Plant Physiology Preview. Published on October 18, 2013, as DOI:10.1104/pp.113.222448
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RUNNING TITLE
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Unreduced pollen formation in apomicts
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CORRESPONDING AUTHOR
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Timothy F. Sharbel
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Department of Cytogenetics and Genome Analyses, Leibniz Institute of Plant Genetics and
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Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany
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Telephone number: +49 (0)39482 5608
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Email adress: [email protected]
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Research area: Genes, Development and Evolution
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TITLE
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The conserved chimeric transcript UPGRADE-2 is associated with unreduced pollen
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formation and is exclusively found in apomictic Boechera
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AUTHORS
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Martin Mau1, José M. Corral1, Heiko Vogel2, Michael Melzer1, Jörg Fuchs1, Markus
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Kuhlmann1, Nico de Storme3 , Danny Geelen3 and Timothy F. Sharbel1,4
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Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben,
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Germany
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Genomics Research Group, Department of Entomology, Max Planck Institute for Chemical
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Ecology, D-07745 Jena, Germany
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Coupure links 653, 9000 Gent, Belgium
Plant
Production,
Faculty
of
Bioscience
Engineering,
Ghent
University,
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ONE-SENTENCE SUMMARY
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A conserved chimeric genomic sequence found exclusively in apomictic Boechera generates a
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putative long non-protein coding transcript with secondary structure folding capability, and is
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highly expressed in antherheads characterized by meiotically unreduced pollen formation.
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FINANCIAL SOURCE
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(1) Apomixis research group by the Leibniz Institute of Plant Genetics and Crop Plant
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Research (IPK)
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(2) EU COST action “Harnessing Plant Reproduction for Crop Improvement” (FA0903)
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CORESPONDING AUTHOR
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Timothy F. Sharbel
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Email adress: [email protected]
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ABSTRACT
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In apomictic Boechera meiotic diplospory leads to the circumvention of meiosis and
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suppression of recombination to produce unreduced male and female gametes (i.e.
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apomeiosis). Here we have established an early flower developmental staging system and
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have performed microarray-based comparative gene expression analyses of the pollen mother
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cell (PMC) stage in seven diploid sexual and seven diploid apomictic genotypes to identify
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candidate factors for unreduced pollen formation. We identified a transcript unique to
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apomictic Boechera called UPGRADE-2 (BspUPG-2), which is highly-upregulated in their
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pollen mother cells. BspUPG-2 is highly conserved among apomictic Boechera genotypes but
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has no homologue in sexual Boechera or in any other taxa. BspUPG-2 undergoes post-
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transcriptional processing but lacks a prominent open reading frame. Together with the
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potential of stably forming miRNA-like secondary structures, we hypothesize that BspUPG-2
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functions as long regulatory non-coding mRNA-like RNA. BspUPG-2 has apparently arisen
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through a three-step process initiated by ancestral gene duplication of the original BspUPG-1
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locus, followed by sequential insertions of segmentally duplicated gene fragments, with final
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exonization of its sequence structure. Its genesis reflects the hybridization history which
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characterizes the genus Boechera.
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INTRODUCTION
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Haploid gamete formation is an important step during the diplohaplontic plant life cycle
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which marks the switch from sporophyte to gametophyte. During female and male
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sporogenesis, the diploid megaspore mother cell (MMC) and pollen mother cell (PMC)
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respectively undergo one reductional and equational mitotic division to form four haploid
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daughter cells. During subsequent male spore development (microgametogenesis), each of the
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four microspores undergoes two mitotic divisions to form a tricellular pollen grain containing
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two mature reduced gametes (generative nuclei) and one vegetative nucleus.
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Despite a relatively simple structure, the male gametophyte is regulated by complex
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gamete-specific molecular programs which are characterized by (1) enrichment of transcripts
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responsible for DNA repair, cell cycle and chromosome organization, (2) underrepresentation
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of the RNA-processing machinery (Pina et al., 2005; Borges et al., 2008), and (3)
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developmental stage-specific transcript sets whose activities vary temporally from the
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uninuclear microspore towards the mature pollen grain (Mascarenhas, 1989; Honys and
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Twell, 2004).
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Loss of function studies of male and female meiosis have revealed cell cycle defects (Yang
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et al., 2003), effects on homeologous chromosome recombination (Lu et al., 2008), and
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defects in meiotic cell fate (Sundaresan et al., 1995; Yang et al., 1999), all of which lead to
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reduced meiocyte fertility or death. Other mutations lead to unreduced (2C) gametes
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(Brownfield and Köhler, 2010).
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Apomeiosis is a naturally occurring form of sensu stricto unreduced egg formation, which
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is the first step of gametophytic apomixis, or asexual (clonal) seed formation in plants
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(Nogler, 1984; Grimanelli et al., 2001). During apomixis the apomeiotically-derived egg-cell
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undergoes parthenogenetic (i.e. without fertilization) development, while endosperm develops
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with (pseudogamous) or without (autonomous) fertilization (Nogler, 1984; Grimanelli et al.,
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2001). Apomixis bears the potential to fix hybrid genotypes over generations and is therefore
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of high interest to agronomy (Grimanelli et al., 2001; Grossniklaus, 2001)
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Apomixis research can be divided into studies of de novo induction of apomixis-like
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processes in sexual plants, or naturally-occurring apomixis (Savidan et al., 2001). Mutant
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screening and attempts to introgress apomixis into sexual taxa have been hindered by its
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molecular genetic complexity (d’Erfurth et al., 2008; Hörandl and Temsch, 2009; Marimuthu
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et al., 2011). Comparative mapping strategies in natural apomicts, with an emphasis on
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aposporic species, have identified candidate factors, although proof of function in crop plants
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is still pending (Guerin et al., 2000; Albertini et al., 2004; Matzk et al., 2005; Schallau et al.,
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2010). Beside theories of apomixis inheritance through a single dominant locus (Mogie, 1988)
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or via a complex of physically-linked coadapted genes (van Dijk et al., 1999), the
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hybridization-derived floral asynchrony (HFA) theory proposes deregulation of sexual genetic
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pathways via asynchronous expression of duplicated gene sets as a mechanism for apomixis
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induction (Carman, 1997). Reproductive deregulation was recently supported by
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heterochronic expression patterns during ovule development in sexual and apomictic
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Boechera (Sharbel et al., 2010), pointing to a network of epigenetic and post-transcriptional
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regulation during germline specification (Twell, 2010).
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The approximately 110 species comprising the North-American genus Boechera Á. Löve
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& D. Löve (Brassicaceae) exist as diploid sexual or diploid and allopolyploid apomictic forms
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(Böcher, 1951; Al-Shehbaz and Windham, 1993+), the latter of which are highly correlated
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with interspecific hybridization (Sharbel et al., 2009; Sharbel et al., 2010; Beck et al., 2011).
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Diploid apomixis is rare, and facilitates comparisons between sex and apomixis without the
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added complexity of differing ploidy (Sharbel et al., 2009; 2010). Apomictic Boechera taxa
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are characterized by Taraxacum-type pseudogamous diplospory (Rollins, 1941; Böcher,
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1951). Besides the formation of unreduced female gametes, apomictic Boechera also produce
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unreduced pollen, as is demonstrated by the fact that diploids and triploids produce seeds
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almost exclusively with hexaploid (6C = [4Cmaternal] + [2Cpaternal]) and nonaploid (9C = [6Cm]
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+ [3Cp]) endosperm (Voigt et al., 2007; Aliyu et al., 2010; Voigt-Zielinski et al., 2012).
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Hence, strong selection pressure to maintain a balanced 2 maternal : 1 paternal genome ratio
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(i.e. endosperm balance number, EBN, Johnston et al. (1980)) during endosperm formation
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apparently characterizes apomictic Boechera (Johnston et al., 1980; Voigt et al., 2007; Aliyu
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et al., 2010). While we have thus far focused our transcriptome comparisons on sexual and
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apomictic ovules (Sharbel et al., 2009; Sharbel et al., 2010), strong selection for genetically
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balanced endosperm during apomictic seed formation has led us to study unreduced pollen
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formation.
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Hence, we have undertaken a microarray-based transcriptome comparison of a single stage
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of antherhead development differentiating reduced and unreduced pollen formation between
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diploid sexual and diploid apomictic Boechera. Here, we describe the isolation and
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characterization of a differentially-expressed candidate gene, BspUPG-2 (Boechera species
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UPGRADE-2, unreduced pollen grain development), which is associated with unreduced
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pollen formation and is potentially part of a homology dependent gene silencing mechanism.
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BspUPG-2 has apparently arisen through gene duplication and subsequent insertions of genic
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and non-genic fragments in the hybrid apomictic genome. Its putative molecular function in
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apomictic genomes of Boechera is discussed.
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RESULTS
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Apomictic Boechera produce (FDR type) unreduced gametes
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One major mechanism which leads to unreduced pollen formation is the meiotic nuclear
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restitution which comprises a failure during the first (FDR) or second meiotic division (SDR).
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Meiotic chromosome behavior (Ross et al., 1996) was examined to determine the exact
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mechanism by which unreduced pollen is formed in facultative and obligate apomictic
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Boechera, and showed that pollen formation differed between apomicts and sexuals (Fig. 1,
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Table S1). The sexual B. stricta ES 612.1 showed expected homologous chromosome pairing,
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with juncture of non-sister chromatids leading to seven bivalents (Fig. 1A). Subsequent two
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nuclear divisions lead to a tetrad with four haploid nuclei (Figs. 1B-G). In contrast, the
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aneuploid obligate apomictic B. polyantha (ES 776.2, 2n = 2x = 15) and the euploid B.
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lignifera (ES 753, 2n = 2x = 14) exhibited complete or partial chromosomal asynapsis
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resulting in univalents at metaphase I which do not (or only partially) segregate during
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meiosis I (Figs. 1I, J and P, Q). As a result, metaphase II plates were frequently fused (Figs.
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1K and R) in contrast to their perpendicular orientation in the sexual reference accession (Fig.
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1D), leading to the generation of dyads with balanced and unreduced chromosome numbers
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(Figs. 1L, M and S, T). Low levels of chromosomal synapsis leading to tetrad formation were
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also observed in all examined diploid obligate apomicts (Figs. 1N and U). Compared to
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obligate apomicts the facultative apomictic B. divaricarpa ES 514 exhibited higher levels of
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nuclei with bivalents (Figs. 1W and Y) which proceeded through both meiotic cell divisions
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(Figs. 1A’, C’ and E’), generating tetrads with four haploid cells (Fig. 1G’). Nonetheless,
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nuclei with univalents were frequently observed (Figs. 1V, X and Z) whose sister chromatids
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were equally separate at metaphase II (Fig. 1B’) to develop balanced dyads (Figs. 1D’ and
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F’). Meiotic chromosome counts of all apomicts strongly support first division restitution
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(FDR) without crossover. Univalents remain together during meiosis I and disjoin in meiosis
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II, where sister chromatids are equationally separated to opposite poles, leading to two
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balanced diploid chromosomes sets in dyads.
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Approximately 28 000 meiocytes from single antherheads of six apomictic and a sexual
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reference genotype were counted to quantify meiotic product frequencies. The sexual
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reference genotype produced the expected high frequency of tetrads (88%), whereas the tetrad
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frequency varied among all tested apomicts (0 to 87%) between individuals of one genotype
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and even between different flower buds from the same individual (Fig. 2A, Table S2). Low
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levels of triads and tetrads were observed for apomictic genotypes ES 805.2 (0.55% and
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0.35%, respectively) and ES 776.2 (0.00%, respectively), both of which showed high levels of
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monad and dyad formation. However, surprisingly high levels of triads and tetrads were
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found for other individuals of both, obligate (300.9 - 24.98% and 57.98%; ES 753 - 32.78%
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and 62.16%) and facultative apomicts (ES 514 - 11.22% and 87.00%; ES 524.2 - 15.67% and
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64.53%, Fig. 2A). The formation of high levels of balanced dyads confirmed that unreduced
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pollen in apomictic Boechera is produced by a defect in chromosome segregation during
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meiosis I. However, unequal sister chromatid segregation during meiosis II, resulting in the
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formation of extra nuclei were low (in total 0.19% meiocytes with micronuclei and 0.02%
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polyads) and mainly detected in the apomict ES 753 (0.88% meiocytes with micronuclei, Fig.
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S1).
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Considering the highly variable pattern of meiocyte frequencies between individuals and
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between flowers of single specific apomictic genotypes (Fig. 2A), only plants producing
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exclusively unreduced pollen were chosen for comparative gene expression analyses. Plant
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selection was therefore based upon measurements of nuclear DNA content of pollen and seed
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nuclei for all individuals (examples in Fig. S1). Interestingly, flow cytometric seed screen
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(FCSS) data of all tested obligate apomictic individuals suggests successful fertilization with
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only unreduced pollen, although also some individuals produce high levels of reduced pollen
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(embryo to endosperm ratio of 2C [1Cm + 1Cp]: 6C [4Cm + 2Cp]; Fig. 2A, Fig. S1; Table
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S3).
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Staging of meiosis in Boechera
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To optimize for comparative gene expression analyses between reduced (sex) and
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unreduced (apomeiosis) pollen, the morphological development of antherheads was quantified
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through staging (sensu Sharbel et al., 2010). Analyses of early flower development from
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about 780 flower buds in six diploid Boechera genotypes (Figs. 2B and C, Fig. 3, Tables 1
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and S3) showed a linear relationship between bud length and flower organ size (e.g. stage S4
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to S12 antherheads: R2apo = 0.87, F = 1273.50, P < 0.001; R2sex = 0.95, F = 4237.23, P <
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0.001). Minor variations among genotypes, but no overall differences between sex and
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apomixis were detected (i.e. stages predict similar organ size classes; general linear model for
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regression line slopes (b) of stage S4 to S12 antherheads: H0: bapo=bsex; R2=0.95, F(44, 8) =
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0.68, P = 0.804; Fig. S2).
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We focused on four major histodifferentiation steps of male gametophyte development
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which are characterized by their pre-meiotic, meiotic and post-meiotic appearance (Fig. 1,
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Table 1). Despite some marker-specific variations in correlation strength, flower bud length
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predicts the gametophytic stage of anthers with relatively high accuracy (N = 455; Sp - 100%,
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PMC - 100%, Me - 52.27% and Msp - 58.24%, Fig. 2B). Antherhead length predicts the
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gametophytic stage of anther development, except for meiotic and microspore stages in
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sexuals, which overlapped in antherhead size class (one-way ANOVA with Tukey-HSD post
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hoc test, between *P < 10-2 and ***P < 10-9, Fig. 2C). The correlation between antherhead
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length and gametophyte stage led to the selection of 400 ± 30 µm antherheads, a stage which
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is significantly enriched for PMCs being close to meiosis in both sexual and apomictic
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genotypes, for collection of total RNA (***P < 0.001, Fisher’s exact test, Table S4; decision
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to take the PMC stage detailed in Materials and Methods section).
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Identification of the apo-specific UPGRADE transcript
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In search for candidate transcripts which are differentially expressed between the sexual
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and the apomictic microsporogenesis program, we performed pairwise comparisons of RNA
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expression levels in microdissected antherheads from 7 sexual and 7 apomictic genotypes
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using a custom Agilent microarray (detailed in “Material and Methods”). Using a foldchange
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threshold ≥ 2 and corrected P-value ≤ 0.05, while allowing one outlier sample, five
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microarray probes were identified to be highly upregulated in all apomicts, except in genotype
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ES 753 (B. lignifera), exhibiting absolute fold change (AFC) levels between 48.74 and 849.31
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compared to sexuals (Fig. S3; Table S5).
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Homologous cDNAs corresponding to the five candidate array probes were identified from
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a flower-specific cDNA library (Table S5; Sharbel et al. (2009)). A BLASTN (Altschul et al.,
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1997) search of these cDNAs against the whole GenBank nucleotide collection
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(http://www.ncbi.nlm.nih.gov/genbank/) revealed hits for only two of the candidate probes;
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probe Sharb1199059 (EMBL/GenBank/DDBJ No. ERS317552) was homologous to an
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Arabidopsis thaliana S-locus lectin protein kinase involved in pollen recognition, and probe
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Sharb0501554 (EMBL/GenBank/DDBJ No. ERS317556) matched two SRK genes (S-12 and
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S-15 type) encoding a Brassica oleracea S-locus receptor kinase involved in pollen self-
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recognition specificity (Table S5).
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Quantitative real-time RT-PCR confirmed that 4 of the 5 microarray probes had expression
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profiles corresponding to the microarray data (Fig. S4; Tables S6 and S7). The single probe
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which could not be qRT-PCR validated (Sharb1199059; lectin homolog) was furthermore
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characterized by a negative BAC screen result (Table S8), and was not considered for further
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analysis. With exception of the apomictic outlier sample (ES 753), the four microarray probes
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were highly upregulated in apomictic antherheads. In contrast to sexuals, in which the relative
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mRNA levels were close to the detection limit, ubiquitous expression of all probe sequences
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was detected in apomictic somatic and reproductive tissues. Moreover, for microarray probes
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Sharb0931225, Sharb0501554 and Sharb0690829 expression was substantially higher in
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antherheads compared to levels in leaf tissue (one-way ANOVA with Tukey-HSD post hoc
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test, P = 0.022, P = 0.008 and P = 0.027, respectively; Fig. S4; Table S7).
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Rapid amplification of cDNA ends (RACE) was performed using gene specific primers
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from microarray probe Sharb0690829 (Table S5) to obtain full-length cDNA including the 3`-
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and 5`-cDNA ends. Although total RNA of antherheads and whole flower tissue from two
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sexuals and eight apomicts was used, 5`- and 3`-RACE generated similar DNA fragments
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exclusively from apomicts, except for the apomictic outlier ES 753 (Fig. S5).
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All 3`- and 5`-RACE fragments from five apomicts were cloned and sequenced, and led to
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the identification of a single polyadenylated full-length transcript, BspUPG (Boechera species
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unreduced pollen grain development (UPGRADE); EMBL/GenBank/DDBJ No. HF930769;
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Fig. 4) with about 2648 nt length (for B. divaricarpa ES 524), excluding the poly(A)
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sequence. The microarray probes Sharb0931225, Sharb0501554 and Sharb0690829 were
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found in the most 3`-exon of the BspUPG transcript (Fig. 4). A comparison of different
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BspUPG full-length cDNA variants with genomic DNA by isolation of 3`-RACE and the
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various 5`-RACE fragments in different Boechera genotypes (Fig. S5, white arrows) showed
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that the gene has an overall length of 3156 nt (for B. divaricarpa ES 524) and contains two
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putative alternative splicing sites, 61 bp (Intron1) and 303 bp size (Intron2), in addition to a
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144 bp intron common to all apomicts (Intron3, Figs. 4 and S5). The U2-dependent classified
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introns (5`-GT/3`-AG splicing site; Simpson and Filipowicz (1996)) are located towards the
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5`-end of the transcript.
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UPGRADE is a chimeric gene encoding a transcript without translational capacity
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BspUPG fragments are homologous to sequences in Arabidopsis located on chromosomes
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1, 4 and 5, which encode respectively an Elongation factor TU/EF1-A protein (EFTU/EF1-A;
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AT4G02930), a RNA recognition motif-containing protein (RNAR; AT5G19960), and the
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HRD3 protein (HMG-coA Reductase Degradation) which is homologous to components of
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the yeast HRD1 complex (AT1G18260; Fig. 4). From these candidates, AtHRD3, which plays
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a central role in endoplasmic reticulum (ER)-associated protein degradation, exhibits the
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highest similarity to BspUPG, with fragments of 145 nt (83% identity, P-value < 5E-31), 118
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nt (87% identity, P-value < 6E-30) and 47 nt in length (89% identity, P-value < 0.0002).
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The transcript carries a poly(A)-tail with some variation in length and initiation position. In
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search for regulatory motifs on BspUPG, a putative near upstream element (NUE, AATAAA)
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of a polyadenylation signal, which is common in plants (Hunt, 1994), was identified 31 nt
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upstream of the polyadenylation site. Furthermore, a single CpG island was detected at the 5`-
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end of the transcript locus (+140 to 312 nt, C+G >60%; Figs. 4 and S6C). Sequences that are
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rich in the CpG/CpNpG patterns are predominantly nonmethylated and tend to be associated
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with genes which are frequently switched on (Deaton and Bird, 2011). A screen for putative
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regulatory features in the upstream sequences (between +47809 and +48921 nt on Assembly
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2, PlantPAN database, Chang et al., (2008); http://plantpan.mbc.nctu.edu.tw/) detected a
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single tandem repeat (-726 to -985 nt, 82 nt consensus size, 3.2 copies; Fig. 4) and multiple
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transcription factor binding site (TFBS; Table S9).
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Coding Potential Calculation of BspUPG (for ES 524; Kong et al. (2007)), excluding the
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constitutive intron 3, revealed a single short open reading frame (ORF) for each of both
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strands. The sense strand gave an ORF with 196 nt (66 amino acids, +895 to +1090 nt, coding
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potential score (cps) = -0.0298) and the reverse complement strand revealed a 202 nt sized
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ORF (68 aa, +1958 to +2159 nt, cps = -1.180). The lack of an ORF length typical for a protein
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encoding mRNA suggested a long non-protein-coding mRNA-like RNA (lncRNA) product of
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BspUPG (ORF typically >100 aa; Kondo (2007)).
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The genomic sequence information of the candidate locus was augmented by a screen of a
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B. divaricarpa bacterial artificial chromosome (BAC) library. Colony-PCR using target-
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specific primers from the chromosome walking approach (detailed in “Material and
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Methods”; Table S10) led to the identification of 12 Boechera BAC clones which hybridized
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with one or several probes simultaneously (Table S8). None of the clones were positive for
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probe Sharb1199059, whereas three single hits for probe Sharb0425060 and nine triple hits
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for probes Sharb0931225, Sharb0501554 and Sharb0690829 were detected, which confirmed
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the results from the RACE experiment (Fig. 4). Restriction digests of the twelve BACs
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suggested partial overlap of their DNA inserts. Sanger sequencing was thus performed on
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BAC clones A4O22, E7K5, C8B11 and F8G11, each being positive for three of five
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microarray probes (Sharb931225, Sharb501554 and Sharb690829). The C8B11 BAC
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sequence contig (Assembly 1, EMBL/GenBank/DDBJ No. HF954100), which could not be
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aligned together with the sequence contigs of the other three BAC clones, reached 57 458 bp
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length with 33.5% average GC content which increased to 40.1% in genic regions. BAC
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sequence contigs from clones A4O22, F8G11 and E7K5 overlapped and were assembled into
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the 58 769 bp Assembly 2 (EMBL/GenBank/DDBJ No. HF954101), with 32.9% average GC
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content increasing to 42.1% in genic regions.
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Annotation of Assembly 1 identified one transposon-related gene, five protein-encoding
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genes and two fragments of protein-encoding genes, all of which (except for TER4) were
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homologous to genes located on Arabidopsis chromosome 1 (Figs. 5A, S6A and B; Table
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S11). In contrast, Assembly 2 contained a higher level of transposon-related genes (3) and
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gene fragments (9) whose homologs were found on all Arabidopsis chromosomes except for
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chromosome 3 (Figs. 5A, S6A and C; Table S11). Comparison of both assemblies showed
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that both fragments share several highly similar sequences; e.g. for the two protein-encoding
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genes MtN21 and RRP4, for a fragment of the protein-encoding gene MBOAT and for the 3`-
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end of BspUPG. Allowing for rearrangements, Assembly 1 and Assembly 2 aligned along
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orthologous regions covering 27.15 kb of Assembly 1 and 46.20 kb of Assembly 2. Synteny
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gaps in Assembly 2 contained transposon-related genes (e.g. TER1 and TER2) and insertions
389
composed of short protein-encoding gene fragments flanked by inverted repeat (IR)
390
sequences (e.g. IR 7; Figs. 5A and S6C; Table S12), suggesting that parts of Assembly 2 arose
391
via partial duplications of Assembly 1. Hence, considering the partial presence of the
392
candidate gene at both loci, we subsequently labeled the original locus in Assembly 1,
393
BspUPG-1, and the duplicated variant on Assembly 2, BspUPG-2 (Figs. 5A and S6).
394
Both apomictic and sexual gene variants were aligned with Assembly 1 and Assembly 2
395
(i.e. BAC DNA sequences), allowing for rearrangements. The 3`-end of all gene variants from
396
sexuals and apomicts, here named locally collinear block 1 (LCB1 in violet, see definition in
397
“Materials and Methods”), shares high similarity with both assemblies. The 5`-ends of
398
apomictic gene copies (LCB2 in brown) were only identified on Assembly 2 (Figs. 5A, B and
399
S6). Unlike apomictic gene variants (now BspUPG-2, see nomenclature above), the 5`-ends
400
(LCBs 3 to 5) of variants from sexuals (now BspUPG-1) share high sequence similarity with
401
Assembly 1, but also exhibit a mosaic distribution of their LCBs relative to the duplicated
402
locus of Assembly 2 (Figs. S6 and S7).
403
404
Transcriptional activity of BspUPG-2 is restricted to apomictic Boechera
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13
405
The transcriptional functionality of BspUPG-1 and BspUPG-2 was inferred by mapping
406
sexual and apomictic cDNA (Sharbel et al., 2009) independently onto both, and revealed that
407
only the apo-specific LCB2 is transcribed in both sexuals and apomicts (see sexual/apomictic
408
cDNA reads, Fig. 5C). In contrast, apomictic cDNA maps to LCB1 and LCB2, including the
409
reads with homology to the candidate microarray 60-mer oligonucleotide probes
410
(Sharb931225, Sharb501554 and Sharb690829, Fig. 5C). No cDNA maps to any of the
411
remaining LCBs. PCR on genomic DNA using specific primers for both loci demonstrates the
412
presence of BspUPG-1 in sexuals and apomicts, but absence using cDNA as a PCR template.
413
In contrast genomic copies and transcripts of BspUPG-2 were solely identified in apomicts
414
(Figs. 5D and E).
415
BspUPG-1 is flanked by four genes centred in the centromeric region on the lower arm of
416
Arabidopsis chromosome 1 (Figs. 5A and S8, Table S11; ~ 0.57 megabases distance to T3P8-
417
sp6, Hosouchi et al. (2002)). Interestingly, two genetic markers (Bst006701 and BSTES0032)
418
from the synthetic F2 linkage map of sexual B. stricta (Schranz et al., 2007), both of which are
419
homologous to the Arabidopsis locus identifiers At1G51310 and At1G43245, flank BspUPG-
420
1. Both markers span the interval of genomic block C1, which localizes BspUPG-1 on the
421
BstLG1 linkage group of B. stricta for which very low levels of recombination were detected
422
(Schranz et al., 2007). The putative location of the duplicated BspUPG-2 is unknown.
423
424
BspUPG-2 has high secondary structure folding potential
425
Apart from other lncRNA types in plants, some lncRNA act as precursors for small non-
426
protein-coding RNAs (ncRNAs or npcRNAs) such as microRNAs (miRNAs) or endogenous
427
trans-acting small interfering RNAs (tasiRNAs), and may regulate translation in cis or trans
428
(Hirsch et al., 2006; Pikaard et al., 2008). A search for miRNA binding sites of known
429
Arabidopsis (www.mirbase.org, default parameter, 1 mismatch) and Boechera miRNAs
430
(Amiteye et al., 2011; Amiteye et al., 2013) revealed no mature miRNAs matching the
431
candidate transcript. Considering this, in addition to the obvious lack of a long ORF on
432
BspUPG-2, a bioinformatics approach was used to examine whether thermodynamically
433
stabile npcRNA structural elements could be predicted from its genomic sequence (detailed in
434
“Protocol S1”).
435
As shown in Figure 6A, the BspUPG forward and reverse strand contains eight regions
436
(i.e. overlapping windows are combined to determine the total length of a candidate region,
437
Table S13) characterized by significant Z-score values, suggesting that parts of BspUPG are
438
able to form stable secondary structures (Figs. 6A and S9). These candidate regions span only
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
14
439
exons, comprising together 25% and 27% of the total length of each strand of BspUPG
440
respectively, whereby npcRNAs 1/6, 2/7 and 4/8 cover similar regions on both strands of the
441
genomic sequence. The predicted secondary structures were classified based upon their (1)
442
minimal folding free energy, (2) adjusted minimal folding free energy (AMFE), (3) minimal
443
folding free energy index (MFEI), and (4) A+U content (Seffens and Digby, 1999; Bonnet et
444
al., 2004; Zhang et al., 2006). Interestingly, all detected secondary structures met most of the
445
criteria for miRNA precursors (Figs. 6B and S9, Table S14). Comparable with known pri-
446
miRNAs in Boechera (Amiteye et al., 2011; Amiteye et al., 2013) and other plant species
447
(Zhang et al., 2006), npcRNAs 1 to 8 demonstrate a lower minimal folding free energy
448
(ranging from -18.27 to -73.72 kcal/mol), have an elevated A+U level (ranging from 56.70 to
449
70.00%), and, with exception for npcRNA 5 (0.70), have a MFEI greater than 1.07 which is
450
much higher compared to transfer RNAs (tRNA, 0.64), ribosomal RNAs (rRNA, 0.59), or
451
random messenger RNAs (mRNA, 0.65).
452
A BLASTN search of the GenBank nucleotide and TAIR10 Genes databases showed that
453
three npcRNAs are significantly similar to known protein-coding genes (i.e. potential
454
regulatory targets). Two of these potential targets, npcRNA 5 and 8, encode for proteins with
455
ribosomal functions. The 70 nt-sized npcRNA 5 (position +1090 nt and +1160 nt on the
456
reverse strand of BspUPG) covers 75% of the third exon of the elongation factor EFTU/EF-
457
1A protein homolog in Arabidopsis, which binds tRNAs in a GTP-dependent reaction to the
458
acceptor site of ribosomes (AT1G18260, 92.86% similarity, P = 7.00E-24, Figs. 6B and C,
459
Table S14). A fragment of the npcRNA 8 maps to a homolog in Arabidopsis lyrata and A.
460
thaliana which is associated with a U3 small nucleolar RNA involved in the processing of
461
pre-rRNA (AT3G06530, P = 0.008). The third, npcRNA 3, maps to exon 25 of the myosin XI
462
B protein-coding gene (AT1G04160, P = 0.01), which belongs to a class of myosins
463
potentially involved in the process of spindle/phragmoplast alignment during cytokinesis
464
(Hepler et al., 2002), and whose mutation led to abnormal pollen development in Oryza
465
sativa, suggesting a crucial role of myosin XI B in pollen formation (Jiang et al., 2007).
466
467
BspUPG-2 is highly conserved in apomictic Boechera
468
Within-individual (i.e. allelic) polymorphisms were not identified for BspUPG-2 among
469
nine tested apomicts. Furthermore, both the original locus (BspUPG-1) and its duplicated
470
variant (BspUPG-2) are highly conserved at the 3`-end between all tested genotypes, which
471
decreases markedly towards the 5`-end (Fig. 7A, Table S15). Sequence conservation is high
472
between all apomicts (i.e. BspUPG-2), including the transcriptional outlier ES 753, with the
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15
473
highest nucleotide divergence between genotypes ES 753 and ES 514 (99.33% ± 0.13
474
similarity), while BspUPG-2 in all other apomicts shares complete sequence identity (Fig.
475
7A). In contrast, sexual sequences can be split into two subgroups based on low sequence
476
conservation at the 5`-end of BspUPG-1 (39.95% ± 0.02 similarity), one representing B.
477
stricta genotypes (99.69% ± 0.05 similarity between B. stricta genotypes), and the second
478
representing the remaining sexual genotypes (98.87% ± 0.30 similarity between non-B. stricta
479
genotypes; Fig. 7A, Table S15).
480
Among all indels which were detected between BspUPG-1 and BspUPG-2 (indels, <50
481
nucleotides, Albers et al. (2011), Table S16), a single 27 nt sequence was exclusively present
482
in BspUPG-1 but not in BspUPG-2 (Fig. 7A, indel 9). Interestingly, Chellappan et al. (2010)
483
identified a new 27-nt small RNA-species that is associated with AGO4 to regulate gene
484
expression at the transcriptional level by directing DNA methylation to some of their target
485
loci in trans. Hence, indel 9 was tested as a small RNA binding site by northern blot
486
screening of small RNAs from sexual and apomictic pooled flower buds using specific sense
487
and antisense probes (detailed in “Protocol S2”; Fig. S10). Expression of the highly
488
conserved plant miRNA167, which was used as positive control, was observed in all flower
489
tissues, whereas no signal was detected for any indel 9-specific probe in both apomictic and
490
sexual flower tissues (Fig. S10).
491
Together, the lack of intra-individual allelic variation and the absence of BspUPG-2 at any
492
other locus in both sexuals and apomicts (Fig. 5) point to a homo- or hemizygous state for
493
BspUPG-2 in apomicts.
494
495
BspUPG-2 arose via genome rearrangements
496
Two lines of evidence were used to validate the proposed fusion of the 5`- (LCB1) and 3`-
497
ends (LCB2) of the transcriptionally active BspUPG-2. A BLASTN search between BspUPG-
498
2 and the complete genomic Sequence Read Archive (SRA) of a sexual B. stricta identified a
499
gap at the juncture between all reads mapping to LCB1 and to LCB2, while all reads per LCB
500
overlapped, suggesting their separate genomic origins in the sexual genotype (position +1820
501
nt; Fig. 7C). We confirmed this result by amplifying a 478 bp-fragment in sexual genotypes
502
using primers corresponding to a fragment of the BspUPG-2-specific LCB2. Additionally,
503
this fragment is mapped in sexual and apomictic Boechera by cDNA belonging to the
504
Boechera homologue of AtHRD3 (BspHRD3; Figs. 4 and 7B).
505
BspHRD3 was used to test whether the 5`-end of BspUPG-2 derived directly from parental
506
genes or from putative duplicated variants. Although BspHRD3 cDNA is conserved with
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
16
507
Arabidopsis HRD3 (93.0%), variation (i.e. insertions) between the transcript and the detected
508
genomic copy, which are not caused by RNA splicing, was identified (Fig. 7B). This would
509
point to two different genomic variants of BspHRD3: (1) one from which the transcript could
510
be detected but not its genomic copy and which lacks the insertions, and (2) a second variant
511
from which the genomic copy could be sequenced but not the transcript and which includes
512
the inserts (Fig. 7B). Interestingly, comparing the genomic sequence of BspHRD3 with
513
BspUPG-2, a 550-bp sized fragment was identified as being homologous to BspUPG-2,
514
including these insertions (Fig. 7B; 93.6% similarity, blue boxes denote insertions), thus
515
pointing to the integration of a fragment of one of the two genomic variants of BspHRD3 into
516
BspUPG-2.
517
As gene duplication is strongly correlated with insertions and pseudogenization (Ohno,
518
1970; Kaessmann, 2010), we tested for duplication by mapping a set of sequence tags from an
519
array-based comparative genome hybridization (aCGH) experiment in sexual and apomictic
520
Boechera (Aliyu et al., in review) against BspUPG-2 (Fig. 7C). In total, 79 array probes from
521
the CGH experiment mapped to BspUPG-2. Whereby most probes mapping onto BspUPG-2
522
show no copy number variations (CNV) in apomicts (96% with the remaining 4% showing
523
depletion), 68% of the tags were depleted in at least one sexual genotype. Approximately half
524
of the depleted sequence tags in sexual genotypes were situated at the 3`-end of BspUPG-2,
525
representing the start of LCB1. The remaining depleted sequence tags were distributed
526
towards the 5`-end of BspUPG-2 between +0 to +1081 nt. To summarize, both LCB1 and the
527
extreme 5`-end of BspUPG-2 is present in fewer copies in sexual compared to apomictic
528
genomes, whereas the middle part of BspUPG-2 shows no variation in copy number in either
529
reproductive mode. The duplication of LCB1 in apomictic genomes is explained by its
530
presence in both assemblies. The duplication of the 5`-end in apomictic genomes is evidenced
531
by the presence of duplicated source gene insertions covering almost exactly the region
532
represented by depleted sequence tags in sexuals (HRD3: +199 to +731 nt, RNAR: +732 to
533
+948 nt, EFTU: +1041 to +1191 nt; Figs. 5 and 7C).
534
Since transposable elements (TEs) are considered to drive genome rearrangements,
535
including duplications, after interspecific hybridization, Assembly 1 and Assembly 2 were
536
screened for Viridiplantae transposable elements. In total, 100 and 113 TEs mapped onto
537
Assembly 1 and Assembly 2 respectively, many of which are simple repeats with low
538
sequence complexity. Excluding all simple repeats left 68 and 73 TEs which mapped onto
539
Assembly 1 and Assembly 2 respectively, with about half of them identified in Arabidopsis
540
(27 and 35 respectively; Fig. S11). The major repeat families are copia-like (18 and 16) and
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
17
541
gypsy-like (8 and 9) LTR retrotransposons, followed by DNA transposons En-Spm (9 and 12),
542
MuDR (11 and 7), hAT (7 and 7) and Helitrons (6 and 9). Proportions of the TE superfamilies
543
do not vary significantly between Assembly 1 and Assembly 2, but do when compared to
544
their distribution across the whole Arabidopsis genome. Copia-like LTR retrotransposons
545
represent the largest proportion of Arabidopsis TEs (57.70%) and those on the Boechera BAC
546
clone assemblies (26.47% and 21.92% respectively). Nonetheless, copia-like LTR
547
retrotransposons are more prominent across the whole Arabidopsis genome compared to both
548
Boechera assemblies (one-tailed Fisher’s exact test, LTR/Copia: P(Assembly1) = 1.83E-07,
549
P(Assembly2) = 5.23E-10), in contrast to DNA transposons of the En-Spm and hAT class and
550
non-LTR retrotransposons of the LINE/L1 class, which are more prominent on both Boechera
551
assemblies (one-tailed Fisher’s exact test, DNA/En-Spm: P(Assembly1) = 4.21E-10,
552
P(Assembly2) = 3.83E-14, DNA/hAT: P(Assembly1) = 2.00E-06, P(Assembly2) = 3.24E-06,
553
LINE/L1: P(Assembly1) = 0.001, P(Assembly2) = 0.008, Fig. S11, Tables S17 and S18).
554
Together these results (Figs. 5 and 7) indicate a duplication of BspUPG-1 fragments from
555
the original locus on Assembly 1 to form the basis of BspUPG-2, which subsequently
556
underwent sequential insertions of genome fragments derived from at least two unlinked
557
genomic regions. Thereby, LCB1 was inserted between LCB2 and LCB4. The newly formed
558
locus was then the insertion target for the duplicated variant of at least one functional gene
559
(e.g. BspHRD3), with subsequent exonization leading to a gain of BspUPG-2 transcriptional
560
activity (Fig. 7D).
561
562
563
DISCUSSION
564
565
High quantitative variation for unreduced pollen formation in apomictic Boechera
566
High variability in pollen morphology and unreduced pollen formation, in addition to
567
tolerance to deviations from the sexual endosperm balance number have been described for
568
some Boechera taxa (Böcher, 1951, 1954; Voigt et al., 2007; Aliyu et al., 2010; Voigt-
569
Zielinski et al., 2012). Despite this variability, castration experiments (Böcher, 1951) and
570
extensive flow cytometric analyses of seeds (Aliyu et al., 2010) strongly support selection
571
pressure for the maintenance of unreduced pollen development to fulfill endosperm balance
572
requirements in diploid apomicts.
573
Here we have performed detailed quantitative analyses of anther growth and
574
microsporogenesis to identify the optimal developmental stage for comparative expression
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
18
575
profiling reduced (sexual) and unreduced (apomictic) pollen formation in 14 genotypes. A
576
strong correlation between microsporogenesis and anther growth was evident at the pre- and
577
postmeiotic stages, whereas it was difficult to identify antherhead lengths corresponding to
578
the meiotic stage (Fig. 2B). Considering spatial and temporal variability of expression profiles
579
in reproductive tissues (Mascarenhas, 1989; Honys and Twell, 2004), the identification of a
580
specific stage of antherhead development and length characterized by PMCs at the onset of
581
meiosis, enabled targeted expression profiling of the meiotic stage which differentiates
582
reduced (sexual) and unreduced (apomeiotic) pollen formation (Table S4). Variable levels of
583
apomeiosis frequencies have been reported for Boechera (Aliyu et al., 2010), and here both
584
obligate and highly facultative apomicts were analyzed. No evidence for sex- or apomixis-
585
specific flower morphological variation was found, although meiosis and the appearance of
586
microspores are developmentally-uncoupled in apomicts relative to sexuals (Figs. 2A and S1).
587
This latter observation supports the hybridization-derived floral asynchrony (HFA) theory,
588
which describes the temporal shift (heterochrony) between both reproductive modes (Carman,
589
1997) which has also been identified on the transcriptomic level (Sharbel et al., 2010).
590
Meiotic chromosome behavior of apomicts producing unreduced pollen (the same used for
591
expression profiling) was primarily asynaptic (Fig. 1), as has previously been shown
592
(asyndetic, sensu Böcher (1951)). Although ultimately producing higher levels of dyads
593
compared to sexuals, the same apomicts demonstrated variability in terms of monad, dyad,
594
triad and tetrad formation (Fig. 2A), mirroring reported genotype-specific variability for
595
meiotic chromosome synapsis potential in both diploid and polyploid Boechera (Böcher,
596
1951; Naumova, 2001; Kantama et al., 2007). Interestingly, variation between individuals of
597
the same clonal lineage for dyad and tetrad formation was also apparent in the majority of
598
tested apomictic genotypes (Fig. 2A), and hence phenotypic variability for pollen formation
599
exists despite genetically clonal reproduction. In this light, the observed sexual allopolyploid
600
progeny from both diploid and triploid obligate apomicts (e.g. ES 753, B. divaricarpa;
601
Schranz et al. (2005)) could be explained by fertilization with reduced self pollen rather than
602
with pollen from another plant (Aliyu et al., 2010), since Boechera is a highly selfing system
603
(Roy, 1995). On a broader scale, our data indicate that fixation of the genotype via apomixis
604
does not necessarily lead to phenotypic stability of male meiocytes.
605
While these data are consistent with previous work (Kantama et al., 2007; Voigt et al.,
606
2007), the lack of correlation with apomeiosis expression (e.g. egg cell formation as measured
607
by FCSS; Fig. S1, Table S1) suggests separate mechanisms leading to unreduced male versus
608
female gametes in Boechera, and supports independent genetic control for at least some of the
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19
609
developmental steps required to form apomictic seeds (van Dijk et al., 1999; Noyes and
610
Rieseberg, 2000; Matzk et al., 2005).
611
The analyses of seed production, anther development, meiosis and pollen formation have
612
shown that (1) unreduced pollen formation occurs via first division restitution (FDR) in
613
apomictic Boechera, (2) this mechanism is not fully penetrant, and (3) that selection pressure
614
for a balanced endosperm in apomictic Boechera leads to the almost exclusive contribution of
615
unreduced pollen to endosperm formation.
616
617
BspUPG-2, unreduced pollen and stable apomixis in Boechera
618
The detailed phenotypic analyses and selection of a specific antherhead stage which
619
differentiated reduced versus unreduced PMCs, in conjunction with a microarray-based
620
analysis of diploid sexual versus diploid apomictic Boechera genotypes, led to the
621
identification of BspUPG-2. Assuming all other developmental aspects of sexual and
622
apomictic antherheads to be the same, the transcriptional activity of this locus is directly
623
correlated with meiotic non-reduction during male sporogenesis, and was furthermore qRT-
624
PCR validated in 14 biological replicates (i.e. 7 sexual and 7 apomictic genotypes) and 3
625
technical replicates (i.e. microarray probes Sharb0931225, Sharb0501554 and Sharb0690829
626
mapping to different regions of the same locus) to show strong apomeiosis-specific
627
upregulation of BspUPG-2 in reproductive tissues of apomictic Boechera (Fig. S4, Table S7).
628
It is unclear why genotype ES 753 was an outlier (Fig. S5), despite sharing a highly-similar
629
BspUPG-2 sequence with other apomictic genotypes. We suspect that genotype-specific shifts
630
in anther development (as observed in other samples) may have led to sampling of pollen
631
formation outside of the developmental window which characterized other genotypes.
632
Flow cytometric analyses of over 20 000 single seeds have demonstrated that virtually all
633
apomictic Boechera are characterized by balanced endosperm (6C = [4Cmaternal] + [2Cpaternal])
634
in diploids and 9C = [6Cm] + [3Cp] in triploids), leading to the hypothesis that both
635
meiotically-unreduced egg and pollen are required for stable apomictic seed production
636
(Aliyu et al., 2010). Similar to the analyses here which lead to the identification of BspUPG-
637
2, a second approach identified APOLLO, a similarly-conserved factor which is correlated
638
with unreduced egg cell formation in apomictic Boechera (Corral et al., submitted in parallel
639
to this manuscript). We tested the hypothesis that both factors should be required to form
640
stable apomictic seed by analyzing for the presence of both factors in the same 73 genotypes
641
examined in Aliyu et al. (2010). Both factors are highly co-correlated (BspUPG-2 - 93.33%;
642
APOLLO - 100%; Corral et al, submitted to Plant physiology in parallel this manuscript, Fig.
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
20
643
8) with highly-facultative and obligate apomictic genotypes (Aliyu et al., 2010), thus
644
supporting our hypothesis.
645
646
BspUPG-2 is a long pri-miRNA with potential regulatory functions in trans
647
Although the apo-specificity of BspUPG-2 attests to its novelty, the lack of homologs in
648
other species and a missing ORF typical for a protein-coding gene leaves the question open
649
whether BspUPG-2 acts in a complementary fashion to increase the penetrance of an existing
650
predisposition for environmentally-induced unreduced gamete formation in sexual genotypes
651
(sensu Aliyu et al. (2010)), or whether it is part of a completely novel pathway.
652
It is unclear whether BspUPG-2 is a key genetic factor or one of several tightly linked
653
factors which control different aspects of unreduced pollen formation (i.e. apomeiosis).
654
Furthermore, modifier genes (Bicknell et al., 2000), genetic background and/or environmental
655
conditions (Nogler, 1984) could explain variation in the level of apomictic trait expression
656
(refer also to Fig. 2A and 8). The detection of the apo-specific indel 9 is one example of a
657
potential co-regulatory target site in BspUPG-2, although no homologous sRNAs were
658
detected (Figs. 7A and S10). Alternatively, the four detected mRNA isoforms (i.e. potentially
659
caused by alternative splicing; Figs. 4 and S5) could regulate transcript abundance (as is
660
predicted for at least 25% of all alternative exons; Stamm et al. (2005)) leading to the
661
observed variation in terms of reduced and unreduced pollen formation in both facultative and
662
obligate apomicts (Fig. 2A).
663
Nevertheless, its tissue-specific expression pattern in addition to conservation at the
664
nucleotide level strongly supports a developmental role for the lncRNA BspUPG-2. Given its
665
chimeric structure, the novel lncRNA BspUPG-2 could have attained neofunctionalization in
666
the context of apomeiosis for pollen development through a number of mechanisms
667
(Kaessmann, 2010), similar to the lncRNAs BcMF11 from Brassica campestris (Song et al.,
668
2007; Song et al., 2012) or Zm401 in maize (Ma et al., 2008). BspUPG-2 could conceivably
669
have a regulatory function via a homology-dependent gene silencing mechanism (HDGS,
670
reviewed in Meyer and Saedler (1996)), such as posttranscriptional gene silencing of related
671
genes in trans (Eamens et al., 2008), which has been found for chimeric Helitron and Pack-
672
MULE RNAs in maize (Jiang et al., 2004; Morgante et al., 2005), or via the modulation of
673
DNA methylation patterns, such as reported for lncRNA-like loci which are associated with
674
polycomb components and histone modifications (de Lucia and Dean, 2011).
675
Consistent with this are the sequence fragments homologous to three different functional
676
parental genes across the 5`-end of BspUPG-2, which putatively could serve as targets for
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
21
677
HDGS (BspHRD3, RNAR and EFTU/EF-1A; Figs. 4, 5A and 7B). Thereby, BspUPG-2 could
678
belong to a novel class of miRNA-containing lncRNAs which serve as a matrix (i.e. as long
679
primary miRNA (pri-miRNA)) for several unidentified precursor miRNAs (pre-miRNA,
680
Altuvia et al., (2005)) or endogenous trans acting short interfering RNAs (ta-siRNA). Such
681
regulatory elements are processed from the excised intronic region of a spliced transcript, or
682
from the full-length unspliced transcript, and have been detected in vegetative developmental
683
pathways (Peragine et al., 2004; Vazquez et al., 2004; Hirsch et al., 2006) as well as in
684
generative tissues (e.g. in mature pollen; Grant- Downton et al. (2009b)).
685
Computational analysis of RNA folding detected several sections of BspUPG-2 which
686
were predicted to form with high probability non-random and stable secondary structures
687
(Fig. 6A, B and S8, Table S13), and which fulfill most criteria for pre-miRNAs (e.g. elevated
688
A+U content, Table S14). Interestingly, these potential pre-miRNAs were not detected in
689
previous screens (Amiteye et al., 2011; Amiteye et al., 2013), which may reflect (1) its tissue-
690
specific and short-term upregulation at the onset of male meiosis, or (2) limitations of
691
homology-based analyses considering that BspUPG-2 is unique to apomictic Boechera. The
692
detection of a stable secondary structure in a region which is homologous to a known protein-
693
coding gene with translation elongation activities during polypeptide synthesis at the
694
ribosome, and activities in signal transduction (i.e. npcRNA 5 similar to GTP binding
695
Elongation factor Tu/EF-1A family protein; E-value=7.00E-24, GO:0003746, AT4G02930)
696
could be a first indication for a HDGS function of BspUPG-2. For example, a homologous
697
factor to EFTU/EF-1A has been implicated with meiosis progression in Xenopus (Bellé et al.,
698
1990; Peters et al., 1995), while one of the other two sequence fragments of the 5`-end of
699
BspUPG-2 is also homologous to a gene with nucleotide binding function (RNAR;
700
GO:0003723).
701
702
High sequence conservation and hemi-or homozygous status imply a selective advantage
703
of BspUPG-2
704
Importantly, BspUPG-2 is a chimera of non-genic (e.g. LCB1) and genic regions (e.g.
705
HDR3, RNAR, EFTU), in which genic regions were derived from partial duplicated variants
706
of their parental genes (e.g. BspHRD3, homologous to AT1G18260, i.e. a pseudogene derived
707
through duplication; Ohno (1972), Figs. 7C and D). Although the long-term survival of
708
chimeric genes is described to be rare (Bennetzen, 2005), BspUPG-2 exhibited an
709
unexpectedly high degree of conservation in DNA sequence (Fig. 7A, Table S15) and
710
expression (Fig. S4) between apomictic Boechera representing different taxa and geographic
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
22
711
origins. In contrast, higher sequence variation was identified for BspUPG-1 in sexuals (e.g.
712
5`-end; Fig. 7A, Table S15). Thus BspUPG-2 appears to be under selective maintenance
713
(sensu Casillas et al. (2007)), which would be consistent with its assumed role in unreduced
714
pollen formation for balanced endosperm in apomicts (Aliyu et al., 2010).
715
The hemi- or homozygous status of BspUPG-2 reflects dominant inheritance as a proposed
716
characteristic for an apomeiosis controlling locus (refer to Grossniklaus et al. (2001), Table
717
1), as has been found for example in Pennisetum (Ozias-Akins et al., 1998). Hemizygosity
718
would reflect an extended period of low to no recombination in its genomic location, and
719
would be consistent with the distribution of TEs at both the original and duplicated locus (Fig.
720
S11). The similar abundance of TEs from Gypsy-type and En-Spm superfamilies, which insert
721
preferentially into gene poor regions (i.e. heterochromatic regions, Fiston-Lavier et al. (2012))
722
and which are enriched in both assemblies compared to their abundance within the total
723
number of Arabidopsis TEs (Fig. S11) points to a co-localization of the original and
724
duplicated locus of BspUPG in the same pericentromeric region (Fig. S9), or to regions with
725
similar characteristics (i.e. interstitial heterochromatic regions comparable to hk4S in
726
Arabidopsis; Fransz et al. (2000)).
727
We hypothezise that the genesis of BspUPG-2 is associated with the recurrent interspecific
728
hybridization which is highly correlated with origins of apomictic Boechera (Schranz et al.,
729
2005; Beck et al., 2011). Assuming co-localization of BspUPG-2 with BspUPG-1 and the
730
formation of highly heterochromatic regions in apomicts as protected zones (e.g. Het- and
731
Del-chromosomes, Kantama et al. (2007)), the genome-wide effects of hybridization (Comai,
732
2000) could have provided the mechanism through which BspUPG-2 originated and gained
733
regulatory function.
734
To summarize, despite variability for unreduced pollen formation in apomictic Boechera, a
735
single novel transcription unit (BspUPG-2) is consistently upregulated in apomictic flower
736
tissues at the PMC stage, and its chimeric sequence structure might reflect the interspecific
737
hybridization history of this genus. We hypothesize that BspUPG-2 arose via sequential
738
(segmental) duplication-insertion events involving at least five loci, from which three
739
originated from transcribed genes (Fig. 7D). In this regard, it is interesting that only the
740
apomixis-specific duplicated locus (BspUPG-2) shows transcriptional activity, while the
741
original locus (BspUPG-1), which is present in both sexuals and apomicts, does not (Fig. 5E).
742
Whereas many studies have focused on mutation accumulation and deregulation with respect
743
to origins of apomixis elements (e.g. Tucker et al. (2003); d’Erfurth et al. (2008)), the
744
emergence of novel genes in apomicts has not been appreciated, although various identified
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
23
745
apomixis-associated loci suggest species-specific apomixis factors (Grossniklaus et al., 2001)
746
for which gains in function are hypothesized (Vielle-Calzada et al., 1996).
747
748
749
750
CONCLUSION
751
The identification of the novel apo-specific BspUPG-2 however, supports the HFA theory,
752
which proposes that apomeiosis, and in a broader perspective apomixis, originates from
753
hybrid-specific “genome collisions” and associated induction of gene duplication and TE
754
activation (Carman, 1997). BspUPG-2 could have a potential application in stabilizing the
755
endosperm balance in the course of the implementation of apomixis into crop plants. How
756
BspUPG-2 has undergone neofunctionalization to develop a trans-regulatory function
757
remains to be clarified.
758
759
760
761
MATERIALS AND METHODS
762
763
Plant material and cultivation conditions
764
Several sets of the same diploids, ten apomictic and twelve sexual Boechera, were used for
765
all experiments (Table S1; note recent taxonomic information, (Koch, 2010)). Ten seeds per
766
genotype were cultured on moist filter paper in sealed Petri dishes and vernalized at 4°C in
767
the dark for two weeks until germination. Seedlings were transplanted to plastic pots
768
(11x11x13 cm) containing autoclaved substrate, and grown in a phytotron without
769
insecticides and herbicides under long-day conditions (16 h light and 8 h dark, 21°C).
770
771
Measurement of relative nuclear DNA content and reproductive mode
772
Relative nuclear DNA content (referred to as ploidy) in leaf, seed (Matzk et al., 2000) and
773
pollen (De Storme et al., 2013) was quantified using leaf tissue from a diploid sexual B.
774
stricta (ES 558.2; Table S1) as an external control. Leaf and seed material (20 seeds per
775
individual plant) were chopped with a razor blade in a drop of Galbraith’s buffer containing 4
776
µg/ml DAPI (Galbraith et al., 1983). Tissue-specific ploidy measurements were performed on
777
a FACSAria II (BD Biosciences, Franklin Lakes, NJ, USA) equipped with a 375 nm near UV
778
laser. Data were measured using the FACSDiva Software (v6.1, BD Biosciences) and
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
24
779
analyzed
using
WinMDI
v2.9
(The
Scripps
Research
Institute,
780
http://facs.scripps.edu/software.html); “C” refers to DNA content of a haploid anaphase cell,
781
and “x” the basic chromosome number.
782
783
Microscopy analysis of gametophyte stages
784
Pollen developmental stages were defined (Fig. 3; Regan and Moffatt (1990)) from each of
785
twelve flower bud size stages (S1-S12) which differed in 100 µm length increments (sensu
786
Smyth et al. (1990) and Sanders (1999)). For dissection of flower buds at stage S3 and
787
antherheads at flower developmental stages S8 – S12 under a Zeiss Discovery V20 (Carl
788
Zeiss, Jena, Germany), stereomicroscope sterile glass needles bent to an angle of appr. 100-
789
120° and tip size of 50-100 µm using a Narishige PC-10 puller (Narishige Group, Kasuya,
790
Japan) were used. Sample preparation of size-staged buds and antherheads for histology and
791
light microscopy was carried out according to Grasser et al. (2009).
792
The decision to take the PMC stage for comparative gene expression profiling is based on
793
two criteria: (1) apomeiosis candidate genes should affect early meiotic stages (Grimanelli et
794
al., (2001), and (2) in previous analyses the spike of gene expression change between sexual
795
and apomictic ovules was detected in the microspore mother cell (Sharbel et al., 2010), a
796
stage which corresponds to the PMC in antherheads .
797
798
Cytochemical analysis of fixated Boechera anthers
799
Cytological observations of meiosis and PMCs were made from sexuals and apomicts at
800
flower bud stages S9 to S10. Meiotic chromosome spreads of anthers were prepared
801
according to Ross et al. (1996) with minor changes, and observed under a Zeiss Axioplan 2
802
imaging microscope (Carl Zeiss). Photographs were taken with an AxioCam HRc Rev. 2
803
camera under 100-fold magnification using a 49 DAPI BP reflector block. Meiocytes at the
804
tetrad stage were examined by separate squashes of all six antherheads per flower bud, from
805
one sexual and six apomictic genotypes, to determine the number of monads, dyads, triads
806
and tetrads in each antherhead. Anthers were squashed according to Peterson et al. (2010),
807
stained (Alexander’s stain, Alexander (1969)) and examined under a Zeiss Axioplan 2
808
imaging microscope (Carl Zeiss). All available meiocytes per anther were counted, and
809
statistical analyses of meiocyte behaviour and anther size correlations of gametophyte stages
810
were evaluated with SPSS v11.5 (LEAD Technologies, Charlotte, NC, USA).
811
812
RNA isolation, microarray hybridization and qRT-PCR of antherhead tissue
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
25
813
Microdissections and total RNA extractions were prepared using tools and a dissection
814
area cleaned with ethanol, treated with RNAseZap® (Ambion, Carlsbad, CA, USA) and
815
washed with DEPC treated distilled water (DEPC, Diethyl phosphorocyanidate).
816
Approximately 30 antherheads of each genotype, corresponding to the PMC stage, were live
817
microdissected from fresh whole flower buds under a Zeiss Discovery V20 stereomicroscope
818
(Carl Zeiss) using sterile glass needles (see above), and collected in 500 µl RNAlater (Qiagen,
819
Hilden, Germany). 18 µl of RNAse-free DNAse I (Qiagen) digested total RNA extracts
820
(RNeasy® Micro kit, Qiagen) were eluted through RNeasy MinElute Spin® columns (Qiagen)
821
and quantified on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,
822
Wilmington, DE, USA). RNA quality of all samples was assessed with the RNA 6000 Nano
823
LabChip Kit II (Agilent Technologies, Santa Clara, CA, USA) on the Agilent 2100
824
Bioanalyzer. Purified total RNA was stored at -80°C.
825
To ensure optimal cRNA yield after labeling, 200ng total RNA with absorbance readings
826
of A260/A280 >1.8 and A260/A230 >1.8 (recommended by Agilent) were used for the
827
labeling procedure. Approximately 1.5 μg of the generated Cy3-labeled cRNA per sample
828
(One-color Quick Amp Labeling kit, Agilent Technologies) was hybridized for 18 hrs at 65°C
829
to the custom Boechera whole flower-105k-Agilent microarrays, which were scanned at 5μm
830
double pass resolution with an Agilent G2565BA Microarray Scanner. The One-color RNA
831
Spike-in kit (Agilent Technologies) was used to assure optimal microarray processing.
832
Microarray hybridization quality was assessed with Feature Extraction 10.1 software (Agilent
833
Technologies), whereas quantile normalization with baseline to median transformation and
834
gene expression analysis was performed with GeneSpring GX 10 software (Agilent
835
Technologies). Differentially expressed microarray probes were considered validated with
836
p≤0.05 as assessed by an unpaired t-test with a mean difference ≥2-fold. P-values were
837
corrected for the family-wise error rate (FWER) as control for false positives using the
838
Bonferroni method.
839
The real-time PCR reactions, using primers which were designed on the cDNA reads
840
homologous to the candidate microarray probes Sharb0690829 (EMBL/GenBank/DDBJ No.
841
ERS317557), Sharb0501554 (EMBL/GenBank/DDBJ No. ERS317556) and Sharb0931225
842
(EMBL/GenBank/DDBJ No. ERS317555; Primer3 v0.4.0, http://frodo.wi.mit.edu/primer3/)
843
were performed according to Sharbel et al. (2010). Seven biological and four technical
844
replicates were run for each probe and tissue in a 384-well plate together with two
845
endogenous control genes tested on Boechera anther material (ACTIN2 (ACT2) and
846
ELONGATION FACTOR α1 (EFα1), Pellino et al. (2011)), negative template and reverse
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
26
847
transcriptase controls. PCR efficiencies and normalized Ct values of each set of four technical
848
replicates were processed with the Real-time PCR Miner v2.0 software (Zhao and Fernald,
849
2005). Relative quantification and normalized cycle threshold (Ct) values of the amplified
850
targets were calculated separately with reference to the expression levels of each of the two
851
housekeeping genes employing the ∆∆Ct method (Pfaffl, 2001) using a calibrator sample (ES
852
910.2; Table S7). The corresponding mean relative expression ratio for each genotype was
853
calculated with SPSS (v11.5; LEAD Technologies), and significant differences between
854
samples were evaluated using a one-way ANOVA (α = 0.05) with a Tuckey-HSD post hoc
855
test for differences between multiple pairs of means.
856
857
Rapid Amplification of cDNA Ends (RACE)
858
The SMARTer RACE method (Clontech, Palo Alto, CA, USA) was employed to obtain 5`-
859
end and poly(A)-site information from BspUPG-2 and BspHRD3, an Arabidopsis
860
homologous of HRD3 in Boechera. Primers (Primer3 v0.4.0) for BspUPG-2 RACE were
861
derived from microarray probe homologous cDNA read ERS317557 (5`-end primer GSP3:
862
5`-TCTTCGCCATCGTTCATGTTTACTTCCG-3`;
863
TCATCATGTCTTCTTCGCCATCGTTCA-3`), and those for BspHRD3 RACE were derived
864
from
865
TAATGCCCACTGGGGTCGTCATTGT-3`;
866
ACTGGAATTGGGTACTTGTATGTCA-3`). PCR reactions were performed with the
867
Advantage 2 PCR Kit (Clontech) and PCR fragments were cloned (pCR4-TOPO TA; Life
868
Technologies) and Sanger sequenced (see previous section).
the
LCB2
of
BspUPG-2
3`-end
(5`-end
3`-end
primer
primer
primer
GSP4:
5`-
GSP11:
5`-
CON234X14_L:
5`-
869
870
Computational analysis of RNA folding probabilities of BspUPG
871
Structural RNAs are usually characterized by an unusual thermodynamic stability and a
872
conserved secondary structure. The minimum folding energy (MFE) as a measure of
873
thermodynamic stability for a sequence (i.e. negative values indicate that a sequence is more
874
stable) was calculated using the RNAfold and RNAz version 1.0 software for Windows of the
875
Vienna RNA package (Hofacker et al. (1994), http://www.tbi.univie.ac.at/ivo/RNA). The
876
presence of statistically significant secondary structures of BspUPG-2 was monitored using
877
Z-score values as described by Crespi et al. (1994) and Bonnet et al. (Bonnet et al., 2004)
878
using similar thresholds to Kavanaugh and Dietrich (2009). All significant secondary
879
structures of BspUPG-2 were classified according to Zhang et al. (2006). The “npcRNA
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
27
880
[number]”-names were chosen to follow the naming convention established by previous
881
investigators (Hirsch et al., 2006).
882
883
BAC probe preparation and screening of Boechera BAC library
884
Candidate 60-mer microarray probes were mapped against Boechera 454 FLX cDNA
885
libraries (Sharbel et al., 2009) using CLC Genomics Workbench v4.5.1 (CLC Bio, Aarhus,
886
Denmark, standard parameters). Flanking regions to the mapped microarray probes were used
887
to design specific primers (Primer3 v0.4.0) for chromosome walking in sexuals and apomicts
888
using a DNA Walking SpeedUp Premix Kit I from Seegene (Seegene, Seoul, Korea, Table
889
S10). Resultant products were transformed into E. coli TOP10 cells using a TOPO TA
890
Cloning Kit for Sequencing (Invitrogen), individual clones were amplified using the
891
TempliPhi™ DNA Sequencing Template Amplification Kit (Reagin, 2003) and Sanger
892
sequenced on an ABI 3730 XL sequencing system. Sequence analysis and assembly was
893
carried out with Lasergene 8 (DNAStar, Madison, WI, USA). The E. coli TOP10 sub-cloned,
894
PCR amplified and finally gel purified DNA walking products (NucleoSpin® Extract II kit,
895
Macherey-Nagel, Düren, Germany) were hybridized against a gridded bacterial artificial
896
chromosome (BAC) library (48 x 384 spotted wells onto a 22 x 22 cm filter membrane,
897
binary vector pCLD04541, Bancroft (1997)). Individually radio-labelled and pooled probes
898
were hybridized as a group using the overgo hybridization method (Ross et al., 1999).
899
Vector inserts of 500ng pure BAC clone DNA extracts were restriction digested to
900
completion with HindIII, BglII and BamHI (Amersham Pharmacia Biotech) at 37°C (HindIII,
901
BglII) and 30°C (BamHI) for 8 h and examined on a 1% agarose gel. Based on their partial
902
overlapping restriction patterns DNA was isolated from four BAC clones (A4O22, E7K5,
903
C8B11 and F8G11) using Nucleobond Xtra Midi Kits (Macherey-Nagel). BAC DNA was
904
randomly sheared (Hydroshear, Digilab, Marlborough, MA, USA) and size-fractionated by
905
agarose gel electrophoresis in ~1kb and ~4-5kb size classes. These fragments were end-
906
repaired, blunt-end ligated into pUC19 (Life Technologies), transformed into E. coli
907
ELECTROMAX DH5α-E electro-competent cells (Invitrogen) and sequenced on an ABI
908
3730 XL automatic DNA sequencer (PE Applied Biosystems). Vector clipping, quality
909
trimming and sequence assembly using stringent conditions (e.g. 95% sequence identity
910
cutoff, 25 bp overlap) was done
911
(http://staden.sourceforge.net/). Assembly of the complete BAC clone sequences was
912
performed using Seqman and the Gap4 algorithm implemented in Staden. Remaining gaps in
913
the contiguous BAC sequences were manually inspected and closed with primer walking or
using Lasergene
8 (DNAStar) and Staden
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
28
914
PCR products crossing the gaps from adjacent contigs. The resulting sequences were
915
assembled using Lasergene 8 (DNASTAR) set to an overlap minimum of 20 bp with 95%
916
identity. All BAC assemblies were annotated using BLASTN and BLASTX searchs against
917
the
918
(http://www.ncbi.nlm.nih.gov/genbank/).
non-redundant
GenBank
nucleotide
and
protein
databases,
respectively
919
920
Processing of DNA sequences and sequence analysis
921
The allelic constitution of BspUPG-2 in nine each sexual and apomictic genotypes was
922
determined by sequencing proof-reading polymerase amplified (Phusion high-fidelity
923
polymerase, Thermo Scientific), gel purified (NucleoSpin® Extract II kit, Macherey-Nagel)
924
and multiply-cloned PCR fragments (CloneJet™ PCR cloning kit, Fermentas) using specific
925
primers
926
TCCGACCTAAATCCTACCAAACTGA-3`; in sexual B. stricta CON234X11L 5`-
927
CAAAAATAAAAGATTTGATGTAGATTGC-3`
928
FLTsexX2L: 5`-GAAGAAAGAGCTACGGCGTGAT-3`; 3`-end primer CON234X5R: 5`-
929
TGCTCAATTTTGAACATCTTATTTGC-3`). Lasergene 8 (DNAStar) was used for
930
assembly and similarity analysis of Phred 20 quality-trimmed sequences. Coding potential
931
was calculated from all six frames of all BspUPG-2 splicing forms using the Coding Potential
932
Calculator software (Kong et al., 2007).
(5`-end
primer
in
apomictic
genotypes
and
in
other
CON234X5L:
sexual
5`-
genotypes
933
Geneious was used for pairwise sequence alignment using CLUSTALW (IUB cost matrix,
934
gap open cost = 15, gap extend cost = 6,66, free end gaps) and maximum-likelihood method
935
comparison (Tamura and Nei, 1993) were conducted in MEGA5 (Tamura et al., 2011) using
936
standard parameters. Pairwise CLUSTALW comparisons in the presence of rearrangements
937
were performed with Mauve (v2.3.1; progressiveMauve, default parameters, Darling et al.
938
(2004)) to detect collinear sequence blocks (LCBs; conserved sequence segments, which are
939
internally free from genome rearrangements) in BspUPG-1 and BspUPG-2 between different
940
genotypes.
941
Local
BLASTN
search
of
the
complete
genomic
SRA
(SRP007750,
942
http://www.ncbi.nlm.nih.gov/sra/, 454 GS FLX) and sequence extraction were conducted with
943
CLC Genomics Workbench (costs = match 1, mismatch 3, existence 5, extension 2; E-value =
944
10; word size 11, filter complexity = yes). For mapping of both, 454 WGS and CNV sequence
945
tags (Aliyu et al., unpublished results) the genomic sequence of BspUPG-2 was divided into
946
eighteen 200 bp bins with 20 bp overlap. All existing CNVs for each single sequence tag are
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
29
947
represented and relative CNV frequencies are considered independently for sexual and
948
apomictic genotypes.
949
BAC sequence assemblies were annotated for transposable elements screening the green
950
plant section (Viridiplantae) of the Repbase repetitive element database (Jurka (1998),
951
http://www.girinst.org/server/Maps/AT/index.html)
952
Programs, einverted and EMBOSS (Rice et al., 2000) were employed to identify inverted
953
repeats with ≥ 80 % matches and the Pipmaker software for simple repeats and CpG islands
954
(Schwartz et al., 2003). LTR analyses of Assembly 1 and Assembly 2 were performed with
955
LTR FINDER software (Xu and Wang, 2007).
using
CENSOR
(Kohany,
2006).
956
957
958
959
SUPPLEMENTAL MATERIALS
960
961
Protocol S1. Computational analysis of RNA folding probabilities of BspUPG-2.
962
Protocol S2. Small RNA Northern blot.
963
Figure S1. Representative meiocytes at tetrad stage and flow cytometric ploidy confirmation.
964
Figure S2. Correlation of flower organs and flower bud length for sexual and apomictic
965
Boechera genotypes.
966
Figure S3. Constantly differentially regulated microarray probes in apomictic compared to
967
sexual Boechera genotypes.
968
Figure S4. Validation of differentially-expressed microarray probes by qRT-PCR.
969
Figure S5. Rapid amplification of cDNA 3`- and 5`-ends of the candidate gene UPGRADE.
970
Figure S6. Percent identity plots showing the original UPGRADE locus and its duplicated
971
variant.
972
Figure S7. Distribution of locally collinear sequence blocks along the BAC clone Assembly 1
973
and Assembly 2.
974
Figure S8. Distribution of Arabidopsis homologous loci corresponding to Assembly 1 and
975
Assembly 2 in Boechera along Arabidopsis chromosomes.
976
Figure S9. Thermodynamically stable non-random secondary structures of BspUPG-2.
977
Figure S10. Northern blot analysis for putative small RNAs homologous to indel 9 at
978
BspUPG-2.
979
Figure S11. Distribution of the TE superfamilies along the original and duplicated
980
UPGRADE locus.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
30
981
Table S1. List of Boechera genotypes with population information used for histological
982
examination of microsporogenesis and candidate gene analyses.
983
Table S2. Analysis of meiocyte constitution at the tetrad stage in diploid sexual and high
984
facultative and obligate apomictic Boechera genotypes.
985
Table S3. High-level 1C-pollen individuals with corresponding flow cytometric seed screen
986
data.
987
Table S4. Frequencies of microspores, meiotic and pollen mother cells relative to antherhead
988
length.
989
Table S5.
990
upregulation in apomictic genotypes.
991
Table S6. QRT-PCR primers used for validation of microarray candidate probes.
992
Table S7. Relative mRNA expression values for four microarray probes in somatic and
993
reproductive tissues.
994
Table S8. Overview of BAC clones carrying one to three candidate microarray probes.
995
Table S9. Transcription factor binding sites upstream of BspUPG-2.
996
Table S10. Primers used for chromosome walking on microarray candidate probes and the
997
source gene BspHRD3.
998
Table S11. Gene annotation of Boechera BAC clone Assembly 1 and Assembly 2.
999
Table S12. Distribution of inverted repeats (IR) on Assembly 1 and Assembly 2.
Microarray probes demonstrating significant absolute fold change (AFC)
1000
Table S13. Detection of candidate regions for stable non-protein-coding RNA secondary
1001
structures of BspUPG-2.
1002
Table S14. Characteristics of thermodynamically stable secondary non-protein-coding RNA
1003
structures with highest Z-score from BspUPG-2 in apomictic Boechera genotypes.
1004
Table S15. Sequence divergence of BspUPG-1 and BspUPG-2.
1005
Table S16. Indels on BspUPG-2 isolates of sexual and apomictic genotypes using a
1006
CLUSTALW multiple sequence alignment.
1007
Table S17. Green plant (Viridiplantae) homologous repetitive DNA element sequences
1008
mapping on Assembly 1.
1009
Table S18. Green plant (Viridiplantae) homologous repetitive DNA element sequences
1010
mapping on Assembly 2.
1011
1012
1013
1014
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
31
1015
ACKNOWLEDGEMENTS
1016
1017
The authors thank H. Block, V. Katzorke, M. Wiesner and I. Walde for technical support. We
1018
thank Prof. I. Schubert for the constructive comments on the manuscript, and to Thomas
1019
Mitchell-Olds and Eric Schranz for seed material. This work was also supported in part by
1020
the COST action HAPRECI “Harnessing Plant Reproduction for Crop Improvement”.
1021
1022
1023
1024
REFERENCES
1025
1026
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apomictic Boechera (H and O). In addition, homologues with close juxtaposition were
1342
occasionally observed in apomictic Boechera (asterisk). Scale bars, 5 μm.
1343
1344
Figure 2. Meiocyte constitution at tetrad stage and correlation of flower bud size and
1345
antherhead length with different gametophyte stages.
1346
(A) Squashes of single antherheads from several individuals per genotype demonstrate
1347
meiocyte constituion at tetrad stage. Roman numbers denote (I) groups of individuals per
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41
1348
genotype primarily (>50%) producing reduced gametes versus (II) groups of individuals per
1349
genotype primarily (> 50%) producing unreduced gametes. (B) Relationship between flower
1350
bud stages S3 to S12 and proportion of anthers at a particular developmental stage of
1351
gametophytes in Boechera. The abbreviations used for each developmental stage are shown in
1352
Table 1. (C) Horizontal bars above boxplots demonstrate significant comparisons between
1353
gametophytic stages per antherhead length within each genotype (*p<0.05; **p<0.01;
1354
***p<0.001, n.s. = not significant), as conducted for a 95% binomial proportion confidence
1355
interval with a one-way ANOVA including Tukey-HSD post hoc test. Despite observations of
1356
antherhead related gametophyte stages for flower bud stages S3 to S12, the correlation
1357
analysis focuses only on S8 to S12.
1358
1359
Figure 3. Light microscopic analysis of Boechera antherhead development and
1360
corresponding gametophyte stages during pollen formation.
1361
(A) Flower buds and appropriate antherheads of different developmental stages S3 to S12 in a
1362
sexual genotype. (B-E) Light microscopy images of semi-thin sections of resin-embedded
1363
antherheads after histological staining displaying the development of the sporocyte into a
1364
mature microspore, used for cytological validation of major gametophyte stages (B – Sp; C -
1365
PMC; D - Me; E – Msp, Table 1). E, endodermis; P, parietal cells; Sp, sporocyte; V, vascular
1366
region; C, connective; T, tapetum; PMC, pollen mother cell; En, endothecium; MC, meiotic
1367
cell; Me, meiosis; Msp, microspores.
1368
1369
Figure 4. Schematic representation of putative splicing forms of BspUPG-2. All four
1370
splice forms could be extracted by end-to-end sequencing in five apomictic Boechera. Exons
1371
are represented by grey boxes and numbers indicate intron-exon boundary positions. BLAST
1372
search results from GenBank nucleotide collection and TAIR10 gene database are given
1373
below.
1374
1375
Figure 5. Original and duplicated UPGRADE locus.
1376
(A) Schematic representation of the gene annotation of Assembly 1 containing the original
1377
BspUPG-1 locus and Assembly 2 containing the duplicated apo-specific BspUPG-2 locus.
1378
Syntenic regions of the two assemblies are shown below. Inverted repeats are denoted as grey
1379
numbered arrowheads. Insertions of genic fragments marked in red letters. (B) Mapping of
1380
genomic sequences of BspUPG-1 in apomictic (e.g. B. divaricarpa, ES 514) and sexual
1381
genotypes (e.g. B. stricta, ES 612.1) onto both BAC clone assemblies identified
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
42
1382
rearrangements of locally collinear blocks (LCBs). Black asterisks denoted indels for the sex-
1383
specific identity of LCB1 and LCB4. LCB5 position on Assembly 1 and Assembly 2 is
1384
displayed in Fig. S5. Black arrows denote primers: 1 - CON234X2L, 2 - CON234X14L, 3 -
1385
CON234X10R, 4 - PC1pol1L, 5 - PC1pol1R, 6 - GSP4, 7 - TSP33R, 8 - CON234X5R, 9 -
1386
Indel9minus. (C) CLC Genomics Workbench (v4.5.1) output file showing different
1387
distribution of separate mapped cDNA reads from sexual and apomictic genotypes onto
1388
BspUPG-2. Both sense-oriented (green) and antisense-oriented (red) cDNA reads are
1389
displayed below grey regions which demonstrate cDNA coverage (in pink). Coloured vertical
1390
bars on cDNA reads show SNPs in comparison with genomic DNA of BspUPG-2. (D) PCRs
1391
with independent primer pairs one located on the LCB1 and one on cDNA mapping positions
1392
of LCB2 in sexual and apomictic genomes illustrate that LCB1 is highly conserved in both
1393
sexuals and apomicts and that LCB2 is also present in sexual genotypes but in a different
1394
position in the genome. Primer locations are shown as numbered black arrows in part (B).
1395
White asterisk denotes missing band for genotype 105.18, which lacks the priming site for
1396
primer 6. (E) PCR with primers (4 and 5) combining LCB1 and LCB2 shows apo-specific
1397
presence of BspUPG-2, whereas BspUPG-1 is present in both sexual and apomictic genomes
1398
(primer 6 and 9). In contrast to BspUPG-1 which is not transcribed in sexuals and apomicts
1399
(primer 6 and 9), BspUPG-2 is solely transcribed from apomicts (primer CON234B2L and
1400
CON234B2R, Table S6). White arrow marks faint band for genotype ES 753 and white
1401
asterisk marks missing band for genotype 105.18, which lacks the priming site for primer 6.
1402
RRP4, Exosome complex component RRP4; MBOAT, membrane bound O-acyl
1403
transferase-like protein; MtN21, nodulin MtN21/EamA-like transporter protein; BspUPG-1,
1404
original locus of candidate gene UPGRADE; TER1-4, transposable element related protein;
1405
TLP5, Tubby-like F-box protein 5; UP1, uncharacterized protein; TIR, TIR-NBS class of
1406
disease resistance protein; UGT, sterol 3beta-glucosyltransferase; NPC1, Niemann-Pick C1
1407
protein; TPR, tetratricopeptide repeat domain-containing protein; DY2A, Dynamin-2A;
1408
GRV2, DNAJ heat shock N-terminal domain-containing protein; HRD3, HRD3-like protein;
1409
RNAR, RNA recognition motif-containing protein; EFTU, Elongation factor Tu; BspUPG-2;
1410
duplicated locus of UPGRADE.
1411
1412
Figure 6. Secondary structure prediction for BspUPG-2.
1413
(A) The Z-score for the sliding window with length between 50nt and 300nt (step size=10) is
1414
plotted vs. position on BspUPG-2. Any window producing a significant Z-score during the
1415
scanning process was considered candidate region for a structural npcRNA. In case that
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43
1416
multiple, overlapping windows of several lengths produced significant Z-scores, the region
1417
encompassed by all the overlapping windows defined the candidate region (represented as
1418
black boxes). Numbers in black boxes are putative npcRNAs having most negative Z-score
1419
(Fig. S8; Tables S13 and S14). (B) Proposed minimum free energy structures (MFE) of
1420
putative npcRNAs with significant BLASTN hits in GenBank and TAIR10 Genes databases.
1421
The structures above are colored by base-pairing probabilities from zero (violet, see legend)
1422
to hundred percent (red). For unpaired regions the color denotes the probability of being
1423
unpaired. (C) Overlap of npcRNA 5 with exon 3 of a GTP binding Elongation factor Tu
1424
family protein (EFTU/EF-1A; AT4G02930). Red letters indicate dissimilarities between
1425
sequences.
1426
1427
Figure 7. Genesis of BspUPG-2 via duplication from the original locus BspUPG-1 and
1428
sequential insertion of genic fragments.
1429
(A) Structural overview of full-length BspUPG-2 in apomictic and sexual Boechera (see
1430
Table 1) adapted and modified from the Progressive Mauve algorithm, exhibits locally
1431
collinear blocks (LCB) between the candidate genes in various genotypes and (B) between
1432
BspUPG-2 and the source genes from which BspUPG-2 hosts fragments (identical colour
1433
display highly similar sequences; black zigzag line denotes truncated sequences due technical
1434
reasons). (C) Distribution of 454 whole genome sequencing (WGS) reads mapping across
1435
sliding windows (bins) of BspUPG-2 versus copy number variations (CNV) in 10 sexual
1436
versus 10 apomictic genotypes (Aliyu et al., unpublished results, details in “Materials and
1437
Methods”; note that “genic inserts” refers to position +199 nt to +1191 nt in (B) and that
1438
WGS reads did not overlap between LCB1 and LCB2). (D) Schematic representation of
1439
parental and chimeric BspUPG genes. Boxed numbers illustrate different LCBs. (+)
1440
Transcription activity and (−) no transcription activity.
1441
1442
Figure 8. Correlation between BspUPG-2 and APOLLO with apomictic Boechera.
1443
The high correlation of BspUPG-2 (primers 4 and 5 as denoted in Fig. 5B) with diploid and
1444
triploid high-level and obligate apomicts (see Appendix in Aliyu et al. (2010)) is also
1445
mirrored by similar results for the female meiosis marker APOLLO (see Corral et al.,
1446
submitted to Plant Physiology in parallel with this manuscript). Numbers above the columns
1447
denote count of tested genotypes per mode of reproduction as determined by Aliyu et al.
1448
(2010).
1449
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44
1450
TABLES
1451
Table 1. Developmental markers of pollen formation corresponding to gametophyte and flower stages.
1452
Developmental Developmental marker and histological eventsc
marker ID
Flower
stage
Size
range
(mm)
Arabidopsis
flower
stagea
Pollen
stageb
S1
0 – 0.1
n/a
n/a
n/a
S2
0.1 – 0.2
0.2 – 0.3
n/a
n/a
n/a
S3
7-8
n/a
Sp
S4
S5
0.3 – 0.4
0.4 – 0.5
9
3
S6
0.5 – 0.6
S7
S8
0.6 – 0.7
0.7 – 0.8
S9
0.8 – 0.9
4
S10
0.9 – 1.0
4–6
1453
n/a
n/a
1454
1455
Sporocytes, premeiotic, anther in differentiation
stage, occurrence of 1° and 2° parietal cells,1456
vascular region initiated.
Pattern of anther defined, all four locules present.
1457
Rapid lengthening of all flower organs, especially
1458
antherheads and filament.
PMC
1459
Pollen mother cell enlarged and clear separated
from tapetum, prior to meiosis, no callose
1460
deposition.
PMC enters pollen meiosis, crushed middle layer,
1461
vacuolated tapetum, initial callose deposition,
binucleate tapetum.
1462
Me
Meiosis, tetrads of microspores, accumulated
callose deposition.
1463
Microspores in interphase, early-vacuolated,
minorities of tetrads or bi-nucleate microspores.
1464
1.1– 1.2
11
6–8
Microspores vacuolated and undergo first mitotic
S12
division, callose degradation, bi-nucleate 1465
microspores.
a
Arabidopsis flower developmental stages taken from Smyth et al. (1990).
1466
S11
1.0 – 1.1
10
5–6
Msp
b
Arabidopsis pollen developmental stage after Regan and Moffatt (1990).
c
Observed histological events characterized according to Sanders et al. (1999).
1467
n/a, not applicable.
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