Chromosome studies and genetic analysis of natural and synthetic apomictic model species

Some plants have gained the ability to produce seed without fertilisation. This alternative to sexual reproduction, known as apomixis occurs most frequently in species of the families of the grasses, roses and composites, and mostly in polyploids and is considered one of the ways to escape from hybrid sterility. An impressive number of apomictic mechanisms have so far been described; most of them with different developmental modes of embryo and endosperm. The trait is potential very promising for producing uniform seeds at high efficiency and low cost, and belongs therefore since long as one to the most wanted grails of the plant breeding community. This PhD thesis points at several fascinating genetical and chromosomal aspects of apomixis using the model species Arabidopsis thaliana and the apomicts of the closely related Boechera holboellii complex. Focus is on two different studies in apomixis research: 1) unravelling chromosome organisation and genomic composition of some of the natural diploid apomicts of Boechera and 2) genetic analysis of AtSERK 1 transformed Arabidopsis for engineering apomictic elements in this sexually propagating diploid.<o:p></o:p></span>Boechera's unsurpassed suitability for research of natural apomicts is its impressive genomic plasticity, large geographical distribution and an endless source of different accessions. Quite exceptional is that apomixis also can occur at the diploid level. The chromosome study involving the two sexual diploid species, B. holboellii and B. stricta (2n=14), and six apomictic accessions of B. holboellii and B. X divaricarpa (2n=14 and 15) demonstrated obvious differences between the sexual and apomictic taxa. DAPI fluorescence and FISH with rDNA probes on mitotic cell spreads clearly uncovered two different aberrant<span class=GramE>chromosome</span>, including a largely heterochromatic chromosome ( Het ) and one much smaller chromosome (<st1:State w:st="on"><st1:place w:st="on">Del</st1:place></st1:State>). Moreover, striking variation between the assumed homologues and great differences between the chromosome portraits of the accessions was observed, suggesting structural karyotype heterozygosity in this material. Additional<span class=GramE>studies</span>on male meiosis of the same sexual diploids and apomicts demonstrated a plethora of meiotic variants, pollen size, seed germination and nuclear contents of the seeds. Some of the apomictic accessions displayed full chromosome pairing and recombination, and reductional meiosis, whereas others demonstrated pairing failure, and skewed chromosome segregations. The course of meiosis and pollen size indicated that both n and 2n gametes were formed. In three accessions we observed chiasmate bonds between the Het chromosome and one of the autosomes; whereas the<st1:State w:st="on"><st1:place w:st="on">Del</st1:place></st1:State>chromosome was associated with two other chromosomes.<o:p></o:p></span>Genome in situ hybridization was carried out to establish the assumed hybrid composition of the apomictic B. holboellii and B. X divaricarpa accessions. In the first part we performed hybridisations with total genomic DNA of the diploid sexual B. stricta as probe and blocked with genomic DNA from B. holboellii . Chromosome complements showed fluorescent signals on the pericentromere regions of only the B. stricta chromosomes so that the parental chromosomes could be distinguished in the hybrid. An additional two-colour genome painting was developed with the simultaneous hybridisation with both B. stricta and<o:p></o:p></span>B. holboellii probes and blocking with total genomic DNA of Arabidopsis thaliana in order to further improve the discrimination of the parental chromosomes. This genome painting study confirmed that chromosomes have evolved repeat differentiation at the pericentromere regions enabling a clear distinction of the B. holboellii and B. stricta chromosomes in the hybrids. This result has made clear that Boechera apomicts are alloploid, with different balanced or unbalanced combinations of B. holboellii and B. stricta chromosomes. Most likely such differences in genomic constitutions result from recurrent diploid-polyploid and polyploid-diploid conversions, the latter with reductional meiosis generating haploid gametes with novel combinations of homoeologous chromosomes.<o:p></o:p></span>This genome in situ hybridisation technique revealed that B. holboellii and B. stricta have undergone dramatic evolutionary changes in the repetitive sequences in the pericentromere regions of their chromosomes, producing species-specific FISH banding. The genome painting also revealed that the Het chromosome has B. stricta repeats suggesting that this highly heterochromatic chromosome originated from a B. stricta autosome, which shows that Het resembles more a Y chromosome than a B chromosome. The Del chromosome demonstrated only a small stricta segment, which at metaphase I was found associated with two B. holboe­llii chromosomes, thus suggesting that this chromosome is likely a holboellii / stricta translocation or recombinant chromosome. The outcome of this molecular cytogenetic study has enabled us to formulate a new model on the origin or apomictic accessions and evolutionary processes of the aberrant chromosomes. Essential in the hypothesis is the assumption of an epigenetic modification in the newborn (allo<span class=GramE>)polyploid</span>hybrid. This modification is accompanied or resulted from chromosome pairing impairments at meiotic prophase, and in the longer term led to isolation from crossover recombination, accumulation of repetitive sequences and heterochromatinization of a part of that chromosome involved. Looking at only these few apomicts it is tempting to believe that the Het chromosome in all accessions originated from the same ancestral pro- Het chromosome and that this chromosome plays a key role in the genetic elements required for the apomictic pathway.<o:p></o:p></span>In the second part of the thesis a screening method is presented to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of Single Sequence Length Polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 ( AtSERK 1) gene expressed under the control of two different promoters: the AtLTP1 and the AtDMC1 in sexual Arabidopsis plants. In only one of the four tested F2 transgenic populations, i.e. , expressing the AtLTP1::AtSERK1 construct we observed two plants (1.8%) with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for number of heterozygous loci, it was concluded that these two putative apomictic plants resulted from either incidental recombination events displaying the genotype of the parent, or that they lost their clonal ability in the next generation. Although this genotype screening in the AtSERK 1 transformant offspring not yet resulted in potential apomicts, further refinement and automation are expected to produce an engineered apomictic system that enables identification of a small number of plants exhibiting heterozygosity fixation in an otherwise fully sexual population. Ultimately, if genes that show the potential to confer apomixis need to be introduced in crop species one will need such screening systems operative in the normal sexual background of the crop. Future experiments of AtSERK 1 may also focus on target genes under control of AtSERK 1 -mediated signalling or in combination with other genes involved in somatic embryogenesis