Genetic diversity within and among 11 juvenile populations of green alder (Alnus crispa) in Canada

Germinated seeds from 11 populations of green alder [ Alnus crispa (Ait.) Pursh] sampled in four Canadian provinces were analysed for electrophoretically demonstrable diversity of 10 enzymes encoded by 15 structural loci. Of these, nine were polymorphic, and on average, 52% of the loci per population were polymorphic. Assuming a diploid model of expression, average level of expected heterozygosity was 0.11 with nearly all populations in Hardy-Weinberg equilibrium for the set of polymorphic loci analysed. No significant inbreeding and associated subpopulation structuring were noted. Rates of gene flow appeared high within and among populations. Although little divergence was observed among populations, genetic and geographical distances between populations were related. Discriminant and cluster analyses revealed a pattern of genetic variation associated with geography. Populations from northern Quebec were poorly differentiated, whereas western populations from Alberta exhibited a larger degree of genetic differentiation. Introgresive hybridization with the sympatric species Alnus sinuata (Regel) Rydberg and partial isolation in the West are suggested as an explanation for this larger differentiation. The occurrence and significance of rare alleles is discussed in relation to the importance of geographical distance in the process of population differentiation in this species.     

ALLOZYME VARIATION WITHIN AND AMONG MATURE POPULATIONS OF SPECKLED ALDER (ALNUS RUGOSA) AND RELATIONSHIPS WITH GREEN ALDER (A. CRISPA)

Vegetative tissues from 14 sexually mature populations of speckled alder (Alnus rugosa (Du Roi) Sprengel) in Québec have been analyzed for electrophoretically demonstrable variation in 9 enzymes encoding a total of 15 loci. Speckled alder demonstrated a diploid-like expression for all polymorphic loci investigated. Populations were in Hardy-Weinberg equilibrium for the 9 polymorphic loci observed. The total expected heterozygosity was 0.173. Analysis of fixation indices indicated a slight deficiency of heterozygotes, relative to panmictic expectations. This was likely due to weak population differentiation, which did not follow any particular geographical trend. Qualitative estimates of interpopulation rates of gene flow were high, and in good agreement with the small pair-wise population genetic distances and among-population fixation index. Levels of genetic diversity and partition of this diversity were similar to previous observations obtained with the sympatric species A. crispa (Ait.) Pursh. However, no phenomena of interspecific hybridization were noted. The average genetic distance between these two alder species was large, with a value of 0.4, emphasizing the different ecological niches colonized by each of the two species. The estimate of divergence time between these two taxa was two million years. It is in agreement with the hypothesis of repetitive spatial isolation of each species in ice-free refugia during the Pleistocene, thus promoting their reproductive isolation. The interspecific divergence noted at the enzyme level allowed for the easy electrophoretic identification of each taxon.     

Temporal variation of the mating system in a natural population of jack pine

Mating system parameters of a northern conifer, Pinus banksiana Lamb., were estimated from allozyme polymorphisms. Seeds analyzed were obtained from serotinous cones of 30 individuals and represented four independent fertilizations in 1975, 1976, 1977 and 1978. Results indicated that a mixed mating system model, with a mean effective outcrossing rate of 88 ± 0.047%, described the mating system of this stand. However, there was an approximately linear increase in the apparent selfing rate from the oldest (1975) to the newest (1978) crop. Two hypotheses could account for these observations. First, there may have been changes in the mating system during the 4-yr period, but linearity of the differences observed in this study may have been due to chance. These changes were, however, independent of the variability of the observed pollen pool. This indicated that they were not a result of different proportions of outcrossed zygotes directly observed. Second, there could have been a more or less constant amount of selfing, followed by a differential loss of viability of selfed and outcrossed zygotes during the period of storage in the cones. Under this hypothesis, selfed zygotes are at a selective disadvantage relative to outcrossed zygotes. No differences in the mating system could be demonstrated among the three crown strata of this stand. There was significant interlocus heterogeneity in the filial generation genotypic distributions and in the estimated outcrossing rates, reflecting the complex nature of forces that can affect single-locus estimates. There was evidence of some additional inbreeding, possibly due to family structures in the stand; however, this was a minor component of the total inbreeding.     

Methods to extract NAD+-malate dehydrogenase efficiently from white spruce needles

The spectrophotometrically-determined activity of NAD⁺-malate dehydrogenase (MDH, EC 1.1.1.37) from white spruce [ Picea glauca (Moench) Voss] needles was assayed with NADH and oxaloacetate. Activity was very low when extracted with only acetate buffer (pH 5.4), phosphate buffer (pH 6.8), or Tris-HCl buffer (pH 8.0). However, activity increased from 1 to over 200 μmol (g dry weight)^-1 min^-1 with the addition of polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) and the detergents, Tween 80, Tergitol 15-S-9 and Triton X-100. Best activity was observed when extracted in a buffer at pH 6.8 and with 1% (v/v) for the three detergents and PEG, and 6% (w/v) for PVP.
MDH activity decreased with age of the needles on the tree. Six-year-old needles contained only about one-fifth of the activity of current year, fully-expanded needles. The main decrease in enzyme activity was observed in one-year-old needles. Protein content obtained from needles extracted with just phosphate buffer (pH 6.8) was very low, but increased greatly when the above chemicals were added to the buffer. In contrast with needles, extracts of vegetative buds contained much higher levels of MDH and protein when extracted with only phosphate buffer (pH 6.8). Although MDH activity in needle extracts declined with storage of the extracts at 4°C in the dark for 6 days, the decrease was least for buffers containing a combination of different protective agents.     

Genetic variation in somatic embryogenic response in open-pollinated families of black spruce

Summary Zygotic embryos from open-pollinated seeds of 20 black spruce (Picea mariana) families were used to investigate the proportion of genotypes that would give rise to embryogenic tissue (ET) and mature somatic embryos. Eighty-five percent of the maternal genotypes gave rise to embryogenic tissue. Within-family rates of ET induction ranged from 0 to 17%, with an average of 8%. The largest proportion of variation was among families, indicating the additive nature of the genetic variation. On a medium with 6% sucrose and 3.7 M ABA, 90% of the embryogenic lines gave rise to abundant (>100/100 mg of ET), well-formed, mature somatic embryos. A medium with 2% sucrose, without 2,4-D, was used to germinate the mature somatic embryos. These were grown in the greenhouse and have now been established in field trials.     

Segregation of allozymes in megagametophytes of viable seed from a natural population of jack pine,Pinus banksiana Lamb.

Summary Segregation ratios of allozymes in haploid female gametophytes obtained from viable seed were studied in a natural population of jack pine, Pinus banksiana. Stability of these ratios was assessed for three levels of the sexually reproductive crown as well as for four years of natural fertilization. Analyses of observed segregation ratios of four of five polymorphic isozyme loci showed good correspondence to the overall 11 ratios expected for simple Mendelian inheritance. Allozymes of glucose-6-phosphate dehydrogenase did not segregate in the expected 11 ratio. In addition, there were significant deviations from the expected segregation ratio for all the loci at some sampling positions on individual trees. Heterogeneity of segregation among trees, strata and years could be the result of pollen pool heterogeneity, segregation distortion and/or recessive lethal and semi-lethal gene combinations resulting in early embryo abortion. These types of segregation deviations in viable seed can affect the estimation of allele frequencies from bulked samples of a small number of individuals, the inference of heterozygosity/homozygosity of parental trees, and estimates of selfing rates.     

GENETIC DIVERSITY AND SPATIAL SUBDIVISION OF POPULUS TREMULOIDES (SALICACEAE) IN A HETEROGENEOUS LANDSCAPE

Trembling aspen (Populus tremuloides Michx.), a clonal angiosperm, is the most geographically widespread tree in North America. It is generally thought that most extant populations in the western interior of Canada and the United States became established shortly after glacial retreat, but sexual recruitment then effictively ceased owing to inimical climatic conditions. Six populations of trembling aspen were studied in the prairie and montane environments of Waterton Lakes National Park, Alberta. Vegetative tissues were analyzed for electrophoretically detectable variation in 13 enzymes encoded by 14 polymorphic loci and three monomorphic loci. All populations maintained high levels of inter- and intrapopulation diversity (P = 0.891; H = 0.319; A = 2.4). The mean fixation index, F, was -0.102, indicating some deviation from Hardy-Weinberg expectations. Genetic differentiation (F_ST = 3.0) was apparent in this ecologically diverse, but geographically small-scale, spatial setting. It is suggested that the maintenance of diversity in the absence of frequent modern-day recruitment, and resistance to further geographic differentiation in this spatially heterogeneous environment reflect occasional seedling establishment through “windows of opportunity” and more importantly, the species' clonal morphology. The phalanx growth form and concomitant physiological integration between ramets combine to spread the risk of death and buffer the effects of selection over time and space.     

Genetics of the polycross

Summary Rates and patterns of male gamete incorporation for a polycross mating design were studied for two independent years of pollination in Norway spruce, Picea abies (L) Karst. Segregation distortion in a subset of maternal clones was documented for one locus. We have proposed a model, involving the existence of a linked lethal allele, which accounts for these observations. Significant temporal and maternal clonal differences were observed in the rates at which single locus and multilocus gametes were incorporated. Striking differences in apparent fertility existed among four clones which produced unique multilocus gametes. One clone, in particular, was shown to be contributing three times as many gametes to the next generation as predicted by the hypothesis of equal clonal male contribution. These deviations from expectation were also detected in the genotypic distributions of the resultant filial generation. Ramifications of these results on family structures in the filial generation, effective size of the male population, and possible bias in inferences of genetic differences and parameter estimation are discussed.     

Enzyme activities during imbibition and germination of seeds of tamarack (Larix laricina)

Activities of six enzymes from extracts of separated embryos and gametophytes of tamarack [ Larix laricina (Du Roi) K. Koch] seeds were assayed at various stages of imbibition and germination. On a per seed part basis, activities of 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), malate dehydrogenase (NAD⁺–MDH, EC 1.1.1.37), isocitrate dehydrogenase (NADP⁺–IDH, EC 1.1.1.42), soluble peroxidase (PER, EC 1.11.1.7), and acid phosphatase (ACP, EC 3.1.3.2) from both the embryo and gametophyte tissues generally increased slowly, following cold stratification for 30 days and imbibition under germinating conditions for 5 days, but then increased at a faster rate with emergence of the radicle and subsequent growth of the seedling. The rate of increase of enzyme activity was highest for PER. Soluble protein levels also increased with imbibition and germination, with about 3 times greater levels present in the gametophyte than in the embryo. Heat inactivation experiments showed that, except for G-6-PD, activities were stable up to 40°C. Inactivation occurred at lower temperatures for G-6-PD, while higher temperatures were required for PER. Incubation of extracts for 7 days at 4°C indicated that loss of enzyme activity was greatest for G-6-PD (3.9% remaining) and least for PER and ACP (94 and 95% remaining, respectively).     

Genetics of the polycross

Summary Seeds from polycrosses with Norway spruce, in which the same sixteen male parents were crossed to a number of female parents in each of two years, were analysed electrophoretically to detect departures from male gamete frequencies expected assuming equal male fertilities, and to detect heterogeneity among female parents in male gamete frequencies in seeds. The data were also used to estimate the fertilities of the polycross trees used as male parents. Significant departures from male gamete frequencies expected assuming equal male fertilities were found in the seed pooled from all crosses. Male fertilities estimated from male gamete frequencies in seed from all crosses also departed significantly from expectation. The results are discussed with respect to assumptions made when estimating general combining abilities and expected response to selection in polycrosses.