A molecular cytogenetic analysis of introgression in Alstroemeria

This thesis describes the results of a molecular cytogenetic investigation of the process of introgression in Alstroemeria . The aim of this study was to transfer chromosomes or genes from one Alstroemeria species into another. For this, two distantly related species, A. aurea and A. inodora , were hybridized and the resulting hybrids were further backcrossed with the species A. inodora . To monitor the process of introgression accurately it was necessary to identify the individual chromosomes of both parental species.In addition to classical karyotyping, reliable criteria for unequivocal identification of all individual somatic chromosomes of the species A. aurea and A. inodora have been developed by applying fluorescence in situ hybridization (FISH) with tandemly repeated DNA probes (Chapter 2). Four probes, two species specific, A001-I (for A. aurea ) and D32-13 (for A. inodora ), and two ribosomal rDNA repeats, pTa71 (18S-25S) and pTa794 (5S), revealed specific banding patterns per chromosome, enabling identification of all individual chromosomes. In contrast to most plant species, both Alstroemeria species contained a large number of rDNA sites (per haploid genome: 20 in A. aurea and 16 in A. inodora ). Remarkably, the physical location of the rDNA sites varied greatly between the two species; only a few were found at similar positions.Besides identification of somatic chromosomes, FISH with the species specific probes was used for the identification of meiotic chromosomes to accurately determine the extent of homoeologous association in the diploid parental hybrid. Multicolour FISH experiments revealed a large number of bivalents per Metaphase I cell (average 6.7 bivalents per MI) of the hybrid A. aurea x A. inodora , which indicated that homoeologous recombination occurs frequently in the hybrid.Successful backcrossing of the highly sterile distant hybrid A. aurea x A. inodora with its parent A. inodora resulted in a small number of first generation backcross plants (BC1). To establish whether and how frequently homoeologous recombination occurred during meiosis in the hybrid, the BC1 plants were analysed with genomic in situ hybridization (GISH). The presence of recombinant chromosomes in all plants provided evidence for homoeologous recombination. All plants were analysed using a combination of FISH with the repetitive probes and GISH that allowed identification of all chromosomes and the construction of detailed karyotypes. In addition, identification of the recombinant chromosomes revealed which of the chromosomes of A. aurea and A. inodora were actually homoeologous.The recombinant chromosomes showed a large number of recombination points, indicating that multiple crossovers had occurred. The distribution of the recombination points indicated that chiasmata are formed along the entire length of the chromosomes. Furthermore, the analysis of recombinant chromosomes revealed structural differences (one translocation and one inversion) between two sets of homoeologous chromosomes. These differences were confirmed by analysis of metaphase I configurations of these chromosomes in the hybrid and the BC1 plants (Chapters 4 and 5).The detailed analysis of the genome constitution of the plants revealed two different types of plants, near diploids (2n=2x+1=17) and triploids (2n=3x=24), indicating that two different types of gametes were functional in the hybrid, viz., n- and 2n-gametes. The GISH and FISH analysis provided evidence that 2n-gametes were functional both from male and female parents and had originated in all cases through a mechanism genetically leading to first division restitution (FDR) (Chapter 4).In order to investigate the fate of the introgressed A. aurea segments in A. inodora , meiosis was analysed using GISH and FISH in the triploid BC1 plants. These plants contained two more or less complete genomes of A. inodora and one of A. aurea as a result of the FDR mechanism (Chapter 4). Metaphase I configurations were studied per set of three homo(eo)logous chromosomes. This analysis revealed, as expected, preferential pairing of the A. inodora chromosomes, whereas the A. aurea chromosomes usually were unpaired. Only low frequencies of homoeologous chromosome pairing were observed, which is in contrast to what was found in the parental hybrid. Interestingly, the recombinant chromosomes generally formed trivalents with their two homo(eo)logues. These chromosomes were only associated at homologous (recombinant) chromosome segments. At later stages of meiosis, normal distribution of the A. inodora chromosomes and the recombinant chromosomes into the gametes was observed, whereas the non-recombinant A. aurea chromosomes were randomly distributed among the gametes, pointing towards the perspective for obtaining addition lines or introgression of alien chromosome segments in Alstroemeria .For breeding, the most important observation, which was made in this molecular cytogenetic study of Alstroemeria , was the high frequency of pairing and recombination between homoeologous chromosomes in the interspecific hybrid and the almost absence of this phenomenon in the further backcross generations. In these backcrosses, preferential pairing between homologous chromosomes was observed. For efficient introgression breeding more emphasis should be given to the production of sufficient BC1 plants and the detection of desired recombinants in this generation.</p

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed

Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids.

Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed