Estimating pollen flow using SSR markers and paternity exclusion: accounting for mistyping.

Highly informative genetic markers, such as simple sequence repeats (SSRs), can be used to directly measure pollen flow by parentage analysis. However, mistyping (i.e. false inference of genotypes caused by the occurrence of null alleles, mutations, and detection errors) can lead to substantial biases in the estimates obtained. Using computer simulations, we evaluated a direct method for estimating pollen immigration using SSR markers and a paternity exclusion approach. This method accounts for mistyping and does not rely on assumptions about the distribution of male reproductive success. If ignored, even minor rates of mistyping (1.5%) resulted in overestimating pollen immigration by up to 150%. When we required at least two mismatching loci before excluding candidate fathers from paternity, the resulting pollen immigration estimates had small biases for rates of mistyping up to 4.5%. Requiring at least three mismatches for exclusion was needed to minimize the upward biases of pollen immigration caused by rates of mistyping up to 10.5%. The minimum number of highly variable SSR loci needed to minimize cryptic gene flow and obtain reliable estimates of pollen immigration varied from five to seven for a sampling scheme applicable to most conifers (i.e. when paternal haplotypes can be unambiguously determined). Between five and nine highly variable SSR loci were needed for a more general sampling scheme that is applicable to all diploid seed plants. With moderately variable SSR markers, consistently accurate estimates of pollen immigration could be obtained only for rates of mistyping up to 4.5%. We developed the POLLEN FLOW (PFL) computer program which can be used to obtain unbiased and precise estimates of pollen immigration under a wide range of conditions, including population sizes as large as 600 parents and mistyping rates as high as 10.5%.

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.

Quantitative trait loci and candidate gene mapping of bud set and bud flush in populus.

The genetic control of bud phenology in hybrid poplar was studied by mapping quantitative trait loci (QTL) affecting the timing of autumn bud set and spring bud flush. The founders of the mapping pedigree were collected from widely separated latitudes to maximize segregating variation for dormancy-related traits in the F(2) generation-the female Populus trichocarpa parent is from Washington State (48 degrees N) and the male P. deltoides parent is from Texas (31 degrees N). Bud set and bud flush timing were measured on the F(2) generation in a replicated clonal field trial. Using a linkage map constructed of AFLP and microsatellite markers, three QTL controlling bud set and six QTL controlling bud flush were detected. Additionally, five candidate genes believed to be involved in perception of photoperiod (PHYB1, PHYB2) or transduction of abscisic acid response signals (ABI1B, ABI1D, and ABI3) were placed on the QTL map. PHYB2 and ABI1B were found to be coincident with QTL affecting bud set and bud flush.

Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies.

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

Transient gene expression of microprojectile-introduced DNA in Douglas-fir cotyledons.

Plasmid DNA containing the reporter gene uidA encoding ß-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was introduced on high-velocity microprojectiles into cultured cotyledons of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. Transient gene expression was measured by counting the number of distinct loci of GUS activity per cotyledon. Contrary to published results on angiosperms, repeated bombardments did not increase expression in Douglas-fir. Expression varied significantly among cotyledons from different seedlings. The amount of time between DNA delivery and treatment of cotyledons with auxins and cytokinins strongly affected GUS expression. The optimal cytokinin pretreatment produced an average of 20 loci per cotyledon. In several experiments, more than 95% of the treated cotyledons exhibited at least some transient expression. Expression remained constant up to three days following DNA delivery into cotyledons.

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons.

The effects of high-concentration, 2-h liquid pulses of N(6)-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 µM BA, and 100 and 200 µM TD. Shoots developed best from buds induced with 300, 400, and 800 µM BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 µM BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.

Chloroplast genomes of two conifers lack a large inverted repeat and are extensively rearranged.

Chloroplast genomes of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and radiata (Monterey) pine [Pinus radiata D. Don], two conifers from the widespread Pinaceae, were mapped and their genomes were compared to other land plants. Douglas-fir and radiata pine lack the large (20-25 kilobases) inverted repeat that characterizes most land plants. To our knowledge, this is only the second recorded loss of this ancient and highly conserved inverted repeat among all lineages of land plants thus far examined. Loss of the repeat largely accounts for the small size of the conifer genome, 120 kilobase, versus 140-160 kilobases in most land plants. Douglas-fir possesses a major inversion of 40-50 kilobases relative to radiata pine and nonconiferous plants. Nucleotide sequence differentiation between Douglas-fir and radiata pine was estimated to be 3.8%. Both conifer genomes possess a number of rearrangements relative to Osmunda, a fern, Ginkgo, a gymnosperm, and Petunia, an angiosperm. Among land plants, structural changes of this degree have occurred primarily within tribes of the legume family (Fabaceae) that have also lost the inverted repeat. These results support the hypothesis that the presence of the large inverted repeat stabilizes the chloroplast genome against major structural rearrangements.