The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar ...P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting "acidless" varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.
Adeno-associated viral vector 9 (AAV9) has recently been shown to penetrate the blood-brain barrier via intravascular administration, making it a good candidate for diffuse gene delivery. However, ...the potential side effects of systemic delivery are unknown. Intrathecal viral vector administration may be more invasive than intravenous injections, but it requires far less vector and it can be performed on an outpatient basis, making it an ideal route of delivery for clinical translation. A total of 12 domestic farm pigs (<20 kg) underwent a single-level lumbar laminectomy with intrathecal catheter placement for AAV9 delivery. Animals were perfused and the tissue was harvested 30 days after treatment. Gene expression was assessed by anti-green fluorescent protein immunohistochemistry. Although a single lumbar injection resulted in gene expression limited to the lumbar segment of the spinal cord, three consecutive boluses via a temporary catheter resulted in diffuse transduction of motor neurons (MNs) throughout the cervical, thoracic and lumbar spinal cords. We now present the first successful robust transduction of MNs in the spinal cord of a large animal via intrathecal gene delivery using a self-complementary AAV9. These promising results can be translated to many MN diseases requiring diffuse gene delivery.
Twenty-four simple sequence repeat (SSR) markers were used to detect molecular polymorphisms among 370 mostly sexually derived Citrus accessions from the collection of citrus germplasm maintained at ...the University of California, Riverside. A total of 275 alleles were detected with an average of 11.5 alleles per locus and an average polymorphism information content of 0.625. Genetic diversity statistics were calculated for each individual SSR marker, the entire population, and for specified Citrus groups. Phylogenetic relationships among all citrus accessions and putative non-hybrid Citrus accessions were determined by constructing neighbor-joining trees. There was strong support for monophyly at the species level when hybrid taxa were removed from the data set. Both of these trees indicate that Fortunella clusters within the genus Citrus but Poncirus is a sister genus to Citrus. Additionally, Citrus accessions were probabilistically assigned to populations or multiple populations if their genotype indicated an admixture by a model-based clustering approach. This approach identified five populations in this data set. These separate analyses (distance and model based) both support the hypothesis that there are only a few naturally occurring species of Citrus and most other types of Citrus arose through various hybridization events between these naturally occurring forms.
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.
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.
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.
Rapid advancements in technologies provide various tools to analyze fruit crop genomes to better understand genetic diversity and relationships and aid in breeding. Genome-wide single nucleotide ...polymorphism (SNP) genotyping arrays offer highly multiplexed assays at a relatively low cost per data point. We report the development and validation of 1.4M SNP Axiom
Citrus HD Genotyping Array (Citrus 15AX 1 and Citrus 15AX 2) and 58K SNP Axiom
Citrus Genotyping Arrays for
and close relatives. SNPs represented were chosen from a citrus variant discovery panel consisting of 41 diverse whole-genome re-sequenced accessions of
and close relatives, including eight progenitor citrus species. SNPs chosen mainly target putative genic regions of the genome and are accurately called in both
and its closely related genera while providing good coverage of the nuclear and chloroplast genomes. Reproducibility of the arrays was nearly 100%, with a large majority of the SNPs classified as the most stringent class of markers, "
" (PHR) polymorphisms. Concordance between SNP calls in sequence data and array data average 98%. Phylogenies generated with array data were similar to those with comparable sequence data and little affected by 3 to 5% genotyping error. Both arrays are publicly available.
•An extensive stability analysis adapted to CHT problems in a full transient flight cycle is performed for the first time. This analysis based on the theory of Godunov-Ryabenkii has been applied to ...two complementary interface conditions (Dirichlet-Robin and Neumann-Robin).•The mathematical expression of the stability bounds and the so-called “optimal coefficients” are provided. These limits have really nothing in common with those obtained from a steady CHT analysis, in companion papers. A new stability analysis is thus fully justified.•Two stability conditions are expressed. The first one is a “fluid” stability condition stating how the fluid grid must be refined in the near-wall region. The other condition is about the temporal amplification factor of the coupled problem, precisely described and summarized in two Tables.•On this theoretical basis, a unified approach is proposed for a full transient flight cycle.•A systematic numerical study – covering all eventualities (low and large Biot numbers) – in a test case is presented. The potential numerical problems (instabilities, oscillations, slow convergence) identified in the literature are encountered and explained. All these problems are clarified and overcome by using the coupling methodology proposed in this paper and in particular the optimal coupling coefficients.•From this study, it is always possible to choose a “natural” method that is unconditionally stable. This is a very positive feature which cannot be reached by a single transmission procedure. The stability behavior is not intuitive and obvious. Understanding the reasons for differences in performance is crucial and implies a detailed analysis taking into account the main physical and numerical parameters.
The purpose of the present study is to describe novel numerical coupling schemes to analyze the transient temperature field in a solid via a conjugate heat transfer procedure. Emphasis is put on the interfacial treatment based on two complementary treatments: Dirichlet-Robin and Neumann-Robin transmission conditions. The numerical methods are first presented on the basis of a stability analysis in an aerothermal model problem. Stability conditions are expressed and the mathematical expression of the most relevant coupling parameters are provided for the first time. Furthermore, an overview of all the coefficients that can be used in a transient thermally-coupled procedure are given and a unified approach for steady and unsteady ramps is proposed. Then, these interfacial schemes are applied to the problem of convective heat transfer over, and transient conduction heat transfer within, a flat plate. A comparative study with realistic operating conditions is carried out, at low and large Biot numbers. It is shown that certain choices of coupling coefficients, even if physically reasonable, may result in non-converging algorithms. This confirms that a model problem provides insight to the behavior of complicated heat transfer cases and constitutes an invaluable aid for generating efficient interfacial schemes. Indeed, the numerical computations demonstrate the efficiency of the numerical schemes based on the main theoretical results. The trends predicted by the model problem are recovered and excellent convergence properties are observed in all cases.
Relationships among 88 accessions representing 45 Citrus species, three man-made hybrids, and six related genera were examined for restriction fragment length polymorphisms (RFLP). Thirty-two Citrus ...and three Microcitrus accessions were also examined by random amplified polymorphic DNA (RAPD) analysis. A measure of relative heterozygosity was estimated based on the mean of the number of fragments per individual per probe-enzyme combination (PEC) divided by total number of fragments per PEC for all non-hybrid Citrus individuals. The presence in a Citrus species of a rare band found also in a related genus was taken as an indication of possible introgression, while the presence of several fragments unique to 1 species was used to indicate non-involvement of that species in hybridization events. Most species that have been described in the literature as hybrids had high heterozygosity indices and no unique fragments. Distance matrices and dendrograms were generated using simple matching coefficient and neighbor-joining cluster analysis. RFLP and RAPD data gave approximately the same results. These data showed C. maxima was affiliated with the papedas C. hongheensis and C. latipes. C. medica clustered with C. indica when only nonhybrid taxa were examined, or among limes, lemons, and relatives when all species were considered. Mandarins did not show strongly supported groupings among themselves, nor with other species. These data showed that several accessions were probably assigned to the wrong species.
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•Studied 47 citrons; many from Yunnan, an area of origin; many novel taxa.•Conducted analysis using microsatellites and single nucleotide polymorphisms.•Heterozygosity observed in ...some. Structure analysis revealed three populations.•Clusters 1 and 2 from China (non-fingered, fingered); cluster 3 from Mediterranean.•A citron-specific microsatellite marker identified.
Citron (Citrus medica L.) is one of the three basic species of the genus Citrus L. that have contributed to the development of cultivated citrus. We analyzed the genetic diversity of 47 citrons (32 from Yunnan Province, China; and 15 of Mediterranean origin) to understand their diversity and relationships within the species. Genetic analysis was conducted using data from microsatellite markers, single nucleotide polymorphisms generated from sequences of a nuclear malate dehydrogenase gene and a chloroplast gene, rps16. Neighbor joining and maximum parsimony analyses were conducted. All three approaches found citron to be monophyletic. Population structure analysis clustered the 47 citrons into three distinct groups. The first group consisted of wild, non-fingered citrons generally having locules, juice sacs and seeds within the fruit. The second cluster consisted mostly of fingered citrons that lacked locules, juice sacs or seeds, and some non-fingered types with smaller locules and vestigial juice sacs, but with seeds. All accessions that clustered in groups I and II originated in China. The third cluster consisted of citrons cultivated in the Mediterranean region. Genetic distance between the clusters from population structure analysis indicated considerable diversity within the species. A citron-specific microsatellite marker was identified and characterized. We observed considerable heterozygosity in certain citrons, contrary to previous reports.
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