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In this chapter, it was confirmed that the mutations mea1-1 and fis2-1 (when carried by the A. thaliana mother) facilitate reproducible generation of genuine A. thaliana x B. pinetorum inter-genus hybrids. This was an unexpected and exciting result as mea and fis2 mutants are maternal gametophytic lethal (Chaudhury et al., 1997; Luo et al., 1999). Currently, it is unknown how the inheritance of mea1-1 and fis2-1 is being rescued in the F1

hybrids. This could be an effect of the B. pinetorum genome or epigenome. Alternatively, it may be an effect of the genomic imbalance, particularly a paternal genomic excess. Given that mea1-1 and fis2-1 are being successfully transmitted, it is possible that an autonomous endosperm has initiated and stabilized to nourish the hybrid genome, although this has not been determined. Currently, it is unknown how mea1-1 and fis2-1 are partially alleviating post-zygotic hybridisation barriers between A. thaliana and B. pinetorum.

Both MEA and FIS are components of the endosperm specific FIS PRC2, which regulates genomic dosage imbalances in the endosperm (Mozgova and Hennig, 2015; Rodrigues and Zilberman, 2015). Interploidy crosses demonstrate dosage imbalances have differing effects based on the parental excess (Scott et al., 1998). A maternal genomic excess can give rise to viable seeds, whereas a paternal excess leads to predominate seed abortion as the endosperm fails to cellularise (Scott et al., 1998; Josefsson et al., 2006; Nowack et al., 2007; Kradolfer et al., 2013a). As the F1 hybrids are four genome plants, there is a genomic

paternal excess (one A. thaliana: three B. pinetorum). Many studies have shown that a paternal excess has a negative effect on seed development, as FIS PRC2 target genes become de-regulated and inhibit endosperm cellularisation. For instance, AGAMOUS-LIKE 62 (AGL62) is a negative regulator of endosperm cellularisation (Kang et al., 2008) and fis2 mutations cause increased AGL62 expression that causes endosperm cellularisation failure

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(Hehenberger et al., 2012). Similarly, the paternally expressed genes PHERES1 (Josefsson et al., 2006) and ADMETOS (Kradolfer et al., 2013b) exhibit FIS PRC2 de-regulation under paternal genome excess (increased maternal allele expression) that contributes to hybrid seed death through endosperm failure. Both have an expected role in regulating the expression of AGL genes. Recent studies have identified more paternally expressed genes involved, with a likely role in increasing up-regulation of pectin degradation genes that prevent endosperm cellularisation (Wolff et al., 2015). In genomic imbalances, these genes, among others, become deregulated due to the failure of the FIS PRC2. This could be due to limited transcript levels of MEA and FIS2 (Erilova et al., 2009). Both MEA and FIS2 are maternally expressed (Kinoshita et al., 1999; Luo et al., 1999; Vielle-Calzada et al., 1999) and the maternal MEA has high specificity to silence (auto regulate) the paternal MEA over other targets (Erilova et al., 2009). In paternal genomic excess hybrids, the FIS PRC2 is able to maintain paternal MEA imprinting, however fails to regulate other targets. In the absence of a functional FIS PRC2, it could be assumed that endosperm cellularisation and thus seed development would not be successful. However, this is not the case; unknown factors outside FIS PRC2 activity can supress de-regulated imprinted genes.

Alternatively, it is possible that the hybrids are developing with an autonomous, diploid endosperm, which has no paternal contribution. A similar phenomenon has been demonstrated in experiments that have utilised mutations in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CDKA;1) (Nowack et al., 2007; Shirzadi et al., 2011). Heterozygous CDKA;1/cdka;1-1 mutants perturb the second pollen mitosis, whereby pollen can successfully fertilise the egg cell, however fertilisation of the central cell is unsuccessful (Iwakawa et al., 2006; Nowack et al., 2006). Although a second mutant sperm cell is delivered to the central cell, successful fertilisation does not occur; karyogamy does not take place and the central cell remains diploid (Aw et al., 2010). However, endosperm proliferation does occur. Subsequent seed development also occurs, however, the endosperm remains under-developed which leads to seed abortion. Interestingly, crossing a homozygous mea/mea mutant with CDKA;1/cdka;1-1 rescues seed development; a viable seed is produced from a diploid endosperm (Nowack et al., 2007). It remains unknown how this rescue occurred. CDKA;1/cdka;1-1 fertilised seeds have approximately 600 genes significantly decreased in expression in the absence of a paternal genome, including imprinted MADS-box transcription factors (Shirzadi et al., 2011). It is possible that a diploid endosperm has supported the inter-genus embryo, whereby paternally expressed genes that

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limit hybridisation are not present. For future study, it would be highly beneficial to investigate the ploidy level of the endosperm and monitor endosperm development.

To date, there have been no reports of mea and fis2 mutations alleviating post- zygotic hybridisation barriers. However, utilising met1 pollen in interploidy hybridisation experiments, Schatlowski et al. (2014) produced a high frequency of viable triploid seeds with a tetraploid endosperm. In these experiments, where the tetraploid endosperm is under increased paternal dosage, paternal CG hypomethylation bypasses the interploidy hybridization barrier, as deregulated FIS PRC2 target genes are repressed by de novo methylation (Schatlowski et al., 2014). Accordingly, the study suggests a functional FIS PRC2 is not required for interploidy hybridisation as other epigenetic modifications can substitute FIS PRC2 activity. Similarly, other studies demonstrate that hypomethylated pollen avoids mea1-1 and fis2-1 seed arrest, whereby a maternal FIS PRC2 is not required (Luo et al., 2000). Therefore, methylation also controls some genes that have key roles in seed development. Furthermore, exacerbation of de-regulated paternally imprinted genes can be combated by adopting other forms of regulation outside FIS PRC2 activity.

Another hypothesis is that lncRNAs have roles in alleviating post-zygotic hybridisation barriers. For instance, lncRNAs may potentially impose regulation by de novo DNA methylation. Pol IV and Pol V lncRNAs are integral components of the RdDM pathway (Wierzbicki et al., 2008; Li et al., 2015a). Other lncRNAs have novel regulatory mechanisms that can influence a 5 kb locus (Ariel et al., 2014). From Chapter Three, it was discovered that mea and fis2 mutations contain over 800 lncRNAs in siliques that cannot be detected in other ecotypes, crosses or other studies. mea and fis2 mutations also induced significant changes in the expression of over 500 lncRNAs. In Chapter Two, lncRNAs were identified that are potentially imprinted. Investigation of lncRNAs in post-zygotic hybridisation barriers is unexplored and a long term aim of this project.

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4.4.3 Conclusions

In this chapter, 33 putative F1 hybrids between A. thaliana Ler and B. pinetorum were

confirmed by PCR and GBS. All putative hybrids tested containing the genomes of both parents. The F1 hybrid genomes were predominantly stable, however one hybrid had a

confirmed deletion of an A. thaliana chromosome. More than 90% of the F1 hybrids were

confirmed to inherit the mutant mea1-1 or fis2-1 allele. Overall the data implies that the mutations mea1-1 and fis2-1 are responsible for partially alleviating hybridisation barriers between A. thaliana and B. pinetorum. How this occurs is relatively unknown, but is an exciting phenomenon for further research.

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CHAPTER FIVE: General discussion