Heredity 70 11993) 628—633 Received 30 October 1992 Genetical Society of Great Britain Cytology of Vicia species II. Banding patterns and chromatin organization in Vicia atropurpurea Desf R. CREMONINI, S. FUNARI, I. GALASSO* & D. PIGNONE* Dipartimonto di Scierize Botaniche, Via L. Ghini 5, Università di Pisa, 56726 Pisa, arid *Istjtuto del Germoplasma, C.N.R., Via Amendola 125, 70700 Ban, Italia Bandingtechniques on metaphase chromosomes of Vicia arropurpurea were carried out and nuclear DNA content in root meristematic cells was determined by cytophotometric analysis. Feulgen absorptions at different thresholds of optical density provided evidence of the organization of the chromatin in interphase nuclei and permitted an evaluation of the cytophotometrically determined heterochromatin amount. These results corroborated the analysis of the banding patterns. Keywords:banding,chromosomes, fluorochromes, heterochromatin, interphase nucleus structure, Vicia species. chromatin, expressed as the number and/or area of Introduction chromocentres, has been used as a parameter for Theheterochromatin of higher plants has often been heterochromatin determination (Havelange & Jeanny, studied for taxonomical and phylogenetical relation- 1984; Patankar & Ranjekar, 1984a; Hilliker & Appels, ships. Indeed, changes in genome size accompanying 1989; Samod etal.,1992). evolution and/or speciation are mainly due to an The genus Vicia comprises about 120 species and increase or decrease of the highly repeated DNA speciation in this genus is accompanied by variation component that can be partly represented by cyto- both in chromosome size and number and nuclear logical heterochromatin. C-banding is generally sup- DNA content. posed to distinguish euchromatin and constitutive In previous papers Cremonini and co-workers have heterochromatin by cytological techniques (Sumner, reported biochemical and cytophotometric data on 1990). Interphase chromocentres formed from consti- relationships between Vicia species belonging to the tutive heterochromatin also show a positive staining Faba section (Cremonini cx' al., 1 992a; Frediani cx' a!., reaction, but facultative heterochromatinized chromo- 1992). In this connection it is of interest to analyse the somes do not give positive C-banding (Verma, 1988). nuclear DNA content, the banding patterns and the Other banding techniques are useful to localize and chromatin organization evaluated by cytophotometry differentiate different types of heterochromatin such as at different thresholds of optical density of Vicia centromeric heterochromatin, NOR-associated hetero- arropurpurea and compare these results with the data chromatin and noncentromeric heterochromatin. Some from Viciafaba. fluorescent dyes or combinations of fluorescent dyes and appropriately chosen counter-stains can be used to Materials and methods characterize heterochromatin cytologically and to dis- criminate C-bands enriched in AT or GC base pairs Chromosome banding (Schweitzer 1979, 1981). The structural organization of the interphase Seeds of V. atropurpurea were soaked in running tap nucleus has long been studied (Nag!, 1979; Nag! & water overnight and germinated in Petri dishes at 22°C Fusenig, 1979; Anamthawat-Jonsson & Heslop- until secondary roots were about 1 cm long. Root Harrison, 1990) and the amount of condensed meristems were pre-treated with a paradichloroben- zene saturated solution for 2 h at 12°C in a water bath. Correspondence: Professor R. Cremonini, Dipartimento di Scienze After washing, they were fixed in ethanol—acetic acid Botaniche, Università di Pisa, Via L. Ohini, 5,1-56126Pisa, Italy. (3:1, v:v) for 24 h. The root tips were squashed in a NUCLEAR ORGANIZATION OF V/CIA A TROPURPUREA 629 drop of 45 per cent acetic acid and the coverslips content. The number of bands was limited and their removed by the dry ice method and dried overnight. distribution was mainly centromeric (Fig. la). Also the For C-banding the technique outlined by Galasso et interphase chromocentres were limited in number and a!. (1992) was used with the reduction of HC1 treat- extent. Three chromosomes showed telomeric bands ment to 2.5mm.H33258 banding was carried out by on the short arms. The satellited chromosome was staining the slides in a 2 1ug ml1 stain solution at pH7 highly heterochromatic, showing differential staining at Mcllvaine buffer for 10 mm; after a wash in the same both telomeres and at interstitial sites. buffer the slides were dried and mounted. For chromo- Fluorochrome staining allowed us to distinguish mycin A3 and DAPI the technique of Galasso & three different classes of heterochromatin whose dis- Pignone(1991)was followed. tribution is summarized in Fig. 1(b). Six chromosomes possessed type 1 while the satellited chromosome showed all the three types (Fig. 2 a—d, 2 b—e). Type 1 of Cytophotometry heterochromatin reacted positively to C-banding but Forcytophotometry the technique outlined by did not show any reaction to either fluorochrome. On Cremonini et a!. (1992a) was used. Squashes of the the Sat-chromosome it was present at the telomeric root tips of Vicia faba were concurrently stained for and centromeric regions of the satellited arm. Type 2 each group of slides and used as internal standards; reacted positively to C-banding and to CMA3, but Vicia faba was used to convert relative Feulgen arbi- showed quenched fluorescence after DAPI staining: on trary units into picograms of DNA. Feulgen DNA the Sat-chromosome the region of the satellited absorption in early prophase was measured at a wave- arm involved in the secondary constriction showed this length of 550 nm using a Leitz MPV 3 microscope pattern. Type 3 was shown by both DAPI and C-band- photometer equipped with a mirror scanner and an ing, but showed reduced fluorescence after CMA3 HP8 5 computer. staining: on the Sat-chromosome this banding pattern With the same instrument and at the same wave- was present at the centromeric and the telomeric length, the Feulgen DNA absorptions of chromatin regions of the non-satellited arm. None of these classes fractions with different condensation levels, in 4C showed a differential reaction after H33258 staining interphase nuclei, were determined by measurements (Fig. 2c). of one nucleus at different thresholds of optical density according to the method of Frediani et a!. (1992) and Cytophotometry Cremonini eta!.(1992a). The instrument does not read those parts of the nucleus where the optical density is Thenuclear DNA content of early prophases and the lower than the preselected limit. The surface area was surface area of interphase nuclei are summarized in also determined. The results of this analysis are Table 1. From the analysis of hydrolysis curves the reported as a percentage of Feulgen absorption in com- optimal time was 7 mm where the highest DNA parison with the initial value of 100. content was recorded. The values of the thresholds of optical density were mathematically elaborated in order to obtain the exact position of the inflexion point in the curves and it was possible to discriminate between two areas of integral 'It I '7 calculation using Simpson's rule. The residual Feulgen — absorption at the inflexion point represents the cyto- photometrically determined condensed chromatin. - The integral calculation was carried out on the best-fit curves obtained by three curves of optical density for each sample. III liii Results DUl aiinri HID Banding r: Iiiiiiliil I___E1 Thekaryotype of Vicia atropurpurea: with one sate!- Fig. 1 (a) Karyotype of Vicia atropurpurea after BSG lited, three submetacentric and three subtelocentric C-banding; (b) ideotype of the same species: interrupted lines chromosomes it resembled that reported by Chooi represent inconstant bands, the hatchings reflect different (1971). C-banding showed a low heterochromatin reaction to banding techniques. 630 ft CREMOMNIETAL. Fig.2Vicia atropurpurea chromosomes stained with Chromomycin A3 (a—d), DAPI (b—e) and Hoechst 33258 (c). The symbol (v) indicates the chromatin associated to the NOR bright fluorescent after CMA and pale after DAPI; the symbol (Y) indicates chromatin segments pale fluorescent after CMA and bright fluorescent after DAPL The results of measurements made on 4C interphase content and having the same surface area. At threshold nuclei at different thresholds of optical density are 6 of optical density Vicia atropurpurea showed 73.71 reported in Table 2 and in Fig. 3. In order to compare per cent of Feulgen absorption and Vicia faba 94.11 the values of the curves of optical density we have per cent. By increasing the thresholds of optical chosen, in each sample, interphase nuclei having density, when the instrument reads only the optically Feulgen absorption values corresponding to 4C dense, more condensed chromatin, the Feulgen NUCLEAR ORGANIZATION OF V/CIA A TROPURPUREA 631 Table 1 Nuclear DNA content of early prophases in the 00 a root meristems and surface area of interphase nuclei (4C) in Vicia samples. Each nuclear DNA content and surface area I are the mean of fifty determinations carried out in five root 80- meristems I Vicia Nuclear DNA content Surface area 060 . (pg, mean S.E.) (urn2 S.E.) 0- species 0)0 0 U atropurpurea 14.71 1525.21 24.20 40 - A (2n= 14) 0' I A a faba 53.31* 3300.09±51.03 (2n= 12) 20- *From Bennett and Smith (1976). I . I 3 9 5 21 27 33 Table 2 Percentages of Feulgen absorption (mean S.E.) at Thresholdsof optical density different thresholds of optical density of interphase nuclei in Fig.3 Percentage