Epigenetic aspects of sexual and asexual seed development.

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Generation of an integrated Hieracium genomic and transcriptomic resource enables exploration of small RNA pathways during apomixis initiation

Background: Application of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts. Results: A genomic and transcriptomic resource was developed for Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified. Conclusions: The established Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium. research into apomixis initiation in Hieracium.David S. Rabiger, Jennifer M. Taylor, Andrew Spriggs, Melanie L. Hand, Steven T. Henderson, Susan D. Johnson, Karsten Oelkers, Maria Hrmova, Keisuke Saito, Go Suzuki, Yasuhiko Mukai, Bernard J. Carroll, and Anna M. G. Koltuno

Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate

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

A Genome-Wide Survey of Imprinted Genes in Rice Seeds Reveals Imprinting Primarily Occurs in the Endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots