Molecular tools for population and ecological genetics in coniferous trees

We have been isolating AC/GT and AG/CT SSRs from the Norway spruce (Picea abies K.) nuclear genome. We isolated several hundreds positive clones from a small-insert genomic library and following sequence analysis we designed primers for 36 of them, 24 containing AG and 12 AC SSRs. After testing them on a panel of spruce individuals 25 of the primer pairs producted a single-locus hypervariable pattern, with the remaining ones giving either a single monomorphic product (18) or very poor amplification (19) or amplification of multiple bands (38). Segregation in accordance with a simple Mendelian model of inheritance was demonstrated for all the loci amplified with the primer pairs giving a simple variable pattern. We screened a panel of 19 spruce trees at these loci. The average number of alleles per locus was 14 and expected heterozygosity 0.80, with up to 23 alleles per locus and heterozygosities exceeding 0.94. This shows that nuclear SSRs can be very useful markers in the population genetics of trees even though the overall efficiency of the marker identification process is quite low due to the high percentage of primer pairs producting complex or "dirty" patterns. We attribute this phenomenon to the high complexity of the spruce genome. Other methods, including the construction of libraries highly enriched for SSR sequences, that we developed in order to make SSR retrival and typing easier and faster will be discussed. We recently extended the use of PCR amplified SSR markers to the chloroplast genome. We demonstrated that mononucleotide poly(A/T) stretches are frequent in the chloroplast genomes of plants and show high levels of between and within population variation, making them ideal tools for cytoplasmic population genetic overcoming the difficulties in finding within species variation that are frequently encountered when analysing the cpDNA molecule by RFLPs or PCR-RFLPs. We will present results of the analysis of mediterranean pine species populations by using a set of cpSSRs that are distributed over the whole cpDNA molecule and discuss the possible applications of such markers for studying gene flow and for paternity analysis.