# Analysis of expression patterns of candidate genes for bud set and cold tolerance in scots pine (Pinus sylvestris 1.)

Komlan Avia, Katri Kärkkäinen, Outi Savolainen

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Komlan Avia, Katri Kärkkäinen, Outi Savolainen. Analysis of expression patterns of candidate genes for bud set and cold tolerance in scots pine (Pinus sylvestris 1.). IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery, Jun 2011, Arraial d'Ajuda, Brazil. , BMC Proceedings, 2011, 10.1186/1753-6561-5-S7-P3 . hal-02947464

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# IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery

Arraial d'Ajuda, Bahia, Brazil. 26 June - 2 July 2011

Edited by Dario Grattapaglia

Published: 13 September 2011

These abstracts are available online at http://www.biomedcentral.com/1753-6561/5?issue=S7

## INTRODUCTION

### A1

IUFRO Tree Biotechnology 2011: "From genomes to integration and delivery"

Dario Grattapaglia¹,²

¹EMBRAPA Genetic Resources and Biotechnology - Estação Parque

Biológico, 70770-910, Brasília, DF, Brazil; ²Graduate Program in Genomics and Biotechnology - Universidade Católica de Brasília - SGAN 916 Modulo B, 70790-160 Brasília, Brazil

BMC Proceedings 2011, 5(Suppl 7):A1

Forest trees have unquestionably entered the genomic era. The updated version of the *Populus* genome, the recently released *Eucalyptus grandis* genome and the concerted efforts towards the generation of genome sequences for spruces (*Picea* sp.) and pines (*Pinus* sp.) by several groups worldwide, are fueling a multitude of inter-disciplinary studies and applications in sustainable forest production and conservation. Time now calls for the integration of scientific fields with an increased sense of urgency for delivery of effective biotechnologies.

The IUFRO (International Union of Forestry Research Organizations) Tree Biotechnology biannual conference has established a solid tradition for over 20 years as the official meeting of the IUFRO working group 2.04.06 – Molecular biology of forest trees. This conference has convened scientists and foresters interested in the genetics, genomics, molecular biology and physiology of forest trees, and the application of this knowledge to tree improvement and conservation. The Tree Biotechnology Conference has undoubtedly been the premiere international forum where the most cutting edge research in tree biotechnology developed both in academia and industry is presented. "From genomes to integration and delivery", this was the theme chosen for the 2011 edition of the IUFRO Tree Biotechnology Conference, first time to be held in South America. Our intention was to promote a more integrated and applied dialogue on tree biotechnology and genomics, beyond the mainstream discussion of the fundamental advances on the genetic mechanisms that underlie tree phenotypes.

In nine scientific sessions some of the current advances of genomics applied to forest conservation, tree physiology, stress response, molecular breeding, in vitro and propagation technologies, wood development and genetically modified (GM) trees were highlighted. With 340 registered participants, the Conference brought to Brazil most of the world's brain power in forest tree genomics and biotechnology. An outstanding team of international scientists shared their results and visions on the present and future of this fast moving area of forest science, while a brilliant group of young scientist and students delivered a very energetic and diverse collection of high-quality scientific presentations. Forty two countries were represented at the Conference with almost 100 different

laboratories from tens of Universities, research institutions and private companies.

During the seven days of the Conference 26 invited lectures, 63 oral and 185 poster presentations were delivered, totaling 274 papers made available as extended abstracts into this BMC Proceedings supplement. The special workshop on the hot topic of "Genomic Selection in tree breeding" and the several reports on whole-genome studies, made this conference edition inaugurate a deliberate effort towards a better integration between the quantitative genomics, the "single-gene" and the system biology approaches to more efficiently unravel the complex relationships between genotypes and phenotypes in forest trees. A field trip to the forest plantations, nurseries and mill of VERACEL Cellulose was a definite highlight and a welcome break from the scientific sessions, providing an overview of some of the advances and challenges facing the translation of research into plantation forestry.

In closing this introductory statement, acknowledgements are due to the outstanding financial support provided by the competitive grants of the Brazilian Ministry of Science and Technology through the National Research Council (CNPq) and the Ministry of Education through its agency for graduate studies (CAPES). Major support was also provided by EMBRAPA (Brazilian Corporation of Agricultural Research), and VERACEL Cellulose, the host organizations, together with an exceptional suite of private sponsors. Besides the organizations that backed this conference and an active Scientific Committee involved in abstract review a number of people were involved in the organization and logistics. The conference would not have been possible without the valuable contributions of all these players.

Given the rewarding feedback received after the Conference, the original goal of providing an exceptional mix of science, social activities and field exploration in a relaxed atmosphere was truly accomplished. The IUFRO Tree Biotechnology Conference 2011 made a significant contribution to advance the forest biotechnology research community one step ahead on the challenging task of moving from gene and genome discoveries to the delivery of valuable technologies into sustainable forestry.

## S1. POPULATION GENOMICS, CONSERVATION AND ADAPTATION

### I1

Missing heritability and missing Fst of candidate genes: why does gene variation differ from trait variation in trees?

Antoine Kremer
INRA, 69 Route d'Arcachon, Cestas 33610, France

E-mail: antoine.kremer@pierroton.inra.fr

BMC Proceedings 2011, 5(Suppl 7):l1

Molecular footprints of phenotypic variation are usually explored by two different approaches in trees. On the one hand, association genetics is
seeking for statistical correlation between SNPs and traits owing to the
significant heritability that is usually observed in progeny tests. On the
other hand, detection of outlier Fst values of single SNPs has also been
implemented to account for the very large differentiation of traits that
are observed in provenance tests. The rationale of both approaches is
based on the extensive within or between population genetic variability
that has been widely recorded in fields tests. While the cataloguing of
candidate genes has steadily increased in trees, so has the inventory of
their diversity in natural or breeding populations. There is now a rapidly
growing body of experimental results showing a very large discrepancy
between the expectations of both approaches and the SNP variation that
is monitored in candidate genes. Most association studies show that
single SNPs explain usually less than 5% of the phenotypic variation of
the trait, while Fst values are at best of the same value than neutral
markers. In my presentation I will explore the reasons of the decoupling
between trait and gene variation, by focusing on the multilocus structure
of traits, as compared to the monolocus SNP information. Indeed,
association studies and Fst outlier detection are essentially based on
single locus approaches, while traits are multidimensional structures.
There are at least three properties of multilocus structures that will be
investigated: cumulativity, interactions and covariation of gene effects.
I will show that cumulativity and interactions may explain the discrepancy

in the case of association studies, while covariation of gene effects (at the
between population level) explain the missing Fst of genes underlying
adaptive traits. These conclusions are supported by experimental results,
theoretical background and simulation predictions.

I2

Population and conservation genomics of forest trees: seeing the forest
for the trees

Andrew Eckert

Department of Ecology and Evolution, University of California at Davis, Davis, CA, 95616, USA

E-mail: ajeckert@ucdavis.edu

BMC Proceedings 2011, 5(Suppl 7):I2

**Background:** Forest trees exhibit striking adaptations to the environments in which they grow. A long history of quantitative genetic experimentation has established the genetic basis for many traits, which are likely adaptive since many of them are also correlated with environmental heterogeneity. The genes underlying these traits, however, have largely remained elusive. Recent applications of high-throughput sequencing and genotyping technologies to natural populations of forest

<figure><img src="image_2.png" /></figure>
trees have identified several promising candidates for genes underlying complex and adaptive traits (reviewed by [1]). The diversity of analytical approaches employed in those studies, however, begs the question of the generality of reported results. Here, I exploited the diversity of analytical approaches used previously to identify genes underlying adaptive traits for two conifer species to assess the logical consistency among results generated from different conceptual frameworks.

**Materials and methods:** Data sets comprised of genotyped single nucleotide polymorphisms (SNPs) were gathered for two North American

conifers (Fig. 1): loblolly pine (Pinus taeda L.) and coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii). These data sets were generated through resequencing of diversity panels (n = 18-24 megagametophytes) for a sample of expressed sequence tag (EST) unigenes (n = 7,535) and cold-hardiness related candidate genes (n = 121), respectively. Genotyping was performed using Illumina's Infinium (loblolly pine) or GoldenGate (coastal Douglas-fir) array technologies. For each data set, associations to phenotypes and environmental variables were gathered from the literature or performed as described elsewhere

<figure><img src="image_3.png" /></figure>
(see references in [1]). For each associated gene, I asked two questions: (1) Are genes associated to phenotypes more often also associated to environmental variables than randomly chosen genes? (2) Are associated genes also outliers for nucleotide diversity and site-frequency spectrum based statistics, nucleotide divergence or $F_{ST}$ more often than randomly chosen genes? Permutation tests were used to assess whether or not observed patterns were different than those produced by chance.

**Results:** A total of ~30 genes were associated with cold-hardiness phenotypes or with climate data for coastal Douglas-fir, while ~850 associated genes were identified for loblolly pine. Genes associated with phenotypes for coastal Douglas-fir were more often associated to environmental variables than randomly chosen genes (P = 0.005), which was only moderately apparent for loblolly pine (P = 0.067). Genes associated to phenotypes or environmental variables were not more likely to be outliers for nucleotide diversity, site-frequency spectrum based statistics, nucleotide divergence or $F_{ST}$ for Douglas-fir (P > 0.05). There was some evidence, however, for non-neutral evolution for associated genes using a statistic based on the McDonald-Kreitman table [2]. In this case, associated genes had too many extreme values for the direction of selection (DoS) statistic than expected from randomly resampling the available genes (P < 0.05). Associated genes on average had skewed site-frequency spectra for loblolly pine, especially for synonymous sites, as well as too many extreme values for the DoS statistic. Further classification of genes for loblolly pine into functional categories revealed striking trends indicative of non-neutral processes underlying some of the associations (Fig. 2). A total of 11 gene categories were consistent with positive (n = 7) or negative (n = 4) selection. In both cases, the frequency of associated loci increased for these categories and the synonymous site frequency spectra became more skewed. Many associations for loblolly pine may thus reflect linked selection, with molecular phenotypes (e.g. gene expression) accounting for all the associations in gene categories indicative of negative selection and environmental associations (e.g. aridity) being largely located in gene categories consistent with positive selection.

**Conclusions:** Many of the genes associated to phenotypes for both species were also correlated to environmental variables and exhibited patterns of non-neutral evolution. Thus, associated genes are prime targets for conservation efforts. The questions posed here, however, make the strong assumption that genes associated to phenotypes or environmental variables should also show non-neutral patterns of evolution. This is not always expected to be the case [3,4], yet the lack of consistency is often interpreted as such and is one explanation for those genes or sets of genes reported here as lacking non-neutral signals. The search for logical consistency among analytical approaches, however, often focuses on uninformative patterns. To illustrate this point, I employed a novel environmental association approach that correlates genetic divergence to environmental change and show that most of the site-frequency spectrum based outliers for coastal Douglas-fir [5] are correlated to change in climate variables but not to extant climate patterns. Taken together these results illustrate that non-neutral genes are often identified during association analyses, that departures from neutrality for genes driving associations are not only those due to recent directional selection, and that further work is needed to understand the population genetic processes underlying associations between genotypes, phenotypes and the environment.

**Acknowledgements:** I would like to thank J. L. Liechty, B. N. Figueroa G. Rosa, and J. L. Wegrzyn for bioinformatics and computational support. I would like to thank all of the collaborators on the ADEPT2 project (http://dendrome.ucdavis.edu/NealeLab/adept2/). This work was funded by the National Science Foundation (IOS-PGRP-0501763, IOS-PGRP 0638502) and the United States Department of Agriculture (NRI Plant Genome 04-712-0084).

## References

1. Neale DB, Kremer A: Forest tree genomics: Growing resources and applications. *Nat Rev Genet* 2011, 12:111-122.

2. Stoletzki N, Eyre-Walker A: Estimation of the neutrality index. *Mol Biol Evol* 2011, 28:63-70.

3. Le Corre V, Kremer A: Genetic variability at neutral markers, quantitative trait loci and trait in a subdivided population under selection. *Genetics* 2003, 164:1205-1219.

4. Pritchard JK, Pickrell JK, Coop G: The genetics of human adaptation: hard sweeps, soft sweeps, and polygenic adaptation. *Curr Biol* 2010, 20: R208-R215.

5. Eckert AJ, Bower AD, Pande B, Jermstad KD, Krutovsky KV, St. Clair JB. Neale DB: Association genetics of coastal Douglas fir (*Pseudotsuga menziesii* var. *menziesii*, Pinaceae). I. Cold-hardiness related traits. *Genetics* 2009, 182:1289-1302.

## I3

### Tracing the history of South-American neotropical savannas and seasonally dry forests evidences from comparative phylogeography

Rosane Collevatti¹*, Suelen Rabelo¹, Joao Nabout², Jose Diniz-Filho¹

¹Universidade Federal de Goias, Brazil; ²Universidade Estadual de Goias, Brazil

E-mail: rosanegc68@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):I3

**Background:** The Neotropical Seasonally Dry Forests (SDF) are tree-dominated ecosystems that occur in disjunct areas of fertile soils throughout the Neotropics. The hypothesis that the vicariance of a formerly continuous seasonal woodland formation, which may have reached its maximum extension during a dry-cool period 18,000-12,000 bp (the LGM), the Pleistocenic Arc Hypothesis, has been raised to explain the disjunct distribution [1]. Alternatively, based on the distribution of contemporary SDF species in Amazonia Forest and palynological data, Pennington [2] also proposed that SDF may have expanded into Amazonia Basin during the Pleistocene, with rain forest and montane taxa largely confined to gallery forest. In addition, a number of studies based on the fossil pollen record now available show that during the early Holocene period (until ca. 6000-5000 14C B.P.), the climate was drier in most of the South American savannas and distribution of savanna-like vegetation in Central and Southeast Brazil was more extensive in early compared with the late Holocene [3-5]. In southeastern Brazil, the current vegetation exist in the region only in the latest Holocene period (since 970 or 600 B.P. for some regions) under the current wet climatic conditions, with an annual dry season of about 4 months. Hence, the fossil record shows that savanna expansion in the Quaternary, especially in southeastern Brazil was characterized mainly by herbaceous and grass savanna which were favored by the drier and highly seasonal climate. It is possible that arboreal savanna taxa became restricted to sites with moist climatic conditions, which served as refugias. We are interested in test these hypotheses using the genus *Tabebuia* as model group. We have chosen five species based on the pattern of geographical distribution: *T. aurea* and *T. ochracea*, from savanna vegetation (cerrado sensu stricto), *T. impetiginosa*, T. roseo-alba from SDF, *T. serratifolia*, widely distributed in Mata Atlantica, SDF, riparian forests and Amazonia. Here, we present the results based on the phylogeography of *T. impetiginosa*.

**Methods:** We first generated a phylogenetic hypothesis based on sequences from three non-coding regions of cpDNA and ITS from nuclear rDNA. At least 16 individuals from 14 populations were sampled and sequenced for three chloroplast intergenic spacers (1635bp). We also sequenced individuals of *T. ochracea*, *T. chrysanta*, *T. chrysotricha* and *Cybsta* *antisiphilitica*, as outgroups to estimate coalescence time and better understand the biogeographical history of *T. impetiginosa*. Time to most recent common ancestor was estimated based on coalescent analysis implemented in BEAST 1.4.7 software [6]. For distribution modeling, we used 77 records of *T. impetiginosa* and four climatic variables (mean annual rainfall and variability, average temperature of the warmest and coldest months) derived from four different coupled Atmosperic-Oceanic Global Circulation Models (AOGCM): CCSM3, CSIRO, HADCM3 and ECHAM. Five different niche models were used: the BIOCLIM, Euclidean Distances (EUCL) Mahalanabis Distances (MAHAL), Genetic Algorithm for Rule Set Production (GARP), and Maximum Entropy (MAXENT). For each of the NM, a total of 750 different models were generated, using distinct combinations of dataset partition and variable selection The paleoclimate scenarios were based on the GCM Genesis 2 [7].

**Results and discussion:** We have no evidences of a recent connection of the SDF in the LGM. Coalescent analyses showed that *T. impetiginosa* populations probably originated at ~7 Myr BP, but populations started to diverge only ~2 Myr BP, with the divergence of two major clades, one that comprises the populations from the East and West boundaries of Cerrado Biome, and the other that corresponds to the forests from Central, West and Northeast Brazil. Major divergences in this last clade coincide with the Quaternary glaciations. Our results strongly support that the disjunct distribution of *T. impetiginosa* may be derived from vicariance and that
long distance gene flow is unlikely, since we found a deep genealogy with alopatic lineages and a remarkable differentiation among populations (AMOVA, Arlequin v. 3.11) [8]. Paleodistribution modeling together with coalescent simulation showed no connection among SDFs in the LGM. Thus, glaciations may have most likely caused population divergence and differentiation due to retreat and range shift of an ancient forest widely distributed throughout the Central, Southwest and Northeast Brazil. Hence, we hypothesize that the isolation of an ancient forest due to a global cooling and drying during the glaciations of Pliocene/Pleistocene is responsible for the disjunct distribution of the SDF and *T. impetiginosa*. Our results also indicated that the last glaciation in Pleistocene were most likely responsible for local differentiation because lineage diversification within localities coincides with ~ 100 kya BP but without important demographic effects because populations showed a constant effective population size and no exponential growth or shrink. In conclusion, the range shift of *T. impetiginosa* toward the Northwest and the lack of a connection among SDFs in the LGM support the hypothesis of a kind of SDF in Amazonian Basin during Pleistocene glaciations but did not support the Pleistocenic Arc Hypothesis.

**Acknowledgments:** Phylogeography analysis of Cerrado tree species have been continuously supported by competitive grants of CNPq to RGC. Our research program integrating macroecology and molecular ecology has been continuously supported by grants to the research network GENPAC (Geographical Genetics and Regional Planning for natural resources in Brazilian Cerrado) supported by CNPq/MCT/CAPES (project # 564717/2010-0 and 563624/2010-8).

## References

1. Prado DE, Gibbs PE: Patterns of species distributions in the dry seasonal forests of South America. *Ann Missouri Bot Gard* 1993, 80:902-927.

2. Pennington RT, Prado DA, Pendry C: Neotropical seasonally dry forests and Pleistocene vegetation changes. *J Biogeogr* 2000, 27:261-273.

3. Salgado-Labouriau ML, Barberi M, Ferraz-Vicentini KR, Parizzi MG: A dry climatic event during the late Quaternary of tropical Brazil. *Rev Paleobot Palyn* 1998, 99:115-129.

4. Behling H, Hooghiemstra H: Neotropical savanna environments in space and time: Late Quaternary. *Interhemispheric climate linkages* Oxford: Academic Press: Markgraf V 2000, 307-323.

5. Behling H: Late glacial and Holocene vegetation, climate and fire history inferred from Lagoa Nova in the southeastern Brazilian lowland. *Veget Hist Archaeobot* 2003, 12:263-270.

6. Drummond AJ, Rambaut A: BEAST: Bayesian Evolutionary Analysis by Sampling Trees. BMC Evolutionary Biology 2007, 7:214.

7. Thompson SL, Pollard D: Greenland and Antarctic mass balances for present and doubled atmospheric CO2 from the GENESIS version-2 global model. *J Climate* 1997, 10:871-900.

8. Schneider S, Roessli D, Excoffier L: Arlequin Ver. 2000: A software for population genetic data analysis. Switzerland: Genetics and Biometry Laboratory, University of Geneva 2000.

# S2. LINKAGE AND ASSOCIATION MAPPING

## I4

**Genomics-based breeding in forest trees: are we there yet?**

David Neale

University of California-Davis, USA

E-mail: DBNeale@ucdavis.edu

BMC Proceedings 2011, **5**(Suppl 7):I4

Efforts to develop genetic marker based approaches to breeding forest trees began in the late 1980s. Approaches based on first generation of markers, allozymes, were not feasible due to the very limited number of markers (<50). The first DNA-based markers, RFLPs, brought more hope as moderately dense genetic maps could be constructed to scan the genome and map quantitative trait loci (QTLs). This approach was quite effective toward mapping QTLs in many forest tree species but the approach could not be brought to application in tree breeding due to low levels of linkage disequilibrium (LD) in forest tree breeding populations and recombination between flanking markers and QTLs with each generation. The next generation of DNA markers based on the

polymerase chain reaction (PCR), RAPD, AFLP and SSR, did not solve the LD and recombination problem, even though more markers were available and throughput increased.

The situation began to change in the early 2000s with the availability of automated DNA sequencing technology and single nucleotide polymorphism (SNP) genetic markers. Now association studies could be performed where SNPs within candidate genes controlling complex traits could be identified and thus "solving" or minimizing the LD and recombination limitation. This approach to complex trait dissection has been widely applied in forest trees and the early approach of QTL mapping in segregating populations has been mostly abandoned. The association genetic approach has been used to find candidate gene SNPs associated to a broad array of quantitative traits of interest (wood properties, growth, abiotic stresses and disease resistance). However, like QTL mapping before, individual SNP x trait associations only account for a small proportion of the variation (generally less than 2-3% of the total phenotypic variance) and the total variation explained by all markers is generally less than 50%. In human genetics, this situation is called the "missing heritability" and there has been great debate over whether this problem can ever be solved in complex trait dissection in humans. In a few agricultural systems however, notably dairy cattle, researchers are now accounting for nearly all the heritable variation.

Efforts to realize genomics-based breeding in conifer tree improvement in the US have culminated under a collaborative research project funded by the USDA. The Conifer Translational Genomics Network Coordinated Agricultural Project (CTGN CAP) has successfully brought genomics-based breeding to application in the four major cooperative forest tree breeding programs in the US. The project has completed the basic research on allele discovery of economic traits in conifers, conducted the translational research, and provided education and training to tree breeders to realize this goal during the four-year project period (2007 to 2011). Genomics-based breeding can now be applied to the production of over 1.3 billion loblolly pine, slash pine and Douglas-fir seedlings planted annually in the US.

The CTGN CAP obtained high-density single nucleotide polymorphic (SNP) genotypes (over 7000 SNPs) on nearly 10,000 loblolly pine, slash pine, and Douglas-fir trees from the breeding cooperatives. These data were used to estimate molecular breeding values that can now be used to make selections as an alternative to breeding values based on mature traits that can take many years to the time of evaluation and can be expensive to measure in field tests. The CTGN CAP was the first to apply the advanced Illumina Infinium SNP-genotyping technology that is now being used in the breeding of most major crops such as corn, wheat, tomato, potato, and many more.

The CTGN CAP culminates a long-term investment by the USDA in this team of researchers and their work on the technological advancement of tree breeding in the US. USDA's efforts began more than 20 years ago with funding of a series of grants under the National Research Initiative Plant Genome Program and continued under the CSREES IFAFS and AFRI programs. Recently, NIFA has awarded a grant to members of this team to conduct full genome sequencing in loblolly pine, sugar pine, and Douglas-fir. The full genome sequence will soon allow tree breeders to practice genomics-based breeding using genetic variation from the entire genome (genomic selection), and thus minimizing or even eliminating the missing heritability problem in forest trees. The partnership between USDA research programs and this team of researchers has resulted in a modernization of tree breeding technology in the US that will secure US competitiveness in the production of forest products.

## I5

**Identification of genes and alleles influencing wood development in Eucalyptus**

Simon Southerton®, Shannon Dillon, Bala Thumma

CSIRO Plant Industry, Australia

E-mail: simon.southerton@csiro.au

BMC Proceedings 2011, **5**(Suppl 7):I5

The goal of many forest tree breeding programs is to increase the quantity and quality of wood products from plantations. Due to their outcrossing breeding systems, long generation times and relatively short history of domestication, breeding populations of most forest trees
closely resemble the wild state. Consequently, vast stores of genetic variation are available for selection. Because wood traits are under polygenetic control (quantitative), genetic improvement will rely on selection of multiple alleles, each of relatively small individual effect. Marker-assisted selection may enhance tree breeding programs by enabling informed selection of parents for crossing; fixing desirable alleles in the homozygous state; increasing selection intensity through screening large numbers of individuals; enabling early selection in seedlings and by reducing phenotyping costs. The low linkage disequilibrium found in most forest trees makes them ideally suited to candidate gene-based association mapping approaches for marker discovery. This approach seeks to find alleles which affect phenotype and that remain linked to the trait across populations and over many generations. This methodology is well suited to tree breeding programs which aim to maintain a broad genetic base i.e. programs with a large number of families.

We are using association studies to identify genes and allelic variation that influences wood fibre properties in *Eucalyptus nitens*. Candidate genes are being selected on the basis of their known involvement in cell wall synthesis pathways expected to impact wood traits. Single nucleotide polymorphisms (SNPs) are identified in candidate genes by sequencing in a number of unrelated individuals. Selected SNPs are being genotyped across large unrelated *E. nitens* populations that have been extensively phenotyped for wood properties including cellulose and lignin content, pulp yield, MFA, and density. Several SNPs significantly associated with wood properties have been identified and subsequently validated in other provenance or mapping populations growing in different environments. Selected SNPs are being investigated further to determine whether or not the SNP is the causative polymorphism and how the polymorphism influences the trait. DNA markers identified in this research may be used to complement existing index selection strategies in *E. nitens* breeding programs. Strategies for exploiting SNPs for marker-assisted selection in seedling-based breeding programs will be discussed.

## I6

### Gene mapping in white spruce (*P. glauca*): QTL and association studies integrating population and expression data

John MacKay¹, Brian Boyle², Walid El Kayal³, Marie-Claire Namroud²,
Trevor Doerksen⁴, Janice Cooke³, Nathalie Isabel⁵, Jean Beaulieu⁴,
Philippe Rigault⁶, Paul Bicho⁷, Jean Bousquet²,¹

¹Université Laval, Arborea, Centre for Forest Research and Institute for Systems and Integrative Biology, Québec, Québec, Canada; ²Université Laval, Arborea, Centre for Forest Research and Institute for Systems and Integrative Biology, Québec, Québec, Canada; ³University of Alberta, Department of biological Sciences, Edmonton, Canada; ⁴Natural Resources Canada, Laurentian Forestry Center and Canadian Wood Fibre Centre, Québec, Québec, Canada; ⁵Natural Resources Canada, Laurentian Forestry Center, Québec, Québec, Canada; ⁶Gydle Inc., Québec, Québec, Canada; ⁷FPInnovations, Vancouver, British Columbia, Canada

E-mail: john.mackay@sbf.ulaval.ca

*BMC Proceedings* 2011, **5**(Suppl 7):I6

**Background:** Connecting phenotype with genotype is the basis for developing forest genetic applications such as marker assisted selection (MAS). Quantitative Trait Locus (QTL) mapping and genetic association mapping (or linkage disequilibrium (LD) are two major approaches to find genes that control phenotypes of interest in forest trees. Quantitative trait loci (QTL) and association mapping experiments in white spruce (*Picea glauca* [Moench] Voss) aimed to identify genes linked to or associated with growth, adaptation, and wood property traits.

Gene mapping in conifer trees presents us with specific challenges, including very large genome sizes, low level of linkage disequilibrium, and large effective size in breeding populations. Therefore, association mapping experiments have relied on testing candidate gene targets rather than genome-wide association scans. We have explored different approaches to utilize gene expression and SNP outlier data to identify candidate genes, to help to explain the findings of gene mapping experiments and provide a broader understanding of observed phenotypic variations.

**Results:** Growth and phenology: The genomic architecture of bud phenology and height growth was investigated by assessing QTLs across

pedigrees, years, and environments (1). A total of 11 distinct QTLs for bud flush, 13 for bud set, and 10 for height growth were localized on a linkage map highly-enriched in gene markers. Nearly 50% of the QTLs were stable across environments and/or years and 20% were replicated between populations. The proportion of phenotypic variance explained by QTLs ranged from 3% to 22.2%, and QTLs accounted for up to 70% of trait variance. These outcomes were integrated with findings from studies aimed identifying local adaption genes and gene expression associated with bud formation.

A genome-wide scan of 534 SNPs localized in 345 expressed genes was used to detect genes putative linked to local adaptation (2). We identified 5.5% of genes as outliers with FST at the 95% confidence level, and 14% of genes as candidates for local adaptation with a Bayesian method. The list of candidate genes and outliers includes sequences which co-localized with the QTLs for bud phenology.

A bud set roadmap was constructed by comprehensive microarray and qRT-PCR analysis of dormancy transition in bud, stem, needle, and root tissues over a time course, under short and long days (3). Tissue expression profiles were used to identify genes expressed only or preferentially in developing buds, which we hypothesize to play a more prominent role in bud formation. A core group of genes likely involved in the initiation of bud formation included about 100 of the bud-prominent genes and several sequences encoding potential regulatory proteins. Several of the bud set roadmap genes including bud-prominent genes co-localized with QTLs for the time of bud set.

**Wood properties:** Wood physical traits were assessed using SilviScan technology in a population of 1700 trees comprising 215 open-pollinated families. In a pilot study, we tested for associations between single nucleotide polymorphisms (SNP) in 550 candidate genes and wood traits (4). We found 13 SNPs significantly associated with wood traits. The phenotypic variance explained reached up to 11% with approaches combining several SNPs.

Most association studies of wood properties have tested candidate genes that are highly expressed in secondary xylem, hypothesizing that genes that are preferentially or strongly expressed during wood formation are more likely to control wood properties. However, this hypothesis had not been tested. The genotyped sequences included genes with diverse expression profiles. Their transcript accumulation profiles were determined in trees grown under controlled conditions with a large-scale custom oligonucleotide microarray representing 25,094 different spruce genes. Of the 550 genes tested for association, 29% accumulated preferentially in secondary xylem compared to both secondary phloem and needles, but as many genes (29%) were phloem preferential. Xylem-preferential RNA accumulation was found for 10 of the 13 genes harbouring SNPs significantly associated. Our findings confirm that expression data were relevant for selecting candidate genes but not all of the genes containing significant SNPs were xylem preferential.

Transcript accumulation was also studied in secondary xylem of trees from the provenance-progeny trial, to further characterize the genes containing SNPs significantly associated with wood traits. In some cases, significantly different transcript levels were found among the different SNP genotypes. Xylem-preferential RNA accumulation was shown for the majority of these genes, which indicates that. Our results suggest that differential expression may be associated with SNP genotypes.

**Large-scale genotyping:** A meta-analysis was used to integrate data from multiple experiments in order to identify and assign priorities to approximately 5000 candidate genes for association mapping experiments. The candidate gene selection considered the above gene expression data, findings from transcriptomic investigations of gene regulation, studies investigating transcriptional variation within mapping populations trees, and outlier data related to local adaptation. A large-scale genotyping chip was developed and data were obtained for 7000 SNPs from nearly 2500 genes.

**Conclusions:** This report describes QTL mapping and genetic association mapping results. We have illustrated ways in which gene expression and population data may be of value in these approaches, whether they are used to select candidate genes or to characterize the physiological processes underlying marker-trait associations.

## References

1. Pelgas B, Bousquet J, Meirmans PG, Ritland K, Isabel N: QTL mapping in white spruce: gene maps and genomic regions underlying adaptive traits across pedigrees, years and environments. *BMC Genomics* 2011, 12:145.
2. Namroud M-C, Guillet-Claude C, Mackay J, Isabel N, Bousquet J: Molecular evolution of regulatory genes in spruces from different species and continent: heterogeneous patterns of linkage disequilibrium and selection but correlated recent demographic changes. *J Mol Evol* 2010, 70:371-386.

3. El Kayal W, Allen CCG, Ju CJ-T, Adams E, King-Jones S, Zaharia Li, Abrams SR, Cooke JEK: Molecular events of apical bud formation in white spruce, *Picea glauca*. *Plant Cell Environ* 2011, 34:480-500.

4. Beaulieu J, Doerksen T, Boyle B, Clément S, Deslauriers M, Beauseigle S, Blais S, Poulin P-L, Lenz P, Caron S, et al. Association genetics of wood physical traits in the conifer white spruce and relationships with gene expression. *Genetics* 2011, 188:197-214.

# S3. GENOMICS ASSISTED BREEDING

## I7

### Capturing and genotyping the genome-wide genetic diversity of trees for association mapping and genomic selection

Matias Kirst¹*, Márcio Resende², Patrício Munoz³, Leandro Neves³
¹School of Forest Resources and Conservation, University of Florida,
P.O. Box 110410, Gainesville, FL 32611, USA; ²Genetics and Genomics
Graduate Program, University of Florida, P.O. Box 110690, Gainesville, FL
32611, USA; ³Plant Molecular and Cellular Biology Graduate Program,
University of Florida, P.O. Box 110690, Gainesville, FL 32611, USA
E-mail: mkirst@ufl.edu
*BMC Proceedings* 2011, **5**(Suppl 7):17

**Background:** Growing demand for food and fiber, and a rapidly changing climate will require that plant breeders accelerate the improvement of germplasm adapted to new sources of biotic and abiotic stress. In trees, the threat from climate change is more evident and the solutions more challenging than in any other plant species, due to the complexity and cost of breeding programs, and the long breeding cycles. Therefore, the discovery of genetic polymorphism that can be exploited for early selection of better adapted and productive individuals is essential. Quantitative trait loci (QTL) analysis provided an initial glimpse at the architecture of complex traits, but limited transferability across populations and resolution hampered the adoption of markers in tree breeding programs. Recently, association studies have become the method of choice for detection of markers implicated in trait variation, because of higher resolution, population transferability and allelic diversity captured relative to the QTL approach. However, in tree species, association studies have been largely constrained to sampling the genetic diversity in a limited fraction of the genome, and in small populations. Evidence from genome-wide association studies (GWAS) in humans and advanced crops clearly show that larger populations, and the sampling of regulatory variants and rare alleles is critical to dissect the genetic control of complex traits for marker-assisted breeding (MAB). As the limitations of QTL and GWAS approaches become evident, "hybrid" intermediate strategies that combine the advantages of both methods have emerged. Notably, genomic selection has become an alternative to MAB. Genomic selection (GS), which relies on developing genome-wide marker-based models that predict the genetic value of progeny, will be particularly valuable for early selection in tree breeding programs. However, the implications of GS may also be highly valuable to identify mating designs that generate progeny with optimal allelic combinations for superior growth and wood properties, and adaptive capabilities.

**Methods:** To address limitation of recent association studies in forest tree species we have adopted an approach that combines targeted sequence-capture followed by high-throughput sequencing, to genotype eastern cottonwood (*Populus deltoides*) and loblolly pine (*Pinus taeda*) populations. To identify genetic polymorphisms that regulate biomass productivity, wood quality, and disease resistance, we have optimized methods of sequence-capture targeted regions of the genome for unbiased, high-throughput and low cost recovery of coding and regulatory sequences. This establishes the foundation for GWAS in both species, addressing a critical limitation of these studies in tree genomes – i.e. sampling of regulatory variants and rare alleles.

While the work described above resolves part of the challenges of association studies, the use of GWAS will be most useful for discovery of genes to be targeted for genetic modification, rather than MAB. This is

the case because the fraction of the total genetic variation explained by association studies is likely to remain small, considering the limitations of the existing populations. In an effort to implement marker-based breeding, we recently completed the first assessment of the utility of GS in an experimental population of loblolly pine, where we explored the contribution of factors such as age of model estimation and site location on the accuracy of prediction models. Furthermore, the incorporation of non-additive effects to prediction models has been evaluated for improvement of their accuracy.

**Results and conclusions:** Two association studies are currently underway, in loblolly pine and *Populus deltoides*, where a large fraction of the coding and regulatory sequences of their genomes are being re-sequenced for SNP detection. Existing results from sequence capture indicate that the majority of targeted regions can be effectively captured, even in genomes of very high complexity, such as that of loblolly pine. In parallel we have now verified the suitability of applying genomic selection to a breeding population of loblolly pine. Estimates generated for prediction models developed for this population indicates that accuracies that are comparable to traditional phenotypic selection can be obtained. Therefore, considering the significant reduction in the breeding cycle length due to early identification of elite genotypes, the increase in efficiency per unit of time in the selection response of genomic selection is almost twice as high, compared to traditional breeding. By combining GS with advanced methods of vegetative propagation, breeding and seedling production, a breeding cycle can be reduced from decades to less than 5 years. While early selection of superior genotypes is an obvious application of GS to tree improvement programs, prediction models can be utilized to guide the mating design of future breeding cycles to favor stacking of favorable alleles over multiple generations. Towards this end, we have initiated crosses aimed at generating families that are predicted to have exceptional adaption, as well as biomass growth and quality properties for bioenergy, pulp and paper and timber production.

**Acknowledgements:** This work was supported by the University of Florida Genetics and Genomics Graduate Program, the Plant Molecular and Cellular Biology Graduate Program, the Forest Biology Research Cooperative (FBRC), the US Department of Agriculture National Institute of Food and Agriculture Plant Breeding and Education Program (award no. 2010-85117-20569) and the National Science Foundation (award no.DBI-0501763).

## I8

### Genomic selection in loblolly pine - from lab to field

Fikret Isik*, Ross Whetten, Jaime Zapata-Valenzuela, Funda Ogut, Steven McKeand
Cooperative Tree Improvement Program, North Carolina State University, Raleigh, USA
E-mail: fisik@ncsu.edu
*BMC Proceedings* 2011, **5**(Suppl 7):18

**Background:** Tree breeding is logistically complex and expensive, and breeders have long sought to use molecular markers to accelerate breeding. A candidate gene approach based on testing for association between the presence of DNA sequence variation in or near candidate genes, and phenotypic variation in a population has long been explored [1,2]. However, using candidate gene approach (QTLs) has not been successful in breeding [3,4]. QTL-trait associations detected in one genetic background are often not observed in other families, because of recombination of genes during the segregation and low levels of linkage disequilibrium in the population. A new technology called genomic selection (GS) is revolutionizing dairy cattle breeding. In GS, marker effects are first estimated in a large training population (>500) with both phenotypic and genotypic data. Subsequently, estimated marker effects are used to predict breeding values in validation populations for which marker genotypes but not phenotypes are available [4]. Several dairy cattle breeding companies now routinely use GS to select and market bulls. The success of GS in cattle breeding is largely based on bovine genome sequencing and discovery of thousands of SNP markers. GS application, if successful, will have a great impact on forest tree breeding because of their complex and logistically difficult breeding programs. Although, there have been several simulation studies examining the effective population size, linkage disequilibrium, and heritability on the predicted accuracy of
GS in tree breeding [5], GS has not yet been demonstrated for forest trees using empirical markers data, mainly due to lack of sufficient dense markers.

**Methods:** Biallelic SNP markers provided by the CTGN project (http://dendrome.ucdavis.edu/ctgn/) were used for genotyping. A population of 149 cloned full-sib offspring of loblolly pine (Pinus taeda L.) was phenotyped. Fitting 3406 informative SNP markers simultaneously, we estimated genome-wide breeding values and compared them with breeding values based on pedigree model. Variances explained by the marker additive and dominant effects were obtained.

**Results:** The accuracy of the genomic estimated breeding values ranged from 0.30 to 0.83 for growth and wood quality traits. Lignin and cellulose content had great accuracy values from GS compared to growth traits. The accuracies were comparable with breeding values that were calculated based on the traditional pedigree model. If we take into account time needed to complete progeny testing, GS would be more efficient than classical progeny testing for some traits. The marker additive effects explained 18% and 23% for lignin and cellulose, respectively. Variances could not be determined for height and volume, because the Gibbs sampler failed to converge, even after five million iterations. We speculate that observed accuracies in this study trace familial linkage rather than historical LD with trait loci, because of small population size and relatively deep pedigrees. The markers are sampling the haplotypes and thus constructing the pedigrees rather than explaining phenotypic variance. Nevertheless, the results are promising, and we expect that with decreases in genotyping cost, GS has a potential to fundamentally change tree breeding in the near future.

**Challenges of GS applications in tree breeding:** Despite promising results from some early work based on empirical data, there some challenges to overcome to routinely use GS in tree breeding. Conifers have genome size with a range between 18,000 and 40,000 Mbp [6]. Their populations have low levels of LD which decays rapidly. LD in loblolly pine decays to less than r²=0.25 within 2000 bp [7]. Low LD is due to genetic recombination over the evolutionary history of the species and causes inconsistency of QTL-marker association. Large genome size and historically low LD require large numbers of dense markers to explain a considerable amount of phenotypic variation in complex traits. Another challenge is the lack of genetic maps in forest trees. With a few exceptions, the genomes of forest trees have not been sequenced, and thus precise locations of SNP markers are lacking, which hinders the use of haplotypes. Using haplotypes reduces the dimensions of the data and thus requires much smaller computing resources to analyze. More importantly, with haplotypes, larger variation between trees can be obtained using allelic combinations, although larger training populations are required to adequately sample the effects of all the haplotypes.

High marker genotyping cost is the major obstacle in applications of GS in forest trees. More cost efficient genotyping technologies, such as genotyping-by-sequencing and restriction digestion are being explored to reduce cost of markers. On the other hand, advances in computer power have made it possible to analyze large amount of complex data, but bioinformatics challenges still remain to analyze sequence data and SNP marker calling.

Further research is needed in development of training models and calibration of prediction model. The number of generations that statistical models can be used before losing accuracy remains to be determined in forest trees. Another question is the validity of models across different populations. In cattle breeding, lower accuracies of GS for dairy versus beef cattle remains a challenge. For some tree species, GxE interaction could be an issue to be addressed; observed marker-trait association observed in one population may not hold in another environment.

**Conclusions:** We are currently working on construction of realized genomic relationship matrix based on SNP markers to use in predictions of breeding values. This method provides flexibility in terms of fitting common environmental effects in mixed models. We expect that decreases in marker genotyping costs will make GS in pine breeding feasible in the near future. Our group will work on pilot projects with forestry companies in the southern US, and plans are underway to revise breeding strategies to incorporate genomic selection.

References

1. Tabor HK, Risch NJ, Myers RM: Candidate-gene approaches for studying complex genetic traits: practical considerations. *Nat Rev Genet* 2002, 3:391-397.

2. Hayes BJ, Bowman PJ, Chamberlain AJ, Goddard ME: Invited review: *Genomic selection in dairy cattle: progress and challenges. J Dairy Sci* 2009, 92:433-443.

3. Goddard ME, Hayes BJ: Genomic selection. *J Anim Breed Genet* 2007, 124:323-330.

4. Meuwissen THE, Hayes BJ, Goddard ME: Prediction of total genetic value using genome wide dense marker maps. *Genetics* 2001, 157:1819-1829.

5. Grattapaglia D, Resende M: Genomic selection in forest tree breeding. *Tree Genetics and Genomes* 2010, 7(2):241-255.

6. Morse AM, Peterson DG, Islam-Faridi MN, Smith KE, Magbanua Z: Evolution of genome size and complexity in Pinus. *PloS ONE* 2009, 4(2):e4332, doi:10.1371/journal.pone.0004332.

7. Brown GR, Gill GP, Kuntz RJ, Langley CH, Neale DB: Nucleotide diversity and linkage disequilibrium in loblolly pine. *PNAS* 2004, 101:15255-15260.

S4. REPRODUCTION, GROWTH AND DEVELOPMENT

I9

Salicylate metabolism in Populus

Chung Jui Tsai¹*, Wenbing Guo¹, Benjamin Babst¹, Batbayar Nyamdari¹, Yinan Yuan², Raja Payyavula¹, Han-Yi Chen¹, Xue Liangjiao¹, Kate Tay¹, Vanessa Michelizzi¹, Scott Harding¹

¹University of Georgia, USA; ²Michigan Technological University, USA

E-mail: cjt sai@uga.edu

BMC Proceedings 2011, 5(Suppl 7):I9

Phenolic metabolites that contain salicylic acid (SA)-like moieties are major non-structural constituents in *Populus* leaves, shoots and roots. These so-called phenolic glycosides (PGs) are taxonomically limited to the Salicaceae, where they are known to mitigate insect and animal herbivory. SA itself is an important signaling molecule in plant defense and abiotic stress responses, and is derived from the isochorismate or phenylpropanoid pathways, depending on the species [1,2]. Hyper-accumulation of SA reduces growth in *Arabidopsis* [3], while high levels of PG have been associated with reduced growth in *Populus* [4]. Although the common PGs, salicin, salicortin, and their derivatives contain a salicyl moiety, a direct metabolic relationship between PG and SA in *Populus* has not been shown. We have therefore been using stable isotope tracers, cell culture feeding, functional genomics and transgenic perturbation to probe the relationship between SA, PG and growth in *Populus*.

Stable isotope incorporation confirmed a hydroxycinnamate-benzoate origin of the salicyl moiety of PGs, and provided the first empirical evidence that the bioactive hydroxycyclohexenone moiety of salicortin is also a phenylpropanoid derivative [5]. Therefore it is unlikely that PG biosynthesis involves the salicylic acid pathway. To further address any possible involvement of the isochorismate pathway in PG biosynthesis, isochorismate synthase (ICS) was characterized. ICS is present as a single-gene in most sequenced plant genomes, but is duplicated in *Arabidopsis*. In *Arabidopsis*, chorismate-derived SA is the obligatory route for defense signaling, mediated by *AtICS1* in response to various biotic and abiotic cues. In contrast, the single-copy *Populus* ICS is not stress-inducible, and is involved in biosynthesis of phyloquinone for photosynthetic electron transport [2]. We found no evidence of ICS involvement in SA or PG biosynthesis in transgenic *Populus*, pointing to lineage-specific evolution of the ICS-derived SA pathway in *Arabidopsis*.

We transformed *Populus* with the bacterial genes for the biosynthesis and degradation of SA for a more in-depth investigation of the possibility of an SA interaction with PG regulation. SA-hyperaccumulating and SA-deficient lines were generated by expressing a salicylate synthase and a salicylate hydroxylase, respectively. SA was converted to SA-glycoside (SAG) and gentisic acid-glucoside (GAG) in the hyperaccumulating lines. SAG and GAG were very low in wild-type and were not detected in the SA-deficient lines. Despite these clear differences, no changes in PG levels were detected. These results argue against SA as potential precursor of PGs. Survival and establishment of rooted cuttings were negatively affected by SA-hyperaccumulation. Once established, however, growth rates were similar among plant lines, in sharp contrast to SA-over-producing *Arabidopsis*. SA-hyperaccumulating lines exhibited altered thermal tolerance, based on electrolyte leakage assays and metabolite profiling.
Together, our results provide support for a phenylpropanoid origin of PGs, and argue against the involvement of SA in PG biosynthesis. The data is consistent with complementary roles for SA and PG in *Populus* fitness.

A number of other experiments were conducted to investigate the regulation of PG homeostasis in *Populus*. Administration of a putative PG precursor salicyl alcohol to cell cultures yielded the glucoside, salicyl. Salicyl alcohol-feeding also altered the partitioning of carbon into condensed tannin (CT) biosynthesis, another quantitatively important phenylpropanoid product [6]. Salicyl alcohol-feeding induced expression of several glycosyltransferases (GT1), as well as genes associated with sucrose catabolism, glycolysis and the Krebs cycle, presumably to accommodate the increased glycosylation. Conversely, transcript levels of most of the flavonoid pathway genes were reduced, consistent with reduced CT synthesis. Given that glycosylation was the only PG biosynthetic step occurring in the fed-cells, the results suggest that glycosylation of phenolic products can play a role in regulating the tradeoff between competing chemical defenses, e.g., PGs and CTs.

Correlation analysis was used to identify PG-coregulated genes from a number of inductive treatments. One such candidate, *SUT4* encoding a tonoplast sucrose transporter, was of particular interest because of the reported salicyl transport activity of its *Arabidopsis* ortholog. RNAi-silencing of *SUT4* altered sucrose distribution between source and sink organs, and reduced PG accrual by 20-30%, accompanied by altered biomass partitioning [7]. Chemical and molecular analyses revealed a complex network linking PG accrual and carbohydrate homeostasis.

PG-coregulated GT1 members were also targeted for characterization, due to the prominent role of glycosylation in PG biosynthesis. We identified a novel salicyl alcohol glycosyltransferase (UGT71L1). Over-expression of UGT71L1 in transgenic *Populus* led to a ~2-fold increase in concentrations of salicin and salicortin in roots. The quantitative effect on secondary metabolism is significant, as PGs are the second most abundant non-structural phenolics, after CTs, in the roots of this poplar clone. UGT71L1 is phylogenetically distinct from the well-characterized SAGTs. It belongs to a *Populus*-specific clade with no close homologs in *Arabidopsis*, *Vitis*, *Medicago* and *Oryza*, consistent with the limited taxonomic range of PGs. Overall, the experiments described highlight the important contributions of sugar transport and phenolic glycosylation to PG homeostasis, while also showing that SA and PGs make independent but complementary contributions toward the maintenance of *Populus* fitness.

## References

1. Wildermuth MC, Dewdney J, Wu G, Ausubel FM: Isochorismate synthase is required to synthesize salicylic acid for plant defence. *Nature* 2001, 414:562-565.

2. Yuan Y, Chung J-D, Fu X, Johnson VE, Ranjan P, Booth SL, Harding SA, Tsai C-J: Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis. *Proceedings of the National Academy of Sciences* 2009, 106:22020-22025.

3. Mauch F, Mauch-Mani B, Gaille C, Kull B, Haas D, Reimmann C: Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase. *Plant J* 2001, 25:67-77.

4. Hwang SY, Lindroth RL: Clonal variation in foliar chemistry of aspen: Effects on gypsy moth and forest tent caterpillar. *Oecologia* 1997, 111:99-108.

5. Babst BA, Harding SA, Tsai C-J: Biosynthesis of phenolic glycosides from phenylpropanoid and benzenoid precursors in *Populus*. *Journal of Chemical Ecology* 2010, 36:286-297.

6. Payavula R, Babst B, Nelsen M, Harding S, Tsai C-J: Glycosylation-mediated phenylpropanoid partitioning in *Populus tremuloides* cell cultures. *BMC Plant Biology* 2009, 9:151.

7. Payavula RS, Tay KHC, Tsai C-J, Harding SA: The sucrose transporter family in Populus: The importance of a tonoplast PtaSUT4 to biomass and carbon partitioning. *Plant Journal* 2011, 65:757-770.

## I10
Division of labor: multiple and specialized controls of vegetative growth and development in a poplar tree

Victor Busov¹*, Yiru Chen¹, Jiqin Gou¹, Steven Strauss², Yordan Yordanov¹, Christine Zawaski¹

¹Michigan Technological University, USA; ²Oregon State University, USA
E-mail: vbusov@mtu.edu

BMC Proceedings 2011, 5(Suppl 7):I10

Plants size, shape and adaptability are determined in large by their capacity to sustain, spur, redirect or arrest localized and whole-plant growth. We show that GA signaling and metabolism determines the level and extent of shoot and root growth. Increase of GA concentrations or signaling leads to increased shoot growth but suppresses root development. These GA-related responses are underpinned by sets of highly specialized in their functions enzymes and signaling factors and cross-talk with other hormonal pathways. The differential effects of GA on root and shoot growth and development are likely associated with a regulatory mechanism responding to optimum and stress conditions. Ongoing work in the laboratory employs genomics and genetics approaches to more thoroughly understand poplar growth and development under stress conditions including nitrogen and water limitations. We use genetic networks analysis and forward genetics approaches to identify key regulators of poplar roots response to stress. Besides overall growth, trees show incredible repertoire of spatiotemporal regulation of growth in relation to control of organ size, growth periodicity and tropic responses. We have identified and characterized novel regulators of these processes in poplar.

## I11
Regulation of shoot-system development in Populus

Amy Brunner¹*, Xiaoyan Sheng¹, Joesph Edwards¹, Takeshi Fujino¹, Chieh-Ting Wang¹, Stephen DiFazio²

¹Department of Forest Resources and Environmental Conservation, Virginia Tech, Blacksburg, VA 24061, USA; ²Department of Biology, West Virginia University, Morgantown, WV 26506, USA

E-mail: abrunner@vt.edu

BMC Proceedings 2011, 5(Suppl 7):I11

Shoot phenology interacts with crown morphology to determine leaf production and duration over the growing season and throughout a tree's life span; and thus, this interaction has a major role in determining whole-tree photosynthesis and biomass yield. For several years, the pattern of shoot meristem activity determines *Populus* crown architecture, but after the first onset of flowering the specification of meristem identity (inflorescence vs. vegetative) also has a major influence on crown form. To study how shoot meristem activity and identity are regulated in *Populus*, we are focusing on two pathways that have been extensively characterized in annual plants: 1) the MORE AXILLARY BRANCHING (MAX) genes that are involved in the synthesis and perception of upwardly mobile stigrolactones that inhibit axillary bud outgrowth, and 2) members of the TERMINAL FLOWER1 (TFL1)/FLOWERING LOCUS T (FT) and APETALA1(API1)/FRUITFULL(FUL) gene families that regulate flowering. We are taking a multi-pronged approach that includes field and controlled-environment study of poplar transgenics, transcriptome analyses, cell-specific expression studies, and QTL and association mapping.

QTL mapping in a hybrid poplar pedigree localized three of seven poplar MAX genes in QTL for bud set and two MAX genes in sylleptic branching QTL. All MAX transgenics were established in a field trial and phenotyping of phenology, crown morphology, and growth is in progress. Greenhouse studies of poplar transgenics with the seven different poplar MAX gene promoters driving the reporter gene GUS show that most of the MAX genes are expressed in vascular tissues. However, vascular expression patterns are different, especially in stems undergoing secondary growth. Surprisingly, none of the RNAi transgenics showed differences in branching in the greenhouse under standard growing conditions. High nitrogen levels can induce sylleptic branching in poplar and greenhouse experiments with MAX transgenics indicate a possible role for one MAX gene in nitrogen-induced sylleptic branching. Ongoing studies of the effects of decapitation of the shoot apex with and without subsequent auxin application to the cut stem indicate that, as predicted based on studies in annual plants, the poplar MAX RNAi transgenics show a reduced response to apically-applied auxin.

Recent work has revealed that poplar *FT1* and *FT2* have diverged in regulation and function with *FT1* promoting the transition to flowering in response to winter temperatures, whereas warm temperatures and long days promote *FT2* expression and shoot growth [1]. In *Arabidopsis*, *FT* activates *AP1*, a key regulator of flower initiation. Expression and transgenic studies in poplar indicate that similar to the two poplar *FTs*, poplar *AP1* subfamily members have roles in both flowering and bud set in response to short-days and abiotic stress. Ongoing work is aimed at defining the roles of *FT* and *AP1* modules in these processes and to
delineate the roles of the five different poplar *AP1/FUL* genes. Finally, *TFL1* acts opposite to *FT* to promote vegetative meristem identity in Arabidopsis, and *AP1* and *TFL1* repress each other's expression. The poplar *TFL1* homologs, *CEN1* and/or *CEN2* have roles in maintaining vegetative identity and in the dormancy-growth transition [2] and we are currently working to define the individual roles of the poplar *CENs* and their relationships to *FT* and *AP1* activities.

**Acknowledgements:** This work was supported by National Research Initiative Competitive Grants Program grant #2008-35301-19167 from the USDA National Institute of Food and Agriculture and the US Department of Energy's Bioenergy Sciences Center.

## References

1. Hsu CY, Adams J, Kim H, No K, Ma C, Strauss SH, Drnevich J, Vandervelde L, Ellis JD, Rice M, Wickett N, Gunter LE, Tuskan GA, Brunner AM, Page GP, Barakat A, Carlson JE, dePamphilis C, Luthe DS, Yuceer MC: *FLOWERING LOCUS T* duplication coordinates reproductive and vegetative growth in perennial poplar. *PNAS* 2011 in press.

2. Mohamed R, Wang CT, Ma C, Shevchenko O, Dye SJ, Puzey JR, Etherington E, Sheng X, Meilan R, Strauss SH, Brunner AM: *Populus CEN/TFL1* regulates first onset of flowering, axillary meristem identity and dormancy release in *Populus*. *Plant J* 2010, 62:674-688.

# S5. BIOTIC AND ABIOTIC INTERACTIONS

## I12

### The genomics of poplar-rust interactions to improve tree resistance against fungal disease

Louis-Philippe Hamel<fnref n="1" />, Meriem Benchabane<fnref n="2" />, Ian T. Major<fnref n="2" />,
Marie-Claude Nicole³, Jen Sheen³, Armand Séguin<fnref n="2" />*

E-mail: armand.seguin@nrcan.gc.ca

BMC Proceedings 2011, 5(Suppl 7):I12

With their long life cycle, trees must have accurate mechanisms to perceive microbial invasion and elaborate signalling networks in order to activate

the appropriate defense response through transcriptional reprogramming. Transcriptional activators and repressors participate in the tight regulation of the stress response, which is key to minimise the fitness costs associated with an activated response. With the availability of its whole genome sequence, ease of growth and clonal propagation, and routine transformation, poplar (*Populus* spp.) is considered a model tree species for genomics research and also a good system in forest pathology [1]. Moreover, genomes of a cortege of associated microorganisms are being sequenced including a tree pathogen, *Melampsora* poplar rust. We pursued various approaches to identify poplar genes involved in the interaction with the biotrophic *Melampsora* rust pathogen.

Recent transcriptome analyses from our lab have shown that the expression of genes encoding several transcription factors are up-regulated during infection by *Melampsora* rust. Similarly we have also shown that mitogen activated protein kinases (MAPKs) are associated with poplar disease resistance against *Melampsora* rust [2]. New data obtained from various experimental approaches have directed our focus on two important families of transcription factors; the jasmonate ZIM-domain (JAZ) and Cys2/His2 zinc-finger protein (ZFP) families. We have identified a novel MAPK-interacting partner, *PtiZFP1*, which belongs to the C2H2 ZFP family of transcriptional EAR repressors. The JAZ family of transcriptional repressors were recently identified as key negative regulators of jasmonate (JA) responses.Transcript analyses show that some ZFP and JAZ members exhibit hormone-related expression profiles and up-regulation by rust infection. This up-regulation of JAZ and ZFP transcripts after rust infection strongly suggests that a hormonal response including JA is a key component of the poplar defence response against *Melampsora*. Late and sustained kinetics of *PtiZFP1* and specific JAZ up-regulation suggest that the corresponding proteins may be required for late regulation of defense mechanisms.

Several recent data have shown that proteolytic cleavage of transcriptional repressors is a general mechanism used by plants to activate gene induction. This mechanism is now well documented for jasmonate signaling, which depends on proteasome-mediated degradation of the JAZ repressors [3,4]. In the present work, we also obtain clear evidence that MAPKs promote degradation of *PtiZFP1* through the 26S proteasome. Our work suggests that *PtiZFP1* and JAZs are part of *Melampsora* specific hormone-related responses, and by correlation as putative transcriptional repressors, participate in the regulation of the transcriptional responses downstream of these stress hormones. We hypothesize that the observed gene induction play a negative feedback loop needed to replace *PtiZFP1* and JAZ proteins that were degraded following through the 26S proteasome. Newly synthesized repressor proteins could contribute to defense attenuation and therefore prevent a runaway response.

<figure><img src="image_4.png" /><figcaption>**Figure 1(abstract I12)** Proposed model of *PtiZFP1* function in the plant defense response.</figcaption></figure>

<footnote n="1">¹Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, Québec, Canada, G1V 4C7 and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114, USA;</footnote>

<footnote n="2">²Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, Québec, Canada, G1V 4C7; ³¹Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Québec, Québec, Canada, G1V 4C7; ⁴Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114, USA</footnote>
Based on our data, we propose a model (Figure 1) where two important families of transcription factors play a predominant role in poplar defense response to the biotrophic fungus *Melampsora*. The activation of poplar MAPKs would lead to *PtiZFP1* degradation and as a result to transcriptional activation of defense-related genes. In parallel we also observed that several genes involved in the JA response (including JAZ) are induced following *Melampsora* infection. This model highlights the importance of the 26S proteasome in regulating protein pools of transcriptional repressor. For future studies we intend to uncover which *cis* elements and target genes are recognized by *PtiZFP1* and perform functional approaches to uncover JAZ interacting proteins in poplar.

During normal conditions, direct transcriptional repressors such as *PtiZFP1* inhibit the expression of stress-related genes. Following stress perception, a MAPK signaling cascade is activated leading to phosphorylation of *PtiZFP1*. Phosphorylation targets *PtiZFP1* for degradation via the 26S proteasome (P26S), thus relieving repression of defense genes. Defense signaling in turn activates *PtiZFP1* gene transcription in order to replenish normal *PtiZFP1* protein levels and complete a regulatory cycle necessary to attenuate the defense response. A similar regulatory cycle exists for jasmonic acid (JA) signaling. JA biosynthesis is induced by stress which then promotes proteasome-mediated degradation of JAZ proteins. These indirect transcriptional repressors sequester the bHLH MYC2, thus inhibiting expression of defense genes under non-stressed conditions. Upon release, MYC2 ensures both positive and negative feedback loops by activating *LOX3* and JAZ genes respectively. Confirmed and hypothetical pathways are presented in bold and dashed lines respectively.

## References

1. Duplessis S, Major J, Martin F, Séguin A: Poplar and pathogen interactions: insights from *Populus* genome-wide analyses of resistance and defense gene families and gene expression profiling. *Crit Rev Plant Sci* 2009, 28:309-334.

2. Boyle B, Levée V, Hamel L-P, Nicole M-C, Séguin A: Molecular and histochemical characterisation of two distinct poplar Melampsora leaf rust pathosystems. *Plant Biol* 2010, 12:364-376.

3. Chung HS, Niu Y, Browse J, Howe GA: Top hits in contemporary JAZ: An update on jasmonate signaling. *Phytochemistry* 2009, 70:1547-1559.

4. Memelink J: Regulation of gene expression by jasmonate hormones. *Phytochemistry* 2009, 70:1560-1570.

## I13
### Adaptation of forest trees to climate change

Christophe Plomion¹*, Ivan Scotti², Sylvain Delzon¹, Jean-Marc Gion³
¹INRA, UMR BIOGECO, France; ²INRA, UMR ECOFOG, France; ³CIRAD, UMR BIOGECO, France
E-mail: plomion@pierronot.inra.fr
*BMC Proceedings* 2011, **5**(Suppl 7):l13

There is an urgent need to determine the adaptive potential of forest trees given their importance in ecosystem functioning and the associated ecological and economic services they provide. Indeed, underestimating rapid adaptation could lead to unnecessary recommendations such as the use of non-native (and perhaps non-adapted) genetic material for reforestation. Overestimating adaptive potential could have detrimental consequences if population decline massively and prove enable to regenerate.

A critical step to study individual's and specie's responses to variation in climate is to examine the basis of adaptation (at the phenotypic and molecular levels) under natural conditions and across multiple years. In this context, these sessile and long lived species have emerged as a model system for studying adaptation into the wild. Whether forest trees will be able to adapt in situ fast enough to outstrip the rate of such modifications relative to their longevity is still largely debated and remains an open question.

The objectives of this presentation are three fold:

i/ provide an overview of the concept of ADAPTATION: a process whereby a genotype or a population becomes better suited to its habitat. Adaptation to climate change can occur through phenotypic plasticity (a mechanism by which individuals can withstand large environmental fluctuation without genetic change) and evolution (i.e. response to natural selection pressure when the level of standing genetic diversity or diversity introduced by gene flow permits it).

ii/ provide some clues (from historical and contemporary data) on how closely can forest tree adaptation be expected to track climate change as rapid as envisioned for the future. They have high phenotypic plasticity

which allows them to tolerate wide environmental variations during their lifetime. Besides, there are some evidence against the idea that rapid environmental changes overwhelms evolutionary processes preventing adaptation to local environments.

iii/ review how genes that matter for adaptation can be detected using 'omics technology in combination with population and quantitative genetics approaches.

# S6. WOOD FORMATION

## I14
### A systems biology approach to understanding the regulation of lignin biosynthesis

Vincent Chiang
Forest Biotechnology Group, North Carolina State University, USA
E-mail: vincent_chiang@ncsu.edu
*BMC Proceedings* 2011, **5**(Suppl 7):l14

Lignin is a complex phenolic structural component of the secondary cell walls of all vascular plants. It is an irreversible end point of a major metabolic pathway in plant secondary metabolism. Lignin is fundamental to the adaptation of plants to land, the evolution of vascular transport and the resistance of plants to pests and pathogens. Lignin is a major barrier to the utilization of biomass for energy, for papermaking, and for forage digestibility due to the interaction of lignin with cellulosics in the plant cell secondary wall.

The past research on lignin biosynthesis is substantial, creating one of a few opportunities in higher plants to integrate genomics, proteomics, biochemistry, chemistry and modeling to develop a comprehensive understanding of biosynthesis and structure of a major component of morphology and adaptation. We are conducting a systems biology study on regulation of lignin biosynthesis in wood formation. We seek to build models to quantitatively illustrate how the entire pathway is organized and regulated and to reveal regulatory and metabolic flux control mechanisms, leading to lignin quantity and structures. We use the model woody plant, *Populus trichocarpa* (Nisqually-1), and the systems approach including advanced quantitative methods of genomics, proteomics, metabolomics, biochemistry and structural chemistry, to provide a comprehensive analysis of the regulation of lignin biosynthesis. A perturbation strategy is used to systematically knock down the expression of all pathway and regulatory genes known to be involved in lignin biosynthesis during wood formation, and the effects on lignin biosynthesis (gene transcripts, proteins, metabolites, quantity and structures) analyzed using advanced genomic methods available. This information forms the foundation of statistics-based mechanistic modeling and lignin quantity/structural predictions for a quantitative model of lignin biosynthesis. Details of systems data generation, data analyses and model development will be presented. Substantial data have been generated for gene-specific (amiRNA & RNAi) transgenic *P. trichocarpa*, enzyme kinetics, protein regulation and numerical modeling, and stable-isotope-dilution based absolute quantitation of proteins and of metabolites. These results will be discussed. Our long term goal is a predictive model of lignin biosynthesis and quantity/structure for greater understanding of the plant response to environmental stress and for more precise strategies to improve plant productivity and the production of energy, biomaterials and food.

## I15
### Ethylene signaling via Ethylene Response Factors (ERFs) modifies wood development in hybrid aspen

Judith Felten¹, Jorma Vahala², Jonathan Love¹, András Gorzsás¹, Lorenz Gerber¹, Manoj Kumar¹, Jaakko Kangasjärvi², Björn Sundberg¹*
¹Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden; ²Department of Biosciences, Division of Plant Biology, University of Helsinki, PO Box 65, FI-00014 Helsinki, Finland
E-mail: Bjorn.Sundberg@genfys.slu.se
*BMC Proceedings* 2011, **5**(Suppl 7):l15

**Background:** The phytohormone ethylene (ET) has the potential to regulate secondary growth of plants and wood formation in trees.
Application of exogenous ethylene or its *in planta* precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to wood forming tissues of hybrid aspen (*Populus tremula* x *Populus tremuloides*) enhances xylem growth [1]. In the same study it was demonstrated that stimulation of enhanced xylem formation (tension wood, TW) at the upper side of leaning stems is mediated by endogenous ET. The production of endogenous ET in TW forming tissues is further supported by the increase of ACC oxidase gene transcript and enzyme activity on the TW side [2].

The ET perception and signal transmission cascade in Arabidopsis has been linked to the transcriptional activation of Ethylene Response Factors (ERFs) [3,4]. As transcription factors, ERFs regulate the expression of various specific downstream target genes by binding to cis-elements in their promoters [5]. We hypothesize that ERFs participate in xylem development through ethylene signaling and that they are involved in ET responses during TW formation.

**Results and conclusions:** We identified 169 *ERF* genes in the *Populus trichocarpa* genome versions 2.0 and 2.2 using regular expression method and pfam search for the ERF domain. These ERFs grouped into 11 distinct groups, similar to ERFs identified in Arabidopsis and rice [6]. Using qPCR we showed that a majority of the *ERF* transcripts were detectable in stem tissues of in vitro or greenhouse grown hybrid aspen. The responsiveness of all *ERF*s to short term ACC treatments (10h, in vitro plants) and to short or long term ethylene treatments (24 or 2 weeks, greenhouse grown plants), was assessed by qPCR. Most of the *ERF*s responded to at least one of the treatments, mostly by increased transcript accumulation. We identified *ERF*s that were specifically induced within the early ACC/ET- or the late ET-response. In addition, some *ERF*s showed prolonged induction up to 2 weeks of ET treatment. These different transcript patterns indicate that different *ERF*s may be involved in distinct, temporally distinguished processes during the ACC/ET-induced secondary xylem growth response.

According to their expression and capacity to be induced by ET or ACC, we selected 26 *ERF* candidates and investigated whether those were responsive to endogenous ET-signals in leaned stems during TW formation. We compared the induction of those 26 candidate *ERF*s in TW with *ERF* accumulation during long-term (2 weeks) ET treatment. Interestingly, a significant overlap of *ERF* induction in both conditions was found. From the 20 *ERF*s that were induced after long-term ET treatment, 16 had an increased transcript abundance during tension wood formation. This indicates that on an *ERF* transcript basis, tension wood formation is largely comparable to a long-term ET treatment.

Based on the transcript data, twenty ERFs were selected for overexpression in hybrid aspen cambium/xylem under the *pLMX5* promoter [1]. Successful overexpression of the selected *ERF*s in transgenic plants was confirmed and two to seven lines of each overexpressed ERF were phenotyped in a greenhouse trial. In general, *ERF* overexpression caused only mild alterations of overall plant stature (height and radial growth). Only overexpression of one *ERF* candidate led to a severe dwarf phenotype with thin stems, reduced fiber and vessel size, reduced height growth and smaller leaves. The absence of any striking phenotypes in all other plants suggests that other regulators in addition to the overexpressed *ERF* s may be necessary to mimic the enhanced growth response observed during ET/ACC/TW mediated stimulation of secondary xylem growth.

A Fourier-Transformed Infrared spectroscopic and Pyrolysis GC-MS based screening of five lines for each of the 20 overexpressed ERFs revealed that five *ERF* s led to changes in cell wall composition in xylem tissues when overexpressed. This suggests that these ERFs have the ability to modify cell wall composition in wood forming tissues and may regulate the expression of cell-wall biosynthesis genes. This hypothesis is now under further investigation.

References

1. Love J, Björklund S, Yahala J, Hertzberg M, Kangasjärvi J, Sundberg B: Ethylene is an endogenous stimulator of cell division in the cambial meristem of *Populus*. *Proc Natl Acad Sci U S A* 2009, 106(14):5984-5989.

2. Andersson-Gunnerås S, Hellgren JM, Björklund S, Regan S, Moritz T. Sundberg B: Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood. *Plant J* 2003, 34(3):339-349.

3. Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR: Activation of the ethylene gas response pathway in Arabidopsis by the

nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. *Cell* 1997, 89(7):1133-1144.

4. Solano R, Stepanova A, Chao Q, Ecker JR: Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-SENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. *Gene Dev* 1998, 12(23):3703-3714.

5. Ohme-Takagi M, Shinshi H: Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. *Plant Cell* 1995, 7(2):173-182.

6. Nakano T, Suzuki K, Fujimura T, Shinshi H: Genome-wide analysis of the ERF gene family in Arabidopsis and rice. *Plant Physiol* 2006, 140(2):411-432.

I16

How sweet it is – making the most of carbohydrate metabolism

Shawn Mansfield

Department of Wood Science, University of British Columbia, Vancouver, Canada

E-mail: shawn.mansfield@ubc.ca

BMC Proceedings 2011, 5(Suppl 7):I16

Like all plants, trees constantly monitor endogenous and environmental cues, and use this information to make adjustments in resource allocation, both to maximize resource acquisition and to minimize exposure to deleterious phenomena. To accomplish this, trees possess mechanisms that integrate and interpret the information provided by internal and environmental signals. Ultimately, these phenomena are controlled at the level of gene expression, which consequently translates into the accumulation of a variety of soluble metabolites, including carbohydrates. Trees are relatively “plastic” in their ability to allocate resources, and such plasticity has an adaptive significance in that it allows trees to (i) match resource allocation with resource acquisition, (ii) acquire new resources more effectively, and in some instances (iii) avoid adverse conditions. The inherent plasticity in resource acquisition can have a profound effect not only on the development and physiology of trees, but also on the industrial harvesting and utilization of the terminal lignocellulosic resource.

Photosynthetic carbon capture by trees represents a major sink for atmospheric CO₂, ultimately terminating in the synthesis of a secondary plant cell wall – a complex matrix of polysaccharides intricately linked to lignin. Therefore, engineering plant where resource allocation is directed to vegetative biomass and fibre properties should have an effect on the plant cell wall traits. As such we have targeted the biosynthetic processes governing sucrose metabolism by mis-regulation of key enzymes associated with the catabolism and anabolism of sucrose in attempts cellulose production – both quantity and quality. This paper will discuss the results of these efforts, and illustrate the altered performance of such plants for industrial use.

S7. PROPAGATION AND IN VITRO
TECHNOLOGIES

I17

From genes towards products and the significance of gene delivery

Magnus Hertzberg

SweTree Technologies AB

E-mail: Magnus.Hertzberg@SweTree.com

BMC Proceedings 2011, 5(Suppl 7):I17

SweTree Technologies is a plant and forest biotechnology company providing products and technologies to improve the productivity and performance properties of plants, wood and fiber for forestry and fiber related industries.

In the area of Biotech Trees, we have been testing and developing genes and biotech trees. Since 2002 we have successfully screened well over one thousand genes in our gene testing program. The program has been mainly directed towards increased growth, improved wood properties and stress tolerance. The current outcome of this program will be presented. The primary gene developing program is performed in hybrid aspen for which we have a very efficient transformation protocol.
However for practical tree biotechnology the genes and DNA constructs have to be transformed into the best commercial clones. Therefore we have been setting up efficient transformation protocols for both *Eucalyptus* and *Populus* elite clones. Another crucial part of a practical tree biotechnology program is to test the GM trees with commercially interesting genes under field conditions. We have initiated field trials and plan to have over 35 different types of transgenic trees in field trials by the end of this year. These field experiments are performed by SweTree or in collaboration with academic partners. The importance and requirements for elite clone transformation and field trials will be discussed in the presentation.

## I18

**Hybrids and mini-cutting: a powerful combination that has revolutionized the *Eucalyptus* clonal forestry**

Teotónio Francisco Assis

ASSISTECH LTDA

E-mail: assisteo@terra.com.br

BMC Proceedings 2011, 5(Suppl 7):I18

**Background:** Eucalypt canker disease (*Chrysoporthe cubensis*) was the main driver of the concept of eucalypt clonal forestry in Brazil. Initially clonal forestry was based on outstanding spontaneous hybrids resistant to canker disease. Since the recognition that cloning superior inter-specific hybrids could be an important general strategy, several crosses were made for many different purposes, especially pulp & paper, charcoal and more recently veneer and solid wood. This strategy has been responsible for a significant advance in the forest productivity of *Eucalyptus*. The underlying foundation for this strategy has been the exploitation of heterosis observed in most inter-specific crosses.

Due to its importance, hybridization and cloning currently constitute a key component of almost all forest-based industrial plantations in Brazil. Significant progress has taken place in the last few years with the different techniques used to carry out controlled crosses and cloning.

**Controlled crosses:** The fact that *Eucalyptus* flowers are hermaphrodite and protandrous makes the traditional method of controlled crosses difficult to perform. This method is based on the exploitation of protandry, which involves emasculation and isolation, prior to pollination. It needs several visits resulting in a technique of low operational yield.

After discovering that different treatments in stile and stigma during anthesis could immediately induce flower receptivity [3], it was developed the OSP (One Stop Pollination) technique [4]. Later it was found that this receptiveness could be achieved just before anthesis [5]. Combining these two discoveries a new technique was developed [2], called AIP (Artificially Induced Protogyny). This technique consists of the artificial "transformation" of protandry into protogyny, obtained by cutting the top of the floral bud operculum together with the upper third of the stile. This is done during the pre-anthesis stage, i.e., when the flower is still closed. Pollen can be applied immediately after induction. Additionally the concept of indoor breeding orchards was developed, precluding the need to isolate individual flowers, umbels or branches, enabling the isolation of whole plants in a collective way.

The combined use of these technologies (AIP and indoor breeding orchards) currently allows large scale controlled crosses, and enables the production of highly superior full-sib families, a task formerly considered technically and economically difficult in *Eucalyptus*. Operational productivity improved from 35 to 400 pollinated flowers/person/hour.

**Cloning techniques:** Cloning techniques have been significantly enhanced in the last years. The first major leap occurred in 1992, when micro-cutting was invented [1] and later mini-cutting. The micro-cutting is a rooting method, where the propagules are obtained exclusively from shoot apices, originated from micro-propagated plants. Mini-cuttings, on the other hand, come from axillary sprouts of rooted stem-cuttings. After the establishment of the mini-clonal hedges, the two techniques are identical, varying only in the origin of the initial source of propagules. Micro-cutting and mini-cutting are the most modern concepts for large scale cloning of *Eucalyptus* species. They are implemented using hydroponic systems such as sand bed with drip irrigation or intermittent

flooding with very specific nutritional parameters that allow commercial scale production of sprouts. Although significant progress has been made in these robust and functional systems, new approaches to clone *Eucalyptus* species are opening important opportunities to improve operational results and also contemplating recalcitrant species in the general hybridization & cloning strategy. One of these new approaches is cloning selected trees from lignotuber tissue. Tested originally at Arcelor Mittal Bioflorestas, cloning from lignotubers constitute a new advance to be soon incorporated in the operational procedures. Another promising approach is to cover mini-stumps with mini-tunnels. Preliminary results of this method at Aperan Bioenergia have shown that productivity, rooting rate and root quality are improved, reducing the formation of callus.

After more than thirty years of experience on deriving clones from inter-specific hybrids, this general strategy, together with the evolution of silvicultural practices, have been the major drivers of the progress experienced in forest productivity in Brazil. The evolution of pulp productivity obtained by cloning superior hybrids has been impressive. While in 1970 the average MAI-Cellulose was 6 ton/ha/year it now reaches 12 ton/ha/year. New projections now work with the possibility of achieving a potential productivity of about 16 ton/ha/year by 2015. One of the key elements for this further jump in productivity is the rapidly increasing use *E. globulus* in hybrid breeding programs. *E. globulus* has superior wood qualities such as low lignin content and high S/G ratio (one of the highest in woody plants) that result in easier cooking, low specific consumption (high wood density and pulp yield) and good bleaching ability. The use of adapted species and clones in crosses with *E. globulus* is yielding a new breed of elite hybrid clones for subtropical and tropical areas.

## References

1. Assis T, Rosa OP, Gonçalves SL: Propagação clonal de *Eucalyptus* por microestaquia. Congresso Florestal Estadual, 7 Nova Prata, Anais Santa Maria: UFSM 1992, 824-837.

2. Assis TF, Warburton P, Harwood C: Artificially induced protogyny: an advance in the controlled pollination of *Eucalyptus*. Australian Forestry 2005, 68:27-33.

3. Cauvin B: Pistil treatments for improved fertility in hybridization of *E. gunnii* (Hook). Sexual reproduction in higher plants University of Sienna, Italy: R Cresti, P Gore and E Pacine 1988, 321-325.

4. Harbard JL, Griffin R, Espejo JE: Mass controlled pollination of *Eucalyptus*. Can J For Res 1999, 29:1457-1463.

5. Trindade H, Boavida LC, Borralho N, Feijó JA: Successful fertilization and seed set from pollination on immature non-dehisced flowers of *Eucalyptus globulus*. Annals of Botany 2001, 87:469-475.

## I19

**Opening the gateway to enhanced *Eucalyptus***

Ziv Shani

Futuragene Ltd., Rechovot, Israel

E-mail: ziv@futuragene.com

BMC Proceedings 2011, 5(Suppl 7):I19

*Eucalyptus* is the second most planted tree on earth. Despite this ranking, its mature breeding programs, clonal propagation and deployment and short rotation time make it a leading target for biotechnology improvement. The recently completed *eucalyptus* genome project provides an enormous opportunity for understanding and developing new traits for eucalyptus forestry. The main obstacle for widespread trait enhancement remains the recalcitrance of commercial eucalyptus clones to transformation and thus the lack of a suitable prototyping system for this species. We believe that the future success of trait development in *eucalyptus* will be enhanced by close collaboration between academic centers and industrial partners. Some of FuturaGene's models for academic collaboration will be described in this presentation.

An example of such potential collaborations may involve BRASUZ1, which constituted the germplasm base for the eucalyptus genome project. This key clone was donated by Suzano Pulp and Paper, FuturaGene's parent, to the genome project. We will present our progress with the transformation protocol for BRASUZ1, which could provide a platform for collaborations. We will also present our perspective on the development of improved transgenic eucalyptus, and an outline for such development programs.
# S8. GENOMIC, PROTEOMIC AND METABOLOMIC TECHNOLOGIES

## I20

**The *Eucalyptus grandis* Genome Project: Genome and transcriptome resources for comparative analysis of woody plant biology**

Alexander Myburg¹*, Dario Grattapaglia², Gerald Tuskan³, Jerry Jenkins⁴, Jeremy Schmutz⁶, Eshchar Mizrachi¹, Charles Hefer², Georgios Pappas², Lieven Sterck⁶, Yves Van De Peer⁶, Richard Hayes⁷, Daniel Rokhsar⁷
¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; ²Plant Genetics Laboratory, EMBRAPA Genetic Resources and Biotechnology - EPQB, 70770-910 Brazilia, Brazil; ³Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA; ⁴HudsonAlpha Genome Sequencing Center, 601 Genome Way, Huntsville, AL 35806, USA; ⁵Bioinformatics and Computational Biology Unit, Department of Biochemistry, University of Pretoria, Pretoria, South Africa; ⁶Department of Plant Systems Biology, VIB, Ghent University, Technologiepark 927, 9052 Gent, Belgium; ⁷Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA

E-mail: zander.myburg@fabi.up.ac.za

BMC Proceedings 2011, 5(Suppl 7):I20

**Background:** The International Year of Forests - 2011 [http://www.un.org/en/events/iyof2011/] will be a milestone for forest tree genomics. The draft genome sequence of *Eucalyptus grandis* was released in January 2011 in the USA (Phytozome [http://www.phytozome.net/]) and in Belgium (BOGAS, [http://bioinformatics.psb.ugent.be/webtools/bogas/]). The genome sequencing was funded by the US Department of Energy (DOE) and performed at the DOE Joint Genome Institute (JGI) in collaboration with members of the *Eucalyptus* Genome Network (EUCAGEN, [http://www.eucagen.org]) who contributed genetic materials, linkage maps, EST resources and bioinformatics support. The *E. grandis* genome together with that of *Populus trichocarpa* [1] and other woody plant genomes recently completed (e.g. *Vitis*, *Cacao*, *Prunus*, *Citrus* and *Malus*) will provide excellent opportunities for comparative studies of the unique biology of woody plants. Eucalypts are currently the most widely grown hardwood fibre crop in the world and eucalypt breeding programs will benefit greatly from the new genomic resources. The reference genome sequence of *Eucalyptus*, a foundation tree genus in Australia comprising more than 70% of the native forest estate, will also offer important benefits for ecological and evolutionary biology studies. We report the sequencing, assembly and annotation of the *E. grandis* genome.

**Genome sequencing and assembly:** Whole-genome (8X) shotgun sequencing was performed for a partially inbred (S1), 17-year-old tree of *E. grandis* (est. genome size 640 Mbp, *n* = 11), BRASUZ1 (Suzano, Brazil). A total of 7.7 million Sanger reads (5.4 Gbp) were produced from plasmid, fosmid and BAC libraries. An inbred genotype was selected to circumvent perceived problems with the assembly of a highly heterozygous eucalypt genome. However, microsatellite genotyping showed that BRASUZ1 was much less homozygous than expected, with large parts of the genome remaining heterozygous presumably due to viability selection. This finding was confirmed during the assembly of the S1 genome - approximately 25% of the assembly occurred in two haplotypes of 3-4X coverage, while the remainder of the genome assembled into a single haplotype of 6-7X coverage. Linkage maps with over 2400 DArT and microsatellite markers were subsequently used as a framework for the assembly of 11 large chromosome scaffolds. The chromosome scaffolds contained 88% (605 Mbp) of the draft assembly, with the remainder of the assembly sequence (85 Mbp) in 4941 smaller scaffolds. Based on similarity searches with 1.6 million ESTs from BRASUZ1, it was estimated that 96% of expressed gene loci were included in the 11 chromosome assemblies.

**Genome annotation:** Genome annotation was performed in parallel at the JGI and at the University of Ghent. Both annotation teams used *ab initio* and homology-based annotation approaches supported by over 4 million 454-FLX-Titanium ESTs produced by the JGI, as well as Sanger, 454 and Illumina EST data provided by collaborators. The two annotations revealed that the 11 chromosome scaffolds contain more than 90% of the predicted protein-coding loci (total 44,974 - JGI, 47,974 - UGen). More than 70% of the predicted genes had EST support and 9,961 (18%) alternatively spliced transcripts were detected. The two annotations are

being compared and a joint annotation may be released for the main *E. grandis* genome paper.

**Genome duplication:** The *Vitis* genome [2], representing an early diverging Rosid lineage (Vitales), was found to contain the ancient hexaploidization event shared by Rosids and Asterids, but none of the more recent genome duplications found in the Rosid lineages represented by *Arabidopsis* and *Populus*. A preliminary analysis performed at UGen of genome duplication in *E. grandis* (representing the Rosid order Myrtales) suggested that the *Eucalyptus* genome most likely contains one more recent duplication event, in addition to the paleohexaploidy event.

**Genome resequencing:** *E. globulus* is a temperate eucalypt with superior wood properties compared to *E. grandis* and is viewed as the premier eucalypt species for pulping. The two species occur in different sections (*Maidenaria* and *Latoangulatae*) of the subgenus Symphyomyrtus and their genome sizes differ substantially (*E. globulus* - 530 Mbp, *E. grandis* - 640 Mbp, [3]). The JGI has performed genome-wide resequencing (>30X Illumina PE) of an *E. globulus* clone (X46, Forestal Mininco, Chile). Approximately 75% of the *E. globulus* Illumina reads mapped to the *E. grandis* reference genome and sequence analysis in these regions revealed an average sequence divergence of 1.5% between the two genomes. Other eucalypt genomes currently being resequenced by collaborators will generate a valuable resource for studies of eucalypt genome evolution.

**Transcriptome resources:** The large amount of transcriptome sequence data was produced the project includes 1.9 million xylem and leaf ESTs (454 reads) from BRASUZ1 and 2.1 million 454 reads from *E. globulus* (X46) xylem and leaf tissues. Together with other large 454 datasets (e.g. [4]) and Illumina mRNA-Seq data [5] produced by collaborators, the *Eucalyptus* research community now have access to excellent transcriptome resources some of which are already available in integrated genome and transcriptome browsers (Eucspresso [http://eucspresso.bi.up.ac.za/]).

**Conclusions:** The *E. grandis* genome sequence will be the first reference for the Rosid order Myrtales and will be informative for comparative genomic studies within the Eudicots. It will also deliver powerful tools for the application of genomics in eucalypt breeding programs.

## References

1. Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, et al: The genome of black cottonwood, *Populus trichocarpa* (Torr. & Gray). *Science* 2006, 313:1596-1604.

2. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. *Nature* 2007, 449:463-467.

3. Grattapaglia D, Bradshaw HD: Nuclear DNA content of commercially important *Eucalyptus* species and hybrids. *Can J For Res* 1994, 24:1074-1078.

4. Novaes E, Drost DR, Farmerie WG, Pappas GJ Jr., Grattapaglia D, Sederoff RR, Kirst M: High-throughput gene and SNP discovery in *Eucalyptus grandis*, an uncharacterized genome. *BMC Genomics* 2008, 9:312.

5. Mizrachi E, Hefer CA, Ranik M, Joubert F, Myburg AA: De novo assembled expressed gene catalog of a fast-growing *Eucalyptus* tree produced by Illumina mRNA-Seq. *BMC Genomics* 2010, 11:681.

## I21

**Populus resequencing: towards genome-wide association studies**

Gerald Tuskan¹*, Gancho Slavov², Steve DiFazio², Wellington Muchero¹, Ranjan Pryia¹, Wendy Schackwitz³, Joel Martin³, Daniel Rokhsar³, Robert Sykes⁴, Mark Davis⁵, Michael Studer⁵, Charles Wyman⁵

¹Oak Ridge National Laboratory, USA; ²West Virginia University, USA; ³Joint Genome Institute, USA; ⁴National Renewable Energy Laboratory, USA;

⁵University of California Riverside, USA

E-mail: gio@ornl.gov

BMC Proceedings 2011, 5(Suppl 7):I21

Genome-wide association studies (GWAS) have been used to identify regions of the genome related to various phenotypes in humans, corn, rice and cattle. Successful application of this approach to bioenergy crops such as *Populus* requires 1) an appropriate mapping population, 2) high-quality
phenotypic data and 3) informative genotypic data. With the goal of reducing the recalcitrance of lignocellulosic biomass for economic production of biofuels and understanding basic mechanisms of cell wall formation in Populus we established 4 clonally replicated common gardens experiments each with 1100 native P. trichocarpa genotypes collected from along the northwest coast of the U.S. and Canada. A high-throughput phenotyping pipeline was developed to measure cell wall chemistry, pretreatment response and enzymatic sugar release. Initially 18 genotypes were resequenced to an average 30X depth in order to design a SNP array to test for statistical association using MMAX and PCA methods of testing among ca. 2500 candidate genes. Genetic structure and linkage disequilibrium (LD) was assessed using SSR and SNP markers. Outlying genotypes were excluded from the analyses and estimates of LD were used to design the bead array. Candidate genes were selected based on QTL intervals, expression profiling within developing xylem and expert opinion. MMAX and PCA results revealed similar significant associations for all measured phenotypes and several SNPs within the candidate gene set explain a relatively high degree of the phenotypic variance. As a result, resequencing has continued in order to conduct GWAS in Populus; the complete set of 1100 genotypes will be complete in 2012.

## I22

### Eucalyptus research in the post-genome era

Georgios Pappas¹, Sergio de Alencar, Orzenil B Silva-Junior, Roberto C Togawa, Marilia CR Pappas, Dario Grattapaglia
EMBRAPA Genetic Resources and Biotechnology - EPqB Flnal W5 Norte, Brazilia DF 70770-917, Brazil
E-mail: gpappas@cenargen.embrapa.br
BMC Proceedings 2011, **5**(Suppl 7):I22

With the efforts of the Joint Genome Institute (JGI) through the EUCAGEN Eucalyptus Genome Network reaching the final release of a *Eucalyptus grandis* reference genome (BRASUZ1), it is anticipated that this accomplishment will profoundly shape the future research of this global tree genus [1]. One of the first steps toward this end has been the refinement of the genome annotation. Robust models have been generated for protein-coding genes. Here we report the annotation of other pivotal genome constituents, transposable elements (TE) and micro RNA genes. TEs are not only the most dominant elements but are also major drivers of genome plasticity. Several bioinformatic strategies were employed to perform "de novo" and homology based prediction of repetitive elements [2]in the current genome release (version 1.0). A total of 53 distinct TE families could be identified, with the retrotransposon super-family being widely over-represented, as observed for the majority of plant genomes sequenced.

Micro RNAs (miRNA), key players in post-transcriptional gene regulation, were annotated using a combination of massively parallel sequencing of small RNA libraries and a genome-wide computational screening to ascertain a compatible secondary structure of the precursor. Both experimental and "in silico" evidences enabled the annotation of 206 distinct miRNA loci comprising 36 different mir gene families including several miRNA isoforms. The blueprint provided by a high-quality reference genome, both in terms of sequence completeness and annotation, will leverage efforts to better characterize intra- and inter-specific sequence variation underlying the marked phenotypic differences among the hundreds of species comprising this genus.

Recent advances of DNA sequencing technologies permit a comprehensive interrogation of several other individuals at a fraction of the cost. In this context, we have used Illumina (2x75bp) short read sequencing data of *E. globulus* clone X46 generated by JGI and made available through the EUCAGEN network to carry out two comparative genomics experiments. From 40X raw sequence data provided it was possible to use an equivalent of 20X coverage (~12Gbp). We first attempted to perform a "de novo" assembly using VELVET [3]. A total of 161,000 contigs were obtained the largest one sizing at ~3,5kb. In spite of the easy access and low cost of next generation sequencing technologies, these results suggest that even for relatively small forest tree genomes, current technical and computational limitations preclude comprehensive assembly, likely due to the ubiquitous occurrence of repetitive elements in such genomes. Nevertheless when we mapped the *E. globulus* sequencing data against the BRASUZ1 reference genome, 55% of the reads could be mapped with high confidence. From these, approximately 800,000 high quality single

nucleotide polymorphisms (SNPs) could be identified clearly showing the key role that the reference genome will have for future genomic undertakings. The sheer number of molecular markers discovered in this experiment not only fosters more powerful studies on the evolutionary history and population genomics of eucalypts, but also inaugurates a new era in molecular breeding of species of this genus, providing genome-wide coverage for genomic selection and association studies.

### References

1. Grattapaglia D, Kirst M: Eucalyptus applied genomics: from gene sequences to breeding tools. *New Phytologist* 2008, 179(4):911-929.

2. Flutre T, Duprat E, Feuillet C, Quesneville H: Considering Transposable Element Diversification in De Novo Annotation Approaches. *Plos One* 2011, 6(1).

3. Zerbino DR, Birney E: Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. *Genome Research* 2008, 18(5):821-829.

# S9. BIOSAFETY, CERTIFICATION AND ECONOMICS OF TREE BIOTECHNOLOGY

## I23

### Science, society and biosafety of a field trial with transgenic biofuel poplars

Rebecca Van Acker¹,², Véronique Storme¹,², Geert Goeminne¹,², Bart Ivens¹,², René Custers³, Dirk Aerts⁴, Wim Soetaert⁴, John Ralph⁵, Nicholas Santoro⁶, Jean-Charles Lepleje, Gilles Pilateje, Wout Boerjan¹,²*

¹Department of Plant Systems Biology, VIB, Gent, Belgium; ²Department of Plant Biotechnology and Genetics, UGent, Gent, Belgium; ³VIB Headquarters, Gent, Belgium; ⁴Department of Biochemical and Microbiol Technology, UGent, Gent, Belgium; ⁵D.O.E. Great Lakes Bioenergy Research Center, U. of Wisconsin, Madison, WI, USA; ⁶D.O.E. Great Lakes Bioenergy Research Center, Michigan State U., East Lancing, Michigan, USA; ⁷INRA Centre d'Orléans, Unité Amélioration, Génétique, et Physiologie Forestières, Orleans cedex 2, France

E-mail: woboe@psb.vib-ugent.be

BMC Proceedings 2011, **5**(Suppl 7):I23

**Background:** Global warming, environmental disasters, and increasing oil prices have catalyzed a worldwide trend to use plant biomass as a renewable source for liquid biofuels and bio-based materials. Plant biomass can be processed into fermentable sugars by enzymatic depolymerization of the cell wall polysaccharides, followed by fermentation. However, the presence of lignin in the cell wall constitutes a major recalcitrance factor because it limits the accessibility of polysaccharidases to the cellulose microfibrils. To overcome this hurdle, plant biomass is pretreated in a costly and energy-requiring process. One approach to overcome the recalcitrance problem is to engineer lignin amount or alter its composition to make lignin more susceptible to chemical degradation [1]. Cinnamoyl-CoA reductase (CCR), the enzyme that converts feruloyl-CoA into coniferaldehyde, is considered the first enzyme in the monolignol-specific branch of the phenylpropanoid pathway. Poplar trees down-regulated in CCR have been produced in the early nineties and planted in a field trial in France to produce sufficient wood for small scale chemical pulping tests [2]. These trees had 20% lower lignin levels and relatively more cellulose per gram of wood [2]. Given that lignin is one of the main limiting factors limiting the conversion of plant biomass into fermentable sugars, and that poplar is considered as a promising second generation biofuel crop, we have re-grown these trees in the greenhouse and in the field, and evaluated wood produced from these trees by saccharification experiments.

**Methods:** Cinnamoyl-CoA reductase (CCR) expression was down-regulated in poplar by sense and antisense strategies [2]. Transgenic trees were evaluated for lignin amount and composition [2] and for sugar release by saccharification assays [3]. After obtaining permission from the regulatory authorities, two transgenic lines were planted in a field trial in Belgium and two in a field trial in France, both under short rotation coppice culture to maximize biomass production. Wood was saccharified with and without acid pretreatment.

**Results:** Wood from CCR down-regulated trees that were grown in the greenhouse was saccharified with and without acid pretreatment. Interestingly, wood from the transgenic lines released more glucose in saccharification assays performed without pretreatment than wood from
wild type trees when saccharified with a pretreatment, demonstrating the improved processing of the cell wall polysaccharides by the enzyme cocktail. These promising results prompted us to ask permission for a field trial to verify whether wood from these trees, when grown in an open field, would still be more easily saccharified. After all, when trees are grown in the greenhouse, they do not experience wind, rain and seasons, and they need to be stacked to grow upward. After a one year long calvary to obtain regulatory permission for the Belgian field trial [4], the trees were grown under short rotation coppice culture in replicated clonal blocks in a small plot of a total of ~ 500 trees. The plot has a fence and is surveilled by cameras. In Spring, the trees are regularly monitored for flowering as they are not allowed to flower. Flowering is not expected because the coppice cycles are as short as 3 years. The possible effects of the low lignin trait on rust infection and insects is being monitored, but it is not expected that these effects will be larger than those associated with conventionally bred poplar hybrids or will be specific to the transgenic trees taking the large genetic diversity of the wild poplar germplasm into account. Activist groups that are concerned that these trees pose different risks than conventionally bred hybrids or pose a risk for human health are invited for a discussion with scientists and the biosafety manager at VIB.

After one year of growth in the field, trees were coppiced to allow the development of multiple shoots in the next spring. The basal 20 cm part of the stem was debarked. Wood harvested from wild type trees was white in appearance, but wood from the transgenic trees was red-brown, most likely due to reactions with ferulic acid, derived from the substrate of CCR, in the cell wall [5]. Importantly, the red coloration was not uniform, but varied significantly in a single trunk, and among trunks from clonally propagated trees. Saccharification potential increased to ~50% per gram dry weight. Our data show that reducing lignin amount in trees improves saccharification potential and should reduce the chemical or energy cost of biomass pretreatments. The trees will be harvested again in winter 2012-2013, i.e. three years after the first coppice. This should provide sufficient biomass for evaluation at semi-industrial scale at the pilot biorefinery in the Port of Ghent [6].

**Acknowledgements:** This research is funded in part by the UGent Multidisciplinary Research Partnership 'Biotechnology for a Sustainable Economy', the Association-UGent Hercules program (AUGE/014), the EU-financed program ENERGYPOPLAR (KBBE-2007-3-1-02), the IWT Flanders for a predoctoral fellowship to RVA and the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494).

**References**

1. Vanholme R, Morreel K, Ralph J, Boerjan W: Lignin engineering. *Curr. Opin. Plant Biol* 2008, 11:278-285.

2. Leplé J-C, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B, Naumann A, Kang K-Y, Kim H, Ruel K, Lefèbvre A, Joseleau J-P, Grima-Pettenati J, De Rycke R, Andersson-Gunnerås S, Erban A, Fehrle I, Petit-Conil M, Kopka J, Polle A, Messens E, Sundberg B, Mansfield S, Ralph J, Pilate G, Boerjan W: Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. *Plant Cell* 2007, 19:3669-3691.

3. Vanholme R, Van Acker R, Boerjan W: Potential of Arabidopsis systems biology to advance the biofuel field. *Trends Biotechnol* 2010, 28:543-547.

4. Wout Boerjan Lab:[http://www.vib.be/en/research/scientists/Pages/Wout-Boerjan-Lab.aspx].

5. Ralph J, Kim H, Lu F, Grabber J, Leplé J-C, Berrio-Sierra J, Mir Derikvand M, Jouanin L, Boerjan W, Lapierre C: Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency). *Plant J* 2008, 53:368-379.

6. Ghent Bioenergy Valley: http://www.gbev.org/; Biobase Europpe, http://www.biobaseeurope.eu/.

**Background:** *Eucalyptus* species are among the fastest growing woody plants in the world and represent about 8% of all planted forests (~18 million hectares) grown in 90 countries [1]. Only a limited number of species are grown commercially, and these have been the focus of extensive breeding to improve desirable wood properties such as basic density, cellulose content, fiber length and improved growth. Improved *Eucalyptus* varieties grown in managed plantations as a source of timber and pulpwood have provided socio-economic benefits for both small and large land owners. *Eucalyptus* also provides a cost-effective source of lignocelluloses for the production of energy and advanced biofuels. However, the large scale plantings of the most productive *Eucalyptus* species in the Southeast U.S. are currently limited to regions of central and southern Florida where no hard freezes occur in winter. Plantings of hardwood species to support the traditional forest industry in the Southeastern U.S. are infrequent due to slow growth rates and high plantation establishment and management costs. The biotech Freeze Tolerant *Eucalyptus* would provide an economically viable plantation hardwood for the Southeastern U.S.

**Methods:** Genetically engineered Freeze Tolerant *Eucalyptus* (AGEH427) was developed by the introduction of plasmid pABCTE01 into the EH1 genotype of *E. grandis* x *E. urophylla* using the *Agrobacterium* transformation system described by Cheah (2001) [2]. pABCTE01 contains a CBF2 expression cassette comprised of Arabidopsis cold-inducible promoter rd29A [3] driving the Arabidopsis *CBF2* (C-Repeat Binding Factor) cDNA [4,5]. It also contains a pollen control cassette consisting of a modified *barnase* gene from *Bacillus amyloliquefaciens* under the control of a *Pinus radiata* anther-specific promoter (PrMC2) [6]. Single insert, backbone free transgenic lines were vegetatively propagated to enable 21 replicated field trials established at eight different locations representing USDA Hardiness Zones 8a (potential kill zone), 8b (target freeze-stress zone), and 9a (freeze stress-free zone) across the Southeast U.S. A simple comparison of pre-winter and post-winter live height measurements was used to calculate a percent dieback of the main stem, and provided appropriate assessment of freeze tolerance and growth performance under field conditions. Data were collected over five winter/ growing seasons. Pollen ablation was studied by collecting multiple flowers from field grown transgenic and non-transgenic trees prior to anthesis and microscopic assessment of the frequency of normal pollen.

**Results:** The transgenic line AGEH427, had significant freeze hardiness that enabled it to grow in USDA Hardiness Zone 8b. The growth of AGEH427 was comparable to EH1 non-transgenic control trees when grown under low- or no-freeze challenge conditions (Zone 9a). In the target freeze-challenged environment (Zone 8b), AGEH427 grew better than the non-transgenic control. After the severe winter of 2009/2010 at a site with a minimum temperature of 16.8 °F, the average live height of control trees was approximately 0.3 feet as compared to 52.4 feet for AGEH427. AGEH427 had approximately 10% dieback from the top. In the zone 8a (winter temperatures as low as 8.4 °F), complete dieback and defoliation were observed, with post-winter tree heights for both AGEH427 and the non-transgenic control being only 0.1 ft. AGEH427 also did not produce pollen, whether or not it was grown in freeze challenged locations. No pollen was produced over multiple years, different flowering seasons, different sites, and different physiological ages of trees while the control trees did produce pollen. Additional phenotypic evaluations of AGEH427 in comparison to the control were performed in field studies across a broad range of environmental conditions in the Southeast U.S., and no differences, other than the freeze tolerance and pollen ablation traits were observed. Neither the transgenic nor the control trees demonstrated any tendency to spread beyond a managed plantation, with low seed set and lack of any seeded volunteers observed in the field trials.

**Conclusions:** The cumulative multi-season data obtained from these trials demonstrated conclusively that the freeze tolerant trait in AGEH427 enabled this tropical tree to grow in the subtropical, freeze-prone USDA Hardiness Zone 8b. The productivity of AGEH427 makes it ideal as a short-rotation plantation hardwood in the Southeast U.S. The yield achievable with freeze tolerant *Eucalyptus* is predicted to meet or exceed the required productivity for an economically feasible hardwood plantation to meet traditional forest industry and bioenergy needs [7]. There is no evidence from the literature or from our field trials which indicates that EH1-427 would be invasive or negatively impact endangered species. EH1-427 is currently the subject of a petition for deregulation in the U.S.
## References

1. FAO: State of the World's Forests. Food and Agriculture Organization 2007.

2. Cheah K: Methods for producing genetically modified plants, plant materials and plant products produced thereby. United States Patent 6,255,559 B1 2001.

3. Yamaguchi-Shinozaki, Shinozaki K: Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Genet 1993, 236:331-340.

4. Jaglo-Ottosen K, Gilmour S, Zarka D, Schabenberger O, Thomashow M: *Arabidopsis* CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 1998, 280:104-106.

5. Zhang X, Fowler S, Cheng H, Lou Y, Rhee S, Stockinger E, Thomashow M: Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant *Arabidopsis*. Plant J 2004, 39:905-919.

6. Rottmann W, Norris-Caneda K, Zhang C: Reproductive ablation constructs. United States Patent 7851679 2010.

7. English B, De La Torre Ugarte D, Jensen K, Hellwinckel C, Menard J, Wilson B, Roberts R, Walsh M: 25% Renewable Energy for the United States by 2025: Agricultural and Economic Impacts. University of Tennessee Agricultural Economics 2006 [http://www.agpolicy.org/ppap/REPORT%2025x25.pdf].

In the 1980s, when the production of transgenic plants was first demonstrated and was soon followed by production of healthy transgenic poplars in the US and then in Belgium, many of us, including some of the most conservation minded in forestry science, embraced genetic engineering/genetic modification as an important new technology for forestry. We had this sense of following the yellow brick road to Oz. It seemed that we could finally get beyond the narrow limitations of the slow outbreeding, quantitative breeding system, and begin an era of qualitative genetics to complement the dominant quantitative genetic paradigm. Unfortunately, when we got closer to the Wizard of Oz things looked a bit different than on the famed Yellow Brick Road (Figure 1).

What happened? Most important, the technology, and how it was structured, ran into a social and political buzz-saw. The world was becoming richer and more sensitive, both in terms of environment and respect for life. A diversity of social movements were taking place, powered by the new internet, that was uncomfortable with the notion of transgenics and who controlled them, and a number of organizations with resources and power opposed transgenics at every turn. A patent system was put in place that only very large companies or a few wealthy foundations could afford to negotiate. Rich consumers and vulnerable consumers sensed risk and little or no direct benefit to them, and thus also opposed the technology in large numbers. Regulations were pushed through that made the costs of field research, marketing, permits, international trade, liability insurance, and labeling huge barriers to investment and commercial adoption. Nearly the only crops to survive produced huge economic benefits and had to be pushed through the regulatory system by the multinational companies that consumers feel the least trust in.

Unfortunately, national and international regulations, once in place, are very hard to change—so this legacy will be with us for many years. Moreover, because the regulations treat all transgenes as hazards until

## I25
Transgenic biotechnology in forestry: what a long strange trip it's been

Steven H Strauss
Department of Forest Ecosystems and Society, 321 Richardson Hall, Oregon State University, Corvallis, OR, 97331-5752, USA
E-mail: Steve.Strauss@oregonstate.edu
BMC Proceedings 2011, 5(Suppl 7):I25

<figure><img src="image_5.png" /><figcaption>**Figure 1 (abstract I25)** On the road to transgenic Oz in the early days of plant and forest biotechnology.</figcaption></figure>
"proven safe," they pose severe problems for conducting high quality field trials of most types of transgenic forest trees. This is a result of their wild and feral relatives, low level of domestication, and ability to produce pollen and seed that can move over large distances. Thus, it's very hard to integrate transgenic trees into conventional breeding and field testing programs—which is essential for their development and application. Meanwhile, social perspectives and the structure of research funding has led to extraordinary scrutiny and amplification of every possible risk of

transgenic trees, usually far out of proportion to its significance compared to conventional agriculture and forestry breeding. Thus, the trip has felt much more like a certain Electric Kool-Aid Test (Figure 2), than a normal technological progression, and not at all like the start of the trip to Oz. On the science side, despite our early successes, when we finally got to meet the Wizard, there was also a number of revelations that gave us pause about the robustness of transgenic tree biotechnology, and most remain significant to this day. 1) It was much harder to transform/

<figure><img src="image_6.png" /><figcaption>**Figure 2(abstract I25)** The trip to transgenic Oz was far risker, but also far richer and more informative about ourselves and our society, than we dard to imagine. We will keep on working and innovating with transgenic tools, both on biological and sociopolitical fronts, because our thirst, imagination, and world demands it.</figcaption></figure>
regenerate most commercially important forest tree species/genotypes than we had imagined, and very little in the way of public research funding has been directed to overcome this in a meaningful way. 2) There was a great deal of hype about some of the early successes, such as in the area of growth rate improvement and lignin modification, with insufficient field trials to judge their true merit. Many have not proven themselves, but the results are often hidden by business confidentiality, academic self-interest, and a paucity of high quality field research 3) Beyond 35S-type overexpression for traits like herbicide and Bt-insect resistance, the tools in place for control of gene expression were found to be coarse, unpredictable, and imprecise, likely due to lack of control of insertion location, chromatin state, and poor understanding of RNAi/siRNA mechanisms. 4) While genetic containment would solve a lot of social and regulatory problems, due to high social concerns, short term funding, strict regulations over field trials, and immature technology, it's unclear if we can attain, in the near to middle term, the nearly perfect level of predictable, field-validated containment that appears to be required. 5) The extraordinary advances in genomics and phenomics in conventional breeding, combined with the high costs of regulations, are pushing transgenic applications to focus on the most new, novel, and valuable applications that cannot be attained by conventional or genome-assisted breeding—which are also those for which attaining regulatory approval will be most difficult. 6) Meaningful, field-based ecological studies of fitness and non-target effects, especially of pest-resistant, stress-resistant, bioindustrial, and bioremediation types of transgenic trees, have not been conducted—leaving a wide swath for models and speculations of severe ecological impact that are likely to be vastly overstated—but push regulations toward ever greater stringency. In sum, a great deal of fundamental science, from the genic to the ecological, and technology development to efficiently identify and transfer genes and pathways, remains to be done by the next generation of scientists and practitioners.

Propelled by the ongoing successes of some pioneering companies, I sense that transgenic forest biotechnology, though facing great difficult challenges at present, will "keep on trucking" in the parlance of "The Trip" (Figure 2). This will be driven by science and technology successes that continue at an impressive rate; urgency in finding solutions in the face of severe global economic and environmental problems; and the human thirst for novelty, innovation, and truth.

# KEYNOTE LECTURE PRESENTATION

## K1

**Exponential advances in Forest Genetics: the past 25 years and the next 25 weeks**

Ronald Sederoff

Forest Biotechnology Group, North Carolina State University, USA

E-mail: ron_sederoff@ncsu.edu

BMC Proceedings 2011, 5(Suppl 7):K1

A history of Forest Biotechnology will be briefly reviewed, from the first cloned tree gene, transgenic trees, DNA markers, genetic maps, QTLs, marker assisted breeding to genome sequencing. Perspectives of newer methods and their applications will be discussed, including integration of genetics and RNA-seq, metabolomics, and proteomics. New applications may be anticipated or expected for breeding, vegetative propagation, phenotypic plasticity, epigenetics, symbiosis, adaptation to biotic and abiotic stress and the directed evolution of some forest tree species.

Forest Biotechnology began over 25 years ago with the advent of new technology of molecular genetics able to advance the fundamental and applied science of forest trees. Methods of gene cloning and gene transfer held the promise of bypassing the long generation times that made genetic analysis and breeding so difficult. This vision has come to pass and the presentations of this meeting show how far we have come. Today, we are deeply involved in the next generation of genetic technology, a second revolution, using genomic sciences, based on the technology and concepts derived from whole genome sequencing, with the same objective, advancing the fundamental and applied science of forest trees.

The path ahead appears to have a third phase, based on an extension of the genomic paradigm, to systems biology and predictive modeling, directed to the same basic goals. Each new phase has been highly

integrative, the first bringing molecular genetics into many traditional disciplines. The second phase, integrated areas of molecular and quantitative genetics with high throughput processing and bioinformatics. What appears to be the third phase will extend this paradigm to incorporate the biochemistry of proteomics, the chemistry of metabolomics and the organization, structure and dynamics of cell biology and whole plant physiology. This integrated paradigm further incorporates an engineering perspective and describes the integrated data as predictive mathematical models, subject to experimental test and manipulation. Much of this new biology will be at the interface of biology and systems engineering. Biologists will learn to think like engineers, and engineers will learn a lot of biology. In time, the distinctions between these disciplines may be lost.

What makes a tree a tree? We now know that a relatively small number of genes regulate the woody phenotype, because a small combination of gene mutations can produce a woody form of Arabidopsis. Similarly, small numbers of genes regulate key aspects of tree dormancy. Much remains to be determined regarding the control of tree growth and development, and little is known about the genetic basis of the wide diversity in morphology, metabolism and adaptation. Beyond the descriptive understanding of tree phenotypes and their diversity, lies the evolutionary question, how did the diversity of trees get to be that way.

Comparative genomics has been a powerful tool to derive gene function by comparisons of woody and herbaceous plants, because a large number of functions are conserved. A major barrier to a broad understanding of trees and all other plants still lies in the lack of information on the function of the majority of plant genes. The functions of most genes are known only by superficial annotation based on the time and place of transcript expression, or response to an external stress. Even for many genes of presumed known function, we do not know if that is the only function or the primary function. Even as more genes are subjected to gain of function/loss of function tests, interpretation suffers from effects of redundancy and lethality.

In the near future, genomic scale integration of processes at the levels of molecules, cells, tissues and organisms, as part of a systems biology approach, will bring about progress toward this goal. This goal appears daunting. One challenge comes from the knowledge of epistasis, or genetic interactions, that indicate substantial complexity even for simple traits. QTL analysis provides a general picture of this complexity, with a frequent genetic architecture of a small number of genes of large effects with a larger number of genes with small effects. Thanks to genomics and computers, the quantitative effects of a large number of genes may now be studied at the molecular level as is being done for genomic selection.

Traditionally, new concepts and technology developed in model systems see earlier applications in medicine and agriculture than in forestry. The lag time may be decreasing. The paradigm for genomics was established by the human genome project, where the draft sequence was initiated in 1989 and a first draft released in 2000. The sequence of Arabidopsis was completed in 2000, rice in 2002, and the first tree genome, black cottonwood, completed in 2006. We see application of new technology to traditional problems, in some cases with great success, and in others with great promise.

Only a few years after gene transfer was demonstrated in model plants, an herbicide resistance gene was inserted into poplar and a tree regenerated. Deployment of genetically modified crops has met with resistance, however, today worldwide; there are about 120 million hectares of GM crops, with half in the USA. GM trees are still highly regulated in the US. In China, GM trees have been widely deployed in the last decade. GM trees still face substantial resistance in the US and more so in Europe.

Chestnut serves as an important model for a Genetically Modified Tree. The prospects of development of a blight resistant American chestnut appear quite good based on a combination of backcross breeding; map based cloning and genetic transformation. A genome sequence for Chinese chestnut is in progress (Carlson et al.). The use of gene transfer for chestnut restoration is compelling because of the high value of the species, and because GM may be required to restore the species. The restoration of the American chestnut would have great value to Appalachian forest ecosystems, as a resource of high value wood, and potentially for bioenergy. The restoration would serve as an example for the application of biotechnology and genomics to many valuable species currently threatened by pests and pathogens or climate change.
# S1. POPULATION GENOMICS, CONSERVATION AND ADAPTATION

## O1

**Comparative genomics of resistance of spruce to the white pine weevil in British Columbia**

Kermit Ritland¹*, Sébastien Verne¹, Barry Jaquish², Carol Ritland¹

¹University of British Columbia, USA; ²Ministry of Forests, Lands and Natural Resource Operations, USA

E-mail: kermit.ritland@ubc.ca

BMC Proceedings 2011, 5(Suppl 7):O1

We present results of two large scale comparative studies of the genomic basis of resistance of Interior spruce to the white pine weevil. Both volume growth, the main objective of the spruce breeding program in British Columbia, and white pine weevil resistance, are examined. "Interior spruce" is a species complex involving mainly *Picea glauca* (White spruce) but introgressed with *P. engelmannii* (Englemann spruce), depending upon locality. In the first study, we compared constitutive expression of 17825 genes between 20 resistant and 20 susceptible trees to the weevil; 54 upregulated and 137 downregulated genes were found in resistant phenotypes, with implications discussed in regard to volume growth. In particular, we will be surveying these genes for SNPs that differ between these two classes of trees in the next year. In the second study, we developed a 1536 Illumina SNP chip based upon candidate genes for weevil resistance. In a novel experimental design, we assayed 945 open-pollinated progeny of the Prince George breeding population (176 parents), and 654 open-pollinated progeny of the Prince Rupert breeding population (134 parents); parents were also genotyped. Within each family of 100 progeny, we identified the highest ranked 3 progeny and the lowest ranked 3 progeny, based upon BC Ministry of Forests scores for volume growth and resistance. These were genotyped and used in a novel test analogous to the transmission disequilibrium test to detect both SNP associations and QTLs linked to SNP markers. Discoveries about associations and QTL are discussed, with the added caution about genotyping error. Both studies illustrate how operational tree breeding populations can provide valuable inferences about tree genomics.

## O2

**Genomics of adaptation and wood properties in Populus trichocarpa**

Carl Douglas

University of British Columbia, USA

E-mail: carl.douglas@ubc.ca

BMC Proceedings 2011, 5(Suppl 7):O2

*Populus trichocarpa* (black cottonwood) and *P. balsamifera* (balsam poplar) are native to North America where large natural populations exist. *P. trichocarpa* has an extensive north-south range along the west coast, while *P. balsamifera* has a northerly range across the continent from Alaska to the North American east coast. Both species are well suited for highly productive bioenergy biomass plantations in north-temperate climates. We are investigating the large reservoirs of natural genotypic and phenotypic variation in wild populations of these species, with an initial focus on *P. trichocarpa*, to identify allelic variation underlying optimal biofuel and biomass traits that could be used for accelerated domestication. We sampled genetic variation in *P. trichocarpa* by Illumina transcriptome resequencing of 20 individuals from provenances along a latitudinal gradient from 60°N to 44°N. This analysis revealed extensive nucleotide, gene expression and alternative splicing polymorphism in 10,000 xylem-expressed genes. Alignment of transcript sequences to the *P. trichocarpa* Nisqually-1 reference genome was used to identify over 500,000 SNPs. These SNPs were combined with a larger SNP set generated by whole genome resequencing of multiple *P. trichocarpa* individuals carried out by the US DOE Bioenergy Sciences Center (BESC) to populate a *P. trichocarpa* SNP database. Using the Illumina iSelect process, we generated an Illumina Infinium bead array for genotyping of 34,000 SNPs that tag 3,700 candidate genes selected for their potential involvement in biomass, adaptation, and wood traits. Using this bead array in collaboration with BESC, we obtained candidate gene SNP genotypes for over 700 *P. trichocarpa* individuals grown in common gardens. This analysis of these

data revealed extensive genotypic polymorphism in the targeted gene set. In parallel, we carried out phenotyping of the trees from the wild populations grown in common gardens, quantifying variation in over 85 morphological, growth, physiological, wood chemistry, and wood quality traits. Population genetic analysis of the SNP data, its use for genotype-phenotype association studies to identify candidate gene alleles underlying variation in traits of interest, and the prospects of using the results for marker assisted breeding will be discussed.

## O3

**Lodgepole pine, jack pine, and their hybrids: molecular markers reveal mountain pine beetle host-range expansion into jack pine of the boreal forest**

Catherine Cullingham¹, Sophie Dang¹, Corey Davis¹, Barry Cooke², David Coltman¹, Janice Cooke¹*

¹University of Alberta, Canada; ²Northern Forestry Centre, Canadian Forest Service, Natural Resources Canada

E-mail: janice.cooke@ualberta.ca

BMC Proceedings 2011, 5(Suppl 7):O3

**Background:** Lodgepole pine (*Pinus contorta* Dougl. ex Loud. var. *latifolia*) is found in western North America, extending from the Yukon into British Columbia and Washington, and along the Rocky Mountains and eastern slopes to Colorado [1]. Jack pine (*Pinus banksiana* Lamb.) is a closely related species found east of the Rockies, mainly in Canada's boreal forest from the Northwest Territories to Quebec [1]. Lodgepole and jack pine ranges overlap in northern Alberta and the Northwest Territories, where these species hybridize (Fig. 1). In the absence of reliable molecular markers, morphological characteristics are commonly used to distinguish lodgepole pine, jack pine and hybrids. However, hybrids and pure species can be difficult to visually distinguish, particularly at the presumed periphery of the hybrid zone, which is poorly described.

Mountain pine beetle (MPB; *Dendroctonus ponderosae* Hopkins) is indigenous to western North America. In the current MPB outbreak, more than 14 million hectares of mainly lodgepole pine forests have sustained MPB-caused mortality in British Columbia alone [2]. Following MPB long range dispersal into northwestern Alberta in 2006, MPB has continued its apparently unprecedented eastward spread into the lodgepole x jack pine hybrid zone of Alberta (Fig. 1).

To better define this hybrid zone, we developed microsatellite and SNP markers that distinguish the pure species from hybrids. We then used the microsatellite markers to test the hypothesis that the MPB epidemic has spread into jack pine.

**Materials and methods:** Foliage was collected from 678 trees representing 25 localities in British Columbia, Alberta, Saskatchewan, Ontario, and Minnesota in 2007, 2008 and 2010, including 154 MPB-attacked trees from British Columbia and Alberta.

Eleven microsatellite markers amplifying both lodgepole and jack pine were developed from published loci [3] and loci isolated from a microsatellite enriched (GT<sub>n</sub>/CT<sub>n</sub>) lodgepole pine library. Genotyping and scoring was carried out as described [4]. There were less than 2% missing data, with a 0.8% genotyping error rate. Loci met Hardy-Weinburg equilibrium criteria; pairs of loci in linkage disequilibrium were negligible. Genetic diversity measures were calculated in GENEPOP [5], GenAIEx [6] and HP-RARE [7]. Assignment tests were conducted in NEWHYBRIDS [8] and STRUCTURE [9]. *In silico* species-discriminating SNP detection was carried out using CLC Genomics with lodgepole and jack pine cDNA 454 sequence data, and validated with PCR.

**Results and discussion:** Genetic diversity was high among localities, and was higher in lodgepole than jack pine. Differentiation among localities ($F_{ST}$ = 0.125) and between species ($F_{ST}$ = 0.133) was high. Within species, differentiation among localities was generally low ($F_{STlodgepole}$ = 0.033, $F_{STjack}$ = 0.016).

The efficacy of the microsatellite loci to distinguish pure species and hybrids was tested by assignment of individuals to their correct class with NEWHYBRIDS and STRUCTURE, using five simulated datasets (ten iterations each) containing multiple levels of admixture generated with known pure lodgepole and jack pine as benchmarks. Using $Q_T$ greater than or equal to 0.9 to assign pure species and $Q_T$ less than 0.9 to designate hybrids, we achieved accurate resolution of first and second generation hybrids in simulated datasets using both NEWHYBRIDS and STRUCTURE, but
<figure><img src="image_7.png" /><figcaption>**Figure 1 (abstract O3)** Canadian data for A. pine distribution, B. historical MPB range, C. extent of the current MPB outbreak prior to 2006, D. extent of the current MPB outbreak following dispersal into northern Alberta. MPB data are overlaid on classic lodgepole and jack pine ranges [1]. Pine volume and MPB attack data courtesy of the Canadian Forest Service.</figcaption></figure>

encountered diminishing power with advanced generations. There was very high agreement between NEWHYBRIDS and STRUCTURE; a decision tree was developed to resolve the small number of disagreements. Assignment of the 678 genotyped individuals suggests that the intersection of the ranges classically described for lodgepole and jack pine [1] underestimates the distribution of hybrids. Pure lodgepole and jack pine were identified within the lodgepole x jack pine hybrid zone, with lodgepole pine often occupying higher elevations.

Eight of 154 MPB-attacked trees genotyped with the microsatellite loci were identified as jack pine. This first evidence of MPB mass-attack and reproduction on jack pine in natural stands was presented to key forest managers and decision makers via the National Forest Pest Strategy – a Canadian Forest Service initiative coordinating federal and provincial forest management and policy development – and led to accelerated development of a consensus inter-jurisdictional strategic plan.

We have developed 9 nuclear, 12 chloroplast, and 3 mitochondrial SNP markers that distinguish lodgepole pine, jack pine, and hybrids. These markers are being used to further delineate the hybrid zone, investigate gene flow and introgression, and continue monitoring MPB invasion of the boreal forest.

**Conclusion:** Microsatellite markers reliably distinguishing lodgepole pine, jack pine and their hybrids were used to better delineate Alberta's lodgepole pine x jack pine hybrid zone and to conclusively demonstrate that MPB has undergone host range expansion into jack pine of the boreal forest, a new habitat for this devastating forest pest.

## References

1. Critchfield WB, Little EL: Geographic distribution of the pines of the world. USDA Misc. Pub. 991 1966.

2. Nealis V, Peter B: Risk assessment of the threat of mountain pine beetle to Canada's boreal and eastern pine forests. Report BC-X-417, Canadian Forest Service 2008.

3. Auckland LD, Bui T, Zhou Y, Shepard M, Williams CG: Conifer microsatellite handbook. Corporate Press, Raleigh, N.C. 2002.

4. Cullingham CI, Cooke JEK, Dang S, Davis CS, Cooke BJ, Coltman DW: Mountain pine beetle host-range expansion threatens the boreal forest. Mol. Ecol. 2011, 20:2157-2171.

5. Raymond M, Rousset F: Genepop (Version-1.2) – population genetics software for exact tests and ecumenicism. J Heredity 1995, 86:248-249.

6. Peakall R, Smouse PE: GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 2006, 6:288-295.

7. Kalinowski ST: hp-rare 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol Ecol Notes 2005, 5:187-189.

8. Anderson E, Thompson EA: A model-based method for identifying species hybrids using multilocus genetic data. Genetics 2002, 160:1217-1229.

9. Pritchard JK, Stephens M, Donnelly P: Inference of population structure using multilocus genotype data. Genetics 2000, 155:945-959.

## O4

### Geographic shifts in climatically suitable areas and loss of genetic variability under climate change in a neotropical tree

Jose Alexandre Diniz-Filho<fnref n="1" />, Rosane Collevatti<fnref n="2" />, Lázaro Chaves³, Thannya Soares<fnref n="2" />, João Carlos Nabout⁴, Thiago Fernando Rangel⁵, Dayane Melo⁶, Jacqueline Lima⁷, Mariana Telles<fnref n="2" />

E-mail: diniz@icb.ufg.br

BMC Proceedings 2011, **5**(Suppl 7):O4

**Background:** Many species are expected to suffer a strong shift in geographic ranges due to climate changes in the next fifty years, depending on their ecological tolerance and current demographical parameters, which were in turn shaped by their evolutionary history. These shifts may also cause a change in genetic population structure and variability, because local extinctions or reduction in fitness are not expected to be random in geographical space. Here we used an ensemble forecast approach of Species Distribution Modeling (SDM hereafter, also known as niche modeling) to derive current and future geographic distribution of the Neotropical tree *Dipteryx alata* ("Baru" tree, Fabaceae). We then obtained a series of genetic parameters for the

<footnote n="1">¹Departamento de Ecologia, ICB, Universidade Federal de Goiás, Brazil;</footnote>

<footnote n="2">²Departamento de Biologia Geral, ICB, UFG, Brazil; ³Escola de Agronomia & Engenharia de Alimentos, UFG, Brazil; ⁴UnUCET, Universidade Estadual de Goiás, Brazil; ⁵Departamento de Ecologia, ICB, UFG, Brazil; ⁶Programa de Pós-Graduação em Agronomia, UFG, Brazil; ⁷Programa de Pós-Graduação em Ecologia & Evolução, UFG, Brazil</footnote>
species after generating extinctions in areas of low future habitat suitability.

**Methods:** We obtained a total of 448 occurrences of *D. alata* throughout the Brazilian Cerrado, which were recorded in a grid with 6240 cells of 0.5° of latitude/longitude covering South America. These occurrences were modeled as a function of eight climatic variables (WORLDCLIM), for the current time and projected into 2050, for three different Global Circulation Models (AOGCMs - CCCma, Csiro, HadCm3) [1,2]. Occurrences were modeled using six different SDM techniques [3]. Methods used were BIOCLIM, Euclidian, Gower and Mahalanobis distances, GARP and MAXENT. For each of these methods, models were built using 255 combinations of the climatic variables, each one tested

using 50 cross-validations using True-Skill statistics (models with TSS < 0.7 were excluded). A Principal Component Analysis (PCA) of the estimated frequencies of occurrence, obtained by SDMs and AOGCMs, was used to compare the maps. Variance components of these sources were obtained and mapped [2].

Genetic data for *D. alata* consisted in microsatellites markers analyzed for 25 widely distributed local populations, encompassing species' geographical range. A total of 644 individual trees were genotyped for eight microsatellite loci. Genetic parameters were used to estimate the total amount of polymorphism and genetic diversity currently found in *D. alata*. We estimated the number of alleles per locus, genetic diversity (expected heterozygosity under Hardy-Weinberg equilibrium), and the expected

<figure><img src="image_8.png" /></figure>
heterozygosity under mutation-drift equilibrium. Moreover, we recalculated these parameters by assuming that, under climate change, there will be a displacement of climatically suitable areas for the species and, consequently, that only populations found in regions above certain levels of suitability in the future will persist.

**Results and discussion:** The ensemble forecasting approach reveals that *D. alata* will shift its geographic range and climatically suitable areas from Central towards Southeastern Brazil (Fig. 1). The first principal component explains 74.3% of the variation among maps and, on average, 95% of the variation among them is due to SDMs used.

The changes in the climatically suitable areas in *D. alata* imply a reduction in the genetic parameters. The parameters remain approximately constant up to a 50% threshold, which is the minimum by assuming a majority consensus of frequency of occurrences. However, after this critical threshold there is an abrupt reduction in all parameters, although the magnitude of shift is only about 10% of the current values, on average (Fig. 2). There is a wide variation of shifts direction and magnitude among loci for each parameter, and actually these trends are usually driven by two or three loci.

The wide current range and ecological tolerance of *D. alata* explains why low levels of loss in genetic diversity were observed here, contrasting with previous results for other species (i.e., the "pequi" tree *Caryocar Braziliense*). Because of the wide range, currently and in the future, some of the local populations with highest genetic diversity will potentially remain in highly suitable areas in the future, even using very conservative thresholds of 80%. However, it is important to highlight that coalescence analyses and the expected heterozygosity under mutation-drift equilibrium suggest strong population bottlenecks for the species in the recent past, which were corroborated by hind cast projections of the SDMs using paleoclimatic data. This explains the low level of genetic variability in *D. alata*, despite its wide geographic range.

Thus, despite the shift in geographic range size and climatically suitable areas towards Southeastern Brazil and the expected downward shift in the genetic parameters, the analyses performed here do not show a strong loss of genetic diversity in *D. alata*. Even so, it is important to realize that these results are mainly due to the relatively low current genetic variability of the species, probably associated with recent population bottlenecks. In this case, climatic shifts can have more serious adaptive consequences and further investigations are necessary to avoid a false indicative of high

probability of persistence of this species based on a low relative level of loss of genetic diversity.

**References**

1. Araújo MB, New M: Ensemble forecasting of species distributions. *Trends in Ecology and Evolution* 2007, **22**:42-47.

2. Diniz-Filho JAF, Bini LM, Rangel TF, Loyola RD, Hof C, Nogues-Bravo D, Araújo MB: Partitioning and mapping uncertainties in ensembles of forecasts of species turnover under climate change. *Ecography* 2009, **32**:897-906.

3. Elith J, Graham CH: Do they? How do they? Why do they differ? On finding reasons for differing performances of species distribution models. *Ecography* 2009, **32**:66-77.

## O5

### Genomic signatures of selection in response to sulfate air pollution in natural populations of red spruce

Om Rajora*, Stanislav Bashalkhanov

Faculty of Forestry and Environmental Management, University of New

Brunswick, 28 Dineen Drive, Fredericton, NB E3B 5A3, Canada

E-mail: Om.Rajora@unb.ca

BMC Proceedings 2011, **5**(Suppl 7):O5

Rapid environmental changes, such as anthropogenic air pollution, can create significant evolutionary pressures on populations, species and ecosystems. Evolutionary processes occurring in natural populations at very short time scales, especially in response to human-induced environmental changes, are not well understood. Confounding effects of geographic variation and demography cannot be easily separated from signatures of recent selection in natural populations. We investigated the genetic response of declining red spruce (*Picea rubens*) populations at high elevation sites in Southern Appalachians to anthropogenic sulfate depositions. Red spruce seedlings are more sensitive to drought and cold stresses elicited by exposure to anthropogenic sulphate air pollution, than old trees. Genetic variation in seedlings and young trees was significantly reduced in heavily polluted stands. Several candidate genes involved in cold acclimation and calcium metabolism demonstrated signatures of selection corresponding with sulfate pollution levels. SNP allele frequencies at one gene involved in calcium metabolism demonstrated directional selection in response to

<figure><img src="image_9.png" /><figcaption>**Figure 2(abstract O4)** Reduction in genetic parameters of *D. alata* estimated at increasingly levels of environmental suitability estimated for 2050.</figcaption></figure>
anthropogenic sulfate deposition in red spruce growing at severely polluted high elevation sites, which corresponds well with the putative role of this gene in adaptation to acidification stress. Unlike range-wide experimental designs (e.g. the popular F<sub>ST</sub> outlier test) and nucleotide diversity-based association studies, our within-population testing approach disentangled the confounding effects of geographic variation and demography from the genetic effects of recent selection.

**O6**
**Effects of forest fragmentation on the effective and realized gene flow of Neotropical tree species: implications for genetic conservation**

Alexandre Sebbenn
Instituto Florestal de São Paulo, Brazil
E-mail: alexandresebbenn@yahoo.com.br
BMC Proceedings 2011, 5(Suppl 7):O6

Tree species are key organisms of forest ecosystems, due their size and extended life, providing the environmental setting for many other living organisms. The levels of genetic diversity and the effective population sizes are essential issues for the maintenance and survival of tree species populations, because they direct adaptation to the current environment and future environmental changes. Forest fragmentation around the world is a fact, affecting many animal and plant populations in all continents. In Brazil, forest fragmentation and selective logging are the two main problems affecting populations of tropical tree species. For example, the Atlantic Forest was intensively destroyed and fragmented during the last century. Today, only between 11 and 17% of the original areas remain, generally split into small forest fragments or isolated trees in pastures and agricultural lands. In the Atlantic Forest, only 3% of the original area of the Araucaria Forest biome in southern Brazil, remains. Forest fragmentation is especially drastic in tropical forest biomes, due to the very high species diversity, associated to low population density (<1 tree/ha). Thus, after forest fragmentation takes place, the remaining fragmented forest may contain the same species diversity, but the populations are strongly reduced, and, in some cases, less than a dozen reproductive individuals remain. The bottleneck (size reduction of the reproductive population) caused by forest fragmentation associated to spatial isolation of the remaining stands may cause the loss of genetic diversity, an increase in inbreeding and relatedness, reducing the effective population size and blocking seeds and pollen migration inside the stands. These hypotheses have been tested in some tropical tree species of the Araucaria Forest and semicidental Atlantic Forest, including *Araucaria angustifolia*, *Copaifera langsdorffii*, *Myracrodruon urundeuva* and *Hymenaea stigonocarpa*, using microsatellite markers and parentage analysis. More specifically, in these studies we tried to answer the following questions: Can spatial isolation of tree populations following forest fragmentation block seed and pollen migration (gene flow)? What is the distance and patterns of seed and pollen dispersal in populations of tree species living within fragmented stands? Is there intra-population spatial genetic structure (SGS) in the adults and regeneration, and where is it higher, in adults or regeneration after forest fragmentation? Can forest fragmentation really reduce the genetic diversity and effective population size and increase the inbreeding and relatedness within populations? What is the necessary number of seed trees to collect seeds aiming at *ex situ* conservation and environmental reforestation plans with a reference effective population size that provides a minimum evolutionary potential?

Knowledge of these issues is crucial to delineate strategies for *in situ* and *ex situ* conservations, tree breeding and environmental restoration plans. As many tropical trees are long lived individuals, established before forest fragmentation had occurred (<100 years), seeds, seedlings and juveniles, established after forest fragmentation have been included in the samples. Thus, these studies have been based on the sampling of all reproductive trees regenerates (realized seed and pollen dispersal), as well as open-pollinated seed (effective pollen dispersal). All adult trees and regenerates were also mapped (x and y coordinates), the diameter at breast height (dbh) and/or total height measured and in the case of dioecious species, all reproductive trees were also sexed. We used a set of microsatellite loci to arrive to a high exclusion power, to guarantee that each candidate parent displays a unique multilocus genotype for parentage analysis. Our results showed an essentially perfect isolation of seed gene flow, with total absence of seed immigration, probably due to the large distance

separating the remaining forest fragments, associated to an intensive use of surrounding land (agriculture, highways, urban areas, etc). Within populations, although long distance seed dispersal events were detected, seeds generally disperse intensively near the mother trees following a classic isolation by distance model. These hypotheses have also been supported by studies of SGS in both adults and juveniles and mating system, revealing significant rates of mating among relatives. In contrast, substantial realized and effective pollen immigrations have been detected in the studied species (ranging from 4 to 10%), evidencing longer-distance pollen dispersal than seed dispersal. Within populations, although the estimates are downward biased due to a substantial rate of pollen immigration in the stands, pollen generally disperses with high intensity at short distances, in a near neighbour pollen dispersal pattern, determined by the individual flowering patterns and behaviour of pollination vectors. Matings are not random, due to selfing, mating among relatives and correlated mating, resulting in higher inbreeding relatedness and lower effective population size in open-pollinated seeds than expected under a panmictic model. High inbreeding and relatedness and lower effective population size were also seen in the regeneration. Lower genetic diversity has been found in seeds, seedlings and juveniles when compared to the adult population, suggesting the occurrence of genetic drift. Taken together our results in the targeted species has indicated a requirement to maintain the genetic connectivity among the forest fragments using strategies of reforestation in riparian areas and forest corridors. Our results also suggest the need to collect seeds from a large number of trees for *ex situ* conservation, tree breeding and environmental reforestation, due to a typically limited effective population size contained in open-pollinated individuals. This small number of species studied so far does not represent all the genetic, demographic, reproductive and ecological characteristics of the tree diversity in the Atlantic Forest. To provide a stronger support to our hypotheses and results to date, new studies of gene flow, mating system, SGS, genetic diversity and effective population size are underway with other species. Finally, it is never enough to reiterate the importance of conserving tropical tree diversity for future generations so that they can benefit from them as we currently do. Although, *in situ* conservation is the indicated method for this purpose, conserving not only the target species, but also other organism in the same biome using *ex situ* strategies is also necessary due the continued fragmentation and degradation of the Atlantic Forest.

**S2. LINKAGE AND ASSOCIATION MAPPING**

**O7**
**Genetic dissection of transcript, metabolite, growth and wood property traits in an F2 pseudo-backcross pedigree of *Eucalyptus grandis* x *E. urophylla***

Maria Magdalena van Dyk¹*, Anand R K. Kullan¹, Eshchar Mizrachi¹,
Charles A Hefer¹, L'Zanne Jansen van Rensburg¹, Timothy J Tschaplinski²,
Katherine C. Cushman², Nancy E Engle², Gerald A Tuskan², Nicoletta Jones³,
Amulf Kanzler³, Alexander A Myburg¹

¹University of Pretoria, Department of Genetics, Forestry and Agricultural
Biotechnology Institute (FABI), South Africa; ²Biosciences Division, Oak Ridge
National Laboratory, USA; ³Sappi Forests Research, South Africa
E-mail: daleen.vandyk@up.ac.za
BMC Proceedings 2011, 5(Suppl 7):O7

**Background:** *E. grandis* is used extensively for the production of pulp and paper due to its rapid growth, good form and ease of vegetative propagation. *E. urophylla* exhibits tolerance to fungal diseases that limit the growth of *E. grandis* in tropical and subtropical regions. Interspecific hybrids of these two species are, therefore, commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions (e.g., South Africa, Congo and Brazil). These hybrids often exhibit superior growth and wood quality compared to the pure species, but the underlying genetic basis of the observed hybrid superiority remains unclear. Phenotypic variation observed in interspecific mapping populations has been used to identify QTLs in several genetic linkage studies in *Eucalyptus*. However, QTL intervals have generally been wide (20 to 30 cM) and may include several hundred
genes. To bridge the gap between fine mapping and QTL validation studies, the expression levels of genes and metabolites in individuals from the segregating population can be treated as quantitative traits and used for eQTL and mQTL mapping, respectively. Co-localization of wood property, expression and metabolite QTLs will facilitate the identification of positional candidate genes and other components of regulatory networks underlying phenotypic variation.

**Methods:** To identify genetic factors controlling growth and wood property traits in *Eucalyptus*, an F₂ pseudo-backcross mapping family derived from a cross between an F₁ hybrid (GUSAP1, *E. grandis* x *E. urophylla*, Sappi Forest Research) and an *E. urophylla* parent (USAP1), consisting of 555 individuals, was used for genetic linkage map construction using microsatellite and DArT markers. Phenotypic trait assessment included physical measurements of tree diameter and wood density performed on 319 three-year-old individuals. Klason (acid-soluble & -insoluble) lignin and cell wall sugar content were determined for a selection of 100 backcross progeny and used for near-infrared analysis (NIRA) calibration. NIRA predictions for glucose, xylose, arabinose, cellulose and total lignin content, as well as pulp yield were made for all 315 individuals. Total lignin and S:G ratios were also separately measured for the 315 individuals. Immature xylem tissues, collected from 192 backcross progeny, were used for metabolite profiling (ORNL, Oak Ridge, TN) and Illumina mRNA-Seq (PE50, BGI Americas) quantification of transcript levels.

**Results and discussion:** Parental genetic linkage maps were constructed for the F₁ hybrid (GUSAP1, *E. grandis* x *E. urophylla*) and the *E. urophylla* backcross parent (USAP1, Kullan et al. submitted). An integrated parental linkage map, constructed using the cross pollinator (CP) population type in JoinMap®4, was used for QTL mapping as this allows for the detection of the effect of alleles inherited from a single parent and the interaction between the alleles inherited from both parents. In addition to more than 50 growth and wood property trait QTLs (physical measurements, NIRA predictions and wood chemistry traits) and more than 80 metabolite QTLs (mQTLs), representing variation in 22 known metabolites, we will present progress made towards the mapping of gene expression QTLs (eQTLs) based on mRNA-Seq profiling of the first 96 backcross individuals.

**Conclusions:** High-density genetic linkage maps, as constructed during this investigation, enable the accurate identification of genomic regions harbouring QTLs, mQTLs and eQTLs. This is an on-going investigation in which we will integrate different genomics data with the aim of dissecting regulatory networks underlying growth and development in *Eucalyptus*.

## O8

### QTL analysis for growth and wood properties across multiple pedigrees and sites in *Eucalyptus* globulus

Jules S Freeman¹, Brad M Potts¹, Geoff M Downes²,
Saravanan Thavamanikumar², David J Pilbeam³, Corey J Hudson¹,
Rene E Vaillancourt¹*

¹School of Plant Science and CRC for Forestry, University of Tasmania,
Hobart, Tasmania 7001, Australia; ²CSIRO Sustainable Agriculture Flagship,
and Cooperative Research Centre for Forestry, Private Bag 12, Hobart, TAS
7001, Australia; ³Department of Melbourne, Water Street, Creswick, Victoria 3363,
Australia; ⁴Southern Tree Breeding Association Inc., 38 Helen Street, PO Box
1811, Mount Gambier, South Australia, 5290, Australia

E-mail: R.Vaillancourt@utas.edu.au
BMC Proceedings 2011, 5(Suppl 7):O8

**Background:** *Eucalyptus globulus* is the most widely planted species for pulpwood production in temperate regions of the world and there are breeding programs in numerous countries. There is interest in molecular approaches to breeding, particularly marker assisted selection of wood properties. QTL analysis has an important role in identifying positional candidate genes responsible for variation in wood properties. This is one approach to targeting genes which may harbour functional allelic variants (SNPs). The objective of this study was to detect and validate QTL across multiple sites and pedigrees, in order to identify genomic regions and genes affecting growth and wood properties with wide applicability in the species. We also aimed to determine the proportion of QTL which were stable in their expression across sites of contrasting productivity. Such information will be important to exploit the full potential of the impending *Eucalyptus* genome sequences.

**Methods:** Linkage mapping and QTL analysis were conducted in 650 individuals from four separate pedigrees; a clonally replicated outbred F₂ family grown on a single site (112 genotypes), and three F₁ families (180 individuals each) grown at two additional widely separated sites. Trees were assessed for growth, wood density, cellulose content, pulp yield, klason lignin content and the syringyl: guaiacyl (S:G) ratio of lignin at 7 years of age. Chemical traits were estimated using NIR spectroscopy [1]. Saturated linkage maps were constructed in each family, predominantly from Diversity Array Technology (DArT) markers [2] as well as some SSRs, candidate genes and AFLP markers using Joinmap. Common markers were used to construct a consensus map of all families which was used for the QTL analyses. QTL analyses were conducted at various levels (across all families and sites combined, in each family and in each family by site combination) in order to investigate the stability of QTL expression across families and sites.

**Results and discussion:** Ninety eight QTL were detected across all analyses, of which 87 affected wood properties and 11 affected growth. QTL for several different chemical wood properties were co-located, consistent with their high phenotypic correlations. Several of the QTL detected co-located with previously reported QTL in *E. nitens*; *E. globulus*; and *E.grandis* [3-5] and candidate genes including COBL4 [5,6], and CCR [4]. Major QTL were also identified in regions of the genome where no candidate genes or QTL had been previously mapped. Nineteen of the QTL were significant in more than one family, and therefore validated. These validated QTL would be the priority for searches for positional candidate genes in the genome sequence of *Eucalyptus grandis*.

Significant QTL by environment interaction was found for 17 out of the 98 (17%) wood property QTL and for 5 out of 11 (45%) growth QTL. This was consistent with significant family by site interactions exhibited across these sites, particularly for growth. The higher level of QTL by environment interaction for growth compared to wood properties was consistent with quantitative genetic studies in the genus [7]. Nevertheless, there was a surprising proportion of wood property QTL (including validated QTL) which were expressed on one site, but not the other within the same family. While there are few multi-site QTL or association mapping studies in forest trees, in most such studies high levels of QTL (or SNP) by environment interactions have been found [8,9].

**Conclusions:** Comparative mapping demonstrates that the genomes of commercially important eucalypt species (within Symphyomyrtus) are highly syntenic and co-linear (e.g. Hudson in prep), suggesting QTL results may be applicable across eucalypt species. It is therefore important to use transferable markers such as DArTs and SSRs to facilitate comparison of QTL results between studies. Considering the findings of this and previous QTL studies in the genus, growth and wood properties appear to be highly polygenic. Several of the QTL detected co-located with previously reported QTL in *E. nitens*; *E. globulus*; and *E.grandis* and also with some well characterised wood property candidate genes. However, the specificity of many QTL to particular families and sites in this study reinforces the importance of considering the effect of site and genetic setting may complicate the application of marker assisted selection (MAS) or genomic selection.

### References

1. Downes G, Meder R, Hicks C, Ebdon N: Developing and evaluating a multisite and multispecies NIR calibration for the prediction of kraft pulp yield in *eucalyptus*. *Southern Forests* 2009, 71:155-164.

2. Sansaloni C, Petroli C, Carling J, Hudson C, Steane D, Myburg A, Grattapaglia D, Vaillancourt R, Kilian A: A high-density diversity arrays technology (DArT) microarray for genome-wide genotyping in *Eucalyptus*. *Plant Methods* 2010, 6:1-11.

3. Thumma B, Southerton S, Bell J, Owen J, Henery M, Moran G: Quantitative trait locus analysis of wood quality traits in *Eucalyptus nitens*. *Tree Genet Genomes* 2010, 6:305-317.

4. Thamarus K, Groom K, Bradley A, Raymond C, Schimleck L, Williams E, Moran G: Identification of quantitative trait loci for wood and fibre properties in two full-sib pedigrees of *Eucalyptus globulus*. *Theor Appl Genet* 2004, 109:856-864.

5. Grattapaglia D, Bertolucci FL, Penchel R, Sederoff RR: Genetic mapping of quantitative trait loci controlling growth and wood quality traits in *Eucalyptus grandis* using a maternal half-sib family and RAPD markers. *Genetics* 1996, 144:1205-1214.

6. Thumma B, Matheson B, Zhang D, Meeske C, Meder R, Downes G, Southerton S: Identification of a cis-acting regulatory polymorphism in a Eucalyptus COBRA-like gene affecting cellulose content. *Genetics* 2009, 183:1153-1164.
7. Muneri A, Raymond C: Genetic parameters and genotype-by-environment interactions for basic density, pilodyn penetration and stem diameter in *Eucalyptus globulus*. *Forest Genetics* 2000, 7:317-328.

8. Jermstad K, Bassoni D, Jech K, Ritchie G, Wheeler N, Neale D: Mapping of quantitative trait loci controlling adaptive traits in coastal douglas fir. *III. Quantitative trait loci-by-environment interactions*. *Genetics* 2003, 165:1489-1506.

9. Novaes E, Osorio L, Drost D, Miles B, Boaventura-Novaes C, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin T, Peter G, Kirst M: Quantitative genetic analysis of biomass and wood chemistry of *Populus* under different nitrogen levels. *New Phytol* 2009, 182:878-890.

## O9

### SNP discovery and association mapping in Eucalyptus pilularis (blackbutt)

Timothy Sexton¹*, Robert Henry², Chris Harwood³, Dane Thomas⁴,
Luke McManus⁵, Carolyn Raymond⁶, Michael Henson⁷, Mervyn Shepherd¹
¹CRC for Forestry, Centre for Plant Conservation Genetics, Southern Cross
University, Lismore, New South Wales, 2480, Australia; ²Queensland Alliance for
Agriculture and Food Innovation, The University of Queensland Brisbane,
Queensland, 4072, Australia; ³CSIRO Ecosystem Sciences, Private Bag 12, Hobart,
Tasmania, 7001, Australia; ⁴South Australian Research and Development
Institute, Wine Innovation Central Building, Waite Research Precinct, corner of
Hartley Grove and Paratoo Road, Urrbrae, 5063, Australia; ⁵CRC for Forestry,
Forest Molecular Biology and Genetics Group, The University of Melbourne,
Creswick, Victoria, 3363, Australia; ⁶Centre for Plant Conservation Genetics,
Southern Cross University, Lismore, New South Wales, 2480, Australia;
⁷Eucalyptus.biz Limited, Rosemead, Polesden Lane, Ripley, Surrey, GU23 6DX, UK
E-mail: sexton.timothy@gmail.com
BMC Proceedings 2011, 5(Suppl 7):O9

**Background:** This research explores the universality of genetic variation in genes controlling wood formation across the genus *Eucalyptus*.

Breeding and deploying Eucalyptus for improved wood quality is constrained by the delay before wood traits can be measured reliably. Marker assisted selection (MAS) offers a way to make earlier selection of wood properties, by selecting Single Nucleotide Polymorphisms (SNPs) in the DNA which can predict specific phenotypic traits (genotype-phenotype links). SNPs shared between species (trans-specific SNPs) may have broad application to multiple species. When the species are more distantly related, ancient SNPs shared between subgenera (trans-subgeneric SNPs) are likely to be of adaptive importance and persist in separate lineages due to balancing selection [1].

**Sample and methods:** The focal species of this investigation was *Eucalyptus pilularis* of the subgenus *Monocalyp tus*, one of the most important species for solid wood production in Australia. As part of the development of suitable SNP markers for *E. pilularis*, two other important species were included for comparative purposes. The first comparison was made with the closely related *Eucalyptus pyrocarpa*, which grows in parapatry with *E. pilularis* and is distinguished by minor morphological characteristics including a larger leaf and capsule size [2]. More distant comparisons were made between both *Monocalyp tus* species and the alternate *Symphyomyrtus* subgenus, represented by *Eucalyptus globulus* subspecies *globulus*.

DNA was pooled from 30 individuals representing the natural geographical range of each of three species. A total of 34 genes were amplified from each pool and sequenced on an Illumina GAIIx. SNPs were identified in individual species pools and classified as trans-specific if they shared the same reference position in two or more species.

The association study focused on *E. pilularis* and utilized 561 destructively sampled trees from a nine-year-old progeny trial established by Forests NSW at Hannam Vale, near Port Macquarie in New South Wales, Australia (Latitude 31°40', Longitude 152°33'). This sample represented the genetic diversity of 284 open-pollinated families, collected across 37 provenances that encompassed a large part of the natural distribution of this species. We tested 40 traits covering growth, wood chemistry (lignin, cellulose, hemi-cellulose complexes), and wood dimensional stability, strength and stiffness [3]. Eight SNPs were genotyped in pectin methylesterase 6 and 7 (*PME6* and 7) using Sequenom iPLEX gold chemistry. Association testing was performed using a General Linear Model (GLM) in TASSEL where 1,000 permutations were applied. Kinship between family members was a problem and was addressed by the inclusion of a kinship matrix as

covariate data within the model. The large population size used also allowed for validation of results in smaller subsets of the population containing only single representatives from each family. Previous studies have reported no geographical population structure for the provenances included in the breeding program [4].

**Results and discussion:** Among the three species 6,852 SNPs were identified in total. Pairwise comparisons between species representative of divergent subgenera revealed that 20-23% of SNPs were shared. These trans-subgeneric SNPs are likely to have persisted in separate lineages for tens of millions of years following the split of the *Monocalyp tus* and *Symphyomyrtus* subgenera and may be indicative of adaptive variation maintained by "balancing selection". The primary signatures of balancing selection were significantly higher proportions of trans-subgeneric SNPs in exons and promoters, compared to introns and 3' untranslated regions. Further analysis revealed that ten of the 34 genes investigated were likely to be influenced by balancing selection with a significantly higher proportion of trans-subgeneric SNPs and high nucleotide diversities. *PME6* and *PME7* were two of the genes considered likely to be influenced by balancing selection. Association testing revealed several significant correlations between SNPs in these genes and solid wood properties of *E. pilularis*. *PME6* was primarily associated with shrinkage of drying timber. This is consistent with a role for pectin as a hydrophilic polysaccharide, and patterns of methyl-esterification are known to affect the water holding capacity of pectin gels. *PME7* was primarily associated with cellulose and pulp yield, and inversely with lignin content. Selection of specific alleles in these genes may enable identification of trees with superior wood quality for breeding and deployment. For some SNPs, the heterozygote was superior, indicating that selection to fix one allele in the breeding population would not be appropriate. In combination with haplotype blocks, a heterozygote advantage at one or more *PME7* loci may explain the absence of one homozygous class at all SNPs investigated in this gene. Alternatively the absent homozygous genotypes may be disadvantaged during early life stages.

**Conclusions:** Using comparative genomics we provided direct DNA-based evidence that many candidate genes for wood formation are under balancing selection and therefore of adaptive importance. Such knowledge of genetic variation maintained by balancing selection could be critical to ensure that selection for alleles linked to desirable phenotypes does not compromise the maintenance of adaptability in the breeding population. This concept needs to be tested, and our experiments provide a number of suitable markers which could provide the basis for further investigation. Each of these informative SNPs in both *PME* genes could be used for MAS, subject to cost-benefit analysis. Given the large number of trans-subgeneric SNPs identified in these genes, many informative SNPs are likely to transfer to other species, facilitating the development of SNP markers in other commercial species within the diverse *Eucalyptus* genus.

### References

1. Klein J, Sato A, Nagl S, O'hUigin C: Molecular Trans-species Polymorphism. *Annual Review of Ecology and Systematics* 1998, 29(1):1-21.

2. Boland DJ, Brooker MIH, Chippendale GM, Hall N, Hyland BPM, Johnson RD, Kleinig D, McDonald MW, Turner JD: *Forest trees of Australia*. Melbourne: CSIRO Publishing, 5 2006.

3. Raymond CA, Henson M, Pelletier M-C, Boyton S, Joe B, Thomas D, Smith H, Vanclay J: *Improving dimensional stability in plantation-grown *Eucalyptus pilularis* and *E. dunnii*. Melbourne, Victoria: Forest and Wood Products Australia, Project # 06.3017 2008, 1-65.

4. Shepherd M, Sexton TR, Thomas D, Henson M, Henry RJ: Geographical and historical determinants of microsatellite variation in *Eucalyptus pilularis*. *Canadian Journal of Forest Research* 2010, 40(6):1051-1063.

## O10

### Comparative genomics of resistance to spruce to the white pine weevil in British Columbia

Kermit Ritland¹*, Sébastien Verne¹, Barry Jaquish², Carol Ritland¹
¹University of British Columbia, USA; ²Ministry of Forests, Lands and Natural Resource Operations, USA
E-mail: kermit.ritland@ubc.ca
BMC Proceedings 2011, 5(Suppl 7):O10

We present results of two large scale comparative studies of the genomic basis of resistance of Interior spruce to the white pine weevil.
Both volume growth, the main objective of the spruce breeding program in British Columbia, and white pine weevil resistance, are examined. "Interior spruce" is a species complex involving mainly *Picea glauca* (White spruce) but introgressed with *P. engelmannii* (Englemann spruce), depending upon locality. In the first study, we compared constitutive expression of 17825 genes between 20 resistant and 20 susceptible trees to the weevil; 54 upregulated and 137 downregulated genes were found in resistant phenotypes, with implications discussed in regard to volume growth. In particular, we will be surveying these genes for SNPs that differ between these two classes of trees in the next year. In the second study, we developed a 1536 Illumina SNP chip based upon candidate genes for weevil resistance. In a novel experimental design, we assayed 945 open-pollinated progeny of the Prince George breeding population (176 parents), and 654 open-pollinated progeny of the Prince Rupert breeding population (134 parents); parents were also genotyped. Within each family of 100 progeny, we identified the highest ranked 3 progeny and the lowest ranked 3 progeny, based upon BC Ministry of Forests scores for volume growth and resistance. These were genotyped and used in a novel test analogous to the transmission disequilibrium test to detect both SNP associations and QTLs linked to SNP markers. Discoveries about associations and QTL are discussed, with the added caution about genotyping error. Both studies illustrate how operational tree breeding populations can provide valuable inferences about tree genomics.

## O11

### Using the *Eucalyptus* genome to understand the evolution of plant secondary metabolites in the *Myrtaceae*

Carsten Kulheim¹, Hamish Webb, Suat Hui Yeoh, Ian Wallis, Gavin Moran, William Foley

Australian National University, Australia

E-mail: carsten.kulheim@anu.edu.au

BMC Proceedings 2011, 5(Suppl 7):O11

**Background:** *Eucalyptus* trees (family Myrtaceae) are well-known for their high foliar content of several classes of secondary metabolites and these have a strong effect on the feeding patterns of several species of marsupials and at least some insects. Best known are the essential oils, which is mostly a mixture of terpenoids, but there are also significant concentrations of flavonoid and formylated phloroglucinol compounds. There is extensive quantitative and qualitative variation within and between species of Myrtaceae in these chemical groups and all appear to be under strong genetic control with heritabilities (H²) between 0.3 and 0.9. As well as being important ecologically, the terpenes in particular are valued as industrial and medicinal products and Australia supports a strong essential oil industry focused on *Eucalyptus* and *Melaleuca* foliar oils.

**Results and discussion:** The *Eucalyptus grandis* genome provides the opportunity to discover the genetic makeup of the biosynthetic pathways for secondary metabolites. We present data from pathways leading into the biosynthesis of terpenes, flavonoids and lignins. The homology of genes and gene families were investigated and compared to a variety of other species including poplar (*Populus trichocarpa*), grape (*Vitis vinifera*) and apple (*Malus x domesticus*). For example, terpene syntheses (the gene family responsible for the final step in the terpene biosynthesis) has 120 members in the genome of *Eucalyptus grandis*, compared to 44 and 99 in poplar and grape, respectively (Table 1). Genes of the biosynthetic pathways for secondary metabolites were mapped to the *Eucalyptus grandis* genome and their location was compared to a number of

quantitative trait loci (QTL) studies that investigated variability in secondary metabolites and wood properties in eucalypts. This approach allowed the discovery of candidate genes for a large number of QTL. Understanding the genetic basis of variations in quantitative traits provides insights into ecosystem function and at the same time may help breeders in the essential oil industry. We have characterized trait associations with polymorphisms from *Eucalyptus globulus*, investigating 200 SNPs and roughly 40 traits ranging from terpenoids to terpene-adducts to flavonoids and to tannin-related traits. We discovered several significant trait associations between allelic variants in the chloroplastic MEP pathway and monoterpenes and between the cytosolic MVA pathway and sesquiterpenes, as well as one allelic variant in a prenyl pyrophosphate synthase that associates with the ratio of monoterpenes to sesquiterpenes. Loci with significant associations were mapped to the *Eucalyptus grandis* genome and compared to published QTL datasets that investigated similar traits. These results represent the first species wide analysis of the molecular basis of quantitative variation in secondary metabolites in any tree.

**Conclusions:** The publicly available genome sequence of *Eucalyptus grandis* is a great resource that can be applied to a variety of questions including the genetic make-up of gene families for biosynthesis of plant secondary metabolites, genome organization of these genes and evolution of traits such as resistance to herbivores or the ability to re-sprout after fire. Combining studies of association genetics, QTL studies together with the genome sequence helps to shed light on the underlying control mechanisms of phenotypic variation.

## O12

### Functional variability of two lignification genes in *Eucalyptus urophylla*

Eric Mandrou¹, Frédéric Mortier², Marie Denis², Gilles Chaix², Emilie Villar³,

Christophe Plomion⁴, Jean-Marc Gion²

¹INRA-CIRAD, France; ²CIRAD, France; ³CIRAD-INRA, France; ⁴INRA, France

E-mail: gion@cirad.fr

BMC Proceedings 2011, 5(Suppl 7):O12

Lignin quantity and composition are major components of wood quality in eucalypts breeding programs. Optimizing these traits can have a huge economical impact for charcoal and pulp production. However, little is known about the genetic determinism of these traits. Our objective is to establish efficient early selection criteria to identify ideotypes for these traits using gene based markers. To reach this goal, we first estimated genetic parameters for lignin quantity and composition using a factorial design comprising 16 founders and 328 progenies of *E. urophylla*. We found high heritability for both Klason Lignin (hÅ²=0.85) and Syringyl to Guaiacyl ratio (S/G: hÅ²=0.62). Then, nucleotide and haplotype diversities were described for two linked genes involved in the lignification process (CCR and ROP1). High levels of nucleotide diversity and rapid decay of linkage disequilibrium were observed in a sample of 16 trees. A SNP by SNP association mapping was carried out in the 328 progenies using a mixed linear model (available in Tassel). A total of 4 SNP (3 in CCR and one in ROP1) were significantly associated with S/G ratio explaining between 1 and 1.8% of the trait variation. Finally, the additive effects of haplotypes (defined by all SNPs) were estimated using a Bayesian approach of the mixed model including the pedigree (MCMCglmm package available in R software). This analysis confirmed the existence of two distinct additive effects for CCR and ROP1 on S/G ratio, suggesting the existence of two S/G QTLs for these two linked genes in *E. urophylla*.

<table><caption>**Table 1(abstract O11) Number of TPS loci in annotated genomes and putative loci in *E. grandis***</caption><thead><tr><th></th><th>A. thaliana</th><th>P. trichocarpa</th><th>O. sativa</th><th>V. vinifera</th><th>E. grandis</th></tr></thead><tbody><tr><td>TPS-a (sesqui)</td><td>23</td><td>21</td><td>31</td><td>42</td><td>53</td></tr><tr><td>TPS-b (mono)</td><td>6</td><td>13</td><td>0</td><td>30</td><td>38</td></tr><tr><td>TPS-b (hemi)</td><td>0</td><td>3</td><td>0</td><td>3</td><td>11</td></tr><tr><td>TPS-c, -e, -f</td><td>3</td><td>5</td><td>13</td><td>8</td><td>2</td></tr><tr><td>TPS-g (mono)</td><td>1</td><td>2</td><td>2</td><td>16</td><td>16</td></tr><tr><td>Sum of TPS genes</td><td>33</td><td>44</td><td>46</td><td>99</td><td>120</td></tr></tbody></table>