Application and comparison of large-scale solution-based DNA capture-enrichment methods on ancient DNA

The development of second-generation sequencing technologies has greatly benefitted the field of ancient DNA (aDNA). Its application can be further exploited by the use of targeted capture-enrichment methods to overcome restrictions posed by low endogenous and contaminating DNA in ancient samples. We tested the performance of Agilent's SureSelect and Mycroarray's MySelect in-solution capture systems on Illumina sequencing libraries built from ancient maize to identify key factors influencing aDNA capture experiments. High levels of clonality as well as the presence of multiple-copy sequences in the capture targets led to biases in the data regardless of the capture method. Neither method consistently outperformed the other in terms of average target enrichment, and no obvious difference was observed either when two tiling designs were compared. In addition to demonstrating the plausibility of capturing aDNA from ancient plant material, our results also enable us to provide useful recommendations for those planning targeted-sequencing on aDNA

Molecular Strategies for Gene Containment in Transgenic Crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops

Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues

Mutation in SUMO E3 ligase, SIZ1, Disrupts the Mature Female Gametophyte in Arabidopsis

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance

Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome

<p>Abstract</p> <p>Background</p> <p>Apomixis, asexual seed production in plants, holds great potential for agriculture as a means to fix hybrid vigor. Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis. Understanding the molecular mechanism regulating aposporous initial specification will be a critical step toward elucidation of apomixis and also provide insight into developmental regulation and downstream signaling that results in apomixis. To discover candidate transcripts for regulating aposporous initial specification in <it>P. squamulatum</it>, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, <it>P. squamulatum </it>(accession PS26), and an apomictic derived backcross 8 (BC₈) line containing only the Apospory-Specific Genomic Region (ASGR)-carrier chromosome from <it>P. squamulatum</it>. Toward this end, two transcriptomes derived from ovules of an apomictic donor parent and its apomictic backcross derivative at the stage of apospory initiation, were sequenced using 454-FLX technology.</p> <p>Results</p> <p>Using 454-FLX technology, we generated 332,567 reads with an average read length of 147 base pairs (bp) for the PS26 ovule transcriptome library and 363,637 reads with an average read length of 142 bp for the BC₈ovule transcriptome library. A total of 33,977 contigs from the PS26 ovule transcriptome library and 26,576 contigs from the BC₈ovule transcriptome library were assembled using the Multifunctional Inertial Reference Assembly program. Using stringent <it>in silico </it>parameters, 61 transcripts were predicted to map to the ASGR-carrier chromosome, of which 49 transcripts were verified as ASGR-carrier chromosome specific. One of the alien expressed genes could be assigned as tightly linked to the ASGR by screening of apomictic and sexual F₁s. Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.</p> <p>Conclusions</p> <p>Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.</p

Ribosomal RNA of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

The nucleolar activity of Hyacinthus orientalis L. embryo sac cells was investigated. The distributions of nascent pre-rRNA (ITS1), 26S rRNA and of the 5S rRNA and U3 snoRNA were determined using fluorescence in situ hybridization (FISH). Our results indicated the different rRNA metabolism of the H. orientalis female gametophyte cells before and after fertilization. In the target cells for the male gamete, i.e., the egg cell and the central cell whose activity is silenced in the mature embryo sac (Pięciński et al. in Sex Plant Reprod 21:247–257, 2008; Niedojadło et al. in Planta doi:10.1007/s00425-012-1599-9, 2011), rRNA metabolism is directed at the accumulation of rRNPs in the cytoplasm and immature transcripts in the nucleolus. In both cells, fertilization initiates the maturation of the maternal pre-rRNA and the expression of zygotic rDNA. The resumption of rRNA transcription observed in the hyacinth zygote indicates that in plants, there is a different mechanism for the regulation of RNA Pol I activity than in animals. In synergids and antipodal cells, which have somatic functions, the nucleolar activity is correlated with the metabolic activity of these cells and changes in successive stages of embryo sac development

Genomic Analysis of Parent-of-Origin Allelic Expression in Arabidopsis thaliana Seeds

Differential expression of maternally and paternally inherited alleles of a gene is referred to as gene imprinting, a form of epigenetic gene regulation common to flowering plants and mammals. In plants, imprinting primarily occurs in the endosperm, a seed tissue that supports the embryo during its growth and development. Previously, we demonstrated that widespread DNA demethylation at remnants of transposable elements accompanies endosperm development and that a subset of these methylation changes are associated with gene imprinting. Here we assay imprinted gene expression genome-wide by performing high-throughput sequencing of RNA derived from seeds of reciprocal intraspecific crosses. We identify more than 200 loci that exhibit parent-of-origin effects on gene expression in the endosperm, including a large number of transcription factors, hormone biosynthesis and response genes, and genes that encode regulators of epigenetic information, such as methylcytosine binding proteins, histone methyltransferases, and chromatin remodelers. The majority of these genes are partially, rather than completely, imprinted, suggesting that gene dosage regulation is an important aspect of imprinted gene expression

Transcriptional activity of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of incorporated 5′-bromouracil, the total RNA polymerase II pool and the hypo- (initiation) and hyperphosphorylated (elongation) forms of RNA Pol II. The first stage, which lasts from the multinuclear stage to cellularization, is a period of high transcriptional activity, probably related to the maturation of female gametophyte cells. The second stage, encompassing the period of embryo sac maturity and the progamic phase, involves the transcriptional silencing of cells that will soon undergo fusion with male gametes. During this period in the hyacinth egg cell, there are almost no newly formed transcripts, and only a small pool of RNA Pol II is present in the nucleus. The transcriptional activity of the central cell is only slightly higher than that observed in the egg cell. The post-fertilization stage is related to the transcriptional activation of the zygote and the primary endosperm cell. The rapid increase in the pool of newly formed transcripts in these cells is accompanied by an increase in the pool of RNA Pol II, and the pattern of enzyme distribution in the zygote nucleus is similar to that observed in the somatic cells of the ovule. Our data, together with the earlier results of Pięciński et al. (2008), indicate post-fertilization synthesis and the maturation of numerous mRNA transcripts, suggesting that fertilization in H. orientalis induces the activation of the zygote and endosperm genomes

Molecular Foundations of Reproductive Lethality in Arabidopsis thaliana

The SeedGenes database (www.seedgenes.org) contains information on more than 400 genes required for embryo development in Arabidopsis. Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development