Proc. Natl. Acad. Sci. USAVol. 93, pp. 2708-2713, April 1996Plant Biology

The genomic and physical organization of Tyl-copia-likesequences as a component of large genomes in Pinuselliottii var. elliottii and other gymnosperms

(genome evolution/Pinus/retrotransposon)

A. KAMM*, R. L. DOUDRICKtS, J. S. HESLOP-HARRISON*, AND T. SCHMIDT**Karyobiology Group, Department of Cell Biology, John Innes Institute, Colney Lane, Norwich, NR4 7UH, United Kingdom; and tSouthern Institute of ForestGenetics, Southern Research Station, Forest Service, U.S. Department of Agriculture, 23332 Highway 67, Saucier, MS 39574-9344

Communicated by J. Heslop-Harrison, Leominster, Herefordshire, United Kingdom, December 13, 1995 (received for review October 21, 1995)

ABSTRACT A DNA sequence, TPE1, representing theinternal domain of a Tyl-copia retroelement, was isolatedfrom genomic DNA ofPinus elliottii Engelm. var. elliottii (slashpine). Genomic Southern analysis showed that this sequence,carrying partial reverse transcriptase and integrase genesequences, is highly amplified within the genome of slash pineand part of a dispersed element >4.8 kbp. Fluorescent in situhybridization to metaphase chromosomes shows that theelement is relatively uniformly dispersed over all 12 chromo-some pairs and is highly abundant in the genome. It is largelyexcluded from centromeric regions and intercalary chromo-somal sites representing the 18S-5.8S-25S rRNA genes. South-ern hybridization with specific DNA probes for the reversetranscriptase gene shows that TPE1 represents a large sub-group of heterogeneous Tyl-copia retrotransposons in Pinusspecies. Because no TPE1 transcription could be detected, itis most likely an inactive element-at least in needle tissue.Further evidence for inactivity was found in recombinantreverse transcriptase and integrase sequences. The distribu-tion of TPE1 within different gymnosperms that containTyi-copia group retrotransposons, as shown by a PCR assay,was investigated by Southern hybridization. The TPE1 familyis highly amplified and conserved in all Pinus species ana-lyzed, showing a similar genomic organization in the three-and five-needle pine species investigated. It is also present inspruce, bald cypress (swamp cypress), and in gingko but infewer copies and a different genomic organization.

Retrotransposons that proliferate by reverse transcription ofRNA intermediates are a feature of all eukaryotic genomesexamined and the major class of mobile genetic elements inplants (1). Because of their structure, two classes of retro-transposons are distinguished: those flanked by long terminalrepeats (LTR) and non-LTR retrotransposons. Since the firstTy-copia elements were detected in plants [Tal inArabidopsisthaliana (L.) Heynh. (2), Tntl in Nicotiana tabacum L. (3)] theyhave been found across a broad phylogenetic spectrum and allmajor lineages of plants including Chlorophyta, Bryophyta,Pteridophyta, as well as Gymnospermae (1, 4, 5). Most of theseTyl-copia elements were identified by using a PCR assaydesigned to detect copia-like reverse transcriptase gene se-quences. Tyl-copia reverse transcriptase gene sequences havebeen identified from Pinus thunbergii Parl. and Pinus coulteriD. Don by PCR (4, 5). A few Tyl-copia group elements havebeen characterized in detail: Tal of A. thaliana, Tntl of N.tabacum, Tstl of Solanum tuberosum L. (6) Barel of Hordeumvulgare L. (7), and Hopscotch of Zea mays L. (8).

Sequence analyses of PCR fragments of reverse transcriptasegenes revealed very high degrees of sequence heterogeneity even

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within a single species, which is put down to the high copy numberof Tyl-copia retroelements detected in plants (9, 10), in contrastto the limited diversity and copy number seen in Saccharomycescerevisiae and Drosophila melanogaster (11, 12). The degree ofsequence divergence is linked generally to phylogenetic relation-ships, implying that sequence divergence during vertical trans-mission of Tyl-copia retrotransposons along evolving plant lin-eages has been a major factor in their evolution (1, 4, 5). Somesignificant exceptions indicate that horizontal gene transfer ofTyl-copia elements needs also to be considered (9, 13, 14).

Conifers are commercially important and inherently inter-esting because they dominate many terrestrial ecosystems.Despite the enormous genome size of gymnosperms [>20000Mbp for Pinus species (15, 16)], little is known about thestructure and composition of the nuclear genome of anygymnosperm. With the exception of chromosome numbers,which are very conserved (2n = 24) (17), there are fewinvestigations of the relatively high percentage of repetitivesequences in the genomes of gymnosperms. Reassociationkinetics data (18, 19) showed 75% of the genome to berepetitive DNA. A retrotransposon element, IFG7, was iso-lated from Pinus radiata D. Don and described. This Ty3-gypsyclass element, showing a different gene order compared to theTyl-copia elements, is highly amplified in the genome.§ Ge-netic linkage maps based on restriction fragment length poly-morphism and random amplified polymorphic DNA markershave been constructed for different pine species (20-24).

In the present work, we aimed to examine the presence andgenomic organization of Tyl-copia elements in Pinus speciesand particularly in slash pine.¶We also aimed to investigate theimportance of the retrotransposon as a component of theenormous and relatively conserved genomes of gymnosperms.

MATERIALS AND METHODSPlant Materials, DNA Extraction, Cloning, and Sequencing.

Total genomic DNA was extracted from needle tissue (10 gfresh weight) from the plant species listed with authorities inTable 1, following the protocol from Wagner et al. (25).Genomic slash pine DNA was shot-gun cloned into pUC18(26), and highly repetitive sequences were isolated. Clonesselected for investigation were sequenced in both directions onan automated 373A DNA sequencer (Applied Biosystems).One clone, named TPE1, had homology to Tyl-copia se-quences (see Results) and was used as a probe.

Abbreviation: DAPI, 4',6-diamidino-2-phenylindole.*To whom reprint requests should be addressed.§Kossack, D. S., Barrios, M. & Kinlaw, C. S., International Union ofForestry Research Organizations Molecular Genetics Meeting, Sep-tember 30-October 4, 1990, Lake Tahoe, NV (abstr.).¶The sequence reported in this paper has been deposited in theGenBank data base (accession no. Z50750).

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Proc. Natl. Acad. Sci. USA 93 (1996) 2709

Table 1. Gymnosperm species used for experimentsGenus Subgenus Species Common name Source

Pinus L. Pinus P. echinata Mill. Shortleaf pine Harrison City, MSP. elliottii Engelm. var. elliottii Slash pine Harrison City, MSP. palustris Mill. Longleaf pine Harrison City, MSP. caribaea Morelet Caribbean pine Puerto RicoP. oocarpa Schiede Puerto RicoP. banksiana Lamb. Jack pine Oneida County, WIP. massoniana Lamb. Masson pine Harrison City, MSP. resinosa Ait. Red pine Oneida County, WI

Strobus Lemm. P. strobus L. White pine Oneida County, WIPicea Diet. P. abies (L.) Karst. Norway spruce Oneida County, WI

P. glauca (Moench.) Voss. White spruce Oneida County, WITaxodium Rich. T. distichum (L.) Rich. Baldcypress Harrison City, MSGingko L. G. biloba L. Gingko Harrison City, MS

All samples were from the collection of Southern Institute of Forest Genetics, U.S. Department of Agriculture, Saucier, MS.

DNA Labeling and Southern Hybridization. The nonradio-active chemiluminescence method ECL (Amersham) was usedfor DNA labeling, hybridization, and detection. Southern blotswere prepared using standard protocols (27). The hybridiza-tion, with a DNA concentration of 10 ng/cm2 of membrane,was done overnight with a stringency of 90%.

A copiaTalTntBarelpinelCpinelTpine2TgingkoTPE1

copiaTalTntlBarelpinelCpinelTpine2TgingkoTPE1

copiaTalTnt 1BarelpinelCpinelTpine2TgingkoTPE1

Fluorescent in Situ Hybridization. TPE1 was labeled withbiotin-11-dUTP (Sigma) by PCR. pTa71, carrying rRNA-encoding DNA and intergenic spacers (28), was labeled withdigoxigenin-11-dUTP (Boehringer Mannheim) by nick trans-lation. Chromosome preparation and in situ hybridization weredone by the procedures of Doudrick et al. (29). Briefly,

INE N- Q E H- NNF- s s B- G K P- BI5Es- GK L-PDP- R.D P- wi,s

B copiaTalTntlBarelHopscotchTPE1FTPE1R

copiaTalTntlBarelHopscotchTPE1FTPE1R

qVVY3YII RR L g[g MR Qp] V gg S YV [K C L ! I I G L 1g e N L K D A Y I [ E H | I E

GLRE R [q[J jTY SS H IIR HI F L R L F G AHLR V SIT E] F Q w E K-- L N A H F T I H HvTrv rIDi A8|El| nEK If ToHU Y EMMSmC g A Q

- -IESllil* EtNCGE]YV -| KNFg|DA |KjH[r|Tv|gigTjPc L 57SEIRIThVITE:KMDAMRH L I R T

FE1 T i ! GT GIKVQ S Cc f H A i R H MEI VrG LQVSCg |H A RH ivGJIRELRiM KIIBI nT P K ..l~ M RHS T g N K X]h I S -[MR E T

FIG. 1. Alignments of predicted amino acid sequences across conserved domains of different Tyl-copia retrotransposons. Dashes show gapsthat were introduced to optimize the alignment. Stop codons are marked by asterisks (*). Homologous amino acids including the TPE1 sequenceare boxed. (A) Alignment of the slash pine element TPE1 to reverse transcriptases of Tyl-copia retrotransposons from Drosophila melanogaster(copia), Arabidopsis thaliana (Tal), Nicotiana tabacum (Tntl), Hordeum vulgare (Barel), Pinus coulteri (pineIC), Pinus thunbergii (pinelT, pine2T)and Gingko biloba (gingko). KTAFLHG und YVDDM sequences correspond to the oligonucleotide primers. (B) Alignment of two slash pineelement sequences from TPE1 (TPE1F and TPE1R) to integrases of Tyl-copia retrotransposons from D. melanogaster (copia), A. thaliana (Tal),N. tabacum (Tntl), H. vulgare (Barel), and Zea mays (Hopscotch).

KTAFLNG T L K E E R L Q -I S C N S D N [l l N A I Y QKTAFLHG E L IE B L Y M E Q P E G C I S E D G E N K V C.L L k i| Y' IQ

KTAFLHG IE Q G F E M A FG Mx lv C KID NMS:i YG 'LQKAAFLNG L KEELYlMM Q P F EDP K N A N KACLQ G IYGV QKTAFLHG A I K EEVYVEV I QPLGIFIE Q D R D T Y CR A LYG I QKTTFLHIG0 E E E B IYSIKQ |P|K G A M |||GS |EL C K|i gL|Y|G|L KKTAFLNG VI VEVYIEQ p EVFE F SSESH C S R A LYG IK QKTAFLNG VI EEVlYI EQ P Q GFEAHI RE S H CR Ki A LYGl QKITFLHR E M i E E Y S E i *

AAR C F E F E Q A L K C E V N S S V j R C I Y I LDK - G NR Q W N K R N R M I D Q N I S E H D A C V Q V S E -

A R Q W M F S FMKS QTYLKTYS D PC K R F S E -

ASS WNKR GE-VI KAFGFIQVVG ES C I Y KVSG-A PRA WN ERMD S YL M K S N IAI P N L YF KVV-E-

P' Rw| Q B DilI) t FBI L F T S S V D i F M Y [F E - G -

A PA W YT R IS Y F T jL G F S K S E VD P N LYQ IVV-E-

APRA YSRIDTYLHQIGFEKSE [t SNLYYIpEv-Q-AP R: A W Y S R I D Y L H Q K S ElDii S N L LY Ii V - G -

. wE[JRW ]H R R Y my M - c -

I Y V L 'YVDDVL LS L YVDDMI I L L IL YVDDMA F L I L YVDDIL I L V L YVDDMI LcV L YVDDML I LYVDDML I E V L YVDDMT S L VL YVDDI

Plant Biology: Kamm et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

seedling root tips for chromosome preparations were treatedwith colchicine and fixed in alcohol/acetic acid (3:1). Anenzyme mixture containing cellulase and pectinase was used tosoften the root tips that were then squashed on chromicacid-cleaned slides. The hybridization mixture containing theprobe was denatured and added to the chromosome prepara-tions; both were denatured in an Omnislide thermal cyclingmachine (Hybaid, Middlesex, U.K.) at 80°C for 8 min. Afterhybridization overnight at 37°C, washes were carried out with82% stringency. Sites of hybridization were detected usingstreptavidin-Cy3 conjugate (Sigma) for biotin-labeled probesand fluorescein isothiocyanate-conjugated sheep anti-digoxigenin antibody (Boehringer Mannheim) for digoxige-nin-labeled probes. Slides were counterstained with DAPI(4',6-diamidino-2-phenylindole), mounted in antifade solu-tion, and photographed with a Leica epifluorescence micro-scope with appropriate filters.Northern Analysis. Poly(A)+ RNA was isolated from pollen

and needles of slash pine using oligo(dT)25-coated magneticbeads according to the instructions of the manufacturer (Dy-nal, Oslo). Northern blots were prepared as described bySambrook et al. (27). TPE1 was labeled by random primingwith [32P]dCTP.PCR Assay. The internal domain of reverse transcriptase

genes from gymnosperm species was amplified using flankingprimers and PCR programs described by Flavell et al. (30).Computer Analysis. The FASTA program of the Genetics

Computer Group package was used for homology searcheswithin the EMBL/GenBank data base (release 83, 1995). Theputative peptide sequence was generated by the same packageusing the program MAP. Alignments were manually optimized.

RESULTSIsolation and Characterization of a Tyl-copia Retrotrans-

poson Sequence from Slash Pine. A highly repetitive sequencewas isolated from a genomic library from slash pine. Thesequence is 1663 bp long and was named TPE1. A homologysearch using TPE1 as query sequence revealed close similarityto the reverse transcriptase and integrase genes of Tyl-copiaretrotransposons from P. thunbergii, A. thaliana, N. tabaccum

A B

and others. Fig. 1 presents alignments of parts of the putativeTPE1 peptide sequence with some previously determinedreverse transcriptase and integrase sequences of Tyl-copiaplant retrotransposons such as Tal, Tntl, Barel, Hopscotch,and sequences isolated from P. thunbergii, P. coulteri, and G.biloba (4), as well as the copia element from D. melanogaster.In general, most of the identity was found at positions that wereconserved in the majority of the compared retroelements.TPE1 can be identified as an internal part of a Tyl-copiaretrotransposon, carrying reverse transcriptase and integrasegene sequences. We infer that a recombination event withinTPE1 led to a compound structure of this element. The reversetranscriptase gene, following the integrase gene within thesame reading frame, is destroyed by insertion of a partialintegrase gene sequence, encoded on the opposite strand ofTPE1 and, hence, lying in inverted orientation. This resultindicates that the element cloned in TPE1 is defective. Fur-thermore, putative stop codons were found within the TPE1sequence, and the introduction of frameshifts was required toenable an alignment with peptide sequences of other Tyl-copiaretrotransposons.Genomic Organization and Heterogeneity Within Pinus

Species. The genomic organization of TPE1 was analyzed bySouthern hybridization to genomic DNA digests of threerelated three-needle Pinus species [Section Pinus SubsectionAustrales Pinus (31)], slash pine, P. palustris (longleaf pine),and P. echinata (shortleaf pine) (Fig. 2A). Strong signals wereobserved in all digests showing that the TPE1 family is highlyrepeated within the genomes. None of the digests revealeddifferences in the hybridization pattern between the species.The hybridization pattern in Apa I digests revealed a strongsmear over the whole track up to high molecular weights (lanes7-9) indicating the presence of TPE1 in many different andprobably methylated genomic loci, presumably dispersedamong other sequences. Other digests showed the conserva-tion of the TPE1 sequence family by the presence of fragmentsbetween 0.2 and 4.8 kbp in all species and also showing that 4.8kbp is the minimum size of the full-repeat TPE1.PCR generated a population of diverged reverse transcriptase

gene fragments representative of the Tyl-copia elements in slashpine. The PCR product was used for Southern hybridization to

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

m.......... :: _ Zw: r !' - -

-- '-:::::~:; ·aiir·i:::::

FIG. 2. Genomic organization of Tyl-copia elements within the genome of shortleaf pine, slash pine, and longleaf pine. (A) Genomic organizationof the TPE1 family. Southern blots of genomic DNA digested with Hae III (lanes 1-3), HinfI (lanes 4-6), Apa I (lanes 7-9), BamHI (lanes 10-12),EcoRI (lanes 13-15) were probed with TPE1. Lambda HindIII-digested DNA was used as DNA size marker (M). (B) Rehybridization of theSouthern blot described aboveA with a population of diverged reverse transcriptase gene sequences of Tyl-copia elements from slash pine, isolatedby PCR.

2710 Plant Biology: Kamm et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 2711

FIG. 3. Localization of a Tyl-copia retrotransposon family and the 18S-5.8S-25S rRNA genes along chromosomes of slash pine by fluorescentin situ hybridization. (A) DAPI staining of metaphase chromosomes of slash pine (2n = 2x = 24). (B) The same metaphase after in situ hybridizationwith 18S-5.8S-25S rRNA genes visualized by yellow-green fluorescence. (C) Detection of the Tyl-copia retrotransposon TPE1 (red fluorescence)on the same metaphase chromosomes. Arrow shows an example of the relatively large exclusion from DAPI-negative intercalary region (arrowin A) harboring 18S-5.8S-25S rRNA genes (arrowed in B).

investigate the heterogeneity of the TPE1 family in three three-needle pine species. Hybridization revealed a strong and complexpattern (Fig. 2B), indicating that Tyl-copia retrotransposons area large component of the genomes of the three pine species. TheTPE1 Southern hybridization pattern (Fig. 2A) is a subset of thepattern revealed by a heterogeneous population of reverse tran-scriptase gene sequences. The most prominent bands are sharedby TPE1 and PCR-amplified sequences from the internal part ofthe reverse transcriptase gene. Hence, it was evident that TPE1represents a major family of Tyl-copia retrotransposons formingone large subgroup of heterogeneous Tyl-copia retrotransposonsin slash, longleaf, and shortleaf pines. In addition, bands >4.8 kbpwere found, indicating a larger repeat size of other families ofTyl-copia elements than found for the TPE1 sequence family.

1 2 3 4 5 6 7 8 9 10 11 12 M

E^^M'*^Ik lriM

FIG. 4. Distribution of TPE1 in several species of Pinus and othergymnosperms. Southern blot of Dra I-digested genomic DNA of P.echinata (lane 1), P. elliottii var. elliottii (lane 2), P. palustris (lane 3),P. caribaea (lane 4), P. oocarpa (lane 5), P. banksiana (lane 6), P.massoniana (lane 7), P. resinosa (lane 8), P. strobus (lane 9), Picea abiesand Picea glauca, mixed (lane 10), T. distichum (lane 11) and G. biloba(lane 12) was hybridized with TPE1. Lambda HindIII-digested DNAwas used as DNA size marker (lane M).

Chromosomal Localization of Tyl-copia Retrotransposonsin the Genome of Slash Pine. From Southern hybridization itwas evident that there has been a substantial amplification ofthe TPE1 family in the slash pine genome. The chromosomaldistribution of Tyl-copia elements in slash pine was investi-gated by fluorescent in situ hybridization to metaphase chro-mosomes (2n = 2x = 24) using biotin-labeled TPE1 as a probe(Fig. 3C). Hybridization revealed that this element is dispersedrelatively uniformly over all 12 chromosome pairs and repre-sents a major component of the slash pine genome. It is largelyexcluded from DAPI-negative centromeric and intercalaryregions harboring the major and minor 18S-5.8S-25S rRNAgenes, as visualized by double in situ hybridization with digoxi-genin-labeled rRNA genes (Fig. 3B).

Distribution in Different Pinus Species and Gymnosperms.The distribution of TPE1 within different gymnosperms wasinvestigated by Southern analysis. TPE1 was used for Southernhybridization of digested DNAs from various Pinus and Piceaspecies, Taxodium distichum (bald cypress or swamp cypress)and G. biloba (gingko). Fig. 4 shows that TPE1 is highlyamplified in all species of pine analyzed (lanes 1-9). A strongsmear over the whole range with three strong bands could bedetected. It is noteworthy that, although TPE1 revealed thesame structure and dispersed genomic organization within allpine species, the strength of hybridization differed. While alltwo- and three-needle pine species (Section Pinus) show stronghybridization and a very similar pattern (lanes 1-8), signifi-cantly less signal was observed in P. strobus (Section Strobus),a five-needle pine species (lane 9). TPE1 is relatively highlyamplified also in spruce, but much less hybridization signalcould be detected in bald cypress and gingko, indicating eithermany fewer copies or considerable lower homology in thesespecies. TPE1 hybridization also showed a different genomicorganization in spruce, bald cypress, and gingko, so we verifiedthe presence of Tyl-copia elements among these species by aPCR assay. Sequences of the expected size (-260 bp) wereamplified (data not shown). No differences in size weredetected, indicating the presence and conservation of thereverse transcriptase domain of Tyl-copia retroelements in thespecies.

DISCUSSIONWe have isolated a highly repetitive DNA sequence, TPE1,from slash pine and used fluorescent in situ hybridization tomap physically these elements on slash pine chromosomes.

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Proc. Natl. Acad. Sci. USA 93 (1996)

Alignments of parts of the putative TPE1 peptide sequencewith known Tyl-copia plant retrotransposons revealed identityat most positions that were conserved in the majority of theretroelements compared, and hence TPE1 was identified as a

retroelement of the Tyl-copia type from slash pine, carryingreverse transcriptase and integrase gene sequences (Fig. 1).So far, little is known about the transposition activity of plant

Tyl-copia retrotransposons because it is difficult to assess theirtransposition and mobility. Numerous mutations within theTPE1 sequence such as putative stop codons, interruptedreading frames, and a disrupted reverse transcriptase genecaused by recombination lead to the assumption that thiselement is defective. Moreover, most of the Tyl-copia elementsare inactive in terms of retrotransposition in slash pine-atleast in needle tissue because no transcripts of TPE1 could bedetected by Northern analysis. This result reflects the commonsituation observed for most plant retrotransposons that werefound to be transcriptionally inactive. In contrast to yeast andDrosophila, where transcription of retrotransposons occurs inmost tissues during the normal life cycle, plant Tyl-copiaelements are usually transcribed poorly. In plants, transposi-tion of the Tyl-copia retroelements Tntl and Ttol fromtobacco has been detected under some conditions but seemsto be strongly regulated by control of transcription (6, 32, 33).Few investigations have shown the chromosomal distribu-

tion of retroelements. Bisl shows quite uniform hybridizationalong all barley (4400 Mbp) chromosome arms, but it is absentor relatively rare in the centromeric heterochromatin andnucleolus organizer regions (34). A similar distribution wasdetected for the Tyl-copia retrotransposons in Vicia faba(13,000 Mbp) (10), whereas a less uniform pattern withabsence or presence at a reduced density at some chromosomalregions, in particular at centromeric and intercalary hetero-chromatin and rRNA loci, was observed for the Tbv Tyl-copiaelements from Beta vulgaris (758 Mbp) (35). Exclusion fromheterochromatic and nucleolus organizer regions, as alsofound with TPE1, seems a feature of many plant Tyl-copiaretrotransposon.

Detailed studies of elements within individual species re-

vealed that, despite maintenance of the overall structure, a

population of many different, but related, sequences are

present within its genome (36, 37). Flavell et al. (30) havecharacterized 31 Tyl-copia clones in potato that could beclearly grouped into six related subfamilies, and diversitiesbetween them up to 75% have been observed. The degree ofsequence heterogeneity shows no correlation with plant divi-sions, and therefore the source of this heterogeneity cannot bea property of any division (1). Theoretical studies, proposingthat sequence heterogeneity is positively correlated with copynumber of elements, were confirmed by investigations withinthe genus Vicia (10).The two sections of the genus Pinus, Pinus and Strobus, had

become distinct taxa by the early Cretaceous period [136million yr ago (38)], so the relatively high conservation of theTPE1 element is noteworthy. The position of P. resinosa is ofinterest: it is native to North America but now normally placedin Subsection Sylvestres Loud. with P. massoniana and otherEurasian pines. The distinct differences in Southern hybrid-ization between the two and the similarity of P. resinosa to theNorth American pines suggests that taxonomic affinities basedon morphology, cone serotiny, and crossing experiments forSubsection Sylvestres might need reconsideration. Klaus (39)has proposed creating Subsection Resinosae, in Section Pinusfor P. resinosa, a suggestion supported by the retrotransposonhybridization data. TPE1 is highly amplified in spruce, but inmany fewer copies in bald cypress and gingko, and a differentgenomic organization was observed in these species than inpine species: the accepted phylogeny of the species correlateswith the order of similarity of signal pattern and intensity forTPE1.

From Southern and in situ hybridization it was evident thatthere has been amplification of the Tyl-copia-like sequences inthe genomes of all the Pinus species analyzed (genome sizestypically 20,000-25,000 Mbp). The high amplification andgenomic distribution of the TPE1 family, dispersed amongother sequences but excluded from particular chromosomalregions, is consistent with the amplification of Tyl-copiaelements seen in the angiosperms. Within the gymnospermsthe divergence of the Tyl-copia sequences follows taxonomicgroupings, as in angiosperm groups where chromosome num-ber is less conserved and genome size is both smaller and morevariable.

A.K. was supported by a grant of U.S. Department of Agriculture-Forest Service (19-94-040). T.S. acknowledges financial support byEuropean Union fellowship (ERB4001 GT931647).

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