The thesis is devoted to the investigation of the genome variability of Antarctic hairgrass D. antarctica, originating from the southern edge of species distribution in the maritime Antarctic, as in nature, as well under the cultivation in experimental in vitro conditions. It is established that the karyotype of most of D. antarctica plants from the Argentine Islands region of maritime Antarctic (Ukrainian Antarctic Station "Vernadsky Research Base") have a typical chromosome set of 2n=26, and consists of 13 pairs of chromosomes of 3-10 µm in size. Moreover, new forms of chromosome polymorphism, plants with hypotriploidy, mixoploidy and B chromosomes, were found for the first time. The 2C DNA content (pg) of D. antarctica plants, which differ by the number of chromosomes, was determined. The DNA amount of diploid plants was in average 10.88 pg, the 2C of specimen with B chromosomes was within the range of values obtained for diploids (10.86 pg), and hypotriploid was characterized by 1.5 times the value of diploids (16.46 pg). The absence of dependence between the chromosome number variability of investigated D. antarctica plants and the level of their molecular genetic differences by the used PCR markers was revealed. It was established that differences between diploids and hypotriploid, or genotype with B chromosomes, do not exceed the level of molecular genetic variability revealed between the diploids only. The structural features of the D. antarctica karyotype were investigated: the localisation of the 5S rRNA and 45S rRNA genes, telomeric and centrometric repeating sequences were determined. The differences in the localisation of rRNA genes in the karyotypes of plants with diploid and hypotriploid chromosome set were revealed. The nature of hypotripoid genotype and structural integrity of B chromosomes have been confirmed by the FISH technique. Karyotype variability detected in hypotriploid and genotype with B chromosomes confirms the instability of D.antarctica genome and demonstrates possible changes of its structure. The preservation the genetic characteristics of plants during microclonal propagation and in vitro long-term cultivation was shown. The absence of difference at the molecular-genetic and cytogenetic levels was detected as between clones of plants each group of individual genotypes, as between clones and parent plants: plants originating from the diploid ancestor remained diploid, mixoploids retained the cells with different chromosome numbers, but their ratios varied depending from passage. The peculiarities of cytogenetic variability in the D. antarctica callus tissues obtained from plants with different chromosome number were determined for the first time. The instability by the number of chromosomes was determined: proportion of cells with different level of ploidy varied from passage to passage in each of the studied callus variant. The modal class was formed by diploid and/or cells with near-diploid chromosome set, regardless the karyotype of ancestor (diploid, with B chromosomes, hypotriploid). Obtained data indicated the presence of chromosomal variability in the callus tissues cells at the early stages of cultivation, and confirm the preservation the cytogenetic features in diploid genotypes of D. antarctica. It was demonstrated that ability to regenerate is inherent both for diploid and hypotriploid genotypes. The diploid cells prevailed in all investigated regenerants regardless of the ploidy of donor plant. Obtained results indicate that the diploid cells in a genetically heterogeneous cell population show enhanced ability to form regenerants and enhanced genomic instability of hypotriploid genotype.
This research is a part of complex assessment the state of vegetation in the maritime Antarctic region, and the frame for further monitoring the status of Antarctic ecosystems, which are under the impact of human activity and climate change in this part of the planet. The usage of cytogenetic, molecular and biotechnological approaches enabled to obtain data about the variability and selection in marginal populations of D.аntarctica, and also allowed to investigate the adaptation of natural, model and artificial cell systems to extreme existence conditions.
