Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species

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1 The Plant Journal (2007) 49, doi: /j X x Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species Ki-Byung Lim 1,2,, Tae-Jin Yang 1,3,, Yoon-Jung Hwang 1,2, Jung Sun Kim 1, Jee-Young Park 1, Soo-Jin Kwon 1, Jin-A Kim 1, Beom-Soon Choi 1, Myung-Ho Lim 1, Mina Jin 1, Ho-Il Kim 1, Hans de Jong 4, Ian Bancroft 5, YongPyo Lim 6 and Beom-Seok Park 1,* 1 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu , Korea, 2 Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul , Korea, 3 Brassica Genomics Team, National Institute of Agricultural Biotechnology (NIAB), Rural Development Adminstration (RDA), Suwon , Korea, 4 Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands, 5 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK, and 6 Department of Horticulture, Chungnam National University, Daejeon, Korea Received 7 June 2006; revised 23 August 2006; accepted 30 August *For correspondence (fax þ ; pbeom@rda.go.kr). These authors contributed equally to this study. Summary We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S 25S rdna sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the pericentromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. Keywords: centromere retrotransposon, peri-centromere retrotransposon, centromere tandem repeat, heterochromatin, genome evolution, FISH. Introduction The genus Brassica includes the most extensively cultivated crops of the Brassicaceae (Cruciferae) family, which include vegetables such as Korean cabbage, cabbage, cauliflower, broccoli and turnip, oil crops including oilseed rape, and various mustards. The three Brassica species usually referred to as the diploid species have distinctive chromosome numbers: x ¼ 8 for B. nigra (termed the B genome), x ¼ 9 for B. oleracea (termed the C genome) and x ¼ 10 for B. rapa (termed the A genome). Three species are usually referred to as allotetraploid species: B. juncea (which contains both A and B genomes), B. napus (which contains both A and C genomes) and B. carinata (which contains both B and C genomes). These three species all exhibit diploid genetics and were formed by hybridization of the diploid species (U, 1935). 173 Journal compilation ª 2006 Blackwell Publishing Ltd

2 174 Ki-Byung Lim et al. Large-scale collinearity has been found between the genetic linkage maps of several Brassicaceae species (Kowalski et al., 1994; Lagercrantz, 1998; Lukens et al., 2003; Paterson et al., 2001; Schmidt et al., 2001). Recent studies have shown that the tribe Brassiceae, which contains approximately 240 species including the Brassica species, is the descendant of a common hexaploid ancestor with a genome similar to that of Arabidopsis thaliana. The three genomes of this common ancestor diverged from each other shortly after the divergence of the Arabidopsis and Brassica lineages, i.e million years ago (Lysak et al., 2005; Yang et al., 2006). Deletion-mediated genome reduction was detected in the triplicated blocks of B. oleracea (O Neill and Bancroft, 2000), B. rapa (Park et al., 2005; Yang et al., 2005a, 2006) and B. napus (Rana et al., 2004), when compared with the corresponding genomic regions of A. thaliana. The centromere is a dynamic and rapidly evolving structure (Ventura et al., 2001; Wong and Choo, 2001) and consists largely of highly repetitive DNA sequences. In most species it has a distinctive chromatin structure and is easily distinguishable under the microscope (Csink and Henikoff, 1998; Lim et al., 2005). Satellite DNA and retrotransposons are the most abundant DNA elements found in plant centromere regions (Jiang et al., 2003). Centromeric repeats often extend over several hundreds of thousands or millions of base pairs. Characterized repeats are composed of bp tandem repeat motifs. These include the 180 bp pal1 satellite in A. thaliana (Nagaki et al., 2003b; Round et al., 1997; Thompson et al., 1996), the 176 bp CentBr tandem repeats in Brassica (Lim et al., 2005), the bp CentO satellite in rice (Cheng et al., 2002; Zhang et al., 2004), the 156 bp CentC satellite in maize (Ananiev et al., 1998) and the 169 bp satellite in Medicago truncatula (Kulikova et al., 2001, 2004). Although the repeat length is similar between taxa, their sequence composition can be very different, even between closely related species. For example, the 180 bp pal1 repeat of A. thaliana does not share sequence homology with the 176 bp CentBr of B. rapa and B. oleracea (Harrison and Heslop-Harrison, 1995; Lim et al., 2005; Xia et al., 1993, 1994). In contrast to the centromeric tandem repeats, the sequences of the centromere-specific retrotransposons (CR) are conserved between related species. For example, CRR in rice (Cheng et al., 2002; Dong et al., 1998), CRM in maize (Nagaki et al., 2003a; Zhong et al., 2002) and cereba in barley (Hudakova et al., 2001) are common centromere retrotransposons in each genus. The genome sequences of A. thaliana and rice revealed that their centromeres have a common structural organization, which comprises extensive tracts of centromeric tandem repeats interrupted by various retrotransposons (Copenhaver et al., 1999; Kumekawa et al., 2000, 2001; Zhang et al., 2004). Peri-centromeric heterochromatin blocks may play an important role in maintaining chromosome structure (Henikoff et al., 2001). In plants, stretches of peri-centromeric heterochromatin (ranging to up to tens of megabases) have been found to consist of pericentromere-specific retrotransposons such as Athila in A. thaliana (Kumekawa et al., 2000) and PCRT in tomato (Yang et al., 2005b). We previously defined the distribution of the known repeat sequences such as CentBr and rdnas in the chromosomes of B. rapa (Lim et al., 2005). Here we use sequence data from B. rapa genomic bacterial artificial chromosomes (BAC) clones to help us to further characterize the molecular organization of the centromere and peri-centromere repeat components. Genome-wide analysis of the occurrence of repetitive elements provides insights into the composition of, and evolutionary processes affecting, the rapidly evolving centromeric regions in the genus Brassica. Results Characterization of centromeric repeats of B. rapa using BAC end sequences As a first step towards genome characterization, we analyzed the sequences derived from the ends of BAC clone inserts (BAC end sequences; BESs) from three libraries. Using BLASTN alignment, we found that the libraries varied greatly in the proportions of clones that contain the 176 bp CentBr tantem repeats. These were represented in 30% of the BESs from the KBrH library (Park et al., 2005), 2.7% of the 8631 BESs from the KBrS library and 1.1% of the BESs from the KBrB library. We attribute the high proportion of CentBr repeats (which contain a HindIII cleavage site) observed in the KBrH library to the use of a partial HindIII-cleaved genomic DNA for construction of the library; the KBrS and KBrB libraries were constructed using genomic DNA cleaved with Sau3AI and BamHI, respectively. The 3080 CentBr-containing BESs identified from the KBrH library came from 1881 BAC clones, 1199 of which contain CentBr sequences at both ends and 682 of which contain CentBr sequences at one end only. Although the majority of BESs contained only the CentBr array, 475 contained a CentBr array interrupted by other sequences. A more detailed analysis of the 3080 BESs containing CentBr sequences is available in Table S1. Sequencing of BAC clones containing centromeric repeat sequences We selected for further characterization two of the BAC clones that contain CentBr sequences at one end only: KBrH015B20 (hereafter 15B20), in which the CentBr array was interrupted by additional sequences, and KBrH001P13 (hereafter 01P13), in which the CentBr array was not interrupted by additional sequences.

3 Centromeric retrotransposons of Brassica species 175 Fluorescent in situ hybridization (FISH) showed that clone 01P13 contains sequences that hybridize to the centromeres of all ten chromosomes, and clone 15B20 contains sequences that hybridize only to the peri-centromeric heterochromatin of chromosomes 1, 3, 4 and 5. We fully sequenced these two BAC clones. In total, we obtained bp of sequence (leaving two small gaps) from 15B20 (GenBank accession number AC166740) and completely sequenced the bp insert of 01P13 (GenBank accession number AC166739). Two classes of 176 bp centromeric tandem repeats had previously been described in B. rapa: CentBr1 and CentBr2, which share up to 86% sequence identity (Lim et al., 2005). Both BAC clones contain CentBr repeats: 3.5 copies in 15B20 and 23.5 copies in 01P13. These CentBr arrays are highly homologous to each other (> 97% sequence identity) and are most similar to CentBr2 (approximately 93% sequence identity). They are interrupted by insertions of retrotransposons, as summarized in Figure S1. Sequence characterization of centromere-specific BAC clone 01P13 In order to investigate the sequence composition of centromeric regions, we analyzed the sequence of BAC 01P13. The clone contains 23.5 copies of the CentBr array, three retrotransposons (a complete and two truncated ones), and a tandem repeat, as shown schematically in Figure 1. A complete retrotransposon, named centromeric retrotransposon of Brassica (CRB), is inserted into the 17th unit of the CentBr array. The retrotransposon is 6010 bp in length, and the 599 bp long terminal repeats (LTRs) are identical. A 5 bp target site duplication (GTCAT) may be identified. The internal sequence begins with the primer-binding site of a trna and ends with a polypurine tract. It contains a singleexon gene encoding a Ty1-copia-like polyprotein of 1545 amino acids. Two highly conserved, but nested and truncated CRBs are inserted in the 24th unit of the CentBr array. The third CRB contains two copies of a tandem repeat of 805 bp, which we term TR805. Sequence characterization of peri-centromere-specific BAC clone 15B20 In order to investigate the sequence composition of pericentromeric regions, we analyzed the sequence of BAC 15B20. The clone contains various repetitive sequences as summarized in Figure 2. The major constituents are retrotransposons, nine of which account for 60% of the total sequences. In addition, there are 3.5 copies of the CentBr tandem repeat, 5S 25S rdna sequences and a degenerate tandem repeat sequence that we term TR238. The TR238 repeats total 37.8 kb in size and show significant homology with the intergenic repeat of rdna (GenBank accession number S78172; Da Rocha and Bertrand, 1995). Figure 1. Schematic representation of BAC clone KBrH001P13. (a) Dot plot analysis of the bp sequence. (b) Schematic representation of the sequence. Two blocks of the 176 bp centromeric tandem repeat (CentBr) are represented as open arrowheads. An 805 bp tandem repeat (TR805) is found in the middle of the third centromerespecific retrotransposons of Brassica (CRB). Three LTR retrotransposons, named CRB, are found as nested insertions in the middle of the CentBr arrays at the 17th and 24th CentBr units, and 5 bp target site duplications are present between the insertion points [expanded view in (c)]. The sequence similarity of the 24 CentBr arrays is represented in Figure S1. Most of the retrotransposons are Ty3-Gypsy elements, which we term peri-centromeric retrotransposon of Brassica rapa (PCRBr). Based on sequence similarity, the retrotransposons could be grouped into four families. The families comprise six copies of PCRBr1, a copy of PCRBr2, a copy of PCRBr3, and a truncated PCRBr4. An overview of the characteristics of these elements is presented in Table 1. The non-truncated elements have recognizable target site duplications and almost identical LTRs. The PCRBr1 members are bp in size, and can be further classified into two subfamilies, which share partial LTRs and internal sequence, as shown in Figure 3. The PCRBr3 appears to be a solo LTR, based on its sequence characteristics and flanking 5 bp target site duplication. The highly degenerated PCRBr4 shows significant similarity with the peri-centromere Athila retrotransposon of A. thaliana. This is likely to be the most ancient element, and may have inserted before divergence of the Arabidopsis and Brassica lineages. The retrotransposons form a mosaic structure: PCRBr1a-1 and PCRBr2 interrupt two separate sites of TR238 blocks, and three

4 176 Ki-Byung Lim et al. (a) (b) (c) Figure 2. Schematic representation of the bp sequence of BAC clone KBrH015B20. (a) A small block of CentBr tandem repeats and four large blocks of degenerate tandem repeats of an approximately 238 bp unit (TR238) are represented as red and black boxes, respectively. Gray boxes show significant homology with 5S and 25S rdna. Nine Ty3-gypsy-type long terminal repeat (LTR) retrotransposons are represented as different colored boxes. Based on sequence homology, the nine retrotransposons are grouped into five families named peri-centromere-specific retrotransposons (PCRBr1a), PCRBr1b, PCRBr2, PCRBr3 and PCRBr4, and are colored light green, light blue, pink, red and dark blue, respectively. LTR regions are denoted as boxed triangles. (b) The sequence that remains after excising the retrotransposons. The CentBr array, TR238 arrays, intergenic sequence of rrna, 5S rrna and 25S rrna are represented schematically as red triangles, black triangles, light gray boxes, a blue box and a purple box, respectively. The insertion points of the retrotransposons and their actual positions in the bp sequences are indicated by lines. (c) The rdna sequence (Genbank accession number D10840) is compared with the homologous sequence showing the related empty site by nested insertion of a bp sequence (arrow) composed of three members of the PCRBr family (1a-2, 1b-1 and 3) and flanking target site duplication (red letters). Five nucleotides for the start and end sequences for 5S and 25S rrna gene are represented as blue and purple letters, respectively. Numerals denote the actual positions of each sequence. Table 1 Sequence homology between the 5 and 3 ends of retrotransposons found in this study Name Size (bp) Length of long terminal repeats (LTR) (bp) 5 3 Similarity between LTRs (%) PCRBr elements, PCRBr1a-2, PCRBr1b-1 and PCRBr3, make up a complex nested insertion into 25S rdna sequences (GenBank accession number D10840). The abundance of repetitive sequences in B. rapa and B. oleracea BAC clone CRB P13 PCRBr1a B20 PCRBr1a B20 PCRBr1a B20 PCRBr1b B20 PCRBr B20 PCRBr B20 We analyzed available BES and whole-genome shotgun sequence (GSS) data to assess the abundance of five major classes of repetitive sequences (CentBr, CRB, PCRBr, 5S and 45S rdna). As summarized in Table 2, approximately 29% of B. rapa BESs and approximately 9% of the B. oleracea GSSs showed sequence homology with these repeats. Excluding the rdna sequences, the Brassica-specific elements CentBr, PCRBr and CRB were present in 4.6, 4.7 and 11.2% of B. rapa BESs and in 0.8, 0.5 and 1.9% of B. oleracea GSSs. These results indicate that Brassica-specific repetitive elements are abundant components of Brassica genomes. Localization of repetitive sequences in the genomes of diploid Brassica species We localized CRB, CentBr, PCRBr, TR805 and TR238 sequences in the genomes of the diploid Brassica species (B. rapa, B. nigra and B. oleracea) by conducting FISH with element-specific probes. The results are illustrated in Figures 4 6 and summarized in Table 3. The CRB probe hybridized at the restricted centromere of all three species. The TR805 probe co-localized with the CRB probe. Signals from the CentBr probes were detected in B. rapa and B. oleracea, but no signals were detected in B. nigra. In B. rapa, the CentBr1 probe hybridized to the centromere regions of eight chromosomes (1, 3, 4, 6, 7, 8,

5 Centromeric retrotransposons of Brassica species 177 Figure 3. Comparison of peri-centromere-specific retrotransposons (PCRBr1) elements. Four sequences of PCRBr1 retrotransposons are compared. Long terminal repeat regions and internal sequence are denoted as boxes and lines, respectively. The regions showing significant homology are color-coded. Table 2 Appearance of major repeats in the genomes of Brassica rapa and B. oleracea Sequence source Query number Number of hits with major repeat sequences (%) a CentBr PCRBr CRB 45S rdna 5S rdna B. rapa BES total (4.6) 4177 (4.7) 9925 (11.2) 6736 (7.6) 576 (0.6) KBrB (1.1) 3596 (5.2) 9021 (12.9) 5728 (8.2) 450 (0.6) KBrH (30.2) 76 (0.7) 533 (5.2) 203 (2.0) 1 (0.0) KBrS (2.7) 505 (5.9) 371 (4.3) 805 (9.3) 125 (1.4) B. oleracea (0.8) 2824 (0.5) 9979 (1.9) (5.4) 2244 (0.4) a Cross-match option: minimum match 10; minimum score 20. CRB, centromere-specific retrotransposons of Brassica; BES, BAC end sequences; PCRBr, peri-centromeric retrotransposon of B. rapa. Figure 4. Detection of centromere-specific repeats on the late metaphase chromosomes. (a) fluorescent in situ hybridization (FISH) with CentBr (red) and centromere-specific retrotransposons of Brassica (CRB) (green) on the meiotic chromosome complement of Brassica rapa, B. nigra and B. oleracea. Bars ¼ 5 lm. (b) Fiber-FISH with CentBr (red) and CRB (green). Bar ¼ 10 lm. 9 and 10) and the CentBr2 probe hybridized to the centromere regions of two chromosomes (2 and 5). In B. oleracea, the CentBr1 probe hybridized to the centromere regions of all nine chromosomes (although at lower signal intensities compared with the results in B. rapa), and the CentBr2 probe hybridized to the centromere regions of five chromosomes (5, 6, 7, 8 and 9). The PCRBr probe hybridized to large pericentromeric blocks in three chromosomes of B. rapa (1, 4 and 5), but hybridization was not detected in B. oleracea or B. nigra. Four chromosomes of B. rapa (1, 3, 4 and 5) have similar morphological structures, including large peri-centromeric heterochromatin blocks that are located in the long arms, below the major centromeric constriction. The PCRBr probe hybridized to the large peri-centromeric heterochromatin on three of these four chromosomes (1, 4 and 5) as shown in Figure 5(b). However, the TR238 probe hybridized to the peri-centromeric regions of all four chromosomes, including chromosome 3, which contains extensive tracts of 45S and 5S rdna. Extended-fiber FISH using PCRBr and TR238 probes showed an interspersed hybridization pattern, as shown in Figure 5(c). Localization of repetitive sequences in the genomes of the allotetraploid Brassica species We localized CRB, CentBr, PCRBr, TR805 and TR238 sequences within the genome of the allotetraploid species by conducting FISH with element-specific probes. These species are: B. juncea (which contains the A genome from a B. rapa progenitor and a B genome from a B. nigra progenitor), B. carinata (which contains the B genome from a B. nigra progenitor and the C genome from a B. oleracea progenitor)

6 178 Ki-Byung Lim et al. and B. napus (which contains the A genome from a B. rapa progenitor and the C genome from a B. oleracea progenitor). The results are summarized in Figure 7. Although we were unable to identify unambiguously which chromosomes in the allotetraploids correspond to their diploid counterparts, the numbers of chromosomes in each species hybridizing with the CRB and CentBr probes corresponds to their expected additive content of A and C genome chromosomes that hybridize to these sequences in the diploids. However, PCRBr, which in the diploids hybridized only to chromosomes 1, 4 and 5 of B. rapa, hybridizes to additional chromosomes in the allotetraploids. These include 45S rdna positions in A-genome chromosome 2 and C-genome chromosome 2, and, in B. napus only, to A-genome chromosome 3. All of these positions are marked by red arrowheads in Figure 7(c). Discussion Figure 5. Multi-color fluorescent in situ hybridization (FISH) of the major (peri)centromeric repeats in Brassica rapa and B. oleracea. (a) CentBr1 (green) and CentBr2 (red), and (b) peri-centromere-specific retrotransposons (PCRBr) (green) probes were hybridized on the same cell complement. Bars ¼ 5 lm. (c) Fiber FISH with PCRBr (green) and TR238 (red) on the fiber DNA indicating that these repeats are also intermingled. Bar ¼ 10 lm. Figure 6. Idiogram of Brassica rapa and B. oleracea based on the measurement of mitotic metaphase complements. Chromosome nomenclature followed Lim et al. (2005) for B. rapa, and is by chromosome length order for B. oleracea. Chromosome 2 of both species is identified as the nucleolar organizer region-bearing chromosome, based on the B. rapa chromosome nomenclature (Lim et al., 2005). CentBr and CRB are centromere components in the genus Brassica At least two classes of 176 bp CentBr repeats are present in B. rapa and B. oleracea. However, we found that the CentBr sequences of B. rapa did not hybridize to the chromosomes of B. nigra. An analysis of sequences in public databases revealed that Sinapis arvensis and Raphanus sativus (both members of the Brassiceae, so closely related to the Brassica species we have studied) have CentBr-like tandem repeats (176 and 175 bp units, respectively), but they show less than 70% sequence similarity with CentBr (Harrison and Heslop- Harrison, 1995; Kapila et al., 1996; Lim et al., 2005; Xia et al., 1994). Corresponding centromeric repeats have not yet been identified in B. nigra, but the lack of hybridization with the CentBr sequences indicates that they will be extensively divergent from those found in B. rapa. The results of FISH on extended fibers revealed that CRB and CentBr are intermingled in the centromere regions of B. rapa and B. oleracea. About 85% of BAC clones that harbor a CentBr array in their end sequences also hybridize to a CRB-specific probe (shown in Figure S2 and summarized in Tables S2 and S3). These results confirm that both CRB and CentBr are major components of centromere regions. However, whereas CRB appears to have been maintained as a common centromere component in Brassica species, CentBr sequences are less widespread, being restricted to the clade containing B. rapa and B. oleracea. Their occurrence in B. rapa and B. oleracea, but not B. nigra, is consistent with the phylogenetic relationships of these species, B. rapa and B. oleracea being more closely related to each other than they are to B. nigra (Cheng et al., 2002; Kurata et al., 2002; Li et al., 2003; Nonomura and Kurata, 1999, 2001). A. thaliana contains a family of LTR retrotransposons that are homologous to CRB with 73% similarity for their internal

7 Centromeric retrotransposons of Brassica species 179 Table 3 Number of chromosomes examined by fluorescent in situ hybridization that possess the major (peri)centromerespecific repeats in basic Brassica species Species (genome) Genome type CentBr1 CentBr2 CRB TR805 PCRBr TR238 B. rapa (A), n ¼ 10 A B. nigra (B), n ¼ 8 B B. oleracea (C), n ¼ 9 C B. juncea (AB), n ¼ 18 A B B. carinata (BC), n ¼ 17 B C B. napus (AC), n ¼ 19 A C All repeats were derived from B. rapa ssp. pekinensis cv. Chiifu. CentBr1 and 2, centromere tandem repeats of B. rapa 1 and 2; CRB, centromeric retrotransposon of Brassica; PCRBr, peri-centromeric retrotransposon of B. rapa; TR238 and TR805, tandem repeats of 238 and 805 bp. nucleotide sequence. These are listed in Table 4. Our interpretation is that CRB was present in the common ancestor of Arabidopsis and Brassica, and was most likely amplified to form a structural component of centromere regions in a hexaploid ancestor of Brassica after the divergence of the Arabidopsis and Brassica lineages, but before divergence of the Brassica species. This resembles the dynamic centromere emergence described in maize chromosome 4 (Page et al., 2001), and in the X chromosome of Lemur catta and Homo sapiens (Ventura et al., 2001; Wong and Choo, 2001). It remains a possibility, however, that CRB could have been a centromere component in an ancestor of A. thaliana, but has been lost by an unknown mechanism. PCRBr is an A-genome-specific peri-centromeric retrotransposon Based on the FISH results with PCRBr and TR238 probes in the three diploid Brassica species, we conclude that these sequences are abundant only in B. rapa. They have accumulated to high copy numbers in the four chromosomes (1, 3, 4 and 5) that have large peri-centromeric heterochromatin blocks (Lim et al., 2005). This amplification must have occurred relatively recently, i.e. since the divergence of the B. rapa and B. oleracea lineages approximately 4 million years ago (Rana et al., 2004). Peri-centromeric retrotransposons occupy 84% of a 326 kb sequenced peri-centromeric region of tomato. One member of a retrotransposon family, PCRT1a, is present in the majority of peri-centromeric heterochromatin blocks of tomato chromosomes (Yang et al., 2005b). More than 100 PCRT1a homologues have been identified in rice, sorghum and maize, but not in Arabidopsis (Yang et al., 2005b). Similarly, large numbers of rice retrotransposons share high homology with the polyprotein of PCRBr1a, although fewer than 10 homologues have been detected in the Arabidopsis genome. These data suggest that peri-centromeric retrotransposons have been differentially accumulated in peri-centromeric heterochromatin blocks of different species. Genome expansion in Brassica Previous studies have indicated that approximately 50% of the B. rapa genome and approximately 60% of the B. oleracea genome consist of Brassica-specific sequences (Ayele et al., 2005; Yang et al., 2005a, 2006). Transposons were found to be major contributors to the genome expansion in Gramineae (Lai et al., 2004) as well as in the Brassicaceae (Zhang and Wessler, 2004), but the processes seem to differ. Comparative genomic analysis of orthologous regions in Gramineae reveals that the larger genomes contain longer collinear sequences, with the expansion being attributable to insertions of transposons (Bennetzen and Ramakrishna, 2002; Ilic et al., 2003; Kellogg and Bennetzen, 2004). However, the collinear sequence regions of the B. rapa genome were generally smaller ( fold) than the counterpart Arabidopsis sequence (Yang et al., 2005a, 2006), even though the B. rapa genome is three times larger than that of A. thaliana (Johnston et al., 2005). In the Brassica species, genome enlargement has occurred partly by polyploidy (Lysak et al., 2005; O Neill and Bancroft, 2000; Park et al., 2005; Schmidt et al., 2001; Yang et al., 2006). Our results show that the genome has also been enlarged by the accumulation of tandem repeats and retrotransposons, including CentBr, TR238, TR805, CRB and PCRBr, although these are largely restricted to the centromeric or peri-centromeric heterochromatic regions. Experimental procedures BAC sequencing Shotgun sequencing was performed on two B. rapa BAC clones, KBrH015B20 (15B20) and KBrH001P13 (01P13) as described previously (Yang et al., 2004, 2005b). The structure of each repeat element was characterized by pairwise sequence comparison using PipMaker (Schwartz et al., 2000), Blast2 analysis ( subsequent BLASTN, BLAST-X ( and RepeatMasker (

8 180 Ki-Byung Lim et al. Figure 7. Fluorescent in situ hybridization with centromere-specific retrotransposons of Brassica (CRB) (a), CentBr (b) and peri-centromere-specific retrotransposons (PCRBr) (c) on the mitotic chromosome complements of Brassica juncea, B. carinata and B. napus. Arrows in (c) represent the detection of PCRBr probe, which was not detected in B. rapa. Arrowheads and numbers in (c) indicate the positions of signals, chromosome number and genome. Red arrowheads represent relatively weak signals at the position of 45S rdna of chromosome 2. Table 4 CRB-like retrotransposons in the genome of Arabidopsis Feature Similarity (%) Match in CRB-like TSD Match in Arabidopsis (bp) TSD GenBank accession number Solo LTR AGAGG AGAGG NC_ Solo LTR TAAGT TAAGT NC_ Solo LTR TATAA TATAA NC_ Complete Truncated GGTAC NC_ Tri LTR , Degenerate , CATTG NC_ TSD, target site duplication; CRB, centromere-specific retrotransposons of Brassica; LTR, long terminal repeats. Similarity (%), nucleotide sequence similarity with the complete CRB-like retrotransposon in of NC_003076_4 (Arabidopsis pseudo chromosome 5 sequence) which show 73% sequence similarity with the internal sequence of CRB element.

9 Centromeric retrotransposons of Brassica species 181 maskerhelp.html), and finished by manual inspection. Gene annotation was achieved using the web-based gene prediction program FGENE-SH Arabidopsis ( berry.phtml). Sequence analysis Preliminary sequence data for B. oleracea was obtained from the TIGR website ( These, and BAC end sequences (BESs) obtained from three B. rapa BAC libraries (KBrH, KBrB, KBrS; were analyzed for distribution of centromeric repeats. CROSS_MATCH (Ewing and Green, 1998; Ewing et al., 1998) was used to select BAC end sequences containing the 176 bp CentBr1 and CentBr2 centromeric tandem repeat. Plant materials The plant lines used were B. rapa ssp. pekinensis cv. Chiifu (accession number IT212910), B. oleracea ssp. alboglabra (accession number IT164995), B. nigra (accession number IT134983), B. juncea (accession number IT021702), B. carinata (accession number INIA ) and B. napus (accession number IT021711). B. carinata was kindly provided by Dr Gòmez-Campo, Univ. Politecnica de Madrid, Spain; the other lines were supplied by Rural Development Administration (RDA) Gene Bank, Korea. Fluorescence in situ hybridization Mitotic and meiotic chromosome preparations were prepared as described previously (Lim et al., 2005). DNA (1 2 lg) of BAC clones was labeled with either biotin-16-dutp or digoxigenin-11-dutp by nick translation (Roche Diagnostics GmbH, Mannheim, Germany), and FISH was performed as described by to Lim et al. (2005). The chromosome preparations were re-used up to three times for FISH detection with different probes. The used slides were de-probed by washing with 2 SSC for 10 min and 4 SSC containing 0.2% Tween-20 for 1 h, followed by fixation by dipping in 4 SSC containing 70% formamide at 70 C for 2 min, then finally dehydration by dipping in serial alcohol solutions (70%, 90% and 100%). DAPIstained metaphase chromosomes were measured and used for karyotype analysis and chromosome nomenclature according to declining length order (Lim et al., 2005). The BDB method was employed in some cases to confirm the centromere position, in addition to FISH (Lim et al., 2005). Slides were examined under an Axioplan 2 imaging photomicroscope (Carl Zeiss, Göttingen, Germany) equipped with epifluorescence illumination and filter sets for DAPI, FITC and rhodamine fluorescence. Images were captured using a Sensys CCD camera (Roper Scientific GmbH, Ottobrunn, Germany). Image processing and threshold was performed using GENUS IMAGE analysis software (Applied Imaging Corporation, Newcastle, UK). DAPI images were sharpened with a 7 7 Hi-Gauss high-pass spatial filter to accentuate minor details and heterochromatin banding of the chromosomes. All fluorescence images were pseudo-colored, and optimal brightness and contrasts were achieved using ADOBE PHOTOSHOP. 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