) BMC Genomics BioMed Central Research article Open Access Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites Robert Kofler*1,Christian Schlotterer?,Evita Luschutzky3 and Tamas Lelley! Address:'University of Natural Resources and Applied Life Sciences,Department for Agrobiotechnology IFA-Tulln,Institute of Biotechnology in E-mail:Robert Kofler'-robert@koflerorat:Christian Schlotterer.christian schloetterer@vu-wien ac at: Evita Luschutzky-Evita.Luschuetzky@umweltbundesamtat;Tamas Lelley-tamas.lelley@boku.ac.at: 'Corresponding author Published:17 December 2008 Received:7 May 2008 BMC Genomics2008.9:612dot10.1186/1471-2164-9-612 Accepted:17 December 2008 This article is available from:http://www.biomedcentral.com/1471-2164/9/612 2008 Kofler et al:licensee BioMed Central Ltd. This is an Open Access article dist s of the Creative cor nse(http://creativecommons.or/icenses/by2.0) roduction in an Abstract Background:Compound microsatellites are a special variation of microsatellites in which two or more individual microsatellites are found directly adjacent to each other. Until now,such composite microsatellites have not been investigated in a comprehensive manner. Results:Our in silico survey of microsatellite clustering in genomes of Homo sapiens,Maccaca mulatta.Mus musculus.Rattus norvegicus,Ornithorhynchus anatinus,Gallus gallus,Danio rerio and Drosophila aste revealed an u expected high abundance of compound mic About 4-25%of all microsatellites could be categorized as compound microsatellites.Compound microsatellites are approximately 15 times more frequent than expected under the assumption of a random distribution of microsatellites.Interestingly,microsatellites do not only tend to cluster but the adjacent repe types of compo mic telites have very similar otifs:in most case (>90%)these motifs differ only by a single mutation(base substitution or indel).We propose that the majority of the compound microsatellites originates by duplication of imperfections in a microsatellite tract.This process occurs mostly at the end of a microsatellite,leading to a new repeat type and a potential microsatellite repeat track. Conclusion:Our findings suggest a more dynamic picture of microsatellite evolution than previously believed.Imperfections within microsatellites might not only cause the "death"of microsatellites they might also result in their"birth". I Background attracted much attention during the last decade and Microsatellites or simple sequence repeats (SSR)are notably resulted in various genetic marker systems [4-6]. DNA stretches consisting of a tandemly repeated short DNA motif (s 6 bp).Due to the special mutation mechanism of microsatellites terme edDNA replication According to Chambers et al.7]the following categories of micr osatellites can be dist nguished:Pure,Inter slippage",these sequences often exhibit length hyper- pure,Compound,Interrupted compound,Complex anc variability with respect to the number of motifs being Interrupted complex.In this survey we mainly refer to repeated reviews:[1-3]l.Owing to this hypervariability Compound and Interrupted compound microsatellites. and an ubiquitous presence in genomes,microsatellites This has to be distinguished from the term microsatellite Page 1 of 14 (page number not for citation purposes)
BMC Genomics Research article Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites Robert Kofler*1 , Christian Schlötterer2 , Evita Luschützky3 and Tamas Lelley1 Address: 1 University of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology IFA-Tulln, Institute of Biotechnology in Plant Production, Konrad Lorenz Straße 20, 3430 Tulln, Austria, 2 Institut für Popluationsgenetik, Veterinärmedizinische Universitat Wien, Josef Baumann Gasse 1, 1210 Wien, Austria and 3 Umweltbundesamt, Spittelauer Lände 5, 1090 Wien, Austria E-mail: Robert Kofler* - robert@kofler.or.at; Christian Schlötterer - christian.schloetterer@vu-wien.ac.at; Evita Luschützky - Evita.Luschuetzky@umweltbundesamt.at; Tamas Lelley - tamas.lelley@boku.ac.at; *Corresponding author Published: 17 December 2008 Received: 7 May 2008 BMC Genomics 2008, 9:612 doi: 10.1186/1471-2164-9-612 Accepted: 17 December 2008 This article is available from: http://www.biomedcentral.com/1471-2164/9/612 © 2008 Kofler et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Compound microsatellites are a special variation of microsatellites in which two or more individual microsatellites are found directly adjacent to each other. Until now, such composite microsatellites have not been investigated in a comprehensive manner. Results: Our in silico survey of microsatellite clustering in genomes of Homo sapiens, Maccaca mulatta, Mus musculus, Rattus norvegicus, Ornithorhynchus anatinus, Gallus gallus, Danio rerio and Drosophila melanogaster revealed an unexpected high abundance of compound microsatellites. About 4 – 25% of all microsatellites could be categorized as compound microsatellites. Compound microsatellites are approximately 15 times more frequent than expected under the assumption of a random distribution of microsatellites. Interestingly, microsatellites do not only tend to cluster but the adjacent repeat types of compound microsatellites have very similar motifs: in most cases (>90%) these motifs differ only by a single mutation (base substitution or indel). We propose that the majority of the compound microsatellites originates by duplication of imperfections in a microsatellite tract. This process occurs mostly at the end of a microsatellite, leading to a new repeat type and a potential microsatellite repeat track. Conclusion: Our findings suggest a more dynamic picture of microsatellite evolution than previously believed. Imperfections within microsatellites might not only cause the "death" of microsatellites they might also result in their "birth". 1 Background Microsatellites or simple sequence repeats (SSR) are DNA stretches consisting of a tandemly repeated short DNA motif (≤ 6 bp). Due to the special mutation mechanism of microsatellites termed "DNA replication slippage", these sequences often exhibit length hypervariability with respect to the number of motifs being repeated [reviews: [1-3]]. Owing to this hypervariability and an ubiquitous presence in genomes, microsatellites attracted much attention during the last decade and notably resulted in various genetic marker systems [4-6]. According to Chambers et al. [7] the following categories of microsatellites can be distinguished: Pure, Interrupted pure, Compound, Interrupted compound, Complex and Interrupted complex. In this survey we mainly refer to Compound and Interrupted compound microsatellites. This has to be distinguished from the term microsatellite Page 1 of 14 (page number not for citation purposes) BioMed Central Open Access
BMC Genomics 2008,9:612 http://www.biomedcentral.com/1471-2164/9/612 custer as used by grover and Sharma isl which refers to standardized:see Additional file 11.All microsatellite rich regions.However,although microsatel bp (se satellites have bee pends on the distance sep the reason for much depate.Imperfections in them microsatellites.In this work,microsatellites being tellite tract If they accumulate in a microsatellite tact.they have eve termed individual microsatellites being part of such a m tellites cSs the impac (CSSR-%)with a given d ranging the satellites have a composite motif.Despite their abundance species yet been studied in a of 50 bp an infle tion point could b wn about thei ent (ng boundary for the ween two panly due to Thy the 2.2 Freque compound microsatellite density in n of bp.Rode d in different arrangements.The AGmic satellite might b mpound microsatellites (Table 1) cated 5'or 3'to th AC lite and eith vhereas I melanogaste and latipes had the lowest on the s ed he me DNA and as t cor tained an exceptionally high cSSR-%in tract of the AC microsatellite.For these reas fou introduced by Kofle cds (able.la mnogaster this proportion was e Here we provide the non-coding sequences (Table 1).The impact of of h setti compoun microsate ully sequence 。 ence cds)the 5 and (Table S2). es can be on the untranslated rately.We analyzed the genomes of 2.3 Distri of compound microsatellites within the andtinus).a bird (Gallus gallus).a fish otsnot homocou Danio rerio)and a insect (Drosophila melanogaster).We within genomes.For example,in H. sapiens and show tha 250 an m are par 1 evolutionary mechanisms leading to the observed high therefore investigated the distribution of compound frequency of compound micrsoatellites. microsatellites along the c 2 Results window si of MbP andst si ofM 2. Distance between microsatellites Page 2 of 14 page number not for citation purposes)
cluster as used by Grover and Sharma [8] which refers to microsatellite rich regions. However, although microsatellites have first been described more than twenty years ago [9], their evolution is still not fully understood [2, 3]. In particular imperfections within microsatellites have been the reason for much debate. Imperfections in the microsatellite tract are thought to interfere with replication slippage by limiting microsatellite size expansion [10-12]. If they accumulate in a microsatellite tract, they have even been proposed to cause the "death" of a microsatellite [13]. The complementary concept, the "birth" of a microsatellite was first introduced by Messier [14]. However, compound microsatellites, i.e. two or more microsatellites being found in close proximity, have been frequently reported in diverse taxa ranging from humans to plants [10, 15-19]. Weber [10] estimated that, about 10% of the human microsatellites have a composite motif. Despite their abundance, compound microsatellites have not yet been studied in a comprehensive manner and very little is known about their origin and evolutionary dynamics. This lack of knowledge about compound microsatellites is partly due to the difficulties involved by their identification using computer aided approaches. The analysis of compound microsatellites is additionally confounded by the fact that two microsatellites can be arranged in several different combinations [16, 20]. For instance, the two microsatellites [AC]n and [AG]m can be found in four different arrangements. The [AG]m microsatellite might be located 5' or 3' to the [AC]n microsatellite and either the poly-TC or the poly-AG tract of the [AG]m microsatellite might be found on the same DNA strand as the poly-AC tract of the [AC]n microsatellite. For these reasons, four different motif standardizations were introduced by Kofler et al. [20] [see also Additional file 1]. Here we provide the first comprehensive survey of compound microsatellites in the fully sequenced genome of eight eukaryotic species. We surveyed the entire genomes as well as the coding sequence (cds) the 5' and the 3' untranslated region (5'-UTR and 3'-UTR) separately. We analyzed the genomes of five mammals (Homo sapiens, Maccaca mulatta, Mus musculus, Rattus norvegicus, Ornithorhynchus anatinus), a bird (Gallus gallus), a fish (Danio rerio) and a insect (Drosophila melanogaster). We show that 4 – 25% of all microsatellites are part of compound microsatellites and discuss the possible evolutionary mechanisms leading to the observed high frequency of compound micrsoatellites. 2 Results 2.1 Distance between microsatellites We define a compound microsatellite as an aggregation of at least two microsatellites with different motifs [partially standardized: see Additional file 1]. All identified microsatellites have a minimum length of 15 bp (see Material and Methods). Whether two or more adjacent microsatellites account as a compound microsatellite depends on the distance separating these microsatellites. In this work, microsatellites being separated by less than a maximum threshold dmax were classified as compound microsatellite. For brevity, we termed individual microsatellites being part of such a compound microsatellite cSSR and the percentage of these microsatellites cSSR-%. We determined the impact of dmax by measuring the proportion of microsatellite which could be classified as compound microsatellites (cSSR-%) with a given dmax (Fig. 1). As expected, the number of compound microsatellites increases with dmax, but the increase is not linear. While we observed species specific differences, the overall pattern is that around a dmax of 50 bp an inflection point could be found, indicating a different behavior (Fig. 1). One difference between cds and whole genome is that for cds an upper boundary for the distance between two microsatellites exists, i.e. the total length of the cds. 2.2 Frequency of compound microsatellites We quantified the compound microsatellite density in the different genomes by setting dmax to 10 bp. Rodents and D. rerio had the highest proportion of microsatellite being classified as compound microsatellites (Table 1) whereas D. melanogaster and O. latipes had the lowest. Interestingly, for coding sequences no major differences were observed between the species (Table 1). Only R. norvegicus contained an exceptionally high cSSR-% in the cds (Table 1). In D. melanogaster this proportion was higher for coding sequences than for genomic sequences, indicating a more pronounced clustering in the cds than in non-coding sequences (Table 1). The impact of different SSR-search settings on the frequency of compound microsatellites can be found in Additional file 2 (Table S2). 2.3 Distribution of compound microsatellites within the genome of H. sapiens The distribution of microsatellites is not homogeneous within genomes. For example, in H. sapiens and M. musculus an increase in microsatellite density toward the ends of the chromosomes was reported (in 2). We therefore investigated the distribution of compound microsatellites along the chromosomes. The SSR and the compound microsatellite densities were calculated with an overlapping sliding window approach using a window size of 5 Mbp and a step size of 1 Mbp. Consistent with previous results, we show that the distribution of microsatellites varies along the chromosomes as well BMC Genomics 2008, 9:612 http://www.biomedcentral.com/1471-2164/9/612 Page 2 of 14 (page number not for citation purposes)
BMC Genomics 2008.9:612 http://www.biomedcentral.com/1471-2164/9/612 whole genome 100 200 100 200 300 Figure 1 e of the cSSR-% as between chromosomes of H.sapiens(Fig2)Generally 2.4 Parameters governing compound microsatellite atellites Nou some chromosome specificpattemn could be detected.While or most chr romosomes the peaks nosome and recon bination 2【ested spondence could besee Also on some chromosomes,the compound microsatellite pattem seems tobe morepronoun M.were used for this analysis. mo ve frequency of ng ch g2) Table I:Frequency of compound microsatellites in the whole genome and in the coding sequence (cds) whole genome coding sequence c2 CSSR3 mds cds mI c2 CSSR3 mds cds 冲m 59792 12984 496 90 253 45 . 133 327 325 237 3769 8s und n :] tta:M.mus.:Mus musculus:R nor:Rattus norvegicus:O.anat.:Ornithorhynchus anotinus:G gal.:Gallus gallus: Page 3 of 14
as between chromosomes of H. sapiens (Fig. 2). Generally, the distribution of compound microsatellites follows very closely the distribution of microsatellites. Nevertheless, some chromosome specific pattern could be detected. While for most chromosomes the peaks in compound microsatellite density follows the microsatellite density, on chromosome15onlya relativelyweak correspondence couldbe seen. Also on some chromosomes, the compound microsatellite pattern seems to bemore pronounced than themicrosatellite pattern (e.g. chromosome 8). Finally, the spacing between the lines indicating the microsatellite and compound microsatellite density differs among the chromosomes of H. sapiens, suggesting that the relative frequency of compound microsatellites differs among chromosomes (Fig. 2). 2.4 Parameters governing compound microsatellite density Differences in compound microsatellite density can be caused by the parameters 'SSR density', 'species', 'chromosome' and 'recombination'. We tested which of these parameters has a significant influence on compound microsatellite density. Due to the scarcity of species with sequenced Y-chromosomes only H. sapiens, Pan troglodytes and M. musculus were used for this analysis. We observed that the parameters 'SSR-density' (CatReg: p < 0.001), 'species' (CatReg: p < 0.001) and 'chromosome' dmax cSSR - percentage [%] cSSR - percentage [%] dmax 0 100 200 300 400 0 10 20 30 40 50 60 H. sapiens M. mulatta M. musculus R. norvegicus O. anatinus G. gallus D. rerio D. melanogaster 0 100 200 300 400 0 10 20 30 40 50 60 whole genome cds Figure 1 Influence of dmax to the cSSR-%. Table 1: Frequency of compound microsatellites in the whole genome and in the coding sequence (cds). whole genome coding sequence species m.1 c.2 cSSR3 %4 m.d.5 c.d.6 m.1 c.2 cSSR3 %4 m.d.5 c.d.6 H. sap. 1 169 530 59 792 129 848 11.1 413.0 21.1 4 965 104 233 4.7 77.4 1.6 M. mul. 1 178 381 61 407 134 455 11.4 445.3 23.2 3 638 64 139 3.8 71.3 1.3 M. mus. 1 574 180 173 535 398 361 25.3 617.9 68.1 3 995 95 202 5.1 72.5 1.7 R. nor. 1 307 474 133 120 291 304 22.3 527.8 53.7 1 883 92 226 12.0 92.6 4.5 O. anat. 133 984 1 913 3 969 3.0 327.2 4.7 1 535 16 34 2.2 42.8 0.5 G. gal. 233 896 8 532 17 989 7.7 237.5 8.7 1 889 36 77 4.1 58.3 1.1 D. rerio 1 048 258 94 159 225 069 21.5 688.1 61.8 3 215 86 180 5.6 72.0 1.9 D. mel. 44 600 714 1 457 3.3 376.9 6.0 4 168 105 213 5.1 145.6 3.7 1 total number of microsatellites in DNA sequence space 2 total number of compound microsatellites in DNA sequence space 3 number of individual microsatellites being part of a compound microsatellite 4 percentage of individual microsatellites being part of a compound microsatellite (cSSR-%) 5 microsatellite density [m./Mbp] 6 compound microsatellite density [c./Mbp] H. sap.: Homo sapiens; M. mul.: Macaca mulatta; M. mus.: Mus musculus; R. nor.: Rattus norvegicus; O. anat.: Ornithorhynchus anatinus; G. gal.: Gallus gallus; D. rerio: Danio rerio; D. mel.: BMC Genomics 2008, 9:612 http://www.biomedcentral.com/1471-2164/9/612 Page 3 of 14 (page number not for citation purposes)
BMC Genomics 2008,9:612 http://www.biomedcentral.com/1471-2164/9/612 h Chr.3 Chr.4 hr. 39 Chr 10 Chr.12 Chr.14 Chr 15 Chr 16 17 Chr.19 Chr.X Chr.Y compound SSR density [#/Mbp SSR density [#/Mbp] not sequenced (poly-N tracts) Figure 2 Compound microsatellite density in the chromosomes of .sapiens compared to the microsatellite density on the calculated with an sliding window approach using a window size of 5 Mbp and a step size of I Mbp. Page 4 of 14 (page number not for citation purposes)
Chr. 1 Chr. 2 Chr. 3 Chr. 5 Chr. 7 Chr. 10 Chr. 13 Chr. 17 Chr. X Chr. Y Chr. 18 Chr. 20 Chr. 21 Chr. 22 Chr. 14 Chr. 15 Chr. 16 Chr. 11 Chr. 12 Chr. 8 Chr. 9 Chr. 6 Chr. 4 Chr. 19 700 0 350 40 10 25 40 10 25 40 10 25 40 10 25 40 10 25 40 10 25 40 10 25 40 10 25 700 0 350 700 0 350 700 0 350 700 0 350 700 0 350 700 0 350 700 0 350 not sequenced (poly-N tracts) SSR density [#/Mbp] compound SSR density [#/Mbp] Figure 2 Compound microsatellite density in the chromosomes of H. sapiens compared to the microsatellite density. Regions which have not yet been sequenced are designated yellow. The scale of the compound microsatellite density is on the left hand side and the scale of the SSR density on the right hand side. The SSR and the compound microsatellite density were calculated with an sliding window approach using a window size of 5 Mbp and a step size of 1 Mbp. BMC Genomics 2008, 9:612 http://www.biomedcentral.com/1471-2164/9/612 Page 4 of 14 (page number not for citation purposes)
BMC Genomics 2008.9:612 http://www.biomedcentral.com/1471-2164/9/612 ever,very large compound microsatellites,containing few exceptions the cds contains more than four SSRs SSRs Isee Additional file 2 Table 571.To test whethe analysis and correlated the density of microsatellites tha compound microsatellites originate from a nesting of oud ot be dassi ficd ascom C 0.001).chromosome(CatReg:0.001)and 'SSR compoundte)have a significantinen tellite density. To dete mine the Additional file 2:Table S11.which suggests that most influence of recc om ination,we C7emigfeomPondmosaiditsdonotoignate encesin recombination rate and found o significan 2.6 Aggregation of microsatellites bination map published by Kong et al.[21]and compared the recombination frequencies with chance,we deter the respect to an assumed random distibution of micro satellites in the genome. Isee Additional file 3 and Additional file 41 2.5 Compound microsatellite complexity oncept of SSR-couples.SSR-couples ple,the compound microsatellite [AC][AGlo contain compound microsatellite.For example a tri-SSR com- lauter ui-ssR co microsatellites(=87%)contain only two cSSRs(Table 2). sidered [partially standardized:see Additional file 1]. Table 2:Compound micro complexity in the whole genome and in the cds. Ce. 6 25 099 4 Z0420000 0 00040 All values are in count Page 5 of 14
(CatReg: p < 0.001) have a highly significant influence on the compound microsatellite density. These three parameters are highly correlated with the compound microsatellite density (CatReg: R2 = 0.94). Additionally, the relative contributions (rc) of these parameters to the regression could be identified. We found that 'species' (rc = 0.36) and 'chromosome' (rc = 0.38) have the strongest influence and that SSR density has a moderate influence (rc = 0.26). Because compound microsatellites are a subset of the total microsatellite repertoire, we modified our analysis and correlated the density of microsatellites that could not be classified as compound microsatellites with compound microsatellites. Again, 'species' (CatReg: p < 0.001),'chromosome' (CatReg: p < 0.001) and 'SSR density' (CatReg: p < 0.001) have a significant influence on compound microsatellite density and are highly correlated (CatReg: R2 = 0.93) with the compound microsatellite density. To determine the influence of recombination, we compared two groups of chromosomes (Y-chromosomes with chromosomes other than Y) with extreme differences in recombination rate and found no significant influence (CatReg: p = 0.214). To further test the influence of recombination we used the human recombination map published by Kong et al. [21] and compared the recombination frequencies with the compound microsatellite density and found only a very weak correlation (Linear regression: R2 = 0.03) [see Additional file 3 and Additional file 4] 2.5 Compound microsatellite complexity Compound microsatellites might contain different numbers of individual microsatellites (cSSRs). For example, the compound microsatellite [AC]9 [AG]10 contains two whereas the compound microsatellite [AC]11 [AG]7 [AC]9 three cSSRs. We call the former 'di-SSR' and the latter 'tri-SSR' compound microsatellite. Most compound microsatellites (≈ 87%) contain only two cSSRs (Table 2). The number of identified compound microsatellites decreases rapidly with an increasing complexity. However, very large compound microsatellites, containing more than eight cSSRs, can be found in many species (Table 2). We found the largest compound microsatellite in D. rerio chromosome 17, having 40 cSSRs. Only with a few exceptions the cds contains more than four cSSRs (Table 2). The complexity of compound microsatellites in the 5'-UTRs and 3'-UTRs is higher, but rarely exceeds three cSSRs [see Additional file 2: Table S7]. To test whether compound microsatellites originate from a nesting of microsatellites, i.e. secondary microsatellites emerging in the tract of primary microsatellites, we analyzed the percentage of tri-SSR compound microsatellites having the pattern: [m1]n1 [m2]n2 [m1]n3 where m1 and m2 are the motifs of the individual cSSRs [partially standardized: see Additional file 1]. In all eight species about 33% of the tri-SSR compound microsatellites exhibit this pattern [see Additional file 2: Table S11], which suggests that most (67%) tri-SSR compound microsatellites do not originate by a nesting of microsatellites. 2.6 Aggregation of microsatellites To test whether the occurrence of compound microsatellites can be attributed to mere chance, we determined whether microsatellites tend to aggregate with respect to an assumed random distribution of microsatellites in the genome. For simplicity we confine this analysis to pairs of adjacent microsatellites and introduce the technical concept of SSR-couples. SSR-couples are each two adjacent microsatellites being separated by less than 10 bp (dmax), which can be part of a more complex compound microsatellite. For example a tri-SSR compound microsatellite could be viewed as two overlapping SSR-couples. SSR-couples containing two microsatellites with an identical motif were not considered [partially standardized: see Additional file 1]. Table 2: Compound microsatellite complexity in the whole genome and in the cds. whole genome cds c.c.:1 2345678 ≥ 9234 ≥ 5 H. sap. 51 997 6 096 1 198 335 106 41 7 12 81 21 2 0 M. mul. 52 796 6 565 1 389 433 155 49 10 10 53 11 0 0 M. mus. 137 237 26 551 6 561 2 080 652 241 99 114 84 10 1 0 R. nor. 113 077 16 505 2 632 607 170 78 19 32 72 11 5 4 O. anat. 1 791 105 13 4 0 0 0 0 14 2 0 0 G. gal. 7 782 610 115 17 6 2 0 0 32 3 1 0 D. rerio 71 280 15 703 4 163 1 641 592 336 143 301 78 8 0 0 D. mel. 685 29 0 0 0 0 0 0 102 3 0 0 1 compound microsatellite complexity Complexity refers to the number of individual microsatellites constituting the compound microsatellite. All values are in counts BMC Genomics 2008, 9:612 http://www.biomedcentral.com/1471-2164/9/612 Page 5 of 14 (page number not for citation purposes)