Molecular Markers
Universal markers from the cytoplasmic (PCR-RFLP) and nuclear (AFLP) genomes will be
used to assess levels of genetic diversity for these two genomes in selected tree species.
Seed flow will also be examined using chloroplast DNA markers. Since chloroplast DNA is
predominantly inherited clonally through the maternal generation in angiosperms, it is an
ideal marker to use for tracing maternal lineages and monitoring seed flow within
populations.
Pollen flow will be monitored by means of biparentally inherited hypervariable nuclear
DNA markers (SSRs) specifically developed for the project.
All Markers will be developed by VIB in collaboration with other partners
Nuclear DNA
Molecular methods will be developed that identify polymorphisms at numerous loci
distributed throughout the nuclear genome. The AFLP (amplified fragment length
polymorphism) method selectively amplifies restriction fragments from total genomic DNA.
Different enzyme and primer combinations will be tested and the most appropriate and most
universal set of enzymes and primers will be selected.
Using the selected set of enzymes and primers, representative AFLP fingerprints will be
generated for each species. The banding patterns will be digitized and stored in a
computer database together with other relevant molecular and genetic information about the
tree species. In this way a reference guide will be made that will be available to all
partners and easily accessible through information networks.
Cytoplasmic DNA
'Universal' primers that amplify non coding regions will be used to examine molecular
variation in the chloroplast DNA. Amplified products will be digested by restriction
enzymes in order to identify existing polymorphism. If polymorphism is not detected, an
alternative will be to use CFLP (cleaved fragment length polymorphism) analysis on the
amplified fragments. Another approach will be to investigate hypervariable genomic
regions, using primers recently developed to detect microsatellite motifs in chloroplast
DNA.
Development of molecular tools for mating system and gene flow analysis
The objective is to identify highly polymorphic DNA markers specifically developed for
each study species. It is hoped that a technique similar to SSRs will be used that doesn't
require the screening of genomic DNA libraries. The method proposed, SAMPL (selective
amplification of polymorphic loci), is an extension of the AFLP technique. Once SAMPL
markers have been identified in one species, they will be converted into STS (sequence
tagged sites) markers and tested in the other species to verify whether they are
universally applicable. Since two-base repeated regions are numerous in tropical trees,
the identification of hypervariable regions should be straightforward.
