Plant Molecular Biology 35: 17–23, 1997. 17c 1997 Kluwer Academic Publishers. Printed in Belgium.

Are rice chromosomes components of a holocentric chromosome ancestor?

Graham Moore, Luis Aragon-Alcaide, Michael Roberts, Steve Reader, Terry Miller andTracie FooteCereals Research Department, John Innes Centre, Norwich Research Park, Colney Norwich, NR4 7UH, UK

Key words: Luzula, centromeres, Ph1, pairing, ancestral genome

Abstract

Comparative genomics reveals that cereal genomes are composed of similar genomic building blocks (linkageblocks). By stacking these blocks in a unique order, it is possible to construct a single ancestral ‘chromosome’which can be cleaved to give the basic structure of the 56 different chromosomes found in wheat, rice, maize,sorghum, millet and sugarcane. The borders of linkage blocks are defined by cereal centromeric and telomeric sites.However, a number of studies have shown that telomeric heterochromatin has neocentromeric activity, implyingthat linkage blocks are in fact defined by centromeric-like sites with conserved sequences. The structure of theancestral cereal genome thus resembles a holocentric chromosome, which is the chromosome structure shared bythe closest relatives of the Gramineae, the Cypericeae and Juncaceae.

Introduction

Comparison of the organisation of genomes of relatedspecies (comparative genomics) sheds light on chro-mosome evolution. Organisation of the genome struc-tures found in present day species should enable usto predict the genome structure of the ancestral pro-genitor. Gross evolution of the ancestor genome couldoccur either through random or non-random breakageat specific sites (Figure 1). After separation of spe-cies, the genomes would be subject to rearrangementsand deletions that are specific to these genomes. Con-straints on the way in which genomes evolve couldreflect a requirement to maintain sets of genes linkedbecause of gene regulation or because of structuralarrangements for pairing during meiosis.

Speciation

The closest relatives of the Gramineae, the Jun-caceae and Cypericeae, possess holocentric chromo-somes, which have dispersed centromeres (Figure 2).Moreover, within the Juncaceae, Luzula spp. (rushes)possess chromosomes that can naturally, or by irra-diation, be cleaved into smaller viable chromosomes.

In hybrids between parents possessing these smallerchromosomes and those possessing the larger chro-mosomes, the smaller chromosomes align and pairwith the larger chromosomes at meiosis [1]. It isnot clear whether the breakage of the larger chro-mosomes in these species occurs randomly along thechromosome or at specific sites such as the dispersedcentromeres. Nevertheless, these observations suggestthat telomeres are not driving pairing in these spe-cies as the two sets of chromosomes have differentnumbers of telomeres. This implies that sites oth-er than telomeres (centromeres and interstitial sites)are determining chromosome recognition in Luzulaspecies and possibly closely related cereals. Cleav-age of chromosomes into smaller viable chromosomessimilar to that occuring in Luzula species could bea major part of the mechanism of genome evolutionof the Gramineae. The question therefore arises as towhether the ancestral genome of the Gramineae willresemble a holocentric chromosome in structure. Thesequences at the ‘diffuse’ centromeric sites in holo-centric chromosomes will be related as they are attach-ment sites for microtubules. If Gramineae chromo-somes have evolved from holocentric chromosomes,there are a number of implications: the sequences at

GR: 201001942, Pips nr. 138656 BIO2KAP

*138656* pl3891si.tex; 16/07/1997; 16:49; v.7; p.1

18

Figure 1. A schematic representation of the two possible mechanisms of evolution from the ancestor progenitor genome during speciation. Theblocks denote chromosome regions in the progenitor genome.

their centromeres may be conserved; their chromo-somes may possess several potential sites for attach-ment of microtubules; a system would have evolvedfor suppressing the activity of potential sites; this sup-pression system could have led to the evolution of bar-coding or discrimination of chromosomes based on theactivity of these sites.

The genomes of species such as maize, rice andwheat which have been isolated by more than 60 mil-

lion years can be used to assess this issue [2, 3]. Thesecereals now have markedly different genome sizes andnumbers of chromosomes. However the gene orderis maintained in these genomes both at the geneticaland physical levels (reviewed in depth by Devos andGale in this Edition). Comparisons between the gen-omes of closely related species such as barley, rye, andwheat or maize and sorghum indicate conservation ofgene order at the genetic level [4–11]. Studies of less

pl3891si.tex; 16/07/1997; 16:49; v.7; p.2

19

Figure 2. The classification of subclass Commelinidae with the monocots. Those families that contain species possessing holocentric chromo-somes are indicated.

closely related species such as rice and wheat, andrice and maize reveal that there is extensive conser-vation of gene order despite gross differences in gen-ome size [12–16]. Comparisons at the physical levelbetween rice and wheat and rice and barley show thatfor the regions studied gene order is maintained [17–18] (reviewed in detail by Kilian and colleagues inthis Edition). An extensive comparison at the phys-ical level of rice chromosome 9 and wheat chromo-some group 5 reveals that the markers on rice chromo-some 9 detect markers on wheat chromosome group5. However, there are regions on wheat chromosomegroup 5 which have accumulated additional markerswhich do not map collinearly [19]. Comparison ofrice, maize and sorghum at the physical level suggest arelatively high level of rearrangements [20]. The phys-

ical mapping data indicates that since the separationof the rice, maize and sorghum genomes, there havebeen duplications, inversions and other chromosomalrearrangements. These complicate an analysis of thebasic chromosome evolution of the genomes involved,and have to be ‘ironed out’ before the broad picturecan be revealed.

If the genetic maps of rice chromosomes are dis-sected into rice linkage segments (like Lego blocks),it is possible to reconstitute the genetic maps of wheatand maize chromosomes using these segments (Fig-ure 3). This analysis reveals the basic tetraploid natureof the genome of maize, and its possible derivationfrom the hybridisation of two parents with chromo-somes formed from different arrangements of ricelinkage segments. Although the designation of dif-

pl3891si.tex; 16/07/1997; 16:49; v.7; p.3

20

ferent sorghum linkage groups varies, it is possibleto assemble all the published data on its genome andrelate it to the genetic maps of sugarcane and maize.The genetic maps of sorghum, Fox-tail millet and sug-arcane chromosomes can therefore also be assembledfrom the same rice linkage segments [21] (Figure 3).Thus the same conserved linkage groups in rice canbe used to represent the genomes of all these species.The basis of this analysis is that the majority of genescontained within a segment in rice also occur in thesame segment in wheat, maize and the other cerealgenomes. The occurrence of conserved linkage seg-ments in these genomes implies that the process ofcereal genome evolution has not been random but isthe result of cleavage and fusion at specific sites.

Ancestral ‘cereal chromosome’

Using rice linkage segments, it is possible to predictthe generalised genome structure of the ancestral grasswhich gave rise to wheat, barley, maize, sorghum andrice some 60 million years ago [22]. The outcomeof this analysis is that the primeval grass may havehad just one chromosome pair (Figure 3). By breakingthis chromosome in different places and by creatinginversions, the two sets of maize chromosomes, thewheat and barley chromosomes, and sorghum and mil-let chromosomes can all be recreated using the sameset of rice segments.

Sites of Chromosome breakage and fusion

The above analysis suggests that the putative ances-tral chromosome can be cleaved to create the basicchromosome structures found in the major cereals oftoday. What however are the sites of breakage andfusion? The recent mapping of rice centromeres [23]provides an indication. Locating the rice, wheat andmaize centromere sites on the ancestral chromosomereveals that the borders of the linkage segments aredefined by centromeric and telomeric sites [24]. Stud-ies have reported that telomeric heterochromatin inmaize, rye, wheat and Bromus can under certain con-ditions function as neocentromeres (reviewed in 25).It is possible, therefore, that the borders of linkagesegments are in fact defined by centromeric-like sites[24].

Recently, a family of conserved sequences locatedat the centromeres of all rice, maize, wheat, barleyand rye chromosomes has been identified [26]. Cer-tain members of this family also detect the telomer-

ic heterochromatin [26]. The implication is that thesame family of sequences is present at the borders ofthe linkage segments and the ancestral chromosomeresembles a holocentric chromosome. Cleavage of theancestral chromosome to create the chromosome struc-tures found in the major cereals is reminiscent of thecleavage of Luzula chromosomes into smaller viablechromosomes. It will be interesting to ascertain wheth-er sequences related to the family of cereal centromeresequences are also found in the Luzula genome andwhether these sequences are the breakage sites to createthe smaller chromosomes. If kinetochores are mountedincorrectly on the opposite centromeric side to the poleto which the microtubules are going to pull the chro-mosome, the segregation of chromosomes could resultin breakage of the chromosome at the centromere as inwheat monosomics.

Centromeres and pairing

Centromeres are required for chromosome segrega-tion. It can now be postulated that they also havea role in chromosome evolution as sites of break-age and rearrangements. Modern cereal chromosomesare monocentromeric, possessing a single functionalcentromeric site, but may also possess ancestral centro-meric sites. Ancestral centromeric sites could maintainthe potential to attach microtubules which would allowthem to orientate or make initial contact with homo-logues during the early stages of meiosis. Is thereexperimental evidence that cereal centromeres mayalso have a role in chromosome pairing? Meiotic pair-ing of two barley homologues in a wheat genetic stockhas been studied during meiosis [27]. Confocal micro-scopy 3-D reconstructions of anther meiocytes revealthat pre-meiotic interphase centromeres are already‘paired’ either in non-homologousor homologous con-figurations at pre-meiotic interphase [27].The chromo-somes with the centromeres still in pairs align and theninteractions occur along their length as meiosis pro-ceeds. Finally the telomeres cluster in a bouquet andsynapsis occurs. Even at these later stages when thechromosomes have decondensed there are still bridgesbetween the homologues. It is possible that the recog-nition at these pre-meiotic stages is protein-based incontrast to the stages of leptotene onwards which arebased on DNA recognition between homologues. Thestudies of Holm [28, 29, 30] using ultrastructural ana-lysis of spread silver stained meiotic nuclei of wheatat leptotene onwards indicate that DNA interactions at

pl3891si.tex; 16/07/1997; 16:49; v.7; p.4

21

Figure 3. The evolution of the ancestor progenitor genome to modern cereal genomes based on linkage blocks. The structure of the genome ofthe ancestral progenitor is shown. The chromosomes of rice, maize and wheat are shown as linkage blocks.

leptotene involve a number of chromosomes which areresolved to bivalents at late zygotene.

In wheat, a series of loci have been identi-fied (primarily Ph1, Ph2, and a Ph1 suppressor)which either suppress or promote homoeologous pair-

ing between the three ancestral genomes of wheat(reviewed by Sears (31)). It is likely that these genesare involved in stabilizing polyploid cereals. A recentstudy indicates that homoeologous pairing is associ-ated with structural changes at centromeric sites in nine

pl3891si.tex; 16/07/1997; 16:49; v.7; p.5

22

different pairing conditions [32]. Clearly such struc-tural changes could also effect other sites involved inpairing such as the interstitial sites. In fact some ofhomoeologous pairing genes may have evolved froma requirement to suppress potential centromeric sites.The interstitial pairing sites on homologues have to bebrought into close proximity in order to interact. Thisimplies microtubule involvement and the movement ofcomplementary pairing sites on both homologues. Theuse of different pairing sites could provide a mech-anism for discriminating chromosomes. Studies haveshown that spindle inhibitors effect chromosome pair-ing [33]. It will be interesting to determine whetherthere is a correlation between ancestral centromericsites and interstitial pairing sites.

Nevertheless, it is clear that the pairing of homo-logues is determined at an early stage of pre-meioticinterphase when centromeres are in pairs and the chro-mosomes start to align. However several studies haveindicated that the Ph1 locus also effects recombin-ation between homologous and homoeologous seg-ments (34, 35). This implies that the Ph1 locus com-prises two genes, the first (as in the case of Ph2 and thePh1 suppressor) effecting centromeric structure andhence chromosome alignments and a second able todistinguish mismatches of sequences in heteroduplexesformed between homologous and homoeologous chro-mosomes. An obvious candidate for the later case isone of the mut S genes [36].

Is rice a good model genome for cereals?

The rice genome should be considered a model gen-ome because of its small size, its low level of repet-itive sequences (some of which are conserved acrossthe Gramineae (reviewed in 37)), the genomic toolsavailable (YACs, BACs and markers) and the geneticalinformation described in a number of papers in thisEdition. Analysis of the rice genome has allowed us toreveal the basic evolutionary units of the cereal gen-omes. Within the major cereals, it possesses the highestnumber of differentiated chromosomes and hence func-tional centromeric sites. These centromeric sites havepermitted us to delimit the major rearrangements inthe cereal genomes (which are indicated by Devos andGale in this Edition). The extension of comparativemapping of rice with species outside the Gramineae,perhaps with rushes or sedges, might reveal the totalnumber of potential centromere sites in the rice gen-ome and hence the total number of sites available for

major rearrangements and for use in the control ofchromosome pairing.

References

1. Nordenskiold H: Tetrad analysis and course of meiosis in threehybrids of Luzula campestris. Hereditas 47: 203–238 (1961).

2. Wolf KH, Gouy M, Yang Y-W, Sharp PM, Li WH: Date ofthe monocot-dicot divergence estimated from chloroplast DNAsequence data. Proc Natl Acad Sci USA 86: 6201–6205 (1989).

3. Martin W, Gierl A, Saedler H: Molecular evidence for pre-cretaceous angiosperms origins. Nature 339: 46–48 (1989).

4. Devos KM, Atkinson MD, Chinoy CN, Francis HA, HarcourtRL, Koebner RMD, Liu CJ, Masojc P, Xie DX, Gale MD:Chromosomal rearrangements in the rye genome relative tothat of wheat. Theor Appl Genet 85: 673–680 (1993).

5. Devos KM, Millan T, Gale M: Comparative RFLP maps ofhomoeologous group 2 chromosomes of wheat, rye and barley.Theor Appl Genet 85: 784–792 (1993).

6. Whitkus R, Doebley J, Lee M: Comparative genome mappingof sorghum and maize. Genetics 132: 1119–1130 (1992).

7. Melake Berhan A, Hulbert SH, Butler LG, Bennetzen JL:Structure and evolution of the genomes of Sorghum bicolorand Zea mays. Theor Appl Genet 86: 598–604 (1993).

8. Periera MG, Lee M, Bramel-Cox P, Woodman N, Doebley J,Whitkus R: Construction of an RFLP map in sorghum andcomparative mapping in maize. Genome 37: 236–243 (1994).

9. Chittenden LM, Schertz KF, Lin Y-R, Wing RA, Paterson AH:A detailed RFLP map of Sorghum biocolor x S. Propinquumsuggests ancestral duplication of the sorghum chromosomesor chromosomal segments. Theor Appl Genet 87: 925–933(1994).

10. Grivet L, D’Hont A, Dufour P, Hamon P, Rogues D, GlaszmannJC: Comparative genome mapping of sugarcane with otherspecies with the Andropogoneaetribe. Heredity 73: 500–508(1994).

11. Grivet L: Marquage moleculaire chez la canne a sucre (Sac-charum spp.); decomposition d’une structure genetique com-plexe et application a l’amelioration varietale. PhD Thesis,University de Paris XI Orsay (1995).

12. Ahn S, Tanksley SD: Comparative linkage maps of the riceand maize genomes. Proc Natl Acad Sci USA 90: 7980–7984(1993).

13. Ahn S, Anderson JA, Sorrells ME, Tanksley SD: Homoeolog-ous relationships of rice, wheat and maize chromosomes. MolGen Genet 241: 483–490 (1993).

14. Kurata N, Moore G, Nagamura Y, Foote T, Yano M, Minobe Y,Gale M: Conservation of genome structure between rice andwheat. Bio/Technology 12: 276–278 (1994).

15. Devos K, Chao S, Li QY, Simonetti MC, Gale MD: Relation-ship between chromosome 9 of maize and wheat homoeolog-ous group 7 chromosomes. Genetics 138: 1287–1292 (1994).

16. Van Deynze AE, Dubcovsky J, Gill KS, Nelson JC, SorrellsME, Dvorak J, Gill BS, Lagudah ES, McCouch SR, Appels R:Molecular-genetic maps for group 1 chromosomes of Triticeaespecies and their relation to chromosomes in rice and oat.Genome 38: 45–59 (1995).

17. Kilian A, Kudma DA, Kleinhofs A, Yano M, Kurata N, Stef-fenson B, Sasaki T: Rice-barley synteny and its application tosaturation mapping of barley Rpg1 region. Nucleic Acids Res23: 2729–2733 (1995).

pl3891si.tex; 16/07/1997; 16:49; v.7; p.6

23

18. Dunford RP, Kurata N, Laurie DA, Money TA, Minobe Y,Moore G: Conservation of fine-scale DNA marker order inthe genome of rice and the Triticeae. Nucleic Acids Res 23:2724–2728 (1995).

19. Foote T, Roberts M, Kurata N, Sasaki T, Moore G: Detailedcomparative mapping of cereal chromosome regions corres-ponding to the Ph1 locus in wheat. Submitted.

20. Chen M, SanMiguel P, De Oliveira AC, Woo S-S, Zhang Het al.: Microcolinearity in sh2-homologous regions of maize,rice and sorghum genomes. Proc Natl Acad Sci (1997) in press.

21. Moore G, Devos KM, Wang Z, Gale MD: Cereal GenomeEvolution, Grasses, line up and form a circle. Current Biology5: 737–739 (1995).

22. Moore G, Foote T, Helentjaris T, Devos K, Kurata N, Gale M:Was there a single ancestral cereal chromosome? Trends Genet11: 81–82 (1995).

23. Singh K, Ishii T, Parco A, Huang N, Brar DS, Khush GS:Centromere mapping and orientiation of the molecular linkagemap of rice. Proc Natl Acad Sci USA 93: 6163–6168 (1996).

24. Moore G, Roberts M, Aragon-Alcaide L, Foote T: Centromericsites and cereal chromosome evolution. Chromosoma in press(1997).

25. Lima-de-Faria A: Molecular evolution and organisation ofchromosomes. Amsterdam Elsevier (1983).

26. Aragon-Alcaide L, Miller TE, Schwarzacher T, Reader SM,Moore G: A Cereal Centomeric sequence. Chromosoma 105:261–268 (1996).

27. Aragon-Alcaide L, Reader SM, Shaw PJ, Beven AF, Miller TE,Moore G: Dynamics of homologous chromosome associationduring pre-meiotic interphase. Submitted.

28. Holm PB: Chromosome pairing and chiasma formation in allo-hexploid wheat, Triticum aestivum analysed by spreading ofmeiotic nuclei. Carlsberg Res Commun 51: 239–294 (1986).

29. Holm PB: Chromosome pairing and synaptonemal complexformation in Hexaploid wheat, monoisosomic and disosomicfor the long arm of chromosome 5B. Carlsberg Res Commun53: 111–133 (1986).

30. Holm PB: Chromosome pairing and synaptonemal complexformation in hexaploid wheat nullisomic for chromosome 5B.Carlsberg Res Commun 53: 91–110 (1986).

31. Sears ER: Genetic control of chromosome pairing in wheat.Ann Rev Genet 10: 31–51 (1976).

32. Aragon-Alcaide L, Reader SM, Miller TE, Moore G:Centromere behaviour in wheat with high and low homoeolog-ous chromosome pairing. Chromosoma in press (1997).

33. Dover GA, Riley R: The effect of spindle inhibitors appliedbefore meiosis on meiotic chromosome pairing. J Cell Sci 12:143–161 (1973).

34. Dubcousky J, Luo MC, Dvorak J: Differentiation betweenhomoeologous chromosomes 1A of wheat and A1 (M) of Trit-icum monococcum and its recognition by thw wheat Ph1 locus.Proc Natl Acad Sci USA 92: 6645–5547 (1995).

35. Luo MC, Dubcousky J, Dvorak J: Recognition of homeologyby wheat Ph1 locus. Genetics 144: 1195–1203 (1996).

36. Cox EC: Recombination, mutation and the origin of species.BioEssays 17: 747–749 (1995).

37. Moore G: Cereal genome evolution: pastoral pursuits with legogenomes. Curr Opin Genet Dev 5: 717–724 (1995).

pl3891si.tex; 16/07/1997; 16:49; v.7; p.7