Cytological and molecular characterization of three gametoclones of Citrus clementina

Abstract Background Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (arrayCGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes

Comprehensive study of the CuF<inf>2</inf> conversion reaction mechanism in a lithium ion battery

Conversion materials for lithium ion batteries have recently attracted considerable attention due to their exceptional specific capacities. Some metal fluorides, such as CuF2, are promising candidates for cathode materials owing to their high operating potential, which stems from the high electronegativity of fluorine. However, the high ionicity of the metal–fluorine bond leads to a large band gap that renders these materials poor electronic conductors. Nanosizing the active material and embedding it within a conductive matrix such as carbon can greatly improve its electrochemical performance. In contrast to other fluorides, such as FeF2 and NiF2, good capacity retention has not, however, been achieved for CuF2. The reaction mechanisms that occur in the first and subsequent cycles and the reasons for the poor charge performance of CuF2 are studied in this paper via a variety of characterization methods. In situ pair distribution function analysis clearly shows CuF2 conversion in the first discharge. However, few structural changes are seen in the following charge and subsequent cycles. Cyclic voltammetry results, in combination with in situ X-ray absorption near edge structure and ex situ nuclear magnetic resonance spectroscopy, indicate that Cu dissolution is associated with the consumption of the LiF phase, which occurs during the first charge via the formation of a Cu1+ intermediate. The dissolution process consequently prevents Cu and LiF from transforming back to CuF2. Such side reactions result in negligible capacity in subsequent cycles and make this material challenging to use in a rechargeable battery.We acknowledge the funding from the U.S. DOE BES via funding to the EFRC NECCES, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001294 (support for Rosa Robert and Lin-Shu Du) and EPSRC via the “nanoionics” programme grant (support for Xiao Hua). Use of the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.This is the final published version of the article. It first appeared at http://pubs.acs.org/doi/abs/10.1021/jp503902z and is posted here under the terms of ACS's Editors' Choice scheme (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)

LRR-RLK family from two Citrus species: Genome-wide identification and evolutionary aspects

Background: Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors. Results: We built integrative protein signature databases for Citrus clementina and Citrus sinensis using all predicted protein sequences obtained from whole genomes. A total of 300 and 297 proteins were identified as LRR-RLKs in C. clementina and C. sinensis, respectively. Maximum-likelihood phylogenetic trees were estimated using Arabidopsis LRR-RLK as a template and they allowed us to classify Citrus LRR- 34 RLKs into 16 groups. The LRR-XII group showed a remarkable expansion, containing approximately 150 paralogs encoded in each Citrus genome. Phylogenetic analysis also demonstrated the existence of two distinct LRR-XII clades, each one constituted mainly by RD and non-RD kinases. We identified 68 orthologous pairs from the C. clementina and C. sinensis LRR-XII genes. In addition, among the paralogs, we identified a subset of 78 and 62 clustered genes probably derived from tandem duplication events in the genomes of C. clementina and C. sinensis, respectively. Conclusions: This work provided the first comprehensive evolutionary analysis of the LRR-RLKs in Citrus. A large expansion of LRR-XII in Citrus genomes suggests that it might play a key role in adaptive responses in host-pathogen co-evolution, related to the perennial life cycle and domestication of the citrus crop species

Fortunella margarita Transcriptional Reprogramming Triggered by Xanthomonas citri subsp. citri

<p>Abstract</p> <p>Background</p> <p>Citrus canker disease caused by the bacterial pathogen <it>Xanthomonas citri </it>subsp. <it>citri (</it>Xcc) <it>has </it>become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative <it>Fortunella margarita </it>Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in <it>Fortunella </it>and citrus at the molecular level.</p> <p>Results</p> <p><b>cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray</b>. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus.</p> <p>Conclusion</p> <p>Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.</p

Preliminary Evidence for Rootstock Effects on HLB Infection Frequency and Disease Severity in Sweet Orange and ‘SugarBelle’ Trees

Evidence is accumulating that root system collapse is involved with HLB-induced tree decline, especially with trees on Swingle and Carrizo.&nbsp; Phytophthora resistance appears to be breaking down in HLB-infected trees on Swingle.&nbsp; Other stresses caused by blight, nematodes, cold, etc. also appear to be interacting with HLB to increase HLB disease frequency and severity.&nbsp; Improved rootstocks could help to mitigate these problems, allowing for sustainable production under appropriate nutrition. We are testing complex hybrid rootstock candidates (diploid and tetraploid) to determine their affect on HLB disease establishment and severity in trees grafted with sweet orange scions; field and greenhouse experiments are underway.&nbsp; Rootstocks differentially translocate nutrients, phytohormones (plant growth regulators), micro-RNAs, small proteins (pathogenesis related?), and other metabolites to the scion.&nbsp; This could have both direct and indirect, quantitative and quantitative affects on scion gene expression, and possibly Liberibacter pathogenesis in citrus – especially with unique complex allotetraploid rootstocks. Data from two young field trials (both with the ‘bad neighbor’ effect) established to evaluate new rootstock candidates, previously not screened for HLB tolerance, will be presented. These include a trial of 3.5 year old trees of ‘SugarBelle’ that is nearly 100% infected with HLB, and a trial of 4.5 year old trees of sweet orange on &gt;50 rootstocks that is approximately 15% infected.&nbsp; Rootstock differences regarding HLB disease frequency and severity are emerging. Complex ‘tetrazyg’ rootstock Orange #19 (Nova+HBPummelo x Cleopatra+Argentine trifoliate orange) is showing more HLB tolerance at both locations.&nbsp;&nbsp; Data on percentages of symptomatic fruit and fruit drop per rootstock will be presented

Preliminary Evidence for Rootstock Effects on HLB Infection Frequency and Disease Severity in Sweet Orange and ‘SugarBelle’ Trees

Evidence is accumulating that root system collapse is involved with HLB-induced tree decline, especially with trees on Swingle and Carrizo.  Phytophthora resistance appears to be breaking down in HLB-infected trees on Swingle.  Other stresses caused by blight, nematodes, cold, etc. also appear to be interacting with HLB to increase HLB disease frequency and severity.  Improved rootstocks could help to mitigate these problems, allowing for sustainable production under appropriate nutrition. We are testing complex hybrid rootstock candidates (diploid and tetraploid) to determine their affect on HLB disease establishment and severity in trees grafted with sweet orange scions; field and greenhouse experiments are underway.  Rootstocks differentially translocate nutrients, phytohormones (plant growth regulators), micro-RNAs, small proteins (pathogenesis related?), and other metabolites to the scion.  This could have both direct and indirect, quantitative and quantitative affects on scion gene expression, and possibly Liberibacter pathogenesis in citrus – especially with unique complex allotetraploid rootstocks. Data from two young field trials (both with the ‘bad neighbor’ effect) established to evaluate new rootstock candidates, previously not screened for HLB tolerance, will be presented. These include a trial of 3.5 year old trees of ‘SugarBelle’ that is nearly 100% infected with HLB, and a trial of 4.5 year old trees of sweet orange on &gt;50 rootstocks that is approximately 15% infected.  Rootstock differences regarding HLB disease frequency and severity are emerging. Complex ‘tetrazyg’ rootstock Orange #19 (Nova+HBPummelo x Cleopatra+Argentine trifoliate orange) is showing more HLB tolerance at both locations.   Data on percentages of symptomatic fruit and fruit drop per rootstock will be presented