Research Project Portals

Assembly, Annotation, and Comparative Analysis of Apomictic Boechera Species Genome

Principal investigator of the project Vladimir Brukhin

Apomixis is asexual way of plant reproduction through seeds, which could be found in more than 400 plant species representing almost 40 families. It is believed that apomixis evolved independently in several taxa from sexual ancestors. Apomixis could be considered as a developmental variation of sexual reproduction in which some steps are lost, reduced, deregulated or desynchronized (Fig.1). The main features of gametophytic apomixis are:

  • Avoidance of meiosis (apomeiosis);
  • Embryo formation via parthenogenesis;
  • Functional endosperm develops either autonomously or pseudogamously (central cell fertilized by sperm cell).

Thus, apomictic and sexual reproduction are closely related and share many regulatory components. Molecular and genetic basis underlying apomixis and amphymixis (sexual reproduction) regulation still remains poorly understood. If apomixis is engineered in crop plants that will revolutionize agriculture as heterosis can permanently be fixed in many consecutive plant generations. So, a better understanding of the molecular basis of apomixis is important. The potential of apomixis as a next generation technology for plant breeding attracts huge interest of scientists to elucidate the molecular and genetic mechanisms of its regulation.

Closely related to the model plant Arabidopsis thaliana, the genus Boechera (Fig. 2) is known to contain both sexual and apomictic species or accessions. Boechera genome hybridogenic origin is supported by the aberrant structure of their chromosomes, as they are often observed as a consequence of hybridization, leading to alloploidy, aneuploidy, the replacement of homeologous chromosomes, and aberrant chromosomes. B. stricta and B. retrofracta are conditionally diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic accessions (Fig. 3). In our research, we are attempting to de novo assemble and to annotate whole genomes and find differences between sexual and apomictic accessions of Boechera. We exploit the Illumina and Pacific Bioscience next generation sequencing platforms. The species B. divaricarpa includes both apomictic and sexual accessions. The apomictic accessions are diploid and produces up to100% seeds with a 2:6 embryo: endosperm ratio, indicative of efficient apomeiosis, parthenogenesis of the unreduced (2n) egg, and pseudogamous fertilization of the central cell with unreduced (2n) pollen. Apomixis usually occurs in polyploid perennial species, while diploid species of the same genus reproduce sexually. However several diploid accessions of Boechera possess a high penetrance of apomixis. Apomictic B. divaricarpa has been the focus of genetic, cytological (Fig. 4), and ecological studies, due to its close relatedness to the model plant Arabidopsis thaliana. We also perform phylogenetic and evolutionary analysis of the apomixis associated genes (Fig. 5). Assembly and correct annotation of highly heterozygous genomes of hybrid apomictic species such as B. divaricarpa genomes will provide a basis to decipher the hybridogenetic events that led to the formation of apomictic Boechera accessions.

Study of genetics and genomics of apomixis facilitates to understanding of the role of heterozygosity in the transition from sexual to the asexual reproduction and also evolution of apomixis as a means of reproductive plasticity, which helps to the survival of the species.

Fig. 1. Schematic representation of sexaual and apomictic development.

The left side of the figure shows the sexual pathway and sporophytic apomixis (adventive embryony). In the sexual pathway the megaspore mother cell (MMC) undergoes two meiotic divisions producing a tetrad of megaspores. Three megaspores degenerate while the functional megaspore gives rise to the embryo sac following three rounds of mitotic divisions (female gametophyte = megagametophyte). The mature embryo sac is 7-celled, 8-nucleate with the egg cell and synergid cells (egg cell apparatus) located at the micropylar pole of the embryo sac and three antipodal cells at the opposite pole. Two polar nuclei fuse to form the diploid nucleus of the central cell. After double fertilisation, one sperm fuses with the egg cell forming the diploid zygote, whereas the second sperm fertilises the central cell producing the first nuclei of triploid endosperm. Synergids participate in the perception of the sperm cells and burst after fertilisation. The mature seed consists of the diploid embryo, the triploid endosperm (nourishing tissue) and remnants of the maternal sporophytic nucellar and integumental tissues form the seed coat. An adventive embryo may develop from the nucellar or inner integumental sporophytic tissues and develop alongside the sexual embryo.

The Right part of the figure shows gametophytic apomixis. Meiosis is bypassed and consequently reduction is avoided. Embryo sac development is initiated from an unreduced diplospore (diplospory) or from an apospory initial cell (apospory). The embryo develops parthenogenetically from the unreduced egg cell while endosperm forms either autonomously or through fertilisation of the central cell (in case of pseudogamy). The mature apomictic seed contains the embryo with a relative ploidy level of 2n, while the endosperm could be of variable ploidy but usually not less than 4n.

 

Fig. 2 Boechera sp. plant and inflorescence

Fig. 3 Circos plot for B. retrofracta and B. stricta (sexual ancestors of apomictic B. divaricarpa). Since both assemblies are performed on a scaffold level, it is difficult to hilghlight any large genome rearrangements. However, this plot is a visual way to represent the scatteredness of both assemblies.

Fig. 4 Apomeiosis during megasporogenesis in B.holboellii s.l. A: Megaspore Mother Cell (MMC); B: Diplospory Diad (Dy); C: Single nuclear Diplosporic Embryo Sac (DES) with the remnants of "megaspore" (“MS”); D: callose in the cell wall of the diplosporic dyad

Fig. 5 Phylogenetic tree of the isoforms of APOLLO locus (exonuclease NEN) that is tightly associated with apomixis in Boechera. Seven Brassucaceae species were chosen to help to analyse evolution of homologs of APOLLO locus of apomictic Boechera species from Corral et al (2013). Sequences of Populus trichocarpa, Vitus vinifera and Glycine max were used as outgroup. The clade related to the APOLLO locus is shown in blue, with apo-alleles shown in red. Numbers near nodes represent corresponding bootstrap support. Branches in the tree are grouped by genes rather than by species, suggesting that the triplication event took place before the separation of the Brassicaceae species. Branch leading to apo-alleles is under positive selection (Ka/Ks 1.4646), which is typical for paralogues that are required to serve a novel function.

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Assembly, Annotation, and Comparative Analysis of Apomictic Boechera Species Genome

Principal investigator of the project Vladimir Brukhin

Apomixis is asexual way of plant reproduction through seeds, which could be found in more than 400 plant species representing almost 40 families. It is believed that apomixis evolved independently in several taxa from sexual ancestors. Apomixis could be considered as a developmental variation of sexual reproduction in which some steps are lost, reduced, deregulated or desynchronized (Fig.1). The main features of gametophytic apomixis are:

  • Avoidance of meiosis (apomeiosis);
  • Embryo formation via parthenogenesis;
  • Functional endosperm develops either autonomously or pseudogamously (central cell fertilized by sperm cell).

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