Cytogenetic Analyses Of Programmed B Chromosome Elimination In Aegilops Speltoides

Some eukaryotes exhibit dramatic differences in genome size between cells of different organs,resulting from the programmed elimination of DNA/chromosomes.Programmed DNA elimination includes chromatin diminution,chromosome elimination,and paternal genome elimination.In plants,only B chromosomes（Bs）of Aegilops speltoides are known to be associated with programmed DNA elimination.Ae.speltoides is an annual diploid species from the Triticeae,with a maximum number of eight Bs in addition to its inherent seven pairs of standard A chromosomes（As）.Although Bs is stable in their numbers in the areal tissue of Ae.speltoides,they undergo precise elimination in roots upon onset of embryo differentiation in yet unknown mechanisms.In this dissertation,to gain insight into regulatory mechanisms underlying root-restricted Bs elimination,Ae.speltoides mutant population was produced by employing X-ray mutagenesis.Then,a systematical screen of de novo generated mutant plants for B chromosome variants in shoots and roots as well as chromosomal distribution of histone protein H3 was carried out with multicolor fluorescent in situ hybridization in the nature population.Finally,functional characterization of candidate proteins potentially involved in Bs elimination was initiated by investigating their chromosomal distribution and dynamic behaviors during cell division in rye plants.The major findings of the present thesis include:（1）To overcome the root-restricted process of B chromosome elimination,X-ray mutagenesis was used to generate mutant plants and different types of restructured Bs were observed.Standard Bs were detected in all analyzed shoots of mutagenized plants,while B-A translocations were only observed in 35.7%of F₁ plants and all Bs variants were stably present in the shoot organs.In total,40 different B variants inconsistently escaped the elimination process in roots.As a result,mosaicism of B chromosome variants was found in roots.Distinct to shoot,Bs variants were randomly observed in the root tissues,suggesting that mutation of critical regulatory genesresponsible for Bs elimination may occur in the A or B chromosome.Only a small B chromosome fragment fused to an A chromosome was stably maintained in roots and shoots across F₁ to F₃ generations.The absence of B-A translocation chromosomes possessing a B-derived centromere in root cells implies that the centromere of the B is a component key to the chromosome elimination process.Results also showed that whereas normal Bs could accumulate in the shoot,all Bs variants did not present in the root.Notably,plants with accumulation of Bs in the root were sterile at the mature stage,indicating that B chromosome elimination may be critical for plant fertility and a strategy for population stability.（2）To address whether the nondisjunction of Bs chromatids is associated with differential chromosomal composition between A and B chromatin,immunostaining was applied to investigate the chromosomal distribution of H3S10ph and CENH3 on the A and B chromosomes during the root chromosomal elimination process at the onset of embryo differentiation stage of Ae.speltoides.Results showed that neither H3S10ph nor CENH3 differs its chromosomal distribution between A and B chromosomes,suggesting that Bs elimination is independent of phosphorylation of Ser10 of H3.（3）To characterize candidate genes potentially involved in the Bs elimination process,three-dimensional immunostaining was performed to investigate the mitotic as well as meiotic chromosomal distribution and dynamics of CENH3,α-tubulin,Nuf2and kinesin in rye.Results showed that Nuf2 expresses consistently during the meiosis and mitosis.In particularly,Nuf2 localizes periphery to CENH3 in the centromere and is associated with tubulin at the metaphase,which acts cooperatively to sustain the cell division.Similarly,kinesin also consistently expresses in the meiosis and mitosis,and more often co-localizes with tubulins,which is indispensable for proper cell division.Furthermore,there was no difference in the distribution of Nuf2 and kinesin on the A and B chromosomes.