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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.

"Fruit trees are economically important species and, as long-living plants, represent an important challenge to understand adaptation to environmental stresses. Detailed functional analyses are often difficult in woody species, because of their biological features and the recalcitrance of some species to plant transformation and/or regeneration, impediments that greatly limit the use of standard genetic and biotechnological approaches for functional genomics studies or plant breeding. Specifically, fruit tree species are difficult to study at genetic and molecular levels (1) because of their perennial nature, (2) the limited information on gene identity and function, and genetic markers directly associated to the control of a character, (3) the unavailability of well-defined molecular genetic linkage maps and (4) the poor development of mapping population and map-based studies, that are generally rather complex in woody plants. Over the last decade, the sequencing of several genomes, coupled with rapid advances in bioinformatics, provided powerful tools for detailed molecular studies on crop plants other than traditional model species. The availability of sequencing data is only a starting point since bioinformatics approaches are not sufficient to define gene roles. To deepen this knowledge, it is necessary to understand how thousands of genes can interact each other to define the structure of a plant and how the metabolic pathways in which they are involved contribute to plant development and adaptation to the environment."

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.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit “hesperidium” is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

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.

The origin and dispersal of cultivated and wild mandarin and related citrus are poorly understood. Here, comparative genome analysis of 69 new east Asian genomes and other mainland Asian citrus reveals a previously unrecognized wild sexual species native to the Ryukyu Islands: C. ryukyuensis sp. nov. The taxonomic complexity of east Asian mandarins then collapses to a satisfying simplicity, accounting for tachibana, shiikuwasha, and other traditional Ryukyuan mandarin types as homoploid hybrid species formed by combining C. ryukyuensis with various mainland mandarins. These hybrid species reproduce clonally by apomictic seed, a trait shared with oranges, grapefruits, lemons and many cultivated mandarins. We trace the origin of apomixis alleles in citrus to mangshanyeju wild mandarins, which played a central role in citrus domestication via adaptive wild introgression. Our results provide a coherent biogeographic framework for understanding the diversity and domestication of mandarin-type citrus through speciation, admixture, and rapid diffusion of apomictic reproduction., To explore the nature of wild and cultivated mandarins, the authors carry out genomic analysis of diverse east Asian citrus. The discovery of a wild species Citrus ryukyuensis native to the Ryukyu islands and a new population of wild mainland Asian mandarin explains the origin and diversity of mandarins and their ability to reproduce apomictically.

Day length is a determinant of flowering time in rice. Phytochromes participate in flowering regulation by measuring the number of daylight hours to which the plant is exposed. Here we describe G123, a rice mutant generated by irradiation, which displays insensitivity to the photoperiod and early flowering under both long day and short day conditions. To detect the mutation responsible for the early flowering phenotype exhibited by G123, we generated an F2 population, derived from crossing with the wild-type, and used a pipeline to detect genomic structural variation, initially developed for human genomes. We detected a deletion in the G123 genome that affects the PHOTOPERIOD SENSITIVITY13 (SE13) gene, which encodes a phytochromobilin synthase, an enzyme implicated in phytochrome chromophore biosynthesis. The transcriptomic analysis, performed by RNA-seq, in the G123 plants indicated an alteration in photosynthesis and other processes related to response to light. The expression patterns of the main flowering regulatory genes, such as Ghd7, Ghd8 and PRR37, were altered in the plants grown under both long day and short day conditions. These findings indicate that phytochromes are also involved in the regulation of these genes under short day conditions, and extend the role of phytochromes in flowering regulation in rice.

Thirty-seven chloroplast molecular markers were used to evaluate the genetic diversity and infer the phylogenetic relationship of 24 Algerian Citrus accessions from the Institut Technique de l′Arboriculture Fruitiere et de la Vigne germplasm bank. The reliability and consistence of the clustering distribution was further asserted including 5 Spanish accessions from the Instituto Valenciano de Investigaciones Agrarias. The accessions were positioned on a phylogenetic tree of the genus Citrus based on previous analyses of the whole sequence of citrus chloroplast. Algerian accessions clustered into two main clades mostly differentiated by the occurrence of either mandarin or pummelo chloroplast types. All 7 mandarins analyzed were grouped in the same clade while the other cluster subdivided in 4 groups, included 1 lumia, 3 lemons, 2 grapefruits and 11 sweet oranges. Algerian grapefruit accessions were grouped together with the pummelos in a single cluster while all sweet oranges formed an independent and homogenous clade. Interestingly, the lemons studied were clustered in 3 different subclusters while Citrus lumia genotype was isolated in a different group. These results suggest that in contrast to the studied Algerian mandarins or sweet oranges, that share all the same mandarin or sweet orange chloroplast haplotype, the high diversity of current lemon accessions is at least partially correlated with the identity of different pummelo progenitors which evolved from a common ancestor. In addition, the data indicate that Citrus lumia is a new type of citrus chloroplast that appears to be phylogenetically related to the chloroplasts of the pummelo and micrantha group.

The genome of any living organism is the foundation upon which every aspect of that organism’s life is built, including all physical characteristics and interactions with the environment, as well as the mechanisms by which the genome sequence content is passed to offspring. The citrus genome underlies and influences every aspect of citrus trees and fruit, and therefore nearly every chapter topic that follows in this book may best be understood when viewed through the lens of genomic comprehension. A brief history of citrus genome sequencing activities is presented in the historical context of rapidly evolving improvements in genome sequencing technologies, including a summary of de novo genome sequence assemblies. Examples of how the abundance of available citrus genome sequence information has informed studies on the nature of progenitor citrus species, and the contemporary citrus species and the admixtures represented in modern citrus cultivars. Further, the specifics of comparative genome analyses and the conclusions drawn on the evolution and domestication of mandarins are provided. Sequencing technology and associated informatics will continue to improve in the coming years, and the application of the new fundamental information to be generated will support and inform future advances in all the subject areas that follow in this book.

In this chapter, we revise the origin of citrus and other related subjects such as their dispersal and evolution that have been a matter of controversy and have remained elusive during the past centuries. Comparative genomic analyses, however, have recently provided evidence that supports the proposal that the center of origin of citrus was the Southeast foothills of the Himalayas, in a region including the eastern area of Assam, northern Myanmar, and western Yunnan. This new vision also suggests that citrus evolved during the Late Miocene through a rapid Southeast Asian radiation correlating with a dramatic weakening of the monsoons provoked by a period of global cooling. The Australian limes and the Japanese Tachibana mandarin split later from mainland citrus during the Early Pliocene and Pleistocene, respectively. Taken together, these findings draw a new evolutionary framework for these fruit crops, a scenario that challenges current taxonomic and phylogenetic thoughts and points toward a reformulation of the genus Citrus.

Background: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. Results: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. Conclusions: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis. Fil: Sabbione, Agustín Andrés. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Daurelio, Lucas Damian. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Veggeti, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Talón, Manuel. Institut Valencià Dinvestigacions Agràries; España Fil: Tadeo, Francisco. Institut Valencià Dinvestigacions Agràries; España Fil: Dotto, Marcela Claudia. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

Background IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like signaling peptides and the associated HAE (HAESA)-like family of receptor kinases were originally reported in the model plant Arabidopsis thaliana (Arabidopsis) to be involved in the regulation of abscission. IDA peptides, as cell-to-cell communication elements, appear to be implicated in many developmental processes that rely on cell separation events, and even in the responses to abiotic stresses. However, the knowledge related to the molecular machinery regulating abscission and other cell separation processes in economically important crops is scarce. In this work, we determined the conservation and phylogeny of the IDA-like and HAE-like gene families in relevant species of the Solanaceae family and analyzed the expression of these genes in Nicotiana benthamiana to identify members involved in abscission, stem growth and response to drought conditions. Results The phylogenetic relationships among the IDA-like members of the species of Solanaceae studied, grouped the NbenIDA1 (A and B) and NbenIDA2 (A and B) pairs of genes with the Arabidopsis prepropeptides related to abscission. In silico analyses of the regulatory elements of the promoter region of IDA-like members showed that these two pairs of genes, NbenIDA1 and NbenIDA2, contained drought and hormonal response elements, although NbenIDA2A lacked the hormonal regulatory elements. Expression analyses showed that both NbenIDA1 genes were upregulated during corolla abscission. NbenIDA1 and NbenIDA2 genes showed tissue differential expression under water stress conditions, since NbenIDA1 genes were highly expressed in stressed leaves while NbenIDA2 genes, especially NbenIDA2B, were highly expressed in stressed roots. In non-stressed active growing plants, nodes and internodes were the tissues with the highest expression levels of all members of the IDA-like family and their putative HAE-like receptors. Conclusion The results suggest that NbenIDA1A and NbenIDA1B are both involved in the natural process of corolla abscission while the NbenIDA1 and NbenIDA2 pair of genes are implicated in the response to water stress. The data also suggest that IDA peptides may be important during stem growth and development. These results add new evidence that the functional module formed by IDA peptides and its receptor kinases, as defined in Arabidopsis, may also be conserved in Solanaceae.

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

Background Rice plants are sensitive to the agro-climate conditions, being photoperiod one of main factor contributing to their adaptation to the region where they are grown. Dissecting the genetic bases underlying diversity in rice populations adapted to specific environmental conditions is a fundamental resource for breeding. In this study we have analysed a collection of japonica varieties adapted to temperate regions to perform association studies with traits of high agronomical interest such as heading date, plant height, number of panicles, panicle length and number of grains per panicle. Results We have performed a genome wide association study using a panel of 1713 SNPs that, based on previous linkage disequilibrium estimations, provides a full coverage of the whole genome. We have found a total of 43 SNPs associated with variations in the different traits. The identified SNPs were distributed across the genome except in chromosome 12, where no associated SNPs were found. The inspection of the vicinity of these markers also revealed a set of genes associated with physiological functions strongly linked to agronomic traits. Of special relevance are two genes involved in gibberellin homeostasis that are associated with plant height and panicle length. We also detected novel associated sites with heading date, panicle length and number of grain per panicle. Conclusion We have identified loci associated with important agronomic traits among cultivars adapted to temperate conditions. Some of these markers co-localized with already known genes or QTLs, but the association also provided novel molecular markers that can be of help to elucidate the complicated genetic mechanism controlling important agronomic traits, as flowering regulation in the non-dependent photoperiod pathway. The detected associated markers may provide important tools for the genetic improvement of rice cultivars in temperate regions.

Citrus, one of the most valuable fruit crops around the world, are severely damaged by biotic stress and huge economical losses are caused by pathogen infections. Non-host response is an essential plant defense mechanism against pathogen attack, however is still not completely characterized and is poorly studied in non-model plants. In previous reports, we characterized C. sinensis non-host response to Xanthomonas campestris pv. vesicatoria (Xcv), in comparison to infection caused by Xanthomonas citri subsp. citri (Xcc). This was described as a hypersensitive response with structural and physiological modifications, and transcriptional reprogramming of pathogen related proteins and transcription factors, among others. Phytohormones serve as key regulators in plant response to stress, by means of interconnected complex pathways. Here, we study the participation of phytohormone pathways during Citrus non-host response to Xcv. Our results indicate a decrease in abscisic acid, cytokinins, gibberellins, brassinosteroids, auxins and nitric oxide, an increase in ethylene and salicylic acid, constant levels of jasmonic acid, whereas polyamine levels change in a very specific pattern. The present work provides a first broad approach to hormone participation during non-host response in a non-model plant of the Citrus genus, also representative of woody plants. Fil: Petrocelli, Silvana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pizarro, María Dolores. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alet, Analía Inés. Universidad Nacional de Rosario; Argentina Fil: De Ollas, Carlos. Universitat Jaume I; España Fil: Talón, Manuel. Instituto Valenciano de Investigaciones Agrarias; Fil: Tadeo, Francisco R.. Instituto Valenciano de Investigaciones Agrarias; Fil: Gómez Cadenas, Aurelio. Universitat Jaume I; España Fil: Arbona, Vicent. Universitat Jaume I; España Fil: Orellano, Elena Graciela. Universidad Nacional de Rosario; Argentina. Instituto de Biología Molecular y Celular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Daurelio, Lucas Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; Argentina

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.

In citrus, the use of rootstocks promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. 'Rangpur' lime (Citrus limonia, Osbeck) is one of the most tolerant rootstock to drought and is largely used by the Brazilian citrus industry. Previous studies have shown that, when grafted with a variety citrus doubled diploid (4x) 'Rangpur' lime rootstock is more tolerant to drought stress than the respective diploid (2x). In the present study, we characterized the water deficit tolerance in 2x and 4x 'Rangpur' lime seedlings. Water deficit was applied respectively for 21 days, followed by recovery irrigation. Physiological and biochemical parameters were measured periodically during the experiment and samples were collected for biochemical analysis. Doubled diploid seedlings were showed to be more water deficit tolerant than 2x. Water deficit caused a greater reduction in stomatal conductance in 2x compared to 4x. The 21st day of stress, leaf water potential in 2x was lower. Analyses of the activity of enzymes involved in detoxification process suggest that the better tolerance of 4x genotypes may be related to better scavenging of reactive oxygen species. (Resume d'auteur)

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.

Tolerance to water deficit in diploid (2x) and doubled diploid (4x) ‘Carrizo citrange’ (Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf) was investigated. Water deficit was applied for 4 weeks. Physiological parameters, including stomatal conductance (g s), photosynthesis (A), transpiration (E), leaf and soil water potentials (Ψ leaf; Ψ soil), and pot water loss, were monitored throughout the stress. Moreover, ABA, H2O2 contents, and the expression of genes involved in ABA biosynthesis (NCED3), regulation of abscisic acid signaling (ABI1), and coding for a catalase enzyme (CAT2) known to favor H2O2 scavenging were monitored. During the experiment g s, A, and E values were most of the time higher in 2x compared to 4x. During the water deficit period, pot water loss decreased faster in 2x compared to 4x, leading to a faster decrease in all physiological parameters in 2x. The higher sensitivity of 2x compared to 4x was correlated with more numerous thinner roots, higher leaf ABA and H2O2 contents, and with the lower leaf water potential. ABI1 and NCED3 expression was not strictly correlated with the ABA content. However, the higher CAT2 expression in 4x was correlated with the lower leaf H2O2 contents. Therefore, the better tolerance observed in 4x ‘Carrizo citrange’ compared to 2x was associated with more limited water consumption and better and H2O2 scavenging.

Summary Plants, when exposed to certain pathogens, may display a form of genotype-independent resistance, known as non-host response. In this study, the response of Citrus sinensis (sweet orange) leaves to Xanthomonas campestris pv. vesicatoria (Xcv), a pepper and tomato pathogenic bacterium, was analyzed through biochemical assays and cDNA microarray hybridization and compared with Asiatic citrus canker infection caused by Xanthomonas citri subsp. citri. Citrus leaves exposed to the non-host bacterium Xcv showed hypersensitive response (HR) symptoms (cell death), a defense mechanism common in plants but poorly understood in citrus. The HR response was accompanied by differentially expressed genes that are associated with biotic stress and cell death. Moreover, 58 transcription factors (TFs) were differentially regulated by Xcv in citrus leaves, including 26 TFs from the stress-associated families AP2-EREBP, bZip, Myb and WRKY. Remarkably, in silico analysis of the distribution of expressed sequence tags revealed that 10 of the 58 TFs, belonging to C2C2-GATA, C2H2, CCAAT, HSF, NAC and WRKY gene families, were specifically over-represented in citrus stress cDNA libraries. This study identified candidate TF genes for the regulation of key steps during the citrus non-host HR. Furthermore, these TFs might be useful in future strategies of molecular breeding for citrus disease resistance. Fil: Daurelio, Lucas Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Romero, Maria S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Petrocelli, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Merelo, Paz. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Cortadi, Adriana Amalia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Talon, Manuel. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Tadeo, Francisco R.. Institut Valencià d’Investigacions Agràries. Centre de Genómica; España Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

10 Pags., 3 Figs., 3 Tabls., The effects of iron (Fe) deficiency on the low-molecular-weight organic acid (LMWOA) metabolism have been investigated in Carrizo citrange (CC) [Citrus sinensis (L.) Osb. x Poncirus trifoliata (L.) Raf.] roots. Major LMWOAs found in roots, xylem sap and root exudates were citrate and malate and their concentrations increased with Fe deficiency. The activities of several enzymes involved in the LMWOA metabolism were also assessed in roots. In the cytosolic fraction, the activities of malate dehydrogenase (cMDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes were 132 and 100% higher in Fe-deficient conditions, whereas the activity of pyruvate kinase was 31% lower and the activity of malic enzyme (ME) did not change. In the mitochondrial fraction, the activities of fumarase, MDH and citrate synthase enzymes were 158, 117 and 53% higher, respectively, in Fe-deficient extracts when compared with Fe-sufficient controls, whereas no significant differences between treatments were found for aconitase (ACO) activity. The expression of their corresponding genes in roots of Fe-deficient plants was higher than that measured in Fe-sufficient controls, except for ACO and ME. Also, dicarboxylate-tricarboxylate carrier (DTC) expression was significantly increased in Fe-deficient roots. In conclusion, Fe deficiency in CC seedlings causes a reprogramming of the carbon metabolism that involves an increase of anaplerotic fixation of carbon via PEPC and MDH activities in the cytosol and a shift of the Krebs cycle in the mitochondria towards a non-cyclic mode, as previously described in herbaceous species. In this scheme, DTC could play an important role shuttling both malate and reducing equivalents between the cytosol and the mitochondria. As a result of this metabolic switch malate and citrate concentrations in roots, xylem sap and root exudates increase., This work was supported by a grant from the Instituto Nacional de Investigaciones Agrarias, Spain (project RTA 04-064).

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.

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na⁺ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na⁺ and K⁺, even at high external Na⁺ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K⁺ uptake under saline conditions.

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).

To reveal specific Cl(-) transport activities in the symplastic pathway, uptake, long-distance transport and distribution of Cl(-) have been investigated in the citrus rootstocks Carrizo citrange (CC, Cl(-) includer) and Cleopatra mandarin (CM, Cl(-) excluder). Using an external concentration of 4.5 mm Cl(-) , both species actively transported Cl(-) to levels that exceeded the critical requirement concentration by one and two orders of magnitude in the excluder and the includer rootstocks, respectively. Both CC and CM modulated Cl(-) influx according to the availability of the nutrient as uptake capacity was induced by Cl(-) starvation, but inhibited after Cl(-) resupply. Net Cl(-) uptake was higher in the includer CC, an observation that correlated with a lower root-to-shoot transport capacity in the excluder CM. The patterns of tissue Cl(-) accumulation indicated that chloride exclusion in the salt-tolerant rootstock CM was caused by a reduced net Cl(-) loading into the root xylem. Genes CcCCC1, CcSLAH1 and CcICln1 putatively involved in the regulation of chloride transport were isolated and their expression analysed in response to both changes in the nutritional status of Cl(-) and salt stress. The previously uncharacterized ICln gene exhibited a strong repression to Cl(-) application in the excluder rootstock, suggesting a role in regulating Cl(-) homeostasis in plants.

Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes, be they transcripts, proteins or metabolites, are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection. In this study, laser microdissection (LMD) was used to isolate epidermal and subepidermal cells from green, expanding Citrus clementina fruit and their transcriptomes were compared using a 20k citrus cDNA microarray and quantitative real-time PCR. The results show striking differences in gene expression profiles between the two cell types, revealing specific metabolic pathways that can be related to their respective organelle composition and cell wall specialization. Microscopy provided additional evidence of tissue specialization that could be associated with the transcript profiles with distinct differences in organelle and metabolite accumulation. Subepidermis predominant genes are primarily involved in photosynthesis- and energy-related processes, as well as cell wall biosynthesis and restructuring. By contrast, the most epidermis predominant genes are related to the biosynthesis of the cuticle, flavonoids, and defence responses. Furthermore, the epidermis transcript profile showed a high proportion of genes with no known function, supporting the original hypothesis that analysis at the tissue/cell specific levels can promote gene discovery and lead to a better understanding of the specialized contribution of each tissue to fruit physiology.