Theor Appl Genet (2000) 101:7-14 0 Springer-Verlag 2000

A. Schmidt * R.L. Doudrick - J.S. Heslop-HarrisonT . S c h m i d t

The contribution of short repeats of low sequence complexityto large conifer genomes

Received: 1 October 1999 / Accepted: 3 November 1999

Abstract The abundance and genomic organization ofsix simple sequence repeats, consisting of di-, tri-, andtetranucleotide sequence motifs, and a minisatellite re-peat have been analyzed in different gymnosperms bySouthern hybridization. Within the gymnosperm geno-mes investigated, the abundance and genomic organiza-tion of micro- and minisatellite repeats largely followstaxonomic groupings. We found that only particular sim-ple sequence repeat motifs are amplified in gymnospermgenomes, while others such as (CAC), and (GACA), arepresent in only low copy numbers. The variation inabundance of simple sequence motifs reflects a similarsituation to that found in angiosperms. Species of thetwo- and three-needle pine section Pinus are relativelyconserved and can be distinguished from Pinus strobuswhich belongs to the five-needle pine section Strobus.The hybridization pattern of Picea species, bald cypressand gingko were different from the patterns detected inthe Pinus species. Furthermore, sequences with homolo-gy to the plant telomeric repeat (TTTAGGG), have beenanalyzed in the same set of gymnosperms. Telomere-likerepeats are highly amplified within two- and three-needle pine genomes, such as slash pine (Pinus elliottiiEngelm. var. elliottii), compared to F! strobus, Picea spe-cies, bald cypress and gingko. R elliottii var. elliottii wasused as a representative species to investigate the chro-mosomal organization of telomere-like sequences by flu-

Communicated by P.M.A. Tigerstedt

A. Schmidt ([XI) . T. SchmidtInstitute of Crop Science and Plant Breeding, OlshausenstralJe 40,D-241 18 Kiel, Germanye-mail: kamm@plantbreeding.uni-kieldeTel.: +49/431/8803288, Fax: +49/431/8802.566

R.L. DoudrickSouthern Institute of Forest Genetics, Southern Research Station,USDA Forest Service, 23332 Highway 67, Saucier,MS 39574-9344, USA

J.S. Heslop-HarrisonKaryobiology Group, Department of Cell Biology,John Innes Centre, Colney Lane, Norwich NR4 7UH, U.K.

orescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromo-somes; they form large intercalary and pericentric blocksshowing that they are a repeated component of the slashpine genome. Conifers have genomes larger than 20000Mbp, and our results clearly demonstrate that repeatsof low sequence complexity, such to (CA),, (GA),,(GGAT), and (GATA),, and minisatellite- and telomere-like sequences represent a large fraction of the repetitiveDNA of these species. The striking differences in abun-dance and genome organization of the various repeatmotifs suggest that these repetitive sequences evolveddifferently in the gymnosperm genomes investigated.

Key words Pinus . Gymnosperms . Simple sequencerepeats . Microsatellites . Minisatellites . Telomere

Introduction

Plant genomes contain several classes of repetitive DNAorganized in tandem arrays. Satellite DNA repeats andrRNA genes have been extensively investigated at themolecular and chromosomal level, respectively, showingthat they range from 150-180 bp or 300-360 bp up toseveral kilobases in length.

However, a large proportion of the plant repetitiveDNA also consists of tandemly arranged repeats whichare much shorter and of lower complexity compared tosatellite DNA and rRNA genes. Monotonous repetitionsof sequence motifs (1-5 bp) are known as simple se- ,quence repeats or microsatellites. They have been foundin many plant species (Weising et al. 1992), andalthough they are ubiquitous elements of eukaryoticgenomes, there are remarkable differences in the abun-dance of microsatellite sequence motifs between plantsand vertebrates (Lagercrantz et al. 1993).

Because of their widespread occurence, hypervari-ability in repeat number and their interspersed multilocusdistribution in eukaryotic genomes, microsatellites arehighly informative markers for plant and mammalian ge-

8

nome analysis and mapping. Although some of these locimay consist of complex or compound microsatellites,the overall simplicity of the basic repeating motifs ismaintained. In plants, microsatellites have been used tomap the genomes of rice (Wu and Tanksley 1993), soy-bean (Akkaya et al. 1992) and cereal species (Becker andHeun 1995). In rapeseed (Brussica napus), chick-pea (Cicer arietinum) and sugar beet (Beta vulgaris),DNA fingerprinting has shown that di-, tri- and tetra-nucleotide repeats are amplified sequences of the ge-nome and are highly informative markers for the differ-entiation of accessions, cultivars and breeding lines(Weising et al. 1992; Poulsen et al. 1993; Schmidt et al.1993).

Another type of short DNA repeats are minisatellites,which are characterized by longer repeating units (lo-50 bp). First isolated from mammalian genomes, mini-satellites have been detected in many plant species(Broun and Tanksley 1993; Winberg et al. 1993;Tourmente et al. 1994). Minisatellite repeats share a GC-rich core motif and are highly polymorphic enablingtheir application as markers for genome mapping. Mini-satellite repeats can be amplified to high copy numbersand make-up a considerable fraction of the repetitiveDNA sequences of plant genomes (Tourmente et al.1994; Metais et al. 1998).

Functional repeats of low sequence complexity arethe telomeres. Occurring at the outermost ends of plantchromosomes, these repeats are highly conserved, form-ing stabilizing (TTTAGGG), stretches at the physicalends of chromosomes that protect the linear chromo-somes from exonucleolytic degradation. Proximal telo-mere repeat copies are less conserved and sequence het-erogeneity increases with distance from the chromosomeends, although the basic structure of these telomere-likerepeats can still be recognized. In Arabidopsis thaliana,it has been reported that telomere-like sequence blocksoccur also in intercalary positions on chromosome I(Richards et al. 1993). By fluorescence in situ hybridiza-tion Fuchs et al. (1995) have shown that intercalaryblocks of telomere-like sequences are found in manyplant genomes.

Conifers are commercially important and inherentlyinteresting because they dominate many terrestrial eco-systems of the world. Despite the large genome sizeof gymnosperms (> 20 000 Mbp for pine species;Wakamiya et al. 1993) little is known about the genom-ic structure and composition of the nuclear genome ofconifer species. With the exception of chromosomenumbers, which are very conserved (2n = 24) (Khoshoo1961), there are few investigations of the undoubtedlyhigh proportion of noncoding DNA in the genomes ofgymnosperms. Reassociation kinetics data revealed75% of the genome to be repetitive DNA (Dhillon1987). For slash pine (Pinus elliottii Engelm. var. el-Ziottii; 2n = 2x = 24) only 18S-5.8S-25s rRNA genesand the Tyl-copia-retrotransposon TPEl have beencharacterized as major classes of repetitive DNA(Doudrick et al. 1995; Kamm et al. 1996) and a Ty3-

gypsy retrotransposon element was characterized ashighly amplified in Pinus radiata (Kossack et al. 1990).A satellite DNA family has been cloned from Piceaspecies and localized along the chromosomes (Brownet al. 1998).

Here we demonstrate that short repeats of simple se-quence structure and low complexity are amplified ingymnosperm species, in particular conifers. We aimed toinvestigate the abundance and genomic organization ofsimple sequence repeats consisting of di-, tri-, and tetra-nucleotide motifs, and a minisatellite repeat in differentgymnosperms by Southern hybridization using syntheticoligonucleotides as probes. By fluorescence in situ hy-bridization on slash pine chromosomes we show that se-quences with homology to the plant telomeric repeat(TTTAGGG), are amplified at many intercalary regionsand are a major component of the P elliottii. var. elliottiigenome.

Plant material, DNA extraction, and in-gel hybridization

Total genomic DNA was extracted from needle tissue (10 g freshweight) following the protocol of Wagner et al. (1987). The plantspecies used are listed in Table 1.

For in-gel hybridization, genomic DNA was digested usingRsaI and separated on 1% agarose gels. Gels were dried in a slabdrver. ore-treated and hvbridized with l%s*PldATP end-labelled

, I 2 .4 d

ohgonucleotides (Ali et al. 1986). The following oligonucleotideswere employed: (CA),; (GA),; (CAC)!,; (GGAT),; (GACA),;(GATA), and the A. thaliana minisatelhte sequence GGAGGT-GGAGGACATGGCGG (Tourmente et al. 1994). The hybridiza-tion and stringent washes were performed at the appropriate T,- 5oC according to Thein and Wallace (1986). Exposure timeswere dependent on the amount of bound radio-labelled probe andvaried, for example, between 5 h for (GA), 26 h for the minisatel-lite motif and 40 h at -80°C using intensifying screens for(GACA).

DNA labelling and Southern hybridization

The nonradioactive chemiluminescence method ECL (Amersham)was used for DNA labelling of pAtT4, a clone containing severalcopies of the A. thaliana telomeric repeat TTTAGGG (Richardsand Ausubel 1988), hybridization and detection. For preparationof Southern blots, 10 ug of genomic DNA were digested usingRsaI, size-fractionated in a 1% agarose gel and transferred onto anylon membrane (Sambrook et al. 1989). The hybridization, witha membrane DNA concentration of 10 rig/cm*, was carried outovernight at a stringency of 90%.

Fluorescence in situ hybridization

Seedling root tips of P elliottii var. elliottii were used for chromo-some preparations. After treatment using colchicine, root tips werefixed in methanol/acetic acid (3: 1). Chromosome preparations andin situ hybridization were done according to Doudrick et al.(1995). pAtT4, carrying the A. thaliana telomeric sequence, waslabelled with biotin-11-dUTP (Sigma) by PCR. The in situ hybrid-ization was performed with the most stringent wash being 40%formamid in 2 x SSC at 42°C. Sites of hybridization were detectedusing a streptavidin-Cy3 conjugate (Sigma). Slides were counter-stained with DAPI (4’,6-diamidino-2-phenylindole).

Results

Short repeats of low sequence complexity ingymnosperm genomes

The existence and distribution of sequences comple-mentary to WA),, (GNs, (CAC),, (GACA),, (GGAV,,(GATA),, as well as a plant minisatellite consensus se-quence, were analyzed in various two- and three-needlepines of the section Pinus, the five-needle pine Pinusstrobus of the section Strobus, and in two Picea speciesand Taxodium distichum (Table 1). Gingko biloba wasused as an out-group, non-coniferous gymnosperm spe-cies. Most of these gymnosperm species are character-ized by extremely large genomes. In order to optimizehybridization experiments, several enzymes commonlyused for fingerprint studies such as RsaI, Hinff, HaeIII,TaqI were employed for the digestion of genomic DNA(data not shown). All restriction patterns resulted in ahybridization smear, and RsaI was chosen for further in-vestigations because it cuts frequently and is not inhibit-ed by methylation.

Hybridization of the dinucleotide repeat (CA), toRsaI-digested genomic DNA revealed that this sequencemotif is equally amplified and present in high copy num-bers in all gymnosperms investigated here. Strong hy-bridization signals visible for all species as a smear overthe whole lane up to 5 kb could be detected, and mostCA repeat copies are present in genomic fragments ofapproximately 0.5-3.0 kb (Fig. 1A).

Rehybridization of the same gel with (GA), resulted ina pattern which is more pronounced in regions of larger re-striction fragments compared to CA. Strong hybridizationwith high-molecular weight fragments up to 20 kb weredetected in all Pinus species. The hybridization signals ofGA are more complex and, despite the smear, many indi-vidual bands were visible. The motif GA was particularlyabundant in the genome of Pinus resinosa (Fig. lB,lane 8). The strength of hybridization with CA and GA

9

showed that these dinucleotide repeats are major compo-nents of the gymnosperm genomes investigated but show adifferent genome organization (cf. Fig. 1A and 1B).

Only a very weak signal could be observed after hy-bridization of (CAC), and (GACA), with all species,suggesting that these sequence motifs are infrequent inthe genomes investigated (Fig. lC, E). We note that(GACA) shows 75% homology to the abundant (GA)and (CA) repeats, demonstrating that hybridization con-ditions clearly discriminated the sequences.

Hybridization of (GGAT), and (GATA), showed me-dium to strong hybridization in the gymnosperm geno-mes investigated (Fig. lD, F). (GGAT), revealed a stronghybridization with a fragment of 1.3 kb which is con-served in most two- and three-needle pine species suchas Pinus echinata, P. elliottii var. elliottii and Pinuspalustris. However, this fragment was not detected inthe two- and three-needle Pinus species l? resinosa andPinus massoniana, nor in I? strobus, Picea species, 7:distichum and G. biloba. Among the investigated gym-nosperm species, the highest copy number of both(GGAT), and (GATA), was detected in T distichum andG. biloba demonstrating that these simple sequence re-peats are very abundant sequences of these genomes. InG. biloba, (GATA), hybridized to multiple fragments be-tween 3.5 kb and approximately 8.0 kb.

In addition to simple sequence repeats, many plantgenomes also contain minisatellite repeats, which arecharacterized by a longer repeating unit and a highCC-rich base constitution. Therefore, we used an oligo-nucleotide representing an A. thaliana minisatellite con-sensus motif to investigate the occurrence of this se-quence in the gymnosperm genomes included in thisstudy. The minisatellite-like motif is abundant in two-and three-needle pines, while it is only moderately am-plified in the five-needle pine, P strobus, in Picea spe-cies, ir: distichum and G. biloba (Fig. 2A). The resultsshow clearly that these sequences form a considerablefraction of Pinus genomes.

Table 1 Gymnosperm species used in this study. All samples were from the collection of the Southern Institute of Forest Genetics, U.S.Department of Agriculture, Saucier (USA)

Lane Genus Sect ion Subsect ion Species Common name Source

L. Pinus Australes Loud.2

89

10111 21 3

Oocarpae Little &Critchfield

Contortae Little &Critchfield

Sylvestres Loud.

Picea Diet.Strobus Lemm. Strobi Loud.

Taxodium Rich.Gingko L.

P echinata Mill.i? elliottii Engelm.

var. elliottiiP. palustris Mill.Pcaribaea MoreletP: oocarpa Schiede

Shortleaf pineSlash pine

Longleaf pineCaribbean pine

R banks&a Lamb. Jack pine

P massoniana Lamb.P resinosa Ait.F! strobus L.P glauca (Moench.) Voss.P. abies (L.) Karst.i!Y distichum (L.) Rich.G. biloba L.

Masson pineRed pineWhite pineWhite spruceNorway spruceBald cypressGingko

Harrison County, MSHarrison County, MS

Harrison County, MSPuerto RicoPuerto Rico

Oneida County, WI

Harrison County, MSOneida County, WIOneida County, WIOneida County, WIOneida County, WIHarrison County, MSHarrison County, MS

A(CA),

BGA),

CWC),

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Wkb)

II WAT), E (GAW, F (GA-W,1 2 3 4 5 6 7 8 910111213 1 2 3 4 5 6 7 8

Fig. 1 A-F Genomic distribution of simple sequence repeats inscverai gymnosperm genomes. Different simple sequence repeats(motif given above each panel) were hyhridixd to RsaI-digestedgenomic DNA of Pinur ecbinatn (I). Pinus el l iort i i var. el l iorri i(2). Pinu.7 paiustrir (3, Pinur corihneo (4% Pinus oocarpn (3,Pinus bnnk.~iana (6). Pinus rnorsoniann (7), Pinus rr.rinosa (R),Pinus strobus (9). Pica &aca (lo), Piceu abies (II), Tnxodiurndisrichum (12) and Giqko h i ioba (13). Lambda HindIlllEroRI-digested DNA was used as the DNA size marker (lane M)

Amplification of telomere-like repeats in pine genomes

Plant genomes, with their large amount of nuclearDNA might contain blocks of the telomeric repeat(TTTAGGG), at intercalary positions (Richards et al.1991).

--21 22

-5 14

-353

- 2 02

-1 37

-083

9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 910111213 M(kb)

'. t *') -21.22

Probe pAtT4, carrying the TTTAGGG tandem repeatsof the A. thnlinna t&mere (Richards and Ausubel1988), was used for Southern hybridization to digestedDNAs from various Pinus and Picra species, 7: disti-chum and G. hiloha. Information for Pinus species wasof most interest, and hence short exposures were needed,otherwise the lanes containing Pirms DNA were over-exposed. The Southern filters showed that the telomericrepeat is highly amplified within two- and three-needlepines of the section Pinus (Fig. 28, lanes l-8). The hy-bridization pattern revealed a smear over the wholerange up to high-molecular weights with some amplifiedfragments, suggesting the presence of the telomere re-peats at different genomic loci with a conserved organi-ration. Hybridization signals larger than 20 kb indicate

A

1 2 3 4 5 6 7 8 9 10111213 M(kb)

-21 2 2

-5 14

-3 53

- 2 02

--137

B

1 2 3 4 5 6 7 8 910111213 M(kb)

-2122

-5 14

- 3 5 3

.

- 2 02

--i 37

1 2

Fluorescence in situ hybridization was used to testwhether telomere-like sequences are located in other re-gions than the physical ends of the P&us chromsomes.As a representative species with economic relevance P.elliotti var. elliottii (2n = 2x = 24) was used for the prep-aration of metaphase chromosomes (Fig. 3A). Weaksites, indicating relatively few copies of the telomere re-peat at the end of chromosome arms, could be detectedby computer-enhanced quantitative pixel analysis. Brightsignals representing extended blocks of telomere-like se-quences were observed at many intercalary regions (redfluorescence in Fig. 3B), consistent with the signalstrength of the Southern hybridization. Up to five signalswere detected per chromosome, visible in pairs on bothchromatids. The strongest amplification at non-telomericsites occurred in the pericentric regions of many chromo-somes (Fig. 3C). Most of the telomere-like DNA sitescorrespond to DAPI-positive regions.

Discussion

We have investigated the occurrence and genomic orga-nization of simple sequence repeats, minisatellite and te-lomere-like sequences and their contribution to the ex-tremely large genomes of various gymnosperms species.

The conifer species analyzed in this study have nucle-ar DNA amounts greater than 20 000 Mbp, and 75% ofthe genomes consist of repetitive sequences (Dhillon1987; Wakamiya et al. 1993; Murray 1998). The hybrid-ization patterns, often detected as a smear includingmany fragments, are presumably a consequence of thelarge genome size, since a very large number of frag-ments contribute to the Southern pattern if the investigat-ed simple repeat motif is present in the genome. Thewide range of molecular weights demonstrates that thesesequences are present at many different loci and arepresumably dispersed among other sequences. This pat-tern is in contrast to the clear-cut polymorphic finger-prints often detected in many angiosperm plant species(Weising et al. 1991; Poulsen et al. 1993; Schmidt et al.1993). However, repetitive fragments were detected inseveral taxa, e.g. with (GA) in Pinus species (Fig. 1B)and with (GATA), in G. biloba (Fig. 1F).

In general, simple sequence repeats are an abundantclass of repeats in plant genomes, but it is not yet clearwhy some sequence motifs are more abundant ormore polymorphic than others (Beyermann et al. 1992;Poulsen et al. 1993; Depeiges et al. 1995). The low copynumber of (CAC), and (GACA), in conifers and G. bi-Zoba is in agreement with the findings in angiospermsand indicates that the structure of large conifer genomesand the organization of simple sequence repeats is tosome extent similar to other higher plants. Studies ofSmith and Devey (1994) showed that the dinucleotidesGA and CA are highly amplified in three other pinegenomes, namely P radiata, Pinus taeda and Pinus syl-vestris. Based on genomic library screening Echt andMay-Marquardt (1997) calculated that GA and CA are

the most abundant simple sequence motifs in P. strobusand f! tueda. Thus, our hybridization data (Fig. 1 A andB) are consistent with previous studies and show that, inparticular, dinucleotides such as GA and CA are majorcomponents of conifer genomes.

The variability in distribution and abundance of sim-ple sequence repeats has been used for taxonomic studiesas well as to study the genetic similarity between speciesor breeding lines in agronomic crops. Within the gymno-sperm genomes investigated in this report, the abundanceand genomic organization of simple sequence repeatslargely follows taxonomic groupings, but unequivocalidentification of single species was not possible. This isin contrast to many fingerprint studies in angiospermgroups where chromosome numbers are less conservedand genome sizes are both smaller and more variable.

Compared to the (CA), hybridization pattern, whereconserved repetitive fragments were absent, the hybrid-ization patterns generated by (GA),, (GGAT),, (GATA),and the minisatellite-motif were more complex, with dis-tinct fragments superimposed on a smear detectable inmost species.

Species of the genus Pinus are taxonomically dividedinto sections and subsections based on morphology (e.g.two- and three-needle, and five-needle species). It isnoteworthy that the two- and three-needle pines of thesubsections Australes, Oocarpae and Contortae (all be-longing to the section Pinus) showed a conservation offragments and similar hybridization patterns (Figs. 1 B,D and Fig. 2A). Z? massoniana and P. resinosa of thesubsection Sylv&ris differed in hybridization withmicro- and m&satellites as well as telomere-like se-quences, from the remaining two- and three-needlepines. Furthermore, :P strobus, a five-needle pine of thesection Strobus, can be distinguished from two- andthree-needle pines by a different minisatellite and telo-mere hybridization pattern, although it is not greatly dif-ferent for the six tested simple sequence motifs. Sincethe two sections of the genus Pinus, Pinus and Strobus,had separated relatively early in conifer phylogeny, thedifferences of the simple sequence repeats between thetwo sections are not surprising. The diverged hybridiza-tion detected in G. biloba and the more distantly relatedconifers, Pinus glauca, Pinus alba and T. distichum, re-flects the taxonomic position of these species. Neverthe-less, both Pinus abies and Z? glauca share some con-served minisatellite fragments.

Despite the ecological and economic importanceof conifers, little is known ‘about the structure and com-position of their nuclear genomes. In Z? elliottii var. elli-ottii, genes encoding the 18S-5.8S-25s rRNA and the5s rRNA are abundant sequences and occur on 10 of the12 chromosome pairs (Brown et al. 1993, Doudrick et al.1995). Other highly repetitive DNA sequences of pinegenomes are retrotransposable elements. The Tyl-copia-like retrotransposon TPEl is distributed over all chromo-somes, but excluded from major rDNA sites, and form alarge proportion of the Z? elliottii var. elliottii genome(Kamm et al. 1996). ZFG7 is an abundant sequence of

1 3

quence has been amplified only in the section Pinus afterseparation of the two sections.

Fuchs et al. (1995) have demonstrated that terminaltelomeric signals are difficult to detect by fluorescencein situ hybridization in Picea species. Similarly, in the \experiments presented here, terminal sites are only de-tectable by computer-enchanced image analyses. Al-though these data indicate that conifer telomeres are rel-ative short, improved hybridization conditions at appro-priate stringencies recently enabled the reliable detectionof telomeres at the termini of slash pine chromosomes(M. Oard, personal communication).

Gene duplication and the formation of complex genefamilies have been widely cited as a cause of plant geno-mes being large, and Kinlaw and Neale (1997) suggestedthat levels of multiplication were greater in conifers thanin other plant species. However, considering the relative-ly low proportion which coding sequences make up of atotal plant genome, it is unlikely that the evolution ofmultigene families alone can explain the enormous sizeof pine nuclear genomes. It is clear that repeats of lowcomplexity such as (CA),, (GA),, (GGAT),, (GATA),, aswell as minisatellite- and telomere-like sequences, re-present a large fraction of the conifer genomes used inthis report.

The fingerprint approach reported here gives a multi-locus survey about the abundance of microsatellite mo-tifs within the genome. Polymorphisms of simple se-quence repeat loci are widely used for plant genomemapping. However, the isolation and molecular charac-terization of DNA fragments containing simple sequencerepeat stretches is an expensive and laborious process, inparticular for large genomes. The results reported hereprovide valuable information for the genome mapping ofPinus species and show which simple sequence repeatmotifs are abundant, and hence suitable for the prepara-tion of enriched libraries, and which are present at lowabundance and more likely to give single-copy markers.

Acknowledgements T. Schmidt acknowledges financial supportby a grant from the USDA Forest Service (SRS 33-G-97-082).J.S. Heslop-Harrison acknowledges EU grant BI04CT962125.

the Q3-gypsy family found in P radiatu (Kossack et al.1990).

There are only a few reports about tandem repeats inconifers, such as satellites with monomers of 150-180bp or 300-360 bp (Brown et al. 1998), in contrast to an-giosperm genomes, where many satellite repeats havebeen cloned and localized along chromosomes by FISH(Kamm et al. 1994; Schmidt and Heslop-Harrison 1998).Molecular cytological studies of P. elliottii var. elliottiirevealed the presence of brightly fluorochrome-stainingregions along the chromosomes. While some of themcould be assigned to rRNA gene blocks (Doudrick et al.1995), there are indications that others are unrelated re-petitive DNA sequences.

Fluorescence in situ hybridization confirmed the sub-stantial amplification of intercalary and pericentric telo-mere-like repeats in the P elliottii var. elliottii pine ge-nome, which was already evident from Southern-hybrid-ization, and,also confirmed their existence as a major ge-nomic component (Figs. 2B and 3B-C). Taking into ac-count that the restriction enzyme RsaI cuts frequently inplant DNA, it is very likely that the large genomic frag-ments on the Southern experiments (Fig. 2B) have an un-usual base composition or are characterized by a low se-quence complexity.

Fluorescence in situ hybridization resulted in signalssimilar to a chromosome-banding pattern that might servefor chromosome identification, perhaps in combinationwith rRNA fluorescence in situ hybridization or fluoro-chrome staining. The telomere-like motif is not restrictedto the end of chromosomes in f! elliottii var. elliottii, andsimilar to many eukaryote genomes ranging from mam-malian species (Moyzis et al. 1988) to A. thaliana(Richards et al. 1991) shows the intercalary and pericen-tric telomeric repeat as a repetitive genomic component.Compiled data about the chromosomal localization of thetelomeric sequence repeats in 44 plant species (Fuchset al. 1995) revealed intercalary telomeric sequences inaddition to the terminal ones in nine of them, and in threespecies also pericentric positions (e.g. in P. sylvestris).

According to Biessmann and Mason (1992), interstiti-al telomeric repeats have developed either as randomshort sequence arrays which may have become extendedby slippage replication, or by attachment via telomeraseor recombination to extrachromosomal linear DNA frag-ments which may then integrate into the genome, or bychromosome rearrangements such as fusions or inver-sions. The morphology and number of chromosomes(2n = 2~ = 24) are extremely conserved in the more than100 extant Pirius species; hence, if fusions of chromo-somes occurred in the genus Pinus, these events must beconsidered as ancient in Pinus evolution and unusual incomparison to other plants. If amplification of telomere-like sequences is not the result of chromosome fusions,the increase in copy number probably still dates backlong ago in the phylogeny of the genus Pinus. Since thetwo sections of the genus, namely section Pinus and sec-tion Strobus, had become distinct taxa in the early Creta-ceous period (136 million years ago), the telomeric se-

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