INTRODUCTION

Automated chromosome analyses have beeninitially implemented in order to improve the ac-curacy and speed of karyotyping in mammaliancells (CASTELMAN and MELNYK 1976). The con-comitant development of microelectronics andrelevant software enabled the design of a com-puter-based chromosome image analyses system(CHIAS). In plants this approach was initiallyutilized for scanning of rye metaphase chromo-somes (FUKUI 1986). Barley chromosomal com-plement was used to reveal the applicability ofCHIAS for standardization of karyotyping andfor screening of in situ hybridization (FUKUI

1988). The utility of chromosome imaging tech-nique in cultured cells and plants treated withradiation or chemical mutagens was envisaged.

CHIAS was proposed as a tool for investiga-tion of the density characteristics of individualplant chromosomes (FUKUI and MUKAI 1988;NAKAYAMA and FUKUI 1997; KATO and FUKUI

1998).Condensation pattern of chromosomes was

also found to be an essential factor for intra-chromosomal distribution of chromosomal al-terations in mammalian genome. It was shownthat condensed chromatin appears to be moreresistant to radiation-induced damage than un-condensed one in CHO cells (SLIJEPCEVICH andNATARAJAN 1994). Clear indication was provid-ed that chromosomes with high gene density arepreferentially repaired in human cells (SUR-RALLES et al. 1997). At present the link betweenchromatin structure, DNA repair and chromo-some fragility is widely accepted (SURRALLES etal. 1998).

Position of the Giemsa bands is thought tobe informative for the intrachromosomal distri-

CARYOLOGIA Vol. 55, no. 2: 99-103, 2002

CHIAS-based positioning of recombination hotspotsand Giemsa bands in a multireconstructedbarley karyotypeG. VENORA1, L. STOILOV2, K. GECHEFF3 and R. CREMONINI4, *

1 Stazione Sperimentale di Granicoltura per la Sicilia, Via Rossini 1, 95041 Caltagirone, Italia.2 Institute of Genetics “Acad. D. Kostoff” Sofia 1113, Bulgaria.3 AgroBioInstitut, 2232 Kostinbrod 2, Bulgaria.4 Dipartimento di Scienze Botaniche, Università di Pisa, Via L. Ghini 5, 56126 Pisa, Italia.

Abstract -The chromosome complement of reconstructed barley karyotype PK88 was analyzed by computer-aided Chromosome Image Analysis System(CHIAS). Fine mapping of Giemsa N-bands and regions with increased meiot-ic recombination activity along each individual chromosome was achieved. It wasalso found that CHIAS-visualized condensation profiles can be utilized as a reli-able criterion for subtle differentiation of hetero- and euchromatin domainswithin a defined chromosomal regions. Application of CHIAS on karyotypes withdistinct chromosome morphology was found to be an appropriate and reliabletool for screening of changes in chromatin compactness and its functional char-acteristics.

Key words: chromosome banding, Hordeum, image analysis, plant chromo-somes, recombination, reconstructed karyotype.

* Corresponding author: fax ++39 050 551345; e-mail:cremonio@dsb.unipi.it

bution of constitutive chromatin domains, gen-erally considered as inert genomic regions. Sincerecombination hot-spots are known to representislands of high gene density in barley and wheat(KUNZEL et al. 2000), the comparison of intra-chromosomal distribution of both of these pa-rameters with the respective densitometric pro-files is supposed to highlight the link betweenchromatin structure and function in a specificchromosomal segments. On the other handknowledge of the precise condensation patternsof individual chromosomes might help to sub-stantiate the influence of chromatin organizationon differential mutation induction. Based on thisassumption we applied CHIAS for precise map-ping of Giemsa bands and regions with highmeiotic recombination activity in barley kary-otype PK88 in order to find out whether andhow their distribution matches with the respec-tive condensation patterns of each individualchromosome.

MATERIALS AND METHODS

Plant materialMultireconstructed barley karyotype PK-88 syn-

thesized via irradiation and intercross methods wasutilized throughout. The karyotype contains translo-cations between chromosomes 1 and 2, 3 and 4, 5 and7 as well as pericentric inversion within chromosome6 (GECHEFF 1989).

Cytological preparationsAfter soaking of the seeds for 1 hr in tap water they

were grown on a moist filter paper in the dark at 24oCuntil the root tips reached about 1,5 cm length. Accu-mulation of the metaphases was achieved either bytreatment with 0.025% colchicine saturated with al-pha-bromonaphtalene or by exposure to ice-cold wa-ter overnight. The material was fixed in ethanol:aceticacid (3:1, v/v). Squashes were made by squeezing of

the root meristematic cells in a drop of 45% aceticacid on a slide surface and brief heating. The cover-slips were removed by liquid nitrogen and the slideswere dried for one week. Staining of the material wasperformed by conventional Feulgen method, includ-ing hydrolysis for 7 min in 1N HCL at 60oC. Theslides were subjected, after staining, to three 10 minwashes SO2 water prior dehydration and made per-manent by mounting in DPX (Fluka). 50 metaphasespreads per variant were selected for further analyses,derived from at least 5 individual slides.

Karyomorphometry and densitometric profilesSlides were analysed according to VENORA et al.

(1991). Karyotype morphometry was assessed by theautomated image analysis system IKAROS 3.40 (Meta-system) software package. On the kariograms obtainedby IKAROS after rectification, using KS400 software,a mid-rib line of a chromatid was also interactivelydrawn. The density distribution along the chromatidwas displayed on the monitor. Sequential data of thegrey values of the pixels were recorded and were sub-jected to the further statistical treatment (mean andStandard deviation). (FUKUI and IIJIMA 1991; FUKUI

1992; KATO and FUKUI 1998; KTOSERUBA et al. 2000).

Intrachromosomal distribution of recombination hot-spots and Giemsa bands

Distribution pattern of recombination hot spotsand Giemsa bands was viewed by subdividing of thewhole genome into 1000 units or miligenomes (KUN-ZEL et al. 2000). Localization of chromosomal regionswith high meiotic recombination activity (<1.0Mb/cM) was derived from the published pattern ofthe normal barley karyotype (KUNZEL et al. 2000) andrecalculated according to the values obtained byCHIAS. Specific distribution of Giemsa N-bandswithin PK88 was based on the previous data of GECH-EFF (1989) and also rearranged according to theCHIAS measurement of the respective chromosomalentities. The designation of chromosomes follows theTriticeae nomenclature for barley (LINDE-LAURSEN etal. 1995). Resulting images were created in Correl-Draw and combined there with the CHIAS-baseddensitometric profiles in a comparative manner.

100 VENORA, STOILOV, GECHEFF and CREMONINI

Table 1 – Karyomorphometric data obtained after CHIAS measurement of the individual chromosomes of karyotype PK 88.

Chromosome Relative Chromosome Long arm Short arm Satellite Arm Ratio ChromosomeNo length % length (µm) (µm) (µm) (µm) long/short type

12 17.40 11.29 a 6.66 4.63 1.44 e m21 12.03 7.81 c 4.45 3.36 1.33 f m34 11.35 7.37 d 5.17 2.19 2.36 a sm43 17.78 11.54 a 7.29 4.25 1.71 d sm57 13.76 8.93 b 5.51 3.42 1.61 d m6i 13.93 9.04 b 4.93 2.51 1.61 1.97 b sm75 13.73 8.91 b 4.81 2.78 1.32 1.73 c sm

Values followed by the same letter are not significantly different (P=0.05) according to Cluster analysis of SCOTT and KNOTT (1974).

RESULTS AND DISCUSSION

Results obtained after CHIAS analyses of thechromosomal complement of karyotype PK 88 aresummarized in Table 1. The data are averaged val-ues calculated from measurement of at least 10metaphase plate per each chromosomes pair. Basedon the respective morphometric indexes idiogram-matic representation of the haploid chromosomalnetwork was created (Fig. 1). The overall karyotypemorphology displays the same characteristics asthose resulting from the previously applied con-ventional N-banding technique (GECHEFF 1989).Such an appearance is a confirmation of the uniquemorphological features of karyotype PK 88 and itspotential for precise cytological marking.

By it vary nature CHIAS-based study of thechromosomal network is supposed to be an ade-quate tool for density characterization of specificchromosomal regions. To reveal this potential weutilized CHIAS for comparison of the overall den-sitometric profiles with the longitudinal distribu-tion of recombination hot-spots and Giemsa N-bands along the individual chromosomal entities(Fig. 2). From the spatial representation of thechromosome images it is evident that standardFeulgen staining generates discernible density char-acteristics of the defined chromosomal domainsand could successfully complement conventionalGiemsa one applied previously for the same pur-poses (FUKUI and KATO 1998). The correspondingdensitometric profiles reflect the density distribu-tion along the chromatid, visualizing both the gen-eral condensation patterns and local fluctuation inthe chromatin compactness. It is important to men-tion that from the plotted density distributions theborders of condensed and dispersed regions with-in each chromosome became easily distinguishable.

Based on CHIAS-obtained numerical data therespective chromosome images of individualchromosomes visualizing the subtle location ofGiemsa N-bands and recombination hot-spots inmiligenomes were interactively drawn based on thescale utilized for densitometric profiles. In generalthe distribution of Giemsa-bands appears to followthe condensation patterns of individual chromoso-mal arms, namely their location as a whole coin-cides with the chromosome stretches of high chro-matin density. As expected, centromeric and nu-cleolus organizer regions appeared to be sites of thelowest chromatin condensation. Giemsa-stained re-gions of the genome are denoted as heterochro-matic ones and the observed correlations make

CHIAS an adequate tool for screening of both in-ert and active genomic regions. The validity of thisstatement was supported also from the fact that re-combination hot-spots were obviously found to re-side in chromosomal segments with low densitycharacteristics, presumably euchromatic ones. Itmight be assumed that joint application of CHIASand karyotypes with suitable chromosomal consti-tution increases substantially the resolution pow-er of the automated chromosome image analyses.

According to the observed relationships it istempting to speculate that a number of parameterscharacterizing local chromosome architecturemight became accessible by our experimental ap-proach. Heterochromatin appearance is thoughtto represent a specific kind of chromatin packag-ing required for gene inactivation (HENNING

1999). It was also shown that maize genes behaveas recombination hot-spots (DOONER and MAR-TINEZ-FEREZ 1997). Thus condensation patterns ofchromosomes obtained by CHIAS can be utilizedas a reliable complement to FISH-based genemapping in plants. Dispersion of repetitive DNAalong the chromosomes could be also visualized.On the other hand studies on the specific appear-ance of plant chromosomes after in situ treatmentwith restriction endonucleases (FREDIANI et al.1987; SCHUBERT et al. 1990) might benefit sub-stantially from CHIAS application. In this respectCHIAS-based fine mapping of chromatin domainsis expected to be relevant approach for our con-

CHIAS ON RECONSTRUCTED BARLEY KARYOTYPE 101

Fig. 1 – Idiogram of the haploid chromosomal complementof karyotype PK 88 constructed according to the morpho-metric parameters obtained by CHIAS.

102 VENORA, STOILOV, GECHEFF and CREMONINI

Figs. 2a-g – In each photo the upper part is reported the intrachromosomal distribution of recombination hot-spots and Giem-sa N-bands versus condensation patterns of PK 88 individual chromosomes. Positioning of the regions with high recombi-nation activity (<1.0 MB/cM as identified by KUENZEL et al., 2000) is denoted in squared regions and black regions are rep-resentative for Giemsa N-bands, heterochromatic regions; Cen: centromeric regions; NOR: nucleolus organizer regions. In thelower part the Feulgen stained chromosome and the densitometric profile are reported: the horizontal axis indicates the greyvalue, the vertical represents the number of pixels from the end of the short arm to the terminal of long arm.

a b

c d

e f

tiguous studies onwards to reveal intrachromoso-mal distribution of DNaseI-sensitive regions andmutation hot-spots resulting from treatment withvarious clastogenic agents in barley genome.

Acknowledgments – We are grateful to V.Manova and D. Telbizova for the valuable technicalassistance. This work was partially supported by theBulgarian National Science Fund, Grant No K-804and by a grant from MURST (Rome).

REFERENCES

CASTELMAN K.R. and MELNYK J. H., 1976 – An au-tomated system for chromosome analyses: final re-port. JPL document 5040-30. Jet Propulsion Lab-oratory, Calif. Inst. Tech., Pasadena, Calif., USA.

DOONER H.K. and MARTINEZ-FEREZ I.M., 1997 – Re-combination occurs uniformly within the bronzegene, a meiotic recombination hot-spot in the maizegenome. Plant Cell, 9: 1633-1646.

FREDIANI M., MEZZANOTTE R., VANNI R., PIGNONE

D. and CREMONINI R., 1987 – The biochemicaland cytological characterization of Vicia faba DNAby means of MboI, AluI and BamHI restriction en-donucleases. Theor. Appl. Genet., 75: 46-50.

FUKUI K., 1986 – Standartization of karyotyping plantchromosomes by a newly developed chromosomeimage analyzing system (CHIAS). Theor. Appl.Genet., 72: 27-32.

–, 1988 – Analysis and utility of chromosome infor-mation by chromosome image analyzing systemCHIAS. Bull. Natl. Inst. Agrobiol., 4: 153-176.

–, 1992 – Chromosome Engineering: Historical per-spective and prospect. In: “Proc. Sec. Jpn. Sympo-sium Pl. Chromos.” Plant Chromosome Researchp. 61-72.

FUKUI K. and IIJIMA K., 1991 – Somatic chromosomemap of rice by imaging methods. Theor Appl.Genet., 81: 589-596.

FUKUI K. and MUKAI Y., 1988 – Condensation patternas a new image parameter for identification of smallchromosomes in plants. Jpn. J. Genet., 63: 359-366.

GECHEFF K.I, 1989 – Multiple reconstruction of bar-ley karyotype resulting in complete cytologicalmarking of the chromosome complement. Theor.Appl. Genet., 78: 683-688.

HENNING W., 1999 – Heterochromatin. Chromoso-ma, 108: 1-9.

KATO S. and FUKUI K., 1998 – Condensation pattern(CP) analysis of plant chromosomes by an improvedchomosome image analysing system, CHIAS III.Chromosome Research, 6: 473-479.

KOTSERUBA V.V., VENORA, G., BLANGIFORTI S., RUFFI-NI CASTIGLIONI M. and CREMONINI R., 2000 – Cy-tology of Vicia species. IX. Nuclear DNA amount,chromatin organisation and computer aided kary-otyping of a russian accesion of Vicia faba L. Cary-ologia, 53: 195-204.

KUNZEL G., KORZUN L. and MEISTER A., 2000 – Cy-tologically integrated physical restriction fragmentlength polymorphism maps for the barley genomebased on translocation breakpoints. Genetics, 154:397-412.

LINDE-LAURSEN I., VON BOTHMER R. and JACOBSENN., 1995 – Karyotype differentiation and evolutionin the genus Hordeum. Kew Chrom. Conf, IV:233-245.

NAKAYAMA S. and FUKUI K., 1997 – Quantitative chro-mosome mapping of small chromosmes by im-proved imaging on CHIAS II. Genes Genet. Syst.,72: 35-40

SCHUBERT I., 1990 – Restriction endonucleases (RE)banding of plant chromosomes. Caryologia, 43:117-130.

SLIJPCECIC P. and NATARAJAN A.T., 1994 – Distribu-tion of radiation-induced G1 exchange and termi-nal deletion break-points in Chinese hamster chro-mosomes as detected by G-banding. Int. J. Radiat.Biol., 66: 747-755

SCOTT A.J. and KNOTT M., 1974 – A cluster methodfor grouping means in the analysis of variance. Bio-metrics, 30: 505-512.

SURRALLES J., PUERTO S., RAMIREZ M.J., CREUS A.,MARCOS R., MULLENDERS L.H.F. and NATARAJANA.T., 1998 – Mut. Res., 404: 39-44.

SURRALLES J., SREBASTIAN S. and NATARAJAN A.T.,1997 – Chromosomes with high gene density arepreferentially repaired in human cells. Mutagene-sis, 12: 437-442.

VENORA G., CONICELLA C., ERRICO A. and SACCAR-DO F., 1991 – Karyotyping in plants by an imageanalysis system. J. Genet. Breed., 45: 233-240.

Received October 8, 2001; accepted November 28, 2001

CHIAS ON RECONSTRUCTED BARLEY KARYOTYPE 103

g