Genomic Distribution of Simple Sequence Repeats in Brassica rapa

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1 Mol. Cells, Vol. 23, No. 3, pp Molecules and Cells KSMCB 2007 Genomic Distribution of Simple Sequence Repeats in Brassica rapa Chang Pyo Hong, Zhong Yun Piao 1, Tae Wook Kang 2, Jacqueline Batley 3, Tae-Jin Yang 4, Yoon-Kang Hur 5, Jong Bhak 2, Beom-Seok Park 6, David Edwards 3, and Yong Pyo Lim* Department of Horticulture, College of Agriculture and Life Science, Chungnam National University, Daejeon , Korea; 1 College of Horticulture, Shenyang Agricultural University, Shenyang , China; 2 Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon , Korea; 3 Primary Industries Research Victoria, Department of Primary Industries, Victorian AgriBioscience Centre, Bundoora 3086, Victoria, Australia; 4 Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul , Korea; 5 Department of Bioscience, School of Bioscience and Biotechnology, Chungnam National University, Daejeon , Korea; 6 Brassica Genomics Team, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon , Korea. (Received December 28, 2006; Accepted March 6, 2007) Simple Sequence Repeats (SSRs) represent short tandem duplications found within all eukaryotic organisms. To examine the distribution of SSRs in the genome of Brassica rapa ssp. pekinensis, SSRs from different genomic regions representing 17.7 Mb of genomic sequence were surveyed. SSRs appear more abundant in non-coding regions (86.6%) than in coding regions (13.4%). Comparison of SSR densities in different genomic regions demonstrated that SSR density was greatest within the 5 -flanking regions of the predicted genes. The proportion of different repeat motifs varied between genomic regions, with trinucleotide SSRs more prevalent in predicted coding regions, reflecting the codon structure in these regions. SSRs were also preferentially associated with gene-rich regions, with pericentromeric heterochromatin SSRs mostly associated with retrotransposons. These results indicate that the distribution of SSRs in the genome is non-random. Comparison of SSR abundance between B. rapa and the closely related species Arabidopsis thaliana suggests a greater abundance of SSRs in B. rapa, which may be due to the proposed genome triplication. Our results provide a comprehensive view of SSR genomic distribution and evolution in Brassica for comparison with the sequenced genomes of A. thaliana and Oryza sativa. * To whom correspondence should be addressed. Tel: ; Fax: yplim@cnu.ac.kr Keywords: Arabidopsis thaliana; Brassica rapa ssp. pekinensis; Genomic Distribution; Microsatellite; Simple Sequence Repeat (SSR). Introduction Simple sequence repeats (SSRs), or microsatellites, are ubiquitous DNA elements in eukaryotic genomes. They consist of tandem repeats of between 1 and 6 base pairs (bp). SSR loci have relatively high mutation rates, ranging from 10 2 to 10 6, which results in alterations in SSR length (Schlötterer, 2000). The instability of SSRs is primarily due to slipped-strand mispairing errors during DNA replication (Ellegren, 2004; Schlötterer, 2000; Tautz and Schlötterer, 1994). The majority of slippage insertions/deletions would be corrected by the mismatch repair system, and only the small fraction of sites that are not repaired lead to SSR mutations (Bachtrog et al., 1999; Eisen, 1999). It has been suggested that the mutation rate of SSRs increases with repeat number (Brinkmann et al., 1998; Goldstein and Clark, 1995; Schlötterer et al., 1998; Schug et al., 1998; Weber and Wong, 1993; Wierdl et al., 1997), with long SSRs in eukaryotic genomes having a mutation bias to become shorter SSRs (Ellegren, 2000; Harr and Schlötterer, 2000; Harr et al., 2002; Schlötterer, 1998; Xu et al., 2000). Kruglyak et al.,(1998) introduced Abbreviation: SSRs, simple sequence repeats.

2 350 Genomic Distribution of SSRs in B. rapa a Markov chain model of SSR evolution that incorporates length-dependent slippage and point mutations interrupting the SSRs. This model suggests that the indefinite growth of an SSR is prevented by the accumulation of base substitutions within the SSR sequence. Hence, species with short SSRs should have a lower SSR mutation rate than species with longer SSRs. The length variability and ubiquitous occurrence of SSRs has led them to be widely used as markers for use in genetic mapping (McCouch et al., 1997; Temnykh et al., 2001; reviewed in Varsheny et al., 2005), linkage and association studies (Abdurakhmonov et al., 2005), phylogenetics (Chung and Staub, 2003; Flannery et al., 2006; Plieske and Struss, 2001), and population studies (Rosenberg et al., 2002). SSRs are found throughout the genome, in both protein-coding and non-coding regions. The density of SSRs varies widely between genomes (Katti et al., 2001; Tóth et al., 2000), and recent evidence suggests a non-random genomic distribution. SSRs have been shown to be more abundant in non-coding regions than in coding regions, with some bias for defined genomic locations (Hancock, 1995; Katti et al., 2001; La Rota et al., 2005; Lawson and Zhang, 2006; Li et al., 2002; 2004; Morgante et al., 2002; Subramanian et al., 2003; Temnykh et al., 2001; Tóth et al., 2000; Zhang et al., 2004). Trinucleotide repeats are the most abundant type of SSR in the protein-coding regions of all taxa. SSRs associated with different regions of a gene, such as 5 or 3 untranslated regions (UTRs), exons, or introns, may play important roles in determining protein function or regulating gene expression (reviewed in Li et al., 2004), so leading to changes in phenotype. SSRs within coding regions may also lead to gain or loss of function via frameshift mutations or expanded toxic mrnas (Li et al., 2002; 2004). For example, the expansion of the trinucleotide GAG repeats in the coding region of the Huntington s disease (HD) gene in humans can lead to Huntington s disease (Li et al., 2004; Zoghbi and Orr, 2000). SSR variation within the non-coding regions of transcribed genes may regulate gene expression by altering transcription, translation, RNA splicing or stability (Li et al., 2004; Meloni et al., 1998; Ranum and Day, 2002; Toutenhoofd et al., 1998). In addition, SSR variation within gene promoters may affect transcription factor binding and alter the level and specificity of gene transcription. The study of SSR distribution within genomes will further our understanding of the evolution of SSRs, including mutation as well as the role of these elements in gene function. Brassica rapa ssp. pekinensis (Chinese cabbage) is a diploid species (2n = 20 as AA genome) and one of six widely cultivated species of Brassica (U, 1935). It has an estimated genome size of 529 Mb (Johnston et al., 2005), approximately four times greater than the sequenced model plant species, Arabidopsis thaliana. B. rapa is an economically important crop, as well as a model plant for studies of polyploidization. It is closely related to A. thaliana, having diverged from a common ancestor million years ago (Yang et al., 1999). In contrast to B. rapa, A. thaliana has a small genome, relatively little repetitive DNA, and a high gene density (Bevan and Walsh, 2005; The Arabidopsis Genome Initiative, 2000). Comparative genetic and physical mapping between Brassica species and A. thaliana has identified co-linear chromosome segments, conserved gene order and a high degree of sequence conservation, with some variation in gene content through deletion or insertion (Lagercrantz, 1998; Lysak et al., 2004; O Neill and Bancroft, 2000; Park et al., 2005; Rana et al., 2004; Yang et al., 2006). However, in comparison with A. thaliana, the diploid Brassica genomes have been extensively triplicated with frequent genomic rearrangements (Lagercrantz, 1998; Lysak et al., 2004; O Neill and Bancroft, 2000; Park et al., 2005; Rana et al., 2004; Yang et al., 2006). In this report, we assess the distribution of SSRs in the B. rapa genome by analyzing 17.7 Mb of genomic sequence derived from bacterial artificial chromosome (BAC)-end sequences and completely sequenced BACs. As BAC-end sequences represent a random sampling of the genome their analysis assists in understanding whole genome structure (Hong et al., 2004; 2006; Messing et al., 2004). In addition, a comparison of SSRs identified within the B. rapa genome with those found within the genome of A. thaliana provides insight into SSR evolution within these two species. Materials and Methods Genomic sequences of B. rapa, consisting of 12,017 HindIII BAC-end sequences (BZ BZ614306, CW CW988843), 12,017 BamHI BAC-end sequences (DX DX022674), and 15 assembled BAC sequences (AC146875, AC155335, AC AC155344, AC155346, AC155348, AC AC166741), were collected from the genome survey sequence (GSS) and high-throughput genomic sequence (HTG) databases of NCBI ( BAC-end sequences were analyzed by open reading frame (ORF) finding and a homology-based database search. ORF finding was performed using the NCBI ORF Finder ( ncbi.nlm.nih.gov/gorf/gorf.html). For homology-based gene identification, BAC-end sequences were compared with sequences in the NCBI non-redundant (NR) protein and dbest databases ( and the annotated Arabidopsis protein database ( using BLAST, with a significant cut-off E-value < BAC sequences were also analyzed using the ab initio gene structure prediction method, FGENESH ( Genomic regions were defined as genic regions (5 -flanking regions adjacent to start codons, 3 -flanking regions adjacent to stop codons, predicted introns, and exons) and intergenic regions. In this study, 5 - and 3 -flanking regions of genes were defined as 200 bp upstream

3 Chang Pyo Hong et al. 351 Table 1. SSR densities in different genomic regions of B. rapa. Genic regions c Repeat types b Intergenic Exons Introns 5 -flanking d 3 -flanking e regions Mono Di Tri Tetra Penta Total Sequence (Mb) f a SSR density = SSR number per 1 Mb of genomic region. b Mono-, di-, tri-, tetra-, and penta-nucleotide repeats. c Genic non-coding regions are 5 - and 3 -flanking regions and introns. d 5 -flanking regions adjacent to the start codon, e 3 -flanking regions adjacent to the stop codon. f Total length (Mb) of each of the predicted genomic regions analyzed. and downstream from the predicted start and stop codons. SSRs were identified using SPUTNIK ( com/sputnik), with the following parameters: (i) SSRs were defined as mononucleotide, dinucleotide and trinucleotide repeats 12 bp, tetranucleotide repeats 16 bp, and pentanucleotide repeats 20 bp; and (ii) no variation (mutation) in repeat motifs was permitted. Frequencies of repeat motifs were calculated for each of the different genomic regions accounting for sequence complementarity (eg. (T) n = (A) n, (CT) n = (AG) n, and (CTT) n = (AAG) n ). Amino acid runs were predicted from trinucleotide SSRs in coding regions defined by ORF finding. Gene ontology (GO) for coding sequences containing trinucleotide SSRs was predicted by comparing them with the protein sequences of A. thaliana ( using BLASTX, and genes were classified using OBE-Edit ( Results and Discussion SSR densities in different genomic regions of B. rapa SSRs were surveyed from B. rapa genomic sequence data consisting of 12,017 HindIII BAC-end sequences (7.7 Mb), 12,017 BamHI BAC-end sequences (8.3 Mb), and 15 sequenced BACs (1.7 Mb). A total of 3,740 SSRs were identified with a frequency of 1 per 4.7 kb. SSR densities within each genomic region were estimated (Table 1). SSR density was greatest in 5 -flanking regions, followed by 3 -flanking regions, introns, intergenic regions, and exons. Compared to the genomes of A. thaliana and O. sativa (Lawson and Zhang, 2006; Zhang et al., 2004), SSR density in B. rapa is relatively low, particularly in intergenic regions. This difference may be due to underestimation of SSR density in B. rapa, or to genome expansion in B. rapa. Our findings that SSR density in B. rapa is greater in non-coding regions than coding regions and that SSRs are more prevalent in the flanking regions of genes parallel the genomic distributions of SSRs in A. thaliana and O. sativa, both of which have an over-representation of SSRs in 5 -flanking regions (Fujimori et al., 2003; Lawson and Zhang, 2006; Li et al., 2002; Morgante et al., 2002; Mortimer et al., 2005; Zhang et al., 2004). Distribution of repeat types in different genomic regions of B. rapa Trinucleotide repeats were the most frequent repeats in the B. rapa genome, representing about 34% of all SSRs identified. However, the abundance of different repeat motifs varied with genomic region. Mononucleotide repeats predominated in intergenic regions, introns, and 3 -flanking regions, dinucleotide repeats predominated in 5 -flanking regions, while trinucleotide repeats were most abundant in exons (Tables 1 and 2). This distribution is similar to that observed for A. thaliana and O. sativa (Lawson and Zhang, 2006; Mortimer et al., 2005; Zhang et al., 2004). Tetra- and pentanucleotide repeats, though generally uncommon, were relatively more abundant in intergenic regions and introns. Mono-, di-, tetra-, and penta-nucleotide repeats were most frequently found in non-coding regions, while trinucleotide repeats were most abundant in exons. The differences of SSR motifs in different genomic regions has previously been demonstrated (Edwards et al., 1998; Field and Wills, 1996; La Rota et al., 2005; Lawson and Zhang, 2006; Li et al., 2004; Metzgar et al., 2000; Morgante et al., 2002; Mortimer et al., 2005; Tóth et al., 2000; Zhang et al., 2004). In particular, the abundance of trinucleotide repeats in exons reflects selection against potential frameshift mutations (Metzgar et al., 2000). Our results suggest that the distribution of SSRs in the genome of B. rapa is non random and biased by natural selection. Distribution of SSR motif sequences in B. rapa The

4 352 Genomic Distribution of SSRs in B. rapa Table 2. The proportion of SSRs in different genomic regions of B. rapa. Repeat Genic regions Intergenic types a Exons (%) Introns (%) 5 -flanking (%) b 3 -flanking (%) c regions (%) Total (%) Mono Di Tri Tetra Penta Total abundance of each SSR motif sequence was assessed for each of the datasets (Fig. 1). The most abundant repeat motifs were (A) n (28.8%), (AG) n (15.4%), (AT) n (13.7%), and (AAG) n (13.3%), reflecting the A/T rich nature of the B. rapa genome. PolyC/polyG repeats were rare, a result also found in other eukaryotic genomes (Katti et al., 2001). In contrast to the genomes of A. thaliana and O. sativa, B. rapa contains more (AT) n dinucleotide repeats, followed in abundance by (AG) n and (AC) n repeats. (AG) n repeats are abundant in A. thaliana and O. sativa, followed by AT and AC repeats (Lawson and Zhang, 2006; Zhang et al., 2004). In humans and Drosophila, (AC) n is the most frequent dinucleotide repeat sequence, followed by (AT) n and (AG) n (Katti et al., 2001). (GC) n repeats are extremely rare in eukaryotic genomes and this is also the case for B. rapa (Katti et al., 2001). Among the trinucleotide repeat motifs, a higher frequency of (AAG) n repeats was identified in B. rapa. The repeats AGG, ATC, ACC, and AGC were also relatively abundant. This is similar to the results in A. thaliana and O. sativa (Hong et al., 2006; Katti et al., 2001; La Rota et al., 2005; Lawson and Zhang, 2006; Morgante et al., 2002; Zhang et al., 2004). Among tetra- and penta-nucleotide repeats in B. rapa and other plant genomes, (AAAN) n and (AAAAN) n, and especially (AAAT) n and (AAAAT) n, are more common than other combinations (Katti et al., 2001; Tóth et al., 2000; Zhang et al., 2004). In an examination of the motif sequence distributions in different genomic regions, the most abundant repeat motifs in intergenic regions and introns were found to be (A) n and (AT) n. The motifs (AG) n and (AAG) n were the most abundant motifs in 5 -flanking regions, (A) n in 3 - flanking regions, and (AAG) n in exons (Fig. 1). In general, this distribution of SSR motifs is similar to those of A. thaliana and O. sativa (Lawson and Zhang, 2006; Zhang et al., 2004). However, (CCG) n repeats are the most frequent SSRs in the 5 -flanking regions and exons of rice (Lawson and Zhang, 2006; Zhang et al., 2004) reflecting the difference in GC content of dicotyledons and monocotyledons (Temnykh et al., 2001). Comparative distribution of SSRs in gene-rich and repeat-rich regions of B. rapa To determine whether SSRs are preferentially associated with gene-rich or repeat-rich regions of the genome in B. rapa, we surveyed SSRs in annotated BACs representing these genomic regions, extracted from the nucleotide database of NCBI. KBrH117M18 (GenBank accession No. AC146875) represents a gene-rich region containing 26 genes and 2 terminal repeat retrotransposon-in-miniature (TRIM) elements (Yang et al., 2007), while KBrH015B20 (AC166740) represents a repeat-rich region derived from peri-centromeric heterochromatin and contains one CentBr1 block (array of 176 bp centromeric satellite repeats), four TR238 blocks (238 bp tandem satellite repeat arrays), 2 rdnas, and 11 TEs (Fig. 2) (Lim et al., 2007). SSRs were 4.2-fold more abundant in the gene-rich region (38 SSRs) than in the gene-poor region (9 SSRs) (Fig. 2). To further support the relationship between SSR abundance and gene density, we performed a regression analysis of gene frequency versus SSR frequency for 15 B. rapa BACs, and found a positive, linear relationship (r = 0.815, P < 0.001) (Fig. 3). These results support the hypothesis of a correlation between SSR density and gene-rich (or nonrepetitive) genomic DNA in plants (Cardle et al., 2000; La Rota et al., 2005; Morgante et al., 2002; Mortimer et al., 2005). In repeat-rich BACs, SSRs were frequently associated with retrotransposons (Fig. 2B). Ramsay et al. (1999) found a similar association between SSRs and repetitive elements such as retrotransposons. Of the SSRs identified within gene poor BACs, three were present in the 5 long terminal repeats (LTRs) of PCRBr4, PCRBr1a-1 and PCRBr1b-1 retrotransposons and two in the 3 LTRs of PCRBr1a-1 and PCRBr1a-3 retrotransposons. The remaining SSRs were in internal sequences of PCRBr2 and PCRBr1a-3 retrotransposons. The most common SSR motif (AG) n was associated with four LTRs and two internal sequences of retrotransposons. Although the Micron family of inverted-repeat transposable elements (MITE) were not found to be associated with SSRs in our study, (AT) n repeats are frequently associated with this TE family in rice (Temnykh et al., 2001). In the human genome a significant positive association also exists be-

5 Chang Pyo Hong et al. 353 A B Fig. 1. Distribution of primary SSR motifs in different genomic regions of B. rapa. SSR motifs corresponding to more than 0.5% of the total population of identified sequences were considered primary SSRs. tween A-rich SSRs and short interspersed elements (SINE) (Nadir et al., 1996), suggesting that these SSRs have been generated by 3 -extension of retrotranscripts, analogous to mrna polyadenylation. Although this association has been reported in a wide range of organisms, a high density of TEs does not always coincide with a high density of SSRs (Schlötterer, 2000), probably due to constraints on retrotransposon structure and evolution in these regions (Ramsay et al., 1999). Biased distribution of codon repeats and associated gene categories in B. rapa Trinucleotide SSRs within exons may encode expressed amino acid runs [(a.a) n ]. A total of 386 predicted amino acid runs were identified in our analysis. The most frequent amino acid runs were serine (Ser) (15.3%), glutamic acid (Glu) (11.9%), aspartic acid (Asp) (10.6%), glycine (Gly) (8.2%), lysine (Lys) (7.3%), and asparagine (Asn) (6.7%) (Fig. 4). The methionine (Met), tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys) runs were very rare or absent (Fig. 4). These results support the hypothesis that the frequency of different trinucleotide SSRs varies as a result of codon usage bias (Katti et al., 2001; Li et al., 2004; Zhang et al., 2004). Although the biased distribution of codon repeats has been demonstrated in several eukaryotic genomes (Katti et al., 2001; Lawson and Zhang, 2006; Li et al., 2004; Tóth et al., 2000; Zhang et al., 2004), the prevalence of specific amino acid runs varies. The most frequent amino acid runs in A. thaliana are [Ser] n, [Pro] n, [Gly] n, [Glu] n, [Gln] n, and [Asp] n, and those in O. sativa are [Ala] n, [Gly] n, [Pro] n, [Ser] n, [Arg] n, and [Glu] n (Lawson and Zhang, 2006). The majority of amino acid runs in the three genomes corresponded to hydrophilic amino acids, with the exception of the proline runs. This observation is consistent with previous results in Drosophila, Caenorhabditis elegans, and yeast (Katti et al., 2001). The functional categories of B. rapa coding sequences containing trinucleotide repeats were investigated by Fig. 2. Relative distribution of SSRs in gene-rich (A) and repeat-rich (B) genomic regions of B. rapa. The genomic regions were derived from two BAC clones annotated previously by Yang et al., (2005), (A) KBrH117M18 (GenBank accession No. AC146875) and (B) KBrH015B20 (AC166740) in GenBank. Retrotransposons in panel (B): a, PCRBr-4; b, PCRBr1b-3; c, PCRBr1a-1; d, 5 LTR of PCRBr1a-2; e, PCRBr1b-1; f, PCRBr- 1a-2; g, Solo-LTR of PCRBr3; h, PCRBr-1a-2; i, PCRBr2; j, PCRBr1a-3; k, PCRBr1b-2 member. Fig. 3. Regression analysis of gene frequency versus SSR frequency in genomic segments of B. rapa. comparison with the Arabidopsis proteome database ( and subsequent Gene Ontology (GO) annotation. The majority of trinucleotide repeat-containing genes belonged to the cellular component (C) category (49.2%), followed by molecular function (A) (36.1%), and biological process (B) (14.7%). Analysis of individual GO categories suggested that trinucleotide repeats occurr most abundantly within genes belonging to the organelle (C5), organelle part (C6), cell (C1), and binding (M2) categories (Fig. 5). The categories of antioxidant activity (M1), interaction between organisms (B4), pigmentation (B6), reproduction (B7), envelope (C2), and

6 354 Genomic Distribution of SSRs in B. rapa Fig. 4. Distribution of amino acid runs encoded by exonic trinucleotide SSRs in B. rapa. membrane-enclosed lumen (C4) were rare or absent (Fig. 5). In contrast, trinucleotide repeats in A. thaliana genes were over-represented by cell, binding, and catalytic activity categories. Catalytic activity, binding, cell, and response to stimulus categories were over-represented in O. sativa genes containing trinucleotide repeats (Lawson and Zhang 2006). Alba et al. (1999) reported an intriguing association between the most common amino acid repeats and components of cell-signaling systems. Based on our results here, we suggest that trinucleotide SSRs in exons may be preferentially associated with cellular component genes in B. rapa. Comparison of SSR abundance in the genomes of B. rapa and A. thaliana A. thaliana and B. rapa diverged from a common ancestor million years ago, with a subsequent triplication of the B. rapa genome resulting in an increase in gene number, as well as genome expansion (Park et al., 2005; Rana et al., 2004; Yang et al., 2006). SSR abundance would thus be expected to be greater in B. rapa due to this genome triplication. To assess this hypothesis we compared SSR abundance in a region of A. thaliana chromosome 5 (3,133,262 bp to 3,234,211 bp) and its homologous region in B. rapa (consisting of four BACs). Three B. rapa BACs represent triplications of the A. thaliana region, KBrH080A08 (AC ), KBrH004D11 (AC155341) and KBrH052O08 (AC155342), while two of them, KBrH052O08 and KBr- H117M18 (AC146875), represent a recent duplication in B. rapa (Yang et al., 2006). 36 SSRs were identified in the genomic region of A. thaliana, compared with 90 SSRs in the homologous segments of B. rapa, suggesting that SSR abundance in B. rapa is 2.5 times greater than in A. thaliana, reflecting the larger genome size of B. rapa. In addition, we compared SSR abundance within the complete A. thaliana genome and a genomic sequence of B. rapa (17.9 Mb). The B. rapa genome is estimated to contain a 110,000 SSRs (529 Mb of genome with one SSR per 4.8 kb). This compares with 36,756 SSRs (one Fig. 5. Gene ontology (GO) annotation of genes containing trinucleotide SSRs in B. rapa. M, B and C indicate the GO annotations for molecular function, biological process and cellular component, respectively. M1, antioxidant activity; M2, binding; M3, catalytic activity; M4, enzyme regulator activity; M5, regulation of biological process; M6, signal transducer activity; M7, transcription regulator activity; M8, translation regulator activity; M9, transporter activity; B1, cellular process; B2, development; B3, growth; B4, interaction between organisms; B5, physiological process; B6, pigmentation; B7, reproduction; B8, response to stimulus; B9, viral life cycle; C1, cell; C2, envelope; C3, extracellular matrix; C4, membrane-enclosed lumen; C5, organelle; C6, organelle part; C7, protein complex. per 3.2 kb) in the A. thaliana genome (Hong et al., 2006). This suggests that SSR abundance is three times greater in B. rapa than that in A. thaliana, reflecting the triplication of genomic segments in B. rapa. Our results demonstrate that the distribution of SSRs in genic and intergenic regions of B. rapa is non-random. Some SSRs are preferentially distributed in peri-centromeric heterochromatic regions. Additionally, we demonstrated that the greater abundance of SSRs in B. rapa compared with A. thaliana is associated with the genome triplication in B. rapa. These results provide insight into the distribution pattern and evolution of SSRs in the genome of B. rapa and provide a basis for comparison with the model plants A. thaliana and O. sativa. Acknowledgments This research was supported by grants from the Rural Development Administration (BioGreen 21 Program) and the Korean Science and Engineering Foundation (R ), Republic of Korea. TWK and JB were supported by Korea Research Institute Bioscience and Biotechnology (KRIBB), Korea Bioinformation Center (KOBIC), the Ministry of Science and Technology (MOST) and BioGreen21 funds. The authors thank Maryana Bhak for editing the manuscript. References Abdurakhmonov, I. Y., Abdullaev, A. A., Saha, S., Buriev, Z. T.,

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