Doctor of Philosophy
Department of Biological Sciences
Lee, L. Slade, The relationship between ploidy, gene expression and vigour in citrus, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 1994. http://ro.uow.edu.au/theses/1062
Autotetraploid Citrus plants exhibit growth retardation compared to their diploid counterparts. Agricultural scientists are attempting to exploit this characteristic to produce dwarfed trees, which offer horticultural advantages over the normally large trees. No conclusive data exist with regard to the dwarfing propensity of tetraploids of various cultivars. Neither is there any information on underlying gene expression effects of autotetraploidy in Citrus cultivars. Tetraploids of Citrus arise apomictically and are therefore somatic and isogenic with their diploid progenitors. Therefore, they provide a useful model for studying the effects of genome duplication. Furthermore, a better understanding of gene expression characteristics in Citrus somatic autotetraploids will result in improved efficiency in tetraploid rootstock breeding programs by aiding the selection of superior germplasm.
In this thesis, differences in gene expression between eight diploid Citrus cultivars and their somatic tetraploid counterparts were investigated, using quantitative methods involving isozyme electrophoresis and laser densitometry. For this purpose, appropriate methods of sample preparation, isozyme electrophoresis and quantification, and DNA measurement were established and verified. Isozymes were electrophoresed on cellulose acetate gels and individual band activities were quantified using a laser densitometer. The effect of genome duplication on quantitative gene expression had not been previously studied for a range of proteins in a number of related genotypes. To compare isozyme activity between diploids and their isogenic tetraploid counterparts it was necessary to compensate for the difference in cell sizes between ploidy levels. This was achieved by expressing sample isozyme activities in terms of their DNA concentrations, which were determined by densitometric and fluorometric methods. In tetraploid plants, many isozyme bands exhibited differing levels of activity compared to diploids of the same cultivar. This effect was repeatable but a very complex situation was apparent in the relationships between individual isozyme bands and between cultivars. When all diploid:tetraploid ratios of isozyme activity per unit DNA for each cultivar were considered collectively, it was found that the majority of the values for each cultivar fell within one of three distinct activity groups. The effect of genome duplication on quantitative gene expression in each cultivar exhibited a strong tendency to either enhanced, reduced or unaffected activities. For any given cultivar, this tendency was uniform across a diverse range of isozymes. For example, isozyme activity per unit DNA was always greater in the tetraploid of the Troyer citrange cultivar compared with its diploid counterpart (for 26 different isozyme bands the ratio of diploid:tetraploid isozyme activity per unit DNA ranged between 0.562 and 0.805). Conversely, the tetraploid of Volkameriana lemon always exhibited reduced isozyme activity, relative to its diploid partner (the diploid:tetraploid ratios for 21 isozyme band activities ranged between 1.412 and 2.034). On the other hand, two mandarin cultivars showed little difference between their ploidy levels. This uniformity of the diploid:tetraploid isozyme activity response of each cultivar suggested that gene expression was modulated by some non-gene-specific or 'extra-genomic' (DeMaggio and Lambrukos, 1974) feature, such as differences in cell morphology between ploidy levels. Very importantly, the assignment of cultivars to the three groups coincided exactly with three distinct phylogenetic groups. This strongly suggested that the non-gene-specific feature above is genetically influenced.
A field trial showed that scions grafted on tetraploid rootstocks were smaller than identical scions grafted on the diploid counterpart rootstocks. This result suggested that tetraploids are physiologically different from their isogenic diploid equivalents. Other field studies compared tree growth characteristics of diploid and tetraploid counterparts of the range of cultivars as examined in the isozyme studies. This work showed cultivar-dependent growth differences between diploids and their tetraploid counterparts. As was the case for their isozyme activity characteristics, growth responses to genome duplication varied between groups of phylogenetically related genotypes. The findings suggested that where there was a marked difference in quantitative gene expression between a diploid and its isogenic tetraploid, vigour may be relatively impaired in the tetraploid. On the other hand, no such vigour effect may occur in genotypes where both ploidy levels exhibit similar quantitative gene expression characteristics.