The thesis is devoted to the study of the phenomenon of somatic reduction and intervarietal polymorphism of homologous chromosomes in A. thaliana and S. cereale plants, on the basis of which two model systems were created to visualize the behavior of parental genomes and the segregation and recombination of homologous chromosomes in vivo and in vitro culture. The research focused on the use of plant models, in vitro culture; induction, molecular mechanisms and genetic effects of somatic reduction; the presence of genetic determinants, external factors and internal agents that cause the occurrence of atypical cell divisions. The possibility to implement the concept of applying somatic reduction phenomenon in homozygous lines obtaining is highlighted.
To create the A. thaliana model hybrid, the transgenic lines of Columbia and Landsberg erecta ecotypes with gusA and gfp reporter genes and nptII and bar selective genes have been obtained, respectively, which provide an opportunity for selection of hybrid plants and allow to distinguish between parental genomes.
A system of molecular SSLP markers has been developed to identify the chromosomes of each ecotype. The SSLP DNA sequences are highly polymorphic in Arabidopsis, have a codominant type of inheritance and are easy to apply. Using the TAIR database a total of 12 SSLP markers were selected performing one marker on each arm of chromosomes 1st to 4th and two markers on each arm of chromosome 5th. Two methods of A. thaliana in vitro cultivation have been tested and optimal conditions for efficient induction of callus and regenerants formation have been determined.
The maximum concentration of PFPA (para-fluoro-L-phenylalanine) was established for induction of somatic reduction in vitro cultured A. thaliana cells, which allows both the callus formation and plant regeneration. As a result, the regenerants were obtained, whose analysis with SSLP marker system showed a loss of heterozygosity in six loci in three plants. A cytogenetic analysis showed the bivalents formation and chromosomal grouping in PFPA treated cells.
Essentially small size of A. thaliana chromosomes for cytogenetic research significantly impairs the visualization and thus the physical evidence of the phenomenon. Unlike A. thaliana, subtelomeric heterochromatin of all rye chromosomes contains several families of tandemly organized DNA sequences, which are characterized by the heterogeneity of the internal organization specific for each monomer of a higher order structure. Monomers of different families have their distinct origin and coexist with the copies of various retroelements families.
Thus, Secale cereale have a great cytogenetic component, namely, large chromosomes and a small number of chromosomes (2n = 14), the presence of large tracks of tandem repeats in subtelomeric heterochromatin and their significant polymorphism in varieties. All these has an advantage for fluorescence in situ hybridization experiments and the ability to discriminate homologous chromosomes. The possibility of identifying individual chromosomes in S. cereale by FISH using probes of different DNA repeats, pSc200, pSc250, pSc119.2, 45SrDNAs, 5SrDNAs (GAA)n, and also the potential for sorting 1R chromosome create powerful tool for obtaining the subgenomic BAC library of a single chromosome. The established polymorphism of the chromosomal localization of repeats (pSc200, pSc250, pSc119.2, 5SrDNA, GAA) in rye varieties Petkus, Imperial, Selgo, Onohoiskaya and Zhyttedaine confirms the possibility of creating on their basis the S. cereale hybrids that would be used as a model system for investigation of the chromosome segregation and recombination and regularity of parental and maternal genomes distribution.
