Report of New B Chromosomes

Cytogenet Genome Res 106:184–188 (2004)DOI: 10.1159/000079285

The occurrence of different Bs in Cestrumintermedium and C. strigilatum (Solanaceae)evidenced by chromosome bandingJ.N. Fregonezi,a C. Rocha,a J.M.D. Torezanb and A.L.L. Vanzelaa

a Departamento de Biologia Geral, CCB, Universidade Estadual de Londrina, Londrina, PR;b Departamento de Biologia Animal e Vegetal, CCB, Universidade Estadual de Londrina, Londrina, Parana (Brazil)

Supported by the Brazilian agencies CAPES, CNPq, Fundaçao Araucaria andCPG-UEL.

Received 10 August 2003; manuscript accepted 9 January 2004.

Request reprints from Andre L. Laforga VanzelaDepartamento de Biologia Geral, CCBUniversidade Estadual de Londrina, CEP 86051-990Londrina, PR (Brazil); telephone: +55 43 3371-4509fax: +55 43 3371-4207; e-mail: andrevanzela@uel.br

ABC Fax + 41 61 306 12 34E-mail karger@karger.chwww.karger.com

© 2004 S. Karger AG, Basel0301–0171/04/1064–0184$21.00/0

Accessible online at:www.karger.com/cgr

Abstract. In this study, we examine the morphology, mitot-ic stability, meiotic behavior and the composition of hetero-chromatin of B chromosomes in Cestrum intermedium andC. strigilatum. The results showed that B chromosome numbershows intraindividual variation in the root meristem, whichseems to lead to a slight rate of B elimination in this somatic

tissue. B chromosomes in both species were similar in size andshape, but differed with regard to the type, size and distributionof heterochromatin. Possible evolutionary pathways for B chro-mosome origin in Cestrum are discussed.

Copyright © 2004 S. Karger AG, Basel

B chromosomes have been reported in many plants and ani-mals, and they have been very well studied in economicallyimportant plant species such as Zea mays (Cheng and Lin,2003) and Secale cereale (see Puertas, 2002). Generally, B chro-mosomes differ from the normal A chromosome complement,in size, form and DNA composition, but are easily recognizedbecause they do not pair and recombine with any of the A chro-mosomes at meiosis. They may or may not be present in certaintissues, individuals, or populations, and probably originatefrom the normal complement by different mechanisms, aschromosome fragmentation, DNA amplification and introgres-sion by interspecific hybridization (see Camacho et al., 2000;Dhar et al., 2002). It is accepted that inter- and intra-individualnumerical variations of B chromosomes occur due to meioticand mitotic instability, and in most cases these chromosomeshave little or no phenotypic effect (Beukeboom, 1994; Puertas,2002).

Bs are partially or totally composed of one or more repeti-tive DNA families whose copy number can be higher in Bs thanin the A chromosomes (Camacho et al., 2000). Different typesof DNA segments have been reported in Bs of plant species,including ribosomal cistrons and Bd49 tandem repeat sequencein Brachycome (Franks et al., 1996; Houben et al., 1997), hete-rochromatin in Medicago (Hossain and Bauchan, 1999), andPREM-1 retroelements and centromeric and telomeric se-quences in Zea mays (Stark et al., 1996; Qi et al., 2002).

Cestrum comprises over 250 tropical and subtropical Amer-ican species (Smith and Downs, 1966). The chromosome num-ber of these species is 2n = 2x = 16, organized mainly in meta-and submetacentric chromosomes, with heterochromatin lo-cated at distal and intercalary positions and near the centro-mere. This heterochromatin was shown to be organized intofour different groups: (i) cold-sensitive regions (CSRs), associ-ated with AT-rich sites; (ii) GC-rich, adjacent to NOR; (iii) GC-rich, not-associated with NOR; and (iv) weak Giemsa C-bands(dots) dispersed along chromosomes (Berg and Greilhuber,1993a, b). This genus is particularly important because theirrepresentatives are widely used in reforestation programs in theSeasonal Semidecidual Forest of North Parana state (SouthernBrazil). During routine cytogenetic studies of tropical tree spe-cies in our laboratory, representatives of five populations oftwo species of Cestrum (C. intermedium Sendtn. and C. strigi-latum R. and P.) were examined, and Bs were found in seed-lings obtained from one tree of each species under natural con-

Cytogenet Genome Res 106:184–188 (2004) 18543

ditions. Both species can be found together in the forests bor-der, but C. strigilatum is more abundant above 600 meters ofaltitude and C. intermedium under that. The aim of this studywas to determine and compare the morphology, mitotic stabili-ty, meiotic behavior and heterochromatin types of Bs in thesetwo species.

Materials and methods

Plant materialSeeds of C. intermedium containing Bs were collected from only one tree

located at the “Parque Estadual Mata dos Godoy”- PEMG - (Londrina, Para-na, Brazil). Similarly, seeds of C. strigilatum were collected from only onetree located at the “Terra Indıgena Sao Jerônimo” – TISJ – (Sao Jerônimo daSerra, Parana, Brazil), about 90 km from Londrina. Both areas are sections ofthe Seasonal Semidecidual Forest of Southern Brazil. Twenty-eight seedlingsof C. intermedium and twenty-five of C. strigilatum were obtained and culti-vated in tubes at a nursery and used for cytogenetic analysis. Approximatelyhalf of the seedlings showed one B, except for C. intermedium that showedtwo individuals with two Bs. Eight seedlings from each species were analysedto investigate intraindividual variation in B number in metaphase spreads.Seedlings were cultivated for flower production. Vouchers of each seed donortree were deposited at the FUEL herbarium.

Conventional stainingRoot tips pretreated with 0.05% colchicine and young anthers were fixed

in Carnoy’s solution (ethanol:acetic acid, 3:1, v:v) for 12–24 h and stored at–20 °C. Chromosome preparations were stained using Feulgen (mitosis) and2% Giemsa (meiosis). Slides were mounted with Entellan (Merck). Cellswere analyzed for the presence of Bs, and grouped as: (i) cells without Bs,(ii) with one, and (iii) with two Bs, only when the presence or absence of Bswas clearly evident.

Cold-sensitive regions (CSRs) stainingRoot tips were collected and kept in a Bristol nutrient solution (Bold,

1949) at 0–2 °C for 24 h, and fixed in Carnoy. Squashes were performed withroot tips in a drop of 45% acetic acid, and slides stained with 2% Giemsa.

Chromosome banding and FISHChromosome banding was performed on root tips softened in 4% cellu-

lase plus 40 % pectinase at 37 °C for 1 h and squashed in a drop of 45% aceticacid. Slides were treated according to Schwarzacher et al. (1980) and stainedwith 2% Giemsa in distilled water, or in 0.5 mg/ml CMA3 and 2 Ìg/ml DAPI.FISH was performed according to Cuadrado and Jouve (1994a) with modifi-cations. The probes utilized were pTa71 and pTa794 (containing 18S-5.8S-26S and 5S rDNA, respectively) labeled with biotin-14-dATP by nick trans-lation. Signals were detected with avidin-FITC and chromosomes counter-stained with 2.5 Ìg/ml propidium iodide.

Slides stained with Giemsa were mounted with Entellan, the slides withfluorochromes were mounted with 50 % glycerol in McIlvaine buffer, andthose used for FISH were mounted with antifade. Photographs were takenwith Kodak Imagelink HQ 25 ISO for conventional staining, Kodak T-Max100 ISO for fluorochrome banding and Kodak Proimage 100 ISO for FISH.

Results

The two Cestrum species had similar karyotypes of 2n =2x = 16, which included mainly metacentric and submetacent-ric type chromosomes, except for the smallest pair which wassubmetacentric in C. strigilatum (Fig. 1A) and more acrocent-ric in C. intermedium (Fig. 1B). Conventional analysis showedindividuals with and without B chromosomes in both species.There was no variation in the morphology among Bs, whichpresented similar size and acrocentric shape (Fig. 1A, B).

Table 1. B chromosome frequency in eight samples of Cestrum inter-medium

Samples Cells without B Cells with 1 B Cells with 2 B Total cells

PEMG-1 0 36 (100%) 0 36 PEMG-6 6 (4.3%) 134 (95.0%) 1 (0.70%) 141 PEMG-7 1 (1.8%) 56 (98.2%) 0 57 PEMG-8 1 (1.7%) 59 (98.3%) 0 60 PEMG-15 3 (5.6%) 51 (94.4%) 0 54 PEMG-18 1 (3.6%) 27 (96.4%) 0 28 PEMG-19 1 (1.1%) 88 (98.9%) 0 89 PEMG-24 0 32 (37.2%) 54 (62.8%) 86

Total 13 483 55 551

Table 2. B chromosome frequency in eight samples of Cestrum strigila-tum

Samples Cells without B Cells with 1 B Cells with 2 B Total cells

TISJ-1 1 (2.8%) 35 (97.2%) 0 36 TISJ-5 0 26 (100%) 0 26 TISJ-10 0 19 (100%) 0 19 TISJ-12 0 24 (100%) 0 24 TISJ-15 1 (3.1%) 31 (96.9%) 0 32 TISJ-20 1 (3.4%) 28 (96.5%) 0 29 TISJ-25 0 21 (100%) 0 21 TISJ-27 1 (4.0%) 24 (96.0%) 0 25

Total 4 208 212

Bs were easily recognized because they were about three timessmaller (2.93 Ìm) than the chromosomes of the normal comple-ment (7.25–10.48 Ìm). In addition, Bs of C. strigilatumappeared as typical univalent in about 80% of pollen mothercells (Fig. 1C).

Somatic cells of both species were analysed to detect intra-and inter-individual variations in the number of Bs (Table 1).In C. intermedium, only one seedling (PEMG-1) showed nointraindividual variation in B number, but the remaining sevenindividuals showed between cell variation, with clear predomi-nance of one of the cell classes. If B number in the modal classwould coincide with the original number of Bs in the zygote, wecould infer that seven of the individuals analysed in C. interme-dium had 1B in the zygote stage, and the remaining individual(PEMG-24) had 2B. The asymmetry of B number distribu-tions, with lower frequency of 2B than 0B classes in 1B seed-lings, and the absence of 3B cells but high frequency of 1B inPEMG-24, suggests a tendency for B chromosome eliminationin this somatic tissue (root meristem). The same was apparentin C. strigilatum, although the tendency to B eliminationseemed to be lower in this species since it occurred in only halfof the individuals analysed (Table 2).

The results obtained from Giemsa C-banding showed thatC. strigilatum (Fig. 1D) and C. intermedium (Fig. 1E) had ter-minal and intercalary heterochromatic blocks in the A comple-ment, and in C. intermedium additional weak centromeric

18644

Cytogenet Genome Res 106:184–188 (2004)

Fig. 1. (A) Metaphase of C. strigilatum with one B. Arrows indicate the submetacentric pair. (B) Metaphase of C. interme-dium with two Bs. Arrows indicate the acrocentric pair. (C) Meiotic metaphase of C. strigilatum, showing an unpaired B.(D) Giemsa C-banding in C. strigilatum. (E) Giemsa C-banding in C. intermedium. Arrows point to the Bs in D and E.(F) Metaphase cold treated in C. strigilatum. Arrow indicates the B. (G) C-CMA3 banding in C. strigilatum. (H) C-DAPI banding inC. strigilatum. (I) C-CMA3 banding in C. intermedium. (J) C-DAPI banding in C. intermedium. Arrows point to CMA3

0/DAPI0 inA and B chromosomes.

bands appeared in some chromosomes. Centromeric bands inC. strigilatum were not seen. Significant differences in the dis-tribution and size of heterochromatic blocks in Bs of the twospecies were observed. In Cestrum intermedium, Bs exhibitedlarge heterochromatic blocks at both terminal positions, oc-

cupying about 50% of the chromosome, and in some prepara-tions, thin intercalary “dots” were also observed (Fig. 1E). Bchromosomes of C. strigilatum exhibited up to three smallintercalary blocks (dots) on the short and long arms, totalingabout 30% of the chromosome (Fig. 1D).

Cytogenet Genome Res 106:184–188 (2004) 18745

Fig. 2. Metaphase of C. intermedium with two Bs after FISH with 45Sand 5S rDNA probes. Large arrows point to Bs without hybridization signals,and small arrows point to 5S rDNA sites on the A chromosomes.

S54

S54

S5 S54

S54

S5

Results of C-CMA3/DAPI banding showed that most of theheterochromatic blocks in the A complement of C. strigilatumevidenced by Giemsa C-banding were GC- or AT-rich. TheseAT-rich blocks were sensitive to cold treatment. Interestingly,the Giemsa C-banded heterochromatin detected in the Bs of C.strigilatum was neither AT- nor GC-rich (Fig. 1G, H). The Acomplement of C. intermedium had pericentromeric AT-richblocks that were not cold sensitive, fine intercalary CSRs asso-ciated with the AT-rich blocks, plus terminal and intercalaryGC-rich blocks. The terminal Giemsa C-banded blocks visual-ized in the Bs of C. intermedium were CMA3

0/DAPI0, in bothsamples with one and two Bs (Fig. 1I, J). Intercalary “dots”visualized with C-Giemsa banding were not evident with fluo-rochromes. CSRs were not detected in the Bs of either species(Fig. 1F). FISH with 45S and 5S rDNA probes showed hybridi-zation signals on A chromosomes but not on B chromosomes inboth species (Fig. 2). Two major signals of 45S rDNA probewere found in terminal regions of a metacentric and a submeta-centric chromosome pair. The latter also showed a proximal 5SrDNA site in the long arm. A minor 45S rDNA site wasdetected at terminal positions of the long arm in another M-SMchromosome pair (Fig. 2).

Discussion

B chromosomes might be present in up to 15% of animaland plant species (Beukeboom, 1994). Detailed studies includ-ing the molecular composition and transmission rate of Bs havebeen conducted in a number of plants (Puertas, 2002), but stud-ies on B chromosomes in the Solanaceae family are scarce, aswell as in other tropical trees. Acrocentric B chromosomes haverecently been described in the Solanaceae Cestrum parqui andin the hybrid C. parqui × C. aurantiacum (Sykorova et al.,2003). These Bs showed overlay of rDNA 5S and 45S at termi-nal/subterminal positions of the short arm, and rDNA 45S atterminal positions of the long arm, besides dots of BR23sequences (a 405-bp segment composed of 9–10 bp minisatel-

lite repeats of 5)-A4–5CTGCT-3)). We provide here the firstreport of the occurrence of B chromosomes in Cestrum inter-medium and C. strigilatum. Bs were found in about half of theindividuals analyzed in each species and the study also revealedchromosome differences in the heterochromatic pattern be-tween the Bs of C. strigilatum and C. intermedium.

It is generally accepted that B chromosome frequency isunstable because Bs do not segregate regularly in meiosis andmitosis, which may lead to B accumulation associated withnondisjunction (Camacho et al., 2000). Samples originatingfrom a donor seed tree of Cestrum intermedium and another ofC. strigilatum, showed some intra-individual variation in thenumber of Bs indicating a slight mitotic instability apparentlycausing some B elimination from somatic tissues. It would beinteresting to analyse whether germ tissues also show this kindof variation and whether it leads to B accumulation or elimina-tion. At this stage of the investigations it is not known whetherthe Bs present in the seedlings came from the male or femaleparent nor whether the presence of Bs is advantageous or dele-terious to individuals carrying them.

Plant genomes are highly dynamic, mainly due to the insta-bility of repetitive DNA associated with amplification, deletionand change in motif position (Guerra, 2000). The dynamics ofrepetitive DNA has been suggested as an important feature inthe appearance and stabilization of B chromosomes in manyorganisms. The higher content of the heterochromatin of Bscould be associated with the intensity of drive (Camacho et al.,2000). Thus, Cestrum is an excellent tool for such studies due tothe presence of Bs and the presence of different types of hete-rochromatin in the A complement, as previously reported byBerg and Greilhuber (1993a, b).

Differences and similarities in the type of heterochromatinbetween chromosomes of the A complement and the Bs, as wellas between Bs of the two species were found. CMA3

0/DAPI0

heterochromatin, which was not cold sensitive, was only de-tected in the B of C. intermedium. This is the first report ofCMA3

0/DAPI0 heterochromatin in the genus Cestrum. It sug-gests a new type of heterochromatin contributing to the forma-tion of Bs. The other differences between the Bs of the two spe-cies were clearly evident in regard to the size and location ofheterochromatin, which was more abundant in C. intermediumthan in C. strigilatum. Apparently, the only similarity betweenthe Bs of C. intermedium and C. strigilatum, besides their samesize and shape observed by conventional staining, was theoccurrence of Giemsa C-banded “dots,” which were not de-tected by fluorochromes nor by cold sensitive treatment. There-fore, it is possible that the heterochromatin found as “dots” inthe Bs is related to those “dots” revealed by Giemsa C-bandingin the A complement of both species. An excellent investigationthat demonstrated the importance of different repetitive DNAsin the B chromosomes of plants was conducted in Brachycomedichromosomatica. In this report, Houben et al. (1999) foundtwo different Bs, one small and another large, which showed atleast one hybridization site for each of the repeated DNAsequences: Bd49, Bdm29 and an inactive rDNA segment,which suggested a possible monophyletic origin for these Bs.

B chromosomes, regardless of size and form, almost certain-ly originate from the standard A chromosome complement

18846

Cytogenet Genome Res 106:184–188 (2004)

(Camacho et al., 2000; Puertas, 2002). Cuadrado and Jouve(1994b) studied different rye plants bearing 0, 1 and 2 B chro-mosomes with some repetitive DNA probes. Based on theirfindings, these authors suggested that different Bs from differ-ent populations present common segments with the A chromo-somes, indicating a possible unique origin from A complement.An excellent contribution in favour of the relationship betweenBs and A chromosomes was reported by Cheng and Lin (2003).These authors isolated Bs by means of micromanipulation andobtained 19 repetitive sequences after PCR amplification, ofwhich just one did not show homology with the A chromo-somes. After having originated from the A complement, Bchromosomes can follow their own evolutionary pathway. Ac-cording to Beukeboom (1994), Bs and As can become lesshomologous due to accumulation of mutations. Unlike rye, theorigin of Bs in these two Cestrum species points to two diver-gent hypotheses. In the first, the occurrence of Bs with similarsize and acrocentric shape in three species of the same genus(including C. parqui), could indicate a common origin for theseBs. Hence, the occurrence of heterochromatin in Bs, differingin type and distribution, could be explained by an independentgeneration of repetitive DNA in C. intermedium and C. strigi-latum, following DNA rearrangements after the formation ofBs. However, it is difficult to explain the maintenance of thesame shape in the Bs of three species after great changes in hete-rochromatin. Perhaps if Bs do follow their own evolutionarypathway this is merely a coincidence. An example of this wasreported in four cytodemes of Brachycome dichromosomatica(see Houben et al., 1999) where the Bs are the same size but inone cytodeme a translocation of the rDNA locus and Bd49position was detected. Other evidence suggests the proposal ofa second hypothesis, that is, the independent origin of Bs. Thefindings that support the latter notion are: (i) the classification

of these species in distinct sections of the genus, according todifferences in morphological features, (ii) the striking differ-ences among Bs in the composition and size of the blocks ofheterochromatin, e.g, the large terminal CMA3

0/DAPI0 blocksin C. intermedium and their absence in C. strigilatum and (iii)the occurrence of 45S and 5S rDNA in C. parqui and itsabsence in C. strigilatum and C. intermedium. Thus, it is possi-ble that these Bs originated independently in these species, andthe similarities in acrocentric form and size were only a coinci-dence. Alternatively, it could indicate the existence of somenucleotypic mechanism that controls and organizes B macro-structure in Cestrum. At the moment, we do not have a definitepreference for one hypothesis over the other, and great effortsare being directed toward the further understanding of theseBs.

This article provides the second account of Bs in the genusCestrum and, in addition, it enhances our knowledge of theimportance of repetitive DNA in the organization of thesechromosomes. However, further studies are warranted to an-swer questions about transmission rate, detailed molecularorganization and evolutionary mechanisms. Bs in Cestrumseem to be excellent material for this type of investigationbecause representatives of two species occur in natural popula-tions, without natural hybrids, and they differ substantiallyfrom A chromosomes in size. The latter physical feature couldfacilitate the identification of Bs in micromanipulation andcrossbreeding procedures.

Acknowledgments

The authors thank Edson Mendes Francisco and Karina L. V. Ramalhode Sa for help with botanical material.

References

Berg C, Greilhuber J: Cold-sensitive chromosome re-gions and heterochromatin in Cestrum (Solana-ceae): C. strigilatum, C. fasciculatum, and C. ele-gans. Plant Syst Evol 185:133–151 (1993a).

Berg C, Greilhuber J: Cold-sensitive regions and hete-rochromatin in Cestrum aurantiacum (Solana-ceae). Plant Syst Evol 185:259–273 (1993b).

Beukeboom LW: Bewildering Bs: an impression of the1st B-chromosome conference. Heredity 73:328–336 (1994).

Bold HC: Bristol’s solution and medium. (1949). Avail-able at: http://www.pai.utexas.edu/research/utex/media/bristol.html.

Camacho JPM, Sharbel TF, Beukeboom LW: B chro-mosome evolution. Phil Trans R Soc Lond B355:163–178 (2000).

Cheng YM, Lin BY: Cloning and characterization ofmaize B chromosome sequences derived from mi-crodissection. Genetics 164:299–310 (2003).

Cuadrado A, Jouve N: Mapping and organization ofhighly-repeated DNA sequences by means of si-multaneous and sequential FISH and C-bandingin 6x-Triticale. Chromosome Res 2:331–338(1994a).

Cuadrado A, Jouve N: Highly repetitive sequences in Bchromosomes of Secale cereale revealed by fluores-cence in situ hybridization. Genome 37:709–712(1994b).

Dhar MK, Friebe B, Koul AK, Gill BS: Origin of anapparent B chromosome by mutation, chromo-some fragmentation and specific DNA sequenceamplification. Chromosoma 111:332–340 (2002).

Franks TK, Houben A, Leach CR, Timmis JN: Themolecular organisation of a B chromosome tandemrepeat sequence from Brachycome dichromosoma-tica. Chromosoma 105:223–230 (1996).

Guerra M: Patterns of heterochromatin distribution inplant chromosomes. Gen Mol Biology 23:1029–1041 (2000).

Hossain MA, Bauchan GR: Identification of B chromo-somes using Giemsa banding in Medicago. J Her-edity 90:428–429 (1999).

Houben A, Leach CR, Verlin D, Rofe R, Timmis JN: Arepetitive DNA sequence common to the differentB chromosomes of the genus Brachycome. Chro-mosoma 106:513–519 (1997).

Houben A, Thompson N, Ahne R, Leach CR, Verlin D,Timmis JN: A monophyletic origin of the B chro-mosomes of Brachycome dichromosomatica (As-teraceae). Plant Syst Evol 219:127–135 (1999).

Puertas MJ: Nature and evolution of B chromosomes inplants: A non-coding but information-rich part ofplant genomes. Cytogenet Genome Res 96:198–205 (2002).

Qi ZX, Zeng H, Li XL, Chen CB, Song WQ, Chen RY:The molecular characterization of maize B chro-mosome specific AFLPs. Cell Res 12:63–68(2002).

Schwarzacher TP, Ambros P, Schweizer D: Applicationof Giemsa banding to orchid karyotype analysis.Plant Syst Evol 134:293–297 (1980).

Smith LB, Downs RJ: Solanaceas, in Reitz PR (ed):Flora Ilustrada Catarinense. Part 1, pp 1–321 (Bi-blioteca Superior de Cultura, Itajaı 1966).

Stark EA, Connerton I, Bennett ST, Barnes SR, ParkerJS, Forster JW: Molecular analysis of the structureof the maize B-chromosome. Chromosome Res4:15–23 (1996).

Sykorova E, Yoong Lim K, Fajkus J, Leitch AR: Thesignature of the Cestrum genome suggests an evolu-tionary response to the loss of (TTTAGGG)n telo-meres. Chromosoma 112:164–172 (2003).