Localization of Single- and Low-Copy Sequences on Tomato … · 1999. 4. 22. · Single-Copy FISH...

Copyright 1999 by the Genetics Society of America

Localization of Single- and Low-Copy Sequences on Tomato SynaptonemalComplex Spreads Using Fluorescence in Situ Hybridization (FISH)

Daniel G. Peterson,* Nora L. V. Lapitan† and Stephen M. Stack‡

*Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia, 30602 and †Department of Soil and Crop Sciencesand ‡Department of Biology, Colorado State University, Fort Collins, Colorado 80523

Manuscript received November 13, 1998Accepted for publication February 1, 1999

ABSTRACTFluorescence 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 arenormally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromo-somes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere withprobe 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 higherresolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreadsof tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that areassociated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs wereidentified on the basis of relative length, arm ratio, and differential staining patterns after combinedpropidium iodide (PI) and 49,6-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 tomatoSC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the numberof base pairs, and the rates of crossing over between these three FISH markers. We also observed thatthe 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 pachytenechromosomes and thereby contributes to our understanding of the relationship between chromosomestructure, gene activity, and recombination.

FOR most plant species, what is known about the Stack 1995). The most direct means of determiningorder of loci on chromosomes is based almost en- the location of genes on chromosomes is through the

tirely on genetic linkage maps. Such linkage maps are use of fluorescence in situ hybridization (FISH). In thisgenerated by producing multi-hybrid crosses and de- technique hapten-labeled DNA probes are hybridizedtermining the relative frequency of recombination be- to chromosomes that have been spread on glass micro-tween genes or molecular markers (Tanksley et al. scope slides, and antibodies or other affinity reagents1989; Paterson 1996). The physical location of genes conjugated to fluorochromes are used to detect directlyon chromosomes is of great interest because chromo- or indirectly sites of hybridization (Trask 1991). FISHsome structure profoundly influences gene activity (see with repetitive sequences as probes has been widely re-Lohe and Hilliker 1995; Wallrath and Elgin 1995; ported for both animal and plant chromosomes, andZuckerkandl and Hennig 1995 for reviews). However, single-copy FISH to mammalian chromosomes is fairlylinkage maps cannot be superimposed on chromosomes routine (e.g., Landegent et al. 1987; Lawrence et al.because map distances are not proportional to physical 1988; Lichter et al. 1991; Heng et al. 1992). Becausedistances (Sturtevant and Beadle 1939; Khush and most techniques used to prepare plant chromosomesRick 1968; Moore and Sherman 1974; Flavell et al. leave overlying debris that interferes with probe penetra-1985). The relative scarcity of genes and crossing over tion, single-copy FISH to plant chromosomes has provenin heterochromatin is partly responsible for this discrep- considerably more difficult (Lehfer et al. 1993; Jiangancy, but crossing over is not evenly distributed in et al. 1995). However, now there are a few laboratorieseuchromatin either (Snape et al. 1985; Dooner 1986; that have overcome this obstacle and are successfullyTsujimoto and Noda 1990; Curtis and Lukaszewski localizing single-copy sequences on plant chromosomes1991; Kota et al. 1993; Stack et al. 1993; Sherman and by FISH (e.g., Leitch and Heslop-Harrison 1993;

Hanson et al. 1995; Jiang et al. 1995; Gomez et al. 1997).FISH is usually performed on mitotic metaphase chro-

Corresponding author: Daniel G. Peterson, Plant Genome Mapping mosomes, but there are reasons to believe that pachy-Laboratory, University of Georgia, Riverbend Research Center, Rm. tene (meiotic) chromosomes may be better substrates.162, 110 Riverbend Rd., Athens, GA 30602.E-mail: [email protected] Each pachytene chromosome (bivalent) is composed of

Genetics 152: 427–439 ( May 1999)

428 D. G. Peterson, N. L. V. Lapitan and S. M. Stack

and TG523 are single-copy markers, Southern blots of TG400two homologous chromosomes that are joined alongindicate that it may be present in more than one copy pertheir entire length by a proteinaceous scaffold calledhaploid genome (S. D. Tanksley, personal communication;

the synaptonemal complex (SC; Moses 1968). Because also see SolGenes web site, http://geneous.cit.cornell.edu/each homologue contains two chromatids, there are solgenes/aboutsolgenes.html/). Transformation of the com-

petent E. coli strain DH5a, plasmid isolations, and verificationfour closely associated copies of each locus available forof RFLP marker size by restriction analysis were performedhybridization on a bivalent. In comparison, there areusing standard methods (Sambrook et al. 1989). The polymer-only two nearby copies of each locus available for FISHase chain reaction was used to concomitantly amplify and

on a metaphase chromosome. In spreads of pachytene label RFLP markers with digoxigenin (DIG; Rashtchian andchromosomes that have been prepared to reveal SCs Mackey 1992), and the resulting probes were used to screen

a Clonetech (Palo Alto, CA) tomato lambda (l) genomic(SC spreads), chromatin extends as a diffuse cloudlibrary (cat. no. FL 1082d). Plating and filter preparation werearound each SC. The loops of DNA extending from theperformed as described in the Clonetech Lambda LibrarySC (e.g., Weith and Traut 1980) appear to be moreProtocol Handbook (PT1010-1). For each plate, an original

accessible to FISH probes than the DNA of condensed and a duplicate filter were prepared. Saline sodium citratemetaphase chromosomes (Moens and Pearlman 1989, (SSC) solutions of various strengths were produced by diluting

a 203 SSC stock solution (3 m NaCl, 0.5 m sodium citrate, pH1990; Heng et al. 1994; Solari and Dresser 1995), and7.0) with deionized water. Filter hybridization and detectionSC spreads can be prepared relatively free of overlyingof positive clones were performed according to the Boehringerdebris. Additionally, pachytene chromosomes are 5–15 Mannheim (Indianapolis) DIG/Genius User’s Manual

times longer than corresponding metaphase chromo- (see http://biochem.boehringer-mannheim.com/prod_inf/somes (Ramanna and Prakken 1967; Stack 1984), so manuals/dig_man/dig_toc.htm/) with modifications as sug-

gested by A. S. N. Reddy and I. S. Day (personal communica-many closely associated loci that are not resolvable bytion). Briefly, nylon filters (Boehringer Mannheim) wereFISH on metaphase chromosomes should be resolvableplaced in standard hybridization buffer [53 SSC, 0.1% N-on pachytene chromosomes. lauroylsarcosine, 0.02% sodium dodecyl sulfate (SDS), 1.0%

Here we report high resolution localization of two w/v Boehringer Mannheim blocking reagent, 0.01 m maleicacid, 0.015 m NaCl, pH 7.5] for 1 hr at 558, incubated overnightsingle-copy sequences and one low-copy sequence onat 558 with gentle shaking in standard hybridization buffertomato SC 11 using FISH. This is the first report ofcontaining 0.01 mg/ml heat-denatured DIG-labeled probe,single-copy FISH to SC spreads and one of only a fewwashed twice in 23 SSC containing 0.1% w/v SDS (558, 5 min

studies in which FISH has been used to study the rela- each wash), washed twice in 0.53 SSC containing 0.1% SDStionship between genetic linkage and chromosome (558, 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),morphology in plants (e.g., Pedersen and Linde-placed in blocking solution (0.1 m maleic acid, 0.15 m NaCl,Laursen 1995; Pedersen et al. 1995). Tomato (Lycopersi-1% w/v blocking reagent) for 1 hr at 208 with gentle shaking,con esculentum Mill., recently renamed Solanum lycopersi- and incubated in a 1:2500 dilution of anti-DIG-alkaline phos-

cum L.) was used for this study because all 12 of its SCs phatase (750 units/ml, Boehringer Mannheim) in blockingare identifiable on the basis of relative lengths and arm buffer for 45 min (208 with gentle shaking). After incubation

with the primary antibody, filters were washed twice in 500ratios (Sherman and Stack 1992, 1995) and becauseml washing buffer (15 min each wash), placed in detectiontomato is a true diploid of agronomic importance (Rickbuffer [100 mm Tris-(hydroxymethyl)-aminomethane, 100 mm1991). Tomato SC 11 was chosen for hybridization be- NaCl, pH 9.5] for 2 min, and incubated in the dark without

cause it is one of the shortest tomato chromosomes, agitation in 200 ml of detection buffer containing 900 ml ofand its corresponding linkage group contains 16 sepa- nitrobluetetrazolium solution (75 mg/ml in 70% dimethyl-

formamide, Boehringer Mannheim) and 700 ml of 5-bromo-rate RFLP loci, several of which are associated with4-chloro-3-indoyl phosphate toludinium salt solution (50 mg/known genes (Tanksley et al. 1992). Combined propid-ml in 100% dimethylformamide, Boehringer Mannheim).

ium iodide and 49,6-d iamidino-2-phenylindole (CPD) After z20 min, positive plaques (small blue “o-shaped” rings)staining was employed to facilitate chromosome identi- began to appear on filters. Filters with a positive plaque in

the same location on both the original and duplicate filtersfication and aid in relating hybridization sites to chro-were thoroughly washed in deionized water, and correspond-mosome structures. Our results suggest that FISH to SCing plaques were removed from petri plates as described byspreads (SC-FISH) can be used to construct comprehen-Sambrook et al. (1989). Secondary and tertiary screens were

sive maps of single-copy sequences on pachytene chro- performed as described above. For each of the three startingmosomes. RFLP markers (TG46, TG400, and TG523), one correspond-

ing lambda clone (designated lTG46, lTG400, and lTG523,respectively) was selected for use in FISH.

Probe preparation: The QIAGEN (Valencia, CA) LambdaMATERIALS AND METHODSMaxi Kit was used to isolate DNA from lTG46, lTG400, and

Screening the lambda tomato genomic library: Plasmids con- lTG523. The DNA was digested with BamHI or EcoRI, andtaining the tomato restriction fragment length polymorphism tomato insert DNA fragments were separated from l arms by(RFLP) markers TG46, TG400, and TG523 were provided by gel electrophoresis. Bands containing tomato insert DNA wereS. D. Tanksley (Cornell University). TG523 flanks the jointless excised from 1% w/v agarose gels, and the QIAGEN Qiaexgene (Wing et al. 1994), and TG400 is linked to the hairless II Kit was used to isolate DNA from agarose. The GIBCO BRLgene (Tanksley et al. 1992). To date, TG46 has not been (Rockville, MD) BioNick Kit was used to label DNA with biotin.closely associated with a particular gene or phenotype. While Each biotin-labeled probe was placed in its own 1.5-ml micro-

centrifuge tube.molecular and genetic evidence strongly suggests that TG46

429Single-Copy FISH to Tomato SC Spreads

Tomato synaptonemal complex spreads: SC spreads were citric acid, 164.7 mm Na2HPO4, pH 7.0) containing 0.5 mg/ml49,6-diamidino-2-phenylindole (DAPI), 1 mg/ml propidiumprepared as described by Peterson et al. (1996) with several

modifications. Briefly, tomato (cv. Cherry) anthers were iodide (PI), or 1 mg/ml PI and 0.5 mg/ml DAPI (i.e., CPD).Slides were washed for 30 sec in deionized water and allowed toplaced in 200 ml of sugar-salt medium [0.56 mm KH2PO4, 0.1

mm acid PIPES, 0.2% w/v potassium dextran sulfate, 1 mm air-dry for 5 min. Ten microliters of freshly prepared antifademedium (aqueous 50 mm Tris, 50% glycerol, 1 mg/ml phenyl-CaCl2, 0.7 m mannitol, 1% w/v polyvinylpyrrolidone (Mr 5

10,000), pH 5.1] containing 3 mg desalted cytohelicase (Sepra- enediamine) was placed on each slide, and a 22- 3 50-mmcoverglass was added. Coverglasses were sealed onto slides withcor, Marlborough, MA). The upper tips of the anthers were

cut off, and anther bottoms were allowed to digest in the dark fingernail polish. Fluorescence and phase-contrast microscopywere performed using an Olympus Provis AX70 microscopefor 10 min. Drops of 45% acetic acid were placed on new

microscope slides (Corning, Inc., Corning, NY), and slides equipped with an Olympus UM-51005 filter cube for simulta-neous visualization of PI and FITC, a U-MNV filter cube forwere wiped dry with a Kimwipe. Slides then were glow-dis-

charged according to Dubochet et al. (1971) except that observation of DAPI and CPD staining, and a U-NG filter cubefor visualizing PI alone. Photographs were taken using a PM-air rather than amylamine vapors was introduced into the

discharge chamber. Dissecting needles were used to squeeze C35DX camera and Kodak Royal Gold 400 film.CPD karyotype: Both freshly fixed SC spreads and SCmicrosporocytes from anthers, and microsporocytes were then

allowed to digest for an additional 10–15 min. A 0.5-ml aliquot spreads that had been used in FISH were CPD stained, andcomplete (or nearly complete) late pachytene sets were photo-of the protoplast suspension was drawn into a siliconized micro-

pipette and gently blown into a 10-ml droplet of hypotonic graphed using phase-contrast and fluorescence microscopy.Phase-contrast and corresponding CPD photographs (magni-bursting medium (0.05% v/v Nonidet P-40 and 0.1% w/v

bovine serum albumin) suspended from the end of a 200-ml fication of prints 32258) were scanned at a resolution of 300dpi into a computer. The chromosome-measuring programpipette tip. The resulting droplet was immediately placed in

the center of a glow-discharged (hydrophilic) microscope Micromeasure 3.01 (available at http://www.colostate.edu/Depts/Biology/Micromeasure/) was used to determine relativeslide, and an additional 10 ml of hypotonic bursting medium

was added. A hand-held nebulizer (Fullam, Latham, NY) was SC lengths and arm ratios from phase-contrast images. TheSC karyotype of Sherman and Stack (1992, 1995) was usedused to immediately give the slide 30 puffs of 4% aqueous

formaldehyde (pH 8.5). The slide was air-dried, fixed in 4% to identify individual-SCs. Adobe Photoshop 4.0 was used todigitally merge phase-contrast and corresponding CPD im-aqueous formaldehyde for 10 min, rinsed twice without agita-

tion in aqueous 0.01% Photoflo 200 (Kodak, Rochester, NY), ages. To describe the position of each CPD band, the distancealong a CPD-banded SC from the center of the CPD band torinsed four times (20 sec each rinse) in distilled water, and

air-dried. the center of the kinetochore was measured and divided bythe length of the entire SC arm. In this way, the position ofFluorescence in situ hybridization: FISH was performed us-

ing a combination of the protocols of Rayburn and Gill each CPD band was expressed as a decimal fraction of thearm length from the centromere. These fractions were(1985) and Anamthawat-Jonsson et al. (1996) with signifi-

cant modifications. Slides with spread SCs were incubated in multiplied by the mean length of the SC arm (Sherman andStack 1995) to give micrometer distances from bands to the23 SSC containing 100 mg/ml RNase A (Sigma, St. Louis) at

378 for 30 min, washed in two changes of 23 SSC (378, 10 centromere (Table 1). Likewise, the length of each CPD bandwas measured and divided by the length of its entire SC tomin each wash), rapidly dehydrated in a graded ethanol series,

and air-dried. Chromosomal DNA was denatured by placing determine the relative proportion of chromosome length con-tained within the band (Table 1).slides in 50 ml of 70% formamide in 23 SSC at 708 for 2.5

min. Slides were rapidly dehydrated in an ethanol series at FISH data analysis: For each late pachytene SC spread (i.e.,spreads with visible kinetochores) showing a FISH signal, a2208 and air-dried. A cocktail containing 75 ml deionized

formamide, 30 ml 50% w/v potassium dextran sulfate, 15 ml phase-contrast and a PI/FITC photograph were taken. Formost sets, a CPD image was also obtained. Phase-contrast and203 SSC, 7.5 ml of 10 mg/ml sheared herring sperm DNA

(GIBCO BRL), and 12 ml of 25 mg/ml biotin-labeled probe(s) FITC/PI photographs were scanned and digitally merged toallow accurate measurement of distances between FISH signalswas heated at 978 for 10 min. Thirty microliters of the heat-

denatured cocktail was placed on a slide, and a 22- 3 50-mm and kinetochores. In all instances, SC 11 was identified onthe basis of relative length and arm ratio according to the SCcoverglass was added. Slides were incubated in sealed humid

chambers at 378 overnight. Coverglasses were gently washed karyotype data of Sherman and Stack (1992, 1995). Inspreads where FISH labeling was observed and a correspond-from slides with 23 SSC at 378. Slides were washed twice in

23 SSC, once in 25% formamide in 23 SSC, twice in 23 ing CPD image was available, the pattern of CPD stainingwas used to confirm the identity of SC 11 (see results).SSC containing 2% Tween-20 (all washes 378, 10 min), and

incubated for 60 min at 378 in blocking buffer (0.1 m maleic To determine the distance between an FITC focus and thekinetochore, the distance along the SC from the location ofacid, 0.15 m NaCl, 1% w/v Boehringer Mannheim blocking

reagent, pH 7.5). Slides were incubated for 1 hr at 378 in 1 mg/ the focus to the center of the kinetochore was measured anddivided by the length of the entire SC arm to express theml mouse anti-biotin (Boehringer Mannheim) in blocking

buffer, washed three times in 23 SSC containing 2% Tween- focus location as a decimal fraction of the arm length fromthe centromere. Fraction distances were multiplied by the20 (378, 10 min each wash), incubated for 1 hr at 378 in

10 mg/ml biotinylated goat anti-mouse (Sigma) in blocking mean length of the SC arm to give micrometer distances fromfoci to the centromere. This is the same technique used bybuffer, and washed three times in 23 SSC containing 2%

Tween-20 (378, 10 min each wash). Slides were then incubated Sherman and Stack (1995) to describe the location of recom-bination nodules on tomato SCs. In cases where an FITC focusin 40 mg/ml mouse anti-biotin fluorescein isothiocyanate

(FITC; Sigma) or 4 mg/ml streptavidin-FITC conjugate in did not lie directly on the SC, a line was drawn from the centerof the FITC focus perpendicular to the axis of the SC. Theblocking buffer for 1 h (378). All slides were washed once in

23 SSC containing 2% Tween-20 (378, 10 min) and twice point of intersection between the line and the SC axis wasmarked as the location of that focus on the SC. In somein 23 SSC at room temperature (10 min each wash).

Counterstaining and microscopy: Slides were incubated for instances, an FITC focus was located in the chromatin distalto the end of the SC. In these cases, a line was extended from15 min in the dark in McIlvaine’s buffer (aqueous 17.7 mm


TABLE 1

Karyotype of the CPD-banded tomato SC complement

Mean proportional Mean micrometer Mean CPD bandSC arms with distance 6 SD to distance 6 SD to Mean proportional length 6 SD inCPD bands a n b kinetochore c kinetochore d band length 6 SD e micrometers f

SC 1S 9 0.00 6 0.01 0.00 6 0.24 0.03 6 0.01 0.86 6 0.25SC 3L 10 0.00 6 0.02 0.01 6 0.11 0.03 6 0.01 0.64 6 0.20SC 6S 9 0.30 6 0.07 1.28 6 0.28 0.04 6 0.01 0.74 6 0.14SC 8S 8 0.33 6 0.05 1.83 6 0.25 0.04 6 0.01 0.76 6 0.19SC 11S 21 0.11 6 0.02 0.82 6 0.18 0.03 6 0.01 0.47 6 0.14

a S, short arm; L, long arm.b n, number of SC spreads examined in which the CPD-banded SC could be positively identified on the basis

of its relative length and arm ratio.c Mean distance from the center of a specific CPD band to the center of the kinetochore expressed as a

proportion of the length of the SC arm in which the band is found. The center of the kinetochore has a valueof 0 while the end of any particular arm has a value of 1.0.

d For a particular CPD band, the proportional distance from the kinetochore multiplied by the mean lengthof the SC arm in which the band is found gives a mean distance (mm) from the band to the kinetochore. Themean arm lengths (from Sherman and Stack 1995) are as follows: 1S, 7.5 mm; 3L, 17.9 mm; 6S, 4.3 mm; 8S,5.5 mm; and 11S, 7.3 mm.

e Mean proportional length of the entire SC occupied by the band.f For a particular CPD band, the proportional length of a CPD band multiplied by the mean length of the

SC on which it is found. Mean SC lengths in micrometers (from Sherman and Stack 1995) are as follows: 1,30; 3, 23.1; 6, 18.5; 8, 18.5; and 11, 16.2.

the end of the SC, a perpendicular line was drawn from the which suggests that kinetochores are highly infiltratedfocus through the extended line, and the point of intersection with chromosomal DNA.was marked as the location of the focus. In PI-stained early pachytene SC spreads of tomato,Initially, all measurements were made from SC spreads la-

heterochromatic regions often appear wider than eu-beled with only one of the three probes. However, after itchromatic regions (Figure 1B). Additionally, hetero-was clearly established that there was a considerable physical

distance between lTG523 and lTG46 and that there was no chromatin stains a bit more intensely than euchromatin.difficulty determining which signal belonged to which probe, In late pachytene spreads, PI-stained SCs have a rathermeasurements were made from these dual-labeled spreads as homogenous appearance with no noticeable stainingwell. No measurements were made from spreads probed with

difference between heterochromatin and euchromatin.any other combination of markers. Statistical analysis of theKinetochores stain with an intensity similar to that ofdistribution of FITC foci for the three probes was performed

using the computer program Instat 1.12a (GraphPad Software, nearby chromatin, again indicating the presence ofSan Diego). DNA in kinetochores.

Dot blot verification of probe identity: To rule out the When early pachytene tomato SC spreads are stainedunlikely possibility that the genomic probes used for FISH

with CPD and examined using a wide UV filter, euchro-were inadvertently switched or cross-contaminated before inmatin and heterochromatin are easily differentiatedsitu hybridization, each DIG-labeled RFLP marker was used

to probe a separate nylon membrane on which a 20-ml drop with heterochromatin fluorescing white and euchroma-(0.5 mg) of each of the biotin-labeled l insert DNAs had tin fluorescing blue (Figure 1C). The differential stain-been dotted. Filter hybridization conditions and colorimetric ing of heterochromatin and euchromatin seen afterdetection of probe hybridization were performed as described

CPD treatment is more striking than differences pro-above.duced by PI staining alone. By late pachynema, hetero-chromatin and euchromatin do not exhibit a predict-able pattern of differential staining (Figure 1D).RESULTS

Throughout pachynema, the NOR (which comprisesDAPI, PI, and CPD staining of tomato SCs: When most of the short arm of chromosome 2) is easily identi-

tomato SC spreads are stained with DAPI, pericentro- fied by its red fluorescence (Figure 1, C and D). Asmeric heterochromatin, euchromatin, and telomeres previously described, the NOR typically exhibits partialare not readily differentiated. However, some bivalents asynapsis (Sherman and Stack 1992; Peterson et al.possess a single, highly localized region that does not 1996), and, as pachynema progresses, the NOR becomesfluoresce, i.e., a DAPI-negative band (Figure 1A). DAPI- more elongate (Figure 1D).negative bands were observed throughout pachynema CPD staining results in a single red “CPD band” withinand diplonema. In late pachytene spreads, kinetochores the pericentromeric heterochromatin of 5 of tomato’s

12 SCs (Figure 1, C and D). The number and generalstain with similar intensity as surrounding chromatin,


Figure 1.—Staining of chro-matin associated with tomatoSC spreads. (A) Early pachy-tene SC set stained with DAPIonly. Several SCs possess a re-gion where DAPI staining is notvisible, i.e., a DAPI-negative band(arrows). No other chromatin-based features are visible. (B)Early pachytene SC spreadstained with PI only. Hetero-chromatic regions (e.g., arrow-heads) appear wider and staina bit more intensely than thedistal euchromatic regions (e.g.,arrows). (C) CPD staining ofan early pachytene SC spread.Pericentromeric heterochro-matin fluoresces white whiledistal euchromatin fluorescesblue. A red-fluorescing chro-matin band (CPD band) is visi-ble within the pericentromericheterochromatin of 5 of toma-to’s 12 SCs (arrows). Thesebands most likely correspondto the DAPI-negative bandsseen in DAPI-stained tomatoSC spreads. The NOR near theterminus of the short arm ofSC 2 fluoresces red and usuallyexhibits partial or completeasynapsis (e.g., diamond-headarrows). (D) Late pachyteneCPD-stained SC spread. Kinet-ochores are visible as thick-enings along SCs (e.g., whiteboxes). On two CPD-bandedSCs, the kinetochores are lo-cated directly over the CPDband (arrowheads). However,on the remaining three CPD-banded SCs a red band is visi-ble outside of the region en-compassed by the kinetochore(arrows). The elongate, asyn-apsed NOR fluoresces red (dia-mond-head arrows). (E) Phase-contrast image of a completespread of late pachytene to-mato SCs. A kinetochore is visi-ble 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 thesame SC spread shown in E. CPD bands are visible on five of the SCs (arrows). (G) Diagram showing the relative position ofCPD 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 orderfrom left to right. Euchromatin is blue, heterochromatin is white, kinetochores are orange, and CPD bands are red. The NORin the short arm of SC 2, which appears red after CPD staining, is colored pink in this idiogram. A constricted area within theNOR marks the general region where SC formation does not occur. Frames that share a common bar: A and B; C and D; andE–G. Bars, 10 mm.

location of CPD bands suggest that they are equivalent and Stack (1992), to construct a CPD karyotype fortomato. CPD bands are found on SCs 1, 3, 6, 8, and 11to the DAPI-negative bands seen in tomato SC spreads

stained with DAPI only (Figure 1A). We used CPD stain- (Figure 1, E–G and Table 1). The CPD bands on SCs1 and 3 lie essentially at centromeric positions while theing coupled with the SC karyotype data of Sherman


bands on SCs 6, 8, and 11 are located at subcentromeric from several experiments, some experiments involveduse of only one probe at a time, and probes used in allpositions in the short arms of their respective chromo-

somes (Figure 1G). An idiogram showing the distribu- FISH experiments were removed from the same threetubes from which samples were taken for dot blot probetion of CPD bands on the tomato SC complement is

shown in Figure 1H. verification (see materials and methods).SCs vs. “prefixed” pachytene chromosomes: In initialFluorescence in situ hybridization: Probes containing

single-/low-copy sequences were obtained by screening attempts to localize specific DNA sequences on meioticchromosomes, we used pachytene chromosomes fixeda tomato l genomic library with the chromosome 11-

associated RFLP markers TG46, TG400, and TG523 before spreading in 1:3 acetic ethanol, i.e., “prefixedpachytene chromosomes,” as substrates for FISH rather(Tanksley et al. 1992). For each RFLP marker, a corre-

sponding positive genomic clone was selected, i.e., than SC spreads. Although, when repetitive sequenceprobes were used, prefixed pachytene chromosomeslTG46, lTG400, and lTG523. Tomato insert DNA from

each clone was isolated (lengths of tomato inserts: were adequate substrates for FISH (e.g., Peterson etal. 1998), localization of single-copy probes on theselTG46, 16,550 bp; lTG400, 10,200 bp; lTG523, 14,000

bp) and labeled with biotin using nick translation. La- chromosomes proved difficult because of high levels ofbackground fluorescence and a scarcity of chromosomebeled insert DNA was hybridized to spreads of tomato

SCs on microscope slides. Slides were treated with sets in which each bivalent could be distinguished, i.e.,less than one completely interpretable set per slide (datamouse anti-biotin followed by incubation in biotinylated

goat anti-mouse. Sites of probe hybridization were de- not shown). When SC spreads were substituted for pre-fixed pachytene chromosomes, significant problemstected using anti-biotin-FITC or streptavidin-FITC con-

jugates. SC 11 was identified on the basis of its relative with background fluorescence were eliminated andchromosome identification was facilitated because eachlength and arm ratio. Additionally, CPD banding analy-

sis was used to confirm the identity of SC 11. The Micro- slide possessed 10 or more SC sets where all 12 tomatoSCs were clearly identifiable (e.g., Figure 1, E–G).measure chromosome measuring program was used to

determine the distance from the centromere to eachFITC focus.

DISCUSSIONEach of the three genomic probes hybridized exclu-

sively to euchromatin on the long arm of SC 11 (Figure CPD staining: If tomato SC spreads are stained witha combination of PI and DAPI, i.e., CPD stained, and2). Little or no background FITC fluorescence was ob-

served, and the efficiency of hybridization (i.e., the per- illuminated with broad-band UV-visible light, structuralfeatures associated with differential chromatin conden-centage of SC spreads where specific labeling of SC 11

could be seen) was high for all three probes (ca. 70%). sation and/or DNA sequence can be visualized. Thesefeatures either are not visible by DAPI or PI stainingOn the basis of the relative locations of FITC foci, the

three probes were positioned in relationship to the cen- alone or are more readily differentiated by CPD stain-ing. Although a variety of dye concentrations weretromere and to one another (Table 2 and Figure 2,

N–P). We refer to the diagram showing the position of tested, best results were obtained when chromosomeswere stained with 1 mg/ml PI and 0.5 mg/ml DAPI (dataeach locus on SC 11 as an SC-FISH map (Figure 2O).

One-way analysis of variance (ANOVA) indicates that not shown).Euchromatin and heterochromatin can be differenti-the difference of the group means for the probes is

highly significant (F 5 146.9, P , 0.0001). Comparison ated by CPD staining in early pachytene SC spreads, butby late pachynema, the two types of chromatin are notof the data for any two individual probes also indicates

that the loci are clearly separated (Bonferroni P value readily distinguished. The explanation for this may bethat the difference in relative condensation of hetero-for any two probes is ,0.001). lTG523 is the most distal,

i.e., farthest from the centromere, of the loci with a near chromatin and euchromatin is decreased during thetransition from early to late pachynema. On the otherterminal location. The probes lTG46 and lTG400 are

located more proximally on SC 11 with lTG400 being hand, CPD bands and NORs remain differentiallystained throughout pachynema (and probably through-closer to the centromere than lTG46. This contradicts

the molecular linkage map of chromosome 11 on which out meiosis).Likewise, a combination of PI and DAPI has beenTG400 is located between TG523 and TG46 (Tanksley

et al. 1992; compare Figure 2N and 2O with 2P). To shown to differentially stain NORs in mitotic and mei-otic metaphase chromosomes of cotton (Hanson et al.rule out the possibility that the probes lTG46 and

lTG400 had been inadvertently switched before FISH, 1995; Ji et al. 1997), mitotic metaphase chromosomesof tomato (T. P. V. Hartman, personal communica-we performed a series of dot blot experiments. These

experiments verified that each biotin-labeled FISH tion), and SC spreads from maize and potato (our un-published observations). Consequently, it seems likelyprobe hybridized exclusively with the RFLP marker orig-

inally used to isolate it. It is unlikely that probes were that the NORs of many, if not all, plants can be visualizedusing CPD staining.switched at the time of FISH because data were obtained


Figure 2.—Single-/low-copy FISHto tomato SC 11. (A) Phase-contrastimage of a late pachytene SC 11. Thekinetochore is clearly visible (arrow-head). (B) Hybridization of lTG523to the long arm of the same SC. Probehybridization is visible as yellow-green(FITC) foci (arrow). The chromo-some appears red because of the PIcounterstain. The kinetochore ismarked by an arrowhead. (C) CPDimage of the same SC. As predicted bythe CPD karyotype, this chromosomepossesses a red CPD band (arrow) inthe short arm near the kinetochore(arrowhead). (D) Phase-contrast im-age of SC 11. The kinetochore is indi-cated by an arrowhead. (E) lTG523hybridization on the SC shown in D.The signal is localized near the end ofthe chromosome (arrow). Note thatthere is little background FITC fluo-rescence, and there are no ectopicsites of hybridization. (F and G)Phase-contrast and PI/FITC image ofa chromosome 11 showing the kinet-ochore (black arrowhead) andlTG400 hybridization (white arrow),respectively. (H and I) Phase-contrastand PI/FITC images of the samechromosome 11. In I, simultaneousdetection of lTG523 (arrowhead)and lTG46 (arrow) is shown. (J–M)Centromere placement and probepositioning on an early pachytene to-mato SC 11. (J) Phase-contrast imageof an SC 11. No kinetochore is visible.(K) lTG523 hybridization (arrow).(L) CPD staining. Heterochromatinappears white and euchromatin blue.A distinct CPD band (arrowhead) isobserved within the pericentromericheterochromatin of the bivalent. (M)Diagram of chromatin features andhybridization sites superimposed onthe phase-contrast image of the SC.In the diagram, euchromatin is blue,heterochromatin is green, the CPDband is red, and FITC foci are yellow.Although a kinetochore is not visi-bleon this SC, the consistent relation-

ship 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, lTG400, lTG46, andlTG523 are represented by the colors blue, red, and green, respectively. (N) Histogram showing the distribution of lTG46,lTG400, and lTG523 FITC foci along tomato SC 11. The number of observed FITC foci is shown on the ordinate axis while adiagram of an average SC 11 is shown on the abscissa. Each vertical bar represents the number of foci observed in a 0.1-mminterval 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 pericentromericheterochromatin. A dotted line extends from the terminus of the long arm of SC 11 to include lTG523 foci that were foundin chromatin extending beyond the end of the SC. (O) SC-FISH map of tomato SC 11. The mean position of each SC-FISHprobe is shown by a vertical bar. All three probes are localized in euchromatin of the long arm. (P) Molecular linkage map oftomato chromosome 11 redrawn from Figure 1 of Tanksley et al. (1992). Regions between adjacent markers are enclosed byhorizontal braces with map distances between markers given above the braces. Note that lTG46 and lTG400 are reversed inrelation to their order on the SC-FISH map. Frames that share a common scale bar: (A–C) bar, 5 mm; (E–I) bar, 5 mm; (J–M)bar, 1 mm.


TABLE 2

Locations of three probes on the long arm of tomato SC 11

Number of Mean locus position 6 SD Distance (mm) fromProbes n a observed FITC foci from kinetochore b kinetochore c 6 SD

lTG46 16 38 0.76 6 0.03 6.78 6 0.28lTG400 12 31 0.62 6 0.04 5.54 6 0.37lTG523 21 58 0.97 6 0.04 8.61 6 0.37

a Number of SC sets from which data were collected for a particular probe. In all sets from which measurementswere made, SC 11 could be positively identified and FISH label was observed only on this SC.

b Mean locus position is expressed as a decimal fraction of the long arm from the kinetochore. The centerof the kinetochore has a decimal fraction value of 0, the terminus of the long arm has a value of 1, and focidistal to the terminus have values .1.

c These values were obtained by multiplying the average locus position by 8.9 mm, which is the mean lengthof the long arm of SC 11 (Sherman and Stack 1995).

Insight into the probable mechanisms by which CPD CPD bands may be equivalent to certain C-bands.A C-band karyotype based on 1:3 acetic ethanol-fixedstaining differentially stains NORs and CPD bands may

be gained by considering the mechanisms by which PI tomato (Moneymaker) pachytene chromosomes showsprominent centromeric/subcentromeric C-bands onand DAPI interact with DNA. PI intercalates between

bases of single- or double-stranded nucleic acid mole- chromosomes 1, 4, 6, 8, and 11 and staining of the NORon chromosome 2 (M. S. Ramanna and L. P. Pijnacker,cules without regard to nitrogenous base composition,

and consequently PI can be used as a quantitative nu- unpublished data). The relative location of majorC-bands on chromosomes 1, 6, 8, and 11 correspondscleic acid dye (Heslop-Harrison and Schwarzacher

1996). DAPI is a double-stranded DNA-specific dye that exactly with locations of CPD bands on these chromo-somes. However, the CPD- and C-band karyotypes differinteracts with DNA by at least two different mechanisms

(see Kapuscinski 1995 for review). In regions where in three ways: (1) There is no corresponding CPD bandfor the major C-band near the centromere of chromo-three or four AT base pairs are located in tandem, DAPI

binds to the minor groove of the DNA, and this results in some 4. (2) A C-band is found near the centromere ofchromosome 3, but it is very small and probably doesa highly fluorescent compound. DAPI also intercalates

between bases, which results in a nonfluorescent com- not account for the relatively large CPD-band we observeon this chromosome. And (3) there are several smallpound. The former reaction is energetically favored in

AT-rich regions while the latter is favored in GC-rich C-bands for which corresponding CPD bands were notobserved. These discrepancies may be caused by differ-areas along a DNA molecule. As an intercalator, the

binding strength of DAPI (in GC-rich areas) is roughly ences in the heterochromatin of the cultivars Cherryand Moneymaker. Also the mechanism of C-bandingequivalent to that of PI. NORs, i.e., rDNA sequences,

are GC-rich compared to most other DNA regions (Mac- (Sumner 1990) differs from the proposed mechanismof CPD staining, so it is not surprising that some CPDgregor and Kezer 1971; Yasmineh and Yunis 1971;

Ingle et al. 1975). As a result, in CPD-stained spreads, bands coincide with C-bands while others do not (andvice versa).minor groove binding of DAPI to rDNA sequences

would be limited. Presumably, DAPI and PI would com- SCs as substrates for single-copy in situ hybridization:Although FISH has been used to localize repetitive se-pete for intercalation sites in the GC-rich rDNA, but

only PI intercalation would result in fluorescence. In quences on spreads of vertebrate SCs (Moens andPearlman 1989, 1990; Heng et al. 1994; Solari andaddition, the PI concentration in the CPD staining solu-

tion is double that of the DAPI concentration, and thus Dresser 1995) and on SC spreads of two plant species(Hasenkampf 1991; Albini and Schwarzacher 1992),PI intercalation would be favored. Consequently, red

(PI) fluorescence predominates in NORs. Similarly, to our knowledge, there have been no reports of single-copy FISH to SCs of any species. Here we demonstrateDAPI-negative bands (seen in DAPI-stained SC spreads)

and CPD bands probably represent other chromosomal that spreads of tomato SCs are well suited for the rigor-ous requirements of in situ detection of single-copy se-regions rich in GC base pairs. The presence of GC-rich

regions in heterochromatin (including GC-rich satellite quences. Because comparable SC spreads can be pre-pared for a variety of plants as well as animals and fungi,sequences) has been reported for other species (see

Sumner 1990 for review). Differential staining between it is likely that this technique can be generally applied.In our experience, background fluorescence is mark-heterochromatin and euchromatin observed in early

pachynema likely reflects differences in both chromatin edly reduced if SC spreads are used as FISH substratesinstead of prefixed pachytene chromosomes. We sus-condensation and GC content.


pect that this phenomenon reflects the time at which matin, tomato should be particularly well suited for genetagging and chromosome walking (Peterson et al. 1998).chromosomes are fixed. Treatment of cells with acetic-

ethanol results in rapid protein crosslinking and fixa- Single- vs. low-copy number sequences: Molecular evi-dence indicates that TG523 and TG46 are located attion of chromosome structure (Sharma and Sharma

1980). Consequently, in prefixed preparations nucleo- single loci on tomato chromosome 11 (Tanksley et al.1992; SolGenes web site, http://geneous.cit.cornell.edu/plasm may be fixed onto chromosomes before squashes/

spreads are made. During FISH, probes and fluoro- solgenes/aboutsolgenes.html/). Our observation thatlTG523 and lTG46 each bind exclusively to a single,chrome-labeled affinity reagents may become trapped

within nucleoplasm surrounding the chromosomes to highly localized region on SC 11 supports this premise.On the other hand, blotting evidence suggests thatproduce high background fluorescence. While diges-

tion of chromosomes with a protease(s) before FISH can TG400 may occur at more than one locus in the tomatogenome (S. D. Tanksley, personal communication; Sol-be used to remove at least some of the contaminating

nucleoplasm from prefixed pachytene chromosomes Genes web site). However, we observed only one highlylocalized site of lTG400 probe hybridization on tomato(Jiang et al. 1995), protease treatment also results in

significant changes in chromosome structure (our expe- SC spreads, and this hybridization site is on the longarm of SC 11. If there is indeed more than one copyrience). In contrast, SCs are spread before fixation. Dur-

ing the spreading process, nucleoplasm around SCs is of TG400 in the tomato genome, this result might beexplained if the copies of TG400 are so closely associateddispersed and chromatin is decondensed, which allows

better access of single-copy probes to chromosomal DNA. that they are not resolvable using our FISH method.Alternatively, it is possible that the lTG400 FISH cloneAlthough we believe that SC spreads possess some

features that make them better suited for single-copy hybridized to a TG400 site on SC 11 and did not recog-nize TG400 loci elsewhere.FISH than prefixed pachytene chromosomes, it should

be noted that other investigators have successfully local- Comparison of the SC-FISH and molecular geneticmaps: While linkage maps show the relative order ofized single-copy sequences on prefixed pachytene chro-

mosomes by in situ hybridization (Shen et al. 1987; Jiang genes along each chromosome, they provide little in-sight into the relationship between genes and chromo-et al. 1995).

CPD banding and FISH: In all cases where single- some structure. This is evident in a comparison of theSC-FISH map (Figure 2O) with the molecular linkagecopy FISH was detected and a CPD photograph was

taken, a CPD band was observed in a position predicted map of chromosome 11 (Figure 2P). On the molecularlinkage map, TG523 is closer to the center of the mapby the CPD band karyotype of SC 11. Although SC 11

can be readily identified on the basis of relative length than it is to the terminus, but on the SC-FISH map,lTG523 is located near the end of the long arm of SCand arm ratio, FISH and CPD results each indepen-

dently indicate that SC 11 was correctly identified in 11. Because TG523 is not the most distal locus on themolecular linkage map, it is likely that there are otherthis study.

Nature of the tomato genome: Because it is possible genes/markers clustered within the more distal subtelo-meric region of the chromosome. With respect to dis-that genomic clones isolated by screening a library with

RFLP markers might contain repetitive elements in ad- tances between markers, all three SC-FISH markers arecontained within a chromosomal region comprisingdition to single-copy DNA, genomic probes are often

“prehybridized” with unlabeled repetitive DNA se- only 19% of the length of SC 11. However, the threeRFLP markers span a region encompassing 35% of thequences before they are placed on chromosomes. Such

chromosomal in situ suppression (CISS) hybridization molecular linkage map (Tanksley et al. 1992).On the SC-FISH map, the order of lTG46 andeffectively prevents highly repetitive sequences in

probes from participating in hybridization with chromo- lTG400 is reversed in relation to their order on themolecular linkage map (Figure 2, N–P). This disagree-somal DNA (Landegent et al. 1987; Lichter et al. 1988,

1990; Jiang et al. 1995, 1996; Zwick et al. 1997). How- ment is not caused by an inability to resolve these mark-ers cytologically, because (with the exception of a singleever, in earlier studies we determined that only 23% of

the tomato genome is composed of repetitive sequences FITC focus) the ranges of FITC foci for the markers donot overlap (Figure 2N). While somewhat unexpected,and that the vast majority of these repetitive sequences

are localized in pericentromeric heterochromatin (see disagreement between the order of loci on tomato mapsis not without precedent (e.g., Schumacher et al. 1995).Peterson et al. 1996, 1998). Consequently, because

there was reason to believe that our genomic probes Relative order of TG46 and TG400: There are severalpossible explanations for the discrepancy between themight not contain repetitive sequences, we tried FISH

without CISS hybridization, and indeed, we found that relative positions of TG46 and TG400 on the SC-FISHand molecular genetic maps. For example, it is possibleeach of the three genomic probes hybridized exclusively

to a different highly localized region on SC 11 in the that (1) the lTG400 probe recognizes a locus on SC 11different from the TG400 locus positioned on the link-absence of suppression sequences. With a relatively

small genome size and little repetitive DNA in its euchro- age map (see above); (2) these loci have been incor-


TABLE 3

Tomato DNA per micrometer of SC in euchromatin and heterochromatin

Proportion of DNA concentrationgenome content a DNA content (bp) b SC length (mm) c (bp/mm)

Euchromatin 0.23 1.91 3 108 136.6 1.40 3 106

Heterochromatin 0.77 6.39 3 108 76.4 8.36 3 106

a Data from Peterson et al. (1996).b Determined by multiplying the fraction of the genome occupied by each type of chromatin by the 1C

genome size (8.30 3 108 bp; Peterson et al. 1998) for tomato.c Data from Sherman and Stack (1995).

rectly positioned on the molecular linkage map; or (3) of the SC-FISH markers and between the SC-FISH mark-ers themselves (Table 4). On the basis of the data inthe order of loci in cv. Cherry tomato differs from the

gene order in the cultivar that was used to prepare the Table 4 and the average positions for the three probesshown in Figure 2O, we estimate that SC-FISH easilymolecular linkage map (i.e., VF36-Tm2a; Tanksley et

al. 1992). permits resolution of loci separated by as few as 1.7million bp with every indication that much closer lociThe first of these possibilities seems unlikely because

the lTG400 probe would have to consistently fail to could be resolved. For comparison, Trask (1991) esti-mated that FISH probes can be mapped to human meta-hybridize to the “RFLP-mapped” copy of TG400 on chro-

mosome 11, yet succeed in hybridizing to a second copy phase chromosomes at a resolution of z3 million bp.On the other hand, substrates other than condensedof TG400 on chromosome 11 overlooked during RFLP

mapping. The homology between the two TG400 copies chromosomes can be used to improve resolution. Forexample, FISH has been used to resolve single-copy(even if limited to the 1200 bp of the RFLP marker

itself) would probably be sufficient to produce some sequences separated by as little as 50 kb in the decon-densed chromatin of interphase nuclei (Lawrence etpositive hybridization at both sites. Consequently, a less

defined distribution of lTG400 FITC foci encompassing al. 1988; Trask et al. 1991). Additionally, Heng et al.(1992) resolved genes as close as 20 kb by FISH to freethe range of lTG46 hybridization sites would be ex-

pected, but this is not observed (Figure 2N). chromatin, and Fransz et al. (1996) resolved sequencesseparated by ,1 kb using stretched, naked DNA as aIn reference to the second possibility, construction

of linkage maps involves sexual crosses and sometimes FISH substrate. Unfortunately, with these latter tech-niques individual chromosomes and structural featuresdifficult and/or complex scoring of recombination

events that could lead to a mistake. However, because such as heterochromatin, centromeres, and telomeresare not readily distinguishable.TG46, TG400, and TG523 have LOD scores of at least

3.0 (Tanksley et al. 1992), it is unlikely that the markers Consolidation of the SC-FISH and recombinationnodule maps: Because of the discrepancy between thehave been positioned incorrectly on the molecular link-

age map (see Tamarin 1999). SC-FISH and RFLP maps for tomato chromosome 11,a meaningful discussion of the relationship betweenPerhaps the most likely explanation for the discrep-

ancy between the SC-FISH and RFLP map is a difference molecular map distances and the number of base pairsbetween the three SC-FISH marker loci is not possible.in locus order between cv. Cherry and cv. VF36-Tm2a

because of a small inversion in chromosome 11. In However, the SC-FISH and recombination nodule mapsfor SC 11 (Sherman and Stack 1995) can be comparedcrosses between our Cherry tomato line and character-

ized translocation lines of cv. Early Fire Ball, no inver- to determine map distances between SC-FISH markers.[Recombination nodules (RNs) are ellipsoidal struc-sion loops were observed in pachytene SC spreads from

the F1 progeny (unpublished observations made during tures associated with SCs during late pachynema, andthere is a 1:1 correlation between RNs and crossoverresearch of Herickhoff et al. 1993). Consequently, if

cv. Cherry contains an inversion in chromosome 11 events (Carpenter 1975; Sherman and Stack 1995;Anderson et al. 1997).] Comparison of the maps indi-relative to cv. Early Fire Ball, either the inversion is

not large enough to produce an inversion loop in the cates that the rate of crossing over per megabase pair(106 bp) is similar in the interval flanked by lTG400 andheterozygote or cv. Cherry and cv. Early Fire Ball have

the same inversion. lTG46 and the interval flanked by lTG46 and lTG523(Table 5). Thus, at least at the resolution of this analysis,Base pair distances between SC-FISH markers: Data

from previous investigations (Peterson et al. 1996, there is no obvious recombination hot/cold spot in oneof these intervals compared to the other (see Segal et1998) were used to calculate DNA densities (1C) in

heterochromatic and euchromatic regions along to- al. 1992 for an example of a recombination hot spot intomato euchromatin).mato SCs (Table 3). This permitted an estimate of the

number of base pairs between the centromere and each Use of CPD banding to position probes on early pachy-


TABLE 4

Calculation of base pairs between different molecular and cytological markers

SC in euchromatin SC in heterochromatin Base pairsMarkers between markers (mm) a between markers (mm) a between markers b

lTG46 and centromere 2.98 3.80 3.60 3 107



lTG523 and lTG46 1.83 0 2.53 3 106

lTG523 and lTG400 3.07 0 4.30 3 106

lTG46 and lTG400 1.24 0 1.74 3 106

a From Sherman and Stack (1995), the long arm of SC 11 (8.9 mm mean total length) has an average of3.8 mm of its length in pericentromeric heterochromatin. To determine the length of the long arm ineuchromatin between a probe and the centromere, 3.8 mm was subtracted from the distance between theprobe and the centromere (see Table 2). Regions between probes are euchromatic.

b Base pairs per micrometer of SC in euchromatin and heterochromatin (as calculated in Table 3) are 1.40 3106 and 8.36 3 106, respectively. The number of base pairs between two markers was calculated by multiplyingthe micrometers in euchromatin between the markers by 1.40 3 106 bp, multiplying the micrometers inheterochromatin between the two markers by 8.36 3 106 bp, and adding the resulting values together.

tene SCs: Because heterochromatin and euchromatin arm to the kinetochore, i.e., the length of the short arm,is [5.8 mm 4 (1.0 2 0.112) 5] 6.5 mm, and the longcan be differentiated in early pachytene SCs, SC-FISH

to early spreads may be useful in determining the chro- arm is (15.0 mm 2 6.5 mm 5) 8.5 mm in length. Conse-quently, in Figure 2M the centromere (purple band)matin background in which a gene/transgene is found.

However, it is difficult to position hybridization sites has been placed 6.5 mm from the end of the short arm.Once the centromere has been positioned, the locationson early pachytene SCs because they do not possess

cytologically discernible centromeres/kinetochores at of FISH hybridization signals can be expressed as frac-tions of the length of the chromosome arm to the cen-the light microscopic level (Stack and Anderson

1986). This problem can be overcome for chromosomes tromere. For example, the two lTG523 FITC foci nearthe terminus of the long arm of SC 11 (Figure 2, K andwith a characterized CPD band(s) because the band can

be used to position the centromere. An example of M) are 7.8 and 7.9 mm from the centromere, respectively.Because the length of the long arm is 8.5 mm (see above),centromere placement in relation to a CPD band is

shown in Figure 2, J–M. The SC 11 in these frames is the two foci lie at points [100 3 (7.8 mm 4 8.5 mm)5] 92.0% and [100 3 (7.9 mm 4 8.5 mm) 5] 93.0%15.0 mm in length. The distance from the end of the

short arm (i.e., the arm lacking a lTG523 FISH hybrid- of the long arm from the centromere.The potential of single-copy FISH to SCs: In this studyization signal) to the CPD band is 5.8 mm. From Table

1, the relative distance between the CPD band and the we demonstrated that single-copy sequences can be reli-ably detected on SC spreads, discovered a discrepancykinetochore of SC 11 is 11.2% of the length of the short

arm. Thus the distance from the terminus of the short between our SC-FISH map and the molecular genetic

TABLE 5

Map distances between markers determined by integrating the data from therecombination nodule map of tomato with the SC-FISH data

Total number of RNs Map units Map units per 106 bp ofMarkers between markers a between markers b DNA between markers c

lTG46 and centromere 219 25.9 0.7lTG400 and centromere 112 13.3 0.4lTG523 and centromere 363.5 43.1 1.1lTG523 and lTG46 144.5 17.1 6.8lTG523 and lTG400 251.5 29.8 6.9lTG46 and lTG400 107 12.7 7.3

a The mean number of RNs observed between two markers based on an analysis of 422 SC 11s (Table A1in Sherman and Stack 1995).

b The map units (percentage crossing over) between two markers were determined by dividing the totalnumber of observed RNs in the interval by the total number of SC 11s from which data were obtained (i.e.,422) and multiplying the quotient by 50 map units (see Sherman and Stack 1995 for more information).

c Map units between markers divided by the number of base pairs between those markers (see Table 4).


heterologous DNA inserts on the mouse meiotic chromosomemap, estimated the distance between molecular markerscore. Chromosoma 103: 401–407.

in base pairs, and integrated the SC-FISH map with the Herickhoff, L., S. Stack and J. Sherman, 1993 The relationshipbetween synapsis, recombination nodules and chiasmata in to-preexisting RN map for tomato. Additionally, our datamato translocation heterozygotes. Heredity 71: 373–385.provide further evidence that there are few repeated

Heslop-Harrison, J. S., and T. Schwarzacher, 1996 Flow cytome-sequences in tomato euchromatin. In broader terms, try and chromosome sorting, pp. 85–106 in Plant Chromosomes—

Laboratory Methods, edited by K. Fukui and S. Nakayama. CRCSC-FISH mapping has considerable potential as a meansPress, Boca Raton, FL.of relating genes to chromosome morphology (espe-

Ingle, J., J. N. Timmis and J. Sinclair, 1975 The relationship be-cially when coupled with differential chromatin-staining tween satellite deoxyribonucleic acid, ribosomal ribonucleic acid

gene redundancy, and genome size in plants. Plant Physiol. 55:techniques such as CPD staining), studying the relation-496–501.ship between chromatin organization and gene expres-

Ji, Y., D. A. Raska, T. D. McKnight, M. N. Islam-Faridi, C. F. Cranesion, relating linkage distances to chromosome struc- et al. 1997 Use of meiotic FISH for identification of a new

monosome in Gossypium hirsutum L. Genome 40: 34–40.ture, and locating sites of transgene insertions. BecauseJiang, J., B. S. Gill, G.-L. Wang, P. C. Ronald and D. C. Ward,SC spreads have been prepared for many plants, ani-

1995 Metaphase and interphase fluorescence in situ hybridiza-mals, and a few fungi, it is probable that SC-FISH map- tion mapping of the rice genome with bacterial artificial chromo-

somes. Proc. Natl. Acad. Sci. USA 92: 4487–4491.ping can be used to investigate the genomes of a wideJiang, J., S. H. Hulbert, B. S. Gill and D. C. Ward, 1996 Interphasevariety of organisms.

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