Cross-species transferability of eastern white pine (Pinus strobus) nuclear microsatellite markers to five Mexican white pines.

We examined cross-species transferability and usefulness of six nuclear microsatellite markers developed in consubgeneric eastern white pine (Pinus strobus) with regard to ecologically and commercially important Mexican white pine species of conservation genetics concern: Pinus chiapensis (Mart.) Andresen, P. flexilis James, P. strobiformis Engelm., P. ayacahuite Ehrenb. Ex Schltdl, and P. ayacahuite var. veitchii (Roezl) G.R. Shaw. Four to six microsatellite loci were found to be polymorphic in different species, with moderate to high informativeness in a relatively small number of samples (PIC/HE=0.25-0.93). This successful transfer sidesteps the time- and resource-consuming development of species-specific microsatellite markers, and will facilitate population and conservation genetic studies and genetic resource management of the less studied Mexican white pines.

Impacts of forest fragmentation on the mating system and genetic diversity of white spruce (Picea glauca) at the landscape level.

We studied the mating system of white spruce (Picea glauca) in a landscape fragmented by agriculture in northern Ontario, Canada. We sampled 23 stands that ranged in size from 1 to >500 trees isolated by 250-3000 m from the nearest other stand. Six polymorphic allozyme loci from four enzyme systems were used to genotype approximately 10 000 embryos from 104 families. We detected no allele frequency heterogeneity in the pollen pool among stands or families (Phi(FT)=-0.025). Overall, estimates of outcrossing were high (t(m)=94% and mean t(s)=91%) but significantly different from unity. Bi-parental inbreeding (t(m)-t(s)=3.2%) was low but significantly different from zero. Allozyme-based outcrossing estimates did not differ significantly among three stand-size classes (SSCs): small (<10 trees), medium (10-100 trees) and large (> or =100 trees). The number of effective pollen donors was high in all SSCs, but was significantly lower in small stands (N(ep)=62.5) than in medium-sized and large stands (both N(ep)=143). The primary selfing rate was significantly higher in medium stands than in large stands. We found no significant difference in genetic diversity measures in the filial (seed) population among SSCs. Overall, these results indicate that white spruce stands in this fragmented landscape are resistant to genetic diversity losses, primarily through high pollen-mediated gene-flow and early selection against inbred embryos. We discuss the importance of using seed data, in conjunction with genetic data, to evaluate the impacts of fragmentation on natural populations.

Intraspecific and interspecific genetic and phylogenetic relationships in the genus Populus based on AFLP markers.

Although Populus has become the model genus for molecular genetics and genomics research on forest trees, genetic and phylogenetic relationships within this genus have not yet been comprehensively studied at the molecular level. By using 151 AFLP (AFLP is a registered trademark of Keygene) markers, 178 accessions belonging to 25 poplar species and three interspecific hybrids were analyzed, using three accessions belonging to two willow species as outgroups. The genetic and phylogenetic relationships were generally consistent with the known taxonomy, although notable exceptions were observed. A dendrogram as well as a single most parsimonious tree, ordered the Populus sections from the oldest Leuce to the latest Aigeiros, a pattern consistent with their known evolutionary relationships. A close relationship between Populus deltoides of the Aigeiros section and species of the Tacamahaca section was observed and, with the exception of Populus wilsonii, between the species of the Leucoides, Tacamahaca, and Aigeiros sections. Populus nigra was clearly separated from its consectional P. deltoides, and should be classified separately from P. deltoides. The AFLP profiles pointed out to the lack of divergence between some species and revealed that some accessions corresponded with interspecific hybrids. This molecular study provides useful information about genetic relationships among several Populus species and, together with morphological descriptions and crossability, it may help review and update systematic classification within the Populus genus.

Genetic diversity impacts of forest fires, forest harvesting, and alternative reforestation practices in black spruce (Picea mariana).

Benchmarks were established for genetic diversity inherent in natural mature populations, and genetic diversity impacts of forest fires, clearcut harvesting and alternative natural and artificial silvicultural regeneration practices were determined in black spruce (Picea mariana). Allozymes of 32 loci were used to determine and compare genetic diversity and genetic relationships of adjacent or nearby four stand types: post-fire natural mature (FNM), post-fire natural young (FNR), post-harvest natural young (HNR) and post-harvest plantation (PLT), of black spruce at each of the four study sites located in two ecoregions in Manitoba: Ecoregion 90-Lac Seul Upland (Eastern) and Ecoregion 158 - Mid-Boreal Lowland (Northern). Both allelic- and genotypic-based genetic diversity parameters, as well as latent genetic potential, were determined. Black spruce populations showed typical moderate to high levels of allozyme genetic diversity. The mean genetic diversity parameters over the 16 black spruce populations sampled were as follows: percent loci polymorphic - 67%, mean number of alleles per locus - 2.52, effective number of alleles per locus - 1.70, observed heterozygosity - 0.222, expected heterozygosity - 0.308, mean number of observed genotypes per locus - 3.65, mean number of expected genotypes per locus - 5.03, genotype additivity (observed) - 116.8, genotype additivity (expected) - 161, genotype multiplicity (observed) - 6.16 x 10(15), genotype multiplicity (expected) - 2.06 x 10(19) and latent genetic potential - 26.12. The four stand types (FNM, FNR, HNR and PLT) had comparable and statistically similar genetic diversity levels at each of the four study sites as well as overall. No significant differences in black spruce genetic diversity levels were observed between the two ecoregions in Manitoba, as well as between the post-fire and post-harvest regenerated stands. No particular order of genetic relatedness among the four stand types was observed. Black spruce populations showed some sort of site-related differentiation in their genetic constitution. Allelic heterogeneity and genetic distances among populations within stand types and among four stand types suggest that the genetic diversity was maintained at the landscape level in black spruce. The results of our study demonstrate that forest fires and currently used clearcut harvesting, and alternative natural and artificial silvicultural regeneration practices, do not adversely affect genetic diversity in black spruce, and that the genetic diversity effects of clearcut harvesting are not significantly different from those due to forest fires in black spruce.

Old-growth red spruce forests as reservoirs of genetic diversity and reproductive fitness.

Old-growth forests are assumed to be potential reservoirs of genetic diversity for the dominant tree species, yet there is little empirical evidence for this assumption. Our aim was to characterize the relationship of stand traits, such as age, height and stem diameter, with the genetic and reproductive status of old-growth and older second-growth stands of red spruce (Picea rubens Sarg.) in eastern Canada. We found strong relationships between height growth (a fitness trait) and measures of genetic diversity based on allozyme analyses in red spruce. The negative relationship between height and the proportion of rare alleles suggests that high proportions of these rare alleles may be deleterious to growth performance. Latent genetic potential, however, showed a significant and positive relationship with height. Stand age was not correlated to height, but was correlated to seedling progeny height. In late-successional species such as red spruce, age and size (e.g., height and stem diameter) relationships may be strongly influenced by local stand disturbance dynamics that determine availability of light, growing space, moisture and nutrients. In larger and older stands, age appeared to provide a good surrogate measure or indicator for genetic diversity and progeny height growth. However, in smaller and more isolated populations, these age and fitness relationships may be strongly influenced by the effects of inbreeding and genetic drift. Therefore, older populations or old-growth forests may represent superior seed sources, but only if they are also of sufficient size and structure (e.g., stem density and spatial family structure) to avoid the effects of inbreeding and genetic drift. Thus, larger and older forests appear to have an important evolutionary role as reservoirs of both genetic diversity and reproductive fitness. Given the rapid environmental changes anticipated (as a result of climate change, increasing population isolation through fragmentation, or following the introduction of exotic pests and diseases) these older populations of trees may have a valuable function in maintaining the adaptive potential of tree species.

Isolation, characterization, inheritance and linkage of microsatellite DNA markers in white spruce (Picea glauca) and their usefulness in other spruce species.

Microsatellite DNA/simple-sequence-repeat (SSR) loci were identified, isolated and characterized in white spruce (Picea glauca) by screening both a non-enriched partial genomic library and a partial genomic library enriched for (AG/TC)n-containing clones. Inheritance and linkage of polymorphic SSR loci were determined in F1 progeny of four controlled crosses. We also assessed the compatibility and usefulness of the P. glauca microsatellite DNA markers in five other Picea species. Twenty-four microsatellites were identified by sequencing 32 clones selected from screens of 5,400 clones from the two libraries. The (AG/TC)n microsatellites were the most abundant in the non-enriched library. Eight microsatellite DNA loci were of the single-copy type, and six of these were polymorphic. A total of 87 alleles were detected at the six polymorphic SSR loci in 32 P. glauca individuals drawn from several populations. The number of alleles found at these six SSR loci ranged from 2 to 22, with an average of 14.5 alleles per locus, and the observed heterozygosity ranged from 0.48 to 0.91, with a mean of 0.66 per locus. Parents of the controlled crosses were polymorphic for five of the six polymorphic SSR loci. Microsatellite DNA variants at each of these five SSR loci followed a single-locus, codominant, Mendelian inheritance pattern. Joint two-locus segregation tests indicated complete linkage between PGL13 and PGL14, and no linkage between any of the remaining SSR loci. Each of the 32 P. glauca individuals examined had unique single or two-locus genotypes. With the exception of non-amplification of PGL12 in P. sitchensis, P. mariana, and P. abies and the monomorphic nature of PGL7 in P. mariana, primer pairs for all six polymorphic SSR loci successfully amplified specific fragments from genomic DNA and resolved polymorphic microsatellites of comparable sizes in P. engelmanni, P. sitchensis, P. mariana, P. rubens, and P. abies. The closely related species P. mariana and P. rubens, and P. glauca and P. sitchensiss could be distinguished by the PGL12 SSR marker. The microsatellite DNA markers developed and reported here could be used for assisting various genetics, breeding, biotechnology, tree forensics, genome mapping, conservation, restoration, and sustainable forest management programs in spruce species.

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.

Microsatellite DNA analysis of genetic effects of harvesting in old-growth eastern white pine (Pinus strobus) in Ontario, Canada.

Microsatellite DNA markers from 13 simple sequence repeat (SSR) loci were used to compare genetic diversity between preharvest pristine and postharvest residual gene pools of two adjacent virgin, old-growth ( approximately 250 years) stands of eastern white pine (Pinus strobus L.) in Ontario. There was concurrence in genetic diversity changes in the postharvest gene pools of the two stands. The total and mean numbers of alleles detected in each stand were reduced by approximately 26% after tree density reductions of approximately 75%. Approximately 18 and 21% of the low-frequency (0. 25 > P > or = 0.01) alleles and 76 and 92% of the rare (P < 0.01) alleles were lost from residual stands A and B, respectively, after harvesting. Multilocus gametic diversity was reduced by 38 and 85% and genotype additivity by approximately 50% in the residual stands after harvesting. Latent genetic potential of each stand was reduced by approximately 40%. Although heterozygosity was reduced (1-5%) in the postharvest residual stands, the reductions were not substantial and not comparable to those using other genetic diversity measures. The reductions in genetic diversity measures were slightly higher than those theoretically expected in postbottleneck populations according to drift theory. In the absence of substantial gene migration that could ameliorate the genetic losses, the ability of the postharvest white pine gene pools to adapt to changing environmental and disease conditions may have been compromised. The microsatellite DNA results for genetic effects of harvesting in old-growth eastern white pine stands were similar to those that we reported earlier from allozyme analysis (Buchert et al. 1997). The results indicate that silvicultural practices should ensure that the gene pools of remaining pristine old-growth stands are reconstituted in the regenerating stands.

Chloroplast DNA variation in Populus. I. Intraspecific restriction fragment diversity within Populus deltoides, P. nigra and P. maximowiczii.

We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of P. deltoides, var deltoides and var occidentalis, showed distinct cp genomes/DNA. Three distinct cp genomes/DNA, separated by a loss or gain of 1 EcoRV restriction site and/or 1 restriction fragment length polymorphism (RFLP), were observed among the individuals of P. deltoides var deltoides. Within P. nigra, cpDNA of var italica was distinct from that of vars nigra and plantierensis by one RFLP and by a loss or gain of one BamHI restriction site. Populus maximowiczii clones of Chinese origin were separated from those of Japanese origin by a gain or loss of one ClaI restriction site in their cpDNA. The estimate of nucleotide substitutions per site in cpDNA was 0.07% between two varieties of P. deltoides, 0.05% between var italica and var nigra or plantierensis of P. nigra, and 0.01% between Japanese and Chinese accessions of P. maximowiczii.

Chloroplast DNA variation in Populus. II. Interspecific restriction fragment polymorphisms and genetic relationships among Populus deltoides, P. nigra, P. maximowiczii, and P. x canadensis.

Restriction fragment analysis was conducted to determine interspecific chloroplast DNA (cpDNA) variation and genetic relationships among Populus deltoides, P. nigra, P. x canadensis (P. deltoides x P. nigra), and P. maximowiczii. Total cellular DNAs of these poplars were digested with 16 restriction endonucleases, and Southern blots of the restriction digests were probed with six different cloned cpDNA fragments from Petunia. P. deltoides, P. nigra, and P. maximowiczii each had a distinct chloroplast genome, separated by many restriction-site and restriction-fragment-length mutations, predominantly in the large single-copy region of the genome. P. x canadensis shared the same cpDNA restriction fragment patterns as P. deltoides var. deltoides. P. nigra was most diverged from P. deltoides, and P. deltoides showed close cpDNA relationships to P. maximowiczii. Nucleotide substitutions per site in cpDNA were 0.0036 between P. deltoides and P. maximowiczii, 0.0071 between P. nigra and P. maximowiczii, and 0.0077 between P. deltoides and P. nigra. We suggest that P. nigra should be classified in a new separate section, the Nigrae.

Chloroplast DNA variation in Populus. III. Novel chloroplast DNA variants in natural Populus x canadensis hybrids.

A rare phenomenon of the occurrence of novel non-parental chloroplast DNA (cpDNA) variants in natural sexual interspecific hybrids between Populus deltoides var deltoides and P. nigra, P. x canadensis is described. Restriction fragment variation of cpDNA in 17 P. x canadensis cultivars was examined and compared with that of representative samples of P. deltoides and P. nigra using 83 combinations of 16 restriction enzymes and six Petunia hybrida cpDNA probes. Twelve cultivars had one to five novel non-parental cpDNA fragments in the chloroplast genome region homologous to the 9.0-kb PstI cpDNA fragment of Petunia from the large single-copy region.

Paternal plastid DNA can be inherited in lentil.

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F1 progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F1 progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F1 plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F1 progeny.

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.).

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F1 and F5 progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F1 progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris 'Laird' and 'Eston' from L.c. ssp. culinaris 'ILL5588', and one restriction fragment polymorphism distinguished L.c. ssp. culinaris 'Laird' and 'Eston' from L.c. ssp. orientalis 'LO4'. Twelve of 13 f1 progeny and all F5 progeny from the intrasubspecific crosses, and all F1 progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f1 plant from an 'Eston' x 'ILL5588' cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F1 progeny.

Mitochondrial DNA variation and genetic relationships of Populus species.

We examined variation in and around the region coding for the cytochrome c oxidase I (coxI) and ATPase 6 (atp6) genes in the mitochondrial genomes of four Populus species (P. nigra, P. deltoides, P. maximowiczii, and P. tremuloides) and the natural hybrid P. x canadensis (P. deltoides x P. nigra). Total cellular DNAs of these poplars were digested with 16 restriction endonucleases and probed with maize mtDNA-specific probes (CoxI and Atp6). The only variant observed for Atp6 was interspecific, with P. maximowiczii separated from the other species as revealed by EcoRI digestions. No intraspecific mtDNA variation was observed among individuals of P. nigra, P. maximowiczii, P. x canadensis, or P. tremuloides for the CoxI probe. However, two varieties of P. deltoides were distinct because of a single site change in the KpnI digestions, demonstrating that P. deltoides var. deltoides (eastern cottonwood) and var. occidentalis (plains cottonwood) have distinct mitochondrial genomes in the region of the coxI gene. Populus x canadensis shared the same restriction fragment patterns as its suspected maternal parent P. deltoides. Nucleotide substitutions per base in and around the coxI and atp6 genes among the Populus species and the hybrid ranged from 0.0017 to 0.0077. The interspecific estimates of nucleotide substitution per base suggested that P. tremuloides was furthest removed from P. deltoides and P. x canadensis and least diverged from P. nigra. Populus maximowiczii was placed between these two clusters.

Maternal transmission of mitochondrial DNA in interspecific hybrids of Populus.

Restriction fragment analysis was conducted to investigate the mode of inheritance of mitochondrial (mt) DNA in F1 progeny of two P. deltoides x P. deltoides, three P. deltoides x P. nigra, and two P. deltoides x P. maximowiczii controlled crosses, and in Populus x canadensis by using 16 restriction endonucleases and two heterologous probes of cloned mtDNA fragments of maize. Five restriction fragment length polymorphisms (RFLPs) of mtDNA differentiated P. deltoides from P. nigra, whereas three RFLPs of mtDNA separated P. deltoides from P. maximowiczii. In all cases, F1 progeny of P. deltoides x P. nigra, and P. deltoides x P. maximowiczii, crosses had mtDNA restriction fragments of only their maternal P. deltoides parents. P. x canadensis had mtDNA restriction fragments of only P. deltoides. F1 progeny of intraspecific P. deltoides crosses also had the same mtDNA fragments as their maternal parent. The results clearly demonstrate uniparental-maternal inheritance of the mitochondrial genome in F1 interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii.

Chloroplast DNA inheritance in Populus.

The inheritance of chloroplast (cp) DNA was examined in F1 hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F1 offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.

Genetic characterization and relationships of Populus alba, P. tremula, and P. x canescens, and their clones.

Isozyme analysis was conducted on individuals of Populus alba L., P. tremula L., and P. × canescens Smith to genetically characterize and differentiate species, hybrids, and individuals, and to determine genetic relationships among them. Thirty gene loci, with 71 alleles, coding for 15 enzymes were observed. Individuals could be identified on the basis of their multilocus genotypes. There were 21 unique multilocus genotypes among 23 P. alba clones. Five P. alba clones from Canada were genetically distinct from each other. Each of the 18 P. tremula and 15 P. × canescens clones had unique multilocus genotypes. Thirteen clones had a unique genotype at a single locus. Percentage of polymorphic loci, average number of alleles per locus, and mean observed heterozygosity were, respectively, 50.0, 1.86, and 0.085 in P. alba, 51.7, 1.66, and 0.096 in P. tremula, and 51.7, 1.86, and 0.157 in P. × canescens. Populus alba and P. tremula were genetically distinct from each other and could be distinguished by mutually exclusive alleles at Aco-3, P. tremula-specific gene Mdh-3, and allele frequency differences at 6 loci. Populus × canescens had allele contributions of P. alba and P. tremula. However, their allele frequencies were closer to those of P. alba than being truly intermediate. The mean genetic identity was 0.749 between P. alba and P. tremula, 0.987 between P. alba and P. × canescens, and 0.817 between P. tremula and P. × canescens. Canonical discriminant analysis of multilocus genotypes separated P. alba, P. tremula, and P. × canescens into three distinct groups and portrayed similar interspecific relationship as above. Our results suggested that the putative P. × canescens individuals consisted of a mixture of F1 hybrids of P. alba and P. tremula and their backcrosses to P. alba.

Associations of genes encoding allozymes peroxidase and superoxide dismutase in poplar and spruce species.

Isozymes of peroxidase (PER) and superoxide dismutase (SOD) were analyzed in vegetative buds or very young leaves of seven species and two interspecific hybrids of Populus, in progenies of seven controlled crosses of three Populus species, and in needles of five Picea species and one putative hybrid. One to three PER, and one or two SOD zones of activity were observed. Electrophoretic mobility (EM) and banding phenotypes of isozymes of one PER locus were identical to those of one SOD locus in vegetative buds of five Populus species and hybrid. In leaves of the four Populus species and hybrid and progenies of controlled crosses, EM and phenotypes of isozymes of two PER loci were identical to those of two SOD loci. In Picea species, EM of isozymes of the only SOD locus was somewhat similar but not identical to that of one PER locus, and isozyme phenotypes of all individuals at the SOD locus were not identical to those at a PER locus. Chi-square tests verified the single-gene Mendelian control of the segregating allozyme variants at each of Per-L1 and Sod-1 in the three Populus species. The results of joint two-locus segregation tests indicated a very tight linkage and no recombination between Per-L1 and Sod-1 in three Populus species. Genes coding for isozymes of one or two PER loci are either presumably the same as, or very tightly linked to, the genes coding for isozymes of one or two SOD loci in the Populus species.