Chromosome-Specific Satellite Sequences in Turritis Glabra

Genes Genet. Syst. (2006) 81, p. 287–290 Chromosome-specific satellite sequences in Turritis glabra

Akira Kawabe*† and Shuhei Nasuda Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho Sakyo-ku Kyoto 606-8502, Japan

(Received 5 May 2006, accepted 25 July 2006)

Two novel repetitive sequence families were isolated from Turritis glabra (2n = 2x = 12). These two repeat families are similar to those of centromeric repeats in Arabidopsis thaliana, are co-localized on one chromosome pair, and differ by about 20% from each other. Phylogenetic analysis revealed that the two repeat families of T. glabra are more similar to each other than to the centromeric repeat families of other Arabidopsis and related species. The relationships of satellite sequences reflected the species phylogeny, indicating that the replacement of satellite sequences has occurred in each species lineage independently, and shared variants could not have existed for a long time between species.

Key words: Arabidopsis, centromere, satellite sequence, Turritis glabra

Centromeric satellite sequences have been reported for diploid species (2n = 2x = 12) and has been used as mate- Arabidopsis thaliana (Maluszynska and Heslop-Harrison, rial for molecular evolution studies as a reference for A. 1991; Murata et al., 1994), and several related species thaliana (Kawabe et al., 1997; Miyashita et al., 1998; (Kamm et al., 1995; Heslop-Harrison et al., 2003; Kawabe Koch et al., 1999, 2000; Kawabe and Miyashita, 2002ab; and Nasuda, 2005; Hall et al., 2005). All the species Lysak et al., 2006).The estimated divergence between T. have similar sequences of 180-bp unit tandem repeats. glabra and A. thaliana is about 20% at synonymous sites A. thaliana has a major centromeric repeat (pAL1) on all of the coding regions, which is similar to the level of diver- ten chromosomes (Maluszynska and Heslop-Harrison, gence between A. thaliana and C. rubella or O. pumilla 1991; Murata et al., 1994). Similarly to A. thaliana, A. (Koch et al., 2000). arenosa and Olimarabidopsis pumila (syn. A. pumila and A plant of T. glabra was sampled from Ohmi-Shira- A. griffithiana) have species-specific 180-bp repeats hama in Shiga prefecture, Japan. Total DNA was puri- (respectively named the pAa and pAg family) on all chro- fied by a modified CTAB method and digested by several mosomes (Kamm et al., 1995; Heslop-Harrison et al., 6-cutter restriction endonucleases. Digestion by only 2003; Hall et al., 2005). In contrast, A. halleri ssp. gem- HindIII or DraI yielded weak ladder pattern bands as mifera and A. lyrata ssp. kawasakiana have at least three expected for tandemly repeated sequences. A basic unit different types of centromeric repeats with chromosome of the ladder bands (about 180 bp) from HindIII-digested specificity (Kawabe and Nasuda, 2005). These three DNA was cloned into pUC18 and sequenced as described families are the pAa family isolated from A. arenosa and by Kawabe and Nasuda (2005). Newly obtained two novel families, pAge1 and pAge2. All these species’ sequences were deposited to DDBJ under Accession num- centromere satellite families differ up to 40% but can be bers AB082536–AB082541. aligned. Capsella rubella, however, has completely dif- Six clones similar to pAL1 of A. thaliana were isolated ferent satellite sequences from other Arabidopsis rela- from HindIII-digested total DNA of T. glabra. All six tives, although the time of its divergence from O. pumila clones have one DraI site, which may cause the ladder is shorter than that between A. thaliana and O. pumila bands produced by DraI digestion. These six sequences (Hall et al., 2005). are 56.9% to 67.8% identical to the pAL1 family sequ- In the present study, satellite sequences similar to the ences of A. thaliana, and can be classified into two groups centromeric 180-bp repeat of A. thaliana were isolated depending on sequence identities. In the present study, from Turritis glabra (syn. Arabis glabra). T. glabra is a the two repeat families are designated pAgl1 and pAgl2. The pAgl1 and pAgl2 sequences are respectively 183 bp Edited by Kiichi Fukui and 179 bp in length and are about 80% (77.5% to 79.8%) * Corresponding author. E-mail: [email protected] † Present address: Institute of Evolutionary Biology, University identical to each other. Within each family, there are 13 of Edinburgh, Ashworth Laboratories King’s Buildings, West and 14 segregating sites in pAgl1 and pAgl2, respectively. Mains Road, Edinburgh EH9 3JT, UK On average, 4.7% and 5.2% nucleotides differ between

288 A. KAWABE and S. NASUDA sequences within pAgl1 and pAgl2, respectively. relatives might occur in each species lineage indepen- The existence of centromeric satellite sequences of dently, and shared satellite families could not be main- other species in the T. glabra genome was tested by PCR tained for a long time. (Fig. 1) as described in Kawabe and Nasuda (2005). PCRs with the pAL1, pAa, pAge1, pAge2, pAg, and C. rubella centromere satellite-specific primers showed no amplification, whereas pAgl1 and pAgl2-specific primers amplified typical ladder fragments. This result indicates that the T. glabra genome has pAgl1 and pAgl2 families as tandem arrays but has no sequences very similar to previously isolated centromere satellite sequences from Arabidopsis species.

Fig. 1. Existence of 180-bp satellite families. PCR products Fig. 2. Phylogenetic relationship of centromeric satellite fami- were electrophoresed on a 2% agarose gel. The primer combi- lies. A phylogenetic tree was constructed by the NJ method nations are indicated at the top of each lane. A 100-bp DNA with Jukes and Cantor distances (MEGA2: Kumar et al., 2000). ladder (Invitrogen) was used as a size marker. Primers for Consensus sequences for each of pAL1 (Martinez-Zapater et al., pAL1, pAa, pAge1 and pAge2 were described in Kawabe and 1986; Simoens et al., 1988), pAa (Kamm et al., 1995; Kawabe Nasuda (2005). Sequences of the primers for T. glabra satellite and Nasuda, 2005), pAge1, pAge2 (Kawabe and Nasuda, 2005), families were 5’-ACC GAT TTT TGA AAA CTT CT-3’ and 5’-CTT and pAg (from A. griffithiana (O. pumila); Heslop-Harrison et CTC CCC CCC CCA CCC AC-3’ for pAgl1 and 5’-ACC GAT TTT al., 2003) and six T. glabra sequences were used. Bootstrap TGT GAA CTT CT-3’ and 5’-AGA AAT TCT CTC CCA CCC AC- probabilities with 500 replications are shown beside the 3’ for pAgl2. Two other primer sets were used for the pAg fam- branches. The scale bar is shown at the bottom left of the tree. ily (5’-CTT CTA CAC TCA CCC ACA CC-3’ and 5’-CTA GGA CTT CTT CTT CAC CTA T-3’) and C. rubella centromere satellite family (5’-TGR GAC CAA ATW TCA ACA AG-3’ and 5’-GAT The genomic organization of the T. glabra satellite AAG AAC CCA AAA CCA TT-3’). sequences was analyzed by fluorescent in situ hybridiza- tion (FISH) (Fig. 3). The two sequence families exist at The phylogenetic relationship of pAL1 type 180-bp cen- condensed chromosomal regions on pachytene chromo- tromeric repeat families shows that the two satellite fam- somes and are co-localized on one set of chromosomes. ilies of T. glabra make a cluster with high bootstrap Although the quality of FISH images is not sufficiently probability and are more similar to each other than to high to draw a firm conclusion, these two satellite fami- other satellite families of other species (Fig. 2). Sequ- lies might be centromere-specific, as expected from the ences from T. glabra make a cluster with the O. pumila similarity of their sequences to other species’ centromere pAg family (average identities to the pAgl1 and pAgl2 satellite sequences. The other five pairs of chromosomes families are 71.2% and 68.8%, respectively) and the other show no signals of the two repeat families. The failure centromere satellite families of other species (A. thaliana to obtain ladder pattern bands with most restriction (pAL1), A. arenosa (pAa), A. halleri (pAa, pAge1, and enzymes suggests that the centromere satellite sequences pAge2), and A. lyrata (pAa, pAge1, and pAge2)) make in these five chromosomes contain no suitable restriction another cluster, as previously reported phylogeny of other enzyme recognition sites. genic regions (Miyashita et al., 1998; Koch et al., 1999, Chromosome-specific satellite sequence families have 2000; Kawabe and Miyashita, 2002b). This observation been reported in both animal and plant species (e.g., suggests that the evolution of satellite sequences reflects Warburton et al., 1993; Choo et al., 1994; Harrison and the divergence of species from which the satellite sequ- Heslop-Harrison, 1995; Gindullis et al., 2001). Like T. ences are obtained. Thus, replacement of older sequ- glabra in the present study, A. halleri and A. lyrata also ences by newly emergent satellites in Arabidopsis have chromosome-specific centromere satellite families Satellite sequences of T. glabra 289

other clade of Arabidopsis-related species studied so far, O. pumila has species-specific 180-bp satellite sequences similar to pAL1 of A. thaliana, but C. rubella has species- specific centromere satellite sequences with no homology to satellite sequences from other Arabidopsis relatives (Hall et al., 2005). In the present study, we found that T. glabra, classified into the same clade as O. pumilla and C. rubella, has two pAL1 type centromere satellite families co-localized on only one chromosome pair. Recent comparative mapping studies of Arabidopsis relatives showed highly conserved chromosome arm structures even very close to centromere regions (Kawabe et al., 2006; Lysak et al., 2006) suggesting that centromeric satellite evolution occurred within established centromere regions. The similarity among 180-bp families from dis- tant species suggests that new sequences are originated from variants of already existing sequences and that replacement of centromere sequences with completely different sequences is a rare event. It would be of interest to know what kinds of satellite sequences exist on the five chromosome pairs of T. glabra that did not show any signals of pAgl1 and pAgl2. Isolation and characterization of other centromeric satellites from T. glabra would make it possible to describe the evolutionary dynamics and the functional conservation of centromere satellite sequences.

We thank T. R. Endo for use of his facilities, M. A. Lysak for comments, and A. Berr for information about A. lyrata ssp. lyrata centromere compositions. This is contribution number 589 from The Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University.

Fig. 3. Organization of satellite DNAs of T. glabra. A) Tetrad REFERENCES stage chromosomes of male meiosis and B) pachytene chromosomes are shown. pAgl1 (red) and pAgl2 (green) were labeled Berr, A., Pecinka, A., Meister, A., Kreth, G., Fuchs, J., Blattner, with digoxigenin (DIG labeling mix, Roche) and biotin (Biotin F. R., Lysak, M. A., and Shubert I. (2006) Chromosome Chemilink, Roche), respectively, and used for FISH analyses as arrangement and nuclear architecture are largely conserved described in Kawabe and Nasuda (2005). DNAs were counter- during evolution of the genus Arabidopsis. Plant J. in stained with DAPI (blue). Scale bars show 5 µm. printing. Choo, K.H., Vissel, B., Nagy, A., Earle, E., and Kalitsis, P. (1994) A survey of the genomic distribution of alpha satel- (Kawabe and Nasuda, 2005). Both A. halleri and A. lite DNA on all the human chromosomes, and derivation of lyrata have at least three different satellite sequence fam- a new consensus sequence. Nucleic Acids Res. 19, 1179– 1182. ilies, which are about 30% different from each other. Gindullis, F., Desel, C., Galasso, I., and Schmidt, T. (2001) The However, the closely related species A. arenosa has just large-scale organization of the centromeric region in Beta one centromere satellite sequence family on all its chro- species. Genome Res. 11, 253–265. mosomes (Kamm et al., 1995). Sequence diversity anal- Hall, S. E., Luo, S., Hall, A. E., and Preuss, D. (2005) Differen- yses of centromere satellite sequences among A. halleri tial rates of local and global homogenization in centromere satellites from Arabidopsis relatives. Genetics 170, 1913– and A. lyrata subspecies revealed some evidence of 1927. recently spread satellite family sequences (Kawabe and Harrison, G. E., and Heslop-Harrison, J. S. (1995) Centromeric Charlesworth unpublished result). Also, the chromo- repetitive DNA in the genus Brassica. Theor. Appl. Genet. some specificities differ between (sub)species (Kawabe 90, 157–165. and Nasuda, 2005; Berr et al. 2006) and even between Heslop-Harrison, J. S., Brandes, A., and Schwarzacher, T. (2003) Tandemly repeated DNA sequences and centromeric local populations (Kawabe and Nasuda, 2006). Thus, the chromosomal regions of Arabidopsis species. Chromosome emergence of new centromere satellite families might be Res. 11, 241–253. a frequent event, and replacement of old satellites should Kamm, A., Glasso, I., Schmidt, T., and Heslop-Harrison, J. S. have occurred very rapidly in each chromosome. In the (1995) Analysis of a repetitive DNA family from Arabidopsis 290 A. KAWABE and S. NASUDA

arenosa and relationship between Arabidopsis species. Kumar, S., Tamura, K., Jacobsen, I., and Nei, M. (2000) Plant Mol. Biol. 27, 853–862. MEGA2: Molecular Evolutionary Genetics Analysis, version Kawabe, A., and Miyashita, N. T. (2002a) Characterization of 2.0. Pennsylvania and Arizona State Universities, Univer- duplicated two cytosolic phosphoglucose isomerase (PgiC) sity Park, Pennsylvania and Tempe, Arizona. loci in Arabidopsis halleri ssp gemmifera. Genes Genet. Lysak, M. A., Berr, A., Pecinka A., Schmidt, R., McBreen, K., Syst. 77, 159–165. and Schubert, I. (2006) Mechanisms of chromosome number Kawabe, A., and Miyashita, N. T. (2002b) DNA variation in the reduction in Arabidopsis thaliana and related Brassicaceae acidic chitinase locus (ChiA) region in Arabis gemmifera and species. Proc. Natl. Acad. Sci. USA 103, 5224–5229. among its related species. Genes Genet. Syst. 77, 167–175. Maluszynska, J., and Heslop-Harrison, J. S. (1991) Localization Kawabe, A., and Nasuda, S. (2005) Structure and genomic orga- of tandemly repeated DNA sequences in Arabidopsis nization of centromeric repeat in Arabidopsis species. Mol. thaliana. Plant J. 1, 159–166. Genet. Genomics 272, 593–602. Martinez-Zapater, J., Estelle, M. A., and Somerville, C. R. (1986) Kawabe, A., and Nasuda, S. (2006) Polymorphic chromosomal A highly repeated DNA sequence in Arabidopsis thaliana. specificity of centromere satellite families in Arabidopsis Mol. Gen. Genet. 204, 417–423. halleri ssp. gemmifera. Genetica 126, 335–342. Miyashita, N. T., Kawabe, A., Innan, H., and Terauchi, R. (1998) Kawabe, A., Innan, H., Terauchi, R., and Miyashita, N. T. (1997) Intra- and interspecific DNA variation and codon bias of Nucleotide polymorphism in the acidic chitinase locus alcohol dehydrogenase (Adh) locus in Arabis and Arabidop- (ChiA) region of the wild plant Arabidopsis thaliana. Mol. sis species. Mol. Biol. Evol. 15, 1420–1429. Biol. Evol. 14, 1303–1315. Murata, M., Ogura, Y., and Motoyoshi, F. (1994) Centromeric Kawabe, A., Hansson, B., Hagenblad, J., Forrest, A., and repetitive sequence in Arabidopsis thaliana. Jpn. J. Genet. Charlesworth, D. (2006) Centromere locations and associ- 69, 361–370. ated chromosome rearrangements in Arabidopsis lyrata and Simoens, C. R., Gielen, J., Van Montagu, M., and Inze D. (1988) A. thaliana. Genetics, 173, 1613–1619. Characterization of highly repetitive sequence of Arabidop- Koch, M., Bishop, J., and Mitchell-Olds, T. (1999) Molecular sys- sis thaliana. Nucleic Acid Res. 16, 6753–6766. tematics and evolution of Arabidopsis and Arabis. Plant Warburton, P. E., Waye, P. E., and Willard, H. F. (1993) Non- Biol. 1, 529–539. random localization of recombination events in human Koch, M. A., Haubold, B., and Mitchell-Olds, T. (2000) Compar- alpha satellite repeat unit variants: implications for higher- ative evolutionary analysis of chalcone synthase and alcohol order structural characteristics within centromeric hetero- dehydrogenase loci in Arabidopsis, Arabis, and related gen- chromatin. Mol. Cell Biol. 13, 6520–6529. era (Brassicaceae). Mol. Biol. Evol. 17, 1483–1498.