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Citrus is an extremely important genus in terms of world fruit production. Despite its economic importance and the small genome sizes of its species (2n = 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.

Several tools have been developed to extract knowledge from the vast amount of data in genomics. The success of the knowledge extraction process depends to arge extent on how easy to learn and use are the tools for bioinformaticians. User interface design is neglected frequently in the genomic tool development process. As a result, user interfaces contain usability problems that make knowledge extraction a complex task. User-Centered Design (UCD) is a design approach that can be applied to improve the usability of interfaces. A fundamental principle of UCD is to design the UI based on users’ knowledge, their needs, objectives, and tasks to ensure that the resulting user interface meets the real user needs. We apply the UCD approach to design, evaluate and improve CitrusGenome, a tool that enables bioinformaticians to extract knowledge from genomic data. Following the UCD process, we first conduct user research to define user needs by applying UCD techniques such as interviews and task analysis. Then, we design a user interface that meets those user needs by using GenomIUm. GenomIUm is a systematic method that guides the design process of user interfaces in the genomic domain. We have performed two UCD iterations and, after each iteration, the user interfaces were validated by bioinformaticians. Some usability problems were found in each iteration. Therefore, we refined the user interface by solving the usability problems and incorporating such solutions into the final design. Finally, bioinformaticians using the refined user interface reported a reduction in the complexity of extracting knowledge from genomic data. We conclude that UCD techniques, together with GenomIUm, can be a useful strategy to design user interfaces that are easier to learn and use in the genomic domain

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.

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.

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.

The genomic domain is a complex data environment that has grown exponentially. Several tools have been developed to extract knowledge from this immense amount of data. Knowledge extraction processes depend to a large extent on how easy and intuitive are the user interfaces of the tools that are used by bioinformaticians. However, genomic tools have frequently ignored the design process of their User Interfaces. Consequently, they have important usability problems that complicates knowledge extraction. User Centered Design (UCD) is a design approach that can be used to improve the usability of genomic tools. It consists on putting the user and its real needs at the center of the design process. Improving the usability of these tools will facilitate knowledge extraction. This paper reports the application of the UCD approach to design a tool that improves knowledge extraction processes in a real world-use case. From a general perspective, UCD consists of “user research” and “design solutions”. The first one was carried out by conducting UCD techniques, including user interviews and task analysis. The second one was carried out by applying GenomIUm, a pattern-based method that guides the design process of genomic user interfaces. As a fundamental part in the UCD approach, the generated user interfaces were validated by expert bioinformaticians who reported that the complexity of extracting knowledge from genomic data was reduced. We conclude that UCD techniques together with GenomIUm can be a useful strategy to design more usable user interfaces in the genomic domain.

We performed genomic analyses on wild species of the genus Citrus to identifymajor 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. 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 secondarymetabolites 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.

The great diversity of the genus Citrus allowed its primary utilization as an ornamental plant before being massively cultivated in many countries for fresh consumption and processing utilization. Citrus fruits are extremely popular worldwide thanks to their physical and chemical attributes they reach during different stages of growth and development. Development of the final fruit size and shape as well as fruit color and flavor, and the accumulation of some compounds beneficial to human health are described and discussed in depth in this chapter. The chapter incorporates updated knowledge on citrus fruit growth and development acquired both by classical agronomic, genetic, and physiological means, and more recent studies using genomic, proteomic, and metabolomic approaches.

Speciation of the genus Citrus from a common ancestor has recently been established to begin approximately 8 Mya 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 thirty thousand retrotransposons were grouped in 10 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 interacts with the internal regulatory mechanisms of the genome controlling the proliferation of mobile elements.

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. Financial support was provided by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA, (Ministerio de Economía, Industria y Competitividad e Innovación, Spain) trough grant # RTA-00071-C06–01.

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

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. The origin, evolution and domestication of Citrus and the genealogy of the most important wild and cultivated citrus varieties. Citrus fruits are one of the most cultivated crops worldwide, yet the evolutionary relationships among citrus species remain uncertain. Daniel Rokhsar, Manuel Talon and colleagues analyse the genomes of 60 accessions that represent a diverse range of citrus species, including 30 newly sequenced citrus genomes. They characterize the diversity and evolution of citrus at the species level and identify interspecific citrus hybrids and admixtures—genetic mixing between previously isolated populations—that could be the result of human activities such as migration and agriculture. The authors identify 10 progenitor species and suggest that citrus originated in southeast Asia, diversifying during the late Miocene epoch through a rapid southeast Asian radiation that correlated with a changing climate, including the weakening of the monsoons. They also find extensive relatedness among mandarins and sweet oranges, showing a complex history of admixture during the domestication of these groups.

International audience; SSRs have been considered as almost ideal markers for genetic diversity analysis. With the increasing availability of sequencing data, SNPs and InDels become major classes of codominant markers with genome wide coverage. We have analyzed the respective values of SSRs, InDels, and SNPs for intra and interspecific Citrus genetic diversity analysis. Moreover, we have compared the diversity structure revealed by markers mined in a single heterozygous genotype (clementine) and markers mined from a large interspecific survey. A random set of markers was selected for each marker class to genotype 48 citrus accessions. SSRs were the most polymorphic markers at the intraspecific level allowing complete varietal differentiation within basic taxa (C. reticulata, C. maxima, C. medica). However, SSRs gave the lowest values for interspecific differentiation, followed by SNPs and InDels, that in average displayed low intraspecific variability but high interspecific differentiation. A clear effect of the discovery panel was observed for SNPs and InDels. The ascertainment biases associated with the clementine heterozygosity mining resulted mainly in an over estimation of within C. reticulata diversity and an underestimation of the interspecific differentiation. In conclusion pluri-allelic SSRs are very useful for intraspecific structure analysis but have limitations for interspecific and phylogenetic studies. SNPs and InDels mined in large discovery panels appear to allow efficient selection of more specialized markers with high potential for phylogenetic diagnosis and to decipher the interspecific mosaic structure of secondary cultivated species or to analyze intraspecific diversity. Several SNP genotyping methods are available for different analysis scales, from high throughput genotyping to small set of data production. With the ongoing resequencing projects, SNPs markers will soon be the most important class of markers for citrus genetics.

The availability of a saturated genetic map of Clementine was identified by the International Citrus Genome Consortium as an essential prerequisite to assist the assembly of the reference whole genome sequence based on a Clementine derived haploid. The primary goals of the present study were to establish a Clementine reference map, and to perform comparative mapping with pummelo and sweet orange. Five parental genetic maps were established with SNPs, SSRs and InDels. A medium density reference map (961 markers for 1084.1 cM) of Clementine was established and used by the ICGC to facilitate the chromosome assembly of the haploid genome sequence. Comparative mapping with pummelo and sweet orange revealed that the linear order of markers was highly conserved. Reasonable inferences of most citrus genomes should be obtained by mapping next-generation sequencing data against the haploid reference genome sequence. Skewed segregations were frequent and higher in the male than female Clementine potentially leading to false interpretation of the genetic determinism of phenotypic traits. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange and identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Introgression of pummelo genome fragments were identified in heterozygosity in each chromosome. Moreover, it appeared that the genome of the haploid Clementine used to establish the citrus reference genome sequence was inherited primarily from the 'Mediterranean' mandarin. The usefulness of this genetic map, anchored in the reference whole genome sequence, is discussed. Citrus Research and Development Foundation 108O568 Ministerio de Ciencia e Innovación: AGL2008-00596-MCI, 2008/121, IPT-010000-2010-43, PSE-060000-2009-8, AGL2007-65437-C04-01/AGR European Commission Citrus Research and Development Foundation itl-bio-03-10122 015453 This work was principally funded by the French ANR CITRUSSEQ project. The European Commission, under the FP6-2003-INCO-DEV-2 project CIBEWU (n°015453), the Spanish Ministerio de Ciencia e Innovación grants, AGL2007-65437-C04-01/AGR and AGL2008-00596-MCI, the Spanish PSE-060000-2009-8 and IPT-010000-2010-43 projects, the Prometeo project 2008/121 Generalidad Valenciana, the Turkish TUBITAK Project No: 108O568; the California Citrus Research Board and UC Discovery grant itl-bio-03-10122 and the Florida Citrus Research and Development Foundation (CRDF), grants #67 and 71 also contributed to the work.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12284-016-0130-5) contains supplementary material, which is available to authorized users.

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.

Abscission events in citrus allow the detachment of entire organs, both vegetative (buds, leaves) and reproductive (flowers, ovaries, fruitlets, mature fruits). This is achieved by the modification of cellular adhesion in a discrete group of specialized cells differentiated in predictable positions, known as abscission zones (AZs). Thus, abscission facilitates several key processes for citrus biology that, in an agricultural context, represent a major limiting factor for crop yield. In this regard, understanding the regulation of abscission is an important item for the citrus fruit industry. Several functional genomic approaches are currently being used to gain insights into the regulation of citrus abscission and to provide potential candidate genes for further biotechnological applications. These include: (a) the development of an expressed sequence tag (EST) dataset from abscission activated AZs and the analysis of ESTs distribution in organs to reveal AZ-preferential gene expression during abscission activation, (b) comparative transcriptomic analysis using cDNA microarrays and laser microdissected tissues (AZ cells, petiole and fruit rind cells flanking AZs), and (c) functional characterization of putative abscission-associated citrus genes in Arabidopsis thaliana and rice (Oryza sativa).

SUMMARY 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.

In recent years we have generated various collections of citrus mutants. One objective of these collections is the selection of genotypes of commercial interest. In the present communication we describe 3 lines derived from clementine (Citrus clementina) through physical mutagenesis, named ‘Clemenverd’, ‘Nero’ and ‘Neufina’. These lines that show enhanced agronomic traits as related to the parental genotype have been registered. ‘Clemenverd’ shows delayed external maturation. ‘Nero’ is an early cultivar of seedless clementine that maintains the excellence of clementine quality. On the other hand, ‘Neufina’ holds well into February the commercial organoleptic qualities of the parental cultivar. ‘Neufina’ also has excellent perdurability in the tree, making it an ideal option to extend the supply of clementines during winter.

5 páginas. 2 tablas.-- 3 referencias.-- Artículo publicado en el XII International Citrus Congress.-- Terol, Javier et al..., Elucidation of genomic sequences is an essential and necessary first step to identify genetic variants associated with agronomic traits of interest. The Citruseq Consortium, a joint effort developed by public institutions and private companies has sequenced the genomes of 150 citrus varieties with the Illumina platform at high average coverage. Paired end genomic DNA libraries of 100 (2x) bp reads and with an average insert size of 500 bp were constructed. The haploid genome of Citrus clementine (www.phytozome.com) was used as reference genome. More than 150 Citrus genomes including rootstocks and species of mandarins, oranges, lemons, grapefruits and limes were sequenced. The consortium also generated a web portal that allows searching and sequence comparisons among approximately 8 million of gene variants and 45 billion of base pairs. Initial analyses suggest that the stored information can help unravel fundamental aspects of biological and evolutionary interest such as the origin of citrus. From the commercial standpoint this information may help to authenticate varieties, to improve marker-assisted breeding and to identify genes of major agronomic interest., This work was funded by the Spanish Ministerio de Ciencia e Innovación PSE-060000-2009-8 and IPT-010000-2010-43 grants. We would like to thank Elena Blazquez,Angel Boix, Juan Vicente Muñoz, Matilde Sancho and Isabel Sanchis for his invaluable help in field and laboratory tasks.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1280-3) contains supplementary material, which is available to authorized users.

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.

Resumen La incertidumbre preside la practica clinica de los profesionales sanitarios, fenomeno al que no permanecen ajenas las enfermeras. La toma de decisiones es una de las principales fuentes de variabilidad en la prestacion de cuidados y el centro de los esfuerzos dirigidos a mejorar la efectividad. Pese a las criticas y limitaciones que la evidencia acusa, no deja de ser una respuesta valida al aumento de la complejidad de las organizaciones sanitarias y de la necesidad de mejorar los resultados de la atencion en terminos de coste-efectividad, coste-beneficio y coste-utilidad. En la enfermeria, la evidencia ha sido conceptualizada como el medio de acercar el conocimiento cientifico y el conocimiento tacito, proporcionando instrumentos que facilitan la toma de decisiones tanto a profesionales como a usuarios. Entre estos instrumentos figuran las guias de practica clinica, pero su uso requiere una adecuada elaboracion, evaluacion e implementacion para que se conviertan en verdaderos motores de mejora de la efectividad de los cuidados. En este articulo se detallan sus principales caracteristicas, en que consisten y como discernir su calidad metodologica para que las enfermeras puedan incorporarlas a su practica habitual.

The muscleblind (mbl) gene encodes protein isoforms Mbl A to Mbl D, which arise by alternative splicing from a common primary transcript. Mbl A, B, and C contain two Zn-finger domains of the type Cys3His, while Mbl D contains only one complete Zn finger. Loss of function mutations in the gene reveal that mbl is involved in both terminal photoreceptor and muscle differentiation in Drosophila. During retina development mbl is essential for rhabdomere differentiation in photoreceptor neuron. Clones homozygous null for mbl completely lack these lightharvesting structures (Begemann et al., 1997). Similarly, the terminal differentiation of the larval body wall muscles is compromised in mbl mutant embryos. In these mutant embryos muscles show sarcomeres with disorganized Z-lines and reduction in the extracellular matrix of indirect muscle attachments (Artero et al., 1998). In addition, biochemical studies aimed at understanding the molecular basis of myotonic dystrophy (DM), a dominant-autosomic disease, identified the human mbl paralogs MBNL and MBLL as critical elements in the DM pathogenesis pathway (Miller et al., 2000). Despite intense research, however, the molecular function of Mbl proteins has not yet been elucidated. The current working models point to a role for MBNL/MBLL proteins in RNA metabolism, possibly during pre-mRNA splicing or mRNA nuclear export. These models are supported mainly by the observation that MBNL/MBLL proteins bind to CUG and CCUG expansions (Miller et al., 2000; Mankodi et al., 2001; Fardaei et al., 2002). To generate both gain of function defects in the fly as well as genetic tools to use in a Drosophila model for the human disease, we made three UAS constructs containing the Drosophila protein isoforms Mbl A and Mbl C and the human MBNL protein isoform encoded by the cDNA KIAA0428, which includes most of the alternatively used exons in the MBNL locus (Fardaei et al., 2002). UAS constructs (Fig. 1) were injected into fly embryos of the genotype white using standard germline transformation methods (Spradling and Rubin, 1982). We established five independent transgenic lines carrying UAS-mbl A, six transgenic lines carrying UAS-mbl C, and three transgenic lines carrying UAS-KIAA0428. These lines were mapped by genetic crosses to the indicated chromosomes (Fig. 1). Direct experimental evidence of Gal4-driven expression for these constructs was obtained by crossing UAS-mbl A and UAS-mbl C to the engrailed-Gal4 driver, which gives a segment polarity pattern of expression clearly different from the endogenous mbl transcription. In situ hybridization in these embryos clearly detected ectopic mbl expression driven by the engrailed promoter (Fig. 2A–C). In addition, expression of the transgenes was targeted to the precursors of adult structures to reveal morphological defects indicative of their biological activity. Targeted expression of Mbl A and Mbl C to the Drosophila eye imaginal disc driven by an endogenous sevenless promoter (sev-Gal4) led to a rough eye phenotype which ranged from a very mild defect in the case of UAS-mbl A to a clearly rough eye in the case of UAS-mbl C (Fig. 2D–F). Expression of UAS-KIAA0428 under similar conditions led to a severely rough eye phenotype and lethality, depending on the transgene used. We used our weakest transgene (UAS-KIAA0428.KD.1) and an incubation temperature of 25°C for these crosses in order to obtain viable offspring and a milder eye phenotype (Fig. 2G). Tangential sections through these rough eyes revealed morphological defects at the cellular level. In all cases we detected ommatidia showing typical planar cell polarity (PCP) defects but also, especially when the UAS-mbl C was overexpressed, photoreceptor loss. In a similar set of experiments, we targeted expression of all three transgenes to the wing imaginal disc using 69B-Gal4 or apterous-Gal4 as drivers (Fig. 2H–K). Offspring from these crosses revealed defects in the mor

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 probabilityor =95%.

Resumen Fundamento La continuidad de cuidados constituye uno de los parametros esenciales de la calidad asistencial. El grado de coordinacion asistencial entre profesionales y entornos resulta trascendental en el cuidado del paciente critico. No siempre se consigue mantener unos niveles optimos de continuidad, y se desconoce cual es la verdadera repercusion que tiene en los pacientes y profesionales, asi como las causas de la discontinuidad en los cuidados. El objetivo del estudio consiste en la construccion y validacion de un instrumento para valorar la continuidad de cuidados. Metodo Estudio lineal, descriptivo, observacional estructurado y retrospectivo. Diseno de un instrumento evaluador de la continuidad de cuidados (GRACO) y validacion de la fiabilidad interobservador en varias unidades de cuidados criticos. Resultados Se realizaron 68 observaciones entre 26 pacientes desde junio a agosto de 1999. Entre los problemas destacaron el dolor agudo de origen isquemico (23,5%), las alteraciones hemodinamicas (10,3%) y las situaciones de compromiso de la via aerea (11,8%). La correlacion interobservador fue elevada: 0,88, p Conclusiones Instrumento con estabilidad aceptable entre observadores. El descenso de concordancias en el apartado de evaluacion obliga a revisar el instrumento en este punto.

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., 栽培されている柑橘類は、野生祖先種から選択されたもの、またはその交雑種であるが、その祖先種の正体、および柑橘類栽培化に対する寄与には依然として議論がある。今回、柑橘類ゲノム（クレメンタインの高精度標準ハプロイドゲノム、ならびにマンダリン、パメロ、スイートオレンジ、およびサワーオレンジのゲノム）の配列解読および比較を行ったところ、栽培種が2つの祖先種に由来することが明らかにされた。栽培パメロは単一祖先種 Citrus maxima から選択されたことがわかったが、栽培マンダリンは祖先マンダリン種 Citrus reticulata に C. maxima から遺伝子移入が行われたものであった。最も広く栽培されている柑橘類であるスイートオレンジは過去に混種した個体の子孫であるが、サワーオレンジは純粋な C. maxima と C. reticulata とのF1雑種であり、このことは、野生のマンダリンが初期の育種生殖系列の要素であったことを意味している。中国の野生「マンダリン」は C. reticulata とは大きく異なっており、別の未確認の野生柑橘種である可能性が示唆される。ゲノム解析で柑橘類の系統発生を理解することにより、分類学的な関係が解明され、配列本位の遺伝的改良が促進される。

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.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25 000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

We have identified two genes from Arabidopsis that show high similarity withCBF1, a gene encoding an AP2 domain-containing transcriptional activator that binds to the low-temperature-responsive element CCGAC and induces the expression of some cold-regulated genes, increasing plant freezing tolerance. These two genes, which we have named CBF2 and CBF3, also encode proteins containing AP2 DNA-binding motifs. Furthermore, like CBF1, CBF2 and CBF3 proteins also include putative nuclear-localization signals and potential acidic activation domains. The CBF2 andCBF3 genes are linked to CBF1,constituting a cluster on the bottom arm of chromosome IV. The high level of similarity among the three CBF genes, their tandem organization, and the fact that they have the same transcriptional orientation all suggest a common origin.CBF1, CBF2, and CBF3 show identical expression patterns, being induced very rapidly by low-temperature treatment. However, in contrast to most of the cold-induced plant genes characterized, they are not responsive to abscisic acid or dehydration. Taken together, all of these data suggest that CBF2 and CBF3 may function as transcriptional activators, controlling the level of low-temperature gene expression and promoting freezing tolerance through an abscisic acid-independent pathway.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; 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.

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.

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.

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 Author 2010. Published by Oxford University Press. All rights reserved.