- Volkaert, Hugo
The Graduate School, University of Maine
The ability to genetically fingerprint trees through the detection of polymorphisms can be a valuable tool in tree breeding. Micro satellites are highly polymorphic in animals and are inherited in a Mendelian fashion. Therefore they were studied as markers for potential use in conifer populations.
Micro satellite loci with AC- and AG-repeats were found in larch and occur about every 690 and 220 kip respectively. For most of the micro satellite loci, primers did not amplify a specific DNA fragment. For one primer pair surrounding a cloned micro satellite, it could be established clearly that two different fragments are amplified from the larch genome. Upon cloning of amplified fragments, multiple base substitutions were found in the sequences flanking the micro satellites repeats. Because of the high rate of base changes and the possibility of multiple primer annealing sites, it is postulated that primers may bind with low specificity at more than annealing sites and compete for efficient amplification.
RAPD makers were also considered as potentially useful source of polymorphism because few DNA amplification products showed a codominant segregation pattern in larch megagametophytes. These amplifications products were cloned sequenced. The two alleles amplified from one tree have almost 2% base substitutions. Cloning of amplified fragments at one locus from eight other trees resulted in eight additional alleles. Amount these ten alleles, base substitutions were found at 15% of the nucleotide positions. Additional polymorphisms were also revealed at other loci through changes in restriction enzyme digestion patterns. Different alleles at these loci can be distinguished by double strand DNA conformational polymorphism. Most base changes are C to T transitions, many at potentially methylated CG and CNG sites.
After conversion to sequences characterized loci, codominant RAPD fragments can be used in population genetic studies. To evaluate the usefulness of these loci to determine the pollen partner in seed orchards, paternity exclusion probabilities were calculated. the exclusion probabilities obtained in a three species, 15 clone experimental orchard range from 0.1 to 0.5 for individual loci within each species, and are comparable to informative isozymes. The Mendelian inheritance of alleles at one locus was tested in a few progeny of a controlled Larix laricina cross.