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New Phytologist on plant evolution Historically, articles in New Phytologist have focused primarily on three areas of plant biology: (1) physiological and developmental processes, (2) how plants cope with environmental stress, and (3) the genetics and physiology of interactions between plants and their symbionts and pathogens. Whilst the journal has also always published high-quality evolutionary studies, in 2003 the Board of Editors made the strategic decision to work toward establishing and developing plant evolutionary biology as a fourth area of focus for the journal. The launch of the Evolution section was marked by the 11th New Phytologist Symposium on ‘Plant speciation’ and subsequent publication of associated papers in a special issue (Vol. 161, No. 1; Rieseberg & Wendel, 2004). Since that time, New Phytologist has continued to foster plant evolutionary research by publishing Tansley reviews (Piazza et al., 2005; Bronstein et al., 2006) and several features on ‘Plant evolutionary ecology’ (Vol. 165, No. 1; Rausher, 2005), ‘Plant evolutionary genomics’ (Vol. 168, no. 1; Mauricio, 2005), ‘Pollination mutualisms in Caryophyllaceae’ (Vol. 169, no. 4; Kephart, 2006) and ‘Heterostyly’ (Vol. 171, no. 3; Mast & Conti, 2006). Response to this initiative has been promising. Over the past year, New Phytologist has received and published many outstanding articles addressing important conceptual issues in plant evolutionary biology. The following sections describe some of these contributions which cover several important areas of plant evolution. New © The Phytologist Authors (2007). (2007) doi Journal : 10.1111/j.1469-8137.2007.00@@@.x compilation © New Phytologist (2007)

Evolution of mating systems Compared with animals, plants exhibit an amazing variety of mating systems: hermapthroditism, monoecy, dioecy, gynodioecy, andromonoecy, etc. Superimposed on this diversity is (1) variability both among and within species regarding whether individuals tend to be primarily selfing or outcrossing, and (2) variation in the morphological and phenological characters that are used to guarantee a particular mating system. It is thus not surprising that a major theme of plant evolutionary biology is elucidating the evolutionary mechanisms responsible for this tremendous diversity. This theme is well represented among recent articles in New Phytologist. Several articles have contributed insights into the evolution and breakdown of self-incompatibility. Stebbins (1974) originally suggested that evolutionary loss of selfincompatibility may be an evolutionary dead end. This

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hypothesis makes intuitive sense because breakdown of incompatibility is often achieved by knockout mutations in one or more genes associated with the self-incompatibility system. The hypothesis has received some empirical support (Takebayashi & Morrell, 2001; Igic et al., 2006), indicating that transitions to self-compatibility are more common than reverse transitions. In a recent article in New Phytologist, however, Ferrer & Good-Avila (2007) found that transitions from self-compatibility to self-incompatibility have been common in the Asteraceae. Their results suggest that self-incompatibility may evolve de novo more frequently than is currently believed. One common explanation for the breakdown of selfincompatibility is that it provides reproductive assurance in species that commonly occur in low-density populations (Stebbins, 1974). Guggisberg et al. (2006) provided evidence in support of Stebbins’ hypothesis by showing that, in Primula, breakdown of distyly is correlated with polyploidy. Polyploid lineages are largely recolonizing populations that occupy previously glaciated areas in North and South America and Europe, situations in which the necessity for reproductive assurance is easily envisioned. Stebbins’ explanation for loss of self-incompatibility also suggests that other mechanisms that confer reproductive assurance may prevent the evolution of self-compatibility. Vallejo-Marín & O’Brien (2007) provide in our pages the first test of this hypothesis and show that, in Solanum, the breakdown of self-incompatibility tends to occur only in species that are not clonal. They argue that clonality provides a form of reproductive assurance that renders self-compatibility unnecessary. A second important issue in understanding the evolution of plant mating systems is accounting for the evolution and maintenance of multimorphic systems such as dioecy and heterostyly. For the evolution of heterostyly, two models have been proposed. Charlesworth & Charlesworth (1979) proposed that avoidance of inbreeding depression is the primary factor that selects first for an incompatibility system, which is then followed by the evolution of morphological heterostyly (the reciprocal placement of anthers and stigmas in different positions in different individuals) to reduce gamete wastage. By contrast, Lloyd & Webb (1992a,b) proposed that the first step in the evolution of heterostyly is the development of a morphological polymorphism that increases pollination efficiency, which is then reinforced by the evolution of self-incompatibility. Pérez-Barrales et al. (2006) attempted to distinguish between these two models by reconstructing the evolution of heterostyly in Narcissus. They found that self-incompatibility coupled with style monomorphy or dimorphy is ancestral in this group, with

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true heterostyly being derived, supporting the Charlesworth and Charlesworth model. Moreover, they also found that shifts from stylar dimorphism to true heterostyly were associated with transitions from fly/butterfly pollination to bee pollination, providing evidence that selection for pollinator efficiency secondarily drives the evolution of heterostylous morphology. The study by Armbruster et al. (2006) provided additional support for the idea that the evolution of heterostyly morphology reflects selection for increased efficiency of pollen transfer. Gynodioecy is a mating system involving the stable persistence of both hermaphrodite and female individuals. One of the commonest explanations for maintenance of gynodioecy is high levels of inbreeding depression, which reduces the fitness of hermaphrodites relative to females (Charlesworth, 1999). Glaettli & Goudet (2006) provided support for this explanation by showing that inbreeding depression for both male and female fertility is substantial in Silene vulgaris. In addition, they demonstrated that progeny produced by selfing contain an excess of females, which also contributes to maintenance of this mating system.

Plant–Enemy coevolution Plants can defend themselves from natural enemies in two different ways: they can evolve to become resistant to enemy attack, or they may evolve to become tolerant (Mauricio et al., 1997). It has been argued that these two types of defence have different implications for plant–enemy coevolution: the evolution of resistance facilitates continued coevolution, while the evolution of tolerance does not because it is not expected to reduce enemy fitness (Stinchcombe, 2002). In one of the first empirical tests of the latter hypothesis, Garrido Espinosa & Fornoni (2006) examined the effects of genetic variation in Datura stramonium for tolerance to its specialized beetle herbivore, Lema trilineata, on beetle growth and survival. Their failure to find any influence of tolerance level on these beetle fitness components supports the hypothesis that the evolution of tolerance often breaks the coevolutionary cycle. In agricultural systems, the dominant paradigm for plant resistance to natural enemies is that resistance frequently involves genes of large effect in both plant and enemy that act in a gene-for-gene manner (Crute & Pink, 1996; Agrios, 1997). While many authors have suggested that this provides a potential paradigm for natural plant–enemy coevolution, very little is currently known about the nature of genetic variation for resistance or virulence in natural plant–enemy systems. Two recent papers in New Phytologist help to fill this lacuna in our knowledge. Kover & Cheverud (2007) performed a standard quantitative trait locus (QTL) analysis of resistance of Arabidopsis thaliana to one of its common bacterial pathogens, Pseudomonas syringae. They found one resistance QTL of major effect accounting for 77% of the

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variation in resistance between two A. thaliana accessions, plus a couple of minor-effect QTLs. Similarly, Kniskern & Rausher (2006) demonstrated that variation in Ipomoea purpurea to the rust pathogen Coleosporium ipomoeae is controlled by segregation of a single Mendelian factor. These two studies thus support the idea that gene-for-gene coevolution may be an appropriate paradigm for coevolution in nature.

Evolutionary genomics The DNA content of haploid genomes varies over several orders of magnitude. Evolutionary biologists are just beginning to come to grips with the causes and consequences of this variation. One possible consequence for which there is some evidence is that genome size variation influences the magnitude of quantitative traits (e.g. Meagher & Vassiliadis, 2005). At present, however, little is understood about how extensive such effects are. However, in a recent New Phytologist report, Beaulieu et al. (2007) demonstrated that there is a moderately strong correlation between genome size and seed mass across 1222 species of gymnosperms and angiosperms. Moreover, they found that very small seeds are never found in species with very large genomes. Because much variation in genome size is thought to be attributable to ‘junk’ DNA such as retrotransposons (Meagher & Vassiliadis, 2005), there is no obvious functional reason to have anticipated such a correlation. Nevertheless, its existence suggests that the evolution of seed mass may be constrained by genome size. These results indicate that we need to entertain seriously the possibility that genome-wide characteristics influence the evolution of individual characters. A central focus of evolutionary genetics and genomics is to understand the types of genetic change that underlie adaptation. One model for adaptive genetic change is the domestication of major crop species. In this context, Li et al. (2006) reported, in a QTL analysis of domestication traits in rice (Oryza sativa), that QTLs of large effect are associated with a number of characters that contribute to more efficient planting and harvesting. While care must be taken in extrapolation from human-imposed selection to evolution in nature, these results support the notion that, under reasonably strong selection, evolutionary change will be accomplished largely by mutations of large effect. Looking ahead, we aim to continue to strengthen the Evolution section which encourages submissions on all aspects of plant evolution, from studies in an ecological context to molecular analyses. We are therefore providing open access to the papers highlighted in this Editorial so that you can enjoy some of the evolutionary studies published in New Phytologist over the past year, and invite you to build on them further. Mark D. Rausher Evolution Section Editor

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References Agrios GN. 1997. Plant pathology. London, UK: Academic Press. Armbruster WS, Pérez-Barrales R, Arroyo J, Edwards ME, Vargas P. 2006. Three-dimensional reciprocity of floral morphs in wild flax (Linum suffruticosum): a new twist on heterostyly. New Phytologist 171: 581–590. Beaulieu JM, Moles AT, Leitch IJ, Bennett MD, Dickie JB, Knight CA. 2007. Correlated evolution of genome size and seed mass. New Phytologist, 173: 422–437. Bronstein JL, Alarcón R, Geber M. 2006. The evolution of plant-insect mutualisms. New Phytologist 172: 412–428. Charlesworth D. 1999. Theories of the evolution of dioecy. In: Gerber MA, Dawson TE, Delph LF, eds. Gender and sexual dimorphism in flowering plants. Berlin, Germany: Springer-Verlag, 33 – 60. Charlesworth D, Charlesworth B. 1979. A model for the evolution of distyly. American Naturalist 114: 467– 498. Crute IR, Pink DAC. 1996. Genetics and utilization of pathogen resistance in plants. Plant Cell 8: 1747–1755. Espinosa EG, Fornoni J. 2006. Host tolerance does not impose selection on natural enemies. New Phytologist 170: 609–614. Ferrer MM, Good-Avila SV. 2007. Macrophylogenetic analyses of the gain and loss of self-incompatibility in the Asteraceae. New Phytologist 173: 401–414. Glaettli M, Goudet J. 2006. Inbreeding effects on progeny sex ratio and gender variation in the gynodioecious Silene vulgaris (Caryophyllaceae). New Phytologist 172: 763 –773. Guggisberg A, Mansion G, Kelso S, Conti E. 2006. Evolution of biogeographic patterns, ploidy levels, and breeding systems in a diploidpolyploid species complex of Primula. New Phytologist 171: 617–632. Igic B, Bohs L, Kohn JR. 2006. Ancient polymorphism reveals unidirectional breeding system shifts. Proceedings of the National Academy of Sciences, USA 103: 1359 –1363. Kephart S. 2006. Pollination mutualisms in caryophyllaceae. New Phytologist 169: 637– 640. Kniskern JM, Rausher MD. 2006. Major-gene resistance to the rust pathogen Coleosporium ipomoeae is common in natural populations of Ipomoea purpurea. New Phytologist 171: 137–144. Kover PX, Cheverud P. 2007. The genetic basis of quantitative variation in susceptibility of Arabidopsis thaliana to Pseudomonas syringae

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(Pst DC3000): evidence for a new genetic factor of large effect. New Phytologist 174: 172–181. Li C, Zhou A, Sang T. 2006. Genetic analysis of rice domestication syndrome with the wild annual species, Oryza nivara. New Phytologist 170: 185–194. Lloyd DG, Webb CJ. 1992a. The evolution of heterostyly. In: Barrett SCH, ed. Evolution and function of heterostyly. Berlin, Germany: Springer-Verlag, 151–178. Lloyd DG, Webb CJ. 1992b. The selection of heterostyly. In: Barrett SCH, ed. Evolution and function of heterostyly. Berlin, Germany: Springer-Verlag, 179–207. Mast AR, Conti E. 2006. The primrose path to heterostyly. New Phytologist 171: 439–442. Mauricio R,. 2005. The ‘bricolage’ of the genome elucidated through evolutionary genomics. New Phytologist 168: 1–4. Mauricio R, Rausher MD, Burdick DS. 1997. Variation in the defense strategies of plants: are resistance and tolerance mutually exclusive? Ecology 78: 1301–1311. Meagher TR, Vassiliadis C. 2005. Phenotypic impacts of repetitive DNA in flowering plants. New Phytologist 168: 71–80. Pérez-Barrales R, Vargas P, Arroyo J. 2006. New evidence for the Darwinian hypothesis of heterostyly: breeding systems and pollinators in Narcissus sect. Apodanthi. New Phytologist 171: 553–567. Piazza P, Jasinski S, Tsiantis M. 2005. Evolution of leaf developmental mechanisms. New Phytologist 167: 693–710. Rausher MD. 2005. Plant evolutionary ecology. New Phytologist 165: 2–5. Rieseberg L, Wendel J. 2004. Plant speciation – rise of the poor cousins. New Phytologist 161: 3–8. Stebbins GL. 1974. Flowering plants. Evolution above the species level. Cambridge, MA, USA: Belknap Press of Harvard University Press. Stinchcombe JR. 2002. Can tolerance traits impose selection on herbivores? Evolutionary Ecology 15: 595–602. Takebayashi N, Morrell PL. 2001. Is self-fertilization an evolutionary dead end? Revisiting an old hypothesis with genetic theories and a macroevolutionary approach. American Journal of Botany 88: 1143–1150. Vallejo-Marín M, O’Brien HE. 2007. Correlated evolution of self-incompatibility and clonal reproduction in Solanum (Solanaceae). New Phytologist 173: 415–421. Key words: mating systems, molecular evolution, phylogenetics, plant–enemy coevolution, population genetics, speciation.

Commentary What are the mechanisms and specificity of mycorrhization helper bacteria? It has long been known that the presence of free-living microbial communities stimulates mycorrhiza formation, and that this effect can also be induced by specific strains of

bacteria isolated from mycorrhizal roots, which have been termed mycorrhization helper bacteria (MHB) (Garbaye, 1994). Although many MHB strains have been isolated and characterized, the mechanisms underlying MHB activity remain to be elucidated. Whereas MHB have been shown to have no particular preference for plant species, they have varying specificities for mycorrhizal fungus strains (Aspray et al., 2006a), although their impacts on growth and colonization of roots by saprotrophic and pathogenic fungi has received little attention. The paper by Lehr et al. (pp. 892–903), in this issue of New Phytologist, provides new evidence for the mechanisms by which one such MHB may

© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org

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operate and also demonstrates that a MHB can promote colonization of roots by the pathogen Heterobasidion abietinum.

‘... , the capacity of bacterial strains to enhance fungus growth in vitro is not a useful indicator of MHB activity, suggesting that effects on the plant are important in determining MHB activity.’

Mycorrhization helper bacteria The mycorrhizosphere is recognized as the soil compartment inhabited and influenced by mycorrhizal fungi (Bending et al., 2006). For most plants this region represents the interface at which plants affect, and are affected by, the soil and its inhabitants. The rapid turnover of mycorrhizal fungus hyphae, the presence of mycorrhizal fungus exudates and mycorrhizal fungus-induced changes to plant exudation may all result in changes to the structure and activity of free-living microbial communities relative to the rhizosphere and bulk soil. Although these interactions can potentially impact the mycorrhizal fungus itself, the plant and soil nutrient cycling processes, knowledge of these impacts and their consequences is poorly understood. MHB are the best studied inhabitants of the mycorrhizosphere. Although most studies of MHB have been conducted in ectomycorrhizal systems, MHB have also been shown to occur in arbuscular symbioses (Duponnois & Plenchette, 2003). In ectomycorrhizal systems, MHB have been isolated from the mycorrhizospheres of many tree–fungus symbioses in nursery, plantation and seminatural situations. MHB strains belong to many different bacterial genera, and strains with the potential to act as MHB are readily isolated from the mycorrhizosphere and appear to have a ubiquitous distribution (Poole et al., 2001). Garbaye (1994) proposed five main mechanisms by which MHB could promote mycorrhiza formation, although to date there has been little robust evidence for the relative importance of any of them. Most evidence for MHB mechanisms has been gathered from in vitro studies of the interactions between mycorrhizal fungi and bacteria or their metabolites in the absence of the host plant, although there is evidence that some MHB require close proximity or contact with the plant to exert MHB effects (Aspray et al., 2006b).

Multiple MHB mechanisms? In their paper, Lehr et al. investigated interactions between Streptomyces sp. AcH505, a MHB of Amanita muscaria and

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Suillus bovinus growing with Norway spruce (Riedlinger et al., 2006), and strains of the root pathogen, Heterobasidion spp. Previous work in the absence of a host plant had demonstrated that Streptomyces sp. AcH505 produced a metabolite, auxofuran, which specifically stimulated growth of A. muscaria but inhibited growth of pathogenic fungi. In the current study, Lehr et al. (2007) found that Streptomyces sp. AcH505 suppressed the plant defence response. Out of 12 Heterobasidion strains tested, the growth of 11 were sensitive to metabolites produced by the MHB and, in the presence of the MHB, could not colonize Norway spruce roots. However, the growth of one Heterobasidion strain appeared not to be affected by the MHB strain, the presence of which stimulated colonization of the root by the pathogen. This increased pathogenicity presumably occurred because of the associated suppression of the host defence response by the MHB. A number of studies have suggested that plant roots produce metabolites which promote growth of mycorrhizal fungi towards the root. These compounds can act by increasing hyphal extension rates and by promoting hyphal branching, thereby directing growth towards and around the root (Akiyama et al., 2005). Furthermore, colonization of roots by compatible mycorrhizal fungi is also known to be associated with reduced or altered expression of host peroxidase (Mensen et al., 1998; Tarkka et al., 2001), which has been interpreted as indicating suppression of the host defence response by the mycorrhizal fungus and induction of changes in root cell wall structure, which facilitate growth of fungus through host tissue and ectomycorrhiza development. The findings of the current study therefore suggest that Streptomyces sp. AcH505 acts as an MHB by expressing a combination of mechanisms which add to existing signalling mechanisms operating between the mycorrhizal fungus and plant symbionts. The metabolite, auxofuran, appears to stimulate presymbiotic growth of the fungus, adding to the action of root-derived metabolites which direct growth of mycorrhizal hyphae towards the root. In addition, the MHB suppresses the host defence response, thereby adding to existing signalling pathways that occur between the mycorrhizal fungus and its host during mycorrhiza formation. Although the factors responsible for suppression of the host defence response by the MHB could not be determined, involvement of the metabolite auxofuran was ruled out, demonstrating that a combination of factors allow this strain to operate as an MHB. However, final proof of the relative importance of these mechanisms in contributing to MHB activity would need to be conducted in systems containing the ectomycorrhizal fungus.

How specific are mycorrhiza helper bacteria? Whether the MHB mechanisms proposed for Streptomyces sp. AcH505 operate in other MHB systems remains to be elucidated. However, the capacity of bacterial strains to enhance fungus growth in vitro is not a useful indicator of


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MHB activity (Garbaye, 1994), suggesting that effects on the plant are important in determining MHB activity. Furthermore, some MHB appear to have specificity for certain strains of mycorrhizal fungus, suggesting that specific metabolite interactions of the type demonstrated for auxofuran produced by Streptomyces sp. AcH505 may operate more widely. MHB show a range of specificities, with some apparently specific to closely related strains of ectomycorrhizal fungus (Duponnois et al., 1993) and others capable of stimulating mycorrhiza formation by diverse ectomycorrhizal fungi (Aspray et al., 2006a), meanwhile, in one case, an MHB isolated from ectomycorrhizal systems was shown to promote formation of the arbuscular symbiosis (Duponnois & Plenchette, 2003). Inoculation of MHB with low specificity into a nonsterile environment containing complex ectomycorrhizal communities is known to have unpredictable consequences for the establishment of ectomycorrhizal fungus species on roots (Aspray et al., 2006a). In the publication of Lehr et al. (2007), this finding is extended, with a strain identified as an MHB also promoting root colonization by a pathogenic fungus. However, further work would be required to determine the relative extent to which root colonization by Heterobasidion spp. and ectomycorrhizal fungi are promoted by the MHB, and whether stimulation of the pathogen would have any consequences for disease development if ectomycorrhizal fungi were present. Nonetheless, the results have implications for the development of MHB as inocula, and further work is required to determine whether MHB could promote root colonization and disease development by other root pathogenic fungi.

Perspectives Although bacteria with the potential to act as MHB appear to occur everywhere, the activities of most MHB have been demonstrated in laboratory or glasshouse situations, and the extent to which they act in undisturbed mycorrhizospheres at natural cell densities and localization patterns within the soil remains to be demonstrated. However, the study of MHB provides insight into the way in which mixed natural microbial communities stimulate mycorrhiza formation, and MHB themselves have potential for biotechnological exploitation (Brule et al., 2001). Furthermore, bacteria inhabit many niches in close proximity to fungi, and interactions of the type shown by MHB could have a widespread distribution in nature (de Boer et al., 2005). For example, some bacterial strains can enhance the pathogenicity of foliar pathogens (Dewey et al., 1999). Genomics tools provide a promising avenue to unravel interactions within the mycorrhizosphere (Schrey et al., 2005). Identification of the mechanisms by which fungi and bacteria interact, and thereby factors controlling specificity, is clearly an important starting point for understanding not only the role of MHB in natural

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systems, but for understanding and exploiting the numerous fungus–bacteria interactions which occur in nature. Gary D. Bending Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK (tel +44(0) 2476575057; fax +44(0) 2476574500; email [email protected])

References Akiyama K, Matsuzaki K, Hayashi H. 2005. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435: 824–827. Aspray TJ, Frey-Klett P, Jones JE, Whipps JM, Garbaye J, Bending GD. 2006a. Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation. Mycorrhiza 16: 533–541. Aspray TJ, Jones EE, Whipps JM, Bending GD. 2006b. Importance of mycorrhization helper bacteria cell density and metabolite localization for the Pinus sylvestris-Lactarius rufus symbiosis. FEMS Microbiology Ecology 56: 25–33. Bending GD, Aspray TJ, Whipps JM. 2006. Significance of microbial interactions in the mycorrhizosphere. Advances in Applied Microbiology 60: 97–132. de Boer W, Folman LB, Summerbell RC, Boddy L. 2005. Living in a fungal world: impact of fungi on soil bacterial niche development. FEMS Microbiology Reviews 29: 795–811. Brule C, Frey-Klett P, Pierrat JC, Courrier S, Gerard F, Lemoine MC, Rousselet JL, Sommer G, Garbaye J. 2001. Survival in the soil of the ectomycorrhizal fungus Laccaria bicolor and the effects of a mycorrhiza helper Pseudomonas fluorescens. Soil Biology and Biochemistry 33: 1683–1694. Dewey FM, Wong YL, Seery R, Hollins TW, Gurr SJ. 1999. Bacteria associated with Stagonospora (Septoria) nodorum increase pathogenicity of the fungus. New Phytologist 144: 489–497. Duponnois R, Plenchette C. 2003. A mycorrhiza helper bacterium enhances ectomycorrhizal and endomycorrhizal symbiosis of Australian Acacia species. Mycorrhiza 13: 85–91. Duponnois R, Garbaye J, Bouchard D, Churin JL. 1993. The fungus-specificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root douglas-fir planting stocks with Laccaria laccata. Plant and Soil 157: 257–262. Garbaye J. 1994. Helper bacteria – a new dimension to the mycorrhizal symbiosis. New Phytologist 128: 197–210. Lehr NA, Schrey SD, Bauer R, Hampp R, Tarkka MT. 2007. Suppression of plant defence response by a mycorrhiza helper bacterium. New Phytologist 174: 892–903. Mensen R, Hager A, Salzer P. 1998. Elicitor-induced changes of wall-bound and secreted peroxidase activities in suspension-cultured spruce (Picea abies) cells are attenuated by auxins. Physiologia Plantarum 102: 539–546. Poole EJ, Bending GD, Whipps JM, Read DJ. 2001. Bacteria associated with Pinus sylvestris-Lactarius rufus ectomycorrhizas and their effects on mycorrhiza formation in vitro. New Phytologist 151: 743–751. Riedlinger J, Schrey SD, Tarkka MT, Hampp R, Kapur M, Fiedler HP. 2006. Auxofuran, a novel metabolite that stimulates the growth of fly agaric, is produced by the mycorrhiza helper bacterium Streptomyces strain AcH 505. Applied and Environmental Microbiology 72: 3550–3557.


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Schrey SD, Schellhammer M, Ecke M, Hampp R, Tarkka MT. 2005. Mycorrhiza helper bacterium Streptomyces AcH 505 induces differential gene expression in the ectomycorrhizal fungus Amanita muscaria. New Phytologist 168: 205–216. Tarkka MT, Nyman TA, Kalkkinen N, Raudaskoski M. 2001. Scots pine

expresses short-root-specific peroxidases during development. European Journal of Biochemistry 268: 86–92. Key words: Heterobasidion, host defence, mechanisms, mycorrhization helper bacteria, specifity.

Meetings Forest genomics grows up and branches out The Forestry Workshop and other talks on forest trees: Plant and Animal Genome XV Conference, San Diego, CA, USA, January 2007 Forest trees have long been a tantalizing target for genetic and genomic studies. They are extremely important from both ecological and environmental standpoints, accounting for a large proportion of the biomass in terrestrial systems and providing some of the most valuable commodities in the world economy. However, trees are notoriously difficult to manipulate genetically because of their large size and long generation time, so progress has historically been slow compared with annual herbaceous plants. The genomics era offers new promise of accelerated rates of gene discovery for forest trees, and new insights into the molecular mechanisms underlying tree development, physiology and adaptation (Brunner et al., 2004). It would seem that forest genomics is entering a new era with new resources and approaches. As demonstrated at the January 2007 Plant and Animal Genome conference in San Diego (http://www.intl-pag.org/), this promise is rapidly being realized in a variety of tree taxa.

‘... the Populus genome may be more similar to ancestral angiosperm genomes than the annual angiosperms that have been sequenced to date’

The tree life style and genome evolution A number of developmental and physiological characteristics distinguish trees from annual plants, including the ability to achieve immense size by extensive development of secondary

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xylem (i.e. wood) and the ability to cope with highly variable biotic and abiotic stresses over a long life span. Studies are beginning to reveal how tree growth habit affects, and is affected by, genome evolution. Analysis of the Populus trichocarpa genome sequence shows a fascinating history of duplication, followed by putative selective retention of specific classes of genes that could be associated with traits advantageous to a long-lived woody perennial (Tuskan et al., 2006). Estimates of substitution rate during Populus evolution suggest that the perennial lifestyle, combined with extensive clonal replication in Populus, may slow the rate of molecular and chromosome-level change during Populus evolution. Thus, the Populus genome may be more similar to ancestral angiosperm genomes than the annual angiosperms that have been sequenced to date. Although the large size of conifer genomes (c. 75–180 times that of Arabidopsis) presents an obstacle to complete genome sequencing, work presented by Emanuele De Paoli (University of Udine, Italy) showed that new and important insights can be gained from sequencing of large genomic regions in gymnosperms. In particular, he looked at the types, distributions and local arrangements of transposable elements in Picea abies (Norway spruce). Increasing evidence supports the importance of intergenic regions, specifically transposable elements in these regions, in genome evolution (Morgante, 2006). Intergenic regions can vary considerably within a species and may have important regulatory roles. Whereas retrotransposon expansion in angiosperms is relatively recent and still evolving, the spruce genome appears more fixed, with estimates of transposon insertion dating long before the major angiosperm genome expansions. The slower genome evolution in poplar and the apparently reduced evolutionary flexibility of the spruce genome are likely to have important implications for adaptive evolution. Another question beginning to be addressed is how the tree life history reflects cooption and modification of genes and mechanisms during land plant evolution. For example, a poplar homolog of the Arabidopsis flowering time gene FT not only affects flowering in poplar but also controls bud set induced by the short days of fall (Bohlenius et al., 2006).


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Class I KNOX homeobox genes regulate the shoot apical meristem in annuals, and Andrew Groover (USDA Forest Service, CA, USA) showed that Populus homologs also regulate the vascular cambium and the differentiation and lignification of cells during wood formation. Noncoding small RNAs that regulate the expression of other genes potentially have a major role in the evolution of gene regulatory networks in trees. Although broadly conserved microRNAs (miRNAs) were initially identified in plants, accumulating evidence indicates that a large proportion of miRNAs are taxa-specific (Rajagopalan et al., 2006). New, faster sequencing technologies, most notably the massively parallel pyrosequencing method developed by 454 Life Sciences (Margulies et al., 2005; referred to as ‘454 sequencing’), has enabled cost-effective deep-sequencing of small RNAs. Ying-Hsuan Sun (North Carolina State University, NC, USA) reported results of 454 sequencing for miRNA discovery in poplar that has already yielded over 90 000 distinct small RNA sequences, including potentially species-specific miRNAs involved in wood formation. The long life spans of trees as well as rates of genome evolution can also affect the genes and pathways for defense responses to insects and pathogens. The critical need for research in this area is best exemplified by the effects of exotic pathogens such as Cryphonectria parasitica, which has virtually eliminated the American chestnut that once dominated eastern hardwood forests. John Carlson (Pennsylvania State University, PA, USA) described using 454 technology to generate American and blight-resistant Chinese chestnut ESTs, and plans to use 454 sequencing for single nucleotide polymorphism (SNP) detection to develop markers for blight resistance. This ground-breaking project will be a closely watched test case of the extent to which genomics-guided approaches can help to restore a native species.

From QTLs to adaptive polymorphisms One of the most successful approaches for identifying the genomic regions that are responsible for phenotypic variation has been quantitative trait locus (QTL) analysis. Such studies have been conducted since the early 1990s in forest trees, and have revealed valuable information about the genetic architecture of a wide variety of complex traits, ranging from well-defined traits such as leaf flavonoid chemistry (Morreel et al., 2006) or disease resistance (Jorge et al., 2005), to extremely complex traits such as growth (Wullschleger et al., 2005) and stress tolerance (Howe et al., 2003). However, since the early days of QTL analysis, there have been doubts about the ultimate utility of this approach in forest trees. QTL mapping in trees is limited to pedigrees in which high linkage disequilibrium (LD) is required for the identification of chromosomal regions containing genes influencing phenotypic traits (Strauss et al., 1992). However, this also means that a large number of genes are present in

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the identified intervals, making cloning of the actual gene influencing the phenotype a daunting task. In recent years, QTL analysis has been successfully combined with other tools from the forest genomics toolbox to narrow the search for candidate genes underlying complex traits. For example, as presented by Gail Taylor (University of Southampton, UK), the European Popyomics project has combined QTL analysis with microarray analyses of gene expression and candidate gene lists to identify genes potentially involved in drought tolerance (Street et al., 2006). Another interesting example of genomics-assisted QTL studies was presented by Gerald Tuskan (Oak Ridge National Laboratory, TN, USA). Populus is dioecious, with separate male and female individuals. Tuskan and colleagues have mapped a locus controlling gender to a portion of the genome with extensive haplotypic diversity, as revealed by the whole genome sequencing project. High-density genetic mapping in this region has demonstrated that recombination is suppressed across this region, which is a typical characteristic of an autosome that is in the process of becoming a heteromorphic sex chromosome (Liu et al., 2004). Amy Brunner (Virginia Tech, VA, USA) reported the use of whole genome microarray analyses across a diverse set of Populus tissues and developmental stages to identify genes that are differentially expressed in developing reproductive buds relative to young vegetative buds. Cross-referencing these genes with the candidate gene lists from the gender intervals has produced a list of 33 candidate genes that can now be functionally characterized for their involvement in gender determination. Association mapping approaches have been developed that take advantage of natural forest populations, which typically have very low LD. In contrast to QTL mapping, genetic markers that show significant association with a phenotypic trait are very close to, or in, the gene influencing the trait, but a larger number of markers must typically be surveyed. Association mapping is thus effective for detecting robust associations between candidate genes and phenotypes (Neale & Savolainen, 2004). The pioneering tree association studies have been conducted primarily in conifers, and major candidate gene association studies are ongoing in loblolly pine (Gonzalez-Martinez et al., 2006), radiata pine (Shannon Dillon, CSIRO, Canberra, Australia), and spruce, driven by Canada’s Arborea project (Pavy et al., 2007). SNP association studies are also becoming a major component of hardwood research programs. For example, the ambitious EVOLVTREE project, as described by Christophe Plomion (INRA, France), will extend work begun in the Popyomics project with Populus and the TREESNIPs project with oak and pine, to examine candidate gene polymorphisms in Populus, pine, and oak. An approach that is in some ways intermediate between QTL analyses and association studies in pure species is the use of hybrid zones to perform whole genome scans for


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associations with phenotypic traits that differ between the hybridizing species (Lexer et al., 2003). Stephen DiFazio (West Virginia University, WV, USA) presented results from a North American hybrid zone between Populus angustifolia and P. fremontii that has a moderate LD that should allow whole genome scans using a moderate number of markers. Meanwhile, populations of pure species adjacent to the hybrid zone have extremely low LD for closely linked loci, thus enabling finer association studies for candidate genes from genomic regions identified in the hybrid zone. Patterns of introgression across such hybrid zones also provide unique insights into the genetic forces maintaining differentiation of the species. For example, fragments that introgress more or less than expected under neutral models co-occur with QTL for leaf chemistry traits that have been demonstrated to have profound ecological effects in these hybrid zones (Whitham et al., 2006). Similar results have recently been reported for a European hybrid zone between P. tremula and P. alba (Lexer et al., 2007).

Conclusions Parallel work in a variety of different angiosperm and gymnosperm taxa is continuing to develop and refine genomic resources for forest trees, and this work is greatly facilitated by recent technological advances. These include the 454 Life Sciences system and emerging platforms from Solexa and ABI, as well as high-throughput SNP genotyping systems such as resequencing arrays and bead array systems. This work is building a strong foundation for comparative tree genomics that will ultimately reveal similarities and differences in the genes and polymorphisms underlying traits important for the tree growth habit, and thus allow adaptive molecular variation to be placed in the broader context of woody plant evolution. The importance of using a combination of approaches and recognizing the conservation of genetic pathways in plant evolution is already being demonstrated in poplar research, and will enable other angiosperm tree taxa to extensively leverage the poplar genome sequence and poplar functional genomics studies. Conifers present the opportunity to study developmental and adaptive traits in trees evolutionarily distant from angiosperms, including identification of ancient shared mechanisms, and those mechanisms that have independently evolved. Sequencing of a significant proportion of a conifer genome would be a substantial benefit not only to conifer research, but also for understanding of the broad patterns of plant evolution and development.

Acknowledgements Many thanks to Christophe Plomion, John MacKay, and Tom Richardson for organizing the Forestry Workshop, and to Chung-Jui Tsai for coorganizing the Populus community microarray workshop.

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Amy M. Brunner1* Stephen P. DiFazio2 and Andrew T. Groover3 1Department

of Forestry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA; 2Department of Biology, West Virginia University, Morgantown, WV 26506, USA; 3Institute of Forest Genetics, Pacific South-west Research Station, USDA Forest Service, Davis, CA 95616, USA (*Author for correspondence: tel +1540 231 3165; fax +1540 231 3698; email [email protected])

References Bohlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O. 2006. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312 : 1040–3. Brunner AM, Busov VB, Strauss SH. 2004. Poplar genome sequence: functional genomics in an ecologically dominant plant species. Trends in Plant Science 9: 49–56. 2005. Gonzalez-Martinez SC, Ersoz E, Brown GR, Wheeler NC, Neale DB. 2006. DNA sequence variation and selection of tag single-nucleotide polymorphisms at candidate genes for drought-stress response in Pinus taeda L. Genetics 172: 1915–1926. Howe GT, Aitken SN, Neale DB, Jermstad KD, Wheeler NC, Chen THH. 2003. From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. Canadian Journal of Botany – Revue Canadienne de Botanique 81: 1247–1266. Jorge V, Dowkiw A, Faivre-Rampant P, Bastien C. 2005. Genetic architecture of qualitative and quantitative Melampsora larici-populina leaf rust resistance in hybrid poplar: genetic mapping and QTL detection. New Phytologist 167: 113–127. Lexer C, Buerkle CA, Joseph JA, Heinze B, Fay MF. 2007. Admixture in European Populus hybrid zones makes feasible the mapping of loci that contribute to reproductive isolation and trait differences. Heredity 98: 74–84. Lexer C, Randell RA, Rieseberg LH. 2003. Experimental hybridization as a tool for studying selection in the wild. Ecology 84: 1688–1699. Liu ZY, Moore PH, Ma H, Ackerman CM, Ragiba MYuQY, Pearl HM, Kim MS, Charlton JW, Stiles JI, Zee FT, Paterson AH, Ming R. 2004. A primitive Y chromosome in papaya marks incipient sex chromosome evolution. Nature 427: 348–352. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Du Dewell SBL, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376–380. Morgante M. 2006. Plant genome organisation and diversity: the year of the junk! Current Opinion in Biotechnology 17: 168–173. Morreel K, Goeminne G, Storme V, Sterck L, Ralph J, Coppieters W, Breyne P, Steenackers M, Georges M, Messens E, Boerjan W. 2006. Genetical metabolomics of flavonoid biosynthesis in Populus: a case study. The Plant Journal 47: 224–237. Neale DB, Savolainen O. 2004. Association genetics of complex traits in conifers. Trends in Plant Science 9: 325–330. Pavy N, Johnson JJ, Crow JA, Paule C, Kunau T, MacKay J, Retzel EF. 2007. ForestTreeDB: a database dedicated to the mining of tree transcriptomes. Nucleic Acids Research 35: D888–D894. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP. 2006. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes and Development 20: 3407–3425.


Mettings Strauss S, Lande R, Namkoong G. 1992. Limitations of molecularmarker-aided selection in forest tree breeding. Canadian Journal of Forest Research 22: 1050–1061. Street NR, Skogstrom O, Tucker J, Rodriguez-Acosta M, Nilsson P, Jansson S, Taylor G. 2006. The genetics and genomics of the drought response in Populus. Plant Journal 48: 321–341. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP, Blaudez D, Boerjan W, Brun A, Brunner A, Busov V et al. 2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science. 313: 1596–1604. Whitham TG, Bailey JK, Schweitzer JA, Shuster SM, Bangert RK, LeRoy CJ, Lonsdorf EV, Allan GJ, DiFazio SP, Potts BM, Fischer DG, Gehring CA, Lindroth RL, Marks JC, Hart SC, Wimp GM, Wooley SC. 2006. A framework for community and ecosystem genetics: from genes to ecosystems. Nature Reviews Genetics 7: 510–523. Wullschleger S, Yin TM, DiFazio SP, Tschaplinski TJ, Gunter LE, Davis MF, Tuskan GA. 2005. Phenotypic variation in growth and biomass distribution for two advanced-generation pedigrees of hybrid poplar. Canadian Journal of Forest Research – Revue Canadienne de Recherche Forestiere 35: 1779–1789.

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new workshop is timely and will hopefully provide a broad forum for the exchange of ideas on how these genomic resources can advance our understanding of fungal biology. For the purpose of the workshop, ‘fungus’ was defined broadly to include oomycetes and other fungal-like organisms. This first collection of talks, highlighted here, succeeded in addressing the objective of illustrating how genome sequences can be exploited to further our understanding of fungal biology. A list of the organisms under discussion is shown in Table 1.

‘... oomycetes may have acquired fungal genes through horizontal gene transfer ...’

Key words: adaptation, forestry, genome evolution, genomics, woody plant. Meetings Mettings XXX

Fungal and oomycete genes galore Fungal Genomics workshop: Plant and Animal Genome XV Conference, San Diego, CA, USA January 2007 The popular Plant and Animal Genome Conference (http:// www.intl-pag.org/) has long been relatively well attended by microbiologists, in part because funding agencies often hold their awardee meetings concurrently (one instance is the NSF/USDA-CSREES Microbial Genome Sequencing awardees workshop). However, since the now defunct Agricultural Microbe Genomes meeting, last held in 2001 (Kamoun & Hogenhout, 2001), there have been few sessions in the program devoted to microbial genomics. This is no longer the case, with the Plant and Animal Genome Conference now hosting the new Fungal Genomics workshop. Although the workshop was scheduled as the last session of the conference (following the Host–Microbe Interactions workshop and just before the banquet and dance party), it kicked off with a roar, as mycologists now have access to dozens of genome sequences, and mycology has, in effect, moved into the postgenomics era (Galagan et al., 2005). The objective of the new workshop is to go beyond generating the sequences to discuss what can be done next, particularly in comparative genomics and global functional analyses. Fungi have more sequenced genomes than any other eukaryotic kingdom, and unlike those of most other eukaryotes, many fungal genomes also are being finished, resulting in a complete accounting of every base. Thus, the

Oomycetes In a somewhat ironic twist, the opening talk focused on oomycetes, a group of organisms that are not ‘true fungi’ but are phylogenetically related to brown algae and diatoms. However, oomycetes share some superficial traits with fungi, such as filamentous growth and heterotrophic lifestyle, and the oomycete research community has long interfaced closely with the fungal community. Recent findings from Nick Talbot’s lab (University of Exeter, UK) suggest that oomycetes may have acquired fungal genes through horizontal gene transfer (Richards et al., 2006), so perhaps the affinity between fungi and oomycetes is more than superficial. Sophien Kamoun (Ohio State University, Wooster, OH, USA) provided an update on the five oomycete genomes that have been sequenced to date (Tyler et al., 2006) (Table 1). The Phytophthora species sequenced are among the most notorious plant pathogens. Phytophthora capsici, P. infestans, and P. sojae infect diverse crops, such as pepper, potato, and soybean, respectively. Phytophthora ramorum affects native woody plants, resulting in environmental damage, and the downy mildew, Hyaloperonospora parasitica, a pathogen of Arabidopsis thaliana, figures prominently in research on this model plant. Kamoun focused on one class of oomycete effectors, the so-called RXLR genes that are delivered inside plant cells to manipulate host defenses (Kamoun, 2006). Using computational approaches, genomewide catalogs of the RXLR effectors were produced, unravelling a complex and divergent set of hundreds of candidate effector genes. Recent work has involved testing for selection among families of recently duplicated paralogous genes and this has provided evidence for positive selection in more


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Table 1 List of fungal and oomycete genomes discussed at the Fungal Genomics workshop and their associated web resources Estimated number of genes


Species

Taxonomy

Genome size (Mbp)

Alternaria brassicicola

Ascomycetes, Pleosporales

30.3

–

Aspergillus flavus Aspergillus oryzae Botrytis cinerea B05.10 Botrytis cinerea T4 Fusarium graminearum Fusarium verticillioides Hyaloperonospora parasitica

Ascomycetes, Eurotiales Ascomycetes, Eurotiales Ascomycetes, Helotiales Ascomycetes, Helotiales Ascomycetes, Hypocreales Ascomycetes, Hypocreales Oomycetes, Peronosporales

36.8 36.7 38.8 – 36.2 41.7 75

12 497 12 735 16 448 – 11 640 14 179 NA

Laccaria bicolor Mycosphaerella fijiensis Mycosphaerella graminicola Nectria haematococca Phanerochaete chrysosporium Phycomyces blakesleeanus Phytophthora capsici Phytophthora infestans Phytophthora ramorum Phytophthora sojae Pichia stipitis Sclerotinia sclerotiorum Stagonospora nodorum

Basidiomycetes, Agaricales Ascomycetes, Dothideales Ascomycetes, Dothideales Ascomycetes, Hypocreales Basidiomycetes, Aphyllophorales Zygomycetes, Mucorales Oomycetes, Peronosporales Oomycetes, Peronosporales Oomycetes, Peronosporales Oomycetes, Peronosporales Ascomycetes, Saccharomycetales Ascomycetes, Helotiales Ascomycetes, Pleosporales

64.9 73.4 41.9 52.4 30 55.9 65 240 65 95 15.4 38 37.1

20 614 – 11 395 16 237 10 048 14 792 12 011 NA 15 743 19 027 5841 14 522 16 597

–, not complete or NA, not available.

Resources Washington University (http://genome.wustl.edu/genome.cgi?GENOME = Alternaria%20brassicicola) http://www.aspergillusflavus.org/) http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID = ao) Broad (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html) Genoscope, in progress with expected release during 2007) Broad (http://www.broad.mit.edu/annotation/genome/fusarium_graminearum/Home.html) Broad (http://www.broad.mit.edu/annotation/genome/fusarium_verticillioides) Washington University (http://genome.wustl.edu/pub/organism/Fungi/Hyaloperonospora_parasitica/assembly/ draft/Hyaloperonospora_parasitica-2.0/) JGI (http://genome.jgi-psf.org/Lacbi1/Lacbi1.home.html) JGI (in progress with expected release during 2007) JGI (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html) JGI (http://genome.jgi-psf.org/Necha1/Necha1.home.html) JGI (http://genome.jgi-psf.org/Phchr1/Phchr1.home.html) JGI (http://genome.jgi-psf.org/Phybl1/Phybl1.home.html) JGI (in progress with expected release during 2007) Broad (http://www.broad.mit.edu/annotation/genome/phytophthora_infestans/Home.html) JGI (http://genome.jgi-psf.org/Phyra1–1/Phyra1–1.home.html) JGI (http://genome.jgi-psf.org/Physo1–1/Physo1–1.home.html) JGI (http://genome.jgi-psf.org/Picst3/Picst3.home.html) Broad (http://www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum/Home.html) Broad (http://www.broad.mit.edu/annotation/genome/stagonospora_nodorum/Home.html)

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than two-thirds of the examined RXLR effector families. This positive selection has, for the most part, targeted the C-terminal region of these effectors consistent with the view that this domain functions inside plant cells and may have been involved in a coevolutionary arms race with host factors. Kamoun also reported on functional analyses, namely applying high-throughput screens to discover effectors with avirulence activities, and this offered Kamoun the opportunity to contribute ‘effectoromics’ to the ever-expanding list of ‘omics’ terms.

Mycosphaerella Gert Kema (Plant Research International, Wageningen, the Netherlands) moved the focus of the workshop on to the ‘true fungi’ by introducing the plant pathogens Mycosphaerella fijiensis and M. graminicola, members of the phylum ascomycota. M. fijiensis is the causal agent of Black Sigatoka disease, the most serious constraint to banana production worldwide, resulting in this pathogen receiving the highest fungicide application of all pathogens globally. Similarly M. graminicola causes septoria tritici blotch, one of the most economically important diseases of wheat worldwide and the largest target for fungicides on this crop. Both of these fungi are in the class Dothideomycetes, a group that includes several other important fungi with sequencing projects, for example Stagonospora nodorum, Pyrenophora tritici-repentis and Alternaria brassicicola. The M. graminicola genome assembly was greatly enhanced by an exhaustive genetic map comprising more than 2200 markers, almost 2000 of which have been sequenced, on 23 linkage groups. Strains of this fungus bear from 18 to 21 chromosomes, among the highest numbers reported among ascomycetes, and also carry some of the smallest chromosomes. However, these are not dispensable as they undergo regular meiosis and do not appear to carry an excessive amount of repetitive DNA. The high-quality genome assembly is close to completion, with the few remaining gaps corresponding mostly to centromeres. The genetic map combined with the high-quality sequence enabled mapping of nine quantitative loci for several traits, including cultivar and host specificity. This pathogen carries a smaller arsenal of cell wall-degrading enzymes compared with species of Aspergillus and Botrytis, suggesting that it might behave more like a biotroph than a necrotroph, especially during the early stages of pathogenesis. Functional analyses have been initiated, for example, the MgHog1 gene is required for filamentous growth on plant surfaces, and a knockout hampers penetration of the host (Mehrabi et al., 2006). Kema also reported on the 7.1× draft genomic sequence of M. fijiensis, which has a genome that is c. 80% larger and more complex compared with M. graminicola, indicated by a double peak of GC content and a higher frequency and diversity of transposable elements.

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Mycotoxins: aflatoxin and fumonisin Continuing with the ascomycetes, Gary Payne (North Carolina State University, Raleigh, NC, USA) focused his talk on comparative genomics of two closely related aspergilli that exhibit distinct ecologies (Payne et al., 2006). Aspergillus flavus is a soil saprophyte that can be an opportunistic pathogen in humans and plants such as maize. This pathogen is economically important because it produces the carcinogenic toxin aflatoxin. Aspergillus oryzae is a domesticated fungus that is also economically important because of its widespread use, particularly in Japan, in the preparation of popular food products such as soy sauce and sake. Obviously, A. oryzae is safe for food consumption; it is not known to be pathogenic, nor does it produce aflatoxin. Interestingly, the two genomes turned out to be very similar, lending credence to the long-held view that A. oryzae is a domesticated strain derived from A. flavus. Payne’s initial comparative analyses focused on genes for secondary metabolism, one of the most astonishing features of fungi (Kroken et al., 2003). Remarkably, A. oryzae has retained an inactive aflatoxin biosynthesis cluster orthologous to that of A. flavus at the distal end of chromosome 3. Both A. oryzae and A. flavus have larger numbers of polyketide synthases ( pks) and nonribosomal peptide synthetases (nrps) relative to other aspergilli. Approximately two-thirds of the pks genes have the same splice site; the remaining third have differing gene structure and, interestingly, one pks gene in A. flavus appears to be deleted in A. oryzae. The next question that Payne and his collaborators are tackling focuses on whether the observed differences are species- or strain-dependent. This will be performed using a combination of PCR analysis, oligonucleotide arrays, and Affymetrix GeneChip analyses. Another fungus which produces mycotoxins and is also a pathogen of maize is Fusarium verticillioides (Gibberella moniliformis). Fumonisins (FB1) are a group of mycotoxins that are renowned for causing a debilitating form of brain cancer in horses and esophageal cancer in humans. Because these mycotoxins are so harmful to human and animal health, they are highly regulated such that contaminated grain cannot be marketed. In addition to F. verticillioides, genomes of three other Fusarium species (F. graminearum, F. oxysporum and F. solani ) will be publicly available. Assembly of the F. verticillioides genomic sequence and automated annotation were completed recently and 87 000 ESTs have also been sequenced. Won-Bo Shim (Texas A & M University, College Station, TX, USA) presented research using the genome to study secondary metabolism in Fusarium species. Analyses of the sequences have revealed that the fumonisin (FUM) gene cluster is a 15-gene regulon with the PKS encoded by FUM1. In total, there are 15 gene clusters that harbor PKS genes in F. verticillioides, two of which, PGL1 (pigment production in perithecia) and GzFUS1 (common mycotoxin), are

© The Authors (2007). Journal©compilation www.newphytologist.org The Authors ©(2007). New Phytologist Journal compilation (2007) www.newphytologist.org © New Phytologist (2007)

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highly conserved with F. graminearum. Five clusters, including FUM, are unique to F. verticillioides among the sequenced Fusarium spp. Shim also reported that the regulation of FB1 production occurs coordinately with conidiation, and, using fcc1, a mutant in a C-type cyclin gene that is affected in FB1 production, has identified genes that are coregulated with the FUM genes. Gene knockouts have revealed both positive and negative regulatory genes. The positive regulators include GBB1, a heterotrimeric G protein beta subunit, a C2H2 transcription factor, and a hypothetical cell membrane named CMG1. The negative regulators are a monomeric G protein, GBP1 (Sagaram et al., 2006), and CPP1, a protein phosphatase 2A catalytic subunit.

Sclerotinia As with the Fusarium genome sequence, a joint community annotation effort has been under way to map the genome of the necrotrophic and broad-host-range pathogen Sclerotinia sclerotiorum. However, this is a homothallic fungus, and therefore a genetic map could not be generated to guide the genome assembly. Instead, an optical map was used to validate and improve the assembly of the 9.9× draft genomic sequence. The mapping of S. sclerotiorum together with Botrytis cinerea, a closely related pathogen for which the genomes of two strains have been sequenced, has more than 40 annotators. Jeff Rollins (University of Florida, Gainesville, FL, USA) reported on how he is using the genome to empower his own research on the regulation of multicellular patterns and cell fate in fungal fruiting bodies (apothecia). For instance, the genome sequence is facilitating the genetic analysis of the effect of light on apothecial development. Genes for putative photoreceptors, such as the white collar ortholog and a cryptochrome-encoding gene (cry1), are being functionally characterized through gene-replacement experiments. The automated gene-call prediction identified 14 522 genes in the S. sclerotiorum genome. This autocalled gene set has been used as the foundation for producing Agilent microarrays. Initial hybridizations with these arrays will focus on validating probe design and investigating the regulation of apothecial development.

Genome annotation The final presentation of the workshop was given by Igor Grigoriev ( Joint Genome Institute ( JGI), Walnut Creek, CA, USA), who has overseen the annotation of many fungal and oomycete genomes, most recently the zygomycete Phycomyces blakesleeanus. The JGI annotation pipeline involves automated gene calls followed by manual curation by community members. So far, more than 15 000 fungal genes have been curated manually by over 150 annotators. JGI typically sequences ESTs to aid the annotation process and is constantly looking for additional data to enhance

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genome annotation. ESTs, proteomics, and microarrays not only help to validate predicted genes but also enable various community-driven activities. Grigoriev showed a number of examples, including preliminary results on metabolic reconstruction for Pichia stipitis, microarray analysis for Laccaria bicolor, and proteomics studies in Phanerochaete chrysosporium. Comparative genomics can also be used to explore genome organization, as was done for Nectria haematococca, and study synteny, as in P. sojae and P. ramorum (Tyler et al., 2006). He also reported on the pioneering approach of combining traditional Sanger dideoxy sequencing with novel methods such as 454 sequencing, as currently performed for the Phytophthora capsici project. A future emphasis at JGI will include developing a critical mass of data and tools for comparative genomics. Finally, Grigoriev reiterated his invitation to all interested scientists to join in webbased community annotations (http://genome.jgi-psf.org/ euk_cur1.html).

Perspectives In retrospect, mycologists should be grateful for the visionary decision by the Broad Institute (at that time, Whitehead Institute) to launch the Fungal Genomics Initiative in 2000 (http://www.broad.mit.edu/annotation/ fungi/fgi/index.html) and to the American Phytopathological Society (APS) for its decision to follow up with a plant-associated microbial sequencing initiative during 2003 (Leach et al., 2003). These initiatives had a catalytic effect on the research community’s interest in genomics, encouraged funding agencies to invest in fungi, and undoubtedly raised interest among other genome centers, such as JGI and Washington University Genome Center, to take up fungal genome projects. Among the 10 fungi or oomycetes on the APS high-priority sequencing list from 2003 (http:// www.apsnet.org/media/ps/MicrobialGenomicsSeqFinal03.pdf), nine are now sequenced or in the pipeline. A more recent boost to the sequencing of fungal genomes was provided by the Community Sequencing Program (http://www.jgi.doe.gov/ CSP/index.html) of the JGI that began during 2005. This program provides free sequencing of organisms suggested by the scientific community. Sequencing fungal genomes has had a notable positive influence on genome science, enabling genomicists to hone their techniques with smaller eukaryotic genomes before tackling larger projects. The time has now come when discoveries from these fungal and oomycete genomes are improving our understanding of these fascinating organisms, which reinforces a sense of excitement about the years to come.

Acknowledgements We thank Gert Kema, Gary Payne, Won-Bo Shim, Jeff Rollins, and Igor Grigoriev for checking drafts of the manuscript.

© www.newphytologist.org The Authors (2007). Journal©compilation The Authors ©(2007). New Phytologist Journal compilation (2007) www.newphytologist.org © New Phytologist (2007)

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Sophien Kamoun1* and Stephen B. Goodwin2 1Department

of Plant Pathology, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691, USA; 2USDA-ARS, Crop Production and Pest Control Research Unit, Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054, USA (*Author for correspondence: email [email protected])

References Galagan JE, Henn MR, Ma LJ, Cuomo CA, Birren B. 2005. Genomics of the fungal kingdom: insights into eukaryotic biology. Genome Research 15: 1620–1631. Kamoun S. 2006. A catalogue of the effector secretome of plant pathogenic oomycetes. Annual Review of Phytopathology 44: 41– 60. Kamoun S, Hogenhout SA. 2001. Agricultural microbes genome 2: first glimpses into the genomes of plant-associated microbes. Plant Cell 13: 451– 458. Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG. 2003. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proceedings of the National Academy of Sciences, USA 100: 15670–15675. Leach JE, Gold SE, Tolin S, Eversole K. 2003. A plant-associated microbe genome initiative. Phytopathology 93: 524–527. Mehrabi R, Zwiers LH, de Waard MA, Kema GH. 2006. MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola. Molecular Plant-Microbe Interactions 19: 1262–1269. Payne GA, Nierman WC, Wortman JR, Pritchard BL, Brown D, Dean RA, Bhatnagar D, Cleveland TE, Machida M, J. 2006. Whole genome comparison of Aspergillus flavus and A. oryzae. Medical Mycology 44: 9–11. Richards TA, Dacks JB, Jenkinson JM, Thornton CR, Talbot NJ. 2006. Evolution of filamentous plant pathogens: gene exchange across eukaryotic kingdoms. Current Biology 16: 1857–1864. Sagaram US, Butchko RAE, Shim W-B. 2006. The putative monomeric G-protein GBP1 is negatively associated with fumonisin B1 production in Fusarium verticillioides. Molecular Plant Pathology 7: 381–389. Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RH, Aerts A, Arredondo FD, Baxter L, Bensasson D, Beynon JL, Chapman J, Damasceno CM, Dorrance AE, Dou D, Dickerman AW, Dubchak IL, Garbelotto M, Gijzen M, Gordon SG, Govers F et al. 2006. Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313: 1261–1266. Key words: evolution, fungi, genomics, mycotoxins, oomycetes. Meetings XXX

Polyploidy: genome obesity and its consequences Polyploidy workshop: Plant and Animal Genome XV Conference, San Diego, CA, USA, January 2007 Polyploidy is a major evolutionary feature of many plants and some animals (Grant, 1981; Otto & Whitton, 2000). Allopolyploids (e.g. wheat, cotton, and canola) were formed by combination of two or more distinct genomes, whereas autopolyploids (e.g. potato, sugarcane, and banana) resulted

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from duplication of a single genome. Both allopolyploids and autopolyploids are prevalent in nature (Tate et al., 2004). Recent research has shown that polyploid genomes may undergo rapid changes in genome structure and function via genetic and epigenetic changes (Fig. 1) (Levy & Feldman, 2002; Osborn et al., 2003; Chen, 2007). The former include chromosomal rearrangements (e.g. translocation, deletion, and transposition) and DNA sequence elimination and mutations, whereas epigenetic modifications (chromatin and RNA-mediated pathways) give rise to gene expression changes that are not associated with changes in DNA sequence. Over time, polyploids may become ‘diploidized’ so that they behave like diploids cytogenetically and genetically. Comparative and genome sequence analyses indicate that many plant species, including maize, rice, poplar, and Arabidopsis, are recent or ancient diploidized (paleo-) polyploids. The consequences of polyploidy have been of long-standing interest in genetics, evolution, and systematics (Wendel, 2000; Soltis et al., 2003). Research interest in polyploids has been renewed in the past decade following the discovery of multiple origins and patterns of polyploid formation (Soltis et al., 2003) and rapid genetic changes in resynthesized allotetraploids in Brassica (Song et al., 1995) and wheat (Feldman et al., 1997). Rapid technological advances have also facilitated genomicscale investigation of polyploids and hybrids (Wang et al., 2006). Many ongoing studies are focused on investigation of: (i) the evolutionary consequence of gene and genome duplications in polyploids; (ii) genomic and gene expression changes in resynthesized allotetraploids; (iii) genetic and gene expression variation in natural populations of polyploids; and (iv) comparison of genetic and gene expression changes in resynthesized and natural polyploids (Wendel, 2000; Osborn et al., 2003; Soltis et al., 2003; Comai, 2005; Chen, 2007). The presentations given at the Polyploidy workshop, Plant and Animal Genome XV Conference (http://www.intl-pag.org/), reflected these current research themes, reporting on ancient polyploidy events in Glycine, expression evolution of duplicate genes in Arabidopsis, gene expression changes in resynthesized Brassica and wheat allopolyploids, hybridization barriers in Arabidopsis, and tissue-specific and stress-induced expression patterns of duplicate genes in cotton and hybrid Populus.

‘... expression of duplicate genes in response to developmental programs is more strongly correlated than that of duplicate genes in response to environmental stresses, suggesting rapid evolution of duplicate genes in response to external factors’


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Fig. 1 Diagram of allopolyploid formation and evolution. A hybrid (not shown) derived from two diploid species can be induced to form a stable allotetraploid via spontaneous chromosome doubling or by colchicine treatment. Alternatively, an allotetraploid can be formed by fusing two unreduced gametes from two diploids or by hybridization of two autotetraploids (not shown) (Chen, 2007). Allotetraploid formation is usually impaired by the hybridization barrier between the two species (red stop sign). Once an allotetraploid is formed, it may undergo rapid genetic changes (e.g. chromosomal rearrangements, loss, and transposition) and epigenetic changes (e.g. chromatin modifications and posttranscriptional regulation). Chromosomes from the two different species are colored orange and green, respectively. The chromosomes (orange or green) in different species are orthologous, and they become homoeologous (orange and green) in the allotetraploid. Over time, allopolyploids may evolve to become diploidized polyploids because of rapid changes in chromosomal structure and sequence composition. In many instances, epigenetic changes predominate in allopolyploids. Interspecific hybridization or allopolyploidy may induce formation of heterochromatin and euchromatin, resulting in gene silencing or activation via transcriptional and post-transcriptional mechanisms. RNA interference induces and maintains heterochromatin formation. These changes in allopolyploids will lead to alteration of gene expression and phenotypic variation. Both genetic and epigenetic changes can be selected by natural or artificial forces that facilitate adaptive evolution of new polyploid species. Solid and dashed arrows indicate observed and predicted changes, respectively.

Duplication of resistance genes Species of Glycine (soybean and relatives) are complex paleopolyploids that underwent at least two rounds of polyploidzation events, estimated to be c. 15 and c. 50–60 million

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years ago (Mya), respectively. To elucidate the complexity of the Glycine genome, Jeff Doyle (Cornell University, Ithaca, NY, USA), a member of the plant genome project led by Roger Innes (Indiana University, Bloomington, USA), reported progress in sequencing two homoeologues of a 1 Mb region that contains several disease resistance gene clusters (R-genes) in two soybean varieties and relatives of soybean. The homoeologous regions were derived from genome duplication which occurred 15 Mya. The gene densities of the two homoeologues in soybean are very different, mainly because of differences in the number of transposable element insertions. The two homoeologues also differ in their R-gene composition, with the gene-poor homoeologue also being degenerate for R-genes. Patterns of R-gene evolution are complex, with apparent recombination among copies and a considerable amount of copy-number variation among lineages. Little of this has been the result of polyploidy, however; only one of over 20 duplication events inferred from phylogenies appears to be related to the 15 Mya duplication, and most expansion has been tandem and much more recent. Variation in R-gene content also occurs among Glycine species, and even between soybean cultivars, suggesting recent and rapid changes. In other regions that do not contain resistance genes, gene densities and repeats tend to be very similar between homoeologues (Schlueter et al., 2006), raising the question of whether the marked differences between homoeologues reported here are the result of evolutionary properties of R-gene clusters. Although much of the change in these two homoeologous regions has occurred recently, it is possible that the divergent evolution of the two homoeologues was set in motion by the polyploid event and has been ongoing subsequently.

Expression evolution of duplicate genes The evolutionary fate of duplicate genes is poorly understood. Theory predicts that duplicate genes will eventually be lost or mutated. However, many gene duplicates are retained in the genome, probably via neofunctionalization or subfunctionalization (Lynch & Force, 2000). To test these hypotheses, Misook Ha (University of Texas at Austin, TX, USA), analyzed expression divergence of c. 2000 pairs of gene duplicates that resulted from a single duplication event that occurred 20–40 Mya (Blanc et al., 2003). The gene expression microarrays measured at various conditions were used to test whether the expression patterns of gene duplicates diverge rapidly compared with the randomly paired genes in response to environmental and developmental changes. The data presented indicate that duplicate genes have a higher similarity of expression patterns than randomly paired genes. Moreover, expression of duplicate genes in response to developmental programs is more strongly correlated than that of duplicate genes in response to environmental stresses, suggesting rapid evolution of

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duplicate genes in response to external factors. To explain these patterns of expression divergence between duplicate genes after whole genome duplication, Ha proposed a model whereby expression of duplicate genes diverges rapidly in response to changes in abiotic and biotic stresses, whereas the expression of duplicate genes diverges relatively slowly in response to developmental changes that are associated with complex biological networks.

Developmental regulation and subfunctionalization of duplicate genes Functional divergence of homoeologous genes is manifested by tissue- or organ-specific expression patterns of duplicate genes, which were first observed in the allopolyploids Brassica and Gossypium (cotton). The silenced rRNAs genes in leaves subjected to nucleolar dominance in Brassica allotetraploids were reactivated in floral organs, suggesting developmentally regulated gene expression (Chen & Pikaard, 1997). Adams et al. (2003) found that developmental regulation of gene expression occurs in 10 out of 40 genes examined in cotton allopolyploids, suggesting tissue-specific regulation of homoeologous genes or subfunctionalization of duplicate genes in allopolyploids. Current work in the Adams laboratory (University of British Columbia, Vancouver, Canada) has focused on using a fluorescence-based semiquantitative assay (snapshot) to distinguish expression differences between homoeologous loci in different tissues and organs and in cold and water submersion stresses. Adams reported that the expression ratios of homoeologous genes change not only in different tissues, but also under different stress (cold and water submersion) conditions. The data from Arabidopsis and cotton suggest that paralogous and homoeologous genes may have similar fates in response to changes in environmental cues and developmental programs.

Genetic and epigenetic changes in resynthesized Brassica allotetraploids Gene expression changes may also be associated with either genetic or epigenetic mechanisms (Osborn et al., 2003; Chen, 2007) (Fig. 1). Robert Gaeta (University of Wisconsin, Madison, WI, USA) reported chromosomal rearrangements and changes in DNA methylation among 50 resynthesized lines of Brassica napus-like plants. There is a correlation between changes in gene expression and chromosomal rearrangements and transposition (insertion of a fragment from one homoeologous chromosome to another). For example, Flowering Locus C expression is dependent on dosage caused by chromosomal rearrangements in 50 allopolyploid lineages. Similar changes were also reported in previous independent studies using resynthesized B. napuslike plants (Pires et al., 2004). Interestingly, the frequency of

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changes in the restriction length fragment polymorphism (RFLP) among 50 lines is relatively low in the first generation following allopolyploid formation but high in the progeny after six generations of selfing. Furthermore, the frequency of DNA methylation changes is fairly constant in selfing progeny. Importantly for those interested in resynthesized polyploids, there is no obvious difference of genomic and gene expression changes in the progeny derived from allotetraploids that are derived from spontaneous chromosome doubling or colchicine-treatment. Chromosomal rearrangements and epigenetic modifications may explain a wide range of morphological changes observed in 50 different lineages of Brassica allotetraploids. As in Arabidopsis allopolyploids (Wang et al., 2006), changes in gene expression are also frequently observed in resynthesized wheat allohexaploids. Bikram Gill (Kansas State University, Manhattan, KS, USA) reported high amounts of gene expression changes using microarray in comparison with wheat diploids, tetraploids, and hexaploids.

From hybridization barriers to the success of allopolyploids Hybridization between the species that are separated for millions of years encounters barriers between alien cytoplasm and nuclear genomes and between two divergent genomes (Comai, 2005; Chen, 2007) (Fig. 1). These barriers are partly reflected by the changes in dosages of maternal and paternal genomes and imprinting patterns of gene expression (Bushell et al., 2003). Comai (University of California at Davis, CA, USA) and colleagues have shown that the expression of PHERES1 and MEDEA is altered in resynthesized Arabidopsis allotetraploids (Josefsson et al., 2006). Although reciprocal crosses of Arabidopsis allotetraploids cannot be made, the data suggest maternal and paternal effects of gene expression on seed fertility in the allopolyploids. Brian Dilkes (University of California at Davis, CA, USA), reported mapping a locus, named after Dr Strangelove (DSL1), in the triploid progeny of Arabidopsis. DSL1 is predicted to be a homologue of TRANSPARENT TESTA GLABRA (TTG2), a WRKY transcription factor. Arabidopsis TTG2 is strongly expressed in trichomes and in the endothelium of developing seeds and subsequently in other layers of the seed coats, and in developing roots. DSL1 does not show imprinting patterns, suggesting that postzygotic barriers and seed fertility may also be affected by proper development of maternal tissues (ovules).

Perspectives Polyploidy is a fascinating biological phenomenon that is a source of the raw genetic materials for adaptive evolution and crop domestication. Polyploid cells are often associated with carcinogenesis in animals, and polyspermy (fertilization


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of more than one sperm into one ovum) usually causes abortive human triploids (McFadden et al., 1993), suggesting why polyploidy is rarer in animals than in plants. The molecular changes observed in various polyploid plant systems will improve our understanding of why polyploid plants are so successful during evolution and why and how plants can tolerate genome obesity (increase in genome dosage) better than animals, especially mammals.

Acknowledgements We thank Keith Adams, Brian Dilkes, Jeff Doyle, Robert Gaeta, and Bikram Gill for providing critical comments to improve the manuscript. The work in the Chen and Soltis laboratories was supported by grants from the National Science Foundation (DBI0501712 and DBI0624077 to ZJC, and MCB0346437 to DES) and the National Institutes of Health (GM067015 to ZJC). Z. Jeffrey Chen1*, Misook Ha1 and Douglas Soltis2 1Section

of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, TX 78712, USA; 2Department of Botany and Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611, USA (*Author for correspondence: tel +512 475 9327; fax +1512-471-2149; email [email protected])

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dominance in polyploid plants: biased expression/silencing of progenitor rrna genes is developmentally regulated in Brassica. Proceedings of the National Academy of Sciences, USA 94: 3442–3447. Comai L. 2005. The advantages and disadvantages of being polyploid. Nature Reviews Genetics 6: 836–846. Feldman M, Liu B, Segal G, Abbo S, Levy AA, Vega JM. 1997. Rapid elimination of low-copy DNA sequences in polyploid wheat: a possible mechanism for differentiation of homoeologous chromosomes. Genetics 147: 1381–1387. Grant V. 1981. Plant speciation. New York, NY, USA: Columbia University Press. Josefsson C, Dilkes B, Comai L. 2006. Parent-dependent loss of gene silencing during interspecies hybridization. Current Biology 16: 1322–1328. Levy AA, Feldman M. 2002. The impact of polyploidy on grass genome evolution. Plant Physiology 130: 1587–1593. Lynch M, Force A. 2000. The probability of duplicate gene preservation by subfunctionalization. Genetics 154: 459–473. McFadden DE, Kwong LC, Yam IY, Langlois S. 1993. Parental origin of triploidy in human fetuses: evidence for genomic imprinting. Human Genetics 92: 465–469. Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA. 2003. Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics 19: 141–147. Otto SP, Whitton J. 2000. Polyploid incidence and evolution. Annual Review of Genetics 34: 401–437. Pires JC, Zhao JW, Schranz ME, Leon EJ, Quijada PA, Lukens LN, Osborn TC. 2004. Flowering time divergence and genomic rearrangements in resynthesized brassica polyploids (brassicaceae). Biological Journal of the Linnean Society 82: 675–688. Schlueter JA, Scheffler BE, Schlueter SD, Shoemaker RC. 2006. Sequence conservation of homeologous bacterial artificial chromosomes and transcription of homeologous genes in soybean (glycine max 1. Merr.). Genetics 174: 1017–1028. Soltis DE, Soltis PS, Tate JA. 2003. Advances in the study of polyploidy since plant speciation. New Phytologist 161: 173–191. Song K, Lu P, Tang K, Osborn TC. 1995. Rapid genome change in synthetic polyploids of brassica and its implications for polyploid evolution. Proceedings of the National Academy of Sciences, USA 92: 7719–7723. Tate JA, Soltis PS, Soltis DE. 2004. Polyploidy in plants. The evolution of the genome. New York, NY, USA: Academic Press. Wang J, Tian L, Lee HS, Wei NE, Jiang H, Watson B, Madlung A, Osborn TC, Doerge RW, Comai L, Chen ZJ. 2006. Genomewide nonadditive gene regulation in arabidopsis allotetraploids. Genetics 172: 507–517. Wendel JF. 2000. Genome evolution in polyploids. Plant Molecular Biology 42: 225–249. Key words: duplicate genes, epigenetics, evolution, gene expression, polyploidy, subfunctionalization.

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