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Localization of Single- and Low-Copy Sequences on Tomato Synaptonemal Complex Spreads Using Fluorescence in Situ Hybridization (FISH)
Daniel G. Peterson, Nora L. V. Lapitan and Stephen M. Stack
Genetics May 1, 1999 vol. 152 no. 1 427-439
Daniel G. Peterson
Nora L. V. Lapitan
Stephen M. Stack
Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.
FOR most plant species, what is known about the order of loci on chromosomes is based almost entirely on genetic linkage maps. Such linkage maps are generated by producing multi-hybrid crosses and determining the relative frequency of recombination between genes or molecular markers (Tanksleyet al. 1989; Paterson 1996). The physical location of genes on chromosomes is of great interest because chromosome structure profoundly influences gene activity (see Lohe and Hilliker 1995; Wallrath and Elgin 1995; Zuckerkandl and Hennig 1995 for reviews). However, linkage maps cannot be superimposed on chromosomes because map distances are not proportional to physical distances (Sturtevant and Beadle 1939; Khush and Rick 1968; Moore and Sherman 1974; Flavellet al. 1985). The relative scarcity of genes and crossing over in heterochromatin is partly responsible for this discrepancy, but crossing over is not evenly distributed in euchromatin either (Snapeet al. 1985; Dooner 1986; Tsujimoto and Noda 1990; Curtis and Lukaszewski 1991; Kotaet al. 1993; Stacket al. 1993; Sherman and Stack 1995). The most direct means of determining the location of genes on chromosomes is through the use of fluorescence in situ hybridization (FISH). In this technique hapten-labeled DNA probes are hybridized to chromosomes that have been spread on glass microscope slides, and antibodies or other affinity reagents conjugated to fluorochromes are used to detect directly or indirectly sites of hybridization (Trask 1991). FISH with repetitive sequences as probes has been widely reported for both animal and plant chromosomes, and single-copy FISH to mammalian chromosomes is fairly routine (e.g., Landegentet al. 1987; Lawrenceet al. 1988; Lichteret al. 1991; Henget al. 1992). Because most techniques used to prepare plant chromosomes leave overlying debris that interferes with probe penetration, single-copy FISH to plant chromosomes has proven considerably more difficult (Lehferet al. 1993; Jianget al. 1995). However, now there are a few laboratories that have overcome this obstacle and are successfully localizing single-copy sequences on plant chromosomes by FISH (e.g., Leitch and Heslop-Harrison 1993; Hansonet al. 1995; Jianget al. 1995; Gómezet al. 1997).
FISH is usually performed on mitotic metaphase chromosomes, but there are reasons to believe that pachytene (meiotic) chromosomes may be better substrates. Each pachytene chromosome (bivalent) is composed of two homologous chromosomes that are joined along their entire length by a proteinaceous scaffold called the synaptonemal complex (SC; Moses 1968). Because each homologue contains two chromatids, there are four closely associated copies of each locus available for hybridization on a bivalent. In comparison, there are only two nearby copies of each locus available for FISH on a metaphase chromosome. In spreads of pachytene chromosomes that have been prepared to reveal SCs (SC spreads), chromatin extends as a diffuse cloud around each SC. The loops of DNA extending from the SC (e.g., Weith and Traut 1980) appear to be more accessible to FISH probes than the DNA of condensed metaphase chromosomes (Moens and Pearlman 1989, 1990; Henget al. 1994; Solari and Dresser 1995), and SC spreads can be prepared relatively free of overlying debris. Additionally, pachytene chromosomes are 5–15 times longer than corresponding metaphase chromosomes (Ramanna and Prakken 1967; Stack 1984), so many closely associated loci that are not resolvable by FISH on metaphase chromosomes should be resolvable on pachytene chromosomes.
Here we report high resolution localization of two single-copy sequences and one low-copy sequence on tomato SC 11 using FISH. This is the first report of single-copy FISH to SC spreads and one of only a few studies in which FISH has been used to study the relationship between genetic linkage and chromosome morphology in plants (e.g., Pedersen and Linde-Laursen 1995; Pedersenet al. 1995). Tomato (Lycopersicon esculentum Mill., recently renamed Solanum lycopersicum L.) was used for this study because all 12 of its SCs are identifiable on the basis of relative lengths and arm ratios (Sherman and Stack 1992, 1995) and because tomato is a true diploid of agronomic importance (Rick 1991). Tomato SC 11 was chosen for hybridization because it is one of the shortest tomato chromosomes, and its corresponding linkage group contains 16 separate RFLP loci, several of which are associated with known genes (Tanksleyet al. 1992). Combined propidium iodide and 4′,6-diamidino-2-phenylindole (CPD) staining was employed to facilitate chromosome identification and aid in relating hybridization sites to chromosome structures. Our results suggest that FISH to SC spreads (SC-FISH) can be used to construct comprehensive maps of single-copy sequences on pachytene chromosomes.
Screening the lambda tomato genomic library: Plasmids containing the tomato restriction fragment length polymorphism (RFLP) markers TG46, TG400, and TG523 were provided by S. D. Tanksley (Cornell University). TG523 flanks the jointless gene (Winget al. 1994), and TG400 is linked to the hairless gene (Tanksleyet al. 1992). To date, TG46 has not been closely associated with a particular gene or phenotype. While molecular and genetic evidence strongly suggests that TG46 and TG523 are single-copy markers, Southern blots of TG400 indicate that it may be present in more than one copy per haploid genome (S. D. Tanksley, personal communication; also see SolGenes web site, http://geneous.cit.cornell.edu/solgenes/aboutsolgenes.html/). Transformation of the competent E. coli strain DH5α, plasmid isolations, and verification of RFLP marker size by restriction analysis were performed using standard methods (Sambrooket al. 1989). The polymerase chain reaction was used to concomitantly amplify and label RFLP markers with digoxigenin (DIG; Rashtchian and Mackey 1992), and the resulting probes were used to screen a Clonetech (Palo Alto, CA) tomato lambda (λ) genomic library (cat. no. FL 1082d). Plating and filter preparation were performed as described in the Clonetech Lambda Library Protocol Handbook (PT1010-1). For each plate, an original and a duplicate filter were prepared. Saline sodium citrate (SSC) solutions of various strengths were produced by diluting a20× SSC stock solution (3 m NaCl, 0.5 m sodium citrate, pH 7.0) with deionized water. Filter hybridization and detection of positive clones were performed according to the Boehringer Mannheim (Indianapolis) DIG/Genius User's Manual (see http://biochem.boehringer-mannheim.com/prod_inf/manuals/dig_man/dig_toc.htm/) with modifications as suggested by A. S. N. Reddy and I. S. Day (personal communication). Briefly, nylon filters (Boehringer Mannheim) were placed in standard hybridization buffer [5× SSC, 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate (SDS), 1.0% w/v Boehringer Mannheim blocking reagent, 0.01 m maleic acid, 0.015 m NaCl, pH 7.5] for 1 hr at 55°, incubated overnight at 55° with gentle shaking in standard hybridization buffer containing 0.01 μg/ml heat-denatured DIG-labeled probe, washed twice in 2× SSC containing 0.1% w/v SDS (55°, 5 min each wash), washed twice in 0.5× SSC containing 0.1% SDS (55°, 15 min each wash), rinsed for 1 min in washing buffer (0.1 m maleic acid, 0.15 m NaCl, 0.3% Tween-20, pH 7.5), placed in blocking solution (0.1 m maleic acid, 0.15 m NaCl, 1% w/v blocking reagent) for 1 hr at 20° with gentle shaking, and incubated in a 1:2500 dilution of anti-DIG-alkaline phosphatase (750 units/ml, Boehringer Mannheim) in blocking buffer for 45 min (20° with gentle shaking). After incubation with the primary antibody, filters were washed twice in 500 ml washing buffer (15 min each wash), placed in detection buffer [100 mm Tris-(hydroxymethyl)-aminomethane, 100 mm NaCl, pH 9.5] for 2 min, and incubated in the dark without agitation in 200 ml of detection buffer containing 900 μl of nitrobluetetrazolium solution (75 mg/ml in 70% dimethylformamide, Boehringer Mannheim) and 700 μl of 5-bromo-4-chloro-3-indoyl phosphate toludinium salt solution (50 mg/ml in 100% dimethylformamide, Boehringer Mannheim). After ∼20 min, positive plaques (small blue "o-shaped" rings) began to appear on filters. Filters with a positive plaque in the same location on both the original and duplicate filters were thoroughly washed in deionized water, and corresponding plaques were removed from petri plates as described by Sambrook et al. (1989). Secondary and tertiary screens were performed as described above. For each of the three starting RFLP markers (TG46, TG400, and TG523), one corresponding lambda clone (designated λTG46, λTG400, and λTG523, respectively) was selected for use in FISH.
Probe preparation: The QIAGEN (Valencia, CA) Lambda Maxi Kit was used to isolate DNA from λTG46, λTG400, and λTG523. The DNA was digested with BamHI or EcoRI, and tomato insert DNA fragments were separated from λ arms by gel electrophoresis. Bands containing tomato insert DNA were excised from 1% w/v agarose gels, and the QIAGEN Qiaex II Kit was used to isolate DNA from agarose. The GIBCO BRL (Rockville, MD) BioNick Kit was used to label DNA with biotin. Each biotin-labeled probe was placed in its own 1.5-ml micro-centrifuge tube.
Tomato synaptonemal complex spreads: SC spreads were prepared as described by Peterson et al. (1996) with several modifications. Briefly, tomato (cv. Cherry) anthers were placed in 200 μl of sugar-salt medium [0.56 mm KH2PO4, 0.1 mm acid PIPES, 0.2% w/v potassium dextran sulfate, 1 mm CaCl2, 0.7 m mannitol, 1% w/v polyvinylpyrrolidone (Mr = 10,000), pH 5.1] containing 3 mg desalted cytohelicase (Sepracor, Marlborough, MA). The upper tips of the anthers were cut off, and anther bottoms were allowed to digest in the dark for 10 min. Drops of 45% acetic acid were placed on new microscope slides (Corning, Inc., Corning, NY), and slides were wiped dry with a Kimwipe. Slides then were glow-discharged according to Dubochet et al. (1971) except that air rather than amylamine vapors was introduced into the discharge chamber. Dissecting needles were used to squeeze microsporocytes from anthers, and microsporocytes were then allowed to digest for an additional 10–15 min. A 0.5-μl aliquot of the protoplast suspension was drawn into a siliconized micro-pipette and gently blown into a 10-μl droplet of hypotonic bursting medium (0.05% v/v Nonidet P-40 and 0.1% w/v bovine serum albumin) suspended from the end of a 200-μl pipette tip. The resulting droplet was immediately placed in the center of a glow-discharged (hydrophilic) microscope slide, and an additional 10 μl of hypotonic bursting medium was added. A hand-held nebulizer (Fullam, Latham, NY) was used to immediately give the slide 30 puffs of 4% aqueous formaldehyde (pH 8.5). The slide was air-dried, fixed in 4% aqueous formaldehyde for 10 min, rinsed twice without agitation in aqueous 0.01% Photoflo 200 (Kodak, Rochester, NY), rinsed four times (20 sec each rinse) in distilled water, and air-dried.
Fluorescence in situ hybridization: FISH was performed using a combination of the protocols of Rayburn and Gill (1985) and Anamthawat-Jónsson et al. (1996) with significant modifications. Slides with spread SCs were incubated in 2× SSC containing 100 μg/ml RNase A (Sigma, St. Louis) at 37° for 30 min, washed in two changes of 2× SSC (37°, 10 min each wash), rapidly dehydrated in a graded ethanol series, and air-dried. Chromosomal DNA was denatured by placing slides in 50 ml of 70% formamide in 2× SSC at 70° for 2.5 min. Slides were rapidly dehydrated in an ethanol series at -20° and air-dried. A cocktail containing 75 μl deionized formamide, 30 μl 50% w/v potassium dextran sulfate, 15 μl 20× SSC, 7.5 μl of 10 mg/ml sheared herring sperm DNA (GIBCO BRL), and 12 μlof25 μg/ml biotin-labeled probe(s) was heated at 97° for 10 min. Thirty microliters of the heat-denatured cocktail was placed on a slide, and a 22- × 50-mm coverglass was added. Slides were incubated in sealed humid chambers at 37° overnight. Coverglasses were gently washed from slides with 2× SSC at 37°. Slides were washed twice in 2× SSC, once in 25% formamide in 2× SSC, twice in 2× SSC containing 2% Tween-20 (all washes 37°, 10 min), and incubated for 60 min at 37° in blocking buffer (0.1 m maleic acid, 0.15 m NaCl, 1% w/v Boehringer Mannheim blocking reagent, pH 7.5). Slides were incubated for 1 hr at 37° in 1 μg/ml mouse anti-biotin (Boehringer Mannheim) in blocking buffer, washed three times in 2× SSC containing 2% Tween-20 (37°, 10 min each wash), incubated for 1 hr at 37° in 10 μg/ml biotinylated goat anti-mouse (Sigma) in blocking buffer, and washed three times in 2× SSC containing 2% Tween-20 (37°, 10 min each wash). Slides were then incubated in 40 μg/ml mouse anti-biotin fluorescein isothiocyanate (FITC; Sigma) or 4 μg/ml streptavidin-FITC conjugate in blocking buffer for 1 h (37°). All slides were washed once in 2× SSC containing 2% Tween-20 (37°, 10 min) and twice in 2× SSC at room temperature (10 min each wash).
Counterstaining and microscopy: Slides were incubated for 15 min in the dark in McIlvaine's buffer (aqueous 17.7 mm citric acid, 164.7 mm Na2HPO4, pH 7.0) containing 0.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI), 1 μg/ml propidium iodide (PI), or 1 μg/ml PI and 0.5 μg/ml DAPI (i.e., CPD). Slides were washed for 30 sec in deionized water and allowed to air-dry for 5 min. Ten microliters of freshly prepared antifade medium (aqueous 50 mm Tris, 50% glycerol, 1 mg/ml phenylenediamine) was placed on each slide, and a 22- × 50-mm coverglass was added. Coverglasses were sealed onto slides with fingernail polish. Fluorescence and phase-contrast microscopy were performed using an Olympus Provis AX70 microscope equipped with an Olympus UM-51005 filter cube for simultaneous visualization of PI and FITC, a U-MNV filter cube for observation of DAPI and CPD staining, and a U-NG filter cube for visualizing PI alone. Photographs were taken using a PM-C35DX camera and Kodak Royal Gold 400 film.
CPD karyotype: Both freshly fixed SC spreads and SC spreads that had been used in FISH were CPD stained, and complete (or nearly complete) late pachytene sets were photographed using phase-contrast and fluorescence microscopy. Phase-contrast and corresponding CPD photographs (magnification of prints ×2258) were scanned at a resolution of 300 dpi into a computer. The chromosome-measuring program Micromeasure 3.01 (available at http://www.colostate.edu/Depts/Biology/Micromeasure/) was used to determine relative SC lengths and arm ratios from phase-contrast images. The SC karyotype of Sherman and Stack (1992, 1995) was used to identify individual-SCs. Adobe Photoshop 4.0 was used to digitally merge phase-contrast and corresponding CPD images. To describe the position of each CPD band, the distance along a CPD-banded SC from the center of the CPD band to the center of the kinetochore was measured and divided by the length of the entire SC arm. In this way, the position of each CPD band was expressed as a decimal fraction of the arm length from the centromere. These fractions were multiplied by the mean length of the SC arm (Sherman and Stack 1995) to give micrometer distances from bands to the centromere (Table 1). Likewise, the length of each CPD band was measured and divided by the length of its entire SC to determine the relative proportion of chromosome length contained within the band (Table 1).
FISH data analysis: For each late pachytene SC spread (i.e., spreads with visible kinetochores) showing a FISH signal, a phase-contrast and a PI/FITC photograph were taken. For most sets, a CPD image was also obtained. Phase-contrast and FITC/PI photographs were scanned and digitally merged to allow accurate measurement of distances between FISH signals and kinetochores. In all instances, SC 11 was identified on the basis of relative length and arm ratio according to the SC karyotype data of Sherman and Stack (1992, 1995). In spreads where FISH labeling was observed and a corresponding CPD image was available, the pattern of CPD staining was used to confirm the identity of SC 11 (see results). To determine the distance between an FITC focus and the kinetochore, the distance along the SC from the location of the focus to the center of the kinetochore was measured and divided by the length of the entire SC arm to express the focus location as a decimal fraction of the arm length from the centromere. Fraction distances were multiplied by the mean length of the SC arm to give micrometer distances from foci to the centromere. This is the same technique used by Sherman and Stack (1995) to describe the location of recombination nodules on tomato SCs. In cases where an FITC focus did not lie directly on the SC, a line was drawn from the center of the FITC focus perpendicular to the axis of the SC. The point of intersection between the line and the SC axis was marked as the location of that focus on the SC. In some instances, an FITC focus was located in the chromatin distal to the end of the SC. In these cases, a line was extended from the end of the SC, a perpendicular line was drawn from the focus through the extended line, and the point of intersection was marked as the location of the focus.
Karyotype of the CPD-banded tomato SC complement
Initially, all measurements were made from SC spreads labeled with only one of the three probes. However, after it was clearly established that there was a considerable physical distance between λTG523 and λTG46 and that there was no difficulty determining which signal belonged to which probe, measurements were made from these dual-labeled spreads as well. No measurements were made from spreads probed with any other combination of markers. Statistical analysis of the distribution of FITC foci for the three probes was performed using the computer program Instat 1.12a (GraphPad Software, San Diego).
Dot blot verification of probe identity: To rule out the unlikely possibility that the genomic probes used for FISH were inadvertently switched or cross-contaminated before in situ hybridization, each DIG-labeled RFLP marker was used to probe a separate nylon membrane on which a 20-μl drop (0.5 μg) of each of the biotin-labeled λ insert DNAs had been dotted. Filter hybridization conditions and colorimetric detection of probe hybridization were performed as described above.
DAPI, PI, and CPD staining of tomato SCs: When tomato SC spreads are stained with DAPI, pericentromeric heterochromatin, euchromatin, and telomeres are not readily differentiated. However, some bivalents possess a single, highly localized region that does not fluoresce, i.e., a DAPI-negative band (Figure 1A). DAPI-negative bands were observed throughout pachynema and diplonema. In late pachytene spreads, kinetochores stain with similar intensity as surrounding chromatin, which suggests that kinetochores are highly infiltrated with chromosomal DNA.
In PI-stained early pachytene SC spreads of tomato, heterochromatic regions often appear wider than euchromatic regions (Figure 1B). Additionally, heterochromatin stains a bit more intensely than euchromatin. In late pachytene spreads, PI-stained SCs have a rather homogenous appearance with no noticeable staining difference between heterochromatin and euchromatin. Kinetochores stain with an intensity similar to that of nearby chromatin, again indicating the presence of DNA in kinetochores.
When early pachytene tomato SC spreads are stained with CPD and examined using a wide UV filter, euchromatin and heterochromatin are easily differentiated with heterochromatin fluorescing white and euchromatin fluorescing blue (Figure 1C). The differential staining of heterochromatin and euchromatin seen after CPD treatment is more striking than differences produced by PI staining alone. By late pachynema, heterochromatin and euchromatin do not exhibit a predictable pattern of differential staining (Figure 1D).
Throughout pachynema, the NOR (which comprises most of the short arm of chromosome 2) is easily identified by its red fluorescence (Figure 1, C and D). As previously described, the NOR typically exhibits partial asynapsis (Sherman and Stack 1992; Petersonet al. 1996), and, as pachynema progresses, the NOR becomes more elongate (Figure 1D).
CPD staining results in a single red "CPD band" within the pericentromeric heterochromatin of 5 of tomato's 12 SCs (Figure 1, C and D). The number and general location of CPD bands suggest that they are equivalent to the DAPI-negative bands seen in tomato SC spreads stained with DAPI only (Figure 1A). We used CPD staining coupled with the SC karyotype data of Sherman and Stack (1992), to construct a CPD karyotype for tomato. CPD bands are found on SCs 1, 3, 6, 8, and 11 (Figure 1, E–G and Table 1). The CPD bands on SCs 1 and 3 lie essentially at centromeric positions while the bands on SCs 6, 8, and 11 are located at subcentromeric positions in the short arms of their respective chromosomes (Figure 1G). An idiogram showing the distribution of CPD bands on the tomato SC complement is shown in Figure 1H.
—Staining of chromatin associated with tomato SC spreads. (A) Early pachytene SC set stained with DAPI only. Several SCs possess a region where DAPI staining is not visible, i.e., a DAPI-negative band (arrows). No other chromatin-based features are visible. (B) Early pachytene SC spread stained with PI only. Heterochromatic regions (e.g., arrowheads) appear wider and stain a bit more intensely than the distal euchromatic regions (e.g., arrows). (C) CPD staining of an early pachytene SC spread. Pericentromeric heterochromatin fluoresces white while distal euchromatin fluoresces blue. A red-fluorescing chromatin band (CPD band) is visible within the pericentromeric heterochromatin of 5 of tomato's 12 SCs (arrows). These bands most likely correspond to the DAPI-negative bands seen in DAPI-stained tomato SC spreads. The NOR near the terminus of the short arm of SC 2 fluoresces red and usually exhibits partial or complete asynapsis (e.g., diamond-head arrows). (D) Late pachytene CPD-stained SC spread. Kinetochores are visible as thickenings along SCs (e.g., white boxes). On two CPD-banded SCs, the kinetochores are located directly over the CPD band (arrowheads). However, on the remaining three CPD-banded SCs a red band is visible outside of the region encompassed by the kinetochore (arrows). The elongate, asynapsed NOR fluoresces red (diamond-head arrows). (E) Phase-contrast image of a complete spread of late pachytene tomato SCs. A kinetochore is visible on each SC (black arrows). One SC is broken into two pieces (white arrow). Likewise, the NOR region has been lost from the end of SC 2 (white arrowhead). (F) CPD staining of the same SC spread shown in E. CPD bands are visible on five of the SCs (arrows). (G) Diagram showing the relative position of CPD bands and kinetochores. Blue lines represent SCs, yellow dots represent kinetochores, and red bars represent CPD bands. The identity (chromosome number) of each CPD-banded chromosome is shown next to an arrow pointing to its kinetochore. (H) Idiogram of the tomato SC complement showing the distribution of CPD bands. SCs 1-12 are shown in consecutive order from left to right. Euchromatin is blue, heterochromatin is white, kinetochores are orange, and CPD bands are red. The NOR in the short arm of SC 2, which appears red after CPD staining, is colored pink in this idiogram. A constricted area within the NOR marks the general region where SC formation does not occur. Frames that share a common bar: A and B; C and D; and E–G. Bars, 10 μm.
Fluorescence in situ hybridization: Probes containing single-/low-copy sequences were obtained by screening a tomato λ genomic library with the chromosome 11-associated RFLP markers TG46, TG400, and TG523 (Tanksleyet al. 1992). For each RFLP marker, a corresponding positive genomic clone was selected, i.e., λTG46, λTG400, and λTG523. Tomato insert DNA from each clone was isolated (lengths of tomato inserts: λTG46, 16,550 bp; λTG400, 10,200 bp; λTG523, 14,000 bp) and labeled with biotin using nick translation. Labeled insert DNA was hybridized to spreads of tomato SCs on microscope slides. Slides were treated with mouse anti-biotin followed by incubation in biotinylated goat anti-mouse. Sites of probe hybridization were detected using anti-biotin-FITC or streptavidin-FITC conjugates. SC 11 was identified on the basis of its relative length and arm ratio. Additionally, CPD banding analysis was used to confirm the identity of SC 11. The Micromeasure chromosome measuring program was used to determine the distance from the centromere to each FITC focus.
Each of the three genomic probes hybridized exclusively to euchromatin on the long arm of SC 11 (Figure 2). Little or no background FITC fluorescence was observed, and the efficiency of hybridization (i.e., the percentage of SC spreads where specific labeling of SC 11 could be seen) was high for all three probes (ca. 70%). On the basis of the relative locations of FITC foci, the three probes were positioned in relationship to the centromere and to one another (Table 2 and Figure 2, N–P). We refer to the diagram showing the position of each locus on SC 11 as an SC-FISH map (Figure 2O). One-way analysis of variance (ANOVA) indicates that the difference of the group means for the probes is highly significant (F = 146.9, P < 0.0001). Comparison of the data for any two individual probes also indicates that the loci are clearly separated (Bonferroni P value for any two probes is <0.001). λTG523 is the most distal, i.e., farthest from the centromere, of the loci with a near terminal location. The probes λTG46 and λTG400 are located more proximally on SC 11 with λTG400 being closer to the centromere than λTG46. This contradicts the molecular linkage map of chromosome 11 on which TG400 is located between TG523 and TG46 (Tanksleyet al. 1992; compare Figure 2N and 2O with 2P). To rule out the possibility that the probes λTG46 and λTG400 had been inadvertently switched before FISH, we performed a series of dot blot experiments. These experiments verified that each biotin-labeled FISH probe hybridized exclusively with the RFLP marker originally used to isolate it. It is unlikely that probes were switched at the time of FISH because data were obtained from several experiments, some experiments involved use of only one probe at a time, and probes used in all FISH experiments were removed from the same three tubes from which samples were taken for dot blot probe verification (see materials and methods).
SCs vs. "prefixed" pachytene chromosomes: In initial attempts to localize specific DNA sequences on meiotic chromosomes, we used pachytene chromosomes fixed before spreading in 1:3 acetic ethanol, i.e., "prefixed pachytene chromosomes," as substrates for FISH rather than SC spreads. Although, when repetitive sequence probes were used, prefixed pachytene chromosomes were adequate substrates for FISH (e.g., Petersonet al. 1998), localization of single-copy probes on these chromosomes proved difficult because of high levels of background fluorescence and a scarcity of chromosome sets in which each bivalent could be distinguished, i.e., less than one completely interpretable set per slide (data not shown). When SC spreads were substituted for prefixed pachytene chromosomes, significant problems with background fluorescence were eliminated and chromosome identification was facilitated because each slide possessed 10 or more SC sets where all 12 tomato SCs were clearly identifiable (e.g., Figure 1, E–G).
CPD staining: If tomato SC spreads are stained with a combination of PI and DAPI, i.e., CPD stained, and illuminated with broad-band UV-visible light, structural features associated with differential chromatin condensation and/or DNA sequence can be visualized. These features either are not visible by DAPI or PI staining alone or are more readily differentiated by CPD staining. Although a variety of dye concentrations were tested, best results were obtained when chromosomes were stained with 1 μg/ml PI and 0.5 μg/ml DAPI (data not shown).
Euchromatin and heterochromatin can be differentiated by CPD staining in early pachytene SC spreads, but by late pachynema, the two types of chromatin are not readily distinguished. The explanation for this may be that the difference in relative condensation of heterochromatin and euchromatin is decreased during the transition from early to late pachynema. On the other hand, CPD bands and NORs remain differentially stained throughout pachynema (and probably through-out meiosis).
Likewise, a combination of PI and DAPI has been shown to differentially stain NORs in mitotic and meiotic metaphase chromosomes of cotton (Hansonet al. 1995; Jiet al. 1997), mitotic metaphase chromosomes of tomato (T. P. V. Hartman, personal communication), and SC spreads from maize and potato (our unpublished observations). Consequently, it seems likely that the NORs of many, if not all, plants can be visualized using CPD staining.
—Single-/low-copy FISH to tomato SC 11. (A) Phase-contrast image of a late pachytene SC 11. The kinetochore is clearly visible (arrowhead). (B) Hybridization of λTG523 to the long arm of the same SC. Probe hybridization is visible as yellow-green (FITC) foci (arrow). The chromosome appears red because of the PI counterstain. The kinetochore is marked by an arrowhead. (C) CPD image of the same SC. As predicted by the CPD karyotype, this chromosome possesses a red CPD band (arrow) in the short arm near the kinetochore (arrowhead). (D) Phase-contrast image of SC 11. The kinetochore is indicated by an arrowhead. (E) λTG523 hybridization on the SC shown in D. The signal is localized near the end of the chromosome (arrow). Note that there is little background FITC fluorescence, and there are no ectopic sites of hybridization. (F and G) Phase-contrast and PI/FITC image of a chromosome 11 showing the kinetochore (black arrowhead) and λTG400 hybridization (white arrow), respectively. (H and I) Phase-contrast and PI/FITC images of the same chromosome 11. In I, simultaneous detection of λTG523 (arrowhead) and λTG46 (arrow) is shown. (J–M) Centromere placement and probe positioning on an early pachytene tomato SC 11. (J) Phase-contrast image of an SC 11. No kinetochore is visible. (K) λTG523 hybridization (arrow). (L) CPD staining. Heterochromatin appears white and euchromatin blue. A distinct CPD band (arrowhead) is observed within the pericentromeric heterochromatin of the bivalent. (M) Diagram of chromatin features and hybridization sites superimposed on the phase-contrast image of the SC. In the diagram, euchromatin is blue, heterochromatin is green, the CPD band is red, and FITC foci are yellow. Although a kinetochore is not visibleon this SC, the consistent relationship between the location of CPD bands and kinetochores allows accurate placement of a "kinetochore" on the SC (black band; see discussion). (N–P) SC-FISH and molecular linkage maps of tomato chromosome 11. In all diagrams, λTG400, λTG46, and λTG523 are represented by the colors blue, red, and green, respectively. (N) Histogram showing the distribution of λTG46, λTG400, and λTG523 FITC foci along tomato SC 11. The number of observed FITC foci is shown on the ordinate axis while a diagram of an average SC 11 is shown on the abscissa. Each vertical bar represents the number of foci observed in a 0.1-μm interval on SC 11. On the SC 11 diagram, heterochromatin is represented by a gold rectangle encompassing the centromere (black circle), while euchromatin is represented by a thin, solid black line extending from both sides of the pericentromeric heterochromatin. A dotted line extends from the terminus of the long arm of SC 11 to include λTG523 foci that were found in chromatin extending beyond the end of the SC. (O) SC-FISH map of tomato SC 11. The mean position of each SC-FISH probe is shown by a vertical bar. All three probes are localized in euchromatin of the long arm. (P) Molecular linkage map of tomato chromosome 11 redrawn from Figure 1 of Tanksley et al. (1992). Regions between adjacent markers are enclosed by horizontal braces with map distances between markers given above the braces. Note that λTG46 and λTG400 are reversed in relation to their order on the SC-FISH map. Frames that share a common scale bar: (A–C) bar, 5 μm; (E–I) bar, 5 μm; (J–M) bar, 1 μm.
Locations of three probes on the long arm of tomato SC 11
Insight into the probable mechanisms by which CPD staining differentially stains NORs and CPD bands may be gained by considering the mechanisms by which PI and DAPI interact with DNA. PI intercalates between bases of single- or double-stranded nucleic acid molecules without regard to nitrogenous base composition, and consequently PI can be used as a quantitative nucleic acid dye (Heslop-Harrison and Schwarzacher 1996). DAPI is a double-stranded DNA-specific dye that interacts with DNA by at least two different mechanisms (see Kapuscinski 1995 for review). In regions where three or four AT base pairs are located in tandem, DAPI binds to the minor groove of the DNA, and this results in a highly fluorescent compound. DAPI also intercalates between bases, which results in a nonfluorescent compound. The former reaction is energetically favored in AT-rich regions while the latter is favored in GC-rich areas along a DNA molecule. As an intercalator, the binding strength of DAPI (in GC-rich areas) is roughly equivalent to that of PI. NORs, i.e., rDNA sequences, are GC-rich compared to most other DNA regions (Macgregor and Kezer 1971; Yasmineh and Yunis 1971; Ingleet al. 1975). As a result, in CPD-stained spreads, minor groove binding of DAPI to rDNA sequences would be limited. Presumably, DAPI and PI would compete for intercalation sites in the GC-rich rDNA, but only PI intercalation would result in fluorescence. In addition, the PI concentration in the CPD staining solution is double that of the DAPI concentration, and thus PI intercalation would be favored. Consequently, red (PI) fluorescence predominates in NORs. Similarly, DAPI-negative bands (seen in DAPI-stained SC spreads) and CPD bands probably represent other chromosomal regions rich in GC base pairs. The presence of GC-rich regions in heterochromatin (including GC-rich satellite sequences) has been reported for other species (see Sumner 1990 for review). Differential staining between heterochromatin and euchromatin observed in early pachynema likely reflects differences in both chromatin condensation and GC content.
CPD bands may be equivalent to certain C-bands. A C-band karyotype based on 1:3 acetic ethanol-fixed tomato (Moneymaker) pachytene chromosomes shows prominent centromeric/subcentromeric C-bands on chromosomes 1, 4, 6, 8, and 11 and staining of the NOR on chromosome 2 (M. S. Ramanna and L. P. Pijnacker, unpublished data). The relative location of major C-bands on chromosomes 1, 6, 8, and 11 corresponds exactly with locations of CPD bands on these chromosomes. However, the CPD- and C-band karyotypes differ in three ways: (1) There is no corresponding CPD band for the major C-band near the centromere of chromosome 4. (2) A C-band is found near the centromere of chromosome 3, but it is very small and probably does not account for the relatively large CPD-band we observe on this chromosome. And (3) there are several small C-bands for which corresponding CPD bands were not observed. These discrepancies may be caused by differences in the heterochromatin of the cultivars Cherry and Moneymaker. Also the mechanism of C-banding (Sumner 1990) differs from the proposed mechanism of CPD staining, so it is not surprising that some CPD bands coincide with C-bands while others do not (and vice versa).
SCs as substrates for single-copy in situ hybridization: Although FISH has been used to localize repetitive sequences on spreads of vertebrate SCs (Moens and Pearlman 1989, 1990; Henget al. 1994; Solari and Dresser 1995) and on SC spreads of two plant species (Hasenkampf 1991; Albini and Schwarzacher 1992), to our knowledge, there have been no reports of single-copy FISH to SCs of any species. Here we demonstrate that spreads of tomato SCs are well suited for the rigorous requirements of in situ detection of single-copy sequences. Because comparable SC spreads can be prepared for a variety of plants as well as animals and fungi, it is likely that this technique can be generally applied.
In our experience, background fluorescence is markedly reduced if SC spreads are used as FISH substrates instead of prefixed pachytene chromosomes. We suspect that this phenomenon reflects the time at which chromosomes are fixed. Treatment of cells with aceticethanol results in rapid protein crosslinking and fixation of chromosome structure (Sharma and Sharma 1980). Consequently, in prefixed preparations nucleoplasm may be fixed onto chromosomes before squashes/spreads are made. During FISH, probes and fluorochrome-labeled affinity reagents may become trapped within nucleoplasm surrounding the chromosomes to produce high background fluorescence. While digestion of chromosomes with a protease(s) before FISH can be used to remove at least some of the contaminating nucleoplasm from prefixed pachytene chromosomes (Jianget al. 1995), protease treatment also results in significant changes in chromosome structure (our experience). In contrast, SCs are spread before fixation. During the spreading process, nucleoplasm around SCs is dispersed and chromatin is decondensed, which allows better access of single-copy probes to chromosomal DNA.
Although we believe that SC spreads possess some features that make them better suited for single-copy FISH than prefixed pachytene chromosomes, it should be noted that other investigators have successfully localized single-copy sequences on prefixed pachytene chromosomes by in situ hybridization (Shenet al. 1987; Jianget al. 1995).
CPD banding and FISH: In all cases where single-copy FISH was detected and a CPD photograph was taken, a CPD band was observed in a position predicted by the CPD band karyotype of SC 11. Although SC 11 can be readily identified on the basis of relative length and arm ratio, FISH and CPD results each independently indicate that SC 11 was correctly identified in this study.
Nature of the tomato genome: Because it is possible that genomic clones isolated by screening a library with RFLP markers might contain repetitive elements in addition to single-copy DNA, genomic probes are often "prehybridized" with unlabeled repetitive DNA sequences before they are placed on chromosomes. Such chromosomal in situ suppression (CISS) hybridization effectively prevents highly repetitive sequences in probes from participating in hybridization with chromosomal DNA (Landegentet al. 1987; Lichter et al. 1988, 1990; Jiang et al. 1995, 1996; Zwicket al. 1997). However, in earlier studies we determined that only 23% of the tomato genome is composed of repetitive sequences and that the vast majority of these repetitive sequences are localized in pericentromeric heterochromatin (see Peterson et al. 1996, 1998). Consequently, because there was reason to believe that our genomic probes might not contain repetitive sequences, we tried FISH without CISS hybridization, and indeed, we found that each of the three genomic probes hybridized exclusively to a different highly localized region on SC 11 in the absence of suppression sequences. With a relatively small genome size and little repetitive DNA in its euchromatin, tomato should be particularly well suited for gene tagging and chromosome walking (Petersonet al. 1998).
Single- vs. low-copy number sequences: Molecular evidence indicates that TG523 and TG46 are located at single loci on tomato chromosome 11 (Tanksleyet al. 1992; SolGenes web site, http://geneous.cit.cornell.edu/solgenes/aboutsolgenes.html/). Our observation that λTG523 and λTG46 each bind exclusively to a single, highly localized region on SC 11 supports this premise. On the other hand, blotting evidence suggests that TG400 may occur at more than one locus in the tomato genome (S. D. Tanksley, personal communication; Sol-Genes web site). However, we observed only one highly localized site of λTG400 probe hybridization on tomato SC spreads, and this hybridization site is on the long arm of SC 11. If there is indeed more than one copy of TG400 in the tomato genome, this result might be explained if the copies of TG400 are so closely associated that they are not resolvable using our FISH method. Alternatively, it is possible that the λTG400 FISH clone hybridized to a TG400 site on SC 11 and did not recognize TG400 loci elsewhere.
Comparison of the SC-FISH and molecular genetic maps: While linkage maps show the relative order of genes along each chromosome, they provide little insight into the relationship between genes and chromosome structure. This is evident in a comparison of the SC-FISH map (Figure 2O) with the molecular linkage map of chromosome 11 (Figure 2P). On the molecular linkage map, TG523 is closer to the center of the map than it is to the terminus, but on the SC-FISH map, λTG523 is located near the end of the long arm of SC 11. Because TG523 is not the most distal locus on the molecular linkage map, it is likely that there are other genes/markers clustered within the more distal subtelomeric region of the chromosome. With respect to distances between markers, all three SC-FISH markers are contained within a chromosomal region comprising only 19% of the length of SC 11. However, the three RFLP markers span a region encompassing 35% of the molecular linkage map (Tanksleyet al. 1992).
On the SC-FISH map, the order of λTG46 and λTG400 is reversed in relation to their order on the molecular linkage map (Figure 2, N–P). This disagreement is not caused by an inability to resolve these markers cytologically, because (with the exception of a single FITC focus) the ranges of FITC foci for the markers do not overlap (Figure 2N). While somewhat unexpected, disagreement between the order of loci on tomato maps is not without precedent (e.g., Schumacheret al. 1995).
Relative order of TG46 and TG400: There are several possible explanations for the discrepancy between the relative positions of TG46 and TG400 on the SC-FISH and molecular genetic maps. For example, it is possible that (1) the λTG400 probe recognizes a locus on SC 11 different from the TG400 locus positioned on the linkage map (see above); (2) these loci have been incorrectly positioned on the molecular linkage map; or (3) the order of loci in cv. Cherry tomato differs from the gene order in the cultivar that was used to prepare the molecular linkage map (i.e., VF36-Tm2a; Tanksleyet al. 1992).
Tomato DNA per micrometer of SC in euchromatin and heterochromatin
The first of these possibilities seems unlikely because the λTG400 probe would have to consistently fail to hybridize to the "RFLP-mapped" copy of TG400 on chromosome 11, yet succeed in hybridizing to a second copy of TG400 on chromosome 11 overlooked during RFLP mapping. The homology between the two TG400 copies (even if limited to the 1200 bp of the RFLP marker itself) would probably be sufficient to produce some positive hybridization at both sites. Consequently, a less defined distribution of λTG400 FITC foci encompassing the range of λTG46 hybridization sites would be expected, but this is not observed (Figure 2N).
In reference to the second possibility, construction of linkage maps involves sexual crosses and sometimes difficult and/or complex scoring of recombination events that could lead to a mistake. However, because TG46, TG400, and TG523 have LOD scores of at least 3.0 (Tanksleyet al. 1992), it is unlikely that the markers have been positioned incorrectly on the molecular linkage map (see Tamarin 1999).
Perhaps the most likely explanation for the discrepancy between the SC-FISH and RFLP map is a difference in locus order between cv. Cherry and cv. VF36-Tm2a because of a small inversion in chromosome 11. In crosses between our Cherry tomato line and characterized translocation lines of cv. Early Fire Ball, no inversion loops were observed in pachytene SC spreads from the F1 progeny (unpublished observations made during research of Herickhoffet al. 1993). Consequently, if cv. Cherry contains an inversion in chromosome 11 relative to cv. Early Fire Ball, either the inversion is not large enough to produce an inversion loop in the heterozygote or cv. Cherry and cv. Early Fire Ball have the same inversion.
Base pair distances between SC-FISH markers: Data from previous investigations (Peterson et al. 1996, 1998) were used to calculate DNA densities (1C) in heterochromatic and euchromatic regions along tomato SCs (Table 3). This permitted an estimate of the number of base pairs between the centromere and each of the SC-FISH markers and between the SC-FISH markers themselves (Table 4). On the basis of the data in Table 4 and the average positions for the three probes shown in Figure 2O, we estimate that SC-FISH easily permits resolution of loci separated by as few as 1.7 million bp with every indication that much closer loci could be resolved. For comparison, Trask (1991) estimated that FISH probes can be mapped to human metaphase chromosomes at a resolution of ∼3 million bp. On the other hand, substrates other than condensed chromosomes can be used to improve resolution. For example, FISH has been used to resolve single-copy sequences separated by as little as 50 kb in the decondensed chromatin of interphase nuclei (Lawrenceet al. 1988; Trasket al. 1991). Additionally, Heng et al. (1992) resolved genes as close as 20 kb by FISH to free chromatin, and Fransz et al. (1996) resolved sequences separated by <1 kb using stretched, naked DNA as a FISH substrate. Unfortunately, with these latter techniques individual chromosomes and structural features such as heterochromatin, centromeres, and telomeres are not readily distinguishable.
Consolidation of the SC-FISH and recombination nodule maps: Because of the discrepancy between the SC-FISH and RFLP maps for tomato chromosome 11, a meaningful discussion of the relationship between molecular map distances and the number of base pairs between the three SC-FISH marker loci is not possible. However, the SC-FISH and recombination nodule maps for SC 11 (Sherman and Stack 1995) can be compared to determine map distances between SC-FISH markers. [Recombination nodules (RNs) are ellipsoidal structures associated with SCs during late pachynema, and there is a 1:1 correlation between RNs and crossover events (Carpenter 1975; Sherman and Stack 1995; Andersonet al. 1997).] Comparison of the maps indicates that the rate of crossing over per megabase pair (106 bp) is similar in the interval flanked by λTG400 and λTG46 and the interval flanked by λTG46 and λTG523 (Table 5). Thus, at least at the resolution of this analysis, there is no obvious recombination hot/cold spot in one of these intervals compared to the other (see Segalet al. 1992 for an example of a recombination hot spot in tomato euchromatin).
Use of CPD banding to position probes on early pachytene SCs: Because heterochromatin and euchromatin can be differentiated in early pachytene SCs, SC-FISH to early spreads may be useful in determining the chromatin background in which a gene/transgene is found. However, it is difficult to position hybridization sites on early pachytene SCs because they do not possess cytologically discernible centromeres/kinetochores at the light microscopic level (Stack and Anderson 1986). This problem can be overcome for chromosomes with a characterized CPD band(s) because the band can be used to position the centromere. An example of centromere placement in relation to a CPD band is shown in Figure 2, J–M. The SC 11 in these frames is 15.0 μm in length. The distance from the end of the short arm (i.e., the arm lacking a λTG523 FISH hybridization signal) to the CPD band is 5.8 μm. From Table 1, the relative distance between the CPD band and the kinetochore of SC 11 is 11.2% of the length of the short arm. Thus the distance from the terminus of the short arm to the kinetochore, i.e., the length of the short arm, is [5.8 μm ÷ (1.0 –0.112) =] 6.5 μm, and the long arm is (15.0 μm –6.5 μm =) 8.5 μm in length. Consequently, in Figure 2M the centromere (purple band) has been placed 6.5 μm from the end of the short arm. Once the centromere has been positioned, the locations of FISH hybridization signals can be expressed as fractions of the length of the chromosome arm to the centromere. For example, the two λTG523 FITC foci near the terminus of the long arm of SC 11 (Figure 2, K and M) are 7.8 and 7.9 μm from the centromere, respectively. Because the length of the long arm is 8.5 μm (see above), the two foci lie at points [100 × (7.8 μm ÷ 8.5 μm) =] 92.0% and [100 × (7.9 μm ÷ 8.5 μm) =] 93.0% of the long arm from the centromere.
Calculation of base pairs between different molecular and cytological markers
Map distances between markers determined by integrating the data from the recombination nodule map of tomato with the SC-FISH data
The potential of single-copy FISH to SCs: In this study we demonstrated that single-copy sequences can be reliably detected on SC spreads, discovered a discrepancy between our SC-FISH map and the molecular genetic map, estimated the distance between molecular markers in base pairs, and integrated the SC-FISH map with the preexisting RN map for tomato. Additionally, our data provide further evidence that there are few repeated sequences in tomato euchromatin. In broader terms, SC-FISH mapping has considerable potential as a means of relating genes to chromosome morphology (especially when coupled with differential chromatin-staining techniques such as CPD staining), studying the relationship between chromatin organization and gene expression, relating linkage distances to chromosome structure, and locating sites of transgene insertions. Because SC spreads have been prepared for many plants, animals, and a few fungi, it is probable that SC-FISH mapping can be used to investigate the genomes of a wide variety of organisms.
This research was funded in part by the United States Department of Agriculture grant 95-37300-1570 to S.M.S. and N.L.V. and Colorado Agricultural Experiment Station grant 1878-670 to S.M.S.
Communicating editor: W. F. Sheridan
Accepted February 1, 1999.
Copyright © 1999 by the Genetics Society of America
Albini S. M.,
Schwarzacher T.
, 1992 In situ localization of two repetitive DNA sequences to surface-spread pachytene chromosomes of rye. Genome 35: 551–559.
Anamthawat-Jónsson K.,
Heslop-Harrison J. S.,
, 1996 Genomic in situ hybridization for whole chromosome and genome analysis, pp. 1–23 in In Situ Hybridization, edited by Clark M.. Chapman and Hall, New York.
Anderson L. K.,
Offenberg H. H.,
Verkuijlen W. M. H. C.,
Heyting C.
, 1997 RecA-like proteins are components of early meiotic nodules in lily. Proc. Natl. Acad. Sci. USA 94: 6868–6873.
Carpenter A. T. C.
1975 Electron microscopy of meiosis in Drosophila melanogaster females. II. The recombination nodule—a recombination-associated structure at pachytene? Proc. Natl. Acad. Sci. USA 72: 3186–3189.
Curtis C. A.,
Lukaszewski A. J.
, 1991 Genetic linkage between C-bands and storage protein genes in chromosome 1B of tetraploid wheat. Theor. Appl. Genet. 81: 245–252.
Dooner H. K.
1986 Genetic fine structure of the Bronze locus in maize. Genetics 113: 1021–1036.
Dubochet J.,
Ducommun M.,
Zollinger M.,
Kellenberger E.
, 1971 A new preparation method for dark-field electron microscopy of biomacromolecules. J. Ultrastructure Res. 35: 147–167.
Flavell R.,
Smith D.,
Thompson W.
, 1985 The distribution of recombination sites, the structure of ribosomal DNA loci and the multiplicity of sequences containing inverted repeats, pp. 1–14 in Molecular Form and Function of the Plant Genome, edited by van Vloten-Doting L., Groot G., Hall T. C.. Plenum Press, New York.
Fransz P. F.,
Alonso-Blanco C.,
Liharska T. B.,
Peeters A. J. M.,
Zabel P.,
. 1996 High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres. Plant J. 9: 421–430.
Gómez M. I.,
Islam-Faridi M. N.,
Woo S.-S.,
Shertz K. F.,
Czeschin D. Jr..,
. 1997 FISH of a maize sh2-selected BAC to chromosomes of Sorghum bicolor. Genome 40: 475–478.
Hanson R. E.,
Zwick M. S.,
Choi S.,
McKnight T.,
. 1995 Fluorescent in situ hybridization of a bacterial artificial chromosome. Genome 38: 646–651.
Hasenkampf C. A.
, 1991 In situ hybridization of Lilium whole mount synaptonemal complex chromosomal preparations. Biotech. Histochem. 1: 210–215.
Heng H. H. Q.,
Squire J.,
Tsui L.-C.
, 1992 High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc. Natl. Acad. Sci. USA 89: 9509–9513.
Tsui L.-C.,
Moens P. B.
, 1994 Organization of heterologous DNA inserts on the mouse meiotic chromosome core. Chromosoma 103: 401–407.
Herickhoff L.,
Stack S.,
Sherman J.
, 1993 The relationship between synapsis, recombination nodules and chiasmata in tomato translocation heterozygotes. Heredity 71: 373–385.
, 1996 Flow cytometry and chromosome sorting, pp. 85–106 in Plant Chromosomes— Laboratory Methods, edited by Fukui K., Nakayama S.. CRC Press, Boca Raton, FL.
Ingle J.,
Timmis J. N.,
Sinclair J.
, 1975 The relationship between satellite deoxyribonucleic acid, ribosomal ribonucleic acid gene redundancy, and genome size in plants. Plant Physiol. 55: 496–501.
Ji Y.,
Raska D. A.,
McKnight T. D.,
Crane C. F.,
. 1997 Use of meiotic FISH for identification of a new monosome in Gossypium hirsutum L. Genome 40: 34–40.
Jiang J.,
Gill B. S.,
Wang G.-L.,
Ronald P. C.,
Ward D. C.
, 1995 Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc. Natl. Acad. Sci. USA 92: 4487–4491.
Hulbert S. H.,
, 1996 Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes. Mol. Gen. Genet. 252: 497–502.
Kapuscinski J.
, 1995 DAPI: a DNA-specific fluorescent probe. Biotechnic Histochem. 70: 220–233.
Khush G. S.,
Rick C. M.
, 1968 Cytogenetic analysis of the tomato genome by means of induced deficiencies. Chromosoma 23: 452–484.
Kota R. S.,
Gill K. S.,
Gill B. S.
, 1993 A cytogenetically based physical map of chromosome 1B in common wheat. Genome 36: 467–475.
Landegent J. E.,
Jansen In De Wal N.,
Dirks R. W.,
Bass F.,
Van Der Ploeg M.
, 1987 Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366–370.
Lawrence J. B.,
Villnave C. A.,
Singer R. H.
, 1988 Sensitive, high resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52: 51–61.
Lehfer H.,
Busch W.,
Martin R.,
Herrmann R. G.
, 1993 Localization of the B-hordein locus on barley chromosomes using fluorescence in situ hybridization. Chromosoma 102: 428–432.
Leitch I. J.,
Heslop-Harrison J. S.
, 1993 Physical mapping of four sites of 5S rDNA sequences and one site of the α-amylase-2 gene in barley (Hordeum vulgare). Genome 36: 517–523.
Lichter P.,
Cremer T.,
Borden J.,
Manuelidis L.,
, 1988 Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224–234.
Chang C. J.,
Call K.,
Hermanson G.,
Evans G. A.,
. 1990 High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247: 64–69.
Boyle A. L.,
, 1991 Analysis of genes and chromosomes by nonisotopic in situ hybridization. Genet. Anal. Tech. Appl. 8: 24–35.
Lohe A. R.,
Hilliker A. J.
, 1995 Return of the H-word (heterochromatin). Curr. Opin. Genet. Dev. 5: 746–755.
Macgregor H. C.,
Kezer J.
, 1971 The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus. Chromosoma 33: 167–182.
Moens P. B.,
Pearlman R. E.
, 1989 Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids. Chromosoma 98: 287–294.
, 1990 In situ DNA sequence mapping with surface-spread mouse pachytene chromosomes. Cytogenet. Cell Genet. 53: 219–220.
Moore C.,
Sherman F.
, 1974 Lack of correspondence between genetic and physical distances in the iso-1-cytochrome c gene of yeast, pp. 295–306 in Mechanisms In Recombination, edited by Grell R.. Plenum Press, New York.
Moses M.
, 1968 Synaptinemal complex. Annu. Rev. Genet. 2: 363–412.
Paterson A. H.
, 1996 Making genetic maps, pp. 23–39 in Genome Mapping in Plants, edited by Paterson A. H.. Academic Press, San Diego.
Pedersen C.,
Linde-Laursen I.
, 1995 The relationship between physical and genetic distances at the Hor1 and Hor2 loci of barley estimated by two-colour fluorescent in situ hybridization. Theor. Appl. Genet. 91: 941–946.
Giese H.,
, 1995 Towards an integration of the physical and the genetic chromosome maps of barley by in situ hybridization. Hereditas 123: 77–88.
Peterson D. G.,
Price H. J.,
Johnston J. S.,
Stack S. M.
, 1996 DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes. Genome 39: 77–82.
Pearson W. R.,
, 1998 Characterization of the tomato (Lycopersicon esculentum) genome using in vitro and in situ DNA reassociation. Genome 41: 346–356.
Ramanna M. S.,
Prakken R.
, 1967 Structure of and homology between pachytene and somatic metaphase chromosomes of tomato. Genetica 38: 115–133.
Rashtchian A.,
Mackey J.
, 1992 Efficient synthesis of biotinylated DNA probes using polymerase chain reaction. Focus 14: 64–65.
Rayburn A. L.,
, 1985 Use of biotin-labeled probes to map specific DNA sequences on wheat chromosomes. J. Hered. 76: 78–81.
, 1991 Tomato paste: A concentrated review of genetic highlights from the beginnings to the advent of molecular genetics. Genetics 128: 1–5.
Sambrook J.,
Fritsch E. F.,
Maniatis T.
, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Schumacher K.,
Ganal M.,
Theres K.
, 1995 Genetic and physical mapping of the lateral suppressor (ls) locus in tomato. Mol. Gen. Genet. 246: 761–766.
Segal G.,
Sarfatti M.,
Schaffer M. A.,
Ori N.,
Zamir D.,
. 1992 Correlation of genetic and physical structure in the region surrounding the I2 Fusarium oxysporum resistance locus in tomato. Mol. Gen. Genet. 231: 179–185.
Sharma A. K.,
Sharma A.
, 1980 Chromosome Techniques—Theory and Practice. Butterworth & Company, London.
Shen D.,
Wang Z.,
Wu M.
, 1987 Gene mapping on maize pachytene chromosomes by in situ hybridization. Chromosoma 95: 311–314.
Sherman J. D.,
, 1992 Two-dimensional spreads of synaptonemal complexes from solanaceous plants. V. Tomato (Lycopersicon esculentum) karyotype and idiogram. Genome 35: 354–359.
, 1995 Two-dimensional spreads of synaptonemal complexes from solanaceous plants. VI. High-resolution recombination nodule map for tomato (Lycopersicon esculentum). Genetics 141: 683–708.
Snape J. W.,
Flavell R. B.,
O'Dell M.,
Hughes W. G.,
Payne P. I.
, 1985 Intrachromosomal mapping of the nucleolar organizer region relative to three marker loci on chromosome 1B of wheat (Triticum aestivum). Theor. Appl. Genet. 69: 263–270.
Solari A. J.,
Dresser M. E.
, 1995 High-resolution cytological localization of the XhoI and EcoRI repeat sequence in the pachytene ZW bivalent of the chicken. Chromosome Res. 3: 87–93.
, 1984 Heterochromatin, the synaptonemal complex, and crossing over. J. Cell Sci. 71: 159–176.
Stack S. M.,
Anderson L. K.
, 1986 Two-dimensional spreads of synaptonemal complexes from solanaceous plants. II. Synapsis in Lycopersicon esculentum (tomato). Am. J. Bot. 73: 264–281.
Herickhoff L. S.
, 1993 Meiotic nodules in vascular plants, pp. 301–312 in Chromosomes Today, edited by Sumner A. T., Chandley A. C.. Chapman and Hall, New York.
Sturtevant A.,
Beadle G.
, 1939 An Introduction to Genetics. W. B. Saunders, Philadelphia.
Sumner A. T.
, 1990 Chromosome Banding. Unwin Hyman, London.
Tamarin R. H.
, 1999 Principles of Genetics. McGraw-Hill, Boston.
Tanksley S. D.,
Young N. D.,
Paterson A. H.,
Bonierbale M. W.
, 1989 RFLP mapping in plant breeding: new tools for an old science. BioTechnology 7: 257–264.
Ganal M. W.,
Prince M. C.,
De Vicente M. C.,
Bonierbale M. W.,
., 1992 High density molecular linkage maps of the tomato and potato genomes. Genetics 132: 1141–1160.
Trask B. J.
, 1991 Fluorescence in situ hybridization. Trends Genet. 7: 149–154.
Trask B. J.,
Massa H.,
Kenwrick S.,
Gitschier J.
, 1991 Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei. Am. J. Hum. Genet. 48: 1–15.
Tsujimoto H.,
Noda K.
, 1990 Deletion mapping by gametocidal genes in common wheat: position of speltoid suppression (Q) and beta-amylase (beta-Amy-A2) genes on chromosome 5A. Genome 33: 850–853.
Wallrath L. L.,
Elgin S. C. R.
, 1995 Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9: 1263–1277.
Weith A.,
Traut W.
, 1980 Synaptonemal complexes with associated chromatin in a moth, Ephestia kuehniella Z. Chromosoma 78: 275–291.
Wing R. A.,
Zhang H.-B.,
Tanksley S. D.
, 1994 Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless. Mol. Gen. Genet. 242: 681–688.
Yasmineh W. G.,
Yunis J. J.
, 1971 Satellite DNA in calf heterochromatin. Exp. Cell Res. 64: 41–48.
Zuckerkandl E.,
Hennig W.
, 1995 Tracking heterochromatin. Chromosoma 104: 75–83.
Stelly D. M.,
. 1997 A rapid procedure for the isolation of Cot-l DNA from plants. Genome 40: 138–142.
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Biosciences for Farming in Africa (B4FA) was set up to help African farmers unlock the continent's huge agricultural potential. Initially we provided training to African journalists so they could communicate balanced, scientifically based information on best practice, innovation and entrepreneurship to decision makers, scientists, educators and farmers, alike.
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Committed to the
Our CompanyCommunity investmentProudly supporting our armed forcesCommitted to the Armed Forces Covenant
We have a proud history of supporting our armed forces – it lies at the very heart of what we do. In 2013, we were the first Defence Company to sign up to the UK Armed Forces Covenant, cementing our commitment to supporting those who serve, have served and their families.
A pledge that together we acknowledge and understand that those who serve or who have served in the armed forces, and their families, should be treated with fairness and respect in the communities, economy and society they serve with their lives.
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We were one of the first six winners of the MoD Employer Recognition Scheme
Gold Award, by which the Defence Relationship Management team recognise an organisation's commitment to the Armed Forces Covenant.
We have held this status continuously since the Scheme began in July 2014.
In 2015, we re-signed the Covenant with an increased number of commitments.
Promote BAE Systems as Armed Forces friendly
Recognise the skills and experience of Service Leavers and Veterans
Promote and provide employment opportunities for Service spouses and partners
Enable Reservist employees to maintain training commitments
Commit to host Reserve recruiting teams at appropriate sites and venues
Adopt a sympathetic and flexible approach to requests for leave from Service spouses
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Regularly review progress against these commitments
Publicise these commitments and invite feedback from the Service community and customers
Here are some of the ways we are delivering on our commitments
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We work with the Careers Transition Partnership to promote our vacancies to ex-service people looking to find a new civilian career. As well as this, we partner with Recruit for Spouses and provide funding and mentoring to help bridge the gap between employers and military spouses.
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How Dreamcloud Winning
Looking to upgrade your old, worn-down mattress to a brand-new one without having to worry about the uber-expensive pricing? Ready to find your first hybrid bed that's remarkably valuable for your budget and packed with extra perks that you may not get anywhere else? Look no further because, in this review, we're going to take a deep dive into the original DreamCloud Hybrid Mattress, one that's already making a lot of noise in the luxury beds market!
Customers and critics have praised this model for its outstanding comfort, impressive pressure relief and long-lasting durability. All of this at a competitively low cost. But, as nice as it seems, how well does it hold up? Keep reading to check our findings from the series of tests that we ran on it, and stay til the end to find out if it's the right one for you!
The DreamCloud: A closer look How Dreamcloud Winning
The DreamCloud Hybrid has a profile of 14 inches. This hybrid design allows for more comfort and support. Its overall construction, which we'll discuss later, allows people with limited budgets the opportunity to upgrade to a better model than their old one.
Although we're only going to focus on the entry-level-type DreamCloud here, it's worth noting that both of those models are made of more expensive material, therefore elevating their comfort levels and price range. This is not meant to discourage you from getting the Original model, by the way, as we're also going to prove later on in this review that it can achieve close to the same luxurious feel as the other two mattresses at a more budget-friendly cost.
It may be unique enough to win hearts, but we can't ignore the criticisms it has faced. Some sleepers have expressed dissatisfaction with the DreamCloud Hybrid, claiming it was too soft and too firm. Some even shared their experience of suffering from back/neck/hip pain from sleeping on it alone.
The product can cause a variety of reactions, which may be mixed depending on how long you use it. How much a sleeper weighs, how old they are, which climate they live in, and what type of position they prefer whenever they rest are among the major factors that will contribute to what they ultimately feel about this product. With that in mind, we can't fully guarantee that the DreamCloud Hybrid may suit all sleep types. However, what we are sure of is that it may be an ideal choice for many who just want overall comfort in their sleep.
Who Might Not Like This Mattress?
If you are looking for a mattress, the first thing you should check is its firmness. How firm or how soft it would turn out to be may either make you want to buy one or turn you off from it entirely. The DreamCloud Hybrid Mattress's comfort level may be advertised to be at 6.5 out of 10 on the firmness scale, but how accurate is its claim? To answer this, we tested out its overall firmness and feel to see for ourselves if DreamCloud is true to its word with its flagship mattress. How Dreamcloud Winning
We sent in four volunteers – each from three different weight groups – to sleep on the DreamCloud Hybrid in different sleeping positions on a two-week venture. Two of them represent the lightweight (130 lbs below) and heavyweight (230 lbs above) sleepers, respectively, while the rest of the team represents the average group (130-230 lbs), although one weighs 155 lbs and the other is close to 225 lbs.
The data collected over the last 14 days suggests that the majority of people could agree that the mattress can accommodate a wide range of body weights and positions. However, not all. If we could rank the DreamCloud Hybrid, we'd give it a solid 7 out of 10 on the firmness scale – medium firm, for sure. It's plush enough to be comfortable, yet supportive enough to contour the body even in different positions. It's also worth noting that the mattress was comfortable for two of the three volunteers immediately upon contact. The other took them several days to adjust.
The DreamCloud Hybrid is a great choice for back sleepers of any weight. Based on what we know so far from our volunteers, no one seemed to have any issue with its firmness given that its construction is versatile enough to support the back well and keep the spine properly aligned. Lightweight sleepers may enjoy this one – not only should they feel supported enough, but its top layers should also give them the feeling of being able to lie on top as comfortably as possible.
Average-weighted individuals may like the DreamCloud Hybrid best as it should give them the perfect balance of relief and hold. The coils beneath the mattress should support their hips and sink into the mattress. Heavyweight sleepers should also feel the same to some extent, but those who weigh around 300lbs or more may not find it suitable since they may sink in too much to the point where pressure is produced. How Dreamcloud Winning
While the DreamCloud mattress may provide most back sleepers with exceptional contouring and spinal alignment, the same can apply to side sleepers, though there are a few notable exceptions. Reactions are mixed for our average-weighted sleepers – our 155-lb volunteer found the bed to be able to support and align their body quite well. Their 225-lb counterpart observed its top layers to be a bit soft under his hips but was able to experience a decent amount of support and comfort.
Heavy folks, particularly those who weigh nearly 300 lbs, may find the DreamCloud Hybrid to be too soft for their liking. Our representative from that group told us her hips were sinking into the mattress often whenever she laid on her stomach, and it reached the point where she found it a bit uncomfortable to sleep in this position. Although stomach sleepers with higher than average weights may have issues with the mattress, lighter people may be able to reap the benefits of this mattress better than others. Considering their build, their hips won't be able to sink in as much, so they should feel as comfortable on their stomachs as with their backs.
Among the three weight groups, those of average weight are more likely to gain an upper hand when it comes to side sleeping on the DreamCloud Hybrid. For folks who weigh between 130-190 lbs, they may receive an ample amount of contouring support, and the pressure along the hips and shoulders won't be as much so they should be good. According to our 155-lb volunteer, the experience was worthwhile as they were able to rest well regardless if they switch to either side or stick to one. It's different for those who are 190 or more.
Both our heavyweight and 225-lb reps have expressed that the mattress was a bit uncomfortable to sleep on sideways. Both experienced a lot of sinking in their hips, shoulders and hips. This can lead to pressure points that could cause minor concurrent pains. While heavier people may find the DreamCloud Hybrid soft, those less than 130 pounds may find it more firm to allow them to sleep well. Our lightweight volunteer can attest to this: when she tried to rest strictly on one side for a prolonged period, she felt as if the top layers were too firm at times, but when it came to sleeping with her sides interchanged, it turned out to be decently comfortable. Minor adjustments can be expected at this rate.
Examining its Pain Relief Capabilities
While our volunteers had a lot of fun testing the DreamCloud Hybrid's firmness, it was also possible to assess how effective it is in pain relief. To do this, we set up a pressure map on top of the mattress, and, once that was done, we asked the four volunteers to lay on the back, stomach, and both sides of their bodies to see if there was any significant pressure difference that we could spot right away.
We believe that the DreamCloud Hybrid can provide excellent pressure relief for most sleepers. Most people, except for those who are over 300 lbs, will find comfort and relaxation right away. The bed's layers, which include the memory foam, are sufficient to support their weight and contour their bodies very well. This results in low pressure throughout the entire body. The mattress creates very few pressure points, which means that back pains are less likely to occur. This is especially important for seniors and people who have back problems.
The DreamCloud Hybrid will produce some pressure points in the stomach area, especially along the hips. Lightweight and average sleepers may be able to rest soundly without the imminent fear of pent-up pressure, but heavyweight ones, especially those whose structure is adjacent to 300lbs, may need to watch out as too much sinking may lead to an increase in pressure points. How Dreamcloud Winning
We found significant pressure points for most body types when it came to side sleep. No matter how much you weigh, it's likely that laying sideways on the surface of the DreamCloud Hybrid may make you prone to varying degrees of discomfort, and it gets much worse if you're heavier. For instance, lighter sleepers may not find the mattress comfortable in this position, while heavier sleepers may feel more pain in their hips and shoulders. This is where the DreamCloud mattress can be a little bit of a problem.
What exactly is the DreamCloud Hybrid Mattress made of? The DreamCloud Hybrid Mattress measures 14 inches in height. It is filled with notable qualities that keep you cool and relaxed throughout the night. Like some of the top-performing hybrid models of today, it's equipped with outstanding cooling capabilities, warding off the body's heat to ensure better sleep.
Cashmere wool has been historically known for being soft to the touch and luxurious in its feel. It's for this reason that DreamCloud chose to incorporate this into the cover of its signature mattress model. The quilt's cloud-patterned design, which is splayed across the mattress, invites the user to lay down on the bed. The material is breathable and has excellent temperature-regulating characteristics to help cool the sleeper at night.
Since its inception, memory foam has been widely recognized to adapt and respond to the body's shape as fluidly as possible and provide much-needed relief from its pressure points. In the case of the DreamCloud Hybrid, it is placed conveniently underneath the quilted top layer. It measures one inch and is just right to support the sleeper, but it doesn't give off a sinking sensation. This material may be notorious for trapping heat, but DreamCloud fixes this by incorporating gel infusion, allowing their body temperature to be cooled down further along with the indented cover.
Speaking of coils, the DreamCloud Hybrid's very foundation comes in the form of a full system of individually wrapped innerspring coils. Contributing to the total height of the mattress at just 8 1/2 inches, each spring is enrobed with a breathable fiber – this way, it should help maximize the bed's cooling capabilities, letting more air in and more heat from the body out. The coils can be responsive to the sleeper's changing movements very well. However, the perimeters of the mattress are strategically strengthened with stronger steel to provide better edge support.
A thick layer of standard high-density foam is found beneath the coil set. Since it serves as part of the foundation that holds the mattress together, it comes as solid and sturdy enough to secure the springs from moving too much, yet also thin enough to allow How Dreamcloud Winning
them to bounce through. It won't feel as if it were there, but you will be able to appreciate the fact that it is there to prevent it from falling apart.
What was the DreamCloud's Performance?
A perfect mattress should be able to adapt to the sleeper's needs. Apart from accommodating their body type and preferred sleep position, other pressing factors should be taken into consideration – this includes a plethora of activities, habits, and even the climate that they are usually accustomed to. Within our two-week testing period, we got to run a series of performance tests to see if the DreamCloud Hybrid has what it takes as an integral staple in the bedroom.
Considering what it's made of and how it functions, the DreamCloud mattress may not fully absorb a person's motions, but it does a decent job, regardless. When we had two of our volunteers test it out for motion transfer, we first noticed that one of them wasn't able to feel another's changing movements as much until they got more intense. It caused little to no disturbance with small movements.
The DreamCloud Hybrid's motion isolation capabilities make it comparable to luxury hybrid beds. Average persons may willingly look past it, but as for any light sleeper with an often restless partner beside them, this model may not be an ideal choice.
DreamCloud Hybrid claims to offer great edge support thanks to reinforced steel coils placed around the edges and perimeter. But how accurate is that claim? We tested it by having volunteers lie on the edges of the bed and we found that the claim was mostly accurate. A mattress with exceptional edge support may allow sleepers to take advantage of the surface without fear of falling or sinking.
The DreamCloud Hybrid is an exception – within our two-week testing period, not a single one of our crew noticed any annoying squeaking coming from within the bed. The way it was constructed or the method of wrapping up each oil may be a contributing factor, but regardless, with this model, noise shouldn't be a huge worry for many. How Dreamcloud Winning
Mobility made easy
Our team discovered that the bed was easy to move around. It would be fine if you were to perform motion-intensive activities on it.
Speaking of motion-intensive, we see a promising amount of potential in the DreamCloud Hybrid as an ideal bed for sexual activities. Folks who engage in sex once in a while may surely enjoy its ability to adapt to their movements swiftly. It makes a noticeable difference in noise, so it is possible to have discreet sex.
Being a medium-firm bed, partners who prefer to do one or three positions may not find enough hugging along the surface to keep them at least secure. The DreamCloud mattress is one of the best beds for sex. How Dreamcloud Winning
The length of this durability will depend on how frequently the user uses it and if there are any additional people who sleep on it. Reflecting on our two-week testing period, we can all agree that the DreamCloud mattress stayed durable enough to not have its ability to provide comfort compromised. That being said, we believe that it should stay this way for at least seven to eight years. How Dreamcloud Winning
The DreamCloud Hybrid Mattress comes packed neatly and compressed in a box. The size of the mattress will determine how heavy it is. The twin is the lightest and Cal King and King are the most heavy. We noticed that many of our team members found it too heavy to transport alone when we opened the Queen-sized version for our tests. So, we added an additional person to ease the burden.
Apart from the mattress, other contents inside the box include a handy booklet for instructions and a free pocket knife that you may use to open it up from the plastic packaging – that is, especially if you don't have one of your own. After you have rolled it out of the box onto your bed frame, it will be fully compressed. You should wait between 24-48 hours. Since you're going to encounter a fresh new mattress in front of you, expect it to emit a slightly off-putting odor.
Models made of synthetic foam layers tend to emit volatile organic compounds (VOCs) collected from manufacturing. Since the DreamCloud Hybrid's foam layers are all CertiPUR-US-certified, the amount should be much less. It is important to let it sit out so that the smell can dissipate after a few days. To speed this process up, we suggest that you do so in a well-ventilated environment by opening up all the windows and doors in the room.
Stellar accommodations aside, one thing that the DreamCloud Hybrid Mattress arguably shines lies in how it's priced. Factoring in the kind of materials used in its inception and the remarkable quality of its framework and foundation, it's no wonder that this model is sold at a range of competitive yet affordable prices.
You may also save money if you order from DreamCloud's official site. You're also rewarded with a free set of bedding, including pillowcases, sheets, and a mattress protector, to help make the set-up process simpler.
DreamCloud Hybrids are currently available in six sizes with prices starting at $799. Check out this table below for additional details inclusive of pricing, dimensions, and weight.
King 76″ W x 80″ L x 14″ H 120.30 LBS $1,499
Want to consider some options? We can help! The DreamCloud Hybrid Mattress may be one of the hottest luxury hybrid models in the market, but there are two other competing alternatives out there that deliver the same promise of comfort and are packed with unique aspects that it may not have. Let's take a look at each of them! How Dreamcloud Winning
The Differences: Saatva is a bit more expensive than the DreamCloud Hybrid thanks partly to its construction – its coil system is more intricate, and the organic cotton-covered Euro pillowtop makes up for a more snug feeling. The former, which has one firmness option, can be adjusted to suit different body types.
The DreamCloud Hybrid's luxuriously crafted medium-firm surface may be inviting for anyone, but not exactly for everyone. We found that the bed does not have enough to hold well for those who weigh as heavy as 300 lbs, more or less. Regardless, it's clear that it has what it takes as a welcoming staple for solo sleepers, couples, and even families!
Additionally, you have much less to lose if you decide to purchase it. You'll be given plenty of exclusive discount rates from DreamCloud's official website, plus exceptionally responsive customer service teams to assist you along the way. For this and all the other good reasons regarding the DreamCloud Hybrid, there's no doubt that we're giving it a resounding 4.5/5 stars. For what it is, it's surely worth the try… and the purchase! How Dreamcloud Winning
FAQ How Dreamcloud Winning
What is DreamCloud's warranty period? And how does it work?
What happens if I decide to return the DreamCloud Hybrid Mattress during the sleep trial period? And does it apply after it ends?
All of DreamCloud's available mattress models are eligible for returns within the 365-night sleep trial. If you are unhappy with your purchase and wish to return it within the 365-night period, the Sleep Concierge team will assist you in arranging for a replacement or refund. You may also opt to exchange it with a replacement model once you receive your cash if you like. How Dreamcloud Winning
Unfortunately, returns after the trial period end are not possible. You wouldn't be eligible for a refund, either.
Where does the DreamCloud Hybrid usually ship to?
DreamCloud doesn't ship products to the United States from its website. This includes Puerto Rico, Guam, and other countries. It does have a Amazon.com certified storefront that allows you to ship any product internationally. How Dreamcloud Winning


text:
Q: How to ignore null/undefined value when mapping on an array? How can I implement this?
I want to map through the plans array and for each item render a list. while This is working, in case if we have null or undefined on a title, we still have 4 Li tag renders which the second one doesn't have any content. but how can I implement this if the title is null/undefined the object get ignored and move to next object and render the rest of the array.
Hope my explanation is clear.
import React from "react";

const App = (props) => {

  const plans = [
    {
      title: "Starter",
      plan: "plan_0",
    },
    { title: undefined,
      plan: "plan_1A",
    },
    {
      title: "Classic",
      plan: "plan_2A",
    },
    {
      title: "Premium",
      plan: "plan_3A",
    },
  ];

  return (

Notes

        {plans.map((plan, i) =>

              {plan.title}

        )}

  );
};

export default App;

at the moment its render like this :

*Starter

*

*Classic

*Premium

Starter

Classic

Premium

A: Just adding a conditionnal statement should do the trick :
    {plans.map((plan, i) =>
        { plan.title &&

                {plan.title}

        }
    )}

A: check to see that plan has a property of title before rendering a list item :)

    {plans.map(
      (plan, i) =>
        plan.title && (

            {plan.title}

        )
    )}

redpajama_set_name: RedPajamaStackExchange


text:
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Asia Pacific Defence Reporter : APDR Feb 2017
LOCKHEED AD TO COME and test requirements. In addition to the weapon adapters, Ferra is also producing and supplying more than 100 other component parts for the F-35 program, being involved in machining of airframe components and the avionics chassis. NORTHROP GRUMMAN U.S . aerospace giant Northrop Grumman is a principal member of the Lockheed Martin-led industry team, which is developing and producing three variants. In the U.S. the company is involved in the development, demonstration and production of the F-35 in a number of ways, including principally the production of the centre fuselage. It is also responsible for the design, development and production of the F-35's AN/APG-81 Active Electronically Scanned Array (AESA) fire control radar, AN /AAQ-37 Distributed Aperture System (DAS), which provides simultaneous modes of situational awareness, infrared search and track, missile warning and navigation not available on any other aircraft, and the AN/ASQ-242 Communications, Navigation and Identification (CNI) avionics suite, which can be dynamically programmed to arm the F-35 pilot with multiple- mission capabilities engineered for seamless transition from one mission phase to the next. Closer to home, Northrop Grumman Australia, a fully owned Australian subsidiary of Northrop Grumman Corporation, has been assigned a significant amount of the MRO&U work for the F-35 program to the Australian defence industry by the JPO. Together with team members, BAE Systems Australia, Rockwell Collins Australia, GE Aviation Systems Australia and Quickstep, Northrop-Grumman will provide the capabilities that underpin six of the 10 MRO&U Repair Technology Categories (RTC). "The assignment of this work to Australia is an endorsement of the health and growth of the Australian defence industry, and an acknowledgement of Northrop Grumman Australia's continued commitment to growing its technical sustainment workforce and capabilities in country," said Ian Irving, chief executive, Northrop Grumman Australia in a press release from November 2016. "We are grateful to our industry teammates, the Australian Joint Strike Fighter Division, the Commonwealth government, and the U.S . JPO for this recognition." THE FUTURE Australian industry participation in the F-35 program has been a success story so far, and the future is expected to continue looking bright with the number of aircraft operating in the region expected to rise. In addition to Australia, both Japan and South Korea have committed to initial purchases of the F-35 and are expected to order more in the future to replace legacy fighter fleets. Closer to Australia, Singapore is also widely tipped to be a future customer, with its Defence Ministry having evaluated the type in detail although it has yet to make a firm order as it has decided that it will not need F-35s till at least 2030. Meanwhile, Canada's vacillation in making its own decision to acquire the F-35, with its current government recently opting to 'kick the can down the road' by acquiring Super Hornets as an interim capability could jeopardise future Canadian industry participation in the program with Australian industry standing ready to plug any gaps. Taken together, these will place the Australian companies involved in regional and global sustainment of F-35 fleets in an advantageous position to secure further contracts down the road. F-35 APDR-FEB-2017.indd 25 30/01/2017 6:39 PM © 2017 LOCKHKHHEED EED EED EED EED MART MAMAMAMARTIN C IN ORPO ORP RATI RATITIATIATI RAT ONONONONNN FOR SECURITY. FOR INDUSTRY. AT LOCKHEED MARTR INN, WEWE'R 'REE EN ENGIGINENEERRINGG AA BEBETTTTERER TTOMOMORORROROW.W . CNY_FG16-16381H_APD 2017-01-25T14:36:42+11:00 25 KHL.indd 1 31/1/17 10:55 pm
APDR Dec16 Jan17
APDR March 2017


at the moment I haven't got the expansion it maybe that I won't be able to get it for a while.
Just so that everyone knows I( can only play on the old maps for now, I'll update this if I get the expansion.
If anyone wants to give me some training send me a message with a time that you'll be able to do it.


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Yemeni-Americans to boycott NY Post over 9/11 front page
NEW YORK — Yemeni-Americans are criticizing the New York Post over its front page last week that featured a photograph of the burning World Trade Center and a quote from Democratic Muslim Rep. Ilhan Omar that some say was dismissive of the Sept. 11 terrorist attacks.
On Sunday, The New York Times reports the Yemeni American Merchant Association announced a formal boycott of the tabloid after Yemini bodega owners in New York had earlier agreed stop selling the newspaper.
Last week's front page featured a quote that came from a recent speech the Minnesota Democrat gave to the Council on American-Islamic Relations, in which she described the 2001 terrorist attack on the World Trade Center as "some people did something."
"We support free speech, but we will not accept the incitement of violence against Muslims," said Debbi Almontaser, the secretary of the board of directors for the merchants.
"What the New York Post is doing is endangering the lives of American Muslims and people of color," Almontaser said.
The merchants are demanding that the newspaper apologize to Omar and to the Muslim-American community in New York.
A spokeswoman for News Corporation, the parent company of the New York Post, declined to comment.
Also on Sunday, House Speaker Nancy Pelosi said she has taken steps to ensure the safety of Omar after President Donald Trump's retweet of a video that purports to show the congresswoman making the remark along with news footing of the hijacked airplanes hitting the Twin Towers. Trump captioned his tweet with: "WE WILL NEVER FORGET!"
Neither Trump's tweet nor the video included Omar's full quote or the context of her comments, which were about Muslims feeling that their civil liberties had eroded after the attacks.
Pelosi was among Democrats who had criticized Trump over the tweet, with some accusing him of trying to incite violence against the Muslim lawmaker. An upstate New York man recently was charged with making death threats against her.
Information from: The New York Times, http://www.nytimes.com
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