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Identification and genomic distribution of gypsy like retrotransposons in Citrus and Poncirus.

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.

Amino Acid Sequence↗

Refinement of the Citrus tristeza virus resistance gene (Ctv) positional map in Poncirus trifoliata and generation of transgenic grapefruit (Citrus paradisi) plant lines with candidate resistance genes in this region.

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it's slow spread in R-2 plant lines or an initial appearance of infection and it's subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it's 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed.

Blotting, Northern↗

[Factors affecting Agrobacterium-mediated genetic transformation of Poncirus trifoliata (L.) Raf].

OBJECTIVE: Establishing an effective system for the Agrobacterium-mediated genetic transformation of Poncirus trifoliata, so as to lay a foundation for improving breeds and introducing foreign genes. METHOD: The explants used for the genetic transformation were the epicotyls from Poncirus trifoliata. The Agrobacterium tumefaciens strain was EHA101, containing vector plasmid PGA482GG. The GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: 20-day epicotyls were suitable for transformation. 2-day precultivation enhanced shoot regeneration. Shoot regeneration frequency was high when the cocultivation lasted 2-3 days. With the presence of acetosyringone during cocultivation, the efficiency of transformation could be greatly enhanced, and the frequency of shoot regeneration rose as high as 27.5%. After the explants were cocultivated with Agrobacterium, 2-day delayed selection could stimulate shoot regeneration. CONCLUSION: An effective genetic transformation system has been established for Poncirus trifoliata on the basis of factors affecting Agrobacterium-mediated genetic transformation.

Acetophenones↗

Comparative analysis on the distribution of heterochromatin in Citrus, Poncirus and Fortunella chromosomes.

Double fluorochrome staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) was used to characterize and compare the distribution of constitutive heterochromatin along chromosomes of Citrus, Poncirus and Fortunella species. Only CMA-positive bands were distinguishable in metaphase chromosomes. Preferential distribution of heterochromatin in terminal regions, mainly of the long arm, and centromeric regions of a few long chromosomes was a common feature of these genera. Heteromorphism between possible homologous chromosomes was present in the majority of species. Citrus and Poncirus revealed some remarkably uniform chromosomes without any intensively fluorescing region, whereas Fortunella cultivars were differentiated by the presence of CMA bands in all chromosomes. Through measurements assisted by a computer, amounts of CMA-positive regions were shown to be highest in Fortunella. Similarities between Citrus and Poncirus suggest little heterochromatin diversification among karyotypes of these genera, whereas Fortunella, with higher amounts and more homogenous distribution of heterochromatin, is more divergent.

Chromomycin A3↗

The chromosomes of Citrus and Poncirus species and hybrids: identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding.

In situ hybridization of 18S-5.8S-25S rDNA probes labeled with biotin or rhodamine and 5S rDNA probes labeled with digoxigenin was used to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L. (2n = 2x = 18), Poncirus trifoliata L. Raf. (2n = 2x = 18), and Citrus x Poncirus hybrids (2n = 2x = 18). Counterstaining with the fluorochromes chromomycin A3 and DAPI uniquely identified many but not all chromosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had five or six sites. Four 5S rDNA sites were detected, mostly linked to 18S-25S rDNA sites. Overall we observed high levels of chromosomal heterozygosity in all accessions examined.

Chromosome Banding↗

Comparison between Poncirus and Citrus genetic linkage maps.

Five genetic linkage maps were constructed for the parents of three progenies: Citrus aurantium (A) x Poncirus trifoliata var. Flying Dragon (Pa), C. volkameriana (V) x P. trifoliata var. Rubidoux (Pv) and a self-pollination of P. trifoliata var. Flying Dragon (Pp). The number of polymorphic markers assayed ranged from 48 for Pa to 120 for A according to the heterozygosity of each parental. As our focus was on genome comparison, most of the markers were newly generated simple sequence repeats. Inter-retrotransposon amplified polymorphisms based on four retrotransposon sequences isolated from Citrus spp were also used to saturate the maps. These polymorphisms were much more frequent in A (53) than in Pa (15) and randomly distributed throughout both genomes. Since comparative genomics and quantitative trait locus analysis applicability depends on the reliability of marker ordering, the causes of variation in marker order were investigated. Around 25% of the markers showed gametal segregation distortions. Segregation distortions were also observed at the zygotic level towards a reduction in the observed frequency of homozygotes from that expected in linkage groups 5 and 7. The presence of balanced lethal factors or gametal incompatibility genes in those genomic regions would explain a zygotic advantage of heterozygotes at these specific regions. Four differences in genomic organization were observed; three are putative translocations and affect homeologous linkage groups 3, 7 and 11, where highly distorted markers are found. Other causes of variation in marker order are also discussed: the introduction of new markers in the map, lowering the LOD score and the mapping software. These results represent the first comparative mapping analysis among Citrus and Poncirus species.

Chromosome Mapping↗

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata).

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.

Base Sequence↗

[Effects of arbuscular mycorrhiza on drought tolerance of Poncirus trifoliata].

This paper evaluated the effects of arbuscular mycorrhizal colonization on the growth and drought tolerance of Poncirus trifoliata in a potted culture under natural water stress and resuming water supply. The results showed that the arbuscular mycorrhizal colonization by Glomus mosseae 93 could significantly increase the plant height, stem diameter, and fresh weight of myocrrhizal plant. By the end of the experiment, the survival percentage of transplanted mycorrhizal plant was 7.1% higher than that of nonmyocrrhizal plant. During natural water stress and resuming water supply, the soluble sugar and soluble protein contents and the SOD, POD and CAT activities of root or leaf were higher or significantly higher in mycorrhizal than in nonmycorrhizal plant, suggesting that arbuscular mycorrhizal colonization could improve the osmoregulatory response of myocrrhizal plant, enhance its protective capability, and depress its cell membrane oxidative damage to lipids. Consequently, the drought tolerance of inoculated Poncirus trifoliata was enhanced. It was considered that the functional mechanism of arbuscular mycorrhizal fungus in enhancing the drought tolerance of host plant was closely related to the protective systems such as SOD, POD, CAT, and soluble protein, etc.

Disasters↗

Screening of potential chemopreventive compounds from Poncirus trifoliata Raf.

Chemopreventive agents induce a battery of genes whose protein products can protect cells from chemical-induced carcinogenesis. In this study, we isolated three different coumarins compounds (1; poncimarin, 2; heraclenol 3'-methyl ester and 3; oxypeucedanin methanolate) from Poncirus trifoliata Raf., and studied whether these compounds increase glutathione S-transferase (GST) expression and activity in the H4IIE cell-line (a rat hepatocyte cell line). CDNB (1-chloro-2,4-dinitrobenzene; GST subtype-nonspecific) and NBD (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; GSTalpha-type-specific) assays revealed that compound 1 most potently increased GST enzyme activities. Western blot analysis using subtype-specific antibodies confirmed that these three coumarins also selectively increased GSTalpha-protein expression, and that compound 1 most actively induced GSTalpha. In contrast, the expressions of the GSTmu and GSTmu subtypes were not altered by these three coumarins. Reporter gene analysis using an antioxidant response element (ARE) containing construct and subcellular fractionation assays, revealed that GSTalpha-induction by compound 1 might be associated with Nrf2/ARE activation. These results suggest that these three coumarin compounds from Poncirus trifoliata Raf possess phase II enzyme inducible functions, and in particular, that poncimarin has chemopreventive potential.

Animals↗

Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.).

A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial.

Blotting, Southern↗

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.

Acclimatization↗

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.

Chromosome Mapping↗

GISH, AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and Poncirus trifoliata.

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2 n=4 x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.

Citrus↗

Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).

Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

Acclimatization↗

A simple chromosomal marker can reliably distinguishes Poncirus from Citrus species.

Several chromosome types have been recognized in Citrus and related genera by chromomycin A(3 )(CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata ("Barnes", "Fawcett", "Flying Dragon", "Pomeroy", "Rubidoux", "USDA") and one P. trifoliata x C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA(+) banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus x Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm.

Brazil↗

Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.).

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F(1) progeny obtained by crossing 'Kiyomi' tangor (C. unshiu x sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F(1) progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.

Agrobacterium tumefaciens↗

Poncirus trifoliata fruit induces apoptosis in human promyelocytic leukemia cells.

BACKGROUND: Substances inducing apoptosis have shown efficacy in the treatment of cancers. Poncirus trifoliata (L.) Raf. (Rutaceae) fruits (PTF) has been used for the treatment of various cancers among Korean Oriental Medical doctors. METHODS: PTF-induced cytotoxicity of human leukemia HL-60 cells was monitored by the MTT assay. The apoptosis was determined by (a) apoptotic morphology in microscopy; (b) DNA fragmentation in electrophoresis and FACS analysis; and (c) activation of caspase-3 and poly-ADP-ribose polymerase (PARP) cleavage assay. RESULTS: The cytotoxic activity of PTF in HL-60 cells was increased in a concentration- and time-dependent manner. PTF caused the cell shrinkage, cell membrane blebbing, apoptotic body and DNA fragmentation. PTF-induced apoptosis is accompanied by the activation of caspase-3 and the specific proteolytic cleavage of PARP. However, PTF did not show cytotoxicity in normal peripheral blood mononuclear cells. CONCLUSIONS: Our novel finding provides evidence that PTF could be a candidate as an anti-leukemic agent through apoptosis of cancer cells.

Apoptosis↗

Prokinetic activity of an aqueous extract from dried immature fruit of Poncirus trifoliata (L.) Raf.

Aqueous extracts from dried immature fruit of Poncirus trifoliata Raf. (Rutaceae) (PF-W) are used as a traditional Korean folk medicine for the treatment of digestive dysfunction. In the present study, PF-W exhibited no significant toxicity even at a dose of 5 g/kg when orally administered to mice. The effect of PF-W on gastrointestinal (GI) motor function was investigated by examining its effect on the serum concentration of orally administered ranitidine, a putative indicator of GI motility, in human subjects. The area under the serum concentration-time curve and the peak serum concentration of ranitidine following an oral administration (300 mg/individual) were decreased by one half as the result of a predose (10 g/individual) of PF-W, except for the time to reach peak serum concentration and the serum half-life at the terminal phase of ranitidine. In rat studies, PF-W had no effect on the apparent permeability of ranitidine across the jejunum or the gastric emptying rate (GER) of phenol red. However, the transit time for charcoal in the intestine was significantly increased by the PF-W pretreatment. The above results are consistent with the hypothesis that PF-W has a unique prokinetic activity, which accelerates the transit of intestinal contents, but has no effect on the GER.

Animals↗