-3.4), and duration of prior replacement therapy (RR 1.08; 95% CI 1.05-1.1). The risk varied significantly depending on the period: using 2002-2007 as the reference period, the RR in 1984-1989 was 3.4 (95% CI 2.6-4.5); in 1990-1995, 1.8 (95% CI 1.3-2.3); and in 1996-2001, 1.4 (95% CI 1.1-1.8; all P < .02). The model remained for 1996-2007, though HCV+ serology was not significant. In conclusion, we showed a significant improvement in recipient survival in Andalusia over time. Correction for worse recipient characteristics suggests continued advances.

Herein we have presented the first report from the Andalusian Kidney Transplant Registry, a Public Health Service Regional Registry in Andalusia, Spain (general population, 8 million). The current analysis was limited to 5599 kidney-alone transplants from deceased donors, grouped into 4 time periods: 1984-1989 (n = 846); 1990-1995 (n = 1172); 1996-2001 (n = 1801); and 2002-2007 (n = 2060). The age of the transplant patients rose over time to 21.7% of recipients of ages >or=60 years in 2002-2007. In the later years we observed an increased incidence of vascular and diabetic causes of end-stage renal disease (ESRD). Patients who underwent retransplantation increased from 2.7% in 1984-1989 to 8.1% in 2002-2007. Time on previous renal replacement therapy (RRT) increased from 33.1 +/- 29 to 48 +/- 53 months. Patient survivals at 1, 5, 10, and 20 years were 96%, 91%, 83%, and 63%, respectively. Censoring for death, graft survivals were 90%, 80%, 67%, and 45%, respectively. Compared with the 1984-1989 period, patient survival improved by about 10% (P < .001) since 1990, remaining stable to 2007. Censored 5-year graft survivals progressively improved from 72% to 77%, 82%, and 85% (P < .001). Upon multivariate analysis, gender, age >39 years, diabetes, and RRT duration were independent predictors of patient survival. Age <18 years, retransplantation, and positive hepatitis C virus serology were independent predictors of lower graft survival. Considering 1984-1989 as the reference time period, both patient and graft mortality risks continuously decreased over the following 3 periods (relative risk [RR] = 0.5-0.4-0.3 for patient mortality; RR = 0.8-0.6-0.5 for graft mortality). In summary, despite an increased number of adverse risk factors, both patient and graft survivals have improved from 1984 to date.

Background: Citrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be crucial for further crop improvements. In this work we report the characterization of three BAC libraries from Clementine (Citrus clementina), one of the most relevant citrus fresh fruit market cultivars, and the analyses of 46.000 BAC end sequences. Clementine is a diploid plant with an estimated haploid genome size of 367 Mb and 2n = 18 chromosomes, which makes feasible the use of genomics tools to boost genetic improvement., Results: Three genomic BAC libraries of Citrus clementina were constructed through EcoRI, MboI and HindIII digestions and 56,000 clones, representing an estimated genomic coverage of 19.5 haploid genome-equivalents, were picked. BAC end sequencing (BES) of 28,000 clones produced 28.1 Mb of genomic sequence that allowed the identification of the repetitive fraction (12.5% of the genome) and estimation of gene content (31,000 genes) of this species. BES analyses identified 3,800 SSRs and 6,617 putative SNPs. Comparative genomic studies showed that citrus gene homology and microsyntheny with Populus trichocarpa was rather higher than with Arabidopsis thaliana, a species phylogenetically closer to citrus., Conclusion: In this work, we report the characterization of three BAC libraries from C. clementina, and a new set of genomic resources that may be useful for isolation of genes underlying economically important traits, physical mapping and eventually crop improvement in Citrus species. In addition, BAC end sequencing has provided a first insight on the basic structure and organization of the citrus genome and has yielded valuable molecular markers for genetic mapping and cloning of genes of agricultural interest. Paired end sequences also may be very helpful for whole-genome sequencing programs.

Background: During the last decade, numerous microsatellite markers were developed for genotyping and to identify closely related plant genotypes. In citrus, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. To optimize the use of these EST-SSRs as genetic markers in genome mapping programs and citrus systematic analysis, we have investigated their polymorphism related to the type (di or trinucleotide) or their position in the coding sequences., Results: Among 11000 unigenes from a Clementine EST library, we have found at least one microsatellite sequence (repeated units size ranged from 2 to 6 nucleotides) in 1500 unigenes (13.6%). More than 95% of these SSRs were di or trinucleotides. If trinucleotide microsatellites were encountered trough all part of EST sequences, dinucleotide microsatellites were preferentially (50%) concentrated in the 5' 100th nucleotides. We assessed the polymorphism of 41 EST-SSR, by PCR amplification droved with flanking primers among ten Citrus species plus 3 from other genera. More than 90% of EST-SSR markers were polymorphic. Furthermore, dinucleotide microsatellite markers were more polymorphic than trinucleotide ones, probably related to their distribution that was more often located in the 5' UnTranslated Region (UTR). We obtained a good agreement of diversity relationships between the citrus species and relatives assessed with EST-SSR markers with the established taxonomy and phylogeny. To end, the heterozygosity of each genotype and all dual combinations were studied to evaluate the percentage of mappable markers. Higher values (> 45%) were observed for putative Citrus inter-specific hybrids (lime lemon, or sour orange) than for Citrus basic true species (mandarin, pummelo and citron) (<30%). Most favorable combinations for genome mapping were observed in those involving interspecific hybrid genotypes. Those gave higher levels of mappable markers (>70%) with a significant proportion suitable for synteny analysis., Conclusion: Fourty one new EST-SSR markers were produced and were available for citrus genetic studies. Whatever the position of the SSR in the ESTs the EST-SSR markers we developed are powerful to investigate genetic diversity and genome mapping in citrus.

Functional genomics technologies have been widely adopted in the biological research of both model and non-model species. An efficient functional annotation of DNA or protein sequences is a major requirement for the successful application of these approaches as functional information on gene products is often the key to the interpretation of experimental results. Therefore, there is an increasing need for bioinformatics resources which are able to cope with large amount of sequence data, produce valuable annotation results and are easily accessible to laboratories where functional genomics projects are being undertaken. We present the Blast2GO suite as an integrated and biologist-oriented solution for the high-throughput and automatic functional annotation of DNA or protein sequences based on the Gene Ontology vocabulary. The most outstanding Blast2GO features are: (i) the combination of various annotation strategies and tools controlling type and intensity of annotation, (ii) the numerous graphical features such as the interactive GO-graph visualization for gene-set function profiling or descriptive charts, (iii) the general sequence management features and (iv) high-throughput capabilities. We used the Blast2GO framework to carry out a detailed analysis of annotation behaviour through homology transfer and its impact in functional genomics research. Our aim is to offer biologists useful information to take into account when addressing the task of functionally characterizing their sequence data.

Background: Improvement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity., Results: A normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis x Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domains, Conclusion: Comparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays.

Background: Cardiovascular disorders are frequently found among chronic renal failure (CRF) patients due to their higher susceptibility to develop atherosclerosis. However, peripheral arterial disease (PAD), that is associated with a high mortality rate, is not usually assessed in these patients. The aims of this study are to find out the prevalence of PAD affecting lower limbs in a population of CRF patients in stages IV/V, and to assess how much PAD determines the 5-year patient survival., Methods: The study population (44 males and 29 females) was aged 58 +/- 15 years. They suffered from advanced CRF (18.6 +/- 6.1 ml/min creatinine clearance), but they were not on dialysis. These patients were sequentially referred initially to the predialysis unit over a period of 14 months. The vascular lesions were assessed by carotid and transcranial ultrasound, as well as by ankle-brachial index test (ABI). Routine 24 h blood and urine laboratory tests were performed for each patient. Cardiovascular morbidity and cardiovascular disease risk factors were evaluated through personal interview., Results: Fourteen patients had an ABI index of less than 0.91 (PAD indicative), 11 of them also suffered from intermittent claudication. PAD affected significantly more males (P = 0.001) and diabetics (P = 0.001). Also, PAD prevalence was significantly higher in patients with a previous clinical record of coronary heart disease (P = 0.001), increased clinical record of cerebrovascular disease (P = 0.005), a thickness of the left ventricular posterior wall (P = 0.03) and lower cardiac ejection fraction (P = 0.02). PAD patients had a significantly different protein intake (P = 0.003), calcium-phosphorus product (P = 0.001), risk of coronary heart disease based on the Framingham score (P = 0.001) and 5-year survival rate (P = 0.004). There were no significant differences for PAD patients in terms of body mass index, creatinine clearance, lipid profile, Ca and P. Multivariate risk factor analysis revealed that a previous clinical record of coronary heart disease and diabetes increased the risk of developing PAD, as defined by ABI < 0.91. After 5 years, 21 patients (29%) had died: 64% of patients that suffered PAD (9/14) and 20% of the non-PAD population (12/59). The Cox proportional hazards model demonstrated that older age and a lower ABI increased the risk of death., Conclusions: The present study, conducted on CRF patients in stages IV and V not undergoing dialysis, showed; (a) that a high percentage of these patients developed PAD (19%) or other vascular pathologies; (b) that there was an associated high mortality rate (29%) after 5 years; (c) that the 5-year mortality rate was significantly higher (P = 0.004) in PAD patients (64 vs 20%).

The GH3 gene family in Arabidopsis, implicated in hormonal homeostasis through the conjugation of indolacetic and jasmonic acids to amino acids, is involved in a broad range of plant growth and development processes. In this work, the analysis of the GH3 family in the genome of Oryza sativa identified 13 hypothetical ORFs. EST analysis and RT-PCR assays demonstrated that 12 of them were active genes. An extensive EST analysis of the GH3 family performed on 26 plant species was used to estimate the minimum number of GH3 genes en each one. The data indicated that the members of the GH3 family progressively increased in the different plant divisions from Chlorophyta (0), Bryophyta (3), and Coniferophyta (4), to Magnoliophyta (7-19). Phylogenetic analyses showed a high degree of conservation between Arabidopsis and rice GH3 proteins and, in general, in the plant kingdom. The data revealed a homology clustering consistent with the functional classification of the Arabidopsis proteins, since most of the 110 sequences analyzed grouped into 2 main clusters, corresponding to the Arabidopsis functional groups I (jasmonic acid adenylation) and II (indolacetic acid adenylation). And additional cluster including group III (non-adenylation ability) was exclusively composed of proteins from Arabidopsis thaliana, Brassica napus and Gossypium hirsutum.

During dorsal closure, the lateral epithelia on each side of the embryo migrate dorsally over the amnioserosa and fuse at the dorsal midline. Detailed genetic studies have revealed that many molecules are involved in this epithelial sheet movement, either with a signaling function or as structural or motor components of the process. Here, we report the characterization of cabut (cbt), a new Drosophila gene involved in dorsal closure. cbt is expressed in the yolk sac nuclei and in the lateral epidermis. The Cbt protein contains three C2H2-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. cbt mutants die as embryos with dorsal closure defects. Such embryos show defects in the elongation of the dorsal-most epidermal cells as well as in the actomyosin cable assembly at the leading edge. A combination of molecular and genetic analyses demonstrates that cbt expression is dependent on the JNK cascade during dorsal closure, and it functions downstream of Jun regulating dpp expression in the leading edge cells.

Summary: We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process., Availability: Blast2GO is freely available via Java Web Start at http://www.blast2go.de., Supplementary Material: http://www.blast2go.de -> Evaluation.

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis allowed us to characterize four novel genes, AtB'epsilon, AtB'zeta, AtB'eta, and AtB'theta;, belonging to the PP2A B' regulatory subunit family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse transcriptase-polymerase chain reaction experiments showed that AtB'epsilon mRNAs are present in all Arabidopsis tissues analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB'zeta, AtB'eta, and AtB'theta; have been identified, indicating that the new genes are actively transcribed. The genomic organization of this family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has been carried out in plants, characterizing B' subunits in a number of different species, and performing a phylogenetic study that included several B' regulatory proteins from animals. Our results indicate that the animal and plant proteins have evolved independently, that there is a relationship between the number of B' isoforms and the complexity of the organism, and that there are at least three main subfamilies of regulatory subunits in plants, which we have named alpha, eta, and kappa.

Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.

The analysis of 460 kb of genomic sequence of Arabidopsis thaliana chromosome III allowed us to identify two new transposable elements named AtC1 and AtC2. AtC1 shows identical long terminal repeats (LTRs) and all the structural features characteristic of the copia-like active elements. AtC2 is also a full copia-like element, but a putative stop codon in the open reading frame (ORF) would produce a truncated protein. In order to identify the copia-like fraction of the A. thaliana genome, a careful computer-based analysis of the available sequences (which correspond to 92% of the genome) was performed. Approximately 300 nonredundant copia-like sequences homologous to AtC1 and AtC2 were detected, which showed an extreme heterogeneity in size and degree of conservation. This number of copies would correspond to approximately 1% of the A. thaliana genome. Seventy-one sequences were selected for further analysis, with 23 of them being full complete elements. Five corresponded to previously described ones, and the remaining ones, named AtC3 to AtC18 are new elements described in this work. Most of these elements presented a putative functional ORF, nearly identical LTRs, and the other elements necessary for retrotransposon activity. Phylogenetic trees, supported by high bootstrap values, indicated that these 23 elements could be considered separate families. In turn, these 23 families could be clustered into six major lineages, named copia I-VI. Most of the 71 analyzed sequences clustered into these six main clades. The widespread presence of these copia-like superfamilies throughout plant genomes is discussed.

We describe a new subfamily of WD repeat proteins characterised by the presence of a C3H zinc finger at the N-terminal part of the protein associated with seven WD40 repeats. We have identified four members of this subfamily in Arabidopsis thaliana, one of them with associated expressed sequence tags (ESTs). We have also identified homologous ESTs in rice, cotton, maize, poplar, pine tree and the ice plant. We do not observe animal homologues, suggesting that this subfamily could be specific for plants. Our data suggest an important role for these proteins. Based on the high sequence conservation within the conserved domains, we suggest that these proteins could have a regulatory function.