Glenn T. Howe

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%.

Molecular genetic analysis of dormancy-related traits in poplars

Abstract We studied the molecular genetics of dormancy-related traits in an F2 family of poplar (Populus) hybrids derived from a cross between a northern genotype of black cottonwood and a southern genotype of eastern cottonwood by mapping quantitative trait loci (QTLs) and candidate genes. Dormancy-related traits included timing of vegetative bud set, fall frost damage, chilling response, timing of vegetative bud flush, and winter survival under field conditions, as well as photoperiodic responses (timing of bud set and number of new leaves) in a warm greenhouse under either a uniform 8-h photoperiod or a naturally shortening photoperiod in the fall. QTL analyses were conducted using a linkage map consisting of AFLP, microsatellite, and candidate gene markers. The candidate genes were chosen because of their potential roles in either photoperiodic perception (PHYB1, PHYB2) or abscisic acid signal transduction (ABI1B, ABI1D, ABI3). Significant QTLs were detected for all dormancy-related traits, except for winter survival, which had a relatively low heritability compared with the other traits. Interestingly, half of the field bud set QTLs did not map near photoperiodic QTLs. This is consistent with the moderate genetic correlation between these traits (0.53 to 0.60) and suggests that genetic differences in photoperiodic responses play only a modest role in explaining genetic differences in the timing of bud set under field conditions. Except for ABI1D, each of the candidate genes tested mapped near one or more of the dormancy-related QTLs. We conclude that molecular markers and QTL analyses can be used to study the genetics of dormancy-related traits, to design more effective breeding programs, and to provide new insights into tree physiology. Nomenclature: Black cottonwood, Populus trichocarpa Torr. & Gray; eastern cottonwood, Populus deltoides Bartr. Ex Marsh.

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata cv. ‘Crandon’) suspension cultures and regeneration of transformed plants is described. Transformants were recovered when suspension cultures were inoculated with Agrobacterium tumefaciens at a density of 107 colony-forming units ml-1, cocultivated for 48 h, and plated to cellulose acetate filters on Woody Plant Medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid and 250 mg l-1 cefotaxime. Levels of cefotaxime greater than 250 mg l-1 were unnecessary for control of residual bacteria and inhibited callus growth. Transgenic plants were regenerated by culturing the transformed callus on media containing 0.11 to 27 μM thidiazuron. In contrast to thidiazuron, N6-benzyladenine had a negative effect on shoot regeneration; the callus became necrotic when we attempted to induce shoots with concentrations of 1.1 to 8.9 μM, and growth was inhibited when concentrations of 0.11 or 0.22 μM were used to regenerate callus from suspension cultures. Following cocultivation of poplar suspension cultures, we recovered transgenic plants containing the maize transposon Ac, and callus containing an insect toxin gene from Bacillus thuringiensis.

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.

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

SummaryThe effects of high-concentration, 2-h liquid pulses of N6-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.

A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation

BackgroundDouglas-fir (Pseudotsuga menziesii), one of the most economically and ecologically important tree species in the world, also has one of the largest tree breeding programs. Although the coastal and interior varieties of Douglas-fir (vars. menziesii and glauca) are native to North America, the coastal variety is also widely planted for timber production in Europe, New Zealand, Australia, and Chile. Our main goal was to develop a SNP resource large enough to facilitate genomic selection in Douglas-fir breeding programs. To accomplish this, we developed a 454-based reference transcriptome for coastal Douglas-fir, annotated and evaluated the quality of the reference, identified putative SNPs, and then validated a sample of those SNPs using the Illumina Infinium genotyping platform.ResultsWe assembled a reference transcriptome consisting of 25,002 isogroups (unique gene models) and 102,623 singletons from 2.76 million 454 and Sanger cDNA sequences from coastal Douglas-fir. We identified 278,979 unique SNPs by mapping the 454 and Sanger sequences to the reference, and by mapping four datasets of Illumina cDNA sequences from multiple seed sources, genotypes, and tissues. The Illumina datasets represented coastal Douglas-fir (64.00 and 13.41 million reads), interior Douglas-fir (80.45 million reads), and a Yakima population similar to interior Douglas-fir (8.99 million reads). We assayed 8067 SNPs on 260 trees using an Illumina Infinium SNP genotyping array. Of these SNPs, 5847 (72.5%) were called successfully and were polymorphic.ConclusionsBased on our validation efficiency, our SNP database may contain as many as ~200,000 true SNPs, and as many as ~69,000 SNPs that could be genotyped at ~20,000 gene loci using an Infinium II array—more SNPs than are needed to use genomic selection in tree breeding programs. Ultimately, these genomic resources will enhance Douglas-fir breeding and allow us to better understand landscape-scale patterns of genetic variation and potential responses to climate change.

Extensive Transcriptome Changes During Natural Onset and Release of Vegetative Bud Dormancy in Populus

To survive winter, many perennial plants become endodormant, a state of suspended growth maintained even in favorable growing environments. To understand vegetative bud endodormancy, we collected paradormant, endodormant, and ecodormant axillary buds from Populus trees growing under natural conditions. Of 44,441 Populus gene models analyzed using NimbleGen microarrays, we found that 1,362 (3.1%) were differentially expressed among the three dormancy states, and 429 (1.0%) were differentially expressed during only one of the two dormancy transitions (FDR p-value < 0.05). Of all differentially expressed genes, 69% were down-regulated from paradormancy to endodormancy, which was expected given the lower metabolic activity associated with endodormancy. Dormancy transitions were accompanied by changes in genes associated with DNA methylation (via RNA-directed DNA methylation) and histone modifications (via Polycomb Repressive Complex 2), confirming and extending knowledge of chromatin modifications as major features of dormancy transitions. Among the chromatin-associated genes, two genes similar to SPT (SUPPRESSOR OF TY) were strongly up-regulated during endodormancy. Transcription factor genes and gene sets that were atypically up-regulated during endodormancy include a gene that seems to encode a trihelix transcription factor and genes associated with proteins involved in responses to ethylene, cold, and other abiotic stresses. These latter transcription factors include ETHYLENE INSENSITIVE 3 (EIN3), ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (EBP), ETHYLENE RESPONSE FACTOR (ERF), ZINC FINGER PROTEIN 10 (ZAT10), ZAT12, and WRKY DNA-binding domain proteins. Analyses of phytohormone-associated genes suggest important changes in responses to ethylene, auxin, and brassinosteroids occur during endodormancy. We found weaker evidence for changes in genes associated with salicylic acid and jasmonic acid, and little evidence for important changes in genes associated with gibberellins, abscisic acid, and cytokinin. We identified 315 upstream sequence motifs associated with eight patterns of gene expression, including novel motifs and motifs associated with the circadian clock and responses to photoperiod, cold, dehydration, and ABA. Analogies between flowering and endodormancy suggest important roles for genes similar to SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL), DORMANCY ASSOCIATED MADS-BOX (DAM), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1).

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

SummaryPlasmid DNA containing the reporter gene uidA encoding β-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.

Components acting downstream of short day perception regulate differential cessation of cambial activity and associated responses in early and late clones of hybrid poplar.

Short days (SDs) in autumn induce growth cessation, bud set, cold acclimation, and dormancy in trees of boreal and temperate forests, and these responses occur earlier in northern than in southern genotypes. Nevertheless, we know little about whether this variation results from differential perception of SDs or differential downstream responses to the SD signal or a combination of the two. We compared global patterns of SD-regulated gene expression in the stems of hybrid poplar (Populus trichocarpa × Populus deltoides) clones that differ in their SD-induced growth cessation in order to address this question. The timing of cessation of cambial cell division caused by SDs differed between the clones and was coincident with the change in the pattern of expression of the auxin-regulated genes. The clones also differed in the timing of their SD-regulated changes in the transcript abundance of genes associated with cold tolerance, starch breakdown, and storage protein accumulation. By analyzing the expression of homologs of FLOWERING LOCUS T, we demonstrated that the clones differed little in their perception of SDs under the growth conditions applied but differed substantially in the downstream responses manifested in the timing and magnitude of gene expression after SD treatment. These results demonstrate the existence of factors that act downstream of SD perception and can contribute to variation in SD-regulated adaptive photoperiodic responses in trees.

Prospects for genetic engineering of insect resistance in forest trees

Abstract Gene transfer and recombinant DNA methods provide opportunities for enhancing insect resistance of forest trees by importing genes from other species and by manipulating native genes to create novel forms of resistance. Current opportunities for enhancing insect resistance include insertion of the toxin gene from the bacterium Bacillus thuringiensis and transfer of proteinase inhibitor genes from other plant species. Work is under way in a number of laboratories throughout the world to insert Bacillus thuringiensis toxin genes into forest tree species. Other strategies, such as the manipulation and transfer of chitinase genes, lectin genes, baculovirus genes, and genes encoding enzymes involved in the production of novel secondary compounds, also hold promise but require more information before their likelihood of success can be judged. Use of genetically engineered, resistant trees should be environmentally safer than controlling insect pests with insecticides. This is primarily because engineered trees affect only species that feed on them, and even then will generally be harmful to only a limited number of insect taxa. The main environmental risk associated with the use of engineered trees is that insects may counter-evolve to overcome their resistance. This would be particularly significant, for example, if counter-evolution to the Bacillus thuringiensis toxin gene in trees also precluded the use of insecticides containing Bacillus thuringiensis . We argue, however, that the risks of serious counter-evolution can be reduced to an acceptable level by maintaining genetic diversity in the forest, using multiple genes for resistance, and employing forest management practices that mitigate the potential for counter-evolution. Genetically engineered resistance should be more effective than the spraying of insecticides because the toxins are delivered as soon as insects begin feeding, can be produced continuously, and are delivered within tissues — thereby contacting insects that are difficult to reach with exterior sprays. We hypothesize that the improved nutrition of coniferous trees provided by intensive forest management should allow them to make heavier investments in novel nitrogen-based defensive compounds such as proteins and alkaloids, and that genetic engineering can help to take advantage of this opportunity. The greatest limitations to the current use of genetic engineering to improve insect resistance of trees are: insufficient knowledge of the molecular biology of insect development, insect pathogenesis, and tree defenses against insects; inefficient systems for insertion of genes into large numbers of tree genotypes; inability to produce sterile trees, necessary to prevent the release of engineered genes into natural or feral populations; concerns about insect counter-evolution to overcome the effects of engineered resistance genes; a lack of public understanding of the true benefits and risks of genetic engineering. In the short term, the greatest benefits from recombinant DNA and genetic engineering technology will be to provide new avenues for understanding tree-insect interactions, and thus new options for combating insect pests.