Research

Telomere-centric genome repatterning determines recurring chromosome number reductions during the evolution of eukaryotes Xiyin Wang1,2,3,4, Dianchuan Jin2,3, Zhenyi Wang2,4, Hui Guo1,5, Lan Zhang2,4, Li Wang2,4, Jingping Li1,6 and Andrew H. Paterson1,5,6,7,8 1

Plant Genome Mapping Laboratory, University of Georgia, Athens, GA 30602, USA; 2Center for Genomics and Computational Biology, Hebei United University, Tangshan, Hebei 063000,

China; 3College of Sciences, Hebei United University, Tangshan, Hebei 063000, China; 4College of Life Sciences, Hebei United University, Tangshan, Hebei 063000, China; 5Department of Plant Biology, University of Georgia, Athens, GA 30602, USA; 6Institute of Bioinformatics, University of Georgia, Athens, GA 30602, USA; 7Department of Crop and Soil Science, University of Georgia, Athens, GA 30602, USA; 8Department of Genetics, University of Georgia, Athens, GA 30602, USA

Summary Author for correspondence: Andrew H. Paterson Tel: +1 706 583 0162 Email: [email protected] Received: 15 April 2014 Accepted: 5 July 2014

New Phytologist (2014) doi: 10.1111/nph.12985

Key words: Arabidopsis, centromere, chromosome number reduction, genome repatterning, grasses, polyploidy, telomere.

 Whole-genome duplication (WGD) is central to the evolution of many eukaryotic genomes,

in particular rendering angiosperm (flowering plant) genomes much less stable than those of animals.  Following repeated duplication/triplication(s), angiosperm chromosome numbers have usually been restored to a narrow range, as one element in a ‘diploidization’ process that re-establishes diploid heredity.  In several angiosperms affected by WGD, we show that chromosome number reduction (CNR) is best explained by intra- and/or inter-chromosomal crossovers to form new chromosomes that utilize the existing telomeres of ‘invaded’ and centromeres of ‘invading’ chromosomes, the alternative centromeres and telomeres being lost. Comparison with the banana (Musa acuminata) genome supports a ‘fusion model’ for the evolution of rice (Oryza sativa) chromosomes 2 and 3, implying that the grass common ancestor had seven chromosomes rather than the five implied by a ‘fission model.’  The ‘invading’ and ‘invaded’ chromosomes are frequently homoeologs, originating from duplication of a common ancestral chromosome and with greater-than-average DNA-level correspondence to one another. Telomere-centric CNR following recursive WGD in plants is also important in mammals and yeast, and may be a general mechanism of restoring small linear chromosome numbers in higher eukaryotes.

Introduction The number of chromosomes into which a genome is packaged is central to transmission genetics, largely determining the complexity of possible multilocus segregation patterns. Genetic linkage preserves co-adapted groups of alleles in natural populations (Presgraves et al., 2009), hinders creation of new gene combinations in crop improvement (Glaszmann et al., 2010; Springer & Jackson, 2010), and provides a powerful research tool (Li et al., 2010; Hamblin et al., 2011). Whole-genome duplication (WGD), or polyploidization, typically doubles chromosome numbers in one generation, exponentially increasing the number of possible segregation patterns and gene linkage relationships. Following polyploidization, chromosome numbers usually decline, sometimes eventually restoring the pre-polyploidy number (Table 1). For example, despite experiencing two genome paleo-duplications and one Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

paleo-triplication, Arabidopsis thaliana has only five gametic chromosomes. Maize (Zea mays) has restored an n = 10 karyotype in only 5–12 million yr (Myr) (Swigonova et al., 2004) after duplication of a 10-chromosome progenitor. Chromosome number reduction (CNR) has largely resulted from chromosome fusion, and biological mechanisms by which this occurs have been discussed. Two chromosomes may merge end to end, during which one chromosome becomes telo- or acrocentric, and another breaks near its centromere (Schubert & Lysak, 2011). Alternatively, one chromosome may merge into another chromosome near the latter’s centromere (Schubert & Lysak, 2011). These mechanisms have been proposed to explain observations in Arabidopsis, Brassica plants, and grasses (Lysak et al., 2006; Luo et al., 2009; Murat et al., 2010; The International Brachypodium Initiative, 2010). However, previous inferences about the proposed mechanisms are still coarse and incompletely explain this complex dynamic process. New Phytologist (2014) 1 www.newphytologist.com

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2 Research Table 1 Chromosome number reduction in plant genomes

Species (common name) Cereals Brachypodium distachyon (purple false brome) Oryza sativa (rice) Sorghum bicolor (sorghum) Setaria italica (foxtail millet) Zea mays (maize) Eudicot plants Carica papaya (papaya) Cucumis sativus (cucumber) Vitis vinifera (grape) Populus trichocarpa (poplar) Malus x domestica (apple) Arabidopsis lyrata (lyrate rockcress) Arabidopsis thaliana (mouse-ear cress) Glycine max (soybean) Brassica rapa (Chinese cabbage) Brassica oleracea (cabbage, etc.)

Extant basic chromosome number

Rounds of WGD/WGT

Ancestral basic chromosome number

Inferred chromosome number if no reduction

5 12 10 9 10

1 (q) 1 (q) 1 (q) 1 (q) 2 (q and 1 WGD)

7q 7q 7q 7q 7q

14 14 14 14 28

12 7 19 19 17 8 5 20 10 9

1 (c) 1 (c) 1 (c) 2 (c and 1 WGD) 2 (c and 1 WGD) 3 (c and 2 WGD) 3 (c and 2 WGD) 3 (c and 2 WGD) 4 (c, 2 WGD, 1WGT) 4 (c, 2 WGD, 1WGT)

7c 7c 7c 7c 7c 7c 7c 7c 7c 7c

21 21 21 42 42 84 84 84 252 252

q, the inferred ancestral grass chromosomes before WGD; c, inferred ancestral eudicot chromosomes before whole-genome triplication (Jaillon et al., 2007; Tang et al., 2008); WGD, whole-genome duplication; WGT, whole-genome triplication.

On the basis of comparative genomics analysis of grass genomes, it was proposed that centromeric/telomeric illegitimate recombination between nonhomologous chromosomes might have led to nested chromosome fusions (NCFs), and that this process may have involved interaction between centromeric and telomeric repeats. This repeat-mediated model has been proposed to describe chromosome number evolution in grasses; however, the model itself has not been subjected to any experimental or theoretical test. Comparative chromosome painting analysis in Arabidopsis and related plants suggested that, during the formation of telo- or acrocentric chromosomes, a large patch of pericentric DNA inversion may occur, which can be revealed in comparative analysis of gene colinearity between different genomes (Schubert & Lysak, 2011). However, the suggested DNA inversion was not observed in proposed chromosome fusions, and the elusiveness of such an inversion has been attributed to a second paracentric inversion to restore gene colinearity (Schubert & Lysak, 2011). Two such events sharing nearby breakpoints are improbable, with no known biological restriction favoring restoration of gene colinearity, and a second inversion at even slightly different breakpoints than the first would also lead to inverted gene colinearity. Both computational simulation and theoretical analysis (see Results below) show such a gene colinearity restoration model is improbable. Further, the above models have not considered chromosome clustering and interaction during meiosis, which may potentially contribute to genomic repatterning. Here, using comparative genomics and computational simulations, we re-analyzed the genome structures of grasses and Arabidopsis, and propose a telomere-centric mechanism to explain why there has been a trend of CNR during the evolution of plants, especially after polyploidizations. Together with additional lines of evidence in other eukaryotes, we further propose New Phytologist (2014) www.newphytologist.com

that the telomere-centric mechanism may be a general principle explaining the evolution of linear chromosomes.

Materials and Methods Plant genome data sets Genomes and their gene annotations were downloaded from the Rice Annotation Project database (version 2.0; http://rapdb.dna. affrc.go.jp/; International Rice Genome Sequencing Project, 2005), the Joint Genome Institute (Sorghum bicolor version 1.0 (http://genome.jgi-psf.org/Sorbi1/Sorbi1.home.html; Paterson et al., 2009) and Brachypodium distachyon version 4.0 (ftp://ftp. jgi-psf.org/pub/JGI_data/phytozome/v4.0/; The International Brachypodium Initiative, 2010)), the Arizona Genomics Institute (maize version 4a.53; http://ftp.maizesequence.org/; Schnable et al., 2009), and the Banana Genome Hub (Musa acuminata; http://banana-genome.cirad.fr; D’Hont et al., 2012). Dot-plot generation Gene CDSs from one plant were searched against its own or another genome sequence(s) using BLASTN. The best, second best, and other matches with E_value > 1e-5 were displayed in different colors, to help distinguish orthology from paralogy, or layers of paralogy as a result of recursive WGDs. Gene families with > 30 members were removed from the analysis. Dot-plots were produced using Perl scripts. Flash cartoon production Flash multimedia cartoons were produced using Adobe Flash language, integrating previous color schemes for grasses (Devos, Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist 2005) and Arabidopsis (Lysak et al., 2006). These color schemes were also used in dot-plots. Repetitive sequence detection Rice repetitive sequences were inferred by running REPEATMASKER (http://www.repeatmasker.org/RMDownload.html) using specific parameters and a core repeat set trained for rice. Statistical significance of homoeologous chromosome fusion The occurrence probability of nested chromosomal fusions between homoeologous chromosomes can be estimated with combinatorial statistics. For example, during formation of the sorghum karyotype, there were 12 chromosomes, merged from 14 ancestral chromosomes, or seven ancestral homoeologous chromosome pairs (see the Results section for evidence). If merged chromosomes are viewed still as independent chromosomal segments, the probability of this event can be estimated. If two merged chromosomes are taken as one, and the order of NCF events is considered, the estimation can be a little different but very similar. The occurrence probability of one out of two NCFs between homoeologous chromosomes can be estimated with combinatory formula (7, 1)(14, 2), where (n, m) is m!/[n! (m – n)!]. In B. distachyon, three out of seven NCF events occurred between homoeologous chromosomes, and the corresponding probability can be estimated by (7, 1)(6, 1)(5, 1)/[(11, 2)(12, 2)(10, 2)]. In maize, three of 10 extant chromosomes contain recently duplicated segments, and the corresponding probability can be estimated by (10, 1)(9, 1)(8, 1)/[(20, 2)(18, 2)(16, 2)].

Research 3 Table 2 Computational simulation of occurrences of repeat-mediated chromosome changes Repetitive sequence ratio

1:1

2:1

4:1

10 : 1

20 : 1

1:5

Centromere-mediated crossover Centromere–telomere crossover Telomere-mediated crossover Nested chromsome fusion

243

436

665

803

905

27

535

459

303

164

94

268

80

43

14

2

0

262

100

62

18

4

1

443

‘TBBOAAT’. If an end–end joining occurred between these two chromosomes, we would get a dicentric chromosome denoted by ‘TBBOBAOAAT’ and a satellite chromosome ‘TBAT’, the latter using ‘A’ and ‘B’ to find the original chromosomes rather than show divided arms. If an NCF occurred, we would get a dicentric chromosome denoted by ‘TAAOBOBAAT’ and a satellite chromosome ‘TBBT’. Different chromosomes were supposed to have the same CT ratio. The CT ratio varies from 20 : 1 to 1 : 5, which can reflect the actual situation of repeat distribution on chromosomes. More extreme distributions in centromeric or telomeric regions would produce more extreme results beyond the present observations on genome repatterning. Each crossover was simulated to occur between two chromosomes, which were determined using a random number generator implemented in Perl. In each computational experiment, a crossover occurred and its type and resulting chromosomes were recorded. The experiment was repeated 1000 times for each predefined CT ratio (Table 2). An algorithm can be formulated as follows. (1) Input: five chromosomes denoted as above and a predefined CT ratio.

Computational simulation of chromosome repatterning We performed two computational simulations to evaluate the models proposed here and those proposed previously to test whether repetitive sequences are sufficient to mediate the process of the repatterning of chromosome sets, as suggested previously (Murat et al., 2010), or there has been crossing-over preferentially involving telomeres and/or centromeres. In the first model, we tested various accumulation ratios of centromeric repeats to telomeric repeats (CT ratios) to investigate how chromosome repatterning would occur, that is, what kind of reciprocal translocation or fusion would probably result, such as inter-centromere crossover, inter-telomere crossover, centromere–telomere crossover, or NCF (Table 2). For simplicity, we hypothesize that crossover between telomeres from the same chromosome would result in NCF. We assumed that there had been five pairs of homoeologous chromosomes, which were denoted as ‘TAAOAAT’, ‘TBBOBBT’, . . ., ‘TEEOEET’, where ‘T’ represents a telomere, ‘O’ represents a centromere, and other letters represent chromosome arms. Crossovers were simulated to occur randomly as to repeat accumulation. If a crossover occurred at the centromeres of the two chromosomes ‘TAAOAAT’ and ‘TBBOBBT’, we would get ‘TAAOBBT’ and Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

(2) Select two chromosomes at random, and a type of crossover occurs randomly as to CT ratio. (3) Output: chromosomes after the crossover. (4) Repeat (2) and (3) 1000 times. (5) Stop.

The second computational simulation assumed preferential crossovers between telomeres and/or centromeres. By specifying whether telomeres or centromeres are involved, we introduced three parameters to partition different cases of chromosome fusions and crossovers. The parameters are telomere involvement ratio (R1), self-telomere-cross ratio (R2), and centromere involvement ratio (R3). R1 describes the likelihood that telomeres are involved. R2 describes the likelihood that there has been a crossover between two telomeres from the same chromosome or from two different chromosomes. R3 describes the likelihood that there has been a crossover of two centromeres or between a centromere and a telomere from different chromosomes. Similarly, we assumed that there had been five pairs of homoeologous chromosomes, and crossovers occur preferentially for telomeres and centromeres. Each ratio varies from 0 to 1. Similarly, random crossovers were realized by using a random number generator to find exchanges of chromosome arms, or NCF. In each computational experiment, a crossover occurred New Phytologist (2014) www.newphytologist.com

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4 Research

and its type and resulting chromosomes were recorded. The experiment was repeated 1000 times for different combinations of repeat ratios. An algorithm can be formulated as follows.

(a)

(b)

(1) Input: five chromosomes denoted as above. (2) For R1 = 0, 0.1, . . ., 1. { For R2 = 0, 0.1, . . ., 1. { For R3 = 0, 0.1, . . ., 1. { Select two chromosomes at random, and a type of crossover

(c)

occurs randomly as to ratios R1, R2, and R3. Repeat this step 1000 times. } } } (3) Output: chromosomes after the crossover. (4) Stop.

Results Inference of grass karyotype evolution Parsimony-based phylogenomic analysis can clarify karyotype evolution. For example, in hypothetical species P and Q that diverged after the whole-genome duplication common to all Poaceae cereals (hereafter denoted cWGD) (Paterson et al., 2004), a single chromosomal inversion in a common ancestor would cause incongruity between paralogous but not orthologous chromosomes. An inversion following P–Q divergence would cause incongruity between both P–Q orthologs and P–P or Q–Q paralogs (Fig. 1). In paleo-duplicated cereal chromosomes, seven breakageinversion events distinguish rice (oryzoids) from sorghum (panicoids), two in a polyploid oryzoid–panicoid common ancestor and five in the panicoid lineage after oryzoid–panicoid divergence (Fig. 1c; Supporting Information Fig. S1). Eight breakage-inversion events in B. distachyon (Fig. S2) may or may not be shared with other pooids not yet sequenced to high contiguity. The rice genome shows no major chromosomal changes since its divergence from other cereals, suggesting that its karyotype closely resembles that of the common cereal ancestor. Five groups of duplicated blocks can be inferred between rice chromosomes (R1–12): R1, 5; R2, 4, 6; R3, 7, 10; R8, 9; R11, 12. Previously, this observation motivated the suggestion that there were five ancestral chromosomes before cWGD and 10 after it, with two splitting into additional chromosomes or segments (Salse et al., 2008; Murat et al., 2010). Key questions regarding the ancestral cereal karyotype are whether rice chromosome 2 (R2) resulted from fusion of the ancestors of R4 and R6, or fission of an R2 duplicate formed R4 and R6; and likewise whether R3 was a fusion of R7, R10 and a segment of R12, or a fission to produce two more chromosomes (a segment merged into R12). The modern R2 and R3 are each New Phytologist (2014) www.newphytologist.com

Fig. 1 Inferring genome structural evolution based on homology between duplicated chromosomes. Suppose that two ancestrally duplicated chromosomes 1 and 2 have been inherited by two extant grass species P and Q. P1 and P2 are, respectively, orthologous to Q1 and Q2, while P2– Q1 and Q2–P1 are paralogs. A structural change specific to grass Q yields the pattern shown in (a); a change predating the divergence of P and Q yields the pattern shown in (b). (c) Comparison of rice (R) and sorghum (S) chromosomes distinguishes evolutionary events in common ancestors (cold-color line boxes) from those specific to sorghum or rice (warm-color line boxes). Quartets of boxes in the same colors help identify an event, in which the solid boxes show orthology, and broken-lined boxes show paralogy. Combinations of a blue box and an arrow show DNA breakages.

mosaics, with segmental correspondence to R6-R4-R6 and R10R7-R12-R7, respectively. We show (Fig. S3) that where gene colinearity deteriorates there is always localized transposon enrichment, a hallmark of NCF (Bennett & Laurie, 1995; Murat et al., 2010; The International Brachypodium Initiative, 2010). Such fusion of one chromosome into another’s pericentromeric region would result in repeat-rich boundaries of colinearity as a remnant of the split ancestral pericentromeric regions. Enrichment of repeats near colinearity breakpoints can still be recognized (after 70 Myr), with two breakpoints having 2–4 times more retrotransposons than average (Fig. S3). The banana genome sequence (D’Hont et al., 2012) provides further evidence of the role of NCF in cereal karyotype evolution. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist Consider R2, R4 and R6 as an example. If R4 and R6 are descendants of different ancient chromosomes from a common ancestor of banana and grasses, their homologous counterparts should be separately located in banana. By contrast, if R2 is a descendent of an ancient chromosome and subsequently spawned R4 and R6, then co-occurrence of segments corresponding to both R4 and R6 would be expected in the banana chromosomes. Chromosome fusions in banana (resulting in only 11 extant chromosomes) after polyploidizations (D’Hont et al., 2012) may lead to some co-occurrence of R4 and R6 homologs in the same banana chromosomes – however, some banana chromosomes or segments correspond only to R4 or R6, not both (Fig. 2). Banana chromosome 7 (Mu7) is homologous to R6 for nearly its full length, but shows no homology with R4. Likewise, Mu6, Mu8 and Mu11 are each homologous to R4 for nearly its full length, but show limited homology to R6 which does not fit with the mosaic structure of R2. Therefore, the most parsimonious interpretation is that R4 and R6 derived from different ancestral grass chromosomes, and formed R2 by fusion. Comparison to banana also implies that R3 formed by a fusion of R7, R10 and a segment of R12. R7, but not R10, has homology to Mu6, Mu7 and Mu9. Likewise, R10, but not R7, has significant homology with Mu1 and Mu11. This implies that R7 and R10 are descendants of two different ancestral monocot chromosomes (Fig. 2). Further, repetitive co-occurrences of segments homologous to R10 and the salient end of R12 in banana chromosomes Mu2, Mu4, and Mu5 suggest that these two elements of the modern R3 are derived from a single ancestral grass chromosome (Fig. 2). In partial summary, comparison of rice and banana chromosomes consolidated our inference that R2 and R3 were each

Research 5

produced by chromosome fusion. Specifically, the formation of each is best explained by NCF, with subsequent inversion in R3 explaining another striking enrichment of repeats by moving part of the ancestral pericentromeric region to the terminus of the long arm. Our model in which R2 and R3 each resulted from fusions of two chromosomes formed in the pan-cereal polyploidy implies that the cereal common ancestor had seven chromosomes (Fig. S5). The inferred grass ancestral karyotype (GAK1–7) of cereals had the following correspondence to the extant rice chromosomes: GAK1: R1, 5; GAK2: R6; GAK3: R4; GAK4: R7; GAK5: R10; GAK6: R8, R9; GAK7: R11, R12. NCF reduced the rice karyotype to 12 (q1–12), also explaining reductions to 10 in the Panicoideae (Fig. S1) and five in B. distachyon (Fig. S2), and > 50% of reductions after maize lineage specific duplication (Fig. S4). For example, GAK3, GAK6, and GAK7 merged to produce Brachypodium chromosome 1 by two serial NCFs (Fig. 3a). The ancestral Panicoideae chromosomes (P3 and P8) of sorghum chromosomes S3 and S8 merged to produce maize chromosome 3 (Fig. 3b). While NCF explains most karyotypic changes among cereal lineages, co-occurrence of broken synteny in maize–sorghum gene homology dot-plots indicates that the ancestral Panicoideae chromosomes P1 and P10 split into two pieces, respectively, one each from P1 and P10 merging to form M5, with the other pieces merging to form M9 (Figs 3c,S4). This observation suggests crossover between two ancestral chromosomes, resulting in reciprocal translocation of chromosome arms (RTA). Reciprocal DNA translocation also explains the production of extant M1 and M10 (Fig. 3d). Collectively, NCF and RTA explain all observed CNRs in the studied species, and possibly the origin of ancestral chromosome pairs q4/q9 and q10/q12 (Fig. S5). q9 and q10 are each acrocentric chromosomes with no discernible gene synteny near one end. We postulate that RTA occurred but that one of the exchanged termini of q9 and q10 was too small to observe, and acrocentry may have eroded any neighboring synteny. A telomere-centric mechanism of genome repatterning

Fig. 2 Dot-plot between rice and banana. Rice and banana chromosomes are, respectively, aligned horizontally and vertically. Red dots show three homologous banana genes best matching a rice gene, and blue dots show other matches. Green boxes highlight cases discussed in the main text. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

We propose a telomere-centric mechanism to underlie the molecular dynamics of karyotype evolution. Key to the mechanism is chromosomal crossing-over, probably during formation of a ‘chromosome bouquet’(Harper et al., 2004) that brings termini into close physical proximity, which may facilitate homologous pairing and synapsis(Niwa et al., 2000; Scherthan, 2001). Intra-chromosomal crossover producing a ring chromosome (RC) (Sodre et al., 2010) may in turn form a major chromosome with two free (‘sticky’) ends and a minichromosome with two telomeres but few genes (Fig. 4a). The mini-chromosome may often be lost with little effect on fitness in plants, although resulting in severe syndromes in humans (Scherthan, 2001). The major ‘sticky-end’ chromosome, containing many genes, is presumably essential (Fig. 5a). One of its ends may attach to another chromosome, possibly New Phytologist (2014) www.newphytologist.com

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6 Research (a)

(b)

(c)

(d)

(e)

(f)

often in a pericentromeric region with repetitive sequences facilitating attachment. This attachment comprises an ‘invasion’, possibly through a classical recombination mechanism after a double-strand break (Barton, 2010), resulting in breakage of the invaded chromosome, with one broken end of each chromosome ligating to one broken end of the other. Spatial proximity would then favor ligation between the other free ends of the chromosomes, nesting the invading chromosome within the invaded one, that is, resulting in NCF, or more precisely NCF mediated by the formation of a satellite RC (Fig. 4a). Further, a crossover proximal to the telomeres may New Phytologist (2014) www.newphytologist.com

Fig. 3 Chromosome fusions during the evolution of grasses and Arabidopsis. Chromosomes, shown as rectangular blocks, are arranged horizontally and vertically to the dot-plot. The color scheme for the chromosomes of grasses mainly follows that of a previous study (Murat et al., 2010), and for Arabidopsis a previous color scheme is adopted (Lysak et al., 2006). Homologous blocks can be classified as primary, resulting from chromosomal orthology (denser and longer), and secondary, resulting from paralogy (homoeology) from ancestral polyploidy. Dashed lines, with colors corresponding to chromosome color schemes, help to show where the merging points were and whether centromeres were preserved or not. Bidirectional arrows on chromosomal rectangles show inversion events. B, Brachypodium distachyon; M, maize; R, rice; S, sorghum; Al, Arabidopsis lyrata; At, Arabidopsis thaliana. (a) Formation of chromosome B1; (b) formation of M3; (c) formation of M5 and M9; (d) formation of M1 and M10; (e) formation of At2 and At3; (f) formation of At4 and At5.

lead to translocation of terminal DNA from one terminus to the other (Fig. 4b). Inter-chromosomal crossover, resulting in RTA, is another mechanism contributing to genome repatterning. Within the chromosome bouquet, crossover near two chromosomal termini might form a major chromosome containing most of their DNA by ‘end–end joining’, and a mini-chromosome containing the telomeres which may be lost (Fig. 4c). End–end joining can explain the origination of three maize chromosomes (Fig. S4). Alternatively, if two chromosomes cross over at some distance from the telomeres, the RTA process may form two novel Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Research 7 (a)

(d)

(b)

(c)

(e)

Fig. 4 Mechanisms of karyotype evolution. Blue or gold bars, chromosomes; red circles, telomeres; green circles, centromeres. (a) Formation of a ring chromosome (RC) during the chromosome bouquet stage, with crossover near the telomeres, resulting in production of a major chromosome having two free ends, and a tiny chromosome with little if any gene content and likely to be lost. The two-free-end chromosome may invade other chromosomes. (b) A variant of RC formation with a break near the telomeric regions results in a terminal-to-terminal translocation. The break may be caused by another crossover and the RC is basically an ‘8’ shape. As in (a), the process will also produce a two-free-end chromosome, and a tiny chromosome likely to be lost. (c) End–end joining of two chromosomes, with crossover at their attached ends, producing two novel chromosomes: a large one that is preserved and a tiny one that may be lost later. (d) Crossover at interstitial locations of two chromosomes, producing two novel chromosomes, with both being preserved; the crossover often occurs in the pericentromeric region(s). (e) Crossover near one telomere of a chromosome may result in an exchange of the telomere with a much larger chromosomal segment.

chromosomes (Fig. 4d). RTA and end–end joining can explain the origins of two pairs of maize chromosomes, M1/M10 and M5/M9, and of cereal ancestral chromosome pairs q4/q9 and q10/q12 (Fig. S1). More frequently than would be expected by chance, the ‘invading’ and ‘invaded’ chromosomes are homoeologs, that is, with more extensive DNA-level correspondence to one another than random chromosomes (Fig. 5b). NCF events between homoeologs account for one of two cases in sorghum (P-value = 0.077) (Fig. S1), and three of seven in Brachypodium (P < 0.0008) (Fig. S2). In maize, the fused chromosomes often shared homology as a result of two rounds of WGD. Three of 10 extant chromosomes contain recent duplicated segments (Fig. S4) (P ~ 0.0002). A Flash video illustrates these changes (Video S1; Fig. S5). In partial summary, these proposed mechanisms can explain all genomic repatternings in the cereal common ancestor following cWGD and those in B. distachyon, wheat (Triticum aestivum), maize, sorghum, and foxtail millet (Setaria italica) (the latter based on comparing a genetic map to the rice sequence (Devos et al., 2000)) lineages following their divergence (Fig. 6). Arabidopsis genome repatterning Telomeric rearrangements also explain most CNRs that differentiate Arabidopsis thaliana from both Arabidopsis lyrata (Kuittinen et al., 2004) and Capsella rubella, the latter two being colinear with one another (Koch & Kiefer, 2005; Yogeeswaran et al., 2005). Reciprocal translocation of arms between Arabidopsis ancestral chromosomes (Lysak et al., 2006) 3 and 5 (AAK3 and AAK5) (Fig. 3e) produced extant A. thaliana chromosome 3 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

(AT3), from most of AAK5 and nearly half of AAK3. A tiny part of AAK5 and the other half of AAK3 merged with AAK4 to produce A. thaliana chromosome 2 (AT2) by end–end joining. Reciprocal translocation of arms between AAK6 and AAK7 (Fig. 3f) produced AT4 and an intermediate chromosome which merged with AAK8 to produce AT5, probably by end–end joining. End–end joining between AAK1 and AAK2 produced A. thaliana chromosome 1. The three end–end joining events might each also have occurred through RTA at their very ends. In each case this would produce a large chromosome and result in loss of a mini-chromosome (Fig. S6), collectively resulting in a reduction in chromosome number from eight to five in A. thaliana. A Flash video illustrates these changes (Video S2; Fig. S7). Computational simulation to evaluate models By considering the potential mediating activity of repetitive sequences and their distribution in centromeres and telomeres, we performed computational simulations to evaluate the models proposed here and those proposed previously. Previous models suggested that centromeric and telomeric repetitive sequences might have mediated the repatterning of chromosome sets. By simulating random crossovers between chromosomes or within a chromosome, we tried to identify genome repatterning outcomes. The likelihood of occurrence of a specific crossover is determined by the relative amounts of repeats in centromeres and telomeres. Our tests indicated that when centromeric repeats were more abundant than telomeric repeats, which is often true, centromere–centromere crossovers would be dominant events (Table 2). Under different repeat accumulation ratios, we found New Phytologist (2014) www.newphytologist.com

8 Research (a)

(b)

Fig. 5 Telomere clustering and genome repatterning. (a) A two-free-end chromosome produced in a process described here may find a DNA region (often pericentromeric regions) to attach to, resulting in breakage of the affected chromosome. If one free end of the first chromosome is ligated with one broken end of the second chromosome, the two chromosomes would each have a remaining free end, and their proximity may increase the chance that they ligate. Therefore, the first chromosome merges into the second one and a large novel chromosome is formed. (b) Similar to (a), but the affected chromosome is the duplicated copy of the two-free-end chromosome.

that NCF cannot become the sole mechanism of genome repatterning, as observed in sorghum and Brachypodium. Therefore, the previous models only based on sharing repeats at centromeres and telomeres cannot fully explain chromosome repatterning. There must be other mechanisms working on the repatterning process. We performed another computational simulation by assuming preferential crossovers between telomeres and centromeres. Under an assumption of preferential crossing-over, we found that NCF or centromere–centromere crossing-over became the dominant, or even the sole, cause of chromosome repatterning (Fig. 7). Such preferential crossing-over can occur when formation of the chromosome bouquet (Ding et al., 2004; Bozza & Pawlowski, 2008) brings both telomeres and centromeres into close proximity, as discussed in the next section.

Discussion We suggest that prior models for genomic repatterning inadequately address the roles and fates of telomeres. ‘Capping’ by New Phytologist (2014) www.newphytologist.com

New Phytologist telomeres helps to protect chromosomes from disorganizing or merging with other chromosomes (Lundblad, 2000a,b; Ferreira et al., 2004). If telomeres are not removed, it is difficult for NCF or end–end joining to occur, as described previously (Murat et al., 2010; Schubert & Lysak, 2011). Homologous chromosome pairing may have contributed to genomic repatterning, with formation of the chromosome bouquet (Ding et al., 2004; Bozza & Pawlowski, 2008) bringing both telomeres and centromeres into close proximity. Telomere clustering, oscillation, and DNA recombination facilitate pairing (Ding et al., 2004). The formation of the chromosome bouquet makes it possible for the chromosomes to recognize their homolog(s), and the telomeric repeat is a primary factor in effective and correct recognition (Ding et al., 2004). Both ends of a chromosome may come near one another and those of other chromosomes during clustering (Barzel & Kupiec, 2008), facilitating formation of an intra-chromosomal ring structure (McClintock, 1939; Sodre et al., 2010) or inter-chromosomal crossover. Greater-than-average DNA-level correspondence between homoeologs may increase their rates of inter-chromosomal crossover. WGD(s) greatly increases spatial and interactive complexity in the nucleus and may increase the rate of crossover, as suggested by frequent DNA lesions and losses observed in artificial polyploids (Kashkush et al., 2002; Albertin et al., 2006), and inferred in paleo-polyploids (Paterson et al., 2004; Wang et al., 2005). Seventy million years after their duplication (or more, by some estimates), chromosomes R11 and R12 (and their orthologs) continue to experience frequent homoeologous recombination near their termini, increasing both DNA loss and mutation (Wang et al., 2011) in a manner that may be a general property of homologous interactions during telomere clustering in polyploids. Our inference that there were seven ancestral grass chromosomes, based on a fusion model for the evolution of R2 and R3, differs from a prior inference that there were only five (Salse et al., 2008; Murat et al., 2010) based on a mixed fusion and fission model. A fission model would involve the creation of neocentromeres and neo-telomeres, which is mechanically difficult and not necessary for the fusion model. Both intra- and inter-chromosome mechanisms could result in an extra centromere(s), with associated chromosomal instability, pairing and disjunction problems, and functional disorder favoring loss of one centromere. In interspecific dot-plots, a preserved centromere will make a gap in both the horizontal and vertical directions between broken colinear regions, whereas centromere loss from an extant chromosome would have no gap in the fused chromosomes (Fig. 3). In all observed cereal NCFs, centromeres of the invading but not the invaded chromosomes were preserved. For example, centromeres from ancestral R3 and R7 were lost in B1, and that of S8 was lost in M3 (Fig. 3a,b). This is consistent with the action of natural selection to protect the genedense (distal/telomeric) regions of the invading chromosome, by removing enormous repeats which may jeopardize genes. Inactivation of centromeres has been observed in maize and barley (Hordeum vulgare) (Nasuda et al., 2005; Han et al., 2006). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 6 Inferred evolutionary history of grass karyotypes. Lines among chromosomes show rearrangement events. NCF, nested chromosome fusion; IV, inversion; CFI, chromosome fission (shown by crosses); CEJ, chromosome end–end joining; RC, formation of ring chromosome; Mya, million years ago; WGD, whole-genome duplication; BEP, the three Poaceae subfamilies Bambusoideae, Ehrhartoideae and Pooideae.

Though invaded centromeres have been largely removed, we can still find some indications of them in the dot-plots. On both sides of locations where the removed centromeres should have been located, the slopes of gene colinearity blocks are often larger than for other regions (Fig. 3a). This is caused by continual repeat accumulation in pericentromeric regions around functional centromeres in extant chromosomes, but not in the corresponding regions near removed centromeres. The dynamics of centromere competition and loss are an interesting subject for further research, perhaps involving a mechanism that continues to affect euchromatin (Woodhouse et al., 2010; Gao et al., 2011). Previous publications have described one (Luo et al., 2009; Murat et al., 2010) or both (Lysak et al., 2006; Schubert & Lysak, 2011) of the NCF and end–end joining models and noted the occurrence of mini-chromosomes (Schubert & Lysak, 2011). However, these reports failed to show how mini-chromosomes formed and how the free-end chromosomes formed and invaded other chromosomes. It was inferred that the formation of a teloor acrocentric chromosome was a necessary process to allow end– end joining (Lysak et al., 2006; Schubert & Lysak, 2011). During the process, the centromere of a metacentric chromosome was inferred to transpose to one chromosome end via a pericentric inversion. It was noted that if two metacentric chromosomes are to merge into one, at least one of them has to become telo- or Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

acrocentric, and during the merging process one telo- or acrocentric chromosome would lose its two telomeres and centromeres. This kind of transposition would avoid the occurrence of dicentric chromosomes, which, although often unstable, can occur and cannot be absolutely avoided during genome reshuffling (Chabchoub et al., 2007; Flegal et al., 2012; Fu et al., 2012). Although telo- or acrocentric chromosomes can form during evolution, we do not think such formation is necessary for end–end joining to occur. As inversion was supposed to have been involved in the process, comparison between an inverted chromosome and its noninverted ortholog would find disturbed gene colinearity. However, we found that, during the formation of Arabidopsis AT2 and AT3 (Fig. 3e) and many extant grass chromosomes, no pericentric inversion could be detected. Previous inference attributed this intact gene colinearity to a subsequent paracentric inversion that restored colinearity (Schubert & Lysak, 2011). However, it is highly improbable that such a paracentric inversion would re-occur precisely at the previous breakpoints. If we assume that inversion occurs randomly, supposing that there are 2000 genes on the affected chromosome, the probability of another inversion to restore the gene colinearity is 4 9 10 6. A simulation test confirmed our inference. Previous publications discussing grass genome reshuffling focused on NCF but omitted RTA or end–end joining (Salse New Phytologist (2014) www.newphytologist.com

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10 Research (a)

(b)

(c)

(d) Fig. 7 Computational simulation of genomic repatterning. Under certain conditions of privileged interactions between telomeres and centromeres, chromosomes were simulated to merge through crossovers at specific locations, resulting in (a) end–end joining, or (b) nested chromosome fusion, exchanging arms through (c) centromere– centromere crossover or (d) centromere– telomere cross-over. The parameters are telomere involvement ratio (R1), selftelomere-cross ratio (R2), and centromere involvement ratio (R3).

et al., 2009; Murat et al., 2010; The International Brachypodium Initiative, 2010). Although events of NCF but not reciprocal translocation of arms or end–end joining were involved in sorghum and Brachypodium genome repatterning, here we found evidence of end–end joining during the evolution of the genomes of the grass common ancestor, maize, and Arabidopsis. Previous publications did not realize the potential dynamics behind the repatterning results. In particular, they did not note the importance of the production of ‘free-end’ chromosomes, which invaded others, and mini-chromosomes, losses of which led to CNR. Both intra- and inter-chromosome reduction mechanisms exploit existing telomeres to construct neo-chromosomes, rather than necessitating the evolution of new ones as sometimes proposed (Zhang & Durocher, 2010; Eckardt, 2011). This principle is consistent with the widespread theme that genomes evolve mainly by reorganizing and/or duplicating existing building blocks rather than creating new ones. This also explains why chromosomal fusion contributes more to genome repatterning than chromosomal fission, the latter leading to unstable chromosomes without telomeres. The losses of mini-chromosomes formed by telomeres have been a necessary step leading to CNR. Telomere-centric models that explain how most plants preserve small chromosome numbers may also explain karyotype evolution of vertebrates (Ijdo et al., 1991) and yeasts (Gordon et al., 2011). Recursive genome duplication renders plant genomes much less stable than those of most eukaryotes, providing New Phytologist (2014) www.newphytologist.com

more information toward understanding rules underlying chromosome structural evolution. Finally, we note that a constraint to this study, and to virtually all such genome comparisons, is that each of the sampled taxa is only represented by one high-quality genome assembly. Major genome structural changes such as those we and others have focused on are thought to be overwhelmingly selected against and therefore extremely rare in the gene pools of most species. However, empirical data are limited and it is of great interest, especially for recently formed polyploids, to investigate the extent to which such structural rearrangements may contribute to within-species polymorphism.

Acknowledgements We appreciate financial support from the China National Science Foundation (30971611 and 31170212), China-Hebei NSF for Talented Young Scholar, China-Hebei 100 Scholars Supporting Project, New Century 100 Creative Talents Project and US National Science Foundation (ACI1339727), received by X.W., and from the US National Science Foundation (MCB-1021718) and the J. S. Guggenheim Foundation received by A.H.P.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 A homologous gene dot-plot for rice (R) and sorghum (S) and the formation of the sorghum karyotype.

Fig. S2 A homologous gene dot-plot for rice (R) and Brachypodium. Fig. S3 DNA distribution and gene synteny. Fig. S4 A homologous gene dot-plot for sorghum and maize and the formation of the maize karyotype. Fig. S5 Grass genome repatterning. Fig. S6 A homologous gene dot-plot for Arabidopsis thaliana and Arabidopsis lyrata to show karyotype formation in the former. Fig. S7 Arabidopsis genome repatterning. Video S1 Dynamic changes during grass genome repatterning. Video S2 Dynamic changes during Arabidopsis genome repatterning. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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