text:
Is your partner one of the faithful zodiac signs or a cheater in disguise?
Is there a person immune to cheating? With temptation lurking everywhere, it is hard not to have a wandering eye. Faithfulness is a quality that everyone seeks in their partners. It comes as no surprise that some of our traits are inherited by the stars. If, in the past, you were cheated on by a partner, try to prevent that by analyzing his zodiac behavior. With this in mind, let us find out which of the star signs are big on fidelity and which ones are cheaters?
Taurus, Leo and Scorpio are signs famous for their loyalty and dependability. They tend to make trustworthy faithful lovers. In case you end up having troubles with one of these star signs, consider that your fault. You want to know why? Because they for sure provided you with lots of warning signs. Being fixed signs of the Zodiac, they're slow to make changes. Once established in a comfortable loving routine, they are generally reluctant to cheat. No matter how great the temptation, they will rarely stray.
Warning about Scorpio: While you'll never find a more steady and devoted partner than a Scorpio, they will only be faithful as long as you are. If you break a Scorpio's trust by cheating on them, you'll lose their loyalty forever. Moreover, you can expect serious revenge in return.
Capricorn is one of the most faithful zodiac signs. What Capricorn seeks is a partner who will be their support and give them a leg up in life. Someone who will help their career and status. When they've finally found that special person who fulfills their practical needs, they will not risk losing them just for the sake of a brief affair. One-night stand is not really their thing.
Virgos like Capricorns are practical where love is concerned. Certainly, this sign doesn't have the reputation of a cheater. Being a workaholic by nature, they simply don't have the time for it. However, when they do love someone, they give their heart and soul to the relationship. And if they get nothing in return, there comes the trouble.
Home and family oriented Cancer will rarely stray in a committed relationship. If they are satisfied on both levels, emotional and material, they would never cheat. Their purpose of living is finding a soul mate they can have a future with. If they think you're the One, they'll never leave you.
On the other side, the zodiac signs most likely to be unfaithful are those for whom personal freedom tends to be a priority in life. Aries can have problems containing their libido. No matter how long they have been in a relationship, they consider themselves single and fancy-free. Like Aries, Gemini and its opposite sign Sagittarius are natural born cheaters. They seek uncommitted relationships and can't stand to be tied down.
Aquarius – sign ruled by Uranus, the planet of experimentation, it's often drawn to open relationships. They like everything that's forbidden. So, our advice is never tell them they must not do it. Only if you give them freedom, they're less likely to feel the need to cheat. However, if you're the possessive type by nature, you could find their love style hard to handle.
Libra, the sign of true love and partnership, is faithful only when they have given you a firm and final commitment. Most Libras never quite know what they want. They have a special talent for attracting the opposite sex very easily. Thus, making it not hard to see why they have a reputation for being unfaithful.
And finally Pieces! They fall in – and out – of love a little too easily and have occasional problems with truthfulness. No matter how well you think you know your Pisces lover, you can never be quite sure what they have done. All the Fishes are experts at covering their tracks.


text:
Researchers discover gene that permanently stops cancer cell proliferation
Researchers at Case Western Reserve University School of Medicine have discovered a mutant form of the gene, Chk1, that when expressed in cancer cells, permanently stopped their proliferation and caused cell death without the addition of any chemotherapeutic drugs. This study illustrates an unprecedented finding, that artificially activating Chk1 alone is sufficient to kill cancer cells.
"We have identified a new direction for cancer therapy and the new direction is leading us to a reduction in toxicity in cancer therapy, compared with chemotherapy or radiation therapy," said Dr. Zhang, assistant professor, Department of Pharmacology at the School of Medicine, and member of the university's Case Comprehensive Cancer Center. "With this discovery, scientists could stop the proliferation of cancer cells, allowing physicians time to fix cells and genetic errors."
While studying the basic mechanisms for genome integrity, Dr. Zhang's team unexpectedly discovered an active mutant form of human Chk1, which is also a non-natural form of this gene. This mutation changed the protein conformation of Chk1 from the inactive form into an active form. Remarkably, the research team discovered that when expressed in cancer cells, this active mutant form of Chk1 permanently stopped cancer cell proliferation and caused cell death in petri dishes even without the addition of any chemotherapeutic drugs.
The biggest advantage of this potential strategy is that no toxic chemotherapeutic drug is needed to achieve the same cancer killing effect used with a combination of Chk1 inhibitors and chemotherapeutic drugs.
Cells respond to DNA damage by activating networks of signaling pathways, termed cell cycle checkpoints. Central to these genome pathways is the protein kinase, called Chk1. Chk1 facilitates cell survival, including cancer cells, under stressful conditions, such as those induced by chemotherapeutic agents, by placing a temporary stop on the cell cycle progression and coordinating repair programs to fix the DNA errors.
It has long been suggested that combining Chk1 inhibition with chemotherapy or radiotherapy should significantly enhance the anticancer effect of these therapies. This idea has serves as the basis for multiple pharmaceutical companies searching for potential Chk1 inhibitors that can effectively combine with chemotherapy in cancer therapy. To date, no Chk1 inhibitor has passed the clinical trial stage III . This led Dr. Zhang's team to look for alternative strategies for targeting Chk1 in cancer therapy.
Future research by Dr. Zhang and his team will consider two possible approaches to artificially activating Chk1 in cancer cells. One possibility is to use the gene therapy concept to deliver the active mutant form of Chk1 that the team discovered, into cancer cells. The other is to search for small molecules that can induce the same conformational change of Chk1, so that they can be delivered into cancer cells to activate Chk1 molecules. The consequence of either would be permanent cell proliferation inhibition and cancer.
This study is published in Cancer Research.

subset: commoncrawl


text:
Fans love Supergirl on the small screen, but now she may be jumping back onto the big screen.
Deadline reports that writer Oren Uziel (Cloverfield Paradox, 22 Jump Street) is working on a script that would bring Supergirl into the DC Universe. The character is currently being played by Melissa Benoist on the CW and previously had a movie back in 1984 starring Helen Slater (who also guest stars on the CW show as Kara's adoptive mom).
According to the report, this film is in such early stages, Warner Bros. hasn't even attached producers to it yet. So questions about how it would fit into the current slate of DC movies are still very much unanswerable. The biggest one would be how, or if, Superman (Kara's cousin from comics lore) would play a role and if that character would still be played by Henry Cavill. Cavill has long expressed interested in returning, and is expected to return for a Man of Steel sequel, but that was before Supergirl was on the table.
There's certainly a change in the air at DC after the release of 2017's Justice League, though. The next three films, Aquaman, Shazam, and Wonder Woman 1984, certainly feel a little lighter than the previous fare. After that, there's the unrelated Todd Phillips Joker movie, the Flash movie (finally), and Birds of Prey, all of which are likely to come before Matt Reeves' Batman film.
Supergirl is a character who hits a nice balance between the Batman v Superman version of DC movies, and what we expect is beginning to take shape from Warner's superhero films.
Let the rampant speculation about who could play the new big screen Supergirl, and who you'd like to direct, begin.


text:
ORIGINAL RESEARCH article
Sec. Plant Genetics and Genomics
Volume 7 - 2016 | https://doi.org/10.3389/fpls.2016.00510
Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes
- 1Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, University of Lisbon, Lisboa, Portugal
- 2Plant Cell Biotechnology Laboratory, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal
- 3Institut National de la Recherche Agronomique, Centre National de Ressources Génomiques Végétales, Castanet-Tolosan, France
- 4RAIZ, Instituto de Investigação da Floresta e Papel, Aveiro, Portugal
- 5Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal
- 6Department of Integrative Plant Biology, Instytut Genetyki Roślin, Polskiej Akademii Nauk, Poznań, Poland
The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH, and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S rDNA locus while the AT-rich heterochromatin pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich heterochromatin, along with genome sizes estimations, support the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich heterochromatin was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1) previously assessed to linkage group 10 (LG10) was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.
Eucalypts (Eucalyptus L'Her) belong to Myrtaceae family and are predominantly native to Australia and to its North islands, where they occur in various habitats. Plants from this genus are highly diverse and display significant adaptability and phenotypic plasticity. They can be giant trees like E. regnans or shrubs and "mallees" like E. pulverulenta (Grattapaglia et al., 2012). The latest taxonomic revision (Brooker, 2000) of the eucalypts recognizes just over 700 species that belong to 13 subgenera including Corymbia K.D. Hill & L.A.S.Johnson (the bloodwood eucalypts) (Hill and Johnson, 1995), and Angophora Cav. (Ladiges, 1997). Most species belong to the subgenus Symphyomyrtus Schauer, which is divided in 14 sections (Brooker, 2000). From all of these sections only three are used in plantation forestry for industrial purposes: sect. Latoangulatae (E. grandis and E. urophylla), and sect. Exsertaria (E. camaldulensis) which grow in tropical and subtropical regions; and sect. Maidenaria (E. globulus) which grows in temperate regions (Grattapaglia et al., 2012). These species and their hybrids are among the world's leading sources of woody biomass and are the main hardwoods used for pulpwood and timber.
The main breeding programs in eucalypts worldwide are focused on improving profit from industrial pulp-wood plantations. Traditionally, key target traits are volume per hectare, wood density, and pulp yield. Secondary wood traits include the quantity or quality of extractives and lignin in the wood that affect the economic and/or environmental cost of pulping (Myburg et al., 2014). Wood properties and final uses are mainly determined by the relative abundance of the three major polymers deposited in the secondary cell walls of xylem cells, namely cellulose (40–50%), hemicellulose (25%), and lignin (25–35%) (Plomion et al., 2001). The identification of major genes involved in lignin biosynthesis provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities (Carocha et al., 2015). The cinnamoyl CoA reductase (CCR) is a key enzyme that converts the hydroxycinnamoyl CoA esters into monolignols, the monomeric units that are incorporated into the lignin heteropolymer. For this reason the CCR gene has been considered a good candidate gene for molecular breeding for wood properties in Eucalyptus (Grattapaglia et al., 2012). The first cDNA encoding CCR was isolated from E. gunnii EguCCR (Lacombe et al., 1997) whereas in E. grandis were reported two genes, EgrCCR1 which has been allocated to the Linkage group (LG) 10 and EgrCCR2 allocated to LG 6 (Carocha et al., 2015). EgrCCR1 was found to be strongly and preferentially expressed in developing xylem, in agreement with its role in developmental lignin biosynthesis (Carocha et al., 2015). Another gene associated with cell growth, differentiation and developmental regulation, is the molecular switch Rho-related small GTP-binding protein EgROP1 or EguRAC1, wich is involved in cell differentiation during secondary xylem formation and might affect lignin composition and quality (Foucart et al., 2009).
The availability of E. grandis genome sequence (Myburg et al., 2014) together with increasingly powerful molecular technologies provide exceptional opportunities for whole-genome comparative and evolutionary studies across species of Eucalyptus. Although there is a huge amount of molecular resources for comparative Eucalyptus studies, including genetic linkage maps constructed with transferable microsatellites (Grattapaglia et al., 2015), QTL mapping (Gion et al., 2015), and microarray-based Diversity Array Technology (DArT) (Grattapaglia et al., 2011; Steane et al., 2011), the knowledge of these genomes at the cytogenomic level is very scarce. The correlation between linkage maps built with several molecular markers and physical chromosomes is not always direct, despite the continued addition of new markers and genes to previous maps. The assembled meta-chromosomes of the E. grandis reference genome sequence were numbered and oriented according to conventional linkage maps using a genome-wide framework of microsatellite and DArT markers (Bartholomé et al., 2015) even though the assigning to linkage maps was only inferred. Only few studies concerning chromosomal counting in several species (Matsumoto et al., 2000; Oudjehih and Abdellah, 2006) and genome size estimations of the economically most important species are available (Grattapaglia et al., 2012).
Comparative cytogenomic analysis is important to understand species evolution through the assessment of genetic divergence between them using genome organization knowledge (Lipman et al., 2013; Cai et al., 2014). Techniques such as fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are exceptional molecular cytogenetic tools to reveal phylogenetic relationships among species in several crops (Maluszynska and Heslop-Harrison, 1993; Fuchs et al., 1998; Hajdera et al., 2003; Srisuwan et al., 2006), but also in trees (Ribeiro et al., 2008). The constitutive heterochromatin (AT-rich or GC-rich DNA) and rRNA genes are the most widely used FISH markers (Siljak-Yakovlev et al., 2014), however modern molecular cytogenetic studies benefit from BAC libraries. FISH using BACs probes has been used to enable successful genome assembly (Chamala et al., 2013; Myburg et al., 2014), to correct chromosome identity (Tuskan et al., 2006; Islam-Faridi et al., 2009), to map genes of interest (Mendes et al., 2011; Silva et al., 2015), and to serve as a supporting tool to select BAC clones for sequencing and tagging gene-rich regions in species with low sequence resources (Książkiewicz et al., 2013). BAC and fosmid clones have also been useful for the integration of genetic and physical maps (Mun et al., 2006) and in comparative genomics and assays of colinearity (Jenkins and Hasterok, 2007; Gu et al., 2009; Zhao et al., 2011; Yang et al., 2014).
In this study we characterized and compared the karyotypes of six eucalypt species belonging to the subgenus Symphyomyrtus: three with high economic importance, E. grandis, E. globulus, and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta, and E. occidentalis. To achieve this we used an integrative approach with estimation of genome sizes, fluorochrome banding, FISH mapping with rDNA markers, and BAC clones.
Materials and Methods
Plant Material
The plant material (Table 1) used in this study was obtained from the seeds collection of the herbarium of the Institute of Agronomy of the University of Lisbon, (Lisbon, Portugal) (LISI). Seedlings were maintained in a growth chamber (22 ± 2°C and photoperiod of 16/8 h of light and dark, respectively). Root tips for chromosome preparations and leaves for flow cytometric analyses were collected from these seedlings. Leaves of the internal reference standard for genome size estimations (Solanum lycopersicum "Stupické") were obtained from seeds kindly provided by the Laboratory of Molecular Cytogenetics and Cytometry (Olomouc, Czech Republic), which were further germinated in pots in a growth chamber with the conditions described above.
Table 1. Taxonomical classification of the plant material, native distribution, seeds provenance, and voucher numbers [LISI designation is in accordance to Index Herbariorum codes (Thiers, 2010)].
Eucalyptus globulus BAC Libraries and Selection of BAC Clones for BAC Landing
The E. globulus BAC libraries EGC-Ba and EGC-Bb were kindly provided by RAIZ Institute (Portugal). The BAC libraries were prepared by Clemson University Genomic Institute (CUGI), from young leaves collected on a elite E. globulus tree (coded TUGAL) grown at Espirra (Portugal), using the CUGI own protocol. Extracted high molecular weight gDNA was restricted with EcoRI (library EGC-Ba) and HindIII (library EGC- Bb) to obtain the appropriate partial digestion conditions (Paiva et al., 2011). For both libraries, pulsed-field gel electrophoresis (PFGE) size-selected restriction fragments were ligated to the EcoRI or HindIII digest pIndigoBAC536 vector. Ligation products were transformed into DH10B T1 phage resistant Escherichia coli cells (Invitrogen, Carlsbad, CA).
Identification of Clones Harboring Genes of Interest
For each library the 36,864 BAC clones were reorganized into 3D-pools at the French Plant Genomic Center (CNRGV) following the procotol used by Paux et al. (2008). The genes of interest were identified by screening these E. globulus BAC 3D-pools by PCR, using specific primers for the E. gunnii Cinnamoyl CoA reductase gene (CCR) (EguCCR; X79566) (F:TGATGAGGTGAACCCAAGAGTA and R:TTTCT CCTGCAAGCTCTTGACA) (Rasmussen-Poblete et al., 2008) and for the E. gunnii Rho-related small GTPbinding protein (EguRAC1; DR410036) (F:AGCGCGTCG AGGTTCATCAA and R: TGGGGAAAGTGTTGCTGGTATA) (Rengel et al., 2009). Chloroplastic and mitochondrial sequences harboring BAC clones were identified as described in Paiva et al. (2011).
BAC Clones Sequencing
Before sequencing, for each selected BAC clone the presence of the gene/loci of interest was rechecked by PCR on one isolated colony. BAC insert sizes of seleced clones were estimated on the electrophoretic profile of NotI enzyme digested BAC DNA fragments and resolved by PFGE (CHEF-DRIII system, Bio-Rad) in a 1% agarose gel in TBE buffer 0.5×, using the conditions described in Paiva et al. (2011). BAC DNA extractions, sequencing library generation, and sequencing were performed by Beckman Coulter Genomics SA. BAC inserts were sequenced by pyrosequencing using a Roche GS FLX Life Sciences instrument (Branford, CT, USA) (Margulies et al., 2005). The MIRA software (http://sourceforge.net/projects/mira-assembler/) was used to perform de novo assembly of the Roche GS FLX reads.
Chromosome Preparations
C-metaphases were induced by treating roots with a saturated solution of α-bromonaphthalene for 3 h at room temperature. Root tips were fixed in fresh ethanol to glacial acetic acid solution (3:1, v/v), and well-spread chromosomes were obtained using the squash technique according to Schwarzacher and Heslop-Harrison (2000) with the enzymatic mixture proper to woody species (Ribeiro et al., 2011).
Fluorochrome Banding
Slides were incubated in McIlvane buffer pH 7.0 + Mg2+ during 15 min followed by an incubation with Chromomycin A3-staining (CMA3) 0.5% during 90 min in the dark at room temperature. The slides were washed in McIlvane buffer pH 7.0 and mounted in 4′,6-diamidino-2-phenylindole (DAPI). DAPI banding patterns were fully revealed after FISH experiments.
Fluorescence In situ Hybridization (FISH)
The following probes were used for FISH: 35S rDNA unit of wheat (pTa71) (Gerlach and Bedbrook, 1979), 5S rDNA unit of E. globulus, and the E. globulus BAC clones EGC_Ba_P37H11 (BAC-CCR1), and EGC_Ba_P22O03 (BAC-RAC1). Clone pTa71 and BAC probes were isolated with NZYMiniprep (NZYtech) and labeled with digoxigenin or biotin using the Nick translation mix (Roche). The 5S ribosomal DNA probe was generated by PCR using forward and reverse primers (F:CCTGGGAAGTCCTCGTGTTG and R:CTTCGGAGTTCTGATGGGAT) in E. globulus DNA labeled with digoxigenin or biotin.
FISH with several probes was performed sequentially. First was performed the BAC "landing" according to Jenkins and Hasterok (2007) with minor modifications: 68% of stringency in all FISH experiments either in self-species or cross-species hybridization. After BAC-FISH metaphases were photographed and the slides were further washed with glacial acetic acid (3:1, v/v), 2xSSC (saline sodium citrate buffer), and increasing ethanol series before hybridization with the rDNA probes according to Alves et al. (2012).
Slides were analyzed with appropriate filters in the epifluorescence microscope Imager Z1 (Zeiss). Images were captured by the monochromatic camera AxioCam Hrm (Zeiss) through the AxioVision software (Zeiss) and assembled by Photoshop CC software.
Karyotype Measurements
Chromosomes were paired by FISH marks, AT-rich bands, and the relative position of the primary constriction, from the biggest to the smallest pair. Measurements of total length and arm ratio were made in chromosomes of five metaphases with the measurement module of the AxioVision software. The nomenclature of the chromosomes was according with Levan and colleagues recommendations (Levan et al., 1964): M (centromere at median point), m (centromere at median region), sm (centromere at submedian region), and st (centromere at subterminal region).
Genome Size Estimation Using Flow Cytometry
The genome size of 10–11 individuals of E. pulverulenta, E. occidentalis, and E. cornuta was estimated using flow cytometry following Galbraith et al. (1983). Briefly, 50 mg of leaves from the sample material and from the reference standard [S. lycopersicum "Stupické"; 2C = 1.96 pg; (Doležel et al., 1992)] were chopped with a razor blade in a Petri dish containing 1 ml of WPB (Loureiro et al., 2007). Due to the high levels of phenolic compounds present in Eucalyptus leaves, we significantly reduced the chopping intensity. The nuclear suspension was then filtered through a 30 μm nylon filter and nuclei were stained with 50 mg ml−1 propidium iodide (PI) (Fluka, Buchs, Switzerland), and 50 mg ml−1 RNase (Sigma, St Louis, MO, USA) was added to the nuclear suspension to prevent staining of double-stranded RNA. Samples were analyzed within a 5 min period in a Partec CyFlow Space flow cytometer (532 nm green solid-state laser, operating at 30 mW; Partec GmbH., Görlitz, Germany). At least 1300 particles per G1 peak were acquired per samples using the Partec FloMax software v2.4d (Partec GmbH, Münster, Germany) (Suda et al., 2007). Despite of the difficulty of analyzing Eucalyptus species using flow cytometry, the average CV value for the G1 peak was below 5% (mean CV value = 4.48% for Eucalyptus spp.; mean CV value for the standard = 3.12%).
The genome size in mass units (2C in pg; sensu Greilhuber et al., 2005) was obtained as follows: Eucalyptus sp. 2C nuclear DNA content (pg) = (Eucalyptus sp. G1 peak mean / reference standard G1 peak mean) * genome size of the reference standard. Differences in genome size among species were evaluated with a One-Way ANOVA (SigmaPlot).
BAC Identification and Sequences Analysis
The BAC library contaminations with chloroplastidial and mitocondrial sequences were respectively, 2.16% and 1.17% for the library EGC_Ba, and 0.20% and 0.77% for the EGC_Bb. The average size of the inserts for both libraries was estimated at 115 kb. Based on the number of clones of each library (36,864 clones for EGC_Ba and 36,864 clones for EGC_Bb) and the size of the E. globulus genome estimated at 1.09 pg/2C (530 Mb/1C) (Grattapaglia and Bradshaw, 1994), the genome coverage of both libraries is 11.2X, and the probability to find a BAC clone harboring a sequence of interest is 99.9% based on the algorithm of Clarke and Carbon (1976).
The 3D-pool approach developed at the French Plant Genomic Center (CNRGV) (Rasmussen-Poblete et al., 2008) revealed to be very efficient in the identification of BAC clones harboring the two genes of interest. Using the primers developed for E. gunnii Cinamoyl-CoA reductase (EguCCR, X79566) (Rengel et al., 2009) and E. gunnii GTPase RAC (EguRAC1, DR410036) (Rasmussen-Poblete et al., 2008) a total of eight and seven BAC clones were identified, respectively. Among these, the BAC clones EGC_Ba_P37H11 and EGC_Ba_P22O03, harboring respectively the EglCCR1 and EglRAC1 genes, were selected for sequencing (Table 2). For EGC_Ba_P37H11 93% of the 64,290 454-reads were assembled into 14 contigs (> 500 bp), with a final assembling size of 138.5 kb. For EGC_Ba_P22O03, also 93% of the 80,593 reads were assembled into 37 contigs (>500 bp), with an assembling size of 141.7 kb. The assembled size of EGC_Ba_P22O03 was similar to that obtained by enzymatic restriction. However, taking into account that the insert of EGC_Ba_P37H11, calculated by enzymatic restriction is about 100 kb, this means that some of the contigs (>500 bp) were overlapping each other.
The presence of both genes of interest was confirmed by Blastn against the non-redundant nucleotide database of NCBI. Besides, EGC_Ba_P37H11 and EGC_Ba_P22O03 sequences mapped as expected at the scaffold J of E. grandis genome (Myburg et al., 2014), in the region containing the bona fide EgrCCR1 (Egr.J03114) (Carocha et al., 2015) and the EgrGTPase-RAC (Eucgr.J02468). EGC_Ba_P22O03 and EGC_Ba_P37H11 sequences were deposited at GeneBank under the accession numbers KU727161 and KU727162, respectively.
RepeatMasker Web Server (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker) vs. open-4.0.5 was used to screen both BAC clone sequences against the DNA sequences in FASTA format against the Repbase-derived RepeatMasker library of repetitive elements (RMLib: 20140131) or against the Dfam database (Dfam: 1.3). The CG content was similar for both BAC clones, 39.20% and 40.92% for the EGC_Ba_P37H11 and EGC_Ba_P22O03, respectively. Contrary to that observed for clone EGC_Ba_P37H11 (only a 48 bp signal of a Ty1/Copia retroelement), the sequence of EGC_Ba_P22O03 revealed the presence of of two larger Ty1/Copia (total 2024 bp) and two Gypsy/DIRS1 (total 853 bp) retroelements and six DNA mutator transposon autonomous regulators, MuDR (357 bp). Moreover, EGC_Ba_P22O03 presented almost the double number of simple repeats (105, total of 3986 bp) of that of the EGC_Ba_P37H11 (55; total of 1823 bp) and also numerous (total 26) low complexity sequences not observed in EGC_Ba_P37H11. The presence of these repetitive elements identified by RepeatMasker could partially explain the high number of contigs (>500) observed for EGC_Ba_P22O03 BAC clone (37 contigs) when compared with that of obtained for EGC_Ba_P37H11 BAC clone (14 contigs).
Karyotype Analysis
All species studied presented 2n = 2x = 22 chromosomes (Figure 1). The mean length of the haploid set ranged from of the studied species is 23.24–20.04 μm in E. grandis and E. pulverulenta, respectively. All karyotypes were composed by small chromosomes with total lengths and arm ratios very similar among species, nevertheless, all chromosomes could be paired and identified and no overlapping was observed between contiguous chromosomes. The karyotypes were mainly composed by chromosomes with the centromere at median region (m) with few pairs with the centromere at median point (M). However, in the species of Bisectae section (E. occidentalis and E. cornuta) one or two pairs, respectively, presented the centromere at subterminal region (sm). In summary, the karyotype formula for E. camaldulensis, E. grandis, and E. globulus is 4M + 18m; for E. pulverulenta is 2M + 20m; and for E. cornuta and E. occidentalis is 2M + 16m + 4sm and 2M + 18m + 2sm, respectively (Table 3).
Figure 1. Fluorescence in situ hibridization with 35S rDNA (red signal) and 5S rDNA (green signal) probes in metaphase chromosomes of E. cornuta (A); E. occidentallis (B); E. camaldulensis (C); E. grandis (D); E. globulus (E); E. pulverulenta (F). Insets showing the 35S rDNA locus (NOR) organization: (D) E. grandis pattern with decondensed domain in the secondary constriction and two condensed domains in both edges (red) of the NOR; Inset in (E) showing a decondensed domain in the secondary constriction and one condensed domain in the distal end (red). Bar = 5 μm.
Genomes sizes of species from Bisectae section, and E. pulverulenta from section Maidenaria were estimated by flow cytometry. These analyses also revealed very similar values among the species of Eucalyptus analyzed, with the average genome size values ranging from 1.25 and 1.26 pg/2C in E. pulverulenta and E. cornuta, respectively, and 1.27 pg/2C in E. occidentalis (Table 4). No statistically significant differences were observed among the species analyzed (P < 0.05).
In order to map the rDNA loci, FISH with 35S rDNA, and 5S rDNA probes was performed in the six Eucalyptus species. All the species presented the 35S rDNA locus on the short arm of the first pair of chromosomes in a pericentromeric position leading to an evident secondary constriction. The 5S rDNA locus was located in the third pair of chromosomes, also positioned pericentromerically in the short arm (Figures 1, 2). Although the distribution pattern of the 35S rDNA was similar, the rDNA organization within the locus was different among species. The inner structure of each nucleolar organizer region (NOR) was clearly identified in the metaphase chromosomes by a large labeled condensed domain in the distal region present in all the species (Figure 1E inset), except from E. grandis. In this species apart from the large condensed site in the distal region, a small condensed site in the proximal region was detected (Figure 1D inset).
Figure 2. Karyograms of E. cornuta; E. occidentallis; E. camaldulensis; E. grandis; E. globulus; E. pulverulenta with FISH markers (35S rDNA (first pair); 5S rDNA (third pair); BAC-CCR1 (ninth pair) above, and DAPI+ banding (lighter blue staining) below. Missing homologous chromosomes were folded over itself in the analyzed metaphases and were unable to be used in these karyograms.
The presence of constitutive heterochromatin was evaluated through CMA3 and DAPI stainings. The GC-rich heterochromatin evaluated by CMA3 banding was restricted to the 35S rDNA loci in all species (Figures 3 and Supplementary Figure S1). The AT-rich blocks detected with the DAPI+ staining could be detected in the telomeric regions of various chromosomes in all species (Figure 2). Still, differences in number and distribution were observed among species, even of the same section, although in all the species studied there was one conserved DAPI+ block detected in the terminal region of the short arm of the nineth pair (Figure 2). E. occidentalis displays more DAPI+ bands, detected in nine chromosome pairs. The DAPI+ co-localization with CMA3 in the distal edge of the secondary constriction is also maintained in almost all species (Figure 3B inset), with the exception of E. camaldulensis (Figure 2). Furthermore, E. globulus presented DAPI+ blocks in both ends of the secondary constriction (Figure 2). E. camaldulensis, E. grandis, and E. globulus have a DAPI+ band co-localized with the 5S rDNA signal (Figure 2). In E. occidentalis the chromosome bearing 5S rDNA presented a DAPI+ band in the short arm, while in all other species, it was located at the end of the long arm (Figure 2).
Figure 3. Chromomycin A3 (CMA3) banding (green) in chromosomes of E. cornuta (A, B), representing the most common pattern, and of E. grandis (C). The CMA positive band is always co-localized with 35S rDNA (red/orange) (B) and with the DAPI+ in E. grandis (inset in B). Bar = 5 μm.
BAC Landing of Genes Involved in Lignin Biosynthesis
FISH with two E. globulus BAC probes, EGC_Ba_P37H11 (BAC-CCR1) and EGC_Ba_P22O03 (BAC-RAC1), presented very distinct patterns. In all Eucalyptus species, BAC-CCR1 probe showed a single locus distal signal on the long arm of a small pair of chromosomes (Figure 4), which presents, in all species, terminal AT-rich blocks in the short arm (Figure 2). BAC-RAC1 probe hybridized in several locations along all the chromosomes of all species, in a disperse or in a cluster manner. Due to the dispersed nature of the signal it was not possible to precisely map the EglRAC1 gene locus (Figure 5). In all the studied species, the BAC-RAC1 signal was more represented in some domains of several chromosomes pairs such as telomeres, (peri)centromeres, and secondary constrictions co-localizing with the 35S rDNA and the 5S rDNA (Figure 5). A detailed analysis of E. grandis karyogram (Figure 6) allowed to precisely map the repetive sequences of BAC-RAC1 in this species: (a) in the first pair, the BAC-RAC1 was confined to the NOR region; (b) in the second pair was clustered at both telomeres; (c) the fourth and seventh pairs contained essentially pericentrometric signals; (d) the six and eleven pairs had dispersed signals only in the long arms, (e) finally, the nineth and tenth pairs had dispersed signal all along the chromosome arms.
Figure 4. Fluorescence in situ hybridization with BAC-CCR probe. E. occidentallis metaphase chromosomes with 35S rDNA (red), 5S rDNA (green), and BAC-CCR (yellow) localizations. Bar = 5 μm.
Figure 5. Fluorescence in situ hybridization with BAC-RAC probe showing disperse pattern (green) along all chromosomes of E. globulus (A) and E. grandis (B). Co-localization (white arrows) with the 5S rDNA (A) and the 35S rDNA condensed and decondensed domains (B). Bar = 5 μm.
Figure 6. Karyograms of E. grandis with 35S rDNA (red) and BAC-RAC1 probe (green) in chromosomes pairs (below) showing the co-localization of both probes in the first chromosome pair.
Comparative Eucalyptus Karyotype Analysis
Comparative karyotype analysis of six Eucalyptus species belonging to the subgenus Symphyomyrtus and to different sections, was achieved using chromosome morphology along with rDNA, BAC landing, and AT-rich as well as GC-rich DNA markers. These markers, together with genome sizes estimations, evidence a high conservation of the genomes within this subgenus.
The counting of chromosomes, especially, small ones, relying only on morphological comparison can lead to wrong conclusions like counting 2n = 24 (Matsumoto et al., 2000; Oudjehih and Abdellah, 2006), instead of the number fixed for the genus 2n = 22 (Grattapaglia et al., 2012). This error is often associated with counting pro-metaphases where the secondary constriction is much spread and the associated arm is very distant from the rest of the chromosome and can be counted as a very small chromosome. FISH with 35S rDNA marker can avoid this situation and even elucidate for pairs in the chromosome sets. FISH markers were essential for unequivocal establishment of chromosome pairs in this study.
Similar karyotype formulas of all species were evidenced, however, slight differences among species of the same section could be detected. Nevertheless, much more divergent karyotypes formulas, obtained exclusively from morphological data, were previously reported (Matsumoto et al., 2000; Mora et al., 2005; Cardemil and Perry, 2007). The small length of Eucalyptus chromosomes together with some differerences in condensation within chromosome pairs and the lack of unequivocal centromeric detection could be responsible for these differences in studies without additional chromosome markers. The Eucalyptus karyotypes studied are mainly composed of strictly metacentric (M) and median chromosomes (m), but submetacentric chromosomes (sm) appeared in E. cornuta and E. occidentalis from section Bisectae. These morphological differences could be due to chromosome rearrangements like deletions and amplifications or translocations of chromosome segments. However, the total chromosome lengths are very similar between species, and therefore, differences in chromosome morphologies rather than be due to large rearrangements seem to be dependent of local expansions of possible repetitive clusters. In fact, data from the E. grandis genome sequence has reinforced the idea that tandem duplications are an important mechanism in genome evolution of this species, since E. grandis has more tandem duplicates and more tandem expanded regions (clusters) than other plant genomes (Myburg et al., 2014).
Although some DAPI+ locations were conserved among species, DAPI banding patterns were unique to each Eucalyptus studied. This diversity may be caused by different redundancy levels of repeated DNA sequences. The differences between genome sizes of the Eucalyptus species are not significantly different. However, E. occidentallis shows a tendency to have a sigthly larger genome. This is in agreement with a larger amount of AT-rich constitutive heterochromatin, seen as DAPI+ blocks, in more chromosomes when compared with the other species studied. Bitonti et al. (1999) had already observed a positive correlation between genome size and the copy number of a given satellite DNA sequence in ten Olea species which was encountered in heterochromatin regions. Also, an example of intraspecific variation involving differences in heterochromatic sequences was described in the subspecies pair Scilla bithynica Boiss. ssp. bithynica, which presents many large C-bands, corresponding to constitutive heterochromatin, and a genome size of 29.2 pg and S. bithynica ssp. radkae, with few small C-bands and a genome size of 22.9 pg (Greilhuber, 1998).
Contrasting to the diversity of DAPI+ banding, we found the same rDNA pattern among the Eucalyptus species: one major pericentromeric 35S rDNA locus and one pericentromeric 5S rDNA locus in chromosome 1 and 3 respectively, evidencing genome conservation in species of the section Symphyomyrtus. However, we found a diferent organization of the 35S rDNA locus among these species: either only one condensed distal domain or two condensed domains in both ends of the NOR locus in E. grandis. These condensed domains, resulting from silent rDNA genes are usually defined as GC-rich constitutive heterochomatin (Guerra, 2000). The rDNA repeats are interspersed with the intergenic sequence (IGS) region, that is enriched in GC (Inácio et al., 2014), and therefore responsible for the CMA3+ staining at the NORs. Moreover, in almost all Eucalyptus species, a DAPI+ band co-localizes with the distal region of the 35S rDNA locus. The co-localization of these marks in the NOR is very rare, since the most common situation is the CMA3+ and DAPI- pattern (Guerra, 2000). This uncommon pattern also occures in Hydrangea aspera ssp. sargetiana (Mortreau et al., 2009) and it has been interpreted as ribosomal clusters interspersed with AT-rich heterochromatin (Colomba et al., 2006). The BAC-RAC1 probe which is enriched in several transposable elements (TEs), shows a strong signal hybridization in these domains. Moreover, high amount of pseudogenes from the 35S cistron with less GC content has been reported in several Eucalyptus genomes (Bayly et al., 2008). To explain the NOR organization we propose that more than one variant of rDNA repeats should be present in the 35S locus. The IGS lengths of some variants can be bigger due to the expansion of the AT-rich region as observed in Fagaceae (Inácio et al., 2014) which would be detected as a positive DAPI staining. Besides, the NOR condensed domains may be enriched in these pseudogenes which could be created by truncation of genes by TEs.
Notably, the 5S rDNA colocalizes with AT rich constitutive heterochromatin in species of different sections like E. camaldulensis, E. grandis, and E. globulus. Normally, this locus is DAPI neutral or could be in some species CMA3+ (Guerra, 2000). However, the same banding pattern was also reported in Hydrangea aspera from group Kawakami (Mortreau et al., 2009) and in Maxillaria species (Cabral et al., 2006). This could have several explanations: other unknown sequences with AT-rich regions may be intermingled, or around the 5S rDNA locus, or the proximity to transposable elements that tend to be silenced by heterochromatization. The association and proximity of transposable elements with genes is very clear in Eucalyptus genomes since the BAC-RAC1 probe is composed of several types of TEs in the vicinity of the gene RAC1 and remarkably, it is co-localized with the the 5S rDNA locus, besides the 35S rDNA. The presence of retroelements (RTEs) in NORs is an uncommon situation, but it has already been referred in the IGS of Musa sp. (Balint-Kurti et al., 2000), and in Allium cernuum (Chester et al., 2010). Also association with the 5S locus and the En/Spm transposon, has already been reported in Poaceae where clusters of this TE were found associated with heterochromatin (Altinkut et al., 2006). The expected silencing of these TEs in the NORs and 5S locus may therefore be a source for the DAPI+ staining in these regions. Besides the localization in NORs, 5S rDNA locus, and telomeres the BAC-RAC1 also showed a disperse signals along the chromosomes giving a pattern already detected in other plant species (Santos et al., 2015). Here we report the first work evaluating by FISH the distribution of repetitive DNA elements in Eucalyptus, and it is in agreement with the distributition of LTR RTE detected in silico (Marcon et al., 2015) where RTEs subfamilies were identified dispersed along chromosomes arms in both gene-rich and repetitive-rich regions. The localization of BAC-RAC1 on subtelomeric domains in several chromosomes agrees with the identification of Copia RTEs near the telomeric regions (Marcon et al., 2015).
CCR1 Gene Location in Eucalyptus Genomes
The assignment of the linkage groups (LGs) to individual chromosomes is of major importance for the integration of genetic and cytogenetic maps. Our integrated results unambigouysly localize the CCR1 gene into the terminal end of the long arm of the chromosome 9, although this gene has been previously assigned to LG10 (scaffold J) in E. grandis (Gion et al., 2015). It is well recognized that the correlation between chromosome size and genetic distances can sometimes be distorted. Whereas, LG are based on the assumption that crossover occurs at random along chromosomes, this is only partly true because genetic recombination is more likely to occur at some regions rather than at others (Han et al., 2011). Heterochromatic regions mostly enriched in RTEs are often associated with suppressed genetic recombination (Wang et al., 2006). Eucalyptus chromosomes 9 and 10 are highly enriched in disperse heterochromatin, detected by FISH with the BAC-RAC1, mainly composed of RTEs, and by DAPI+ staining. In this situation, the genetic recombination randomness along chromosomes may be affected, leading to the non correlation detected. In Lotus japonicus, where the distance calculated by recombination frequencies at the terminal regions is larger than the physical distance of the chromosomes this discrepancy is due to the presence of heterochromatin (Ohmido et al., 2010).
In this work we mapped the rDNA loci, and the cinnamoyl CoA reductase gene, and determined the distribution of some retroelements in six species of Eucalyptus. We also calculated the genome size of three species. CCR1 gene was mapped into chromosome 9 in all species, clearly assign LG10 to physical chromosome 9.
A multi BAC-FISH approach with clones giving single-locus FISH signals is now mandatory to correctly identify Eucalyptus chromosomes, to support the established linkage groups and to better understand the evolution of Eucalyptus genus.
Our integrated results strengthened the conservation of the genomes in Eucalyptus species across four sections of subgenus Symphyomyrtus (Bisectae, Exsertaria, Latoangulatae, and Maidenaria). Thus, this work reinforces the idea that sequence information from the E. grandis genome can be transposed to other Symphyomyrtus species. This work is novel and contributes to the understanding of eucalypts genome organization which is essential to develop advanced breeding strategies for this genus.
Author Contributions
LMC and JP conceived the study and designed the experiments. TR, JL, and RB performed the experiments. TR, LM, and JP wrote the manuscript. HB and JP prepared the 3D BAC pools. CM collected the material and preformed the first analysis of the E. globulus BAC libraries. All authors read and approved the final manuscript.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We are grateful to Paulo Forte, Paula Paes, and Paula Soares for providing the seeds and other plant material. We also thanks of Victor Carocha and Sonia Vautrin for the technical help on BAC libraries. The authors acknowledge FCT (Fundação para a Ciência e a Tecnologia) for supporting this research under the frame work of project "GenEglobwq - Scanning for candidate genes underlying a pulp yield QTL in Eucalyptus globulus" (PTDC/AGR-GPL/66564/2006), BioResources 4 Sustainability unit (GREEN-IT) and FCT LEAF (Linking Landscape, Environment, Agriculture and Food) Unit (UID/AGR/04129/2013). TR was supported by funding from FCT with a Post-Doc grant (SFRH/BPD/64618/2009). JP acknowledge his research contract in the frame of EU-FP7-ERAChairs-PillotCAll-2013 project "Biotalent - The creation of the Department of Integrative Plant Biology" (FP7-REGPOT-621321).
Project reference: PTDC/AGR-GPL/66564/2006, "GenEglobwq - Scanning for candidate genes underlying a pulp yield QTL in Eucalyptus globulus."
Funded under: FCT Fundação para a Ciência e a Tecnologia.
Project reference: FP7-ERAChairs-PilotCall-2013 (FP7-REGPOT-621321) "Biotalent - The creation of the Department of Integrative Plant Biology".
Funded under: EU-FP7 - Seventh Framework Programme.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2016.00510
Supplementary Figure S1. Metaphases of E. occidentalis (A), E. camaldulensis (B), E. globulus (C), and E. pulverulenta (D) with CMA3 green fluorescence in the NORs. Bar = 5 μm.
Altinkut, A., Raskina, O., Nevo, E., and Belyayev, A. (2006). En/Spm-like transposons in Poaceae species: transposase sequence variability and chromosomal distribution. Cell. Mol. Biol. Lett. 11, 214–230. doi: 10.2478/s11658-006-0017-3
Alves, S., Ribeiro, T., Inácio, V., Rocheta, M., and Morais-Cecílio, L. (2012). Genomic organization and dynamics of repetitive DNA sequences in representatives of three Fagaceae genera. Genome 55, 348–359. doi: 10.1139/g2012-020
Balint-Kurti, P. J., Clendennen, S. K., Doleželová, M., Valárik, M., Doležel, J., Beetham, P. R., et al. (2000). Identification and chromosomal localization of the monkey retrotransposon in Musa sp. Mol. Gen. Genet. 263, 908–915. doi: 10.1007/s004380000265
Bartholomé, J., Mandrou, E., Mabiala, A., Jenkins, J., Nabihoudine, I., Klopp, C., et al. (2015). High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly. New Phytol. 206, 1283–1296. doi: 10.1111/nph.13150
Bayly, M. J., Udovicic, F., Gibbs, A. K., Parra, O. C., and Ladiges, P. Y. (2008). Ribosomal DNA pseudogenes are widespread in the eucalypt group (Myrtaceae): implications for phylogenetic analysis. Cladistics 24, 131–146. doi: 10.1111/j.1096-0031.2007.00175.x
Bitonti, M. B., Cozza, R., Chiappetta, A., Contento, A., Minelli, S., and Ceccarelli, M. (1999). Amount and organization of the heterochromatin in Olea europaea and related species. Heredity (Edinb). 83, 188–195. doi: 10.1046/j.1365-2540.1999.00564.x
Brooker, M. I. H. (2000). A new classification of the genus Eucalyptus L'Hér. (Myrtaceae). Aust. Syst. Bot. 13, 79–148. doi: 10.1071/SB98008
Cabral, J. S., Felix, L. P., and Guerra, M. (2006). Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae). Genet. Mol. Biol. 29, 659–664. doi: 10.1590/S1415-47572006000400015
Cai, Z., Liu, H., He, Q., Pu, M., Chen, J., Lai, J., et al. (2014). Differential genome evolution and speciation of Coix lacryma-jobi L. and Coix aquatica Roxb. hybrid guangxi revealed by repetitive sequence analysis and fine karyotyping. BMC Genomics 15:1025. doi: 10.1186/1471-2164-15-1025
Cardemil, E. J., and Perry, B. C. (2007). Comparative analysis of karyotypes from the Strezelecki Ranges race of the complex Eucalyptus globulus Labill. ssp. globulus (Myrtaceae) and a population in Central-Southern Chile. Silvae Genet. 56, 158–162.
Carocha, V., Hefer, C., Cassan-wang, H., Fevereiro, P., Alexander, A., Paiva, J. A. P., et al. (2015). Genome-wide analysis of the lignin toolbox of Eucalyptus grandis. New Phytol. 206, 1297–1313. doi: 10.1111/nph.13313
Chamala, S., Chanderbali, A. S., Der, J. P., Lan, T., Walts, B., Albert, V. A, et al. (2013). Assembly and validation of the genome of the nonmodel basal angiosperm Amborella. Science 342, 1516–1517. doi: 10.1126/science.1241130
Chester, M., Sykorova, E., Fajkus, J., and Leitch, A. R. (2010). Single integration and spread of a copia-like sequence nested in rDNA intergenic spacers of Allium cernuum (Alliaceae). Cytogenet. Genome Res. 129, 35–46. doi: 10.1159/000312959
Clarke, L., and Carbon, J. (1976). A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome. Cell 9, 91–99. doi: 10.1016/0092-8674(76)90055-6
Colomba, M., Vitturi, R., Libertini, A., Gregorini, A., and Zunino, M. (2006). Heterochromatin of the scarab beetle, Bubas bison (Coleoptera: Scarabaeidae) II. Evidence for AT-rich compartmentalization and a high amount of rDNA copies. Micron 37, 47–51. doi: 10.1016/j.micron.2005.06.004
Doležel, J., Sgorbati, S., and Lucretti, S. (1992). Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol. Plant. 85, 625–631. doi: 10.1111/j.1399-3054.1992.tb04764.x
Foucart, C., Jauneau, A., Gion, J. M., Amelot, N., Martinez, Y., Panegos, P., et al. (2009). Overexpression of EgROP1, a Eucalyptus vascular-expressed Rac-like small GTPase, affects secondary xylem formation in Arabidopsis thaliana. New Phytol. 183, 1014–1029. doi: 10.1111/j.1469-8137.2009.02910.x
Fuchs, J., Strehl, S., Brandes, A., Schweizer, D., and Shubert, I. (1998). Molecular-cytogenetic characterization of the Vicia faba genome - heterochromatin differentiation, replication patterns and sequence localization. Chromosom. Res. 6, 219–230. doi: 10.1023/A:1009215802737
Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M., Sharma, D. P., and Firoozabady, E. (1983). Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051. doi: 10.1126/science.220.4601.1049
Gerlach, W., and Bedbrook, J. (1979). Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7, 1869–1885. doi: 10.1093/nar/7.7.1869
Gion, J. M., Chaumeil, P., and Plomion, C. (2015). EucaMaps: linking genetic maps and associated QTLs to the Eucalyptus grandis genome. Tree Genet. Genomes 11, 795. doi: 10.1007/s11295-014-0795-0
Grattapaglia, D., and Bradshaw, H. D. (1994). Nuclear DNA content of commercially important species and hybrids. Can. J. For. Res. Can. Rech. For. 24, 1073–1077.
Grattapaglia, D., Mamani, E. M. C., Silva-Junior, O. B., and Faria, D. (2015). A novel genome-wide microsatellite resource for species of Eucalyptus with linkage-to-physical correspondence on the reference genome sequence. Mol. Ecol. Resour. 15, 437–448. doi: 10.1111/1755-0998.12317
Grattapaglia, D., Silva-Junior, O. B., Kirst, M., de Lima, B. M., Faria, D. A., and Pappas, G. J. (2011). High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species. BMC Plant Biol. 11:65. doi: 10.1186/1471-2229-11-65
Grattapaglia, D., Vaillancourt, R. E., Shepherd, M., Thumma, B. R., Foley, W., Külheim, C., et al. (2012). Progress in Myrtaceae genetics and genomics: eucalyptus as the pivotal genus. Tree Genet. Genomes 8, 463–508. doi: 10.1007/s11295-012-0491-x
Greilhuber, J. (1998). Intraspecific variation in genome size: a critical reassessment. Ann. Bot. 82, 27–35. doi: 10.1006/anbo.1998.0725
Greilhuber, J., Doležel, J., Lysák, M. A., and Bennet, M. D. (2005). The origin, evolution and proposed stabilization of the terms "genome size" and "C-value" to describe nuclear DNA contents. Ann. Bot. 95, 255–260. doi: 10.1093/aob/mci019
Gu, Y. Q., Ma, Y., Huo, N., Vogel, J. P., You, F. M., Lazo, G. R., et al. (2009). A BAC-based physical map of Brachypodium distachyon and its comparative analysis with rice and wheat. BMC Genomics 10:496. doi: 10.1186/1471-2164-10-496
Guerra, M. (2000). Patterns of heterochromatin distribution in plant chromosomes. Genet. Mol. Biol. 23, 1029–1041. doi: 10.1590/S1415-47572000000400049
Hajdera, I., Siwinska, D., Hasterok, R., and Maluszynska, J. (2003). Molecular cytogenetic analysis of genome structure in Lupinus angustifolius and Lupinus cosentinii. Theor. Appl. Genet. 107, 988–996. doi: 10.1007/s00122-003-1303-3
Han, Y., Zhang, Z., Huang, S., and Jin, W. (2011). An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2. BMC Genet. 12:18. doi: 10.1186/1471-2156-12-18
Hill, K. D., and Johnson, L. A. S. (1995). Systematic studies in the eucalypts 7. A revision of the bloodwoods, genus Corymbia (Myrtaceae). Telopea 6, 185–504. doi: 10.7751/telopea19953017
Inácio, V., Rocheta, M., and Morais-Cecílio, L. (2014). Molecular organization of the 25S–18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis. PLoS ONE 9:e98678. doi: 10.1371/journal.pone.0098678
Islam-Faridi, M. N., Nelson, C. D., DiFazio, S. P., Gunter, L. E., and Tuskan, G. A. (2009). Cytogenetic analysis of Populus trichocarpa - Ribosomal DNA, telomere repeat sequence, and marker-selected BACs. Cytogenet. Genome Res. 125, 74–80. doi: 10.1159/000218749
Jenkins, G., and Hasterok, R. (2007). BAC "landing" on chromosomes of Brachypodium distachyon for comparative genome alignment. Nat. Protoc. 2, 88–98. doi: 10.1038/nprot.2006.490
Książkiewicz, M., Wyrwa, K., Szczepaniak, A., Rychel, S., Majcherkiewicz, M., Przysiecka, L., et al. (2013). Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics. BMC Genomics 14:19. doi: 10.1186/1471-2164-14-79
Lacombe, E., Hawkins, S., Van Doorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., et al. (1997). Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J. 11, 429–441. doi: 10.1046/j.1365-313X.1997.11030429.x
Ladiges, P. Y. (1997). "Phylogenetic history and classification of eucalypts," in Eucalypt Ecology: Individuals to Ecosystems, eds J. E. Williams and J. C. Z. Woinarski (Cambridge: Cambridge University Press), 16–29.
Levan, A., Fredga, K., and Sandberg, A. A. (1964). Nomenclature for centromeric position on chromosomes. Heriditas 52, 201–220. doi: 10.1111/j.1601-5223.1964.tb01953.x
Lipman, M. J., Chester, M., Soltis, P. S., and Soltis, D. E. (2013). Natural hybrids between Tragopogon mirus and T. miscellus (Asteraceae): a new perspective on karyotypic changes following hybridization at the polyploid level. Am. J. Bot. 100, 2016–2022. doi: 10.3732/ajb.1300036
Loureiro, J., Rodriguez, E., Doležel, J., and Santos, C. (2007). Two new nuclear isolation buffers for plant DNA flow cytometry : a test with 37 species. Ann. Bot. 100, 875–888. doi: 10.1093/aob/mcm152
Maluszynska, J., and Heslop-Harrison, J. S. (1993). Physical mapping of rDNA loci in Brassica species. Genome 36, 774–781. doi: 10.1139/g93-102
Marcon, H. S., Domingues, D. S., Silva, J. C., Borges, R. J., Matioli, F. F., de Mattos Fontes, M. R., et al. (2015). Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic. BMC Plant Biol. 15:198. doi: 10.1186/s12870-015-0550-1
Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., et al. (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–381. doi: 10.1038/nature03959
Matsumoto, S. T., Marin-Morales, M., Ruas, C. F., and Ruas, P. M. (2000). Cytogenetic analysis of seven species of Eucalyptus L'Her.(Myrtaceae). Caryologia 53, 205–212. doi: 10.1080/00087114.2000.10589197
Mendes, S., Moraes, A. P., Mirkov, T. E., and Pedrosa-Harand, A. (2011). Chromosome homeologies and high variation in heterochromatin distribution between Citrus L. and Poncirus Raf. as evidenced by comparative cytogenetic mapping. Chromosom. Res. 19, 521–530. doi: 10.1007/s10577-011-9203-x
Mora, F., Palma-Rojas, C., and Jara-Seguel, P. (2005). Comparación del cariotipo de Eucalyptus globulus y Eucalyptus cladocalyx (Myrtaceae). Agric. Técnica 65, 20–25. doi: 10.4067/s0365-28072005000100002
Mortreau, E., Siljak-Yakovlev, S., Cerbah, M., Brown, S. C., Bertrand, H., and Lambert, C. (2009). Cytogenetic characterization of Hydrangea involucrata Sieb. and H. aspera D. Don complex (Hydrangeaceae): genetic, evolutional, and taxonomic implications. Tree Genet. Genomes 6, 137–148. doi: 10.1007/s11295-009-0235-8
Mun, J. H., Kim, D. J., Choi, H. K., Gish, J., Debellé, F., Mudge, J., et al. (2006). Distribution of microsatellites in the genome of Medicago truncatula: a resource of genetic markers that integrate genetic and physical maps. Genetics 172, 2541–2555. doi: 10.1534/genetics.105.054791
Myburg, A., Grattapaglia, D., Tuskan, G., Hellsten, U., Hayes, R., Grimwood, J., et al. (2014). The genome of Eucalyptus grandis. Nature 509, 356–362. doi: 10.1038/nature13308
Ohmido, N., Ishimaru, A., Kato, S., Sato, S., Tabata, S., and Fukui, K. (2010). Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes. Chromosom. Res. 18, 287–299. doi: 10.1007/s10577-009-9103-5
Oudjehih, B., and Abdellah, B. (2006). Chromosome numbers of the 59 species of Eucalyptus l'herit. (Myrtaceae). Caryologia 59, 207–212. doi: 10.1080/00087114.2006.10797916
Paiva, J. A. P., Prat, E., Vautrin, S., Santos, M. D., San-Clemente, H., Brommonschenkel, S., et al. (2011). Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries. BMC Genomics 12:137. doi: 10.1186/1471-2164-12-137
Paux, E., Sourdille, P., Salse, J., Saintenac, C., Choulet, F., Leroy, P., et al. (2008). A physical map of the 1-gigabase bread wheat chromosome 3B. Science 322, 101–104. doi: 10.1126/science.1161847
Plomion, C., Leprovost, G., and Stokes, A. (2001). Wood formation in trees. Plant Physiol. 127, 1513–1523. doi: 10.1104/pp.010816
Rasmussen-Poblete, S., Valdes, J., Gamboa, M. C., Valenzuela, P. D. T., and Krauskopf, E. (2008). Generation and analysis of an Eucalyptus globulus cDNA library constructed from seedlings subjected to low temperature conditions. Electron. J. Biotechnol. 11, 4–5. doi: 10.2225/vol11-issue2-fulltext-14
Rengel, D., San Clemente, H., Servant, F., Ladouce, N., Paux, E., Wincker, P., et al. (2009). A new genomic resource dedicated to wood formation in Eucalyptus. BMC Plant Biol. 9:36. doi: 10.1186/1471-2229-9-36
Ribeiro, T., Barão, A., Viegas, W., and Morais-Cecílio, L. (2008). Molecular cytogenetics of forest trees. Cytogenet. Genome Res. 120, 220–227. doi: 10.1159/000121070
Ribeiro, T., Loureiro, J., Santos, C., and Morais-Cecílio, L. (2011). Evolution of rDNA FISH patterns in the Fagaceae. Tree Genet. Genomes 7, 1113–1122. doi: 10.1007/s11295-011-0399-x
Santos, F., Guyot, R., do Valle, C., Chiari, L., Techio, V., Heslop-Harrison, P., et al. (2015). Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses. Chromosom. Res. 23, 571–582. doi: 10.1007/s10577-015-9492-6
Schwarzacher, T., and Heslop-Harrison, J. S. (2000). Practical In situ Hybridization. Oxford: BIOS.
Siljak-Yakovlev, S., Pustahija, F., Vicic, V., and Robin, O. (2014). "Molecular Cytogenetics (FISH and Fluorochrome Banding): resolving species relationships and genome organization," in Molecular Plant Taxonomy: Methods and Protocols, Methods in Molecular Biology, ed P. Besse (New York, NY; Heidelberg; Dordrecht; London: Springer), 309–323.
Silva, S., da, C., Mendes, S., Filho, W., dos, S. S., and Pedrosa-Harand, A. (2015). Chromosome homologies between Citrus and Poncirus —the comparative cytogenetic map of mandarin (Citrus reticulata). Tree Genet. Genomes 11:811. doi: 10.1007/s11295-014-0811-4
Srisuwan, S., Sihachakr, D., and Siljak-Yakovlev, S. (2006). The origin and evolution of sweet potato (Ipomoea batatas Lam.) and its wild relatives through the cytogenetic approaches. Plant Sci. 171, 424–433. doi: 10.1016/j.plantsci.2006.05.007
Steane, D. A., Nicolle, D., Sansaloni, C. P., Petroli, C. D., Carling, J., Kilian, A., et al. (2011). Population genetic analysis and phylogeny reconstruction in Eucalyptus (Myrtaceae) using high-throughput, genome-wide genotyping. Mol. Phylogenet. Evol. 59, 206–224. doi: 10.1016/j.ympev.2011.02.003
Suda, J., Kron, P., Husband, B. C., and Trávníček, P. (2007). "Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology," in Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes, eds J. Doležel, J. Greilhuber, and J. Suda (Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA), 103–130.
Thiers, B. (2010). "A global directory of public herbaria and associated staff," in Index Herbariorum. Available online at: http://sweetgum.nybg.org/ih
Tuskan, G., a, Difazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., et al. (2006). The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596–1604. doi: 10.1126/science.1128691
Wang, Y., Tang, X., Cheng, Z., Mueller, L., Giovannoni, J., and Tanksley, S. D. (2006). Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics 172, 2529–2540. doi: 10.1534/genetics.106.055772
Yang, L., Koo, D. H., Li, D., Zhang, T., Jiang, J., Luan, F., et al. (2014). Next-generation sequencing, FISH mapping and synteny-based modeling reveal mechanisms of decreasing dysploidy in Cucumis. Plant J. 77, 16–30. doi: 10.1111/tpj.12355
Zhao, X., Lu, J., Zhang, Z., Hu, J., Huang, S., and Jin, W. (2011). Comparison of the distribution of the repetitive DNA sequences in three variants of Cucumis sativus reveals their phylogenetic relationships. J. Genet. Genomics 38, 39–45. doi: 10.1016/j.jcg.2010.12.005
Keywords: BAC-landing, CCR1, ROP1, FISH, Eucalyptus, heterochromatin, transposable elements
Citation: Ribeiro T, Barrela RM, Bergès H, Marques C, Loureiro J, Morais-Cecílio L and Paiva JAP (2016) Advancing Eucalyptus Genomics: Cytogenomics Reveals Conservation of Eucalyptus Genomes. Front. Plant Sci. 7:510. doi: 10.3389/fpls.2016.00510
Received: 19 February 2016; Accepted: 31 March 2016;
Published: 22 April 2016.
Edited by:Xiaowu Wang, Chinese Academy of Agricultural Sciences, China
Reviewed by:Robert Hasterok, University of Silesia in Katowice, Poland
Weiwei Jin, China Agricultural University, China
Copyright © 2016 Ribeiro, Barrela, Bergès, Marques, Loureiro, Morais-Cecílio and Paiva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Leonor Morais-Cecílio, firstname.lastname@example.com;
Jorge A. P. Paiva, email@example.com


text:
If you're looking for a little outdoors stoke, check out "YETI Anthem" (from YETI).
From YETI.
"For the restless and determined, 'nowhere' is a state of mind — a drive to find the purest form of shared time, simple pleasures, and personal pursuit, whether that's 15,000 feet above sea level or 1,000 miles past the last stoplight. It's there that you're standing alongside other untamable spirits who stake their truth in going the extra mile. Because like you, they believe nowhere is too far if it's where you'd rather be."
To check out more from YETI, please click here.


text:
October 12, 2018
With whom are you surrounding yourself? Hopefully, you're surrounding yourself with people who help you #becomebetter.
A huge part of growing in your personal and professional walk is being open to listening to and hearing advice from others. Depending on how you receive that feedback and differentiate it to meet your needs, that advice can become more than a helpful hint, a useful strategy, or a pro tip.
In fact, in this series, I'm reflecting upon advice that's been not only a game-changer for me–rather, these words from the wise have changed my life.
Unless you've been completely disconnected from current events, you're aware of today's discourse that was once civil. You remember the good ole days when rhetoric was once meaningful conversation, and people were not expected to loudly interrupt each other. Maybe you've experienced the hateful, sheer vitriol that was once a semi-heated debate between friends over a cup of coffee, for example, only to end in a firm handshake or a sincere hug–till next time.
Those days are gone.
Or are they?
Nearly by definition, we educators are a passionate people. Somewhere inside every educator is a heart to care for kids, their well-being, and their learning. That's quite a responsibility–and an incredible opportunity.
Caring for the Whole Child is like embracing the entire lifelong journey–it's all interconnected–it's all interdependent. We're all in all of this together.
All learners–including educators–have their own backgrounds, experiences, and perspectives, and they inherently bring those with them to their learning spaces. When these ingredients fuel strengths-based leadership and help to flavor learning and make life better for kids, it's a thing of beauty.
Passion is like a wild horse, glorious and graceful, and occasionally prone to race really fast in the wrong direction. If we're not mindful to check it at the door sometimes, unbridled passion becomes inappropriate. And these neigh-gative distractions can drag away school culture. Unchecked expression can run down the daily climate, upset the apple cart, and even cause bucking between colleagues and friends.
How can we rein in our passions while still embracing authenticity?
The answer sounds simple, like it could be fenced-in, yet sometimes it's just beyond our self-control.
Especially in today's world of expressing our disagreements to the extreme, and where many choose not to catch more flies with honey, I would argue that "delivery is [not] 90%"–to me, delivery is 100%. How we treat each other–by way of delivery, tone, and expression–is everything–even more important than the content itself. Guiding our professional passions matters.
You can have all the credibility, knowledge, and experience in the world, but if you don't have the people skills to build relationships and make meaningful connections–then you've got nothing.
If influencing or persuading others, driving change, shaping the future, earning respect, or even just maintaining friendship is your goal, you have to know where the line is, and when, where, why, and how it's okay to cross that line–if at all.
Special thanks to my friend Chris Tuttell for giving me some of the best advice ever:
"It's always right to stand up for what you believe. Just make sure that your passion isn't alienating the very people you're trying to persuade."
It's advice like this from over a year ago that still has me thinking. It's inspired me to research the art of communication, especially language, feedback, and delivery.
I see so many productive debates–without crossing the line. You see, it's not about being right or winning an argument, necessarily, as much as it is about doing the right things for the right reasons in our profession–a very, very sacred profession.
Let's collaborate to agree on those right things, and, together, let's pursue those with the right kind of passion.
Always right to stand up for what you believe! Just make sure your passion isn't alienating people you are trying to persuade. @ChrisTuttell
— Kyle Hamstra (@KyleHamstra) September 16, 2017


text:
About Perfect 10 Auto
Welcome to Perfect 10 Auto,
Your Spring Lake Park dealer.
Perfect 10 Auto is a family run, independent automotive dealer serving the greater Twin Cities and surrounding areas with quality used automobiles. Perfect 10 Auto regards repeat customers and neighbors in the community as our most valuable asset. Our used car dealership has been in business for over 20 years by providing our customers with top notch used automobiles and excellent customer service.
Our clients have high expectations for their vehicles, and equally high expectations about the dealership professionals who serve them. Perfect 10 Auto is widely recognized to be among the best in quality, reliability, value and customer satisfaction in both sales and service.
At Perfect 10 Auto, we are here to make purchasing your vehicle as easy as possible. We partnered with some of the leading financing institutions to make it happen.
Perfect 10 Auto has several different finance options to suit your current credit situation. Competitive interest rates from some of the leading local and nation credit unions!
You see, we get it. At Perfect 10 Auto we understand that bad credit isn't a sign of bad character. We know how tough it can be, especially when you're talking about hefty divorce settlements, losing your job, credit card bills stacking up, and so on.
Our goal is simple: we want to get you financed so that you can get back on your feet. You see, building credit is a process, and buying a car is an investment. Good people shouldn't have to deal with sneaky car salesmen who are only trying to make a buck. Our system works because we do it the right way!
Our Perfect 10 Auto team is fully dedicated to helping people like you. When you choose us, you not only become our customer, you join our family - and we look after our family!
Of course, if you need further help, do not hesitate to contact us and we will do everything we can to get you approved.
Minimum downpayment required, varies depending on the vehicle ---
It's that simple! If you meet these simple requirements your financing is absolutely guaranteed. We require you to put down a down payment, but there are no tricks, no gimmicks- just real financing that will help you establish the strong credit you deserve.
When you're ready, stop on by for a test drive!


text:
I have a list of five things that I'll never be good at: celebrity names, spellings, geography, politics, and directions. I've managed to squeeze my way out of conversations and situations that involve the first four, however, when it comes to directions it's a whole different story. I can't tell you how many times I've managed to get lost. And I hardly mean missing a turn or two. I mean ending up in a different State miles and miles away from the intended destination. One would assume that my car must be full of maps to make up for this weakness of mine. But you'll be wrong. It's almost like I want to be good at being bad at directions! I don't carry any maps, with the exception of a DC map which I just recently acquired. The rest is just little post-it notes with scribbles on them, or from memory (which explains why I get lost all the time). The worst thing is it's hard to hide this fact, especially when my friends or family are wondering why I'm still not there. Or when I miss a plane because I got lost getting to the airport. Or when I miss a dinner reservation. Or…I can really go on for eternity.
Yesterday I added a whole new chapter to my stories of getting lost.
It was Thursday and out of no where I decided to run home instead of taking the metro. I work in downtown DC and live in MD. There is a trail that connects the two and is mostly used by bike commuters. I didn't have anywhere to be that evening, so running 7-8 miles didn't really seem like a big deal. The plan was to run the trail from my office to a metro stop in MD and then take the train to my apartment which is about 5 miles further north. If I left at 5:00pm, I would no doubt be home eating a giant bowl of ice cream by 7:00pm. No big deal!
I happily trotted out of the office in my running gear. Since I hadn't planned this, I was wearing a cotton t-shirt and shorts…not the best thing to run in on a hot humid day. I had my keys, phone, money, IDs, stuffed in the little pockets of the shorts. So my shorts slid down a little further each time I took a step forward. One of the worst things to be stuck with while running is inappropriate or uncomfortable clothing and my choices today qualified. I put clothing higher on the list than bad weather. Weather can be dealt with proper gear. But sloppy wet cotton shirt with heavy shorts wedging between your thighs is just not bearable. Believe me.
Regardless, I was still so happy to get on with this adventure. I plan on getting a bike soon, so this meant I would get a little preview of what my upcoming commute to work might look like. I was running along water on one side and the shade of beautiful trees on the other. What a perfect day (minus the shorts wedging).
Eventually I was getting shoved in the wild growth while the bike commuters zoomed by me. I saw a solution to this a little farther up where a gravel road went parallel to the bike path. I got on the gravel and picked up speed since I didn't have to worry about getting run over. A few skinny trees and plants divided the two paths, but other than that, they went in the same direction. I was so in-tune with my running and music that I missed the part where the gravel road become a whole different trail that goes in a whole different direction, in a whole different state!!
There was still a lot of foot traffic on my trail and so I didn't notice that I was going the wrong way. Unfortunately, people started disappearing and about 4 miles down I was the only one still running. I was a bit spooked but I decided to suck it up and told myself this is what trails look like! I decided to keep running till I came across a map.
Unfortunately there weren't any maps or names or anything on this trail. After another 4 miles of nothing I was getting scared and very thirty! I didn't have my hydration pack or a water bottle (another grave mistake). But I didn't know I was going to run for this long, I was supposed to be out of this wilderness by now. So at around the 9th mile, I took a turn onto a beaten path that seemed to lead towards the noise of the road. Thankfully after about quarter of a mile it took me back to civilization, by which I mean a small liquor store and a post office.
I was in the middle of no where from the look of it! I got a bottle of water, but apparently I needed to buy at least $5 worth of items to use my card. So I bought some Gatorade, a big bottle of water, and a Clif bar. Then in my sweaty, stinky, and mouth-full-of-Clif state, I tried to get directions from the store clerk. I soon found out that these people had no idea where they lived either. They pointed me towards East and asked me to keep walking. The buses had stopped running at this point and the nice old lady on her way home from the liquor store, didn't want to give me a ride. So I took my only option and started walking East with my two big bottles of water and gator (and don't forget the shorts that kept wedging and sliding at the same time).
Lets just keep the story short here by saying that I walked/ran for another 10 miles before I got to a metro stop. By the time I got home it was 9:30pm! My legs felt like noodles and I looked like shit. I can't believe that the poor people on the metro had to look at me and smell me. I was so delirious that I couldn't help but laugh at this situation. I made a call to Devang telling him I was finally home (he was aware of my situation at about the 12th mile), and after eating my bowl of cereal, I was ready for bed. I slept pretty darn well that night.
Some people might wonder why I didn't just take a cab, or call a friend to pick me up. I guess the best way I can explain this is that my sense of pride got in the way. I wanted to say I ran home, and damn it, I wasn't gonna let this little fact of getting lost get in the way. I feel okay this morning. My legs are a little sore, but nothing too bad. I plan on taking the right trail next week and hopefully that story will end on a much happier note.


text:
70's americano mug mars
|In stock
70s ceramics americano mug in the colour mars.
Whether you are a first time lover of our 70s ceramics, or a long term collector, there is always room for more. We are really excited to say that more funky colours, new finishes and rusty designs found their way into our 70s ceramics collection!
colour: Mars
dimensions: 11,5x8,5x8cm
product length/depth (cm): 11,5
product width (cm): 8,5
product height (cm): 8
product weight (gr): 240
material: Stoneware
characteristic: Reactive glaze
food safe: Yes
dishwasher proof: Yes
product capacity (ML): 260
microwave proof: Yes
Differences may appear: Yes
0 stars based on 0 reviews


text:
Cover Reveal for my next book coming May 27, 2020!
Subscribe to my newsletter for the latest news and for EXCLUSIVE ORIGINAL content as well as my food/travel/book tips! smarturl.it/AvivaEmailList
Love on the Ropes: A slow burn, frenemies-to-lovers romantic comedy set in the world of public radio.
Former TOPGUN pilot Clint Davenport is the newly-crowned prince of public radio now that he's hosting America's most popular morning show. The ladies can't get enough of this Flying Ace, but he's all out of runway for the fairer sex. Until he finds himself on the ropes (literally) at a company retreat and the only person who can bring him in for a safe landing is his office frenemy, who seems as comfortable in a climbing harness as he used to be in an F-18.
Erin Hung grew up sheltered in Honolulu, so when her favorite cousin invited her to move to L.A., she made the leap. Four years later this late bloomer has blossomed, but she still has a lot to prove and is gunning for a promotion. She has no time for distractions, especially romantic ones…and there's no way she's interested in that cocky ex-fighter pilot who just got tapped as host. Unfortunately, working with Mr. Cockpit Cockypants is the only way to get her raise.
When work forces them together, it seems like the beginning of a new Cold War, or maybe Clint's cocky charm is just what Erin needs to see there's more to life than just work.
Love on the Ropes is a standalone and book one in the Love in Action series, a spin-off of the BECKONED series.


text:
a brand
employees love
Get in touch
The essence
Our sweet spot, that lies at the cusp of brand and HR. Three pronged: Craft Employee Value Propositions (EVP's). Deliver on the promises to employees. Attract top talent for companies that aspire to be a great place to work and a destination for talent, a strategic approach to this discipline is a must.
Our success stories
Go Big
Late 2017, a global health sciences (clinical research) firm forayed into India with its first Centre of Excellence in Bangalore. They acquired an Indian player that had ~300 employees with a view to grow the centre by 10X in 2 years.
Download →
Know Your Talent
According to recent analysis by Bloomberg News, India is likely to have the world's largest workforce by 2027, with a billion people aged between 15 and 64. As it stands 18% of India's current population of over 1.3 billion falls between the ages of 15-25.
Download →
Living the Values Code
ABC is one of the top liquor companies in the world that acquired a leading Indian Alco – Bev player a couple of years ago. The new leadership in its aspiration to transform the Indian company into 'The most admired company in the consumer goods sector'...
Download →
"The Chosen One"on Engineering Campuses
XYZ hires engineers from the top 30 technical institutes around the country. The company does high-end design work in India, hence needs to recruit the crème de la crème from engineering colleges. The reality was that when they visited colleges...
Download →
Employer brand masterclass
Championing an employer brand requires an assortment of professional skills, it's like being an "all-rounder". This foundational workshop helps you learn how to craft EVP's, embed them within, devise a talent attraction strategy-and finally 'go to market' online and offline
Carpediem inks
The B-Schooler's love affair with Campus Competitions!
Classes, assignments, surprise tests, pre-placement talks & competitions – A typical day at a premium Business School in India. The…
In every way the Apple of my eye
An "Apple" video positioning the company as a place to work was being discussed on one of the LinkedIn groups…
Ride with the reputed brands towards the talent market
This video takes a leaf out of the classical brand association approach. For long brands have ridden on the fame…


text:
It's been an exciting quarter of play at Candlestick Park, with momentum swinging back and forth dramatically between the Green Bay Packers and San Francisco 49ers. Colin Kaepernick has been responsible for spectacular touchdowns for both teams, while DuJuan Harris has come to life and is the difference between the two teams so far. After one quarter of play, Green Bay leads 14-7.
Kaepernick kicked things off with an interception on his first drive, which was returned for a touchdown by Sam Shields. He bounced back with a great second drive, capping it off with a 20-yard run on a crucial 3rd down and 8 play to tie up the score at 7-7.
The Packers went three-and-out on their first drive after attempting three passes -- two unsuccessfully -- but started running the ball on their second offensive possession. Harris was massive on the drive, picking up 36 yards on five carries, including a big first down on 3rd and 1 and a brilliant 18-yard touchdown run.
So far, this game is everything that everyone thought it would be. It's dead even, the offenses are explosive, and the defenses are making occasional big plays even though they're struggling to keep up.


text:
Ronald Bailey
On the C-SPAN Networks:
Ronald Bailey is a Correspondent for Science in the Reason Magazine with seven videos in the C-SPAN Video Library; the first appearance was a 1992 Forum as a Representative for the Cato Institute. The year with the highest average number of views per program was 2008 with an average of 1,595 views per program. Most common tags: Science & Technology, Environment.
Videos: 6 c. March 21, 1992 - Present Representative, Cato Institute
Videos: 1


text:
Ratus is a deep cleansing treatment for women's intimate part. The treatment helps women reduce excessive vagina discharge, eliminating fungi, discomfort and unpleasant odor and itch and enhance intimacy. The breathing technique used to contract the vagina may help tighten and maintain elasticity of the feminine organ muscle while the warm vapor is steam up your intimate […]Continue reading
Monthly Archives: December 2019
Did you know that getting a weekly massage can also have many benefits for your mind and body apart from relaxing you? Let's take a look: Helps Relieve Pain Find yourself popping painkillers for pain relief? Put down those little pills that can cause your body more harm than good, and head on over for […]Continue reading


text:
Facing drug charges of any kind is serious, but the possible consequences of a conviction vary depending on the circumstances of an arrest, how much drugs the person is carrying and what types of drugs are involved. The consequences for operating a meth lab can be different from consequences for possession of a small amount of marijuana, for example. Understanding the legal details associated with any charge lets individuals make informed decisions about defense strategies or pleas.
For a woman and three men in Dallas, Texas, serious charges related to an arrest on Thursday, March 6, have to do with 20 gallons of liquid methamphetamine. According to police, a popular method of transporting the liquid was involved — there was an extra gasoline tank installed on a vehicle that was filled with the stuff. Police say the arrests came about after officers worked with an informant, who is a defendant looking to lighten the sentence in a separate drug case.
According to reports, the informant received information about a truck that would be used to move drugs from Waco to Dallas. The informant was given instructions to pick up the truck and make a drug delivery. The informant picked up the truck, brought it to a location so police could confirm that drugs were present and then drained the drugs from the extra tank into buckets. Following instructions from a contact in Mexico, the informant delivered the drugs to a residence.
Police moved on the house following the delivery. Officers report smelling meth and gasoline, and state there were buckets of liquid meth and some was also found in the bathtub. Other items found in the home included handguns, cash and bags of crystal meth. All four defendants were found in the home and arrested; there were also two small children present.
For defendants facing similar charges, it's important that they understand how their cases will proceed. An experienced criminal defense attorney may benefit such defendants by ensuring that their legal rights are protected.
Source: The Dallas Morning News, "4 arrested after liquid meth smuggled in pickup's gas tank" Kevin Krause, Mar. 06, 2014


text:
Cameron Diaz and Alex Rodriguez reunited as they were spotted together while relaxing on Miami beach.
Alex Rodriguez
Alex Rodriguez & Kate Hudson Reportedly Split Up
A report confirmed that Alex Rodriguez and Kate Hudson have broken up after a year together.
Madonna's Age Ban
Madonna, who was included in the headlines with her divorce, has banned friends from mentioning her age.
Madonna Is Happy That Guy Ritchie Is Moving On
Singer Madonna is reportedly happy that her soon-to-be former-husband Guy Ritchie is moving on. Is Guy Ritchie really moving on?
Madonna: "Intoxicated" With A-Rod?
Madonna is "intoxicated" with her rumored lover Alex 'A-Rod' Rodriguez.
A-Rod Talked About Madonna, Charity & Divorce
Yankees star Alex Rodriguez revealed for the first time on his relationship with Madonna, saying: "We're friends."
Alex Rodriguez Spends Thanksgiving With Family & Madonna
New York Yankees player Alex Rodriguez celebrated Thanksgiving with his family in Miami before heading off to spend time with Madonna just days after her preliminary divorce from Guy Ritchie was granted, according to reports.
Madonna Confirmed Relationship With Alex Rodriguez
Madonna unofficially confirmed her relationship with Alex 'A-Rod' Rodriguez at her show Wednesday night.
Madonna: Grateful With Work, Furious With Alex Rodriguez?
Madonna, who hit the headlines with her divorce, found marital breakup to be tough but she felt grateful that her heavy workload "provides a distraction that keeps me going."
Madonna Reached Divorce Settlement
Madonna, who previously shared an advice to Britney Spears, and Guy Ritchie will be granted a preliminary divorce decree at the High Court in London on Friday, according to the court lists.
Madonna & A-Rod Cozy Rendezvous At Jerry Seinfeld's Home
Madonna and Alex Rodriguez secretly jetted off in two private helicopters for a quick and cozy rendezvous in the Hamptons.
Madonna's Kabbalah Anger Cure
Madonna is having Kabbalah "anger management" counseling to deal with her rage at estranged husband Guy Ritchie.
Madonna: Still 'Catholic' Girl
Madonna's brother Christopher Ciccone insisted that the singer is still "a Catholic girl" and would never have an affair.
Madonna's Representative Dismisses Divorce & Affair Rumors
Madonna's spokeswoman has released an exclusive statement. Her statement rejects the reports that the singer's marriage to her film director husband Guy Ritchie is on the rocks.


text: The full set of copper pendant lamps, above our tableslorierslorier15:47December 20th, 2017 The full set of copper pendant lamps, above our table View in Instagram ⇒ Share this:Click to share on Twitter (Opens in new window)Click to share on Facebook (Opens in new window) Related 0 0 Spin around you plant, and let is tilt to the side that weighs the most NEW: Geographic plate of the terrain in the Netherlands. Now available for €89 0 Comments Leave a reply Click here to cancel the replyYour email address will not be published. Required fields are marked *CommentName * Email * Website Δ


text:
We've been thinking a lot about wellness lately, thanks to the good influence of Cathy Woods, the cowgirl/yogini who will be guiding the upcoming three-day Mind Body Equine retreat for women.
In case you can't make it to the retreat in May, we have plenty of other ways to nurture mind-body wellness all year round at C Lazy U. Sign yourself up for some self-care with Western flair at our elevated mountain getaway.
Therapeutic encounters with the horses
Equine therapy is a very real thing. Interacting with these large, docile creatures works in powerful and healing ways, and traditional riding is a great way to bond with a horse. Another way to feel the positive effects of a horse's companionship is to take a ride on our special feed wagon for the inside scoop on animal husbandry at the ranch. Or simply take a stroll over to the stable to pay the horses a home visit. They love guests! The ranch hands are happy to help you feed treats to the horses and feel the warmth of their friendly nuzzling. Stress levels fall as you commune with our gentle residents of the stable.
Breathe deep in nature
There's no question about it: deep contact with nature soothes the soul. At C Lazy U, we're lucky enough to be surrounded by wilderness and the Rocky Mountain National Park. With an expansive trail system and our seasoned guides, you can choose your own adventure for getting outdoors. In the winter, discover the rhythm and serenity of snowshoeing through a forest of freshly fallen snow. In the summer, stretch your legs on a rejuvenating hike. We'll share trail maps and all sorts of firsthand knowledge to help you pick the best route for your wellness goals.
Check out C Lazy U's 5 Must-See Locations in the Rocky Mountains.
Practice mindfulness with a spa treatment
Mindfulness is about staying present in the current moment. Take in the sensations of the Lazy You Spa consciously, and let our master practitioners lift your mind and body to blissful relaxation. If spa treatments aren't your thing, make time for a soak in the pool's spring-fed hot tub, or simply find your bliss in the luxury of your cabin, soaking in a bathtub or by the cozy fireside.
Combine yoga with nature
Don't forget to pack your yoga pants! Yoga is a part of our wellness culture at the ranch. Look for upcoming yoga retreats on our events calendar, or sign up any time for an on-demand yoga session at our fitness center. Looking for that perfect yoga-in-nature combination? Ask about our special Hike to Yoga excursions. The lookout points will not disappoint.
Take some time off from the kids
A break from the kids equals instant stress relief. Family vacation at C Lazy U strikes a good balance between togetherness time and parental freedom with our Kids Programs. Leave the youngsters to us during the day, then convene again for meals. Kids and teens are in good hands with our expert counselors, who fill their days with action-packed ranch fun. How you fill your days is entirely up to you!
Give yourself some self care at one of the best all-inclusive resort experiences in the world!


text:
I went to Springfest! It's like Oktoberfest, but smaller. This is a festival where you drink beer, eat as much as you can (sausage, cheese, pretzels, etc.), and dance on the benches to traditional german songs. It was so fun seeing everyone get so excited for every song the band played. Even if I didn't know the song or the language is was in, I learned them by the end of the festival and they played plenty of American songs as well! Buying or renting dirndl or lederhosen is optional, but definitely adds to the experience! (The best place we found is called Steindl Trachten located in the center of town) The fun environment and friendly people allowed us to meet so many people from different parts of the world!
Walk in and pick a seat, any seat. we were looking for one when this group of old german men dressed in traditional clothes waved us over to sit at their table in between all of them. We quickly noticed that they were regulars there. Complete with their own vintage beer jugs, and matching pins on their hats, we could tell that they had been friends for quite some time and this was like their normal routine. They proceeded to buy us beers, pretzels, and give us free things as souvenirs. Even though we didn't speak the same language, we quickly became friends and it was sad when we had to leave. This may have been my favorite memory of the trip.
This plaza is filled with history and amazing architecture. It's also a great place to find shopping and food.
The main attraction of this amazing building is the glockenspiel. Every day at 11, noon, and 5 pm the glockenspiel with near life-size figures perform for the crowd gathered around.
A large public garden named for the rolling "english" hills. Rent a bike or rowboat, have a picnic, our just enjoy the outdoors!
Hey, I'm Adrienne! Welcome to this place for me to share my take on travel, style, design, and sustainability. See more about me here!


text:
This article is more than 1 year old
'In Europe you have watches but in Africa we have time'
So get in your car and wait
Reg hack John Oates took time out from the Plymouth-Banjul rally — "just like the Paris-Dakar event, but for poorer, more stupid, people" — to file this report from Mauritania.
Looking as friendly as a man in military fatigues in the middle of a minefield can do the Mauritanian immigration official smiled broadly and said: "In Europe you have watches but in Africa we have time – now get back in your car and wait."
But he was only joking. The first group of this year's Plymouth-Dakar rally made it safely through the minefields which separate Morocco and Mauritania, and through the layers of red tape involved in bringing an ageing car in little more than two hours.
Thanks to a very helpful man called Momo we made it to Nouabidou in one piece and found Achmed – a 60-year-old man who claimed to be the country's first official desert guide.
With his help our group of six cars – only one of which is four-wheel drive — made it across the Sahara in three days. This involved a fair amount of pushing and some frankly terrifying driving. Second gear and maximum revs seems to be the answer.
Our little Citroen van, plastered in El Reg vultures, enjoyed the sand and got us through with no big dramas. The driveshaft is clinging on with the help of a handful of plumber's mastic and the iPod and iTrip still providing music.
New Year's Eve in Mauritania's capital will hopefully involve some alcohol. After six and a half thousand kilometres we deserve a drink. Tomorrow we head for Senegal and then we are nearly in Gambia and our final destination — Banjul, where the van will be sold and the proceeds given to charity. Last year, the rally participants raised £27,000 for charity.
A very big thank you to all our sponsors who made this possible: all at El Reg, Microsoft UK for picking up the sizeable petrol bill, Classic Car Storage for mechanical genius and tools, and numerous generous individuals. ®