c-banded karyotypes of two silene species with heteromorphic sex chromosomes

C-banded karyotypes of two Silene species withheteromorphic sex chromosomes

Aleksandra Grabowska-Joachimiak and Andrzej Joachimiak

Abstract: Mitotic metaphase chromosomes ofSilene latifolia(white campion) andSilene dioica(red campion) were stud-ied and no substantial differences between the conventional karyotypes of these two species were detected. The classifica-tion of chromosomes into three distinct groups proposed forS. latifolia by Ciupercescu and colleagues was consideredand discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. GiemsaC-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainlydistally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromo-somes and some autosome types, and to detect differences between the twoSilenekaryotypes. It was shown thatS. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The twoSilenesex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphicbands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixedbands, and someS. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatinamount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome andkaryotype evolution in these two closely related dioeciousSilenespecies.

Key words: dioecious plant,Silene dioica, Silene latifolia, karyotype, sex chromosomes, heterochromatin, C-banding.

Résumé: Des chromosomes en métaphase mitotique duSilene latifolia (compagnon blanc) et duSilene dioica(compagnon rouge) ont été examinés et aucune différence substantielle n’a été observée quant aux caryotypesconventionnels entre ces deux espèces. La classification des chromosomes au sein de trois groupes, telle que proposéepar Ciupercescu et collègues pourle S. latifolia, a été considérée et discutée. De plus, un nouveau satellite de petitetaille a été observé sur le bras court du chromosome 5 métacentrique. Une coloration des bandes C au Giemsa chezces deux espèces a montré la présence de plusieurs bandes hétérochromatiquess tant fixées que polymorphes et celles-ci étaient principalement situées en position distale ou centromérique. Ces études de coloration des bandes C ontpermis de mieux caractériser les chromosomes sexuels ainsi que certains types d’autosomes et de détecter desdifférences entre les caryotypes des deux espèces deSilene. Il a été montré que leS. latifolia contient davantaged’hétérochromatine polymorphe, principalement au niveau des centromères. Les deux chromosomes sexuels sedistinguent facilement non seulement sur la base de leur taille ou de leur contenu en ADN mais également d’après desdifférences au niveau de leurs bandes C colorées au Giemsa. Tous les chromosomes Y montrent invariablement uneseule bande distale et aucune autre bande, polymorphe ou fixée, n’a été observée chez les deux espèces. Le chromo-some X possède deux bandes fixées en position terminale et certains chromosomes X chez leS. latifolia possèdent unfragment centrique additionnel de taille variable. La quantité et la distribution de l’hétérochromatine, révélées par cescolorations Giemsa, contribuent à une meilleure compréhension du problème de l’évolution du caryotype et deschromosomes sexuels chez ces deux espèces dioïques apparentées au sein du genreSilene.

Mots clés: plante dioïque,Silene dioica, Silene latifolia, caryotype, chromosomes sexuels, hétérochromatine, colorationdes bandes C.

[Traduit par la Rédaction] Grabowska-Joachimiak and Joachimiak 252

Genome45: 243–252 (2002) DOI: 10.1139/G01-143 © 2002 NRC Canada

243

Received 7 May 2001. Accepted 5 November 2001. Published on the NRC Research Press Web site at http://genome.nrc.caon 7 February 2002.

Corresponding Editor: P.B. Moens.

Aleksandra Grabowska-Joachimiak.1 Cytogenetics Group in the Department of Plant Breeding and Seed Science, TheAgricultural University of Cracow,ºobzowska 24, 31–140 Kraków, Poland.Andrzej Joachimiak. Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Grodzka 52, 31–044 Kraków, Poland.

1Corresponding author (e-mail: [email protected]).

I:\gen\gen45\gen-02\G01-143.vpFriday, February 01, 2002 8:45:00 AM

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Introduction

Silene latifolia (white campion,Melandrium album) pos-sesses the most well-studied plant sex-chromosome systemand provides excellent opportunities for studies of sex deter-mination and sex-chromosome evolution in plants. Conven-tional studies on white campion chromosomes have beenconducted for a long time. The most complete informationabout the karyotype structure of this species was presentedby Ciupercescu et al. (1990). Deeper insight into chromo-some structure inS. latifolia and a more complete character-ization of its karyotype are highly desirable. Interestingstudies on these subjects were performed recently, focusingon the localization and characterization of the coding andnon-coding sequences, especially those within the X and Ychromosomes. Some of these studies, performed with mod-ern techniques of molecular cytogenetics, furnished informa-tion concerning Y sequence degeneration (Guttman andCharlesworth 1998; Filatov et al. 2000), localization ofhighly methylated and acetylated chromatin domains(Vyskot et al. 1993, 1998; Siroky et al. 1999), repetitive andtelomeric DNA sequences (Buzek et al. 1997; Scutt et al.1997; Garrido-Ramos et al. 1999; Matsunaga et al. 1999),and replication patterns of chromosomes (Siroky et al. 1994,1999; Vyskot et al. 1998). There also have been attempts atrapid plant sexing on the basis of RAPD markers (Mulcahyet al. 1992) and cytometric DNA estimation (Veuskens et al.1992; Vagera et al. 1994; Doleñel and Göhde 1995). Andro-genic in vitro regenerants have been studied to explain therole of sex chromosomes in sporophytic development andthe degree of Y chromosome degeneration (Veuskens et al.1992; Vagera et al. 1994).

Each of these studies has yielded much essential informa-tion on the karyotype ofS. latifolia, but they also show theneed for further investigation (Moneger et al. 2000; Moneger2001). Some questions concerning molecular cytogenetics,sex chromosome differentiation, and karyotype structure arestill unresolved, for example the character of subtelomericsequences, the level of Y chromosome degeneration, and theamount and distribution of heterochromatin within thekaryotype. Vyskot et al. (1998) suggested thatSilenechro-mosomes possess specific gene-rich euchromatic regionslocalized subtelomerically, but Buzek et al. (1997), Garrido-Ramos et al. (1999), and Matsunaga et al. (1999) have re-ported repetitive DNA sequences for the same chromosomelocations. The level of Y chromosome degeneration is stillunclear in the light of published results on androgenicregenerants inS. latifolia. Veuskens et al. (1992) observedno androgenic embryos lacking the X chromosome amongseveral hundred androgenic progenies of this species thatwere studied, suggesting substantial loss of genetic activityby the Y chromosome. On the other hand, complete in vitroandrogenesis of dihaploid male plants (2n = 24, YY) was re-ported by Vagera et al. (1994), suggesting a lack of degener-ation of essential sporophytically expressed genes on the Ychromosome, and slight X/Y chromosome divergence. Reli-able information on the level of Y chromosome deteriorationwill provide deeper insight into the structure and function ofsex chromosomes inS. latifolia, especially into the possibleevolution of dosage-compensation mechanisms in this spe-cies. Gene dosage regulatory mechanisms are associated

with the evolution of a genetically eroded, highlyheterochromatinized sex chromosome in the heterogameticsex (Charlesworth 1991, 1996). Thus, the occurrence of dos-age compensation is conditioned by the presence and rangeof degeneration of Y-borne genes. If the decrease in Y ge-netic activity in Silene is only slight and affects a few Y-borne genes, unambiguous detection of compensatory mech-anisms on the cytological level can be difficult.

In this study, we analysed the karyotype structure andheterochromatin distribution within the karyotypes of twoclosely related (Desfeux and Lejeune 1996; Desfeux et al.1996) dioeciousSilene species,Silene latifolia and Silenedioica. To our knowledge, the Giemsa C-banded karyotypesof these species have not been presented so far; thus, ourbanding studies provide an addendum to previous data ontheSilenekaryotype. This more-complete information on theheterochromatin amount and distribution in these two spe-cies could help elucidate the character of subtelomericsequences, the degree of Y chromosome degeneration, theaccumulation of non-coding sequences, and the evolution ofsex chromosome systems inSilene.

Material and methods

Plant materialSeeds collected from different natural populations of

S. latifolia and S. dioica near Kraków, Poland (Table 1),were germinated on moistened blotting paper in Petri dishesat room temperature for 3–5 days. The radicles were col-lected, pretreated with ice-cold water for 24 h, and thenfixed in a mixture of glacial acetic acid and absolute ethanol(1:3).

For conventional karyotype studies, fixed root tips wererinsed three times with distilled water, hydrolyzed in 1 MHCl at 60°C for 10 min, and squashed in 45% acetic acid.The cover glasses were removed and the squashes wererinsed in 96% ethanol and left to dry in the open air. Thechromosomes were stained with a 0.1% aqueous solution oftoluidine blue. For densitometric DNA measurements thehydrolyzed material was Feulgen stained with pararosaniline(Sigma, St. Louis, Mo.) according to the procedure de-scribed by Bennett et al. (1998).

Giemsa C-banding procedureThe chromosomes were stained using the slightly modi-

fied C-banding schedule of Jouve et al. (1980). After rinsingin distilled water, radicles were incubated with 45% aceticacid for 1 h. The root tips were cut off and squashed in 45%acetic acid. Squashes were frozen using liquid nitrogen. Af-ter the cover slips were removed, the exposed preparationswere air dried and incubated in absolute ethanol for about24 h, followed by incubation of the preparations in 0.2 MHCl for 2 min at 60°C, rinsing under tap water and in dis-tilled water, incubation in 3% Ba(OH)2 solution for 5 min at38°C, rinsing under warm tap water (until completely clear),incubation in 2× SSC buffer for 1 h at60°C, and staining in2% Giemsa solution (in Sorensen buffer (pH 6.9)) for about45 min.

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Chromosome measurements and karyotype analysisImages were captured and processed using a CCD camera

and Lucia G (Laboratory Imaging Ltd., Praha, Czech Repub-lic) or Multiscan (Computer Scanning Systems Ltd.,Warzawa, Poland) software. For chromosome measurements,one metaphase of high quality per seedling was selected,thus the number of analyzed metaphase plates (Table 1) re-flects the number of analyzed individuals. Female biasamongS. latifolia and male bias amongS. dioicaprogenieswere observed in our material, but almost equal numbers ofmale and female seedlings from each population were cho-sen for precise karyotype analysis.

The relative lengths (rl ) of autosomes were calculated asthe total length (tl) of the haploid autosome (11A) set (rl =(p + q) × 100/11A; p, shorter arm;q, longer arm). Becauseof the considerable X/Y length difference, this methodseems to be more precise than the method proposed byCiupercescu et al. (1990), in which the relative lengths of fe-male or male chromosomes were calculated differently, as apercentage of the female (11A + X) or male (11A + Y) hap-loid set. For further calculations, idiogram construction, andX/Y comparisons, it is more convenient to assume the iden-tity of the female and male autosome set. In the C-bandingstudies, relative heterochromatin amounts are also calculatedwith reference to the total length of the haploid autosomeset. Heterochromatin bands were divided into two groups asfollows: (i) fixed (90–100% frequency in chromosome col-lection) and (ii ) polymorphic (Joachimiak and Kula 1993).For idiogram construction, the fixed band size was calcu-lated as an average of the whole chromosome collection, butthe polymorphic band size was calculated differently (as anaverage of the chromosomes with that specific band).

For determination of the position of the centromere, thearm ratio (r = q/p) and centromere index (i = p × 100/(p +q)) were calculated. Chromosomes were conventionally clas-sified by varying r (according to Levan et al. 1964) andivalues (as recommended by Ciupercescu et al. 1990). Be-cause of the non-uniform contribution of conventionalSilenechromosome types (m and sm) within different metaphases,probably caused by different degrees of chromosome armcondensation (Kakeda and Fukui 1994), an arbitrary, non-conventional, more realistic classification of the isobrachial(r < 1.50,i > 40) and heterobrachial chromosomes was used.Those chromosome types distinguished thusly show morestable frequencies within the analysed material.

DNA measurementsDNA amounts in sex chromosomes of 100 different fe-

male and 100 different male metaphases were determineddensitometrically in arbitrary units. For chromosome identi-fication and further calculations, the absolute sex-chromosome lengths were determined in each Feulgen-stained metaphase. For determination of the existing differ-ences between sex chromosomes, the length and DNA ratiosbetween longer and shorter sex chromosomes within eachmetaphase plate were calculated (XL:XS in females and Y:Xin males).

Results

Conventional karyotypeThe analysed genomes contain metacentric and submeta-

centric chromosomes (Figs. 1a and 1f), but the clear divisionof autosomes into these two major, conventionally classifiedgroups (six metacentrics and five submentacentrics) pro-posed by Ciupercescu et al. (1990) was not confirmed in ourstudy. The autosome complement of both species, as deter-mined in this work, consists of nine pairs of metacentric andtwo pairs of submetacentric chromosomes (Tables 2 and 3).The morphological differences between some autosomes ofintermediate length are small, thus their exact identificationwithin particular preparations could be difficult. Only thelongest homobrachial (1) and heterobrachial (7) chromo-some pairs, and the submetacentric pair (9) with the highestarm ratio coefficient are easily identifiable. InS. dioica(butnot in S. latifolia) the two shortest chromosome pairs (6 and11) are also easily distinguishable from the rest of theautosomes and from each other owing to their length and thedifferences in their centromere positions.

Based on centromere position and its variation, the auto-some complement can be divided non-conventionally intothree chromosome groups: (i) four pairs of metacentric chro-mosomes with low and relatively stable arm ratios (r < 1.25;standard deviation < 0.15); (ii ) one pair (9) of stable sub-metacentrics (r > 1.70 in almost all analysed metaphases);and (iii ) six pairs of chromosomes with more variablecentromere positions. The latter are classified conventionallyby their average arm ratio and centromeric index either assubmetacentrics (one pair in each of the two species, but notthe same pair) or as metacentrics (five pairs). Two of the fiveless-stable chromosome types with the lowest average arm

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Grabowska-Joachimiak and Joachimiak 245

No. of metaphase platesused in karyotype analysis

Species Localities (all near Kraków) NS CS CB 2n

Silene latifolia Wieliczka 60 10 8 24a, 26b

Pychowice 82 10 8 24Sidzina 35 10 7 24

Silene dioica Wieliczka 50 10 8 24Dobranowice 90 10 6 24Tyniec 23 10 5 24

Note: NS, number of seedlings used to establish chromosome number; CS, conventionally stained; CB, C-banded.

aSome plants with extra X heterochromatin.bOne plant with 2n = 26, XY.

Table 1. Material analysed in this study.



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Fig. 1. Classically stained and C-banded chromosomes ofS. latifolia (a–e) and S. dioica(f–i). (a and f) Toluidine blue stained malemetaphases; (b and h) C-banded female metaphases; (c and i) C-banded male metaphases; (d) fragment of C-banded female metaphasewith clearly visible large centric segments of X-heterochromatin; (e) fragment of C-banded male metaphase with sex chromosomes andtwo SAT chromosomes of type 9; (g) fragment of C-banded male metaphase with sex chromosomes and chromosomes 10 and 3 withpolymorphic segments of centric heterochromatin. Scale bar = 5µm.



© 2002 NRC Canada


ratio (r < 1.50) were most frequently observed as meta-centrics, but three others were submetacentrics in manymetaphase plates. From this point of view, our proposeddivision of autosomes into two groups — six homobrachialand five heterobrachial pairs (Table 4) — appears to bemore consistent and is in line with the division ofSileneautosomes into six metacentric (1–6; group A) and five sub-metacentric (7–11; group B) chromosome pairs as proposedby Ciupercescu et al. (1990) and as accepted by other au-thors (Matsunaga et al. 1994, 1999, Siroky et al. 2001).

The are no substantial differences in karyotype structurebetweenS. latifolia and S. dioica(Fig. 1; Tables 2 and 3).Not only are the morphology and distribution of homo-brachial (A) and heterobrachial (B) chromosomes very simi-lar, but the mean absolute lengths of the male and femalegenomes of these two species are also nearly the same(29.76 and 27.85µm in S. latifolia, respectively; 29.51 and27.89µm in S. dioica, respectively). The only major differ-ence is in the length of the Y chromosome. TheS. latifoliaY chromosome is, on average, slightly (0.29µm) longer thanthe S. dioica Y chromosome, but this difference concernsboth chromosome arms and does not affect the arm ratio.

In the conventionally stained preparations, the secondary

ChromosomeRelative length(x±SD, %)

Arm ratio,r (x±SD, %)

Centromere index,i (x±SD, %)

Chromosomeshape Group

1 10.61±1.30 1.15±0.12 46.46±2.82 m A2 9.37±1.13 1.39±0.30 41.83±3.92 m A3 9.26±1.07 1.13±0.09 46.98±1.73 m A4 8.86±1.07 1.40±0.28 41.65±3.74 m A5 8.41±0.90 1.17±0.15 46.01±2.94 m A6 8.07±1.02 1.15±0.13 46.47±2.78 m A7 10.50±1.19 1.56±0.24 39.05±4.41 m B8 9.59±0.96 1.60±0.22 38.48±4.03 m B9 9.31±0.85 2.05±0.37 32.76±5.12 sm B10 8.18±1.02 1.75±0.49 36.31±6.33 sm B11 7.84±0.79 1.66±0.47 37.63±5.43 m BX 15.80±1.86 1.23±0.28 44.81±4.15 m SexY 23.76±4.12 1.10±0.08 47.60±1.54 m Sex

Note: SD, standard deviation; m, metacentric; sm, submetacentric

Table 2. Classification of theSilene latifoliachromosomes.

ChromosomeRelative length(x±SD, %)

Arm ratio,r (x±SD, %)

Centromere index,i (x±SD, %)

Chromosomeshape Group

1 10.31±1.46 1.13±0.10 46.94±2.03 m A2 9.69±0.90 1.21±0.12 45.20±2.21 m A3 9.24±0.79 1.13±0.11 46.96±2.23 m A4 8.73±0.96 1.48±0.36 40.32±4.86 m A5 8.51±0.95 1.32±0.24 43.12±3.37 m A6 7.49±1.07 1.11±0.11 47.40±2.12 m A7 10.37±1.46 1.75±0.54 36.35±6.28 sm B8 9.75±1.18 1.54±0.21 39.38±2.37 m B9 9.52±1.24 2.02±0.46 33.09±5.15 sm B10 9.12±1.13 1.57±0.26 38.92±2.93 m B11 7.27±0.96 1.58±0.32 38.78±3.31 m BX 15.77±1.18 1.26±0.13 44.26±2.20 m SexY 22.54±3.21 1.10±0.10 47.60±1.94 m Sex

Note: m, metacentris; sm, submetacentric.

Table 3. Classification of theSilene dioicachromosomes.

ChromosomeConventionalS.l./S.d.

Proposed hereS.l./S.d. Group

1 m hm A2 m hm A3 m hm A4 m hm A5 m hm A6 m hm A7 m/sm ht B8 m ht B9 sm ht B10 sm/m ht B11 m ht BX m hm CY m hm C

Note: S.l., S. latifolia; S.d., S. dioica; m, metacentric; sm,submetacentric; hm, homobrachial; ht, heterobrachial.

Table 4. Classification ofS. latifolia and S. dioicachromosomesby centromere position.



constrictions (nucleolar organizer regions (NORs)) in SAT(satellited) chromosomes were usually not visible; however,in some metaphase plates single, small-satellited, chromo-somes were evidenced. In both analysed species, three verysimilar satellited chromosome types (5, 7, and 9) were identi-fied (Fig. 2).

Silene X and Y sex chromosomes are of very differentlengths (Fig. 1; Tables 2 and 3) and DNA amounts. In bothspecies, the calculated Y:X length ratio is about 1.5. TheY:X DNA ratio is essentially the same (Table 5). Differencesbetween the two female X chromosomes were also observed,but these differences were small and there was no precisecorrelation between chromosome length and DNA amount(Table 5). In some female metaphase plates the longer Xchromosome possessed less DNA than the shorter X chro-mosome. There were no visible differences in condensedchromatin patterns between male and female cell nuclei, andthe small chromocentres observed in classically stainedpreparations corresponded to the heterochromatic segmentsin the C-banded material. Thus, the different mitotic conden-sation of the sex chromosomes in female cells was probablynot caused by functional condensation (facultative hetero-chromatinization) of one of the X chromosomes during thepreceding interphase. Moreover, the calculated homologuelength ratios (1.04, 1.10, 1.12) for three well-distinguishablepairs (1, 7, and 9) ofS. latifolia autosomes were comparablewith the observed chromosome X length ratios in females ofS. latifolia (1.04) andS. dioica(1.12).

C-bandingThe chromosomes of both species show mainly terminally

and centromerically distributed heterochromatin segments(Figs. 1 and 3). There is only one intercalary band, represent-ing NOR-associated heterochromatin on the shorter arm ofsubmetacentric SAT chromosome 9. In some preparations, thesatellites in these chromosomes were C-band positive (Fig. 1e;Fig. 2). Satellites and intercalary heterochromatin wereobserved only in less-condensed chromosomes; in more-

condensed chromosomes they tended to fuse to one terminallylocated band. In two other SAT chromosome pairs (5 and 7),NOR-associated intercalary heterochromatin was not observed,and the satellites (if visible) were euchromatic (Fig. 2).

AutosomesC-banding provides an opportunity for better characteriza-

tion of some autosome types that appear to be very similarin conventionally stained preparations (Fig. 3). InS. latifolia, the four homobrachial chromosomes with thelowest arm-length ratios (1, 3, 4, and 6) wereheterochromatin rich and showed differently located fixedand (or) polymorphic bands. Two of them (1 and 3) pos-sessed fixed bands on both ends, and the other two showedless-stable polymorphic bands in these positions; only chro-mosome 4 showed a fixed band on the end of the short arm.Each of the homobrachial chromosomes could show poly-morphic bands at the centromeres. Two homobrachial chro-mosomes with higher arm-length ratios (2 and 5) wereheterochromatin poor. A small, terminal, heterochromaticsegment was observed with relatively high (78%) frequencyon the long arm of satellited chromosome 5 only (Fig. 2). Ofthe heterobrachial chromosomes (7–11), only two (9 and 10)showed fixed heterochromatic segments on the shorter arms(9, 10) or at the centromere (9). There was no fixedheterochromatin on the long arm of any of the heterobrachialchromosomes, but additional terminal and (or) centromericbands were observed on chromosomes 8, 10, and 11.

There were some differences between the two C-bandedSilene karyotypes concerning the amount of visible hetero-chromatin (Figs. 1b, 1c, 1h, and 1i), chiefly the presence ofsome fixed and polymorphic bands within particular chromo-some types (Fig. 3).Silene latifoliashows a larger number ofpolymorphic segments of centromeric heterochromatin. Somebanding differences betweenS. latifolia and S. dioica areprobably of diagnostic value: (i) the satellited chromosome 9of S. latifolia shows stable bands on the shorter arm and at thecentromere, and no intercalary and (or) subtelomeric hetero-chromatin on the longer arm, whereas chromosome 9 ofS. dioica shows two stable segments, located on both arms(Figs. 2 and 3); (ii ) the small homobrachial (6) and hetero-brachial (11) chromosomes ofS. dioica are deprived ofheterochromatin and inS. latifolia these chromosomes showheterochromatic bands in different locations, but these bandsare mainly polymorphic (the subtelomeric band on the longerarm of chromosome 6 is observed in 74% of the chromo-somes); (iii ) there are only two chromosome types (3 and 10)with polymorphic bands at the centromere inS. dioica(Fig. 1g). In S. latifolia, eight chromosome types can bearcentromeric heterochromatin, and in chromosome 9 this seg-ment is stable (Fig. 3).

Sex chromosomesSilene spp. sex chromosomes were not only different

lengths, but also showed substantial differences in hetero-chromatin distribution (Fig. 3). The Y chromosome showeda stable heterochromatic segment located in the distal regionof one arm (Figs. 1c, 1e, 1g, and 1i). Proper identification ofthis arm by length was difficult because of the almoststrictly median centromere position in the Y chromosome;however, in the majority of preparations (76%) the banded

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Fig. 2. Satellited chromosomes 5, 7, and 9 ofS. latifolia andS. dioica. TB, classically stained with toluidine blue; CB, C-banded.



arm was longer. There were no visible differences betweenmale sex chromosomes ofS. latifolia and S. dioica, and noother stable or polymorphic heterochromatin segments on Ywere observed in either species.

All of the X chromosomes analyzed contained clearly vis-ible distal C-bands on both arms (Figs. 1b–1e and 1g–1i).Additionally, someS. latifolia X chromosomes showed acentrally located segment of heterochromatin (Figs. 1b and

1d). This kind of heterochromatin was evidenced in plantsfrom one of three analysed populations (Table 1). The extrasegment was highly polymorphic with respect to its presenceand size (Figs. 1b–1e), and probably asymmetrically locatedversus the centromere. The centromeres in C-banded chro-mosomes without centric segments were narrowed andfaintly stained, but their exact localization in relation to thelarger segments was difficult to establish. In some X chro-

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DNA Total length

Species Ratio Average Min./max. Average Min./max.

Silene latifolia XL:XS 1.10±0.09 0.97 / 1.16 1.04±0.13 1.01 / 1.14Y:X 1.46±0.12 1.29 / 1.63 1.48±0.16 1.29 / 1.86

Silene dioica XL:XS 1.05±0.09 0.94 / 1.16 1.12±0.11 1.01 / 1.17Y:X 1.43±0.12 1.33 / 1.80 1.42±0.16 1.31 / 1.88

Note: XL, longer X; XS, shorter X.

Table 5. X1:X2 and Y:X ratios by DNA amounts and total chromosome lengths.

Fig. 3. C-band idiograms ofS. latifolia and S. dioicachromosomes. Black, fixed segments of heterochromatin; grey, polymorphicsegments of heterochromatin.



mosomes, narrowed structures resembling centromeres wereobserved in close proximity to a large heterochromatic seg-ment on the longer-arm side (Fig. 1d). In chromosomes withsmall amounts of this type of heterochromatin, C-positivedots seemed to be located exactly within the primary con-striction (Figs. 1b and 1e). The large pericentric segmentsprobably arose by addition of heterochromatin to the shorterX arm. Chromosomes devoid of this kind of heterochromatinare less metacentric (average arm ratior = 1.33) than chromo-somes with large extra segments (r = 1.13).

Discussion

The presented banding studies throw some light on thecharacter of repeated sequences detected by previous authors(Buzek et al. 1997; Garrido-Ramos et al. 1999; Matsunaga etal. 1999) and provide an understanding of some structuralaspects of theSilenekaryotype, such as existing differencesbetween the X and Y chromosomes, the degree of Y hetero-chromatinization, and better characterization of autosome pairs.

The nature of the distally located heterochromatin can bededuced from the molecular and in situ hybridization studieson S. latifolia performed by Scutt et al. (1997), Buzek et al.(1997), Garrido-Ramos et al. (1999), Siroky et al. (1999),and Matsunaga et al. (1999). Heterochromatin segments ofthis type were occupied mainly by some members of a repet-itive sequence family isolated from either sex chromosomesor the whole genome by Buzek et al. (1997), Matsunaga etal. (1999), and Garrido-Ramos et al. (1999). All of the se-quences (X-43.1, RMY1, andSacI, respectively) are verysimilar, possess short motives resembling the plant conserva-tive telomere sequence, and are classified by the authors assubtelomeric. Distal segments of some chromosomes (5 and7–10) can possess rDNA sequences (Buzek et al. 1997;Siroky et al. 1999, 2001; Matsunaga et al. 1994, 1999).These sequences colocalize with the satellited ends of chro-mosomes 5, 7, and 9, and with the non-satellited ends of theshorter arms of heterobrachial chromosomes 8 and 10. Asshown in our C-banding studies, those segments in hetero-brachial chromosomes 8–10 that were enriched with rDNAsequences are occupied by fixed (9 and 10) or polymorphic(8) heterochromatin, but two other rDNA clusters (in chro-mosomes 5 and 7) are euchromatic. Buzek et al. (1997) andMatsunaga et al. (1999) reported that highly repetitive sub-telomeric sequences and rDNA are located within the same

chromosome domain in only one member of these rDNA-bearing chromosomes (identified by the authors as chromo-some 10). The fluorescent signals of these two sequencespartially overlapped, and it was suggested that the rDNAcluster is more distally located. Matsunaga et al. (1999)showed that repetitive subtelomeric sequences were absenton the longer arm of this chromosome. In our opinion, thischaracteristic heterobrachial chromosome represents a satel-lited chromosome devoid of distal heterochromatin on thelonger arm, which we classified as chromosome 9 (Figs. 2and 3). If so, the rDNA cluster represents the distally locatedsatellite along with the NOR, and the repetitive sequencesare located within the intercalary heterochromatin we de-tected on the shorter arm of this chromosome. Thus, the in-tercalary segment probably has the same molecular structureas the majority of the more distally located segments onnon-satellited chromosomes.

It has been suggested thatSilenechromosomes are cappedwith plant telomere sequences (TTTAGGG)n of the Arabi-dopsistype, and that the highly repetitive sequences detectedare subtelomeric (Buzek et al. 1997; Riha et al. 1998, 2000;Matsunaga et al. 1999). Thus, it is very probable that themajority of terminal Giemsa C-bands identified here are alsosubtelomerically located.

As shown by Giemsa C-banding, many chromosomes inthe Silene karyotype can possess fixed or polymorphicheterochromatic segments at or very near the ends, but somequestions concerning the nature of these segments come intoview. It is broadly accepted that constitutive, positivelyGiemsa C-banded heterochromatin is hypoacetylated andlate-replicating, but Vyskot et al. (1999) and Siroky et al.(1999) showed that the terminal domains ofSilenechromo-somes are hyperacetylated and very early replicating, thusresembling the euchromatic, gene-rich regions of eukaryoticchromosomes.

There is no available information about the molecularstructure of the second, centromerically located class ofSilene heterochromatin. This kind of heterochromatin ishighly polymorphic and more frequently observed inS. latifolia than in S. dioica. Centromeres in highereukaryotes are always located deep in heterochromatin-likestructures, and the minimum amount of this type ofchromatin required for centromere function is about 500 kb(Lohe and Hilliker 1995). The sensitivity of the Giemsa C-band method lies in the range of about 107 bp (Schweizer et

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250 Genome Vol. 45, 2002

Heterochromatin contribution (%)

Subtelomeric Centromeric Intercalary Sum

Silene latifoliaAutosomes 8.07±2.68 2.21±1.34 0.19±0.15 10.47±4.37X chromosome 10.21±3.04 4.28±1.9 — 14.49±5.43Y chromosome 3.78±1.17 — — 3.78±1.17

Silene dioicaAutosomes 6.29±1.80 0.65±0.28 0.17±0.07 7.08±2.67X chromosome 12.84±2.19 — — 12.84±2.19Y chromosome 4.01±1.28 — — 4.01±1.28

Table 6. Amount of heterochromatin in autosomes (calculated as percentage of length ofautosome set) and sex chromosomes (calculated as percentage of X or Y length) ofS. latifoliaand S. dioica.



al. 1987), therefore smaller centromeric clusters of hetero-chromatic sequences are not detectable with this method. Itis highly probable that all centric segments ofSilenechro-mosomes are of similar molecular structure, because thenon-coding sequence families located within particular chro-mosome domains show great homogenization within the plantgenome (Schmidt and Heslop-Harrison 1998).

There is a clear tendency to accumulate centromere-associated heterochromatic sequences inS. latifolia; solidblocks of these sequences are observed as heterochromaticbands in many chromosomes of this species. The highlypolymorphic character of these bands points to their additivenature. Interestingly, the clear tendency to accumulatecentric heterochromatin is observed not only withinS. latifolia autosomes but also within X chromosomes. Insome instances, the blocks of centric X-heterochromatin arethe largest C-positive segments within the karyotype.

The two Silenesex chromosomes are easily distinguish-able by their length differences and also their Giemsa C-banding patterns. In both species, the Y chromosomes in-variably show a terminal (subtelomeric) band on one chro-mosome arm. In most, but not all, male metaphases, thisbanded arm is slightly longer than the non-banded arm. Ex-act identification of the Y arm within a particular malemetaphase by length alone seems problematic, but properGiemsa C-banding resolves this problem. As shown by insitu hybridization studies on meiotic chromosomes under-taken by Buzek et al. (1997) and Farbos et al. (1999), the re-petitive subtelomeric sequences most probably locatedwithin this heterochromatic segment colocalize with the dis-tal Y region homologous to the X chromosome. Thus, thefixed heterochromatic segment identified in our Giemsa C-banding studies can serve as a very useful non-fluorescentchromosomal marker of a Y arm with a pseudoautosomal re-gion, probably in both analysedSilenespecies. It has beensuggested that theS. latifolia Y chromosome also showsheterochromatin near the centromere on thepseudoautosomal chromosome arm (Farbos et al. 1999;Lardon et al. 1999), and that some Y chromosomes can pos-sess distally located repetitive heterochromatic sequences onthe differential arm (Farbos et al. 1999). Surprisingly, nofixed or polymorphic heterochromatin was detected at theselocations in the C-banded Y chromosomes of the analysedspecies. The lack of additional clusters of highly repetitiveX43.1 and RMY sequences on the Y chromosome was alsoshown by Buzek et al. (1997) and Matsunaga et al. (1999).Giemsa C-banding is a good method for cytological visual-ization of heterochromatin within the genome; it works ef-fectively irrespective of the nucleotide sequence. In ouropinion, the lack of visible heterochromatic segments be-yond the pseudoautosomal region of the Y chromosomemight be considered a characteristic feature of this chromo-some.

It has been suggested (Bull 1983; Lucchesi 1978, 1994;Charlesworth 1996; Charlesworth and Charlesworth 1998)that distinct sex chromosomes have evolved from a pair ofhomologues independently in many taxa, sharing commonfeatures including genetic deterioration of the Y chromo-some and mechanisms of dosage compensation. It is alsogenerally believed that Y degeneration results from chromo-somal attrition and accumulation of highly non-coding se-

quences (heterochromatinization). In the two analyzedSilenespecies, no tendency to accumulate heterochromatin withinthe Y chromosomes was detected. In comparison with the Xchromosome and with the autosome set, the Y chromosomesof S. latifolia andS. dioicashow the least amount of hetero-chromatin (Table 6). Furthermore, inS. latifolia, a clear ten-dency to accumulate X-borne heterochromatin wasevidenced. From this point of view, it is far more likely tofind X-specific than Y-specific highly repetitive sequenceswithin the S. latifolia genome.

Acknowledgements

The authors thank Anna Sobieszczanska for methodicaladvice on the C-banding procedure and Joanna Klos fortechnical assistance.

References

Bennett, M.D., Leith, I.J., and Hanson, L. 1998. DNA amounts intwo samples of angiosperm weeds. Ann. Bot.82 (Suppl. A):121–134.

Bull, J.J. 1983. Evolution of sex determining mechanisms. BenjaminCummings, San Francisco, Calif.

Buzek, J., Koutnikova, H., Houben, A., Riha, K., Janousek, B.,Siroky, J., Grant, S., and Vyskot, B. 1997. Isolation and charac-terization of X chromosome-derived DNA sequences from adioecious plantMelandrium album. Chromosome Res.5: 57–65.

Charlesworth, B. 1991. The evolution of sex chromosomes. Sci-ence (Washington, D.C.),251: 1030–1033.

Charlesworth, B. 1996. The evolution of chromosomal sex deter-mination and dosage compensation. Curr. Biol.6: 149–162.

Charlesworth, B., and Charlesworth, D. 1998. Some evolutionaryconsequences of deleterious mutations. Genetica,102/103: 3–19.

Ciupercescu, D.D., Veuskens, J., Mouras, A., Ye, D., Briquet, M.,and Negrutiu, I. 1990. KaryotypingMelandrium album, adioecious plant with heteromorphic sex chromosomes. Genome,33: 556–562.

Desfeux, C., and Lejeune, B. 1996. Systematics of EuromediterraneanSilene (Caryophyllaceae): evidence from a phylogenetic analysisusing ITS sequences. C. R. Acad. Sci. Ser. III Life Sci.319:351–358.

Desfeux, C., Maurice, S., Henry, J.P., Lejeune, B., and Gouyon, P.H.1996. Evolution of reproductive systems in the genusSilene. Proc.R. Soc. Lond. Ser. B Biol. Sci.263: 409–414.

Doleñel, J., and Göhde, W. 1995. Sex determination in dioeciousplantsMelandrium albumand M. rubrum using high-resolutionflow cytometry. Cytometry,19: 103–106.

Farbos, I., Veuskens, J., Vyskot, B., Oliveira, M., Hinnisdaels, S.,Aghmir, A., Mouras, A., and Negrutiu, I. 1999. Sexual dimor-phism in white campion: deletion on the Y chromosome resultsin floral asexual phenotype. Genetics,151: 1187–1196.

Filatov, D.A., Moneger, F., Negrutiu, I., and Charlesworth, D. 2000.Low variability in a Y-linked plant gene and its implications forY-chromosome evolution. Nature (London),404: 388–390.

Garrido-Ramos, M.A., de la Herran, R., Ruiz Rejon, M., and RuizRejon, C. 1999. A subtelomeric satellite DNA family isolatedfrom the genome of the dioecious plantSilene latifolia. Ge-nome,42: 442–446.

Guttman, D.S., and Charlesworth, D. 1998. An X-linked gene withdegenerate Y-linked homologue in a dioecious plant. Nature(London),393: 263–266.

© 2002 NRC Canada




© 2002 NRC Canada

252 Genome Vol. 45, 2002

Joachimiak, A., and Kula, A. 1993. Cytotaxonomy and karyotypeevolution in Phleum sect. Phleum (Poaceae) in Poland. PlantSyst. Evol.188: 17–30.

Jouve N., Diez N., and Rodriguez M. 1980. C-banding in 6×-Triticale × Secale cerealeL. hybrid cytogenetics. Theor. Appl.Genet.57: 75–79.

Kakeda, K., and Fukui, K. 1994. Dynamic changes in the morphol-ogy of barley chromosomes during the mitotic metaphase stage.Jpn. J. Genet.69: 545–554.

Lardon, A., Aghmir, A., Georgiev, S., Moneger, F., and Negrutiu, I.1999. The Y chromosome of white campion: sexual dimorphismand beyond.In Sex determination in plants.Edited by C.C.Ainsworth. Bios Scientific Publishers, Oxford. pp. 89–99.

Levan, A., Fredga, K., and Sandberg, A.A. 1964. Nomenclature forcentromeric position on chromosomes. Hereditas,52: 201–220.

Lohe, A.R., and Hilliker, A.J. 1995. Return of the H-word (hetero-chromatin). Curr. Opin. Genet. Dev.5: 746–755.

Lucchesi, J.C. 1978. Gene dosage compensation and the evolutionof sex chromosomes. Science (Washington, D.C.),202: 711–716.

Lucchesi, J.C. 1994. The evolution of heteromorphic sex chromo-somes. BioEssays,16: 81–83.

Matsunaga, S., Hizume, M., Kawano, S., and Kuroiwa, T. 1994.Cytological analyses inMelandrium album: genome size andfluorescence in situ hybridization. Cytologia,59: 135–141.

Matsunaga, S., Kawano, S., Michimoto, T., Higashiyama, T., Nakao,S., Sakai, A., and Kuroiwa, T. 1999. Semi-automatic laser beammicrodissection of the Y chromosome and analysis of Y chromo-some DNA in a dioecious plant,Silene latifolia. Plant CellPhysiol.40: 60–68.

Moneger, F. 2001. Molecular and evolutionary analysis of a plantY chromosome. C. R. Acad. Sci. Ser. III Life Sci.324: 531–535.

Moneger, F., Barbacar, N., and Negrutiu I. 2000. DioeciousSileneat the road: the reasons Y. Sex. Plant Reprod.12: 245–249.

Mulcahy, D.L., Weeden, N.F., Kesseli, R., and Carroll, S.B. 1992.DNA probes for the Y-chromosome ofSilene latifolia, a dioeciousangiosperm. Sex. Plant Reprod.5: 86–88.

Riha, K., Fajkus, J., Siroky, J., and Vyskot, B. 1998. Developmen-tal control of telomere lengths and telomerase activity in plants.Plant Cell,10: 1691–1698.

Riha, K., McKnight, T.D., Fajkus, J., Vyskot, B., and Shippen,D.E. 2000. Analysis of the G-overhang structures on planttelomeres: evidence for two distinct telomere architectures.Plant J.23: 633–641.

Schmidt, T., and Heslop-Harrison, J.S. 1998. Genomes, genes andjunk: the large-scale organization of plant chromosomes. TrendsPlant Sci.3: 195–199.

Schweizer, D., Loidl, J., and Hamilton B. 1987. Heterochromatinand the phenomenon of chromosome banding.In Results andproblems in cell differentiation. 14. Structure and function ofeukaryotic chromosomes.Edited byW. Hennig. Springer-Verlag,Berlin, Heidelberg. pp. 235–254.

Scutt, C.P., Kamisugi, Y., Sakai, F., and Gilmartin, P.M. 1997. Laserisolation of plant sex chromosomes: studies on the DNA compo-sition of the X and Y sex chromosomes ofSilene latifolia. Ge-nome,40: 705–715.

Siroky, J., Janousek, B., Mouras, A., and Vyskot, B. 1994. Replica-tion patterns of sex chromosomes inMelandrium albumfemalecells. Hereditas,120: 175–181.

Siroky, J., Hodurkova, J., Negrutiu, I., and Vyskot, B. 1999. Func-tional and structural analyses in autotetraploidSilene latifolia.Ann. Bot. 84: 633–638.

Siroky, J., Lysak, M.A., Doleñel, J., Kejnovsky, E., and Vyskot, B.2001. Heterogeneity of rDNA distribution and genome size inSilenespp. Chromosome Res.9: 387–393.

Vagera, J., Paulikova, D., and Doleñel, J. 1994. The development ofmale and female regenerants by in vitro androgenesis in dioeciousplant Melandrium album. Ann. Bot. 73: 455–459.

Veuskens, J., Ye, D., Oliveira, M., Ciupercescu, D.D., Installe, P.,Verhoeven, H.A., and Negrutiu, I. 1992. Sex determination inthe dioeciousMelandrium album: androgenic embryogenesis re-quires the presence of the X chromosome. Genome,35: 8–16.

Vyskot, B., Araya, A., Veuskens, J., Negrutiu, I., and Mouras, A.1993. DNA methylation of sex chromosomes in a dioeciousplant, Melandrium album. Mol. Gen. Genet.239: 219–224.

Vyskot, B., Siroky, J., Hladilova, R., Belyaev, N.D., and Turner,B.M. 1998. Euchromatic domains in plant chromosomes as re-vealed by H4 histone acetylation and early DNA replication.Genome,42: 343–350.



c-banded karyotypes of two silene species with heteromorphic sex chromosomes

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