Molecular cytogenetics in Picea

A fundamental characteristic of a species is its karyotype, a description of each chromosome. In spruce (Picea) and other conifer genera, chromosome identification is hindered by similarities in chromosome size and morphology within a species. Homeologous chromosomes of related species are also generally indistinguishable. The focus of this Ph.D thesis is the development of a new technology in conifer cytogenetics, in situ hybridization, to address these inherent difficulties and to establish procedures for the physical mapping of the spruce genome. In situ hybridization augments conventional cytogenetics with the methods of molecular biology and allows the visualization of defined DNA sequences along metaphase chromosomes and interphase chromatin. The chromosomal locations of three tandem repeated DNA sequences, including the genes encoding the 18S-5.8S-26S ribosomal RNA, those encoding the 5S ribosomal RNA, and a centromeric satellite DNA (SGR-31), were determined in white spruce (Picea glauca (Moench) Voss) and Sitka spruce (P. sitchensis (Bong.) Carr.). A molecular description of the 5S ribosomal RNA genes and SGR-31 in white spruce, comprising their nuclear organization, nucleotide sequence and genomic copy number, was also performed. Combining in situ hybridization data from the 18S-5.8S-26S ribosomal RNA and the 5S ribosomal rRNA genes permitted the first unequivocal identification of each somatic chromosome of a conifer species. All three repeated DNAs were subsequently mapped onto the same metaphase spreads, producing the first cytogenetic maps of white and Sitka spruces. The white spruce map consists of twelve loci, seven corresponding to 18S-5.8S-26S ribosomal RNA loci, one to the 5S ribosomal DNA, and four to SGR-31 sites. Comparison of this map to that of Sitka spruce (five 18S-5.8S-26S ribosomal RNA loci, one 5S ribosomal RNA site, and five SGR-31 loci) revealed that despite the overall uniformity in appearance among homeologous chromosomes, the repeated DNA complement of these closely related spruce genomes is in flux. The prospects for the use of in situ hybridization in conifer genome analysis are immense. It’s greatest contribution, which hinges on improvements in detection sensitivity, will be in the integration of genetically defined linkage groups with the chromosomes on which they reside. Investigating the relationship between genetic linkage, recombination and chromosome structure or the co-linearity of gene sequences and chromosomal synteny among spruce species and other conifer genera, and the eventual cloning of genes based on map position, are now within the possibilities of conifer genetics.Forestry, Faculty ofGraduat