Population substructure in continuous and fragmented stands of Populus trichocarpa.

Population substructure has important implications for both basic and applied genetic research. We used 10 microsatellite markers to characterize population substructure in two ecologically and demographically contrasting populations of the model tree Populus trichocarpa. The Marchel site was a continuous stand growing in a mesic habitat in western Oregon, whereas the Vinson site consisted of three disjunct and isolated stands in the high desert of eastern Oregon. A previous study revealed that pollen-mediated gene flow is extensive in both populations. Surprisingly, model-based clustering, principal components analysis and analyses of molecular variance provided overwhelming support for the existence of at least two intermingled sub-populations within the continuous Marchel population (F(ST)=0.026, P<0.001), which occupied an area with a radius of only about 250 m. Genets in these two sub-populations appeared to have different relative clone ages and phenologies, leading us to hypothesize that they correspond to different seedling cohorts, each established from seeds produced by relatively few mothers. As expected, substructure was stronger in the fragmented Vinson population (F(ST)=0.071, P=0.001), and this difference appeared to result from the more extensive family structure in this population. Using group-likelihood methods, we reconstructed multiple interconnected half-sib families in the Vinson population, with some genets having as many as eight putative siblings. Researchers involved in ongoing and future association studies in P. trichocarpa should account for the likely presence of subtle but practically significant substructure in populations throughout the range of this species.

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa.

Pollen-mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 +/- 0.02) than in the substantially larger and more isolated Vinson site (0.32 +/- 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (Phi FT = 0.253, N = 27 mothers) than in the Marchel population (Phi FT = 0.052, N = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes (sensu Wright 1946) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower-cost alternative to paternity analysis.

Alcohol-inducible gene expression in transgenic Populus.

We tested the efficiency and optimized the conditions for controlled alcohol-inducible transgene expression in Populus using gus as a reporter gene. Specificity of induction, efficiency in different organs, effect of three chemical inducers, and induction methods were tested using up to 10 independent transgenic events generated in two different Populus genotypes. The optimal inducer concentration and the duration of induction period were determined in dose-response and in time-course experiments. Under in vitro conditions, beta-glucuronidase (GUS) induction was efficient both in the aerial parts and in the roots of regenerated plantlets. Among the chemical inducers tested, ethanol was the most effective activator with no apparent phytotoxicity when concentrations were at or below 2%. After 5 days of treatment, fluorometrically-determined the GUS activity could be detected when inducing with ethanol at concentrations as low as 0.5%. Prolonged induction by ethanol vapors significantly increased the GUS activity in leaves from both the tissue culture plants and greenhouse-grown plants.

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations.

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.

Relative Lewis basicities of six AI(ORF)4- superweak anions and the structures of LiA.

The relative Lewis basicities of six Al(ORF)4- ions, Al[OC(CH3)(CF3)2]4-, Al(OC(CF3)3]4-, Al(OCPh(CF3)2]4-, Al[OC[4-C6H4(tBu)](CF3)2]4-, Al(OC(Cy)(CF3)2]4-, and Al(OCPh2(CF3)]4-, have been determined by measuring their relative coordinating abilities towards Li+ in dichloromethane. The relative Li- Lewis basicities of the Al(ORF)4- ions are linearly related to the aqueous pKa values of the corresponding parent HORF fluoroalcohols. The Lewis basicity of Al[OCH(CF3)2]4- could not be measured because two of these anions can coordinate to one Li+ cation. The structures of LiAl[OCH(CF3)2]4 and [1-Et-3-Me-1,3-C3H3N2][Li[Al[OCH(CF3)2)4]2] were determined.

A DEFICIENS homolog from the dioecious tree black cottonwood is expressed in female and male floral meristems of the two-whorled, unisexual flowers.

We isolated PTD, a member of the DEFICIENS (DEF) family of MADS box transcription factors, from the dioecious tree, black cottonwood (Populus trichocarpa). In females, in situ hybridization experiments showed that PTD mRNA was first detectable in cells on the flanks of the inflorescence meristem, before differentiation of individual flowers was visually detectable. In males, the onset of PTD expression was delayed until after individual flower differentiation had begun and floral meristems were developing. Although PTD was initially expressed throughout the inner whorl meristem in female and male flowers, its spatial expression pattern became sex-specific as reproductive primordia began to form. PTD expression was maintained in stamen primordia, but excluded from carpel primordia, as well as vegetative tissues. Although PTD is phylogenetically most closely related to the largely uncharacterized TM6 subfamily of the DEF/APETELA3(AP3)/TM6 group, its spatio-temporal expression patterns are more similar to that of DEF and AP3 than to other members of the TM6 subfamily.

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis.

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.

Zirconium and hafnium hydrogen monothiophosphates, H2Zr(PO3S)2 and H2Hf(PO3S)2. Syntheses and selective ion-exchange properties of sulfur-containing analogues of H2M(PO4)2 (M = Zr, Hf).

The reactions between aqueous solutions of M4+ (M = Zr, Hf) and PO3S3- each result in the precipitation of a white gel that can be dried to a powder. Elemental analysis results for the white polycrystalline product yield a stoichiometry of H2M(PO3S)2. These new compounds are characterized by thermal analysis (DSC, TG-MS), vibrational spectroscopy (FT-IR, FT-Raman), 31P MAS NMR spectroscopy, energy-dispersive spectroscopy (EDS), and powder X-ray diffraction (XRD). On the basis of the characterizations and the results of trialkylamine intercalation experiments, we conclude that the H2M(PO3S)2 compounds have a layered structure that is likely similar to that of alpha-H2Zr(PO4)2.H2O. The interlayer spacing for both H2M(PO3S)2 compounds, determined by XRD, is approximately 9.4 A. Our characterization results suggest that the sulfur atom of each PO3S3- group is preferentially pointed into the interlayer region of the compound and is protonated. Two of the many potentially interesting properties of H2Zr(PO3S)2, its ion-exchange capacity and selectivity, are investigated. H2Zr(PO3S)2 is demonstrated to be an effective and recyclable ion-exchange material for both Zn2+(aq) and Cd2+(aq). Mass balance experiments indicate that the removal of Cd2+(aq) and Zn2+(aq) ions by solid H2Zr(PO3S)2 occurs by an ion-exchange process. Ion exchange results in the formation of the new compounds H0.2Cd0.9Zr(PO3S)2 and H0.50Zn0.75Zr(PO3S)2. The extraction of metal ions is monitored by XRD, vibrational spectroscopy, and elemental analysis. H2Zr(PO3S)2 reversibly intercalates Zn2+(aq) ions through three complete cycles of intercalation and deintercalation without any loss of ion-exchange capacity.

Structure and expression of duplicate AGAMOUS orthologues in poplar.

To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus).

We describe a protocol for Agrobacterium tumefaciens-mediated transformation of hybrid cottonwoods (Populus sections Tacamahaca Spach. and Aigeiros Duby). The protocol has allowed routine transformation of several economically important cottonwood hybrids (Populus trichocarpa Torr. & Gray×P. deltoides Bartr. ex. Marsh. and P. deltoides×P. nigra L.) that were previously difficult to transform. The procedure was applied to 11 different hybrid cottonwood genotypes and one P. deltoides genotype using kanamycin as the selection agent. Additional experiments showed a very strong interaction between auxin preculture and the effectiveness of various cytokinins for induction of shoot organogenesis. The data also demonstrated the superiority of Agrobacterium strain EHA105 over C58 and LBA4404 for T-DNA transfer based on transient assays with a reporter gene.

Weakly coordinating anions HA2-generated from oxoanions A- and their conjugate acids. Coordination equilibria, ionic conductivities, and the structures of [Cu2(H(CH3SO3)2)4]n and [Cu(CO)(H(CF3CO2)2)]2.

The coordination or ion pairing of the hydrogen-bonded anions H(CF3CO2)2- and H(CH3SO3)2- to NEt4+, Li+, Cu+, and/or Cu2+ was investigated. The structure of [Cu2(H(CH3SO3)2)4]n consists of centrosymmetric dimeric moieties that contain two homoconjugated (CH3SO2O-H...OSO2CH3)- anions per Cu2+ ion, forming typical Jahn-Teller tetragonally elongated CuO6 coordination spheres. The oxygen atoms involved in the nearly linear O-H...O hydrogen bonds (O...O approximately 2.62 A) are not coordinated to the Cu2+ ions. The structure of Cu2(CO)2(H(CF3-CO2)2)2 consists of pseudo-C2-symmetric dimers that contain one homoconjugated (CF3COO-H...OCOCF3)- anion per Cu+ ion, forming highly distorted tetrahedral Cu(CO)O3 coordination spheres. Three of the four oxygen atoms in each hydrogen-bonded H(CF3CO2)2- anion are coordinated to the Cu+ ions, including one of the oxygen atoms in each O-H...O hydrogen bond (O...O approximately 2.62 A). Infrared spectra (v(CO) values) of Cu(CO)(CF3CO2) or Cu(CO)(CH3SO3) dissolved in acetonitrile or benzene, with and without added CF3COOH or CH3SO3H, respectively, demonstrate that HA2- anions involving carboxylates or sulfonates are more weakly coordinating than the parent anions RCO2- and RSO3-. Direct current conductivities of THF solutions of Li(CF3CO2) containing varying concentrations of added CF3COOH further demonstrate that Li+ and NEt4+ ion pair much more weakly with H(CF3CO2)2- than with CF3CO2-.

Abundant mitochondrial genome diversity, population differentiation and convergent evolution in pines.

We examined mitochondrial DNA polymorphisms via the analysis of restriction fragment length polymorphisms in three closely related species of pines from western North America: knobcone (Pinus attenuata Lemm.), Monterey (P. radiata D. Don), and bishop (P. muricata D. Don). A total of 343 trees derived from 13 populations were analyzed using 13 homologous mitochondrial gene probes amplified from three species by polymerase chain reaction. Twenty-eight distinct mitochondrial DNA haplotypes were detected and no common haplotypes were found among the species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (HS) averaged 0.22, and population differentiation (GST and theta) exceeded 0.78. Analysis of molecular variance also revealed that >90% of the variation resided among populations. For the purposes of genetic conservation and breeding programs, species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Neighbor-joining phenograms, however, strongly disagreed with those based on allozymes, chloroplast DNA, and morphological traits. Thus, despite its diagnostic haplotypes, the genome appears to evolve via the rearrangement of multiple, convergent subgenomic domains.

Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY.

Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees. The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica).

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5' and 3' ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.

Mitochondrial DNA products among RAPD profiles are frequent and strongly differentiated between races of Douglas-fir.

Racial differentiation and genetic variability were studied between and within the coastal, north interior, and south interior races of Douglas-fir using RAPD and allozyme markers. Nearly half of all RAPD bands scored (13: 45%) were found to be amplified from mitochondrial DNA. They exhibited maternal inheritance among hybrids and backcrosses between the races, and were much more highly differentiated (GST = 0.62 for haplotype frequencies) than were allozymes (GST = 0.26). No evidence of hybridization or introgression was detected where the coastal and interior races come into proximity in central Oregon.

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene.

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trn Y-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3' end of trnY and 25 bp of its 3' flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossing-over.

Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.

We have estimated the time for the last common ancestor of extant seed plants by using molecular clocks constructed from the sequences of the chloroplastic gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the nuclear gene coding for the small subunit of rRNA (Rrn18). Phylogenetic analyses of nucleotide sequences indicated that the earliest divergence of extant seed plants is likely represented by a split between conifer-cycad and angiosperm lineages. Relative-rate tests were used to assess homogeneity of substitution rates among lineages, and annual angiosperms were found to evolve at a faster rate than other taxa for rbcL and, thus, these sequences were excluded from construction of molecular clocks. Five distinct molecular clocks were calibrated using substitution rates for the two genes and four divergence times based on fossil and published molecular clock estimates. The five estimated times for the last common ancestor of extant seed plants were in agreement with one another, with an average of 285 million years and a range of 275-290 million years. This implies a substantially more recent ancestor of all extant seed plants than suggested by some theories of plant evolution.

Chloroplast DNA transgresses species boundaries and evolves at variable rates in the California closed-cone pines (Pinus radiata, P. muricata, and P. attenuata).

We studied phylogenetic relationships among populations and species in the California closed-cone pines (Pinus radiata D. Don, P. attenuata Lemm., and P. muricata D. Don) via chloroplast DNA restriction site analysis. Data on genetic polymorphism within and among 19 populations in the three species were collected using 9 to 20 restriction enzymes and 38 to 384 trees. Because only five clades and extremely low intraclade diversity were found, additional phylogenetic data were collected using a single representative per clade and two outgroup species, P. oocarpa Schiede and P. jeffreyi Loud. In total, 25 restriction enzymes were employed and approximately 2.7 kb surveyed (2.3% of genome). The five clades recognized were Monterey pine, knobcone pine, and the southern, intermediate, and northern races of bishop pine. On the basis of bootstrapping, both Wagner and Dollo parsimony analyses strongly separated the northern and intermediate races of bishop pine from the southern race; knobcone pine from Monterey and bishop pines; and the closed-cone pines from the two outgroups. Approximate divergence times were estimated for the lineages leading to knobcone pine and to the intermediate and northern populations of bishop pine. The position of Monterey pine relative to bishop pine within their monophyletic clade was unresolved. Surprisingly, Montery pine and the southern race of bishop pine were much more similar to one another than was the southern race of bishop pine to its conspecific intermediate and northern races. Both the Monterey and southern bishop pine lineages also evolved severalfold more slowly than did the knobcone pine and intermediate-northern bishop pine lineages.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroplast DNA diversity among trees, populations and species in the California closed-cone pines (Pinus radiata, Pinus muricata and Pinus attenuata).

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [Gst = 84(+/- 13)%]; nucleotide diversity among species was estimated to be 0.3(+/- 0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [Gst = 87(+/- 8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/- 0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.