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Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a “tetraploid-dihaploid-tetraploid” series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003–2007) to reach levels of 85–90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements.

In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a 'tetraploid-diploid-tetraploid' series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid-diploid conversion reverted during the diploid-tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized.

Apomixis, an asexual mode of reproduction through seeds, holds much promise for agricultural advances. However, the molecular mechanisms underlying this trait are still poorly understood. We previously isolated several transcripts representing novel sequences differentially expressed in reproductive tissues of sexual and apomictic plants. Here, we report the characterization of two of these unknown RNA transcripts (experimental codes N17 and N22). Since original fragments showed no significant homologies to sequences at databases, preliminary genomic PCR experiments were carried out to discard possible contaminations. RACE extension on flanking regions provided longer sequences for the candidates and additional related transcripts, which revealed similarity to LTR retrotransposons carrying short transduplicated segments of protein-coding genes. Interestingly, some transduplicated segments corresponded to genes previously associated with apomictic development. Gene copy number estimations revealed a moderate representation of the elements in the genome, with significantly increased numbers in a sexual genotype with respect to an apomictic one. Genetic mapping of N17 showed that a copy of this particular element was located onto Paspalum notatum linkage group F3c, at a central non-recombinant region resembling a centromere. Expression analysis showed an increased activity of N17 and N22 sense strands in ovules of the sexual genotypes. A retrotransposon-specific differential display analysis aimed at detecting related sequences allowed the identification of a complex family, with the majority of its members represented in the sexual genotype. Our results suggest that these elements could be participating in regulatory pathways related to apomixis and sexuality.

Imidazolinones are powerful herbicides that inhibit branched‐chain amino acid biosynthesis by targeting the catalytic subunit of acetohydroxyacid synthase (AHAS). Imidazolinone application in the advanced vegetative or early reproductive developmental stages is associated with male sterility in resistant sunflower (Helianthus annuus L.); however, the underlying mechanism of this sterility remains unknown. This study describes the morphological, cytoembryological, and molecular alterations induced by imazapyr (IM) treatment on reproductive tissues at different developmental stages in two sunflower genotypes, resistant and intermediate resistant, respectively. Pollen and seed physiological variables were compared between the treated and control plants. The number of pollen grains per flower and viable seeds were negatively affected by IM treatment in the intermediate‐resistant genotype, and the biometric traits of early developed disc flower were also significantly different in this genotype. Differential interference contrast microscopy revealed that IM treatment slightly accelerates megagamethophyte development. Anther observations at microsporogenesis using confocal microscopy show that the sporogenous tissue was damaged. Furthermore, the expression profiles of the sunflower AHAS paralogs (ahas1, ahas2, and ahas3) were measured by quantitative polymerase chain reaction in the anthers and pistils of two developmental stages in treated and control plants. Imazapyr treatment in early reproductive growth stages clearly induces divergent expression patterns in the ahas gene family. These findings provide new insight into a novel chemical method for inducing male sterility in sunflowers and enhance our understanding of the effects of AHAS‐inhibitor herbicides in reproductive tissues.

Key messagencRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes.AbstractApomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30–60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.Electronic supplementary materialThe online version of this article (10.1007/s11103-017-0679-4) contains supplementary material, which is available to authorized users.

No abstract

Imazapyr (IM) treatment applied at early reproductive developmental stages have been associated with induced male sterility in sunflower (Helianthus annuus L.). The aim of this work was to determine the effects induced by two different doses of IM-treatment (1X and 2X; 80 and 160 g a.i.•ha -1 ) on pollen grain yield, pollen viability and on the exine surface, in an intermediate resistant (I) and a completely resistant (R) genotype. An anatomical analysis of some stages during the microsporogenesis and microgametogenesis was also achieved by confocal microscopy. Plants of both genotypes survived the herbicide treatments and showed no external injury symptoms. Differential response to different doses of IM was obtained in R and I genotypes.The pollen grains per flower and the viability in seeds were significantly reduced in the I genotype by the 2X treatment; however, the pollen viability of the remaining grains was not altered, and the diameter of the pollen grains was increased by the herbicide treatment. Anatomical and cytological observations of the sporogenous tissue development were made via confocal microscopy. IM treatment induced alterations in sporogenous cells during the initial phases of microsporogenesis in both genotypes, and the 2X dose accelerated the microgametogenesis process, particularly in the I genotype. These findings improved the understanding of the mechanism underlying the gametocide effects of the imidazolinones in sunflower.