The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.     

Genetic Modifiers of dFMR1 Encode RNA Granule Components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP''s translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.THE subcellular localization and regulated translation of stored mRNAs contributes to cellular asymmetry and subcellular specialization (Lecuyer et al. 2007; Martin and Ephrussi 2009). In mature neurons, local protein synthesis at active synapses may contribute to synapse-specific plasticity that underlies persistent forms of memory (Casadio et al. 1999; Ashraf et al. 2006; Sutton and Schuman 2006; Richter and Klann 2009). During this process, synaptic activity causes local translation of mRNAs normally stored in translationally repressed synaptic mRNPs (Sutton and Schuman 2006; Richter and Klann 2009). While specific neuronal translational repressors and microRNAs have been implicated in this process, their involvement in local translation that underlies memory, as well as the underlying mechanisms, are generally not well understood (Schratt et al. 2006; Keleman et al. 2007; Kwak et al. 2008; Li et al. 2008; Richter and Klann 2009). Furthermore, it remains possible that there are neuron-specific, mRNA-specific, and stimulus-pattern specific pathways for neuronal translational control (Raab-Graham et al. 2006; Giorgi et al. 2007).The Fragile-X Mental Retardation protein (FMRP) is among the best studied of neuronal translational repressors, in part due to its association with human neurodevelopmental disease (Pieretti et al. 1991; Mazroui et al. 2002; Gao 2008). Consistent with function in synaptic translation required for memory formation, mutations in FMRP are associated with increased synaptic translation, enhanced LTD, increased synapse growth, and also with enhanced long-term memory (Zhang et al. 2001; Huber et al. 2002; Bolduc et al. 2008; Dictenberg et al. 2008).FMRP co-immunoprecipitates with components of the RNAi and miRNA machinery and appears to be required for aspects of miRNA function in neurons (Caudy et al. 2002; Ishizuka et al. 2002; Jin et al. 2004b; Gao 2008). In addition, FMRP associates with neuronal polyribosomes as well as with Staufen-containing ribonucleoprotein (mRNP) granules easily observed in neurites of cultured neurons (Feng et al. 1997; Krichevsky and Kosik 2001; Mazroui et al. 2002; Kanai et al. 2004; Barbee et al. 2006; Bramham and Wells 2007; Bassell and Warren 2008; Dictenberg et al. 2008). FMRP-containing neuronal mRNPs contain not only several ubiquitous translational control molecules, but also CaMKII and Arc mRNAs, whose translation is locally controlled at synapses (Rook et al. 2000; Krichevsky and Kosik 2001; Kanai et al. 2004; Barbee et al. 2006). Thus, FMRP-containing RNA particles are probably translationally repressed and transported along microtubules from the neuronal cell body to synaptic sites in dendrites where local synaptic activity can induce their translation (Kiebler and Bassell 2006; Dictenberg et al. 2008).The functions of FMRP/dFMR1 in mRNA localization as well as miRNA-dependent and independent forms of translational control is likely to require several other regulatory proteins. To identify such proteins, we used a previously designed and validated genetic screen (Wan et al. 2000; Jin et al. 2004a; Zarnescu et al. 2005). The overexpression of dFMR1 in the fly eye causes a “rough-eye” phenotype through a mechanism that requires (a) key residues in dFMR1 that mediate translational repression in vitro; (b) Ago1, a known components of the miRNA pathway; and (c) a DEAD-box helicase called Me31B, which is a highly conserved protein from yeast (Dhh1p) to humans (Rck54/DDX6) functioning in translational repression and present on neuritic mRNPs (Wan et al. 2000; Laggerbauer et al. 2001; Jin et al. 2004a; Coller and Parker 2005; Barbee et al. 2006; Chu and Rana 2006). To identify other Me31B-like translational repressors and neuronal granule components, we screened mutations in 43 candidate proteins for their ability to modify dFMR1 induced rough-eye phenotype. We describe the results of this genetic screen and follow up experiments to address the potential cellular functions of five genes identified as suppressors of sev-dfmr1.     

Unraveling the Complex Trait of Crop Yield With Quantitative Trait Loci Mapping in Brassica napus

Yield is the most important and complex trait for the genetic improvement of crops. Although much research into the genetic basis of yield and yield-associated traits has been reported, in each such experiment the genetic architecture and determinants of yield have remained ambiguous. One of the most intractable problems is the interaction between genes and the environment. We identified 85 quantitative trait loci (QTL) for seed yield along with 785 QTL for eight yield-associated traits, from 10 natural environments and two related populations of rapeseed. A trait-by-trait meta-analysis revealed 401 consensus QTL, of which 82.5% were clustered and integrated into 111 pleiotropic unique QTL by meta-analysis, 47 of which were relevant for seed yield. The complexity of the genetic architecture of yield was demonstrated, illustrating the pleiotropy, synthesis, variability, and plasticity of yield QTL. The idea of estimating indicator QTL for yield QTL and identifying potential candidate genes for yield provides an advance in methodology for complex traits.YIELD is the most important and complex trait in crops. It reflects the interaction of the environment with all growth and development processes that occur throughout the life cycle (Quarrie et al. 2006). Crop yield is directly and multiply determined by yield-component traits (such as seed weight and seed number). Yield-related traits (such as biomass, harvest index, plant architecture, adaptation, resistance to biotic and abiotic constraints) may also indirectly affect yield by affecting the yield-component traits or by other, unknown mechanisms. Increasing evidence suggests that “fine-mapped” quantitative trait loci (QTL) or genes identified as affecting crop yield involve diverse pathways, such as seed number (Ashikari et al. 2005; Tian et al. 2006b; Burstin et al. 2007; Xie et al. 2008; Xing et al. 2008; Xue et al. 2008), seed weight (Ishimaru 2003; Song et al. 2005; Shomura et al. 2008; Wang et al. 2008; Xie et al. 2006, 2008; Xing et al. 2008; Xue et al. 2008), flowering time (Cockram et al. 2007; Song et al. 2007; Xie et al. 2008; Xue et al. 2008), plant height (Salamini 2003; Ashikari et al. 2005; Xie et al. 2008; Xue et al. 2008), branching (Clark et al. 2006; Burstin et al. 2007; Xing et al. 2008), biomass yield (Quarrie et al. 2006; Burstin et al. 2007), resistance and tolerance to biotic and abiotic stresses (Khush 2001; Brown 2002; Yuan et al. 2002; Waller et al. 2005; Zhang 2007; Warrington et al. 2008), and root architecture (Hochholdinger et al. 2008).Many experiments have explored the genetic basis of yield and yield-associated traits (yield components and yield-related traits) in crops. Summaries of identified QTL have been published for wheat (MacCaferri et al. 2008), barley (Von Korff et al. 2008), rice, and maize (http://www.gramene.org/). The results show several common patterns. First, QTL for yield and yield-associated traits tend to be clustered in the genome, which suggests that the QTL of the yield-associated traits have pleiotropic effects on yield. Second, this kind of pleiotropy has not been well analyzed genetically. The QTL for yield (complicated factor), therefore, have not been associated with any yield-associated traits (relatively simple factors, such as plant height). Therefore, they are unlikely to predict accurately potential candidate genes for yield. Third, only a few loci (rarely >10) have been found for each of these traits. Thus, the genetic architecture of yield has remained ambiguous. Fourth, trials were carried out in a few environments and how the mode of expression of QTL for these complex traits might respond in different environments is unclear.In this study, the genetic architecture of crop yield was analyzed through the QTL mapping of seed yield and eight yield-associated traits in two related populations of rapeseed (Brassica napus) that were grown in 10 natural environments. The complexity of the genetic architecture of seed yield was demonstrated by QTL meta-analysis. The idea of estimating indicator QTL (QTL of yield-associated traits, which are defined as the potential genetic determinants of the colocalized QTL for yield) for yield QTL in conjunction with the identification of candidate genes is described.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to ∼10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.HORIZONTAL gene transfer (HGT) contributes to bacterial evolution by providing access to DNA evolved and retained in separate species or strains (Cohan 1994a,b; Bergstrom et al. 2000; Ochman et al. 2000; Feil et al. 2001; Koonin 2003; Lawrence and Hendrickson 2003; Fraser et al. 2007). Multilocus sequence typing (MLST) has provided strong evidence for frequent transfer and recombination of chromosomal DNA between related bacterial strains within the same species (Maiden et al. 1998; Enright et al. 2002). HGT occurring by natural transformation allows bacteria to exploit the presence of nucleic acids in their environment for the purposes of nutrition, DNA repair, reacquisition of lost genes, and/or acquisition of novel genetic diversity (Redfield 1993; Mehr and Seifert 1998; Dubnau 1999; Claverys et al. 2000; Szöllösi et al. 2006; Johnsen et al. 2009). It can be inferred from observations of the presence of extracellular DNA in most environments that bacteria are constantly exposed to DNA from a variety of sources, without such exposure necessarily producing observable changes in the genetic compositions of bacterial populations over evolutionary time (Thomas and Nielsen 2005; Nielsen et al. 2007a,b).The absence of sequence similarity between the donor DNA and the DNA of the recipient bacterium is the strongest barrier to the horizontal acquisition of chromosomal genes in bacteria (Matic et al. 1996; Vulic et al. 1997; Majewski 2001; Townsend et al. 2003) as illegitimate recombination occurs only at extremely low frequencies in bacteria (Hülter and Wackernagel 2008a). Single-locus transfer models have been extensively applied and have demonstrated a log-linear decrease in recombination frequencies with increasing sequence divergence for Bacillus subtilis (Roberts and Cohan, 1993; Zawadzki et al. 1995), Acinetobacter baylyi (Young and Ornston 2001), Escherichia coli (Shen and Huang 1986; Vulic et al. 1997), and Streptococcus pneumoniae (Majewski et al. 2000). For instance, heterogamic transformation between nonmutator isolates at the rpoB locus of B. mojavensis is undetectable at sequence divergences >16.7% (Zawadzki et al. 1995) and between S. pneumoniae isolates with sequence divergences >18% (Majewski et al. 2000). In A. baylyi, the nonmutator sequence divergence limit for detectable transformation at the pcaH locus of strain ADP1 was found to be 20% (Young and Ornston 2001), and up to 24% overall divergence yielded transformants at 16S rRNA loci in strain DSM587 (Strätz et al. 1996).Several recent studies also show that short stretches (<200 bp) of DNA sequence identity can facilitate additive or substitutive integration of longer stretches (>1000 bp) of heterologous DNA in bacteria (Prudhomme et al. 1991, 2002; de Vries and Wackernagel 2002; Hülter and Wackernagel 2008a). Thus, the uptake of DNA in bacteria can facilitate larger substitutions within gene sequences and the integration of additional DNA material on the basis of recombination initiated in flanking DNA stretches (either at one or both ends) with high sequence similarity (Nielsen et al. 2000). On the other hand, segments of heterologous DNA interrupting the synteny of homologous DNA have also been shown to be a barrier in intraspecies transformation in S. pneumoniae (Pasta and Sicard 1996, 1999).The various studies of the interspecies transfer potential of single genes demonstrate that the immediate local sequence divergence of the transferred locus is of high importance in determining recombination frequencies in hosts up to 20% divergent (at the housekeeping gene level). However, it can be hypothesized that the broader structural, organizational, and biochemical properties of the genome region surrounding a particular locus will determine its transfer potential to more divergent host species (Cohan 2001; Lawrence 2002). The interspecies transfer potential of various genome regions/loci between more diverged species (>20% at the housekeeping gene level) may therefore differ substantially from a log-linear model (determined experimentally for more closely related species) as local gene organization becomes less conserved with evolutionary time. The barriers to gene exchange between divergent bacterial species is likely a combination of inefficient recombination due to both mismatched base pairs (the main determinator in the log-linear model) and conflicting gene order and organization across the local recombining DNA regions. In addition, selective barriers due to negative effects on host fitness of the transferred DNA regions may become increasingly important for the removal of recombination events from the bacterial population. Recent bioinformatics-based genome analysis of E. coli and Salmonella genomes suggests various parts of the bacterial genome may have different suceptibilities to undergo evolutionarily successful recombination leading to temporal fragmentation of speciation (Lawrence 2002; Retchless and Lawrence 2007). Nevertheless, few studies have experimentally tested the effect of variable species and chromosome locations of genes on their transfer potential between bacteria (Ravin and Chen 1967; Ravin and Chakrabarti 1975; Siddiqui and Goldberg 1975; Cohan et al. 1991; Huang et al. 1991; Fall et al. 2007).Here, we determine to what extent genome location contributes to sexual isolation between the recipient A. baylyi strain ADP1 and 19 sequence divergent (24–27% divergent at the mutS/trpE loci) donor Acinetobacter strains and species (carrying a selectable nptI gene in a total of 95 random genome locations).     

Polymorphic Genes of Major Effect: Consequences for Variation,Selection and Evolution in Arabidopsis thaliana

The importance of genes of major effect for evolutionary trajectories within and among natural populations has long been the subject of intense debate. For example, if allelic variation at a major-effect locus fundamentally alters the structure of quantitative trait variation, then fixation of a single locus can have rapid and profound effects on the rate or direction of subsequent evolutionary change. Using an Arabidopsis thaliana RIL mapping population, we compare G-matrix structure between lines possessing different alleles at ERECTA, a locus known to affect ecologically relevant variation in plant architecture. We find that the allele present at ERECTA significantly alters G-matrix structure—in particular the genetic correlations between branch number and flowering time traits—and may also modulate the strength of natural selection on these traits. Despite these differences, however, when we extend our analysis to determine how evolution might differ depending on the ERECTA allele, we find that predicted responses to selection are similar. To compare responses to selection between allele classes, we developed a resampling strategy that incorporates uncertainty in estimates of selection that can also be used for statistical comparisons of G matrices.THE structure of the genetic variation that underlies phenotypic traits has important consequences for understanding the evolution of quantitative traits (Fisher 1930; Lande 1979; Bulmer 1980; Kimura 1983; Orr 1998; Agrawal et al. 2001). Despite the infinitesimal model''s allure and theoretical tractability (see Orr and Coyne 1992; Orr 1998, 2005a,b for reviews of its influence), evidence has accumulated from several sources (artificial selection experiments, experimental evolution, and QTL mapping) to suggest that genes of major effect often contribute to quantitative traits. Thus, the frequency and role of genes of major effect in evolutionary quantitative genetics have been a subject of intense debate and investigation for close to 80 years (Fisher 1930; Kimura 1983; Orr 1998, 2005a,b). Beyond the conceptual implications, the prevalence of major-effect loci also affects our ability to determine the genetic basis of adaptations and species differences (e.g., Bradshaw et al. 1995, 1998).Although the existence of genes of major effect is no longer in doubt, we still lack basic empirical data on how segregating variation at such genes affects key components of evolutionary process (but see Carrière and Roff 1995). In other words, How does polymorphism at genes of major effect alter patterns of genetic variation and covariation, natural selection, and the likely response to selection? The lack of data stems, in part, from the methods used to detect genes of major effect: experimental evolution (e.g., Bull et al. 1997; Zeyl 2005) and QTL analysis (see Erickson et al. 2004 for a review) often detect such genes retrospectively after they have become fixed in experimental populations or the species pairs used to generate the mapping population. The consequences of polymorphism at these genes on patterns of variation, covariation, selection, and the response to selection—which can be transient (Agrawal et al. 2001)—are thus often unobserved.A partial exception to the absence of data on the effects of major genes comes from artificial selection experiments, in which a substantial evolutionary response to selection in the phenotype after a plateau is often interpreted as evidence for the fixation of a major-effect locus (Frankham et al. 1968; Yoo 1980a,b; Frankham 1980; Shrimpton and Robertson 1988a,b; Caballero et al. 1991; Keightley 1998; see Mackay 1990 and Hill and Caballero 1992 for reviews). However, many of these experiments report only data on the selected phenotype (e.g., bristle number) or, alternatively, the selected phenotype and some measure of fitness (e.g., Frankham et al. 1968, Yoo 1980b; Caballero et al. 1991; Mackay et al. 1994; Fry et al. 1995; Nuzhdin et al. 1995; Zur Lage et al. 1997), making it difficult to infer how a mutation will affect variation, covariation, selection, and evolutionary responses for a suite of traits that might affect fitness themselves. One approach is to document how variation at individual genes of major effect affects the genetic variance–covariance matrix (“G matrix”; Lande 1979), which represents the additive genetic variance and covariance between traits.Although direct evidence for variation at major-effect genes altering patterns of genetic variation, covariation, and selection is rare, there is abundant evidence for the genetic mechanisms that could produce these dynamics. A gene of major effect could have these consequences due to any of at least three genetic mechanisms: (1) pleiotropy, where a gene of major effect influences several traits, including potentially fitness, simultaneously, (2) physical linkage or linkage disequilibrium (LD), in which a gene of major effect is either physically linked or in LD with other genes that influence other traits under selection, and (3) epistasis, in which the allele present at a major-effect gene alters the phenotypic effect of other loci and potentially phenotypes under selection. Evidence for these three evolutionary genetic mechanisms leading to changes in suites of traits comes from a variety of sources, including mutation accumulation experiments (Clark et al. 1995; Fernandez and Lopez-Fanjul 1996), mutation induction experiments (Keightley and Ohnishi 1998), artificial selection experiments (Long et al. 1995), and transposable element insertions (Rollmann et al. 2006). For pleiotropy in particular, major-effect genes that have consequences on several phenotypic traits are well known from the domestication and livestock breeding literature [e.g., myostatin mutations in Belgian blue cattle and whippets (Arthur 1995; Grobet et al. 1997; Mosher et al. 2007), halothane genes in pigs (Christian and Rothschild 1991; Fujii et al. 1991), and Booroola and Inverdale genes in sheep (Amer et al. 1999; Visscher et al. 2000)]. While these data suggest that variation at major-effect genes could—and probably does—influence variation, covariation, and selection on quantitative traits, data on the magnitude of these consequences remain lacking.Recombinant inbred line (RIL) populations are a promising tool for investigating the influence of major-effect loci. During advancement of the lines from F₂''s to RILs, alternate alleles at major-effect genes (and most of the rest of the genome) will be made homozygous, simplifying comparisons among genotypic classes. Because of the high homozygosity, individuals within RILs are nearly genetically identical, facilitating phenotyping of many genotypes under a range of environments. In addition, because of recombination, alternative alleles are randomized across genetic backgrounds—facilitating robust comparisons between sets of lines differing at a major-effect locus.Here we investigate how polymorphism at an artificially induced mutation, the erecta locus in Arabidopsis thaliana, affects the magnitude of these important evolutionary genetic parameters under ecologically realistic field conditions. We use the Landsberg erecta (Ler) × Columbia (Col) RIL population of A. thaliana to examine how variation at a gene of major effect influences genetic variation, covariation, and selection on quantitative traits in a field setting. The Ler × Col RIL population is particularly suitable, because it segregates for an artificially induced mutation at the erecta locus, which has been shown to influence a wide variety of plant traits. The Ler × Col population thus allows a powerful test of the effects of segregating variation at a gene—chosen a priori—with numerous pleiotropic effects. The ERECTA gene is a leucine-rich receptor-like kinase (LRR-RLK) (Torii et al. 1996) and has been shown to affect plant growth rates (El-Lithy et al. 2004), stomatal patterning and transpiration efficiency (Masle et al. 2005; Shpak et al. 2005), bacterial pathogen resistance (Godiard et al. 2003), inflorescence and floral organ size and shape (Douglas et al. 2002; Shpak et al. 2003, 2004), and leaf polarity (Xu et al. 2003; Qi et al. 2004).Specifically, we sought to answer the following questions: (1) Is variation at erecta significantly associated with changes to the G matrix? (2) Is variation at erecta associated with changes in natural selection on genetically variable traits? And (3) is variation at erecta associated with significantly different projected evolutionary responses to selection?     

The Effect of Recent Admixture on Inference of Ancient Human Population History

Despite the widespread study of genetic variation in admixed human populations, such as African-Americans, there has not been an evaluation of the effects of recent admixture on patterns of polymorphism or inferences about population demography. These issues are particularly relevant because estimates of the timing and magnitude of population growth in Africa have differed among previous studies, some of which examined African-American individuals. Here we use simulations and single-nucleotide polymorphism (SNP) data collected through direct resequencing and genotyping to investigate these issues. We find that when estimating the current population size and magnitude of recent growth in an ancestral population using the site frequency spectrum (SFS), it is possible to obtain reasonably accurate estimates of the parameters when using samples drawn from the admixed population under certain conditions. We also show that methods for demographic inference that use haplotype patterns are more sensitive to recent admixture than are methods based on the SFS. The analysis of human genetic variation data from the Yoruba people of Ibadan, Nigeria and African-Americans supports the predictions from the simulations. Our results have important implications for the evaluation of previous population genetic studies that have considered African-American individuals as a proxy for individuals from West Africa as well as for future population genetic studies of additional admixed populations.STUDIES of archeological and genetic data show that anatomically modern humans originated in Africa and more recently left Africa to populate the rest of the world (Tishkoff and Williams 2002; Barbujani and Goldstein 2004; Garrigan and Hammer 2006; Reed and Tishkoff 2006; Campbell and Tishkoff 2008; Jakobsson et al. 2008; Li et al. 2008). Given the central role Africa has played in the origin of diverse human populations, understanding patterns of genetic variation and the demographic history of populations within Africa is important for understanding the demographic history of global human populations. The availability of large-scale single-nucleotide polymorphism (SNP) data sets coupled with recent advances in statistical methodology for inferring parameters in population genetic models provides a powerful means of accomplishing these goals (Keinan et al. 2007; Boyko et al. 2008; Lohmueller et al. 2009; Nielsen et al. 2009).It is important to realize that studies of African demographic history using genetic data have come to qualitatively different conclusions regarding important parameters. Some recent studies have found evidence for ancient (>100,000 years ago) two- to fourfold growth in African populations (Adams and Hudson 2004; Marth et al. 2004; Keinan et al. 2007; Boyko et al. 2008). Other studies have found evidence of very recent growth (Pluzhnikov et al. 2002; Akey et al. 2004; Voight et al. 2005; Cox et al. 2009; Wall et al. 2009) or could not reject a model with a constant population size (Pluzhnikov et al. 2002; Voight et al. 2005). It is unclear why studies found such different parameter estimates. However, these studies all differ from each other in the amount of data considered, the types of data used (e.g., SNP genotypes vs. full resequencing), the genomic regions studied (e.g., noncoding vs. coding SNPs), and the types of demographic models considered (e.g., including migration vs. not including migration postseparation of African and non-African populations).Another important way in which studies of African demographic history differ from each other is in the populations sampled. Some studies have focused on genetic data from individuals sampled from within Africa (Pluzhnikov et al. 2002; Adams and Hudson 2004; Voight et al. 2005; Keinan et al. 2007; Cox et al. 2009; Wall et al. 2009), while other studies included American individuals with African ancestry (Adams and Hudson 2004; Akey et al. 2004; Marth et al. 2004; Boyko et al. 2008). While there is no clear correspondence between those studies which sampled native African individuals (as opposed to African-Americans) and particular growth scenarios, it is clear from previous studies that African-American populations do differ from African populations in their recent demographic history. In particular, genetic studies suggest that there is wide variation in the degree of European admixture in most African-American individuals in the United States and that they have, on average, ∼80% African ancestry and 20% European ancestry (Parra et al. 1998; Pfaff et al. 2001; Falush et al. 2003; Patterson et al. 2004; Tian et al. 2006; Lind et al. 2007; Reiner et al. 2007; Price et al. 2009; Bryc et al. 2010). Furthermore, both historical records and genetic evidence suggest that the admixture process began quite recently, within the last 20 generations (Pfaff et al. 2001; Patterson et al. 2004; Seldin et al. 2004; Tian et al. 2006). Recent population admixture can alter patterns of genetic variation in a discernible and predictable way. For example, recently admixed populations will exhibit correlation in allele frequencies (i.e., linkage disequilibrium) among markers that differ in frequency between the parental populations. This so-called admixture linkage disequilibrium (LD) (Chakraborty and Weiss 1988) can extend over long physical distances (Lautenberger et al. 2000) and decays exponentially with time the since the admixture process began (i.e., recently admixed populations typically exhibit LD over a longer physical distance than anciently admixed populations).While it is clear that African-American populations have a different recent demographic history than do African populations from within Africa and that admixture tracts can be identified in admixed individuals (Falush et al. 2003; Patterson et al. 2004; Tang et al. 2006; Sankararaman et al. 2008a,b; Price et al. 2009; Bryc et al. 2010), the effect that admixture has on other patterns of genetic variation remains unclear. For example, Xu et al. (2007) found similar LD decay patterns when comparing African-American and African populations. It is also unclear whether the recent admixture affects our ability to reconstruct ancient demographic events (such as expansions that predate the spread of humans out of Africa) from whole-genome SNP data. Most studies of demographic history have summarized the genome-wide SNP data by allele frequency or haplotype summary statistics. If these summary statistics are not sensitive to the recent European admixture, then the African-American samples may yield estimates of demographic parameters that are close to the true demographic parameters for the ancestral, unsampled, African populations. This would suggest that the differences in growth parameter estimates obtained from African populations cannot be explained by certain studies sampling African-American individuals and others sampling African individuals from within Africa. However, if these statistics are sensitive to recent admixture, then they may give biased estimates of growth parameters.Here, we examine the effect of recent admixture on the estimation of population demography. In particular, we estimate growth parameters from simulated data sets using SNP frequencies as well as a recently developed haplotype summary statistic (Lohmueller et al. 2009). We compare the demographic parameter estimates made from the admixed and nonadmixed populations and find that some parameter estimates are qualitatively similar between the two populations when inferred using allele frequencies. Inferences of growth using haplotype-based approaches appear to be more sensitive to recent admixture than inferences based on SNP frequencies. We discuss implications that our results have for interpreting studies of demography in admixed populations.     

Deletion of a Novel F-Box Protein,MUS-10, in Neurospora crassa Leads to Altered Mitochondrial Morphology,Instability of mtDNA and Senescence

While mitochondria are renowned for their role in energy production, they also perform several other integral functions within the cell. Thus, it is not surprising that mitochondrial dysfunction can negatively impact cell viability. Although mitochondria have received an increasing amount of attention in recent years, there is still relatively little information about how proper maintenance of mitochondria and its genomes is achieved. The Neurospora crassa mus-10 mutant was first identified through its increased sensitivity to methyl methanesulfonate (MMS) and was thus believed to be defective in some aspect of DNA repair. Here, we report that mus-10 harbors fragmented mitochondria and that it accumulates deletions in its mitochondrial DNA (mtDNA), suggesting that the mus-10 gene product is involved in mitochondrial maintenance. Interestingly, mus-10 begins to senesce shortly after deletions are visualized in its mtDNA. To uncover the function of MUS-10, we used a gene rescue approach to clone the mus-10 gene and discovered that it encodes a novel F-box protein. We show that MUS-10 interacts with a core component of the Skp, Cullin, F-box containing (SCF) complex, SCON-3, and that its F-box domain is essential for its function in vivo. Thus, we provide evidence that MUS-10 is part of an E3 ubiquitin ligase complex involved in maintaining the integrity of mitochondria and may function to prevent cellular senescence.THE mus-10 mutant was isolated from a screen aimed at identifying Neurospora crassa strains that were sensitive to MMS and therefore likely to lack proper DNA repair mechanisms (Kafer and Perlmutter 1980). Epistasis analyses involving mus-10 suggested that it belonged to the uvs-6 epistasis group, which functions in recombination repair (Kafer and Perlmutter 1980; Kafer 1983). However, mus-10 did not display several phenotypes common to other members of the uvs-6 epistasis group: chromosomal instability, a high sensitivity to histidine, and the inability to produce viable ascospores in homozygous crosses (Newmeyer et al. 1978; Newmeyer and Galeazzi 1978; Kafer and Perlmutter 1980; Kafer 1981; Schroeder 1986; Watanabe et al. 1997; Handa et al. 2000; Sakuraba et al. 2000). Furthermore, the frequencies of spontaneous and radiation-induced mutation observed in mus-10 were similar to those of a wild-type strain (Kafer 1981). Past efforts to uncover the nature of these discrepancies or the function of the mus-10 gene product have been uninformative.The majority of cellular ATP is produced in mitochondria through aerobic respiration, which couples electron flow through respiratory complexes within the mitochondrial inner membrane with oxidative phosphorylation. Besides their role in ATP synthesis, mitochondria are also involved in many other cellular processes including beta-oxidation (Bartlett and Eaton 2004), calcium homeostasis (Gunter et al. 2004; Rimessi et al. 2008), production of iron-sulfur clusters (Zheng et al. 1998; Gerber and Lill 2002; Lill and Muhlenhoff 2005; Rouault and Tong 2005), and apoptosis (Green 2005; Antignani and Youle 2006; Xu and Shi 2007). Although virtually all mitochondrial proteins are encoded within the nucleus, a small number of proteins are encoded by mitochondrial DNA (mtDNA). The integrity of the mitochondrial genome may affect cell survival as mutations in mtDNA accumulate in patients suffering from severe neurological diseases including Alzheimer''s, Huntington''s and Parkinson''s, as well as several types of cancer (Chatterjee et al. 2006; Higuchi 2007; Krishnan et al. 2007; Reeve et al. 2008). The number of mtDNA mutations also increases with age, suggesting a link between mitochondrial dysfunction and ageing (Cortopassi and Arnheim 1990; Corral-Debrinski et al. 1992; Cortopassi et al. 1992; Simonetti et al. 1992; Reeve et al. 2008). Contrary to the single genome in the nucleus, there are several copies of mtDNA in each mitochondrion. Thus, defects in a few mitochondrial genomes do not necessarily lead to mitochondrial dysfunction. Many patients suffering from mitochondrial diseases exhibit heteroplasmy, a phenomenon in which a mixture of wild-type and mutant mtDNAs exist in a single cell. The ratio of wild-type to mutant mtDNAs is critical in determining the penetrance of the genetic defect, where mutant loads >60% are required to cause respiratory chain dysfunction within an individual cell (Boulet et al. 1992; Chomyn et al. 1992; Sciacco et al. 1994).Even though N. crassa strains are generally deemed immortal if they can be subcultured ∼50 times, a wild-type strain was recently reported to senesce after 12,000 hr of growth, implying that this fungus undergoes natural or programmed ageing (Maheshwari and Navaraj 2008; Kothe et al. 2010). However, replicative life span is also influenced by genetic background as certain mutations can cause progressive deterioration of growth, ultimately leading to death. One such example is the nuclear-encoded natural death (nd), which when mutant causes a senescence phenotype correlating with the accumulation of multiple mtDNA deletions (Sheng 1951; Seidel-Rogol et al. 1989). The deletions of mtDNA in nd occurred between two 70- to 701-bp direct repeats, suggesting that the nd gene product regulates recombination, repair, or replication of mtDNA (Bertrand et al. 1993). Another nuclear mutation, senescence (sen), was isolated from N. intermedia and introgressed into N. crassa (Navaraj et al. 2000). Deletions were also observed in the mtDNA of sen mutants, but unlike those occurring in nd were flanked by 6- to 10-bp repeats typically associated with GC-rich palindromic sequences (D''Souza et al. 2005). The nature of the sequences that flanked the mtDNA deletions in these two mutants supported the existence of two distinct systems of mtDNA recombination in N. crassa: a general system of homologous recombination (system I) and a site-specific mechanism (system II), mediated in part by nd and sen, respectively (Bertrand et al. 1993; D''Souza et al. 2005). The nd and sen mutations have been mapped to linkage groups I and V, respectively, but neither gene has been cloned and the precise function of their gene products remains unclear. Two ultraviolet (UV)-sensitive mutants, uvs-4 and uvs-5, are thought to undergo senescence, but unfortunately, these strains have not been studied in great detail (Schroeder 1970; Perkins et al. 1993; Hausner et al. 2006). Premature senescence has also been observed in cytoplasmic mutants of N. crassa including the E35 and ER-3 stopper mutants that harbor large mtDNA deletions, as well as strains that accumulate mitochondrial plasmids capable of inserting into mtDNA through homologous recombination (de Vries et al. 1986; Akins et al. 1989; Myers et al. 1989; Niagro and Mishra 1989; Court et al. 1991; Alves and Videira 1998).While trying to establish the role of MUS-10 in DNA repair, we discovered that the mus-10 mutant exhibited a shortened life span, an abnormal mitochondrial morphology and mtDNA instability. We cloned the mus-10 gene through its ability to complement the MMS sensitivity of the mus-10 mutant and revealed that it encoded a novel F-box protein. This suggested that MUS-10 is part of an Skp, Cullin, F-box containing (SCF) E3 ubiquitin ligase complex that targets proteins for degradation by the 26S proteasome. The data we present in this article offer proof that an SCF complex can regulate both mitochondrial maintenance and cellular senescence.     

Does Gene Translocation Accelerate the Evolution of Laterally Transferred Genes?

Lateral gene transfer (LGT) and gene rearrangement are essential for shaping bacterial genomes during evolution. Separate attention has been focused on understanding the process of lateral gene transfer and the process of gene translocation. However, little is known about how gene translocation affects laterally transferred genes. Here we have examined gene translocations and lateral gene transfers in closely related genome pairs. The results reveal that translocated genes undergo elevated rates of evolution and gene translocation tends to take place preferentially in recently acquired genes. Translocated genes have a high probability to be truncated, suggesting that translocation followed by truncation/deletion might play an important role in the fast turnover of laterally transferred genes. Furthermore, more recently acquired genes have a higher proportion of genes on the leading strand, suggesting a strong strand bias of lateral gene transfer.GENE insertions and deletions, together with gene translocations play important roles in bacterial genome evolution (Garcia-Vallvé et al. 2000; Ochman and Jones 2000; Tillier and Collins 2000a; Fraser-Liggett 2005). Gene insertions and deletions, as the essential driving forces in influencing gene content (Kunin and Ouzounis 2003), have received a great deal of attention. Various methods have been employed to study gene insertions and deletions previously; for instance, there are studies of population dynamics (Nielsen and Townsend 2004), such as a birth-and-death model of evolution (Berg and Kurland 2002; Novozhilov et al. 2005), phylogeny-dependent studies including parsimony methods (Daubin et al. 2003a,b; Mirkin et al. 2003; Hao and Golding 2004), and maximum-likelihood methods (Hao and Golding 2006b, 2008b). It has been shown that recently laterally transferred genes have high evolutionary rates and high rates of gene turnover (Daubin et al. 2003b; Hao and Golding 2004, 2006b).Gene rearrangement has also been commonly studied as another important driving force that shapes bacterial genomes (for a review, see Rocha 2004). Gene order changes in genomes are history dependent; for instance, fewer gene rearrangements are expected among more closely related species. Gene order within genomes has therefore been used to reconstruct phylogeny (Sankoff et al. 2000; Tamames 2001; Rogozin et al. 2004; Belda et al. 2005). Previous studies have focused mainly on lateral gene transfer (LGT) and gene rearrangement individually, but little is known about any association between laterally transferred genes and gene rearrangements. The study of gene order of laterally acquired genes might shed some light on the understanding of the LGT process.In this study, we have examined gene translocations and lateral gene transfers in closely related genome pairs. It is shown that the proportion of translocated genes among recently acquired genes is always high, while the proportion of translocated genes is always low in ancient genes, suggesting that gene translocation tends to take place in recently transferred genes. The results also reveal that translocated genes have elevated rates of evolution compared with positionally conserved genes and gene truncation is more prevalent in translocated genes. These findings suggest that gene translocation might accelerate the gene turnover of recently transferred genes and/or that genes likely to undergo translocation are those genes more likely to be laterally transferred and dispensable for the genome. Furthermore, the proportion of recently acquired genes is higher on the leading strand, suggesting that laterally transferred genes are biased toward being on the leading strand. After lateral transfer, some genes could be translocated to the lagging strand and some translocated genes are likely to be eliminated during evolution.     

Phenotypic Consequences of Aneuploidy in Arabidopsis thaliana

Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.THE genome of aneuploid individuals contains incomplete chromosome sets. The balance between chromosome types, and the genes they encode, is compromised, resulting in altered expression of many genes, including genes with dosage-sensitive effects on phenotypes. In humans, only a few types of aneuploid karyotypes are viable (Hassold and Hunt 2001), highlighting the deleterious effect of chromosome imbalance. The most commonly known viable form of aneuploidy in humans is Down syndrome, which results from a trisomy of chromosome 21 in an otherwise diploid background. Down syndrome patients exhibit many specific phenotypes, sometimes visible only in a subset of patients (Antonarakis et al. 2004). For phenotypes found in all Down syndrome patients, the penetrance of each phenotype varies between patients (Antonarakis et al. 2004). Despite the increasing amount of information available about the human genome and the availability of a mouse model for Down syndrome (O''Doherty et al. 2005), the genes responsible for most of the phenotypes associated with Down syndrome are still unknown (Patterson 2007; Korbel et al. 2009; Patterson 2009). Recently, detailed phenotypic analyses of as many as 30 aneuploid patients have allowed the identification of susceptibility regions for several specific phenotypes (Patterson 2007, 2009; Korbel et al. 2009; Lyle et al. 2009), but the specific genes remain to be identified. Understanding the physiology of aneuploidy is not only relevant to those individuals with aneuploid genomes but also to understanding cancer since most cancerous cells are aneuploid (Matzke et al. 2003; Pihan and Doxsey 2003; Storchova and Pellman 2004; Holland and Cleveland 2009; Williams and Amon 2009) or the consequences of copy number variation and dosage sensitivity (Dear 2009; Henrichsen et al. 2009).Plants are more tolerant of aneuploidy than animals (Matzke et al. 2003) for reasons that remain unclear. Since the discovery of the Datura trisomic “chromosome mutants” by Blakeslee (1921, 1922), viable trisomics of each chromosome type have been described in numerous species. Trisomics exhibit phenotypes specific to the identity of the triplicated chromosome (Blakeslee 1922; Khush 1973; Koornneef and Van der Veen 1983; Singh 2003). More complex aneuploids, i.e., individuals carrying more than one additional chromosome, can be viable as well and have been observed in many plants species, especially among the progeny of triploid individuals (McClintock 1929; Levan 1942; Johnsson 1945; Khush 1973). Some species appear to be more tolerant of complex aneuploidies than others, suggesting a genetic basis for aneuploidy tolerance (Satina and Blakeslee 1938; Khush 1973; Ramsey and Schemske 2002; Henry et al. 2009). Aneuploid individuals frequently appear spontaneously within polyploid plant populations, presumably due to a failure to equally partition the multiple chromosome sets at meiosis (Randolph 1935; Doyle 1986). These aneuploids exhibit few or subtle phenotypic abnormalities and can often compete with their euploid progenitors (Ramsey and Schemske 1998). Plants therefore provide an excellent opportunity for a genome-wide investigation of aneuploid syndromes: sample size is not limited, phenotypes can be described and assessed in detail, and plant aneuploid populations provide a complex mixture of viable karyotypes.In this article, we report our investigation of the relationship between phenotype and karyotype in populations of aneuploid Arabidopsis thaliana plants. All simple trisomics of A. thaliana have been previously isolated and phenotypically characterized (Steinitz-Sears 1962; Lee-Chen and Steinitz-Sears 1967; Steinitz-Sears and Lee-Chen 1970; Koornneef and Van der Veen 1983), demonstrating that they are tolerated in A. thaliana. We previously reported that aneuploid swarms—populations of aneuploid individuals of varying aneuploid karyotypes—could be obtained from the progeny of triploid A. thaliana individuals (Henry et al. 2005, 2009). Using a combination of a quantitative PCR-based method and flow cytometry, we were able to derive the full aneuploid karyotype of each of these individuals (Henry et al. 2006). We further crossed triploid A. thaliana to diploid or tetraploid individuals and demonstrated that at least 44 of the 60 possible aneuploid karyotypes that could result from these crosses (aneuploid individuals carrying between 11 and 19 chromosomes) were viable and successfully produced adult plants. Taken together, these populations and methods make it possible to explore the basis of aneuploid syndromes in A. thaliana. In this study, we were able to phenotypically characterize at least one individual from 25 different aneuploid karyotypes falling between diploidy and tetraploidy. We demonstrated that specific phenotypes are affected by the dosage of specific chromosome types. The effect of the dosage of specific chromosome types on traits was additive and could be used to predict the observed phenotype. The availability of multiple generations of aneuploid and euploid individuals allowed us to investigate potential long-term effects of aneuploidy as well as parent-of-origin effects on aneuploid phenotypes.