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© 1999 Macmillan Magazines LtdNATURE | VOL 402 | 16 DECEMBER 1999 | www.nature.com 761

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Sequence and analysis of chromosome 2of the plant Arabidopsis thalianaXiaoying Lin*, Samir Kaul*, Steve Rounsley², Terrance P. Shea², Maria-Ines Benito, Christopher D. Town, Claire Y. Fujii, Tanya Mason,Cheryl L. Bowman, Mary Barnstead, Tamara V. Feldblyum, C. Robin Buell, Karen A. Ketchum, John Lee, Catherine M. Ronning,Hean L. Koo, Kelly S. Moffat, Lisa A. Cronin, Mian Shen, Grace Pai, Susan Van Aken, Lowell Umayam, Luke J. Tallon, John E. Gill,Mark D. Adams², Ana J. Carrera, Todd H. Creasy, Howard M. Goodman², Chris R. Somerville², Greg P. Copenhaver², Daphne Preuss²,William C. Nierman, Owen White, Jonathan A. Eisen, Steven L. Salzberg, Claire M. Fraser & J. Craig Venter²

The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA

* These authors contributed equally to this work............................................................................................................................................................................................................................................................................

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130±140 Mb), excellentphysical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype intwo gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch ofuninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scaleduplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencingof nearly 2 Mb within the genetically de®ned centromere revealed a low density of recognizable genes, and a high density anddiverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of acontinuous stretch of 75% of the mitochondrial genome into chromosome 2.

Arabidopsis thaliana (thale cress), a member of the mustard family,is a dicotyledenous ¯owering plant with a diploid number of 10 thathas become a widely used model for the study of plant biologybecause of its small size, short generation time, facile genetics andease of transformation. Whole-genome analysis began ten years agowith the production of cosmid and yeast arti®cial chromosome(YAC)-based physical maps1, while early large-scale sequencingefforts focused on ordered cosmids2. On August 20±21 1996,representatives of six research groups from Japan, Europe and theUSA met in Washington DC to discuss strategies for facilitatinginternational cooperation in completing the sequencing of theArabidopsis genome (the Arabidopsis Genome Initiative (AGI)) bythe year 2004. The group agreed on a workplan to ensure that thesequencing of all ®ve chromosomes was completed except for thedif®cult to sequence repetitive regions: the nucleolar organizerregions (NORs) and centromeres (http://genome-www.stanford.edu/Arabidopsis/AGI/AGI_memo.html)3.

The main molecular and cytological features of the A. thalianachromosomes had been de®ned by the time high-throughputsequencing began (Fig. 1). The NORs in A. thaliana are found onthe upper ends of chromosomes 2 and 4. Each region, 3.5±4 Mb inlength, consists of an array of tandemly repeated ribosomal RNAgenes4. Using a combination of restriction analysis, polymerasechain reaction (PCR) and sequencing, it has been shown that therDNA of the NOR on chromosome 4 immediately abuts thetelomeric repeats with less than 500 base pairs (bp) of interveningsequence5. Restriction analysis is consistent with a similar arrange-ment on chromosome 2, with the rDNA terminating at a differentpoint within the repeat unit. By contrast, the genomic organizationof A. thaliana centromeres is not well characterized. These regionsare heterochromatic6 and contain large tandem arrays of a family of180-bp elements7±9 that are reminiscent of the 170-bp alphoid

repeats found in primate centromeres10. Large blocks of DNAconsisting primarily of 180-bp repeats have been characterized by2D gel electrophoresis and assigned to speci®c centromeres on thebasis of restriction-fragment-length polymorphisms11. The 180-bprepeat block on chromosome 2 is 820±830 kb in size11, which shouldbe considered as a minimum estimate for the size of theunsequenced centromeric region on this chromosome.

Features of chromosome 2Chromosome 2 is acrocentric and was originally estimated to be13.5 Mb in length from the YAC-based physical map12. Sequencingwas initiated using bacterial arti®cial chromosomes (BACs) thathad been placed onto the physical map of chromosome 2 byhybridization to YACs and genetic markers. Subsequently, BACend sequences and BAC ®ngerprint data13 allowed extension fromthese initial seed points and completion of the entire chromosome.A total of 257 BAC and P1 clones (including 5 BACs completed byother groups) were sequenced to produce over 24 Mb of ®nishedsequence, which has been assembled as two contigs terminating inblocks of 180-bp repeats. These repeats represent the inner bound-aries of our ®nished sequence.

The upper (short) arm, measured from the lower end of the NORto the centromeric 180 bp repeats, is 3.6 Mb. The northern-mostBAC (F23H14) in this contig contains a single rDNA repeat unitthat is oriented in the same direction as the telomere-proximalrepeat, suggesting that all the rDNA units are arranged in a head-to-tail fashion running 59 to 39 from the telomere towards thecentromere14. Immediately adjoining the rDNA is about 60 kb ofhighly repetitive sequence containing many transposons. The lower(long) arm from centromere to telomere is 16 Mb. The sequence ofthe southern-most BAC in this contig (F11L15) is joined by a smallPCR fragment to the A. thaliana sequence in pAtT51, a telomere-containing clone previously mapped to TEL2S15 (http://genome-www.stanford.edu/Arabidopsis/ww/Nov98RImaps/index.html).Within this region, a putative transcriptional co-activator gene wasidenti®ed. Because pAtT51 was derived from the Landsberg eco-type, the last 2.5 kb of this contig may not be identical to thesequence in the Columbia ecotype.

² Present addresses: Cereon Genomics, 270 Albany Street, Cambridge, Massachusetts 02139, USA (S.R.,T.P.S.); Celera Genomics Corporation, 45 West Gude Drive, Rockville, Maryland 20850, USA (M.D.A.,

J.C.V.); Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachussets 02114,

USA (H.M.G.); Department of Plant Biology, Carnegie Institution of Washington, 260 Panama St.,

Stanford, California 94305, USA (C.R.S.); Department of Molecular Genetics and Cell Biology, University

of Chicago, Chicago, Illinois 60637, USA (G.P.C., D.P.).

© 1999 Macmillan Magazines Ltd

At 19.6 Mb, the overall length of chromosome 2 (excluding theNOR and centromere) is 45% larger than the original estimate. Thisdifference is due in part to gaps in the YAC-based physical map andto a deletion in one of the YACs (see Fig. 1). Similar increases in theactual physical lengths of the other A. thaliana chromosomesindicate that the genome size is actually 130±140 Mb, rather thanthe earlier estimates of 70±100 Mb.

Using sequence information for 37 markers from the ArabidopsisRecombinant Inbred genetic map (http://genome-www.stanford.edu/Arabidopsis/ww/Nov98RImaps/index.html), the relationshipbetween physical and genetic distances along the chromosomewas examined. As the centromere represents a discontinuity,regression analysis for the two arms was performed separately.The ratios of physical to genetic distance on the short arm andlong arm were not signi®cantly different, with values of 244 kb cM-1

and 223 kb cM-1, respectively (Fig. 2a). Although these two valuesindicate a similar overall rate of recombination along the two armsof the chromosome, local distortions are evident which may re¯ectchromosomal rearrangements between the two ecotypes used toconstruct the mapping population (Fig. 2b).

Gene contentUsing a combination of gene prediction programs and databasesearches, the chromosome was annotated (Fig. 3 (facing page 768);and Table 1). Statistics of the chromosomal composition are listedin Table 2, along with corresponding data from other eukaryoticgenome projects. A graphical distribution of various features isshown in Fig. 4. Of the 4,037 genes identi®ed, 2,078 (51.5%) wereassigned either a de®nite or putative function, 865 (21.4%) werelabelled as unknown genes and 1,094 genes (27.1%) were designatedas hypothetical. In addition, 400 pseudogenes, most of which arerelated to proteins found in retrotransposons and are located nearthe centromere, and 79 genes encoding structural RNAs (73 tRNA, 4tRNA and 2 small nucleolar (snRNA)) were found.

On average, a gene occurs every 4.4 kb, is about 2 kb in length(from start to stop codon) and contains 4.6 exons. More than 50%of the chromosome is therefore intergenic sequence. Twenty-threeper cent of the genes consist of a single exon, whereas the largest

gene (T20F21.17) contains 52 predicted exons and encodes aprotein that is 50% identical to the human ch-TOG protein16. Atleast 1,352 (33.5%) genes are expressed, as re¯ected by the presenceof a matching complementary DNA or expressed sequence tag(EST) entry in GenBank. Genes that are most highly representedby ESTs include a glycine-rich RNA-binding protein, aquaporins,ribulose bisphosphate carboxylase activase, a chlorophyll a/bbinding protein and lipid transfer proteins. The distribution ofgenes and ESTs along the chromosome is shown in Fig. 4b, c.

Classi®cation by biological role or biochemical function of theknown and putative genes shows that most major cellular processesappear to be represented, with genes involved in regulatory func-tions and signal transduction (DNA-binding proteins/transcriptionfactors and protein kinases) comprising the largest functionalgroups (Table 1). These homology-based assignments are consistentwith results from searching the hidden Markov models (HMMs) ofprotein domains in the pFam database17. Of the 4,037 genespredicted to encode a protein, 1,436 match at least one of the 434distinct pFam HMMs (22% of the total HMMs). The mostfrequently seen matches are to the LRR (leucine-rich repeat, 239matches), the pkinase (eukaryotic protein kinase, 161 matches) andthe zf-C3HC4 (zinc ®nger, C3HC4 type, 46 matches) HMMs. Thecellular destinations of the proteins were also predicted. Over 17%(698) contained signal peptides, whereas over 10% (416) werepredicted to be organelle-targeted (135 for the chloroplast and281 for the mitochondrion). In addition, more than 16% of theproteins are probably located in cellular membranes, with 665sequences predicted to have 4 or more transmembrane segments(TMSs). Furthermore, about 25% of the proteins with 1, 2 or 3predicted TMSs (1,438) are predicted to be localized to themembrane.

Gene and chromosomal duplicationsChromosome 2 contains many duplicated genes. More than 60% of

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762 NATURE | VOL 402 | 16 DECEMBER 1999 | www.nature.com

cMMarker

49< er

19< mi421

69< ve018

87< mi79a

3.5< kk1

Telomere

Centromere

Telomere

NOR

∆

{

Figure 1 Features of Arabidopsis thaliana chromosome 2. The chromosome is roughly

23 Mb long (including the NOR), spans a genetic map distance of 90 cM and contains

15% of the unique sequence of the A. thaliana genome. The locations of a number of

genetic markers are shown. The original YAC contigs are represented by the orange line,

and the location of the deletion noted in the text is shown (D).

2,500

500

400

300

200100

2,0001,5001,000

20,000

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8,000

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b

0 40 60 80 10020

0 40 60 80 10020

Genetic map distance (cM)Phy

sica

l/gen

etic

dis

tanc

e (k

b c

M–1

)P

hysi

cal d

ista

nce

(kb

) a

Figure 2 Representation of physical/genetic distance along chromosome 2. a, Physical

distance (in kb) is plotted against position of each marker on the Recombinant Inbred (RI)

map. Data above and below the centromere are treated as separate sets for regression

analysis. Note that genetic distance is measured from the telomere (TEL2N), whereas

physical distance is measured from the bottom of the NOR. b, The ratio of physical to

genetic distance between successive pairs of markers is plotted against map position

for each marker pair on the RI map.


the predicted proteins (2,542 out of 4,037) have a signi®cant match(FASTA, P , 10 2 10) to another A. thaliana protein, with 2,138matching at least one other protein encoded on chromosome 2. Ofthese, 593 are found in 239 tandem duplications that range in sizefrom 2 to 9 genes (average 2.5). A particularly striking example isfound in BAC F16P2, which contains tandem repeats of three genefamilies (Fig. 5). Of the 12 intact copies of a putative short-chaindehydrogenase/reductase gene in this region, 9 are found in tandem.This gene family is most similar to tropinone reductase, a branchpoint in the biosynthesis of tropane alkaloids including suchmedicinally important compounds as atropine, scopolamine andcocaine18,19. Although these alkaloids are not known to occur in theBrassicaceae, at least one of these genes is expressed, suggesting arole in secondary metabolism which has yet to be elucidated. Thissame BAC also contains a tandem array of seven genes encodingglutathione S-transferase (GST), which has been implicated innumerous plant processes including stress and pathogen responses,xenobiotic tolerance and vacuolar sequestration20. In total, 13 GSTgenes have been found on chromosome 2, with 4 of the other genesoccurring as tandem pairs. The GSTs within the long tandem repeatare more closely related to one another than to any other of the 25GSTs known in Arabidopsis, indicating that this expansion is ofrelatively recent origin. There is also a smaller duplication consisting

of three pumilio-like genes, two of which are in tandem. Theproximity of these gene duplications suggests either that this is arepeat-prone region of the chromosome or that they arose as part ofthe same expansion event.

Some of the duplicated genes are found within large chromoso-mal duplications (Fig. 6). A segment of the bottom arm ofchromosome 2 (13.4±14.1 Mb, containing 170 genes) from cloneF20M17 to F4P9 was found to be duplicated on chromosome 1(from clone F19P19 to F3F20) with an inversion in the middle(13.8±13.9 Mb on chromosome 2). Within this region, 57 gene pairs(33%) are duplicated between the two chromosomes. Severalinstances of tandem duplications on just one of the two chromo-somes were also observed. For example, F4P9.8 encodes an auxin-inducible protein that matched two adjacent copies of a similarprotein-coding sequence (F19P19.31 and F19P19.32) on chromo-some 1; and a single copy of a gene encoding an ion-channel proteinon chromosome 1 (YUP8H12.9) matched two adjacent copies onchromosome 2 (T32F6.9 and T32F6.8). Sequence comparison of theproteins in these asymmetric duplications suggests that the tandemduplication events occurred after the large-scale duplication. Anadditional smaller section near the top of chromosome 2 (200±500 kb) shares 16 genes with a region on chromosome 1 (T10B6).An even larger duplication (4.6 Mb) between chromosome 2 (6.7±11.3 Mb, from clone F9O13 to F18A8) and chromosome 4 (fromclone F20O9 to the bottom end of the chromosome) was found. Inthis region, 39% of the genes (430 out of 1,100) are duplicatedbetween the two chromosomes, including some singleton-tandemduplication pairs, as described above. In addition, although thisregion contains megabase-scale rearrangements (translocations orinversions), within each rearranged segment gene order is stillpreserved between the two chromosomes. A small part of thisduplication (45 kb) has been described recently21.

Comparative and evolutionary genomicsComparison of the predicted proteins on chromosome 2 to otherA. thaliana proteins, to proteins from other plants, and to allavailable complete genome sequences provides insight into theevolution of plant genomes. More speci®cally, the timing of geneduplications can be better understood. For example, as most of the2,542 chromosome-2 proteins (83%) that have paralogues withinthe A. thaliana genome are more similar to their paralogue than toany protein in the available completed genomes, it is likely that thesegene duplications occurred after the separation of plants from theanimal and fungal lineages. This analysis also suggests that all plantshave a common set of genes for many functions. Even thoughsequencing in other plants has focused primarily on particular typesof genes, more than 40% (1,656) of the proteins encoded onchromosome 2 have a signi®cant match to a protein previously

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NATURE | VOL 402 | 16 DECEMBER 1999 | www.nature.com 763

Table 1 Classi®cation of chromosome-2 proteins according to role orfunction

Percentage ofchromosome-2

proteins.............................................................................................................................................................................

Regulatory functions 7.8Signal transduction 4.0Cellular structure, organization and biogenesis 3.6Transport and binding proteins 3.6Protein fate 3.0Energy metabolism 2.7Growth and development 2.6Secondary metabolism 2.5Cellular processes 2.4Pathogen responses 2.0Protein synthesis 1.9Fatty acid and phospholipid metabolism 1.5General transcription 1.4Environmental response 1.0DNA metabolism 0.7Central intermediary metabolism 0.7Amino-acid biosynthesis 0.6Purine and pyrimidine base, nucleoside and nucleotide metabolism 0.6Biosynthesis of cofactors, prosthetic groups and carriers 0.4Other categories 6.9Unclassi®ed 1.4Unknown protein 21.4Hypothetical protein 27.1.............................................................................................................................................................................

4,037 genes are included in this analysis.

Table 2 Compositional analysis of Arabidopsis chromosome 2 and comparison with other eukaryotic chromosomes and genomes

Feature A. thaliana* P. falciparum* S. cerevisiae² C. elegans²...................................................................................................................................................................................................................................................................................................................................................................

Overall length Total: 19.6 MbShort arm: 3.6 MbLong arm: 16.0 Mb

0.95 Mb 13.4 Mb 97 Mb

Base composition (%GC) Overall: 35.8%Coding: 43.6%

Non-coding: 32.1%

19.7%24.3%13.3%

38%40%35%

36%§§

Number of genes 4,037 209 6,339 19,099Gene density 4.4 kb per gene 4.5 kb per gene 2.1 kb per gene 5.0 kb per gene

Exon statistics³ 20,352 exons4.6 exons per gene

295 bp per exon

355 exons1.7 exons per gene

728 bp per exon

6,561 exons1.04 exons per gene

§

§5 exons per gene

§

Intron statistics 15,926 introns3.6 introns per gene

178 bp per intron

144 introns1.6 introns per gene

207 bp per intron

§§§

§§§

...................................................................................................................................................................................................................................................................................................................................................................

* Chromosome 2 only.² Complete genome.³ Includes intronless genes.§ Not published.


identi®ed in other plant species. Of these, 48% (803) do not have amatch in any complete genome sequence. In addition, comparisonwith other species indicates that many of the genes on chromosome2 probably originated within the plant lineage. More than 65%(2,745) of the predicted proteins do not show signi®cant similarityto any other completed genome, suggesting that they are unique toplants. Of the remaining proteins (1,293), the majority are mostsimilar to proteins from Saccharomyces cerevisiae (368 or 28%) orCaenorhabditis elegans (633 or 49%). The number of proteins whosebest match is to one of these two proteomes is proportional to thetotal number of proteins encoded in each genome, suggesting that A.thaliana is roughly equally evolutionarily distant from animals andfungi. The remaining 292 proteins (23%) are most similar to bacterialand archaeal proteins, which may be due in part to transfers of genesfrom organellar genomes to the nucleus (see below).

Lateral transfers of genes from organelles to the nucleus have beenwell established22. In most cases, such genes were identi®ed becausetheir products were known to function in the organelle. The

sequence of chromosome 2 allows a reverse type of analysis,identi®cation of nuclear genes that originated in the chloroplastby their evolutionary ancestry. Genes most similar to cyanobacterialgenes relative to genes from other complete genomes were con-sidered to have been acquired from the chloroplast genome. Usingthis methodology, 135 putative chloroplast-derived genes wereidenti®ed. Of these, 71 (52.5%) are predicted to have chloroplast-targeting peptides. The putative functions of these genes includeDNA repair, protein synthesis, regulatory functions, cell division,photosynthesis, transport and oxidation±reduction. As these genesare not found in any of the completed plant chloroplast genomes,most of these gene transfers probably occurred soon after thesymbiosis with the chloroplast evolved. Many other genes are ofunknown function and will be worth pursuing as potential novelchloroplast-targeted functions.

A mitochondrial genome in the nucleusCompletion of the chromosome has revealed a large and

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Trinucleotide compositionvariation

GC content (%)

Gene density

EST density

Tandem gene duplications

tRNA gene density

Transposon density

Intermediate repeats

20304050607080

020406080

02468

0

2

4

6

0

5

10

15

Chromosome position (Mb)

0 5 10 15 20

c

a

b

g

f

e

d

0

10

20

30

Figure 4 Distribution of various features along chromosome 2. The two arms of the

chromosome are represented as a continuous sequence of DNA, beginning immediately

after the NOR (position 1) and continuing to the centromere (position 3,606,929), where it

is joined to the long arm (which terminates with the most telomeric base (position

19,647,005)) by a 120-bp spacer. The various features are plotted relative to the

nucleotide position on the chromosome. a, Chi-squared statistics of trinucleotide

frequency50 and GC composition in a 10-kb sliding window. b, Gene density per 100 kb;

c, EST density per 100 kb. d, Tandem repeats, showing repeat number as well as

location. e, tRNAs per 100 kb. f, Density of transposon-related sequences per 100 kb.

g, Position of pericentromeric repeat classes along chromosome 2. From top to bottom

they are 180 bp, Athila, 106B, 11B7RE, 163A, 164A, 278A, mi167, pAtT27, new repeats

1±4. The GC peak (45%) and the gaps in gene and transposon density from 3.2 to 3.5 Mb

reveal the site of the mitochondrial DNA insertion.


unexpected organellar-to-nuclear gene-transfer event. Within thegenetically de®ned centromere, there is a stretch of 270 kb ofsequence that is nearly identical to that of the Arabidopsis mito-chondrial genome. The authenticity of this insertion in the Colum-bia ecotype was con®rmed by PCR ampli®cation across thejunctions of mitochondrial and unique nuclear DNA, followed bythe sequencing of the corresponding fragments. This insertion is

much larger than any of the previously reported organellar-nucleartransfers23, and is 99% identical to the mitochondrial genome,suggesting that the transfer event was very recent. As the publishedsequence is derived from the C24 ecotype24, it is not possibledetermine whether the observed sequence polymorphisms are dueto divergence of the insertion from the mitochondrial genome or todifferences among ecotypes. The organization of the mitochondrial

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Chromosome 2 coordinate (Mb)

Chr

omos

ome

1 re

lativ

e p

ositi

on(M

b)

13.4 13.6 13.8 14.0

1.0

1.4

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a

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Chromosome 2 coordinate (Mb)

Chr

omos

ome

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e p

ositi

on(M

b)

7.0 8.0 9.0 10.0 11.0

16.0

15.0

14.0

13.0

12.0

17.0

Figure 6 Large chromosomal duplications. Regions of similarity were initially detected

using the MUMmer program after which sequences were aligned using the dds program

with criteria of at least 75% identity over 100 bp. Points in the dot plot represent the

coordinates of each such match. Values show positions (in bp) within each contig

assembled from all available sequence. a, Duplication between chromosomes 1 and 2.

b, Duplication between chromosomes 4 and 2. Arrows indicate the small, previously

described duplication21.

G GG G G G G

T T T T T T T TT T

PP T T

P

26349 30113 33877 37641 41405 45169 48933

101829

75281715176775363989602255646152697

978659410190337865738280979045

127977124213120449116685112921109157105393

Figure 5 Organization of genes on BAC F16P2 showing the three tandem gene

duplications. The display from TIGR Annotator shows the exon±intron structure of the

annotated genes. The glutathione S-transferase and tropinone reductase genes are

labelled G and T, respectively. A smaller duplication of pumilio-like protein (P) is also

present.


DNA in the nucleus differs from that of the published mitochon-drial genome and its predicted alternate forms25; it corresponds toanother possible isoform with an internal deletion (Fig. 7). Thisdeletion may have occurred during or after transfer or mayrepresent an alternate form of the Columbia mitochondrialgenome.

The pericentromeric regionUntil the completion of chromosome 2, it was unclear what featureswould be discovered in the regions around the centromere (thepericentromeric region). In addition to the detection of the mito-chondrial genome insertion, an analysis of this region has revealedthe presence, distribution and diversity of speci®c repetitive ele-ments on a single chromosome. Transposons were identi®ed bysearching for predicted amino-acid sequences that are similar tocharacterized transposases, reverse-transcriptases, polyproteins orother transposon-related coding sequences. Of the 563 transposonproteins annotated, 50% are pseudogenes found either as fragmentsof previously intact elements or as genes interrupted by stop codons,frameshifts or other mutations. A breakdown of the classes oftransposons identi®ed is shown in Table 3. Representative membersof most of the large transposon families seen in other plants(Mutator, En-Spm and Ac-Ds (haT/mariner)) are present. Retro-elements including members of the LINE-like Ta11 elements, long

terminal repeat (LTR) elements in both the Ty3-gypsy and the Ty1-copia family, and the Arabidopsis Athila family represent the largestclass of transposons. This accumulation of retroelements and othertransposons may be due to either preferential insertion into theregions ¯anking the centromere or their elimination from the rest ofthe genome at a higher rate26. In addition, representatives ofpreviously described intermediate repeats as well as some newrepeats were identi®ed (Fig. 4f), a distribution consistent withprevious hybridization data27±30.

As shown in Fig. 4, the number of recognizable genes decreasesclose to the centromere, whereas the number of transposableelements and various classes of intermediate repeats increases.Nevertheless, within this region there are some identi®able genes:a protein kinase (T25N22.14); a plasma membrane proton ATPase(F9A16.7); an ABC transporter (T5E7.1); a DNA-replication licen-sing factor (T12J2.1), an RNA helicase (T12J2.7) and a 40Sribosomal protein S16 (F7B19.13). The RNA helicase, proteinkinase and 40S ribosomal protein appear to be actively expressedgenes, as each is represented in the EST collection.

ConclusionsArabidopsis has been considered to be not only a tractable species forplant biology studies, but also a suitable model from which afundamental understanding of basic plant processes could beapplied to agriculturally relevant species. The analysis of thecomplete sequence of chromosome 2 validates this concept becausemany genes that have previously been characterized in other plantshave been found. As observed with other eukaryotic genomeprojects, however, roughly half of the predicted genes have noknown function. This represents a large, untapped resource ofinformation that will require a combination of knockoutapproaches, antisense technologies and global gene-expressionanalyses to understand their functions.

Both gene duplication and large-scale chromosomal duplicationsare observed, events that apparently occurred after the divergence ofplants from other lineages. These duplications are larger than anyreported in the C. elegans genome31, but less extensive thanthe complete genome duplications proposed for maize32 andS. cerevisiae33. As gene duplication is often accompanied by func-tional divergence34, the identi®cation of such duplications in theplant lineage may help identify new plant functions. Many of theduplications occurred in tandem, generating closely linked genefamilies which may provide some of the allelic diversity thatbreeders and agricultural researchers are seeking. Some newfamilies, such as the family of genes that may encode one step inalkaloid biosynthesis, could provide a reservoir of molecular tools tomodify plant metabolism and productivity in a bene®cial manner.

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D' 1 B 3 C 1 A' 3c

d

100 200 300 Mito (kb)

3430

3230

3330

Chr

2 (k

b)

A

1

B3

C

1

D

3'

D'

A'

1

1

B

C

Possibleinsertionpoint

3

3

ba

A. thalianamitochondrial

genome366,923 bp

Figure 7 Proposed model for insertion of mitochondrial DNA into chromosome 2.

a, Diagram of the major form of the published A. thaliana C24 mitochondrial genome25.

The different colours correspond to the major segments of the genome: A (nucleotide

297,579±44,698); B (48,895±112,146); C (118,737±178,862); D (183,060±

290,989); and two major repeats, 1 (44,698±48,894, 178,863±183,059) and 3

(112,147±118,736, 297,579±290,990). A small repeat (repeat number 2) is not shown.

The orientation of one of the three repeats is inverted relative to the other. The arrows are

used to indicate orientation for comparison with the alternative form. b, Hypothetical

alternative form, generated by in silico rearrangements around some of the repeats.

Arrows and primes indicate inversions. c, Linearized form of genome shown in b if

inserted at the point between repeat 3 and segment D9. d, Dot plot of the linearized

alternate form against the region of chromosome 2 containing the mitochondrial DNA. The

gap in the diagonal indicates that there was a deletion of a large segment (see text).

Table 3 Representaton of transposable elements on chromosome 2

Transposon class Subclass Family Number of openreading frames

.............................................................................................................................................................................

DNA elementsMutator 73

En-Spm/Tam1/Psl 71Ac-Ds 12mariner 2

hAT 1

RetroelementsNon-LTR LINE-likeretrotransposons

Ta11-like 152TSCL 1

LTR retrotransposonsAthila 75Other 141

Ty1-copia-like (Ta1)Ty3-gypsy-like (Tat1)

Retroviral-like Replication protein 28Helicase 7

.............................................................................................................................................................................

Total 563.............................................................................................................................................................................


The completion of chromosomes 2 and 4 (ref. 35), whichaccounts for 30% of the entire Arabidopsis genome, represents alandmark achievement in eukaryotic genomics. The 16-Mb contigon the lower arm of chromosome 2 is the largest sequence assemblypublished to date. Sequencing within the genetically de®ned cen-tromere has shown not only an abundance of degenerate transpos-able elements, but also a number of recognizable intact genes, someof which appear to be expressed. As researchers move on tosequence larger and more complex genomes, it remains to be seenwhether the level of genome closure achieved in this model plantcan be duplicated.Note added in proof : Since this paper was accepted for publication, a23Mb contig from human chromosome 22 has been published(See Nature 402, 489±495 (1999). M

MethodsSequencing

Three libraries made from the Columbia ecotype of A. thaliana were used in sequencingchromosome 2: the TAMU BAC library (clones pre®xed with T36); the IGF BAC library(clones pre®xed with F37); and the Mitsui P1 library (clones pre®xed with M38). ShearedBAC DNA (1.6±2 kb) was ligated to a modi®ed pUC19 vector and transformed intoEscherichia coli. Sequencing reactions were performed using either BigDye primers,BigDye Terminators or Dichlorohodamine Terminators (P.E. Biosystems), and were runon ABI 377 and ABI 3700 sequencers (P.E. Biosystems). Shotgun clones were sequenced togenerate 7±8-fold coverage of each BAC. In total, 590,165 reactions were carried out togenerate the sequence of the chromosome. In collaboration with Celera Genomics, 99,072reactions were run on ABI 3700 sequencers, which accounted for nearly 20% of the overallsequence for the chromosome. The discrepancy rate on these machines, as determined bycomparison of overlapping BACs, was comparable to that of ABI 377 machines (1unresolved discrepancy per 32,000 bases). A base difference is possibly due to residualheterozygosity or to different DNA sources used to make the various libraries. IndividualBACs were assembled from the shotgun sequences using the TIGR Assembler program39.Following assembly into contigs, the Grouper program (TIGR, unpublished software) wasused to link individual assemblies within the BACs. The BACs were then closed using acombination of BAC walking, directed PCR and resequencing of individual shotgunclones. To con®rm the assembly of the entire BAC sequence, predicted and actualrestriction digest patterns were compared. The collinearity of the BACs with theArabidopsis chromosome 2 sequence was con®rmed by alignment of the sequence ofchromosome 2 with markers obtained from the Arabidopsis Recombinant Inbred map(http://genome-www.stanford.edu/Arabidopsis/ ww/Nov98RImaps/index.html) andwith YAC end sequences. Several markers that clearly mapped to single genetic loci wereexcluded from this analysis because they contained repetitive sequence and could not beassigned a unique location on the sequence map.

Two BACs (T5M2 and T5E7) within the genetically de®ned centromere26 were found tocontain long stretches of sequence with very high similarity to the Arabidopsis C24mitochondrial genome (69 kb on T5M2, and 74 kb on T5E7) joined directly to nuclearsequence. Two overlapping BACs provided an additional 122 kb of purely mitochondrialsequence to close the gap, suggesting that the total size of the organellar DNA insertion isabout 270 kb. Veri®cation of the mitochondrial insertion involved PCR ampli®cation ofthe two ¯anking junction sequences from Arabidopsis Columbia genomic DNA. PCRproducts with the expected size were ampli®ed that had sequence identical to the junctionBACs. Similar sized junction PCR products were obtained using additional BACs fromboth the TAMU and IGF libraries. Determination of the exact size and sequence of theinsertion will be facilitated once the sequence of the Columbia mitochondrial genome isknown, and it becomes possible to distinguish between nuclear- and organellar-derivedmitochondrial sequences.

The sequence of chromosome 2 identi®ed by the BAC clone F11L15 did not contain thetelomeric repeat consensus sequence 59-TTTAGGG-39 (ref. 15). However, the sequence ofthe lower telomere of chromosome 2 had been previously identi®ed in the clone pAtT51(ref. 15) (http://genome-www.stanford.edu/Arabidopsis/ww/Nov98RImaps/index.html).pAtT51 was sequenced to identify primer sites that could be used to walk inwards from thetelomere. Southern hybridization using probes derived from F11L15 and pAtT51suggested that the gap between the BAC contig and pAtT51 was less than 1 kb. A 702-bpPCR product that spanned this physical gap was ampli®ed from Arabidopsis Columbiagenomic DNA and sequenced.

Analysis

Annotation of chromosome 2 involved both DNA and protein database searches and geneprediction programs. Gene predictions were made by Genscan40, Gene®nder (P. Green andL. Hillier, unpublished data) and GRAIL41. Splice sites were identi®ed by these programs,by NetPlantGene42 and by alignment with EST and protein sequences using the dds anddps programs43. Predicted protein sequences were searched against a non-redundantamino-acid database and against HMMs of the protein domains from pFAM3 (1,407) andTIGR built HMMs (502) with the HMMer2 program17. tRNAs were identi®ed usingtRNAscan-SE44. Repeats were identi®ed using RepeatMasker (A. F. A. Smit and P. Green,http://ftp.genome.washington.edu/RM/RepeatMasker.html) and a modi®ed version ofthe suf®x tree algorithm used in the MUMmer system45. Output from the gene ®nding andsignal detection programs was displayed using the Annotator genome viewer (L. Zhou,

unpublished data) and manually edited. The accuracy of computational gene ®nders forArabidopsis is quite variable and all gene predictions that are not supported by indepen-dent evidence, such as protein or EST homology, should be regarded as tentative, pendingfurther research.

Putative membrane-spanning proteins were identi®ed using TopPred46. SignalP47 wasused to detect signal peptide sequences, while Predotar (N. Peeters et al., unpublished)searched for organellar targets. In addition, ChloroP48 and Mitoprot49 were used to ®ndproteins targeted for the chloroplast and mitochondria, respectively. The results presentedare the concordance of the predictions.

We analysed the proteome of chromosome 2 by comparing each predicted protein witha database of all proteins from chromosome 2, all available completed genomes and allproteins from A. thaliana and C. elegans using described methods50. To identify genes onother chromosomes that were related to chromosome-2 genes, nucleotide searches werealso used as much of the nucleotide sequence in GenBank is unannotated. Each predictedtranscript was searched against all available genomic sequence using a cut-off of 75%identity over 50% of the transcript length in order to take intron sequences into account.

To search for large-scale duplications within the genome, we assembled pseudomole-cules representing all available sequence of chromosomes 1 and 4. The sequence ofchromosome 2 was then compared with chromosomes 1 and 4 using the MUMmerprogram45 with a word size of 20. Long-range duplications were found by plotting theresults, and a local alignment program dds43 was used to identify matches between theexact repeats within the duplicated regions (stringency .75%). Because of insuf®cientsequence or overlap information, it is dif®cult at the present time to constructchromosome-size pseudomolecules of chromosomes 3 and 5 and assess the possibility ofother large duplications.

Received 5 October; accepted 29 October 1999.

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Acknowledgements

We thank S. Jackson, J. Jiang, K. Meyer, E. Richards and S. Tabata for helpful contributionsin completing the chromosome. In addition, we would like to thank the TIGR faculty,system administrators, sequencing facility, bioinformatics department and administrativestaff. This work was supported by the National Science Foundation, the Department ofEnergy and the US Department of Agriculture.

Correspondence and requests for materials should be addressed to J. C. Venter(e-mail: [email protected]).

articles


Figure 3 Gene map of Arabidopsis thaliana chromosome 2. Predicted gene models are

shown with arrowheads indicating the direction of transcription. Genes are colour coded

according to broad role categories as shown in the key. The region highlighted in pink

indicates the genetically de®ned centromere within which the cross-hatched gene-free

region represents the mitochondrial insertion. The gap indicates the highly repetitive

centromeric region. Note that the NOR is not drawn to scale. Gene identi®cation numbers

follow the recently adopted nomenclature (At2gNNNNN), where At is Arabidopsis thaliana,

2 is chromosome number, g indicates that the feature is a gene and numbering is from top

to bottom of each chromosome in increments of 10.

Q


Puj

a

Dan

iela

Nuc

leol

ar o

rgan

izer

reg

ion

Tel

omer

e

tRN

A

18S

RN

AS

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nce

gap

Mito

chon

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l reg

ion

Gen

etic

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ere

5.8S

RN

A

26S

RN

A

snR

NA

1010

1020

1030

2940

29

20

2000

019

990

1998

019

970

1996

0

1039

0

2381

0

1038

0

2380

0

1037

0

2379

0

1036

0

2378

0

1035

0

2377

0

1034

0

2376

0

1033

0

2375

0

1032

0

2374

023

730

2742

027

430

2744

027

450

2746

027

470

2748

027

490

2750

027

510

6830

6840

6850

6860

6870

6880

6890

6310

6320

6330

6340

6350

6360

6370

6380

6390

6400

6410

1987

019

860

1985

019

840

1983

019

820

3487

0

1981

0

3488

0

1980

0

3489

0

1979

0

3490

0

1978

0

3491

034

920

3493

034

940

3496

0

4387

043

860

4385

043

840

4383

043

820

4381

043

800

1977

019

760

1975

019

740

1973

019

720

3497

0

1971

019

700

3498

034

990

1969

0

3500

0

2163

0

1968

0

3501

0

2162

021

610

3502

035

030

2160

0

3504

0

2159

0

3505

0

2158

0

3506

0

2156

021

550

2154

0

4210

042

090

4208

042

070

4206

042

050

4204

042

030

1967

0

3246

032

470

3248

032

490

3250

032

510

3252

032

530

4113

041

120

4111

041

100

4109

041

080

4107

041

060

3215

0

4105

0

3214

032

130

4104

0

3212

032

100

3209

032

080

3207

032

060

1699

017

040

4103

041

020

4101

041

000

4099

040

980

3205

032

040

3203

032

020

3201

032

000

3199

031

980

3197

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960

4090

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4092

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570

1216

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170

2558

0

1218

0

2559

0

1219

0

2560

025

610

1220

0

2562

0

1221

0

2563

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640

1166

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650

1164

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630

1162

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610

1160

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590

1222

0

1158

0

1223

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240

1225

012

260

1227

012

280

1229

012

300

1231

012

320

5350

2874

028

750

2876

028

780

2879

028

800

4012

040

130

4014

040

150

4019

040

200

4021

0

2134

021

350

2137

021

380

2139

021

400

2141

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420

2143

0

1185

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860

1187

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880

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4780

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1736

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290

1738

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9790

9800

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6740

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6670

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2826

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7260

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7280

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