Plant Genome IV Conference Plant Genome IV Conference
In order for molecular markers to be used in the population genetics of forest trees they have to meet certain requirements such as the ease and speed of genotyping, the codominance of alleles, the reproducibility over time and space, the high information content and the possibility of easily exchanging marker information. After taking into account advantages and disadvantages of each of the systems we decided to focus our attention on the use of simple sequence repeats (SSRs or microsatellites) because they are codominant, reproducible, highly informative and easy to exchange even though they have high development costs. We have been isolating AC/GT and AG/CT SSRs from the Norway spruce (Picea abies K.) nuclear genome. Based on hybridization data we estimate that there is one AC SSR every 400 kb and one AG SSR every 200 kb. When the genome size of spruce is considered (30-40 pg) this corresponds to an extremely large number of SSRs available for analysis. 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 produced 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 tested. We screened a panel of 18 spruce trees at these loci. The average number of alleles per locus was 14 and expected heterozygosity 0.80. This shows that nuclear SSRs can be very useful markers for the population genetics of conifers even though the overall efficiency of the marker identification process is quite low due to the high percentage of primer pairs producing complex or "dirty" patterns. We attribute this phenomenon to the high complexity of the spruce genome and have observed that screening against clones containing highly repeated DNA can make the SSR isolation process more efficient. All the six monomorphic AG clones seem to belong to a highly repeated but diverged sequence family, with homologies between 50 and 90% whose distribution in the genome is currently under study. Other methods, including the construction of libraries highly enriched for SSR sequences, that we developed in order to make SSR retrieval and typing easier and faster will be discussed. We will also present results of the analysis of four spruce populations at 7 SSR loci and discuss levels and distribution of genetic variation and heterozygosity. We also 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 genetics 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 via pollen and for paternity analysis. Preliminary data show that the cytoplasmic diversity is extremely high in Pinus halepensis populations in the eastern Mediterranean area where the centre of origin of the species is supposed to be and is near to zero in the western Mediterranean region.