Thirty-six nuclear-encoded RAPD loci and 20 allozyme loci were studied to compare levels of diversity and differentiation among populations and races of the widespread North American conifer, Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RAPD assays used diploid seed embryo DNA from 22 to 36 trees in each of six populations that sampled the three major races (two populations per race). A comparable allozyme data set for nearby populations was constructed from a published study. RAPDs of organelle origin were excluded by hybridization of blotted RAPD gels with chloroplast and mitochondrial DNA-enriched probes. RAPD and allozyme markers had similar levels of diversity within populations (H S = 0.22<0.03 and 0.16<0.03, respectively) and differentiation among populations (G ST = 0.34<0.07 and 0.29<0.07, respectively). When the allozyme data set was transformed into dominant, biallelic markers to study how RAPDs may bias diversity estimates, resampling studies showed that simulated H S and H T were reduced by half regardless of sample size. Because observed diversity for RAPDs was equivalent to, or higher than, that of allozymes, our simulations suggest that RAPD markers may contain substantially higher levels of inherent, but hidden, diversity. In contrast, the simulations showed that estimates of G ST using RAPDs should not be significantly biased at the population sizes we employed.

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Introgressive hybridization and phylogenetic relationships between Norway, Picea abies (L.) Karst., and Siberian, P. obovata Ledeb., spruce species studied by isozyme loci We analysed patterns of genetic variation at 26 isozyme loci across the area of two main forestforming spruce species in Eurasia, Norway spruce (Picea abies (L.) Karst.) and Siberian spruce (P. obovata Ledeb.). Ten seed samples from distant parts of the P. abies-P. obovata area and from a supposedly wide zone of introgressive hybridization between them were investigated. A very high level of allozyme variation was found in populations of both species. As parameters of gene diversity, the mean number of alleles per locus, percentage of polymorphic loci (95 per cent criterion) and expected heterozygosity averaged 2.8, 61.5 and 0.252 for P. abies and 2.4, 61.5 and 0.213 for P. obovata, respectively. Norway and Siberian spruces turned out to be extremely similar genetically. We did not find any fixed allele differences between them, i.e. there were no diagnostic loci and only a few alleles could be characteristic of some populations. Cluster and multivariate analyses have shown that these two species should be considered as two closely related subspecies or two geographical races of one spruce species undergoing considerable gene exchange. Our genetic data agree with morphological data and confirm the existence of a wide zone of introgressive hybridization between Norway and Siberian spruces -perhaps the widest known among plants. The samples which, according to morphological and geographical data, were taken from presumably 'hybrid' populations showed 'intermediate' genetic characteristics. Clinal variation was suggested for some alleles, and the 'rare allele phenomenon', i.e. higher frequencies of rare and unique alleles, was observed in the 'hybrid' spruce populations.

Twenty-two highly variable SSR markers were developed in Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco] from five SSR-enriched genomic libraries. Fifteen PCR primer pairs amplified a single codominant locus, while seven primer pairs occasionally amplified two loci. The Mendelian inheritance of all 22 SSRs was confirmed via segregation analyses in several Douglas-fir families. The mean observed heterozygosity and the mean number of alleles per locus were 0.855 (SE=0.020) and 23 (SE=1.6), respectively. Twenty markers were used in genetic linkage analysis and mapped to ten known linkage groups. Because of their high polymorphism and unambiguous phenotypes, 15 single-locus markers were selected as the most suitable for DNA fingerprinting and parentage analysis. Only three SSRs were sufficient to achieve an average probability of exclusion from paternity of 0.998 in a Douglas-fir seed orchard block consisting of 59 parents.