# S3. GENOMICS ASSISTED BREEDING

## O13

### Genomic selection in tree breeding: testing accuracy of prediction models including dominance effect

Marie Denis*, Jean-Marc Bouvet

CIRAD - Research unit 108 "Breeding and adaptation of tropical and Mediterranean plants", International campus of Baillarguet TA A-39/C, 34398

Montpellier, Cedex 5, France

E-mail: marie.denis@cirad.fr

BMC Proceedings 2011, 5(Suppl 7):O13

**Background:** The concept of Marker Assisted Selection (MAS) is rapidly evolving in animal and plant breeding. With the advent of high throughput molecular technology, numerous molecular markers distributed throughout the whole genome can be produced to characterize many genetic entries involving new perspectives in methodology of selection. An important research activity has begun in the animal world given the first theoretical framework for a methodology called genomic selection (GS) [1]. Several statistical approaches have been proposed for the prediction of genomic breeding values and numerous results are available that validates the interest of this method in animal breeding. In plants the GS is still limited to very advanced model species involved in genetic improvement and especially from scenario-based simulation [2,3].

In tree breeding the GS could significantly reduce the cost of genetic improvement schemes by limiting the size and number of field experiments; and facilitating the early selection at the nursery stage [4]. If most of the studies on GS have addressed the prediction of breeding value, taking into account the gene additive effects, there is still a lack of analyses dealing with the total genetic value (genotypic value) including both additive and dominance effects. This aspect is important in plant and especially in tree breeding where the goal of some programs is the production of clones or elite families. The aim of this study is to investigate the performance of GS in the context of tree breeding when the selection is based on genotypic value. The proposed approach allows taking into account both additive and dominance effect [5] for each marker in the statistical model. Six scenarios are simulated to test the reliability of the GS in the frame of recurrent selection scheme.

**Methods:** **Simulation:** The data used to evaluate the accuracy of the model have been simulated using HaploSim package in R software [6]. Firstly, populations were simulated for 1000 generations at an effective size of 100 to reach a mutation-drift balance. Fifty parent trees were then selected to start a breeding scheme that was conducted during two generations. At each generation, a progeny test was implemented using a factorial mating design. The fifty percent parents were selected and crossed using circular design to constitute the following generation. At each generation, 670 individuals issued from the mating of 16 females and 34 males were evaluated for clonal selection. The 670 individuals were genotyped for 400 SNP markers equally-spaced across one chromosome of one Morgan corresponding to an efficient marker density.

The broad sense heritability HÅ² was equal to 0.3. A gamma distribution was used to sample the 44 QTL effects.

The additive (breeding), dominance and genotypic values were simulated for each individual. The ratio of dominance to additive variance was equal to 0.1, 0.5 and 1. Six scenarios were evaluated for predicting the genotypic value: three different ratios and two different QTL distributions (high proportion with small or medium effects).

**Analysis model:** Genomic selection consists in following steps: (i) estimation of the effects of all markers in a 'training data set', where the individuals are phenotyped and genotyped; (ii) prediction of the genetic values of other 'evaluation' individuals by combining their marker genotypes with the estimates obtained in step (i).

A Bayesian implementation of the Lasso method with BLR package in R [7,8] was used to estimate the substitution and dominance effects for each of the 400 SNP. This method allows predicting the genotypic value using all markers simultaneously with different variances for each marker effect. We evaluated the performance of the statistical model with and without dominance effects. The training and validation set corresponded, respectively, to the first and second generation containing each 670 individuals. The criterion to compare the different scenarios was the accuracy calculated as the correlation between true and predicted genotypic value. Each simulated data set and analysis was replicated 30 times.

**Results:** The accuracy of two models decreases when the ratio of dominance to additive variance increases whatever the QTL distribution (table 1). In all scenarios GS is superior to basic phenotypic selection. In addition, the model with dominance effects shows a higher accuracy, especially when the variance ratio increases. For the second generation the same trend is observed but the model with dominance effects is more accurate when the ratio of variances is greater than 0.5. A lower accuracy is observed in the second generation; it can be attributed to the low linkage disequilibrium of this breeding population two generations after selection in simulated wild population.

**Conclusions:** The model including dominance effects is more accurate to predict the genotypic value especially when the dominance-additive variance ratio increases. These results are particularly interesting for tree improvement in hybrid populations where dominance effects are marked and clonal varieties are produced (eucalyptus, poplar, for example).

### References

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

2. Bernardo R, Yu JM: Prospects for genomewide selection for quantitative traits in maize. *Crop Sci* 2007, 47:1082-1090.

3. Heffner EL, Sorrells ME, Jannink JL: Genomic Selection for Crop Improvement. *Crop Sci* 2009, 49:1-12.

4. Grattapaglia D, Resende MDV: Genomic selection in forest tree breeding. *Tree Genetics and Genomes* 2011, 7:241-255.

5. Toro MA, Varona L: A note on mate allocation for dominance handling in genomic selection. *Genet Sel Evo* 2010, 42:1-33.

6. Coster A, Bastiaansen J: HaploSim: HaploSim 2009.

<table><caption>**Table 1(abstract O13) Accuracy (se) of GS in the first and second generation without and with dominance effects for six different scenarios and accuracy of phenotypic selection (30 replicates)**</caption><thead><tr><th rowspan="2">ratio</th><th colspan="3">QTL distribution: small effects</th><th colspan="3">QTL distribution: medium effect</th></tr><tr><th>Dominance variance/additive variance</th><th>Phenotypic selection</th><th>Model without dominance</th><th>Model with dominance</th><th>Phenotypic selection</th><th>Model without dominance</th><th>Model with dominance</th></tr></thead><tbody><tr><th colspan="8">First generation</th></tr><tr><td>0.1</td><td>0.52(0.03)</td><td>0.80(0.03)</td><td>0.81 (0.03)</td><td>0.53(0.03)</td><td>0.79 (0.03)</td><td>0.80(0.03)</td></tr><tr><td>0.5</td><td>0.51(0.03)</td><td>0.70 (0.05)</td><td>0.73 (0.04)</td><td>0.53(0.04)</td><td>0.68 (0.04)</td><td>0.73(0.04)</td></tr><tr><td>1</td><td>0.51(0.03)</td><td>0.61(0.06)</td><td>0.69(0.05)</td><td>0.53(0.03)</td><td>0.64(0.04)</td><td>0.71(0.04)</td></tr><tr><th colspan="8">Second generation</th></tr><tr><td>0.1</td><td>NO</td><td>0.63(0.13)</td><td>0.63(0.13)</td><td>NO</td><td>0.62(0.08)</td><td>0.62(0.08)</td></tr><tr><td>0.5</td><td>NO</td><td>0.53(0.13)</td><td>0.55(0.12)</td><td>NO</td><td>0.53(0.11)</td><td>0.54(0.10)</td></tr><tr><td>1</td><td>NO</td><td>0.42(0.13)</td><td>0.48(0.13)</td><td>NO</td><td>0.42(0.10)</td><td>0.47(0.9)</td></tr></tbody></table>

NO: not observed.
7. De los Campos G, Naya H, Gianola D, Crossa J, Legarra A, Manfredi E, Weigel K, Cotes JM: Predicting quantitative traits with regression models for dense molecular markers and pedigrees. *Genetics* 2009, **182**:375-385.

8. De los Campos G, Pérez P: BLR: Bayesian Linear Regression. 2010, R package version 1.1.

O14

Stability of Genomic Selection prediction models across ages and environments

Marcio FR Resende Jr.¹*, Patricio R Muñoz Del Valle², Juan J Acosta³,
Marcos DV Resende⁴, Dario Grattapaglia⁵, Matias Kirst³

¹Genetics and Genomics Graduate Program, University of Florida, Gainesville, FL, USA; ²Plant Molecular and Cellular Biology Graduate Program, Gainesville, FL, USA; ³School of Forest Resources and Conservation, University of Florida, Gainesville, FL, USA; ⁴EMBRAPA Forestry, Colombo, Parana, Brazil; ⁵EMBRAPA Genetic Resources and Biotechnology, Brazilia, DF, Brazil

E-mail: mresende@ufl.edu

BMC Proceedings 2011, 5(Suppl 7):O14

**Background:** A tree breeding program is characterized by long generation intervals which, over time, result in a much smaller number of breeding cycles when compared to annual crops. Moreover, most economically important traits in a tree-breeding program are quantitatively inherited, display low heritability and are expressed late in the life cycle. Genomic Selection (GS) is expected to be particularly valuable for tree species, leading to shorter generation intervals and improved genetic gain over time.

The main factors that affect the accuracy of GS prediction models are the level of linkage disequilibrium (LD) in the training population, the training population size, the heritability of the trait and the number of QTL regulating its variation. However, it is yet largely unknown how stable prediction models are across environments and different ages. This knowledge is critical for tree breeders that wish to use genomic selection in their genetic improvement program.

Here, we report the first assessment of the utility of genomic selection in a conifer species. We developed prediction models for growth traits measured at multiple sites, to evaluate the impact of genotype by environment interactions in their accuracy. Training populations were also measured over multiple ages and models were developed to assess their value in predicting breeding values later in the lifecycle.

**Material and methods:** Here we analyzed a population of 790 to 840 individuals of loblolly pine, clonally replicated in four sites in the southeastern US: Palatka and Nassau (Florida, USA), Cuthbert and B.F. Grant (Georgia, USA). The population is derived from 61 full-sib families, established by crossing 32 parents in a circular mating design. The traits analyzed in this study were diameter at breast height (DBH) measured when trees were three, four and six years old; and total height (HT) measured when trees were one, two, three, four and six years old. All the individuals were genotyped with a total of 3,938 SNPs [1]. Single marker regression association analyses were initially performed treating the markers as fixed effects. The markers that were selected in this association analysis had their effects estimated adjusting all the allelic effects simultaneously using a genomic BLUP procedure [2]. These analyses were performed across all sites, traits and ages and the estimated effects of the markers were validated using a 10-fold cross validation approach. The selection gain of genomic selection was compared to classical phenotypic selection considering a reduced breeding cycle due to early selection.

**Results and discussion:** The accuracies of the prediction models for GS developed using phenotypic data measured in each site at year 6 ranged from 0.65-0.75 for DBH, and 0.64-0.77 for HT. To evaluate the performance of GS relative to traditional breeding methods, we estimated the accuracies of BLUP-based selection [3] and used it as a benchmark for the comparison of the accuracies obtained by GS. The increase in efficiency per unit of time in the selection response of GS was 53-95% higher for DBH, and 58-118% higher for HT, assuming a conservative reduction of 50% in the length of the breeding cycle.

To evaluate if models generated at early ages would predict well the phenotype at mid-rotation, we assessed the accuracy of models developed for HT based on data collected at ages 1 to 4, but validated with measurements from the same populations at age 6. Accelerating model estimation is beneficial because the sooner models that accurately predict

phenotypes at rotation age can be developed, the faster genomic selection can be adopted. However, the models developed for HT early in the rotation (age 1 to 3) had limited accuracy in predicting phenotypes at age 6.

Next, we tested the suitability of models estimated in each individual site, in predicting phenotypes across different sites. The accuracies reduced up to 86% (Table 1) and the decrease parallels the increase in geographic distance between the site for which models were estimated, and the site where they were validated. Therefore, environment × genotype interactions appear to severely affect the transferability of models across breeding zones.

Out of the total number of markers, 177 and 83 were significantly associated with traits across all sites for DBH and HT, respectively. Those were selected for model estimation since they may reflect marker-trait associations that are independent, or at least less affected, by genotype × environment interactions. Initially, models were re-estimated based on data from each site, using only the subset of markers, and re-validated in the same site. Accuracies were only slightly lower (3–9% for DBH, 6–13% for HT) than those obtained when models included the full set of significant makers (Figure 2). Therefore, a significant component of the genetic variance appears to be captured by relatively few markers (~100). Next, prediction models were estimated based on a combine-site model and validated individually in each of the four sites (Figure 1). While accuracies remained relatively high across sites (0.43–0.51 for DBH, 0.46–0.54 for HT), there is a decrease of up to 30% relative to the accuracy obtained when the full set of markers and site-specific specific data was used for model estimation (Figure 1). Therefore, while the same reduced set of markers is relevant for explaining the genetic variation in all sites, the magnitude of their effect changes significantly from site to site.

In conclusion, the results in efficiency demonstrated that incorporating genomic selection would dramatically increase the genetic gains per unit of time of a conifer's breeding program. Moreover, even at relatively low marker density, the accuracy of prediction models could significantly impact the genetic gain efficiency. However, the use of a prediction model should be constrained within a breeding zone once genotype × environment can affect the prediction and reduce the accuracy of those models.

<figure><img src="image_10.png" /><figcaption>**Figure 1(abstract O14)** Accuracy of genomic selection models estimated using the complete (black bar) or subset of markers significant across all sites (white and grey bars) for DBH (upper panel) and HY (lower panel). Prediction models were estimated and validated in each individual site (black and white bars), or estimated based on a combined-site model and validated in individual sites (grey bar).</figcaption></figure>
## References

1. Eckert AJ, van Heerwaarden J, Wegrzyn JL, et al: Patterns of Population Structure and Environmental Associations to Aridity Across the Range of Loblolly Pine (Pinus taeda L., Pinaceae). *Genetics* 2010, 185:969-982.

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

3. Grattapaglia D, Resende M: Genomic selection in forest tree breeding. *Tree Genetics & Genomes* 2010, 1-15.

## O15

### Candidate gene-based association mapping of growth and wood quality traits in *Eucalyptus globulus* Labill

Saravanan Thavamanikumar¹, Josquin Tibbits², Luke McManus¹, Peter Ades¹, Desmond Stackpole³, Sara Hadjigol⁴, René Vaillancourt⁴, Peng Zhu², Gerd Bossinger¹

¹Department of Forest and Ecosystem Science, The University of Melbourne, Water Street, Creswick, Victoria 3363, Australia and Co-operative Research Centre for Forestry, Private Bag 12, Hobart 7001, Tasmania, Australia;

²Department of Forest and Ecosystem Science, The University of Melbourne, Water Street, Creswick, Victoria 3363, Australia. Present address: Department of Primary Industries, Biosciences Research Division, Victorian AgriBiosciences Centre, 1 Park Drive, La, Australia; ³School of Plant Sciences, University of Tasmania, Private Bag 55, Hobart 7001 Tasmania, Australia. Present address: Scion, Te Papa Tipu Innovation Park, 49 Sala Street, Rotorua 3010, Private Bag 3020, Rotorua 3046, New Zealand; ⁴School of Plant Sciences, University of Tasmania, Private Bag 55, Hobart 7001 Tasmania, Australia and Co-operative Research Centre for Forestry, Private Bag 12, Hobart 7001, Tasmania, Australia; ⁵South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong 510650, People Republic of China
E-mail: saro.t@unimelb.edu.au

BMC Proceedings 2011, 5(Suppl 7):O15

**Background:** The identification of polymorphisms that underlie complex phenotypic traits presents new and exciting challenges to molecular genetics. Association mapping, which uses linkage disequilibrium (LD) to map trait variation with nucleotide polymorphisms, has proved suited for this purpose in outcrossed tree species. Association mapping can be undertaken either at a candidate gene level or at the whole-genome level however; until sufficiently dense marker assays are developed the candidate gene approach will remain the most effective way of dissecting complex traits in tree species. To date, this approach has led to the identification of several quantitative trait nucleotides (QTN) that associate with a variety of breeding traits including, early wood specific gravity, percentage latewood [1], microfibril angle [2], cellulose [3]and carbon isotope discrimination [4].

**Aim:** Our research aims to identify single nucleotide polymorphism (SNP) markers using candidate gene-based association mapping that can predict growth and wood quality in *Eucalyptus globulus*.

**Materials and methods:** A *Eucalyptus globulus* provenance-progeny trial, planted in 1989 near Latrobe in north-central Tasmania, Australia, by Gunnis Ltd, was used as the association discovery population. An eight-year-old Southern Tree Breeding Association (STBA) breeding trial growing near Frankland, Western Australia, is being used for validating marker-trait associations. Twenty functional candidate genes for wood and fiber formation, were selected for this study. SNPs were discovered by direct sequencing of PCR products from 11 to 28 trees.

The iPLEX Gold assay (Sequenom Inc.) was used to genotype 98 selected polymorphisms in up to 385 individuals from the discovery population. This subset includes individuals from eight races of *E. globulus*. Linkage disequilibrium between SNPs was estimated using GEVALT [5] and Hardy-Weinberg equilibrium was estimated using FSTAT ver 2.9.3.2 [6] respectively. To account for genetic structure, 18 SSR markers were genotyped in all the discovery samples. Ancestry (Q) co-efficients were estimated using the model-based clustering method as implemented in STRUCTURE [7]. A matrix of pairwise kinship coefficients (K) was calculated as described in Ritland [8], using the software SpaGeDi [9]. Marker-trait associations were tested using a mixed linear model (MLM) [10], which accounted for both population structure and familial relatedness using TASSEL version 2.0.1 [11].

**Results:** At a 5% significance level, only 4 SNPs deviated from Hardy Weinberg expectations in more than one race. With all races pooled, LD between SNPs was very low with only 1.8% of the pairwise comparisons having r² values greater than 0.33. Only where r² is greater than 0.33 is

there sufficiently strong LD to be useful for association mapping [12]. Only one pair of SNPs, *EgCSA3_4186* and *EgMYB2_1380* between genes was found to be in LD (r² > 0.5). When LD was computed within races 1.3 to 2.7% of the pairwise comparison had r² values greater than 0.33.

Of the 98 polymorphisms tested against 12 traits, 33 associated significantly (P < 0.05) with one or more traits giving a total of 62 associations. Individual polymorphisms explained between 0.9 and 3.8% of the phenotypic variation observed. Marker-trait associations found in the discovery population are currently being validated by testing their consistency using the validation population.

**Conclusions:** Candidate gene based association mapping studies are a useful means of dissecting complex quantitative traits in species like *E. globulus* with low LD and high nucleotide diversity. This study, like other tree association mapping studies, shows that the percentage of phenotypic variation explained by a single polymorphism will often be small. This is not unexpected because of the complex nature of most wood quality traits. The small proportion of the phenotypic variation explained so far using association mapping should not detract from its future use in tree breeding, since genotyping costs are expected to fall while throughput increases, thereby facilitating larger scale association mapping efforts in the near future.

**References**

1. Gonzalez-Martinez SC, Wheeler NC, Ersoz E, Nelson CD, Neale DB: *Association Genetics in Pinus taeda* L. I. Wood Property Traits. *Genetics* 2007, 175:399-409.

2. Thumma BR, Nolan MR, Evans R, Moran GF: Polymorphisms in cinnamoyl CoA reductase (CCR) are associated with variation in microfibril angle in *Eucalyptus* spp. *Genetics* 2005, 171:1257-1265.

3. Thumma BR, Matheson BA, Zhang D, Meeske C, Meder R, Downes GM, Southerton SG: Identification of a Cis-Acting Regulatory Polymorphism in a Eucalypt COBRA-Like Gene Affecting Cellulose Content. *Genetics* 2009, 183:1153-1164.

4. Gonzalez-Martinez SC, Huber D, Ersoz E, Davis JM, Neale DB: *Association genetics in Pinus taeda* L. II. Carbon isotope discrimination. *Heredity* 2008, 101:19-26.

5. Davidovich O, Kimmel G, Shamir R: GEVALT: An integrated software tool for genotype analysis. *Bmc Bioinformatics* 2007, 8.

6. Goudet J: FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3.2). 2002. Available from http://www2unilch popgen/software/fstat.htm

7. Pritchard JK, Stephens M, Donnelly P: Inference of Population Structure Using Multilocus Genotype Data. *Genetics* 2000, 155:945-959.

8. Ritland K: Estimators for pairwise relatedness and individual inbreeding coefficients. *Genetical Research* 1996, 67:175-185.

9. Hardy OJ, Vekemans X: SPAGEDI: a versatile computer program to analyse spatial genetic structure at the individual or population levels. *Molecular Ecology Notes* 2002, 2:618-620.

10. Yu JM, Pressoir G, Briggs WH, Bi IV, Yamasaki M, Doebley JF, McMullen MD, Ault BS, Nielsen DM, Holland JB, et al: A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. *Nature Genetics* 2006, 38:203-208.

11. Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ram-doss Y, Buckler ES: TASSEL: Software for Association Mapping of Complex Traits in Diverse Samples. *Bioinformatics* 2007, 23:2633-2635.

12. Ardlie KG, Kruglyak L, Seielstad M: Patterns of linkage disequilibrium in the human genome. *Nat Rev Genet* 2002, 3:299-309.

## O16

### Genomic Selection for growth traits in *Eucalyptus*: accuracy within and across breeding populations

Dario Grattapaglia¹, Marcos Deon Vilela Resende², Márcio Ribeiro Resende³, Carolina Paola Sansaloni⁴*, Cesar Daniel Petrolli⁴, Alexandre Alves Missiaggia⁵, Elisabete Keiko Takahashi⁶, Karina Carnielli Zamprogno⁷, Andrzej Kilian⁸

¹EMBRAPA Genetic Resources and Biotechnology - Estação Parque Biológico, 70770-910, Brazilia, DF, Brazil; ²EMBRAPA Forestry Research, Colombo, PR, 83411-000, Brazil; ³Universidade Federal de Viçosa - Bioagro, Viçosa MG, 36570-000, Brazil; ⁴Universidade de Brazilia - Campus Darcy Ribeiro Braslia, DF, 70910-900, Brazil; ⁵FIBRIA, Rod. Aracruz/Barra do Riacho, km 25, Aracruz, ES, 29197-900, Brazil; ⁶CENIBRA Celulose Nipo Brazileira S.A, Belo Oriente, MG, 35196-000, Brazil; ⁷VERACEL Celulose S.A, Eunápolis, BA, 45820-970, Brazil; ⁸DARt - Diversity Arrays Technology, POB 7141, Yarralumla, ACT, 2600, Australia

E-mail: carosansalon1@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):O16
**Background:** Genomic selection (GS) involves selection decisions based on genomic breeding values estimated as the sum of the effects of genome-wide markers capturing most QTLs for the target trait(s). GS is revolutionizing breeding practice for complex trait in domestic animals. The same approach and concepts can be readily applied to forest tree breeding. Trees also have long generation times and late expressing traits. Differently from association genetics that aims at dissecting complex traits in their discrete components, GS precludes the discovery of individual marker-trait associations and focuses on prediction of performance. By capturing the "missing heritability" of complex quantitative traits beyond the few effect variants that association genetics has so far typically identified, GS might soon cause a paradigm shift in forest tree breeding. In a prior deterministic study we assessed the impact of linkage disequilibrium (modeled by Ne and inter-marker distance), the size of the training set, trait heritability and the number of QTL on the predicted accuracy of GS [1]. Results indicate that GS has the potential to radically improve the efficiency of tree breeding. The benchmark accuracy of conventional BLUP-based phenotypic selection (0.68) was reached by GS even at a marker density ~2 markers/cM when Ne ≤30, while up to 10 markers/cM are necessary for larger Ne. Shortening the breeding cycle by 50% with GS provides an expected increase ≥100% in selection efficiency. To validate these simulation results we carried out a large multi-population proof-of-concept study of GS in tropical Eucalyptus. In this report we present results of this on-going study for two populations and three different quantitative traits.

**Methods:** This study was carried out in two structured populations of Eucalyptus represented by two progeny trials of independent breeding programs. The first was a progeny trial developed by CENIBRA (CEN) with 43 full-sib families generated by an incomplete diallel mating design carried out by intercrossing 11 highly selected *E. grandis* x *E. urophylla* hybrids clones therefore having an effective population size of Ne= 11. The second population was a progeny trial installed by FIBRIA (FIB) with 232 full-sib families involving 120 elite parents, the vast majority F1 hybrids of *E. grandis*, *E. urophylla*, *E. globulus* and *E. maidenii*. For the GS study however only the most phenotypically relevant 75 families were used derived from intercrossing 55 parents therefore limiting the effective population size to Ne= 55. A sample of trees was taken in a stratified way from all families to provide a balanced representation of all families with similar numbers of individuals per family. Bark or leaf samples were collected, DNA extracted and genotypes obtained for 783 and 920 trees from CEN and FIB respectively. Genotyping was carried out using a high-throughput genotyping platform developed earlier [2] that provided 3,120 and 3,564 robustly scored dominant DArT markers. De-regressed phenotypes for Height (H), Diameter at Breast Height (DBH), wood density (WD), pulp yield, lignin content and *Puccinia* rust resistance were obtained. Single marker regression association analyses were initially performed with all the traits, treating the markers as fixed effects. Markers selected in the previous association analysis had their effects estimated adjusting all the allelic effects simultaneously using Random Regression Best Linear Unbiased Predictor. Each population was then divided in a training set comprising 90% of the total number of individuals and a validation set with the other 10%. The estimated effects of the markers were validated using a cross validation approach using random sub-sampling with ten replications so that all individuals had their phenotypes predicted by GS. Details of the overall analytical approach have been described [3,4].

**Results and conclusions:** To date results have been obtained for height, DBH and wood density while the remaining traits are under analysis. Realized accuracies by Genomic Estimated Breeding Values (GEBV) for H and DBH were 0.67 and 0.69 for CEN and 0.62 and 0.54 for FIB while for WD in FIB it was 0.53. These accuracies match or surpass those obtained by conventional BLUP (Best Linear Unbiased Prediction) based phenotypic selection. Not surprising GEBV accuracies were low (~0.18) across populations implying variable genotype-phenotype associations across backgrounds so that population-specific GS models will be necessary. GS-based reduction in breeding time by 50%, i.e. by reducing a breeding generation time from 12 to 6 years should provide gains ≥100% in selection efficiencies. If a reduction from 12 to 3 years using flower induction can be achieved, gains in selection efficiency theoretically could surpass 300%. These are among the first experimental results of GS in forest trees and in plants in general. With the technological advances and declining costs of genotyping methods we anticipate that GS will soon be implemented operationally and revolutionize *Eucalyptus* tree breeding practice.

**Acknowledgments:** Brazilian Ministry of Science and Technology (CNPq Grant 577047-2008-6), FAP-DF NEXTREE Grant 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004.

**References**

1. Grattapaglia D, Resende MDV: Genomic selection in forest tree breeding. *Tree Genetics & Genomes* 2011, 7(2):241-255.

2. Sansaloni CP, Petroli CD, Carling J, Hudson CJ, Steane DA, Myburg AA, Grattapaglia D, Vaillancourt RE, Kilian A: A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in Eucalyptus. *Plant Methods* 2010, 6:16.

3. Resende MDV, Lopes PS, Silva RL, Pires IL: Seleção genômica ampla (GWS) e maximização da eficiência do melhoramento genético. *Pesquisa Florestal Brazileira* 2008, 56:63-77.

4. Resende MDV, Resende MF, Aguiar AM, Abad JIM, Missiaggia AA, Sansaloni C, Petroli C, Grattapaglia D: Computação da Seleção Genômica Ampla (GWS). *Série Documentos da EMBRAPA Florestas* 2010, 209:78.

**O17**

**High-throughput targeted SNP discovery using Next Generation Sequencing (NGS) in few selected candidate genes in *Eucalyptus camaldulensis***

Prasad Suresh Hendre*, Rathinam Kamalakannan, Rathinavelu Rajkumar, Mohan Varghese

ITC R&D Centre, Peenya Industrial Area, No.3, 1st Main, 1st Phase, Bangalore- 560 058, Karnataka, India

E-mail: prasad.hendre@itc.in

BMC Proceedings 2011, 5(Suppl 7):O17

**Background:** The present era of high throughput technologies offer immense promise and innovative applications for SNP discovery and high quality parallel genotyping [1,2]. Using advancements in the next generation sequencing (NGS) technologies, the *en masse* SNP discovery for targeted genomic regions is possible for eucalypts. The river red gum or *Eucalyptus camaldulensis* (*Ec*) is a fast growing, hardy and highly adaptable eucalypt species acclimatized to Indian climatic conditions and these new advancements would aid in developing new tools and techniques for its improvement. In our knowledge, limited efforts have been undertaken to identify SNP markers in eucalypts either by employing RNA sequencing [3] or by using few genes available in the literature [4]. Despite these minuscule efforts, useful SNP markers were discovered in *Cinnamoyl CoA Reductase (CCR)* gene with potential application [5]. Using the recently released whole genome sequence of *E. grandis* (*Eg*), herein we describe targeted SNP discovery in 41 candidate genes by employing Illumina's 72-bases paired end sequencing technology.

**Materials and methods:** The DNA was isolated from a SNP discovery panel consisting 96 individuals from a naturally mating *Ec* population from Australia following standard procedures (modified CTAB method). Twelve primary DNA pools were constituted by mixing equimolar concentrations of eight DNAs @ 10 ng/mL. Forty one genes selected for SNP discovery were identified from *Eg* genome (http://eucalyptusdb.bi.up.ac.za/gbrowse8x) by employing Arabidopsis TAIR 9 gene IDs. Further the primer pairs were designed to amplify the gene fragments. The individual primary DNA pool was amplified (Veriti-ABI) using *Paq* DNA polymerase (Agilent Technologies), all amplicons pooled (figure 1), eluted if necessary (*EcCRE-AHK4*, *ECOBP1*, precipitated using ethanol and dissolved in TE (0.1)).

A paired end library suitable for 72-bases read length was prepared and sequenced on an Illumina GAIIx sequencer and analyzed using bwa and samtools with appropriate parameters (outsourced to Genotypic Technologies Ltd, Bangalore). The SNP data was adjusted for read depth (1/10th SD) and rare allele frequency (<5%). Further approximate equal frequency (EF) blocks were manually estimated by nearest neighborhood (NN) analysis in MS Excel (MS Office 2007), wherein, a block of NN SNPs having frequency difference of less than 0.02-0.03 was considered as single EF block. Web-based gene prediction tool FGENESH (http://linux1.softberry.com) was used for identifying genic regions such as UTRs, exons and introns with *Arabidopsis thaliana* gene model.

**Results and discussion:** Forty one growth and adaptive genes were selected based on literature search [6, TAIR database]. A total of 100.5 kb genomic sequence from *Ec* genome spread over ~1055 Mbp reads was generated (~94% high quality reads with average read depth 6124). A total of 11,329 SNPs were polymorphic within *Ec* and 378 SNPs exhibited
<figure><img src="image_11.png" /><figcaption>Table 1(abstract O17) Results from SNP discovery in 41 candidate genes</figcaption></figure>

<table><thead><tr><th rowspan="2">Predicted gene region</th><th colspan="3">SNP frequency parameters</th><th colspan="4">SNP classification</th></tr><tr><th>SNP count (range)</th><th>SNP Frequency in bases/SNP (range)</th><th>Total length in bp (range)</th><th>Ts</th><th>Tv</th><th>Total</th><th>Ratio(Ts/Tv)</th></tr></thead><tbody><tr><td>5'UTR(n=1)</td><td>4</td><td>40.8</td><td>163</td><td>1</td><td>3</td><td>4</td><td>0.33</td></tr><tr><td>Exons(n=176)</td><td>427 (1-52)</td><td>105.8 (0-1339)</td><td>45,177 (200-3487)</td><td>310</td><td>117</td><td>427</td><td>2.65</td></tr><tr><td>Introns(n=136)</td><td>536 (0-64)</td><td>71.3 (0-472)</td><td>38,210 (68-5079)</td><td>332</td><td>204</td><td>536</td><td>1.63</td></tr><tr><td>3'UTR(n=27)</td><td>54 (0-11)</td><td>81.6 (0-358)</td><td>4,405 (7-674)</td><td>33</td><td>21</td><td>54</td><td>1.57</td></tr><tr><td>Unclassified(n=13)</td><td>69 (0-25)</td><td>62.7 (0-425)</td><td>4,329 (4-425)</td><td>40</td><td>29</td><td>69</td><td>1.38</td></tr><tr><td>Nongenic(n=17)</td><td>101 (0-25)</td><td>82.6 (0-974)</td><td>8,340 (6-1481)</td><td>63</td><td>38</td><td>101</td><td>1.66</td></tr><tr><td>Total(n=370)</td><td>1,191 (1-115)</td><td>84.5 (38.2-974)</td><td>100,624 (634-9864)</td><td>779</td><td>412</td><td>1191</td><td>1.89</td></tr></tbody></table>

Note: n: number of units detected from 41 genes; Ts: transitions, Tv: transversions.

inter-species polymorphism between *Ec* and *Eg*. In addition, 75 insertions and 90 deletions within *Ec* and eight intra-specific deletions in comparison to *Eg* were detected. After appropriate corrections as

described, the 'useful' SNP number reduced to 1,191 which was ~10.5% of the original SNP count (~frequency of 1 per 84.5 bp). Table 1 describes findings from the present analysis of SNPs. A total of 198 putative EF blocks containing 541 SNPs, grossly comparable to LD blocks, with 55, 65 and 34 in exons, introns, exon-intron junctions respectively were detected (rest all were small in numbers) with an average length of ~105 bp (SD: ± 182; range: 1-1234 bp, distribution shown in figure 2; ~3 SNPs/block) and would aid in selection of SNPs. The comparable mean lengths adjusted for the respective amplicon lengths were around 0.014 to 0.016 (SD: ±0.013 to ±0.015) for exons, introns and nongenic regions whereas for intron-exon junctions it was 0.028±0.023, significantly longer than the rest (p=0.03).

**Conclusions:** Herein, NGS (Illumina) platform was successfully used for identifying ~1,200 SNPs in 41 targeted genes in *Ec* which has shed important light on quantitative and qualitative distribution of SNPs. In addition, the analysis of EF blocks also provided important guidelines for selection of SNPs for genotyping.

**Acknowledgements:** The authors acknowledge valuable discussions with Dr. Navin Sharma, Dr. DS Gurumurthy, (both ITC R&D Centre, Bangalore, India) and Dr. BR Thumma, Dr. Simon Southerton, (both CSIRO-Plant Industry, Canberra, Australia) and also the Eucagen website (http://eucalypusdb.bi.up.ac.za/gbrowse8x) for making the *Eg* sequence available.

References

1. Rafalski A: Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 2002, 5:94-100.

<figure><img src="image_12.png" /></figure>
2. Perkel J: SNP genotyping: six technologies that keyed a revolution. *Nature Methods* 2008, 5:447-454.

3. 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.

4. Kulheim C, Yeoh SH, Maintz J, Foley WJ, Moran GF: Comparative SNP diversity among four *Eucalyptus* species for genes from secondary metabolite biosynthetic pathways. *BMC Genomics* 2009, 10:452.

5. Thumma BR, Nolan MF, Evans R, Moran GF: Polymorphisms in Cinnamoyl CoA Reductase (CCR) are associated with variation in microfibril angle in *Eucalyptus* spp. *Genetics* 2005, 171:1257-1265.

6. Busov VB, Brunner AM, Strauss SH: *Genes* for control of plant stature and form. *New Phytologist* 2008, 177:589-607.

## O18

### Mapping and transcriptomic approaches implemented for understanding disease resistance to *Phytophthora cinamomii* in *Castanea* sp

Rita Costa¹*, Carmen Santos¹, Fábio Tavares¹, Helena Machado¹, José Gomes-Laranjo², Thomas Kubisiak³, Charles Dana Nelson³

¹Molecular Biology Laboratory -L-INIA - INRB I.P. Av da República, 2780-159 Oeiras, Portugal; ²Centre for Investigation and Agro Environmental and Biological Technologies (CITAB) - Department of Agronomy, University of Trás-os-Montes e Alto Douro, Apt. 1013, 5001-801 Vila Real, Portugal; ³USDA Forest Service, Southern Research Station, Southern Institute of Forest Genetics, 23332 Old Mississippi Highway 67, Saucier, MS, USA

E-mail: ritalouencocosta@gmail.com

*BMC Proceedings* 2011, **5**(Suppl 7):O18

The European chestnut, *Castanea sativa* Mill, covers a total area of 2.53 million hectares two million of which are chestnut forests, i.e. forests where chestnut is the dominant tree species, being the remaining 0.53 million hectares devoted to fruit production (20.9% of the total chestnut-growing area). Chestnut fruit production in Europe declined considerably during the XX century to the current 200,000 t (almost 300 million euros). This decline arose mainly due to serious diseases and changes in the structure of society. Towards the end of the century, there was a sharp increase in chestnut demand which triggered new planting and the restoration of old orchards throughout Europe, although ink disease (*Phytophthora* spp.), canker blight (*Cryphonectria parasitica* (Murrill) M.E. Barr.) and more recently *Dryocosmus kuriphilus* Yasumatsu, still represent major threats to the species. In Europe, chestnut breeding has been focused on the improvement of cultivars and rootstocks through selection and hybridisation with Asian species resistant to ink disease. Considerable success was obtained using this approach, however little information was acquired until now on the genetic basis of resistance in chestnut.

A set of interspecific crosses were established between European chestnut and Asian species and two separate full-sib pedigrees have been produced, SC (C. *sativa* x C. *crenata*) and SM (C. *sativa* x C. *mollissima*). The goal is to perform DNA marker:trait association analysis to identify QTLs related to ink disease metrics and also to identify putative resistance genes to *P. cinamomii* using a transcriptomic approach. An overview of the study will be presented including the genotyping of the mapping population (parents and progenies) with nuclear SSR markers designed for *Castanea mollissima* and provided by the American team of Fagaceae Genomics Project, for the construction of the first genetic linkage map. The methodologies implemented for the determination of the metrics of resistance of mapping population, for further identification of QTLs, will also be presented.

## O19

### Sequence-based breeding; application to unravel traits of the 6F crops: food, feed, fiber, flowers, fuel and fun

Mark van Haaren
Keygene Inc, the Netherlands
E-mail: mvh@keygene.com
*BMC Proceedings* 2011, **5**(Suppl 7):O19

The genomes of the world's most important crop species differ enormously in size, ploidy levels, repeat composition and germplasm diversity, posing a tremendous challenge to utilize them for breeding and

trait improvement. Keygene's Crop Genome Center addresses this challenge by developing and applying sequence-based methodologies to advance building genome assemblies and elucidating genetic diversity of 6F crops. For example, novel strategies to build very high quality genome sequence assemblies make use of our Whole Genome Profiling (WGP) technology and high parallel local sequence assemblies. For sequence-based genotyping we developed random Sequence-Based Genotyping (rSBG), a technology which incorporates the high-throughput sequencing capacity of the Illumina Genome Analyzer (GA)-II and the genome complexity reduction capabilities of AFLP*. rSBG allows for simultaneous sequence-based marker discovery and detection and is therefore a highly suitable technology for cost efficient, highly flexible, sequence-based genotyping, not requiring prior sequence information and custom reagents. These technologies, together with KeyGene's lead discovery and molecular mutagenesis platforms, significantly increase the rate with which the genetic basis for (complex) traits can be unraveled. In this presentation, I will present recent applications of these technologies in our breeding research programs.

The AFLP*, Whole Genome Profiling and rSBG technologies are covered by patents and patent applications owned by Keygene N.V.
AFLP is a registered trademark of Keygene N.V.

## O20

### The regulation of quantitative variation of foliar terpenes in medicinal tea tree *Melaleuca alternifolia*

Hamish Webb¹*, Kulheim Carsten¹, Lan fear Rob¹, John Hamill², William Foley¹

¹Australian National University, Australia; ²Monash University, Australia

E-mail: hamish.webb@gmail.com

*BMC Proceedings* 2011, **5**(Suppl 7):O20

**Background:** Terpenes are important mediators of plant-environment interactions as well as often being economically important. Terpenes are biosynthesized via two spatially separate pathways: the MEP pathway in the plastid, the site of monoterpane biosynthesis and the MVA pathway in the cytosol, the site of sesquiterpane biosynthesis (McGarvey and Croteau 1995; Eisenreich *et al.* 1998; Rohmer 1999). Studies using transgenic plants have demonstrated that when the MEP genes *dxs* and *dxr* and MVA pathway gene *hmgr* are up-regulated plants exhibit an increase in foliar terpene concentrations (Chappell *et al.* 1995; Wildung and Croteau 2005; Carretero-Paulet *et al.* 2006). However, as yet, the importance of the expression of these genes has not been demonstrated for naturally occurring quantitative variation in foliar terpene concentrations.

**Aims and methods:** *Melaleuca alternifolia* (medicinal tea tree) is a small tree in the family Myrtaceae that is grown in plantations for its valuable terpene-rich leaf oil. We studied the expression of MVA and MEP pathway genes to identify those that are correlated with variation in foliar terpene concentration. Diagnostic primers were designed for seven MEP pathway genes, three MVA pathway genes and three genes downstream of both pathways. Transcript abundance for all 13 genes was then quantified from 48 individuals on the Fluidigm biomark platform. Foliar terpene concentrations were determined by GC of ethanol extracts containing an internal standard.

**Results:** Foliar terpene concentration varied from 5 to 12.2% of dry matter (DM), terpinen-4-ol concentration from 2.1 to 6.6% of DM and bicyclogermacrene concentration from 0.1 to 0.5% of DM. The transcript abundance of five genes within the MEP pathway were correlated significantly with variation in foliar terpinen-4-ol concentrations. The transcript abundance of three MEP pathway genes and one MVA pathway gene were significantly correlated with foliar bicyclogermacrene (the most abundant sesquiterpene) concentrations. However, the expression of the genes within both pathways were co-dependent and showed strong pair-wise correlations within pathways. Therefore, multiple regression models were formulated to account for interactions amongst the genes. A multiple regression model using only the genes in the MEP pathway explained 87% of variation in foliar terpinen-4-ol concentrations (Figure 2). A multiple regression model of the genes within both the MEP and MVA pathways explained 60% of variation in foliar bicyclogermacrene concentrations (Figure 2). Two sub-models using only the genes from either pathway explained significantly less variation in bicyclogermacrene concentrations than the full model (MEP only, r² = 0.5; MVA only, r² = 0.15) and indicated that both pathways contribute to the
<figure><img src="image_13.png" /><figcaption>**Figure 1(abstract O20)** Simplified schematic of the terpene biosynthetic pathway in plants, showing the catalyzing enzymes for each step.</figcaption></figure>

pool of isopentyl pyrophosphate (IPP) used to biosynthesize bicyclogermacrene.

**Discussion:** Several studies have shown that the expression of *dxr* and *dxs* has a strong influence on foliar terpene yield; however, this has only ever been demonstrated in transgenic plants and never on naturally

occurring, quantitative variation in terpene concentrations. Our results show that a large proportion of the quantitative variation of foliar terpene concentration in *M. alternifolia* can be explained by quantitative variation of transcript abundance of genes in the early stages of terpenoid biosynthesis. A number of MEP pathway genes are correlated

<table><thead><tr><th colspan="3">Terpinen-4-ol</th></tr><tr><th>Predictor</th><th>Slope</th><th>P value</th></tr></thead><tbody><tr><td>dxs2</td><td>70.883</td><td>&lt;0.001</td></tr><tr><td>dxr</td><td>37.682</td><td>0.018</td></tr><tr><td>mct</td><td>4.129</td><td>&lt;0.001</td></tr><tr><td>mcs</td><td>53.857</td><td>0.18</td></tr><tr><td>hds</td><td>9.499</td><td>0.585</td></tr><tr><td>gpps</td><td>8.3</td><td>0.019</td></tr><tr><td>l(hds<sup>2</sup>)</td><td>92.068</td><td>&lt;0.001</td></tr><tr><td>l(mcs<sup>2</sup>)</td><td>41.043</td><td>0.125</td></tr><tr><td>mct:gpps</td><td>3.603</td><td>&lt;0.001</td></tr><tr><td>dxr:mcs</td><td>77.482</td><td>0.018</td></tr><tr><td>dxr:hds</td><td>54.508</td><td>0.01</td></tr><tr><td>hds:l(mcs<sup>2</sup>)</td><td>60.705</td><td>0.014</td></tr><tr><td>dxs2:l(mcs<sup>2</sup>)</td><td>52.135</td><td>0.017</td></tr><tr><td>dxs2:mcs</td><td>145.007</td><td>0.001</td></tr><tr><td>Overall R<sup>2</sup></td><td>0.871</td><td></td></tr><tr><td>Model p value</td><td>&lt;0.001</td><td></td></tr></tbody></table>

<table><caption>**Figure 2(abstract O20)** Multiple linear regression models of gene expression against terpinen-4-ol and bicyclogermacrene concentrations.</caption><thead><tr><th colspan="3">Bicyclogermacrene</th></tr><tr><th>Predictor</th><th>Slope</th><th>P value</th></tr></thead><tbody><tr><td>dxs2</td><td>1.708</td><td>0.206</td></tr><tr><td>dxs3</td><td>1.198</td><td>0.159</td></tr><tr><td>dxr</td><td>6.472</td><td>0.004</td></tr><tr><td>cmk</td><td>12.013</td><td>&lt;0.001</td></tr><tr><td>mcs</td><td>7.398</td><td>0.002</td></tr><tr><td>hds</td><td>12.472</td><td>0.005</td></tr><tr><td>pmd1</td><td>2.503</td><td>0.08</td></tr><tr><td>mvk</td><td>2.205</td><td>0.111</td></tr><tr><td>hmgs</td><td>1.228</td><td>0.208</td></tr><tr><td>mcs:hds</td><td>9.575</td><td>0.009</td></tr><tr><td>dxs2:cmk</td><td>12.352</td><td>&lt;0.001</td></tr><tr><td>dxs2:hds</td><td>6.972</td><td>0.001</td></tr><tr><td>Overall R<sup>2</sup></td><td>0.603</td><td></td></tr><tr><td>Model p value</td><td>0.001</td><td></td></tr></tbody></table>
with variation in both the monoterpene terpinen-4-ol as well as the sesquiterpene bicyclogermacrene, which is also correlated with one MVA pathway gene. The multiple regression models for both terpinen-4-ol and bicyclogermacrene, each explained a significant amount of variation in the concentrations of each terpene.

The correlation between monoterpenes and transcripts from the MEP pathway was expected as they both occur in the plastid, however we also found a significant correlation between MEP pathway genes and bicyclogermacrene, a sesquiterpene synthesized in the cytosol. These correlations, as well as the results of the multiple regression analysis, strongly suggests that in *M. alternifolia*, the IPP from the plastid contributes significantly to the pool of IPP used for sesquiterpene biosynthesis.

**Conclusions:** This is the first study that shows the importance of variation in transcript abundance of terpene biosynthesis genes in determining naturally occurring variation in foliar terpene concentrations. The results also suggest that IPP originating in the plastid contributes significantly to the pool of precursors used for sesquiterpene biosynthesis in the cytosol in *M. alternifolia*. These results open the way for future studies to identify markers that can be used to improve early selection of plants with an enhanced yield and thus greater profitability.

## References

1. Carretero-Paulet L, et al: Enhanced flux through the methylenylthritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase. *Plant Molecular Biology* 2006, 62:683-695.

2. Chappell J, et al: Is the reaction catalyzed by 3-Hydroxy-3-Methylglutaryl coenzyme a reductase a rate-limiting step for isoprenoid biosynthesis in plants? *Plant Physiology* 1995, 109:1337-1343.

3. Eisenreich W, et al: The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. *Chemistry & Biology* 1998, 5: R221-R233.

4. McGarvey DJ, Croteau R: Terpenoid metabolism. *Plant Cell* 1995, 7:1015-1026.

5. Rohmer M: The mevalonate-independent methylenylthritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis, including carotenoids. *Pure and Applied Chemistry* 1999, 71:2279-2284.

6. Wildung MR, Croteau RB: Genetic engineering of peppermint for improved essential oil composition and yield. *Transgenic Research* 2005, 14:365-372.

# S4. REPRODUCTION, GROWTH AND DEVELOPMENT

## O21

### A roadmap of apical bud formation in white spruce identifies potential regulators of time to bud set

Janice Cooke¹*, Walid El Kayal¹, Betty Pelgas², Irina L. Zaharia³, Suzanne Abrams³, Nathalie Isabel²

¹University of Alberta, Canada; ²Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Canada; ³Plant Biotechnology Institute, National Research Council of Canada

E-mail: janice.cooke@ualberta.ca

BMC Proceedings 2011, 5(Suppl 7):O21

**Background:** Bud development is an adaptation that temperate forest trees have acquired to survive inclement winter conditions and resume growth the following spring. Bud development is a complex physiological and developmental process comprising bud formation, cold and desiccation tolerance development, and dormancy acquisition [1]. Because bud formation is accompanied by growth cessation, the timing of bud formation represents a trade-off between acquisition of winter hardiness and duration of active growth [2]. This is particularly true for determinate species such as white spruce (*Picea glauca* [Moench] Voss), where preformed stem units contained within the apical bud constitute most of next season's growth. Timing of bud set in white spruce shows genetic variation [3]. Our goal is to identify genes that exert genetic control over time to bud set in white spruce. As first steps towards that goal, we have conducted microarray gene expression profiling of shoot tips during the transition from active growth to dormancy in white spruce, and identified candidate regulators of bud formation by comparing these differentially expressed (DE) genes to results obtained

from QTL analyses of time to bud set conducted using the same genes as molecular markers.

**Materials and methods:** Two year old white spruce nearing completion of active growth were placed in either short day (SD; 8 h days / 16 h nights, 20°C) or continued long day (LD; 16 h days / 8 h nights, 20°C) photoperiods, and samples harvested at 0, 1, 3, 7, 14, 28 and 70 d. After an additional 8-15 weeks in SD, plants were transferred to low temperatures (LT) under continued SD for 3-4 weeks prior to final harvest. Light microscopy, microarrays, SYBR Green qRT-PCR, and hormone analyses were conducted as described [4].

**Results and discussion:** Unlike indeterminate species such as *Populus* spp., white spruce does not require environmental cues such as reduced photoperiod or cooler temperatures to initiate bud formation, although these cues may be required to complete bud development and acquire endodormancy. However, SD hastens and synchronizes bud formation. To reduce biological variation, we conducted most analyses with seedlings under SD. Microarray analyses revealed 4460 DE genes in shoot tips during SD-induced bud formation. These analyses revealed the timing of molecular processes that occur during bud formation, identifying DE groups of genes associated with meristem transitions, identity and maintenance; organogenesis; protein synthesis and turnover; chromatin assembly and remodeling; cell wall biosynthesis and expansion; carbohydrate and lipid metabolism; secondary metabolism; and general stress responses. Changes in gene expression correlated with anatomical changes occurring at the shoot apex during the transition from active growth to endodormancy. Comparison of DE genes in developing buds under SD and LD uncovered ca. 2000 genes that are DE under both photoperiods at comparable phases of bud formation. This common set of genes is likely important for bud formation, particularly during early stages. Many genes DE shortly after transfer of plants to SD are likely photoperiod-responsive but not essential for bud formation. Genes DE only under SD at later stages of bud formation could be involved in processes associated with completion of bud formation and transition to endodormancy, events which do not appear to occur fully under LD. Further comparison of shoot tip, needle and stem transcript abundance profiles during this time course revealed approximately 100 genes that are DE only in developing buds and show greater transcript abundance in developing buds than other tissues. Several genes DE during bud formation – including some genes both preferentially expressed and only DE in buds – are putatively associated with hormone signalling. Quantification of ABA, cytokinins, auxin and their metabolites during SD-induced bud formation revealed distinct profiles for these hormones during bud formation, with shifts in hormone levels coinciding with morphological events such as bud scale development. A small number of genes DE in developing buds fall within QTL regions identified for time of bud set [5]. Some of these genes also show signatures of selection in natural populations of white spruce via identification of Fst outliers [6]. These candidate genes may exert genetic control over time to bud formation, and are currently being studied in more detail. An eQTL experiment and association study are underway to identify additional candidate regulators of time to bud set.

**Conclusions:** This comprehensive study of gene expression changes that occur at the shoot apex during the transition from active growth to dormancy in white spruce has revealed timing of processes important for overwintering preparation, and groups of genes implicated in these processes. Use of these same genes as molecular markers in QTL analyses has enabled us to tentatively identify candidate genes that may exert genetic control over the time of bud formation in white spruce. These results provide new insight into an important adaptive trait, and lay the groundwork for more detailed studies into regulators of time of bud set in conifers.

## References

1. Ruttink T, Arend M, Morreel K, Storme V, Rombauts S, Fromm J, Bhalerao RP, Boerjan W, Rohde A: A molecular timetable for apical bud formation and dormancy induction in poplar. *Plant Cell* 2007, 19:2370-2390.

2. Howe GT, Aitken SN, Neale DB, Jermstad KD, Wheeler NC, Chen THH: From genotype to phenotype: unraveling the complexities of cold adaptation in forest trees. *Can J Bot* 2003, 81:1247-1266.

3. Jaramillo-Correa JP, Beaulieu J, Bousquet J: Contrasting evolutionary forces driving population structure at ESTPs, allozymes and quantitative traits in white spruce. *Mol Ecol* 2001, 10:2729-2740.
4. 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.

5. 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.

6. Namroud MC, Beaulieu J, Juge N, Laroche J, Bousquet J: Scanning the genome for gene single nucelotide polymorphisms involved in adaptive population differentiation in white spruce. *Mol Ecol* 2008, **17**:3599-3613.

## O22

### Greenhouse and field evaluation of transgenic poplars with modified gibberellin metabolism and signaling genes

Venkatesh Viswanath¹*, Cathleen Ma¹, Elizabeth Etherington¹,

Palitha Dharmawardhana², David W Pearce³, Stewart B Rood⁴, Victor B Busov⁵,

Steven H Strauss¹

¹Department of Forest Ecosystems and Society, Oregon State University, 321 Richardson Hall, Corvallis, OR 97331, USA; ²Department of Botany and Plant

Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OR 97331,

USA; ³Department of Biological Sciences, University of Lethbridge, 4401

University Dr. W, Lethbridge, AB T1K 3M4, Canada; ⁴Department of Biological

Sciences, University of Lethbridge, 4401 University Dr. W, Lethbridge, AB T1K 3M4, Canada; ⁵School of Forest Resources and Environmental Science,

Forestry and Wood Products Building, Michigan Technological University, 1400 Townsend Dr. Houghton, MI 49931, USA

E-mail: Venkatesh.Viswanath@oregonstate.edu
BMC Proceedings 2011, 5(Suppl 7):O22

**Summary:** Although there have been many observations of transgenes leading to improved plant growth in greenhouse experiments, there is a paucity of data that have been validated by multiple greenhouse experiments or field studies. Genes that encode regulators of gibberellin metabolic pathways were among the first reported to improve biomass growth in trees, and thus were the logical focus of this study. Poplars were transformed with eight different transgenic constructs designed to modify gibberellin biosynthesis and/or signaling. Dramatic improvements in growth rate were observed for several constructs under greenhouse conditions. However, these were often not seen in repeated greenhouse studies, and were statistically unconfirmed for most constructs in the field. Our results underline the importance of multiple greenhouse and field trials during studies with transgenes that modify hormonal homeostasis.

**Genes studied:** The genes under investigation in this study were GA20-oxidase, PHOR1-1, and SPINDLY. GA20-oxidase catalyzes the conversion of C20-gibberellins to C19-gibberellins through a series of sequential oxidation steps using 2-oxoglutarate as a co-substrate [1]. This enzyme was first isolated from pumpkin cotyledons [2], and is involved in the penultimate step in the production of bioactive gibberellins. The PHOR1-1 gene is a positive regulator that was first isolated from leaves of potato plants subjected to a short day photoperiod (SD) [3]. Altered feedback regulation was observed in transgenic plants produced by antisense inhibition of this gene, indicating the role of the PHOR1-1 protein as an intermediate in the gibberellin signaling pathway. SPINDLY (SPY) is a gene that codes for an O-linked N-acetylglucosamine transferase [4]. In Arabidopsis, it has been

shown to inhibit GA signaling, and thus is considered a negative regulator of GA signaling [5].

**Methods:** Combinations of the three different genes and seven different promoters were used (Table 1).

In addition to constitutive promoters, we used native promoters of poplar genes which were identified by screening whole-genome microarray expression data. Plants transformed with all eight constructs were grown in the greenhouse, while those transformed with six of these constructs were also grown in the field. For four constructs, the greenhouse trials were repeated at least once. In all, 2,066 trees were studied in the greenhouse (286 independent transformations) and 556 trees (94 independent transformations) were studied in the field. Transgenic and non-transgenic controls were included in both the greenhouse and field studies. Height and diameter measurements were recorded in both the greenhouse and the field, and shoot and root biomass were measured on the greenhouse-grown plants. Levels of bioactive gibberellins were also measured on a subset of plants transformed with the two cisgenes (a gene having driven by its own promoter and ending with its own terminator), as well as in plants that had the RGL and the GA2-oxidase promoters driving the PtGA20-oxidase gene.

**Results and discussion:** When considering the best growth rate improvements for constructs that had repeated greenhouse trials, the maximum growth rate improvement seen (the mean of the best two events relative to the control) varied from 50 to 220% i.e., a fractional growth rate improvement ranging from 0.5 to 2.2 (Figure 1).

However, results were not consistent between the different greenhouse trials. The best events showed more than 100% in maximum growth rate improvement (fractional growth rate improvement > 1) in six different greenhouse experiments (Fig. 1); however, these events did not have a similar extent of improvement when the experiments were repeated. Five out of eight constructs produced a statistically significant improvement in growth rate (Figure 1).

Only one out of the six constructs tested in the field produced a statistically significant improvement in volume index, and this was for the RGL promoter driving the PtGA20-oxidase gene. The GA20-oxidase and the PHOR1 cisgenic plants, when grown in the greenhouse, had substantial increases (73-133%) in the levels of bioactive gibberellins in developing shoots compared to control plants, however, there was no significant correlation between gibberellin levels and volume index of each event. The levels of the gibberellin biosynthetic precursors were also measured and found to be greater than the bioactive gibberellin level, suggesting feedback inhibition—a possible mechanism for the lack of association with growth. Our results suggest that physiology-modifying transgenes strongly interact with growth environments. This implies that, as in conventional plant breeding, greenhouse results are of limited value and field trials are essential for evaluation of varieties with improved productivity.

### References

1. Yamaguchi S: Gibberellin metabolism and its regulation. *Annu Rev Plant Biol* 2008, **59**:225-251.

2. Lange T, Hedden P, Graebe JE: Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis. *Proc Natl Acad Sci U S A* 1994, **91**:8552-8556.

3. Amador V, Monte E, García-Martínez JL, Prat S: Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo. *Cell* 2001, **106**:343-354.

<table><caption>Table 1(abstract O22) Summary of constructs used in the transformation experiments</caption><thead><tr><td>Promoter</td><td>Gene</td><td>Terminator</td><td>Number of events studied (GH/Field)</td><td>Total number of trees (GH/Field)</td></tr></thead><tbody><tr><td>PtGA20ox7</td><td>PtGA20ox7</td><td>PtGA20ox7</td><td>38/35</td><td>345/278</td></tr><tr><td>PtPHOR1</td><td>PtPHOR1</td><td>PtPHOR1</td><td>26/3</td><td>327/24</td></tr><tr><td>RGL1-1</td><td>PtGA20ox2-2</td><td>NOS</td><td>34/17</td><td>444/83</td></tr><tr><td>GA2ox1</td><td>PtGA20ox2-2</td><td>NOS</td><td>30/15</td><td>425/75</td></tr><tr><td>B-expansin</td><td>PtGA20ox2-2</td><td>NOS</td><td>24/0</td><td>120/0</td></tr><tr><td>PtCESA1</td><td>PtGA20ox2-2</td><td>NOS</td><td>16/0</td><td>80/0</td></tr><tr><td>35S</td><td>AtSPY</td><td>OCS</td><td>33/12</td><td>163/48</td></tr><tr><td>35S</td><td>HvSPY</td><td>OCS</td><td>33/12</td><td>162/48</td></tr></tbody></table>
<figure><img src="image_14.png" /><figcaption>**Figure 1 (abstract O22)** Results from greenhouse trials of transgenic plants. The constructs are denoted by the promoter used, except in the case of *SPY* genes. Dark bars show the maximum fractional rate improvement (best two transgenic events from best greenhouse experiment) compared to controls. Open bars show the mean fractional growth rate improvement for all transgenic events in that greenhouse experiment. Brackets denote one standard error. A "*" above an open bar indicates statistically significant difference between the transgenic and control groups.</figcaption></figure>

4. Silverstone AL, Tseng TS, Swain SM, Dill A, Jeong SY, Olszewski NE, Sun TP: Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis. *Plant Physiol* 2007, **143**:987-1000.

5. Greenboim-Wainberg Y, Maymon I, Borochov R, Alvarez J, Olszewski N, Ori N, Eshed Y, Weiss D: Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling. *Plant Cell* 2005, **17**:92-102.

## O23

### Enhanced cytokinin signaling stimulates cell proliferation in cambium of Populus

Juha Immanen¹*, Kaisa Nieminen², Jing Zhang¹, Rishi Bhalerao³,
Ykä Helariutta¹

¹University of Helsinki, Finland; ²University of Lausanne, Switzerland; ³Umeå Plant Science Center, Sweden

E-mail: juha.immanen@helsinkifi

BMC Proceedings 2011, **5**(Suppl 7):O23

**Introduction:** Understanding the regulation of radial growth that underlies wood development is of great importance for future use of tree products as a renewable resource. For this purpose, we have performed a detailed analysis of cytokinin function in the regulation of cambial development in a tree stem. Furthermore, we have identified potential cytokinin signaling response genes active during the secondary development in Arabidopsis and currently studying the best candidate genes also in Populus.

**Results and discussion:** In the cambium, expression of cytokinin receptor genes peaks in the actively dividing cells. This indicates that cytokinin signaling pathway is a major regulator of cambial cell proliferation. We have previously shown in transgenic Populus trees that we can slow down the cambial activity by reducing the level of cytokinin signaling in it [1]. To study if cytokinin signaling is truly a rate-limiting process for cambial activity, we have now engineered transgenic Populus trees with an enhanced cambial cytokinin signaling level. In these trees, we were able to see that elevated cytokinin signaling stimulated the cambial cell division activity and led to increased biomass production. Furthermore, based on microarray profiling in Arabidopsis, we have been able to identify several transcription factors functioning downstream of cytokinin signaling during the secondary development. These transcriptional regulators have stimulatory effect on radial growth in Arabidopsis root. We are currently studying if the Populus orthologs of these genes have similar function in the cambium of the tree stem.

**Conclusions:** We have shown that the cambial cell division activity, and through it the production of stem biomass, can be enhanced or decreased

through elevated or decreased cytokinin signaling. Together, our results show that cytokinins are major rate-limiting hormonal regulators of cambial development. Our study provides potential for applied research towards future breeding of faster growing tree cultivars.

**Reference**

1. Nieminen K, Immanen J, Laxell M, Kauppinen L, Tarkowski P, Dolezal K, Tähtiharju S, Elo A, Decourteix M, Ljung K, Bhalerao R, Keinonen K, Albert VA, Helariutta Y: Cytokinin signaling regulates cambial development in poplar. *Proc Natl Acad Sci U S A* 2008, **105**(50):20032-20037.

## O24

### WAT1 (WALLS ARE THIN1) defines a novel auxin transporter in plants and integrates auxin signaling in secondary wall formation in Arabidopsis fibers

Philippe Ranocha¹, Oana Dima², Judith Felten³, Amandine Freydier¹, Laurent Hoffmann¹, Karin Ljung³, Benoit Lacombe⁴, Claire Corratgé⁴, Jean-Baptiste Thibaud⁴, Björn Sundberg³, Wout Boerjan², Deborah Goffner¹*

¹Université de Toulouse; UPS; UMR 5546, Surfaces Cellulaires et Signalisation chez les Végétaux; BP 42617, F-31326, Castanet-Tolosan, France; ²VIB Department of Plant Systems Biology, UGent Department of Plant Biotechnology and Genetics, 9052 Gent, Belgium; ³Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden; ⁴Biochimie et Physiologie Moléculaire des Plantes, CNRS UMR 5004, Institut National de la Recherche Agronomique U386, Montpellier SupAgro, Université Montpellier II, Place Viala, 34060 Montpellier Cedex, France
E-mail: goffner@scsv.ups-tlse.fr

BMC Proceedings 2011, **5**(Suppl 7):O24

**Background:** Our knowledge of signaling mechanisms involved in secondary cell wall (SCW) formation is quite limited. To discover novel markers of SCW, a genomics approach using *Zinnia elegans* xylogenic cultures was undertaken that identified hundreds of gene candidates expressed at the onset of secondary wall formation [1]. *Arabidopsis* homologs and the corresponding T-DNA mutants for each *Zinnia* gene were identified and the panel of *Arabidopsis* cell wall mutants was subjected to developmental and wall-related phenotyping.

**Results and conclusion:** Among the most interesting mutants was *wat1* (*walls are thin1*). The most conspicuous phenotypic feature of *wat1* was the severe reduction (sometimes to the extent of being inexistent) of SCW in xylary and interfascular stem fibers. Interestingly, xylem vessel wall thickness and morphology were not modified by the mutation.
In addition to the SCW phenotype, *wat1* was characterized by 5-Me-tryptophan seedling toxicity, severely decreased auxin transport and content in stems, and massive down-regulation of auxin-related genes. These data led us to the conclusion that WAT1 acts as an upstream regulator of SCW deposition in fibers, presumably through an auxin-mediated mechanism [2].

Bioinformatic analysis of WAT1, annotated as 'homolog to a *Medicago truncatula* nodulin gene, MtNOD21', suggested that WAT1 encoded a putative transporter belonging to the Plant Metabolite Exporter family [3]. WAT1:GFP fusion protein experiments localized WAT1 on the tonoplast, confirming the prediction that WAT1 is a membrane protein. Although WAT1 is plant-specific, it shares structural similarities with bacterial amino acid transporters in that it consists of ten transmembrane domains encompassed within a tandem Domain of Unknown Function 6 (DUF6). To characterize WAT1 function, we recently tested its capacity to transport tryptophan and/or auxin in both yeast and *Xenopus* oocytes. Neither WAT1-expressing yeast cells nor *Xenopus* oocytes were able to facilitate radiolabeled Trp import or export. However, we have been able to demonstrate that WAT1 facilitates auxin import in both expression systems. These results clearly place WAT1 among the ranks, along with PINs, AUX/LAXs and ABCB/MDR/PGPs, as a novel, bona fide auxin transporter in plants.

This study constitutes the first functional characterization of any of the 46 members of the WAT1 gene family in *Arabidopsis* and our hope is that this discovery will help pave the way in identifying the functions of other family members. Moreover, the *wat1* mutant will be an ideal tool to address the question as to how auxin subcellular homeostasis plays a role in fiber SCW formation in *Arabidopsis*. Our current efforts to understand poplar WAT1-mediated auxin signaling in wood formation in trees will also be discussed.

## References

1. Pesquet E, Ranocha P, Legay S, Digonnet C, Barbier O, Pichon M, Goffner D: Novel markers of xylogenesis in *Zinnia elegans* are differentially regulated by auxin and cytokinin. *Plant Physiol* 2005, 139:1821-1839.

2. Ranocha Ph, Denancé N, Vanholme R, Freydier A, Martinez Y, Hoffmann L, Köhler L, Pouzet C, Renou JP, Sundberg B, Boerjan W, Goffner D: *Walls Are Thin (WAT1)*, an *Arabidopsis* homolog of *Medicago truncatula* NODLIN21, regulates auxin homeostasis and is required for secondary wall formation in fibers. *Plant J* 2010, 63:468-483.

3. Saier MH, Tran CV, Barabote RD: TCDB: the Transporter Classification Database for membrane transport protein analyses and information. *Nucleic Acids Res* 2006, 34 part 16:181-186.

## O25

### Molecular dissection of an adaptive epigenetic memory mechanism in norway spruce

Igor Yakovlev¹*, YeonKyeong Lee², Bjørn Rotter³, Tore Skrøppa²,
Jorunn Elisabeth Olsen², Øystein Johnsen², Carl Gunnar Fossdal¹

¹Norwegian Forest and Landscape Institute, PO box 115, 1431, Ås, Norway;
²Norwegian University of Life Sciences, Department of Plant and
Environmental Sciences, 1432, Ås, Norway; ³GenXPro GmbH, Franksfurter
Innovationszentrum (FIZ), Altenhöferallee 3, 060438 Franskfurt am Main,
Germany

E-mail: yai@skogglandskap.no

BMC Proceedings 2011, 5(Suppl 7):O25

In Norway spruce, environmental conditions during the reproduction can greatly influence progeny performance. We found that the temperature during post meiotic megagametogenesis (zygotic embryogenesis) and seed maturation shift the growth cycle program of the embryos in the seeds, resulting in significant and long lasting phenotypic changes in the progeny. Traits that are affected include the timing of dehardening and bud burst in the spring; leader shoot growth cessation in the summer, and bud set and cold acclimation in the autumn. All processes are advanced or delayed in correspondence with the temperature during female reproduction. Colder reproductive environment advance bud set and cold acclimation during autumn and dehardening and bud burst during spring in their progenies. Temperature dependent difference in timing of terminal bud formation in identical clones was equivalent to a 4-6° latitudinal ecotypic difference. The progeny "remember" the temperatures and photoperiod prevailing during zygotic embryogenesis

and seed maturation and this memory, affecting the climatic adaptation in this species, is an epigenetic phenomenon.

This phenomenon is not only of evolutionary significance but has clear practical implications. This memory can help the conifer to cope with the anticipated rapid change in climatic conditions. It will have importance for the deployment of seedlings produced in seed orchards containing clones that are translocated to warmer sites, and it may be used to produce seedlings that have specific adaptive properties. So, it is possible to produce distinct phenotypes (epitypes) in Norway spruce, however this type of long lasting effects is not well documented in other organisms so far.

The molecular mechanism behind this striking epigenetic memory phenomenon is not yet unraveled but transcriptional changes are clearly involved. In epigenetically different progenies, transcriptional analysis revealed that seedlings from full-sib families produced at different embryogenesis temperature under long and short day conditions differed. Suppressive subtracted cDNA libraries revealed significant differences in their transcriptomes. Using qRT-PCR, microRNA pathways genes PaDCL1 and 2 and PaSGS3 as well as transposons related genes are differential expressed in the epigenetically different progenies with phenotypic differences in bud burst and bud set.

MicroRNAs (miRNAs) are endogenous small RNAs that can exert epigenetic gene regulatory impacts. We have examined the possible role of miRNA in the epigenetic phenomena, and found that Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel non-conserved miRNAs. From concatemerized small RNA libraries from seedlings from the same parents, originated from seeds developed in a cold and warm environment from a family with distinct epigenetic effects, contrasted to one from a family with little response, miRNAs potentially involved in this epigenetic memory was identified. Most of the miRNAs target unknown genes or genes with no known function. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in progenies with distinct epigenetic difference in bud set, but not in the progenies from a non-responding family, making them excellent candidate miRNAs. The differentially expression of specific miRNAs in genetically identical but epigenetically different progeny indicate their putative participation in the epigenetic regulation.

Epigenetic mechanisms influence phenotype through altered regulation of gene expression that is mitotically (and sometimes meiotically) propagated. Understanding the mechanisms involved in the initiation, maintenance, and heritability of epigenetic states is an exiting aspect of research in current biology. Epigenetic regulation may be realized through several interconnected molecular pathways including DNA methylation, histone modification and chromatin remodeling, small non-coding RNAs and transposable element regulation.

Among spruce ESTs we found 64 homologs of genes described as involved in DNA methylation, histone modification and chromatin remodeling and small RNA biogenesis in other plant species. In general, known epigenetic mechanism related genes are very well represented in the spruce genome. We analyzed the transcription patterns of these genes using RT-PCR in epigenetically different zygotic embryogenic samples on different stages of development and in seedlings, originated from full-sib families clearly differed in epigenetic response. The largest difference in gene expression of selected genes was found at the earlier stages of embryogenesis while in seedlings a low number of these genes were differentially expressed. Most of the known epigenetic mechanism related genes in seedlings were steadily expressed in all studied samples independently of their epitype.

To get a deeper analysis of epigenetic related transcriptome we used high-throughput sequencing (RNA-seq and miRNA-seq) in cooperation with GenXPro GmbH. Using MACE (massive cDNA 3'end sequencing) deep mRNA sequencing on the Illumina GSII platform, we analyzed the genes differentially expressed in *P. abies* during early stages of embryonic development. We selected genes which could be involved into epigenetic response by comparison warm and cold originated "embryonic epitypes" from the same full-sibs family somatic embryos developed in cold (18°C) and warm (30°C) environmental conditions. Additionally, for more distinct analysing of the large amount of "no database hit" reads we sequenced one normalised library using 454 Titanium GS FLX sequencing to get reference transcript set of expressed genes. The sequencing data is currently under processing and we are going to discuss main results here. To proceed with our initial study of miRNAs in spruce, we used Illumina/ SOLEXA sequencing to identify small RNAs expressed at the same epigenetic responsive family developed in warm and cold environment
progenies following short-day treatment. The identification of novel miRNA candidates are in progress and the confirmation of conserved and novel miRNA by qRT-PCR analysis will be presented.

## O26

### Dual function of auxin during leaf abscission in poplar

Xu Jin¹,²*, Urs Fischer¹,²

¹Swedish Agricultural University, Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Umeå, Sweden; ²University of Göttingen, Forest Botany and Tree Physiology, Göttingen, Germany
BMC Proceedings 2011, 5(Suppl 7):O26

**Background:** Leaf abscission is an important trait for biomass production and seasonal acclimation in deciduous trees of the temperate region. Various plant hormones are involved in the timing of abscission. For example, ethylene signaling is required to induce hydrolysis of cell walls, while an auxin gradient [1] was suggested to act upstream of ethylene on the onset of leaf abscission. Besides pharmacological application of auxins on cut surfaces of explants, experimental evidence for such a gradient is however lacking. In addition to its function in temporal control, auxin has also been suggested to be a positional signal specifying the cells of the abscission zone [2].

**Methods:** We established an experimental system on intact Populus trees, which allows us to induce abscission synchronously under controlled conditions. Leaf blades were bagged in aluminum foil and abscission was recorded daily. Cumulative abscission followed a sigmoidal curve for dark-induced leaves, whereas control leaves in transparent bags of the same weight as the aluminum foil bags were not separated from the stem. Abscission was preceded by senescence in the petiole but not in the leaf.

**Results and conclusions:** Local auxin applications directly onto the abscission zone, as well as onto the distal end of the petiole, delayed dark induced abscission indicating that auxin could range not only as a short but also as a long distance signal. Similarly, an inhibitor of polar auxin transport retarded separation from the plant body. By contrast, auxin applied onto mature abscission zones only delayed abscission slightly in comparison to auxin applications before the development of an abscission zone. Taken together this points to a distinct function of auxin in early stages of abscission. Interestingly, we found shortly after dark-induction a new auxin response maximum on the abaxial side of the petiole, highlighting the incipient abscission zone. This auxin response maximum progressively moved from the abaxial to the adaxial side of the petiole, preceding the maturation of the abscission zone, presumably providing positional information for the formation of the abscission zone. Microarray data identified the auxin efflux carriers PIN1 and PIN5, as well as a novel auxin transporter, to be down-regulated after dark induction. Immunolocalizations of those carriers will reveal if their subcellular localization and expression can explain the novel auxin response maximum.

### References

1. Louie DS, Addicott FT: Applied auxin gradients and abscission in explants. *Plant Physiology* 1970, 45(6):654-658.

2. McManus MT, Thompson DS, Merriman C, Lyne L, Osborne DJ: Transdifferentiation of mature cortical cells to functional abscission cells in bean. *Plant Physiology* 1998, 116(3):891-899.

# S5. BIOTIC AND ABIOTIC INTERACTIONS

## O27

### Biomarkers and gene copy number variation for terpenoid traits associated with insect resistance in Sitka spruce: An integrated genomic, proteomic, and biochemical analysis of (+)-3-carene biosynthesis

Joerg Bohlmann¹*, Dawn Hall¹, Jeanne Robert¹, Christopher Keeling¹, Dominik Domanski², Alfonso Lara Quesada¹, Sharon Jancsik¹, Michael Kuzyk², Britta Hamberger¹, Christoph Borchers³

¹Michael Smith Laboratories, University of British Columbia, Canada;

²University of Victoria - Genome BC Proteomics Centre, Canada; ³University of Victoria - Genome BC Proteomics Centre and Department of Biochemistry, University of Victoria, Canada

E-mail: bohlmann@msl.ubc.ca

BMC Proceedings 2011, 5(Suppl 7):O27

Conifers have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. The large diversity of terpenoid metabolites is determined by the size and composition of the terpene synthase (TPS) gene family, and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (*Picea sitchensis*) to white pine weevil (*Pissodes strobi*). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (82-95% amino acid sequence identity) (+)-3-carene synthase (*PsTPS-3car*) genes. Transcript profiling identified one *PsTPS-3car* gene (*PsTPS-3car1*) which is expressed in both genotypes, one gene (*PsTPS-3car2*) expressed only in resistant trees, and one gene (*PsTPS-3car3*) expressed only in susceptible trees. The *PsTPS-3car2* gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring substantiated this pattern of differential expression of members of the PsTPS-3car family on the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3as a factor for the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by PsTPS-3car gene copy number variation, variation of gene and protein expression, and variation of catalytic efficiencies.

### Reference

1. Hall DE, Robert JA, Keeling CI, Domanski D, Qesada AL, Jancsik S, Kuzyk M, Hamberger Br, Borchers CH, J Bohlmann: An integrated genomic, proteomic, and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (*Picea sitchensis*) genotypes which are resistant or susceptible to white pine weevil. *The Plant Journal* 2011, 65:936-948, doi: 10.1111/j.1365-313X.2010.04478.x

## O28

### Integrative approach involving RNA-Seq, foliar traits and growth measurements revealed genotype-specific plasticity on *Eucalyptus* subjected to seasonal water shortage

Emilie Villar¹*, Christophe Plomion², Jean-Marc Gion¹

¹CIRAD, France; ²INRA, France

E-mail: villar@pierroton.inra.fr

BMC Proceedings 2011, 5(Suppl 7):O28

**Background:** In the context of climate change, water availability will become an important limiting factor threatening biomass production, particularly in intensively managed forest tree plantations. The complexity of drought stress response calls for integrative approaches combining physiological, anatomical and molecular investigations at different scales ranging from particular cells to the whole plant. Such a strategy was developed for two eucalyptus clones used in industrial plantations in the Republic of Congo and known for their differences in term of productivity and water-use efficiency.

**Methods:** The two genetic units used in this study consisted in one more productive hybrid of *E. urophylla* *E. grandis* (18-50), and one less productive hybrid obtained from open-pollination of *E. alba* (1-41). These genotypes were subjected to two watering regimes (irrigated during two dry seasons, versus non-irrigated) in open field conditions. Growth parameters such height, diameter at breast height, stem, leaf and root biomasses as well as physiological parameters, such carbon discrimination and specific leaf area, were regularly measured over two growing seasons. At the end of the second dry season (i.e. 14 months-old trees), polyphenols accumulation and anatomical measurements were performed on mature leaves. RNA of shoot apices from trees representing the four conditions (i.e. 2 genotypes subjected to 2 watering treatments) were extracted and used to create non-normalized libraries. Using 454/Roche sequencing, we performed digital expression analysis to highlight differentially expressed transcripts showing genotype, treatments and/or genotype by treatment interaction effects. Expression of candidates genes revealed by this first analysis were assessed by RT-qPCR on shoot apices and on mature leaves.

**Results and discussion:** Growth parameters and stem biomass analysis revealed that the two studied genotypes presented similar degrees of phenotypic plasticity in response to watering treatment during the
dry season. Nevertheless, comparison of growth increments during wet seasons suggested that the more productive genotype had higher ability to recover from water shortage during the dry seasons and to use favorable conditions to increase its above-ground biomass. We found that the more productive genotype displayed greater plasticity of foliar traits in the non-irrigated treatment during the dry season, suggesting that it could adjust more quickly its leaf parameters when rainfalls resume.

On mature leaves from trees subjected to water deficit, we identified mechanisms established to limit water losses by cuticle reinforcement and stomatal density decrease, to preserve leaf turgor by increase of collenchyma layer, to enhance hydraulic conductance with xylem vessel diameter decrease, and to preserve cells from reactive oxygen species through accumulation of hydroxycinnamates. Those adjustments were more intense for the more productive genotype, suggesting that this genotype may entailed higher cost strategies to preserve its leaf functions.

On shoot apices, 454-sequencing provided a catalogue of 129,993 unigenes (49,748 contigs and 80,245 singletons) from the initial sequencing of 398Mb corresponding to 1.14Mreads. Digital expression analysis identified 1,280 contigs displaying differential expression between the two genotypes, 155 contigs between treatments, and 274 contigs displaying genotype x treatment interaction effects. The more productive genotype mainly under-expressed genes related to photosynthetic activity during the dry season, activating genes to reallocate resources through major changes in primary metabolism, whereas the less productive genotype displayed lower levels of variation in gene abundance between treatments. This analysis allowed to reveal a set of candidate genes that could be related to phenotypic variation observed in mature leaves.

**Conclusions:** The productivity of the two genotypes used in this study differed essentially in terms of ability to recover from water shortage. These differences could be explained by different strategies of investment in preservation of leaves. In the environmental gradient tested, it seemed that higher investments enabled genotype 18-50 to increase its leaf resistance, and subsequently to recover quickly high growth performance once environmental conditions become favorable. Genes that potentially underlie such physiological plasticity were revealed through integrative approaches. More investigations are now needed to valid their putative roles in phenotypic plasticity in response to water deficit. Forward genetic approaches could be used to screen the variability of gene expression and other more-integrated phenotypic traits, the nucleotide diversity of these candidate genes and their covariations on a larger genetic background.

## O29
### Influence of water deficit on the induced and constitutive responses of pines to infection by mountain pine beetle fungal associates

Adriana Arango-Velez, Miranda Meents, Jean Linsky, Walid El Kayal, Eri Adams, Leonardo Galindo, Janice Cooke
Department of Biological Sciences, University of Alberta, Canada
E-mail: adrianaa@ualberta.ca
BMC Proceedings 2011, 5(Suppl 7):O29

**Background:** The ongoing outbreak of mountain pine beetle (MPB; *Dendroctonus ponderosae* Hopkins) and its associated pathogenic fungi (e.g. *Grosmannia clavigera* [Robinson-Jeffrey and Davidson] Zipfel, de Beer and Wingfield) in western North America has resulted in the loss of more than 13 million hectares of pines since 1999 in British Columbia alone [1]. MPB has principally attacked lodgepole pine (*Pinus contorta* Dougl. ex Loud. var. *latifolia*) in British Columbia. However, since 2006 MPB has spread into northern Alberta, where lodgepole pine hybridizes with jack pine (*Pinus banksiana* Lamb.) [2]. Few studies have compared lodgepole and jack pine defense responses, but given that lodgepole pine and MPB share a co-evolutionary history [1] whereas jack pine is a new host for MPB [2], it is reasonable to expect that differences might exist. Some regions affected by the current outbreak have experienced drought conditions during the last decade. Water deficit can limit carbon assimilation, potentially increasing tree susceptibility to MPB and their symbiotic fungi [3]. We are testing the hypotheses that lodgepole and jack pine defenses against MPB and *G. clavigera* differ, and that water deficit affects these responses.

**Materials and methods:** The relationship between water availability and tree defense was evaluated in (1) two year old lodgepole and jack pine

seedlings in growth rooms, and (2) ca. sixty year old lodgepole x jack pine hybrids in naturally regenerated, thinned stands. Soil relative water content was monitored using time-domain reflectometry. Seedlings were subjected to watering or water deficit for one week prior to wounding or wounding plus *G. clavigera* inoculation. Mature trees were either watered or water limited via tarps for six weeks before wounding plus *G. clavigera* inoculation. In both experiments, water deficit conditions were maintained throughout the time course.

Tree physiological status was evaluated by measuring gas exchange and stomatal conductance using a LiCor 6400, stem hydraulic conductivity using a low-pressure flow meter and safranin dye xylem perfusion, and HPLC. Defense responses were assessed by lesion measurements histochemistry, and qRT-PCR.

**Results and discussion:** Stomatal conductance and photosynthesis significantly decreased under water deficit for both lodgepole and jack pine seedlings, but seedling hydraulic conductivity was not affected. The mild water deficit applied to the mature trees reduced stomatal conductance and photosynthesis, but not significantly.

Stem lesions are a means of killing and compartmentalizing invading organisms [4]. *G. clavigera*-induced lesions developed more slowly in jack pine than lodgepole pine seedlings. Stem hydraulic conductivity decreased in inoculated lodgepole but not jack pine seedlings, likely because of greater tracheid occlusion caused by increased fungal growth and/or resin production in lodgepole pine [5]. Water deficit reduced lesion development rates at early time points in inoculated lodgepole and jack pine seedlings, as well as in mature trees at 5 weeks post-inoculation. Lesion length has been considered an indicator of tree defense capacity [6], with longer lesions reported to reflect increased release of toxic and/or inhibitory substances [7]. Accordingly, we interpret slower lesion development to indicate a slower defense response. Our results suggest (1) more rapid defense responses to *G. clavigera* in the co-evolved lodgepole pine host than in the new jack pine host, and (2) defense responses are slowed by water limitation.

We then examined the effect of water deficit on transcript abundance corresponding to genes classically associated with drought and defense responses. We conducted qRT-PCR transcript abundance profiling of secondary phloem from mature lodgepole x jack pine hybrids. We first profiled four *aquaporin* and five *DREB* genes, families associated with water stress responses. Although the mild water deficit did not significantly alter expression of these genes, expression of one *aquaporin* and one *DREB* decreased in response to *G. clavigera* inoculation. We then profiled five *chitinase* and four *terpene synthase* defense-associated genes. Expression of two *chitinases* was significantly induced by water deficit but not *G. clavigera*. Expression of other *chitinases* significantly increased in response to fungalinoculation, but the response was attenuated by water deficit. Expression of one *terpene synthase* significantly increased with fungal inoculation, but this response was also attenuated under water deficit. In contrast, water deficit increased constitutive expression of another *terpene synthase*. Higher constitutive expression of the *monoterpene synthase* under mild water stress suggests a pre-emptive defense via higher biosynthesis of volatile monoterpenes. Microarray and qRT-PCR analyses of the lodgepole and jack pine seedling experiment are underway.

**Conclusion:** Our analyses suggest that defense responses of jack pine differ from those of lodgepole pine. Molecular analyses are underway to further characterize these differences. Both constitutive and induced defense responses are modulated in pines by water deficit, and this response appears to be gene-specific. This study shows evidence of cross talk between the water stress and defense responses of pine trees.

### References

1. Raffa KF, Aukema BH, Bentz BJ, Carroll AL, Hicke JA, Turner MG, Romme WH: Cross-scale drivers of natural disturbances prone to anthropogenic amplification: the dynamics of bark beetle eruptions. *BioScience* 2008, 58:501-517.

2. 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.

3. McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, Plaut J, Sperry J, West A, Williams DG, Yepez EA: Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? *New Phytol* 2008, 178:719-739.
4. Franceschi VR, Krokene P, Christiansen E, Krekling T: Anatomical and chemical defenses of conifer bark against bark beetles and other pests. *New Phytol* 2005, 167:353-376.

5. Joseph G, Kelsey RG, Thies WG: Hydraulic conductivity in roots of ponderosa pine infected with black-stain (*Leptographium wagneri*) or annosus (*Heterobasidion annosum*) root disease. *Tree Physiol* 1998, 18:333-339.

6. Paine TD, Stephen FM: Influence of tree stress and site quality on the induced defense system of loblolly pine. *Can J For Res* 1987, 17:569-571.

7. Raffa KF, Berryman AA: Physiological aspects of lodgepole pine wound response to a fungal symbiont of the mountain pine beetle *Dendroctonus ponderaseae*. *Can Entomol* 1983, 115:723-734.

**O30**

*Heterobasidion spp, triggers non-specific defence responses in bark of Norway spruce*

Malin Elfstrand¹*, Jenny Arnerup¹, Karl Lundén¹, Marie Danielsson²,
Marten Lind¹, Ake Olson¹, Anna-Karin Borg-Karlsson², Jan Stenlid¹

¹Dept of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden; ²Ecological Chemistry Group, Department of Chemistry, KTH, Stockholm, Sweden

E-mail: Malin.Elfstrand@slu.se

*BMC Proceedings* 2011, **5**(Suppl 7):O30

Norway spruce [*Picea abies* (L.) Karst.] is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is *Heterobasidion parviporum* (Fr.) Niemelä & Korhonen. The completed genome sequence of *H. irregularis* opens up new possibilities to understand the interactions between *Heterobasidion* spp. and spruce. However to date there are no completed genome sequencing projects of conifer genomes available for complementing studies. To achieve a better understanding of induced transcriptional defence responses in Norway spruce upon *Heterobasidion* spp. attack, we compared transcriptional responses in bark to *H. parviporum* infection to the response to wounding using cDNA-AFLP and transcriptome sequencing.

In an initial study bark samples were harvested at 3, 7 and 14 days post inoculation (dpi) and untreated bark was used as negative control. About 2500 transcribed derived fragments (TDFs) generated by cDNA-AFLP were screened. 199 TDFs were investigated further based on band intensity in the inoculated bark in relation to either untreated bark or wounded bark. Out of these, 119 TDFs had a putative homology and a consistent band intensity pattern between replications. A majority of these TDFs showed homology to genes known to associate with defence e.g. *3-deoxy-d-arabino-heptulosonate 7-phosphate synthetase (DAHP)*, *Pathogenesis-related protein 1 (PR1)*, *Lipoxygenase (LOX)*, *ACC-synthase (ACS)*, *ACC-oxidase (ACO)* and *Jasmonate ZIM-domain 1 (JAZ1)*. Many of these are found in Salicylic acid- or Jasmonic acid/ethylene-signalling pathways. The majority of the TDFs showed a similar expression pattern for all treatments but samples inoculated with *H. parviporum* generally showed an enhanced reaction (induction/repression) compared to wounding alone. Expression patterns were confirmed by qPCR in material treated with wounding and inoculation with *H. parviporum* or *Phlebiopsis gigantea*. Our data suggest that infection with *H. parviporum* in Norway spruce induces a broad defence, with many similarities to non-specific defence responses in angiosperms. Additionally signs of reallocation of carbon from primary to secondary metabolism were evident.

With this information at hand we analysed four Norway spruce genotypes with either high or low susceptibility to *Heterobasidion* spp. [1] sampled 0, 5, 15 and 28 dpi with *H. annosum*. The bark phenol-composition was profiled in each sample. The 500,000 454-reads were assembled into 17,228 contigs that assembled in 14,364 putative transcript units (PTU) using the sequence assembler software Newbler™ (http://www.454.com). The assembled reference file was annotated with the software Blast2Go and the PTUs were submitted to differential expression analysis. Data on associations between gene expression levels and phenol composition in bark upon *H. annosum* inoculation and level of susceptibility will be presented.

**Reference**

1. Karlsson B, Swedjemark G: Genotypic variation in natural infection frequency of Heterobasidion spp. in a *Picea abies* clone trial in southern Sweden. *Scand J Forest Research* 2006, 21:108-114.

**O31**

*Transcriptome sequencing of *Eucalyptus camaldulensis* seedlings subjected to water stress*

Bala Thumma
CSIRO Plant Industry, Australia
E-mail: reddy.thumma@csiro.au
*BMC Proceedings* 2011, **5**(Suppl 7):O31

Water stress limits plant survival and production in many parts of the world. Identification of genes responding to water stress conditions will underpin efforts to breed plants better adapted to drought. We studied the effect of water stress on *Eucalyptus camaldulensis* seedlings derived from three natural populations. Physiological and growth traits were measured and gene and allelic expression in leaves was examined by RNA sequencing (RNA-seq). Water stress had a significant impact on all the physiological and growth traits, while differences between the populations were not significant. Genes differentially expressed in leaves were identified by de novo assembly and by *ab initio* transcriptome mapping using the *Eucalyptus grandis* reference genome sequence. Gene ontology (GO) enrichment tests with 2,500 significantly differentiated genes revealed 128 stress-related gene categories were up-regulated while 28 gene categories belonging to photosynthesis and other metabolic processes were down-regulated under stress treatment. More than 190,000 single nucleotide polymorphisms (SNPs) and small indels were detected and 4,053 of these revealed differential allelic expression between control and drought stressed seedlings. Allelic expression of 70% of these variants was correlated with total gene expression. These variants may be cis-acting variants or in high linkage disequilibrium with such variants. The SNPs and indels identified in this study form a useful resource for further testing in association studies.

**O32**

*The relationship between intra-specific variation in the *Populus* transcriptome, stomatal development, and the metabolome in response to drought*

Erin T Hamanishi¹*, Sherosha Raj², Olivia Wilkins², Barb R Thomas³, Shawn D Mansfield⁴, Aine L Plant⁵, Malcolm M Campbell⁶

¹Faculty of Forestry, University of Toronto, 33 Willcocks Street, Toronto, Ontario M5S 3B3 Canada; ²Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2 Canada; ³Alberta-Pacific Forest Industries Inc. P.O. Box 8000 Boyle, Alberta, T0A 0M0, Canada; ⁴Department of Renewable Resources, University of Alberta, 731 General Services Building, Edmonton, AB T6G 2H1, Canada; ⁵Department of Wood Science, University of British Columbia, 4030-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada; ⁶Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada; ⁷Centre for the Analysis of Genome Evolution and Function; Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada

E-mail: erin.hamanishi@utoronto.ca

*BMC Proceedings* 2011, **5**(Suppl 7):O32

Drought is one of the most significant factors limiting tree growth. Trees in the genus *Populus* are particularly noted for their drought sensitivity; therefore, understanding the mechanisms by which these economically and ecologically important forest trees respond to drought is of paramount importance. The ability of *Populus* trees to contend with drought is dependent on the responsiveness of the genome, and in turn, the ability of the transcriptome to appropriately remodel growth, metabolism and development. Amassing evidence indicates that different species of *Populus* have divergent mechanisms and adaptations to contend with drought stress; however, individuals within a given species also display divergent drought responses. In order to investigate the intra-specific variation underpinning the drought response, we examined six genotypes of *P. balsamifera*. Using Affymetrix Poplar GeneChips, we found a positive correlation between the magnitude of drought-induced changes in the transcriptome and the capacity of the genotype to maintain growth. Surprisingly identifiable differences at the transcriptome were observed, and similar responses were observed within the metabolome. Although common drought responses could be identified within the species, the complexities of these responses must be taken into consideration when defining species- or genus-level drought responses.
O33

New insights in the molecular events underlying actinorhizal
nodulation in the tropical tree *Casuarina glauca*

Claudine Franche¹*, Hassen Gherbi¹, Meriem Benabdou², Sergio Svistoonoff¹,
Valerie Hocher¹, Didier Bogusz¹

¹Rhizogenese, Institut de Recherche pour le Développement, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France; ²Laboratoire de Génétique, Biochimie et Biotechnologies Végétales, Département de Biologie et d'Ecologie, Université Mentouri, Route Ain El Bey, Constantine, Algérie
E-mail: claudine.franche@ird.fr

BMC Proceedings 2011, 5(Suppl 7):O33

**Background:** Actinorhizal root nodules result from the interaction between a nitrogen-fixing actinomycete called *Frankia* and roots of dicotyledonous trees and shrubs belonging to 8 plant families and 25 genera. Most actinorhizal plants are capable of high rates of nitrogen fixation comparable to those found in legumes. As a consequence, these trees are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. This facility for adaptation has drawn great interest to actinorhizal plants, particularly to several species of *Casuarinaceae* such as *Casuarina glauca*, which can be used for fuelwood production, agroforestry, and land reclamation in the tropics and subtropics.

The basic knowledge of the symbiotic association between *Frankia* and actinorhizal plants is still poorly understood, although it offers striking differences with the *Rhizobium*-legume symbiosis [1]. Recently, the development of genomics in some actinorhizal plants such as *C. glauca* [2], together with the possibility to obtain transgenic actinorhizal plants following *Agrobacterium* gene transfer [3], offer new approaches to understand the molecular basis of the actinorhizal process. We will highlight recent progress in the molecular knowledge of the early stages of the actinorhizal symbiosis. A comparative analysis of the symbiotic pathway in actinorhizal trees and legumes will be presented.

**Methods:** Plant material. *Casuarina glauca* Sieb. Ex Spreng seeds were obtained from B&T World Seeds (Aigues-Vives, France) and germinated in sterile conditions as described [4].

Genetic transformation of Casuarina glauca. Composite plants of *C. glauca* were obtained following the genetic transformation by *Agrobacterium rhizogenes* A4RS containing the appropriate binary vector [5]. For promoter analyses, binary vectors were derived from pBIN19 and for RNAi experiments, hairpin constructs were cloned into the pHKN29 vector using standard molecular methods.

Nodulation by *Frankia*. Composite and control plants were nodulated with a suspension of *Frankia* Cc13 [4].

**Results and discussion:** *CgSymRK* is involved in the actinorhizal signal transduction pathway: The development of genetic and genomic tools for the model legumes *M. truncatula* and *Lotus japonicus* has greatly facilitated the cloning of genes required for root symbiosis [6]. Some of these genes were found to be involved in the establishment of both rhizobia and mycorrhiza symbioses, and designated as common SYM genes constituting the common SYM pathway. They include genes encoding a leucine-rich-repeat (LRR) receptor kinase (SymRK), cation channels, nuclear pore complex proteins, a calcium and calmodulin-dependent protein kinase (CCaMK) and a nuclear coiled protein. The question was raised whether some of these symbiotic genes were shared in the signal transduction pathway in response to *Frankia* factors and rhizobial Nod factors.

To answer this question, a functional study of *CgSymRK*, a gene isolated from *C. glauca*, homologous to the receptor-like kinase gene *SymRK* required for nodulation and mycorrhization in legumes, was undertaken. Downregulation of *CgSymRK* resulting from a RNA interference approach revealed that the frequency of nodulated RNAi-*CgSymRK* plants was reduced 2-fold compared to control *C. glauca* plants [7]. In addition, a range of morphological alterations was observed in the down-regulated *CgSymRK*-nodules. Additional experiments revealed that *CgSymRK* was also necessary for the establishment of the symbiosis with the arbuscular mycorrhiza *Glomus intraradices*. Therefore, the function of *SymRK* is conserved between legumes and actinorhizal plants.

**Search for other candidates of the signalling pathway:** Our group has developed the first genomic platform to identify plant genes involved in the symbiotic process between *Frankia* and *C. glauca* [2]. Based on the

comparison with legumes sequences, ESTs sharing significant homologies with genes from the Nod signalling pathway were identified. Functional analyses of the candidate orthologues to the *CCaMK* and *NIN* genes are in progress. *CCaMK* is a calcium and calmodulin-dependent protein kinase that is presumed to decode and transduce Nod-factor specific calcium spiking response in legumes. Preliminary experiments suggest that *CgCCaMK* is necessary for both nodulation and endomycorrhization in *C. glauca*, thereby suggesting a conservation of the whole common SYM pathway in the actinorhizal plants.

**Conclusion:** Although several actinorhizal genera contain species that are economically important in forestry and land regeneration, progress has been slower in our knowledge of the actinorhizal symbiosis than that of the legume rhizobium interaction. The availability of a method for the genetic transformation of Casuarinas has resulted in a major breakthrough since it opened the way to functional gene analysis in the actinorhizal host plant. Deep sequencing analyses are now in progress in *C. glauca* and mining these data will undoubtedly contribute to dissecting the molecular dialogue between *Frankia* and the host plant in the different stages of development of the actinorhizal nodule.

References

1. Franche C, Lindström K, Elmerich C: Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants. *Plant and soil*, 2009, 321:35-59.

2. Hocher V, Auguy F, Argout X, Laplaze L, Franche C, Bogusz D: Expressed sequence-tag analysis in Casuarina glauca actinorhizal nodule and root. *New Phytol* 2006, 169:681-688.

3. Svistoonoff S, Gherbi H, Nambiar-Veetil M, Zhong C, Michalak Z, Laplaze L, Vaissayre V, Auguy F, Hocher V, Doumas P: Contribution of transgenic Casuarinaceae to our knowledge of the actinorhizal symbioses. *Symbiosis* 2010, 50:3-11.

4. Franche C, Diouf D, Le QV, Bogusz D, N'diaye A, Gherbi H, Gobé C, Duhoux E: Genetic transformation of the actinorhizal tree Allocasuarina verticillata by *Agrobacterium tumefaciens*. *Plant J* 1997, 11:897-904.

5. Gherbi H, Nambiar-Veetil M, Zhong C, Félix J, Autran D, Girardin R, Vaissayre V, Auguy F, Bogusz D, Franche C: Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata. *Mol. Plant Microbe Interact* 2008, 21:518-524.

6. Madsen LH, Tirichine L, Jurkiewicz A, Sullivan JT, Heckmann AB, Bek AS, Ronson CW, James EK, Stougaard J: The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. *Nat. Commun* 2010, 1:1-12.

7. Gherbi H, Markmann K, Svistoonoff S, Estevan J, Autran D, Giczey G, Auguy F, Péret B, Laplaze L, Franche C, Parniske M, Bogusz D: SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. *Proc. Natl. Acad. Sci. U.S.A* 2008, 105:4928-4932.

O34

Potential use of the *Eucalyptus globulus* vacuolar pyrophosphatase 1 (EgEVP1) to generate drought and salt resistant plants

Erwin Krauskopf¹*, Maria Cecilia Gamboa²

¹Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile;

²Fundacion Ciencia para la Vida, Santiago, Chile

E-mail: ekrauskopf@unab.cl

BMC Proceedings 2011, 5(Suppl 7):O34

Desertification is a phenomenon that affects vast territories worldwide usually caused by high soil salt concentrations, being salt and water stress one of the main problems that affect plant growth. Part of the strategy developed by the plant to face these stresses consists on the compartmentalization of cytoplasmic Na⁺ within the vacuole, by a coordinated expression of a H⁺ pump that uses pyrophosphate as a source of energy (known as vacuolar pyrophosphatase) and a Na⁺/H⁺ exchanger, both located on the vacuole membrane. The vacuolar pyrophosphatase has been described in various plant species, much of which are of commercial importance, such as rice, wheat and corn, along some woody species such as poplar and apple. In our laboratory we isolated the cDNA of the *Eucalyptus globulus* vacuolar pyrophosphatase (EVP1), establishing by real-time PCR that its transcript expression increases when *Eucalyptus globulus* plants are subjected to water and salt stress. An analysis of its genomic sequence allowed us to identify four introns and a promoter region of approximately 2Kb. The fusion of
the GUS reporter gene to the promoter region as well as to the first EVP1 intron allowed us to establish for the latter a possible regulatory function, in addition to identifying the promoter tissue-specific expression in *A. thaliana* plants. Finally, *A. thaliana* transgenic plants generated by overexpressing EVP1 produced seeds that exhibited a higher germination rate in high salt conditions compared to the wild-type plants. Furthermore, adult transgenic plants had more leaves in the rosette and a higher tolerance to salt and water stress.

Funded by: PFB-016 and DI-UNAB

# S6. WOOD FORMATION

## O35

### Overexpression of walldof transcription factor increases secondary wall deposition and alters carbon partitioning in poplar

Isabel R Gerhardt¹*, Silvia B Filippi², Vagner Okura³, Jaime Coutinho², Ana P Rizzato², Karina Gui², Nathália Vessali², João HM Pontes², Thiago Cordeiro², Solange M Silva², Alexandre F Garcia², Paulo Arruda³

¹Canavialis Alellyx, Campinas, SP, Brazil, Present address: Embrapa Forestry, Colombo, PR, Brazil; ²Canavialis Alellyx, Campinas, SP, Brazil; ³Canavialis Alellyx, Campinas, SP, Brazil, Present address: State University of Campinas, Campinas, SP, Brazil

E-mail: isabel.gerhardt@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):O35

**Background:** Of the total worldwide biomass produced by land plants, 70% is represented by cell walls [1]. In angiosperm trees, the fixed carbon is accumulated mainly in the secondary walls of xylem cells, which turns xylem cell wall formation a major carbon sink in trees [2]. The understanding of mechanisms that regulate carbon flux to the synthesis of cell wall components is a fundamental goal to maximize the amount of lignocellulosic biomass.

Several families of transcription factors have been shown to be important regulators of secondary wall biosynthesis, but how plants control and direct carbon partitioning among wall components still remains unclear [3,4].

In an attempt to identify transcription factors that participate in the regulation of carbon flux into secondary wall formation, a systematic search for poplar transcription factors expressed in a wood-preferred manner was performed. We have used the collection of over 400,000 ESTs from *Populus* sp. available at GenBank to determine the tissue-specific pattern of genes encoding transcription factors. Among these, a DOF (DNA-binding with one finger) domain transcription factor family member (which we named *WALLDOF*) that presents a cambium-preferred expression profiling was selected for further characterization.

DOF proteins are plant-specific transcription factors suggested to participate in the regulation of biological processes such as light-regulated gene expression, photosynthetic carbon assimilation, accumulation of seed-storage proteins, germination, response to phytohormones, guard cell-specific gene expression, flavonoid metabolism, and lipid biosynthesis [5,6].

In this work, we report that overexpression of *WALLDOF* in transgenic poplar is capable of increasing secondary wall deposition and altering carbon partitioning in poplar stems.

**Methods:** The collection of over 400,000 ESTs from *Populus* sp. available in GenBank was searched for the tissue-specific pattern of expressed genes. For this purpose, ESTs from 100 cDNA libraries were grouped into 15 representative tissues/organs including apical shoot, bark, cambial zone, catkins, cultured cells, flower buds, leaves, mixed tissues, petioles, roots, seeds, shoot meristem, shoots, vegetative buds and xylem (including xylem and wood libraries). A set of clusters generated by CAP3 was searched for those composed of at least 60% of EST reads from libraries representing poplar cambial zone and stem tissues.

One cluster representing a DOF transcription factor family member with a cambium-preferred expression profiling was identified. Using a cDNA synthesized from total RNA from stems of *Populus deltoides* plants, the full-size ORF of the *WALLDOF* gene was cloned under the control of a 1.0 kb fragment of a *Populus deltoides* cinnamate 4-hydroxylase (C4H) promoter. Wild-type aspen hybrid (*Populus tremula* x *Populus alba*) was transformed with *Agrobacterium tumefaciens* carrying the construct.

**Results and conclusions:** We obtained 12 transgenic poplar lines that were transferred to soil and grown in the greenhouse. After two months, they were harvested and screened for changes in lignin deposition by

phloroglucinol staining of transverse stem sections. The initial screen showed that the intensity of the staining was higher in seven of twelve lines, suggesting a stronger lignification in those plants. Based on the initial lignin screen, four of the seven transgenic lines were randomly chosen for in-depth characterization. Overexpression of *WALLDOF* in transgenic poplar leads to a dramatic thickening of secondary wall and a reduction in the lumen area, which resulted in an increase of as much as 78% of the area occupied by wall in fiber cell cross-sections. Wall thickening at the expense of lumen area also resulted in higher wood density in transgenic lines. Moreover, *WALLDOF* overexpressing plants presented increased cellulose content, a general reduction in hemicellulose carbohydrates, mainly mannose and arabinose, and alteration in lignin composition. Starch content in stems was also reduced in transgenic plants.

Aiming to figure out which genes could be related to the altered phenotype, we proceed with a xylem transcriptome analysis of *WALLDOF* events using Affymetrix Genechip Poplar Genome Array. We have identified 825 genes that are differentially expressed, including a set of genes that participate in carbon and nitrogen metabolism.

The results indicate that *WALLDOF* is involved in carbon flux regulation and carbon partitioning through cell wall formation, and can contribute for increasing biomass production.

### References

1. Pauly M, Keegstra K: Cell wall carbohydrates and their modification as raw materials for biofuels. *Plant J* 2008, 54:559-568.

2. Coleman HD, Samuels AL, Guy RD, Mansfield SD: Perturbed lignification impacts tree growth in hybrid poplar - a function of sink strength, vascular integrity and photosynthetic assimilation. *Plant Physiol* 2008, 148:1229-1237.

3. Zhong RQ, Ye ZH: Regulation of cell wall biosynthesis. *Curr Opin Plant Biol* 2007, 10:564-572.

4. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M: Quantitative genetic analysis of biomass and wood chemistry of *Populus* under different nitrogen levels. *New Phytol* 2009, 182:878-890.

5. Kim H-S, Kim SJ, Abbasi N, Bressan RA, Yun D-J, Yoo S-D, Kwon S-Y, Choi S-B: The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis. *Plant J* 2010, 64:524-535.

6. Yang X, Tuskan GA, Cheng MZ: Divergence of the Dof gene families in poplar, *Arabidopsis*, and rice suggests multiple modes of gene evolution after duplication. *Plant Physiol* 2006, 142:820-830.

## O36

### Boundary issues in regulation and evolution of wood formation

Yordaan Yordanov*, Victor Busov

Forest Resources and Environmental Science, Biotechnology Research

Center, Michigan Technological University, USA

E-mail: ysordan@mtu.edu

BMC Proceedings 2011, 5(Suppl 7):O36

Wood has played an indispensable part in the rise of human civilization, evolution of modern plants and has cornerstone importance for the sustained long term health and stability of managed and unmanaged ecosystems. Despite its importance, the molecular mechanisms involved in wood formation are still poorly understood.

Wood formation initiates in a lateral meristem known as vascular cambium which consists of meristem cells (cambial initials) organized in single-cell radial file that form continuous cylinder around the stem. Cambium initials divide to produce xylem, phloem, and ray mother cells that in turn undergo several rounds of divisions followed by differentiation into respective cell types. Because of their importance to regulation the quantity and quality of wood production these processes are of substantial theoretical and applied interest but are poorly understood and thus very difficult to manipulate. The progression from meristem to fully differentiated cells is a complex process requiring highly organized transition from different developmental states, most prominently, loosing the pluripotency of meristem cell and committing to a specific cell/tissue fate (e.g., xylem, phloem or rays). Because of the importance of this transition plants establish developmental boundaries that insulate cells in different stages of this progression. We have recently shown that plant-specific transcription factors of the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family play key role in the establishment and function of these boundaries [The Plant Cell, 2010, 22: 3662-3677].
The discovery leading to the realization of the importance of LBD proteins in woody development was the identification and subsequent characterization of an activation tagged mutant that shows enhanced woody growth. We have found that the activation of a LBD encoding gene, *PtaLBD1* (*Populus tremula* × *P. alba* LBD1) with uncharacterized previously function in poplar and *Arabidopsis*, is primarily involved in differentiation of secondary phloem and ray cells. Transgenic gain and loss-of-function experiments demonstrated that PtLBD1 is a positive regulator of secondary phloem growth and development. Expression and localization experiments showed predominant expression in the phloem and localization of the transcript on the boundary between the differentiating phloem and the cambium zone. Genes encoding regulators of meristem identity genes like *ARBORKNOX1* (*ARK1*) and *ARBORKNOX2* (*ARK2*) were downregulated while key regulators of phloem initiation like *ALTERED PHLOEM DEVELOPMENT (APL)* gene was upregulated in the activation tagged mutant line. The phenotype, localization and misexpression of *ARK1*, *ARK2* and *APL* genes indicate that LBD1 is a critical component in the establishment and function of the developmental boundary between the vascular cambium and the differentiating phloem. It shows that the boundary genes like LBD have dual function to repress meristem identity but promote proliferation and differentiation. We have also found that LBD1 is downregulated by auxin and is co-expressed with strong repressors of auxin response. Because auxin concentrations peak in the cambium and LBD1 is downregulated by auxin we hypothesize that part of the molecular mechanism that establishes the boundary is through auxin-mediated repression of LBD1 in the cambium zone.

Vascular cambium of most extant plants is bifacial – producing xylem to the inside and phloem to the outside of the stem trunk. We then asked if the boundary on the xylem side of vascular stem is organized and regulated in a similar manner. A broad preliminary survey of the LBD family in *Populus* identified 57 members; microarray results display that four LBD genes (*PtaLBD1*, *PtaLBD4*, *PtaLBD15*, *PtaLBD18*) show predominant expression in stems undergoing wood production with two genes expressed in the phloem (*PtaLBD1* and *PtaLBD4*) and two in the xylem (*PtaLBD15*, *PtaLBD18*). This suggests that a similar regulatory mechanism is in operation during xylem growth and differentiation.

Our studies also shed light on possible mechanisms in the evolution of woodiness and wood anatomy. The LBD proteins are present in most genomes of the sequenced species from the plant kingdom with exceptions of algae. This suggests that the LBD gene family has evolved after the colonization of land plants. Comparative sequence analysis of putative LBD1 orthologs in several sequenced plant genomes, indicates a large and highly significant expansion of the LBD1 gene in *Vitis vinifera*. There were 12 putative LBD1 orthologs. *Vitis* is vine and has a unique wood anatomy. Most notably, the rays are highly multiseriate and secondary phloem is well-developed with multiple growth rings. The intercalation of less-lignified tissues in the xylem of vines and lianas and proportionally more parenchyma tissues is an adaptive feature allowing more stem flexibility. The multiseriate rays and increased secondary phloem production in *Vitis* resembles the poplar transgenics with increased *PtaLBD1* expression. Thus the putative increased LBD1 gene dosage in *Vitis*, well-corresponds to its wood anatomical features and suggests that LBD genes like LBD1 may have played important role in evolution of wood anatomy as an adaptive mechanism reflecting species biology and life habit.

Our findings have broad importance with respect to regulation and evolution of wood formation. They provide tools for manipulation of woody growth and development and provide clues for the mechanisms involved in evolution of wood anatomy with relation to plants' biology and ecology.

**Background:** Xylem functions in conduction of water and minerals throughout the plants, and supports the plant body. One of the features of xylem cells is development of secondary wall structure between plasma membrane and (primary) cell wall. Recently, it is expected that knowledge on xylem development can be utilized for application of improvement of the plant biomass, since most portion of wood, which represents one of important sources of woody biomass, is mainly composed of two types of xylem cells, xylem vessels and fiber cells. Previously we established the *in vitro* transdifferentiation system, in which Arabidopsis suspension cells could synchronously transdifferentiate into xylem vessel elements. A number of genes whose expression is elevated during the transdifferentiation processes have been isolated by using microarray analysis [1]. We revealed that one of the identified genes, which encoded a NAC domain protein, *VND7* (*Vascular-Related NAC Domain Protein7*), plays a pivotal role in promoting the xylem vessel differentiation [1,2].

Recently, to efficiently obtain xylem vessel elements, we used a glucocorticoid-mediated post-translational induction system [3]. The transgenic Arabidopsis plants exhibited transdifferentiation of most of cells into xylem vessel elements, and the plants died. This induction system worked in poplar trees and in suspension cultures of cells from Arabidopsis and tobacco. These data demonstrate that the induction systems controlling VND7 activity can be used as powerful tools for understanding xylem cell differentiation.

**Objectives of the research:** Several studies report that VND7 regulates expression of downstream of some transcription factors, suggesting that existence of transcriptional network regulating xylem vessel differentiation. Here, in order to identify direct target genes of VND7, we performed global transcriptome analysis using Arabidopsis transgenic lines in which VND7 activity could be induced posttranslationally.

**Methods:** We generated a transgenic Arabidopsis plant expressing *VND7-VP16-GR* [3] driven by *CaMV35S* promoter. *VND7-VP16-GR* and *VP16-GR* seedlings were soaked with water containing 10 µM cycloheximide (CHX), a protein synthesis inhibitor, for 2 hours. After removal of the solution, the seedlings were re-soaked with water containing 10 µM CHX, with or without 10 µM dexamethazone (DEX), for 4 hours. Microarray analysis was performed using GeneChip ATH1 Arabidopsis genome arrays. The effector, reporter, and reference plasmids [4], were delivered into the rosette leaves of Arabidopsis by particle bombardment. After overnight incubation, luciferase activity was assayed. For electrophoretic mobility shift assay (EMSA), promoter fragments were labeled with biotin. The poly-His-tagged N-terminal region of VND7 (His-VND7<sup>1-161</sup>) protein was purified from *Escherichia coli*.

**Results:** As shown previously, overexpression of *VND7* induces expression of many genes related to the differentiation of vascular vessels [4]. Microarray analysis revealed that 300 genes are upregulated by more than two-fold in the transgenic *VND7* plant. To identify direct target genes of VND7 among the upregulated genes, we used a glucocorticoid-mediated posttranslational induction system. We generated a transgenic Arabidopsis plant expressing chimera *VND7-VP16-GR* gene under the control of the *CaMV35S* promoter [3]. We subjected *VND7-VP16-GR* seedlings to pre-treatment with CHX, followed by treatment with or without DEX. Microarray analysis using these samples revealed that, among the 300 genes upregulated in the *VND7-YFP* plants, 63 were also upregulated (fold change > 2; FDR < 0.1 [P < 0.026]) in the *VND7-VP16-GR* plants in response to DEX treatment. These genes encode a broad range of proteins such as transcription factors, IRREGULAR XYLEM proteins, and proteolytic enzymes, known to be closely associated with xylem vessel formation [5].

To define the promoter region responsible for the upregulation of gene expression by VND7, we carried out transient reporter assays using the *XCP1* promoter sequence. We constructed reporter plasmids by linking various lengths of *XCP1* promoter sequences to a minimal *CaMV 35S* promoter driving the firefly luciferase gene and the *VND7* gene driven by *CaMV 35S* promoter was used as an effector plasmid. We concluded that the region of *XCP1* promoter between residues -211 and -96 is necessary and sufficient for gene expression induced by VND7 [5].

In order to investigate the direct DNA/protein interaction between the *XCP1* promoter sequence and the VND7 protein, we carried out EMSA. Two distinct regions of the *XCP1* promoter were demonstrated to be responsible for VND7 binding. Furthermore, we also showed that VND7 protein binds to several promoter sequences present in candidate direct target genes [5].

O37

Vascular-related NAC-domain 7 directly regulates a broad range of genes for xylem vessel differentiation

Masatoshi Yamaguchi¹*, Nobutaka Mitsuda², Misato Ohtani³,

Masaru Ohme-Takagi², Ko Kato¹, Taku Demura⁴

¹Graduate School of Bioscience, NARA Institute of Science and Technology, Japan; ²National Institute of Advanced Industrial Science and Technology, Japan; ³RIKEN Biomass Engineering Program, Japan; ⁴Graduate School of Bioscience, NARA Institute of Science Technology, Japan and RIKEN Biomass

Engineering Program, Japan

E-mail: yamagu@bs.naist.jp
**Conclusions:** These findings indicated that VND7 upregulates, directly and/or indirectly, many genes involved in a wide range of processes in xylem vessel differentiation. Interestingly, VND7 directly regulates lots of genes involved in programmed cell death, while most of genes controlling secondary cell wall biosynthesis could be regulated through the MYB transcription factors. Further investigation of consensus DNA binding sequences of target genes will help elucidate the regulation of gene expression in xylem vessel formation. To obtain a view of xylem vessel differentiation, we also need to reveal how the direct target genes function during xylem vessel formation.

References

1. Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T: Transcription switches for protoxylem and metaxylem vessel formation. *Genes Dev* 2005, 19:1855-1860.

2. Yamaguchi M, Kubo M, Fukuda H, Demura T: VASCULAR-RELATED NAC-DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. *Plant J* 2008, 55:652-664.

3. Yamaguchi M, Goué N, Igarashi H, Ohtani M, Nakano Y, Mortimer JC, Nishikubo N, Kubo M, Katayama Y, Kakegawa K, Dupree P, Demura T: VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. *Plant Physiol* 2010, 153:906-914.

4. Yamaguchi M, Ohtani M, Mitsuda N, Kubo M, Ohme-Takagi M, Fukuda H, Demura T: VND-INTERACTING2, a NAC domain transcription factor negatively regulates xylem vessel formation in *Arabidopsis*. *Plant Cell* 2010, 22:1249-1263.

5. Yamaguchi M, Mitsuda N, Ohtani M, Ohme-Takagi M, Kato K, Demura T: VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation. *Plant J* 2011, 66:579-590.

**O38**
**Populus biomass protein-protein interactions and their functions**

Xiaoyan Jia¹, Mingzhe Zhao², Chengsong Zhao², Xiaoyan Sheng¹, Allan Dickerman³, Eric Beers², Amy Brunner¹

¹Department of Forest Resources and Environmental Conservation, Virginia Tech, Blacksburg, VA 24060, USA; ²Department of Horticulture, Virginia Tech, Blacksburg, VA 24060, USA; ³Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24060, USA

E-mail: abrunner@vt.edu
BMC Proceedings 2011, 5(Suppl 7):O38

Proteins are molecular machines that play roles in almost all biological activities through interactions with other molecules such as carbohydrates, lipids, nucleic acids and other proteins. We are mapping protein-protein interactions relevant to woody biomass production by focusing on proteins co-expressed in poplar secondary xylem. In addition to revealing novel regulatory mechanisms important to woody biomass production, mapping the poplar protein-protein interactome will provide fundamental information relevant to xylem differentiation and secondary growth. We have cloned a portion of the poplar biomass ORFeome, specifically 374 ORFs that are upregulated in xylem versus phloem, for use in protein-protein interaction research. Here we summarize the techniques we are using to discover and study protein-protein interactions and results to date.

1. Yeast two-hybrid (Y2H) binary assays: 108,205 Y2H binary assays involving over 300 biomass ORFeome members have been performed and 11 interaction pairs identified. The proportional yield from our binary screen is similar to that represented by the current preliminary binary screen data from the Arabidopsis interactome project.

2. Y2H cDNA library screening: We have used 40 bait proteins to screen our poplar xylem cDNA prey library, and a total of 60 biomass ORFeome members comprising putative regulators of lignocellulose synthesis will be screened in 2011. For the 26 ORFs that are completely through the library screen, we have identified 44 unique interacting sequences. Thus far, the proportional yield of interactors for proteins catalyzing metabolic reactions (such as cellulose synthase, PB138) is much lower than that for regulatory proteins (such as NIMA kinase, PB223). Results from these Y2H screens are available at our project website (http://xylome.vbi.vt.edu/index.html). Additional website information includes a project overview, project objectives, progress to date, sequences for all ORFs and primers, clone availability, and Y2H protocols.

3. Confirmation of selected protein-protein interactions identified by Y2H screens: Selected Y2H interactions are being confirmed by independent methods including bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation/affinity purification using plant transient or stable expression systems. We have developed improved vectors and protocols for reducing background fluorescence during BiFC experiments.

4. Functional analysis: We are characterizing the functions of selected interacting pairs in both poplar and Arabidopsis by ectopically expressing or suppressing genes singly and in combination. For ectopic and/or over-expression experiments we are using 35S, SUC2 (phloem specific) and XCP2 (xylem specific) promoters. Co-expression of 35S::PB15 and 35S::PB129 in Arabidopsis resulted in expanded interfascicular regions containing enlarged fibers compared to fibers in wild-type plant interfascicular regions of the inflorescence stem. Importantly, this phenotype was not observed in transgenics overexpressing just one of these genes, showing the potential of novel interactome data to be translated into alteration of wood phenotypes. Arabidopsis mutants carrying T-DNA insertions within Arabidopsis orthologs of PB15 and PB129 are also being evaluated, and Populus RNA interference (RNAi) lines have been generated to study the effects of silencing PB15 and/or PB129 and their paralogs.

5. Transgenic poplar field trial: We prepared transgenic poplar overexpressing 11 biomass genes as tandem affinity purification-(TAP)-tagged fusions and established these in a replicated field trial, which will be evaluated for overexpression phenotypes and serve as a source of xylem for identification of co-purified proteins.

**O39**
**Identification of laccases involved in lignin polymerization and strategies to deregulate their expression in order to modify lignin content in Arabidopsis and poplar**

Jouanin Lise¹, Berthet Serge¹, Mazel Julien¹, Demont-Caulet Nathalie¹, Ayangma Brice², Le-Bris Philippe¹, Baratiny Davy¹, Leplé Jean Charles², Lapierre Catherine¹

¹JPB, UMR1318 INRA-AgroParisTech, 78026 Versailles, France; ²AGPF, INRA, 45002 Ardon, France

E-mail: jouanin@versailles.inra.fr

BMC Proceedings 2011, 5(Suppl 7):O39

Lignins have a major impact on the agro-industrial uses of plants. Until now, most of the strategies considered for lignin reduction have targeted the monolignol pathway since the genes involved in these metabolic steps have been identified in many plants. Less is known about the other steps and in particular on lignin polymerization in the cell wall. While it is established that peroxidases are involved in the polymerization of lignin precursors, it is not yet clear whether laccases (EC 1.10.3.2) participate in constitutive lignification.

In order to address this issue, laccase genes (AtLAC4 and AtLAC17) that are highly expressed in Arabidopsis stems were studied. AtLAC17 was specifically expressed in the interfascicular fibers while AtLAC4 was expressed in vascular bundles and interfascicular fibers. *Arabidopsis* T-DNA insertion mutants were selected and characterized. Two double mutants were obtained by crossing the *AtLAC17* (*lac17*) mutant with two *AtLAC4* mutants (*lac4-1* and *lac4-2*). The single and double mutants displayed normal growth, except the *lac4-2 lac17* mutant that sometimes had a semi-dwarf phenotype and collapsed vessels. While the single mutants had moderately reduced lignin levels, the stems of *lac4-1 lac17* and *lac4-2 lac17* had lignin content reduced by 20% and 40%, respectively. This lower lignin level improved their saccharification yield. Thioacidolysis revealed that disrupting *AtLAC17* mainly affected the deposition of G lignin units in the interfascicular fibers and that complementation of *lac17* with *AtLAC17* restored the normal lignin profile. This study provides evidence that both *AtLAC4* and *AtLAC17* contribute to the constitutive lignification of *Arabidopsis* stems and that *AtLAC17* is involved in the deposition of G lignin units in fibers, suggesting a role in early lignification (Berthet et al, in press).

The double mutants cannot be obtained for species that are propagated vegetatively such as poplar. In order to produce plants with lower laccase activity and reduced lignin content, we therefore used a miRNA strategy. The overexpression of two miRNA (miR397 and miR408) targeting several laccase genes was tested in different plants including Arabidopsis and poplar. These miRNAs were expressed constitutively under the control of
the CaMV 35S promoter or of lignin-specific promoters such as CAD and C4L in transgenic Arabidopsis and poplar.

Results obtained using of this miRNA strategy in *Arabidopsis* and preliminary results for poplar will be presented.

O40

**An AC-type element mediates transactivation of secondary cell wall carbohydrate-active enzymes by PttMYB021, the Populus MYB46 orthologue**

Ines Ezcurra¹, Camilla Johansson, Prashanth Tamizhselvan, Anders Winzell, Henrik Aspeborg

KTH Biotechnology, 106 91 Stockholm, Sweden

E-mail: ines@biotech.kth.se

BMC Proceedings 2011, 5(Suppl 7):O40

**Background:** The transcription factor MYB46, together with its redundant paralog MYB83, regulates expression of secondary cell wall biosynthesis genes in Arabidopsis [reviewed in 1].

We isolated the Populus MYB46 ortholog, PtxtMYB021 from hybrid aspen (Populus tremula x tremuloides) [2]. Transiently expressed MYB021 transactivated gene promoters of Populus xylan-active enzymes GT43A, GT43B and Xyn10A. Analysis of conserved motifs within these promoters identified the sequence CCACCAAC, which is similar to the AC elements mediating transactivation by MYB transcription factors during lignin biosynthesis, and we showed that this motif is enriched in xylem-specific carbohydrate active enzyme (CAZyme) promoters.

**Methods:** To establish whether the AC-type motif in CAZyme promoters is important for their function, we analyzed loss-of-function and gain-of-function GUS reporter constructs of the GT43A promoter in a transient transcription assay, by co-infiltration with a 35S:MYB021 effector in Nicotiana benthamiana leaves.

**Results and discussion:** We show that mutation of the AC-type element in the GT43A promoter abolishes transactivation by MYB021, whereas an AC multimer confers MYB021 transactivation to a minimal 35S promoter. Others have shown that the recombinant Eucalyptus grandii MYB46/MYB83 ortholog, EgMYB2, directly binds the AC-motif variant CCACCTACC found in the EgCCR gene promoter [3], suggesting that MYB021 directly binds the AC-type motif. We propose that, in angiosperms, the AC-type regulatory element mediates MYB46's transactivation of its target genes. Further, MYB46 activates lignocellulose synthesis in a feed-forward loop with downstream MYBs, involving both direct activation of downstream MYBs and joint regulation of downstream secondary cell wall synthesis targets (Fig. 1). Recent results show direct activation of downstream lignocellulose synthesis targets by higher hierarchy NAC domain regulators [4,5].

Interestingly, the downstream MYB regulators of lignin biosynthesis MYB58 and MYB63 bind the AC element but reportedly do not regulate CAZyme-mediated secondary cell wall polysaccharide biosynthesis [6]. Other downstream MYBs that bind variants of the AC element are the EAR domain lignin repressors, such as EgMYB1 and ZmMYB31 (Table 1). Then, binding of the distinct lignocellulose synthesis MYB transcription factors to particular AC-element variants may be mediated by motif sequence, motif context and/or interactions with cofactors. Also, some AC element variants overlap extensively with each other and with the AC-type element defined

<figure><img src="image_15.png" /><figcaption>**Figure 1(abstract O40)** Regulatory networks in lignocellulose synthesis in angiosperms, shown using Arabidopsis gene nomenclature, involve feed-forward loop circuits. Dashed arrows highlight direct target gene activation by higher hierarchy regulators. White boxes at the bottom indicate target gene programs. For simplicity, some regulators, including ASL19, MYB103 and KNAT7, are omitted. Adapted from [1] and references therein.</figcaption></figure>
<table><caption>Table 1(abstract O40) Sequence overlap in AC motifs</caption><thead><tr><th>Species</th><th>Genbe</th><th>Motif name</th><th>Motif sequence</th><th>MYB protein<sup>†</sup></th><th>Reference</th></tr></thead><tbody><tr><td>Phaseolus</td><td>PAL2</td><td>AC-I</td><td>CCCACCTACC</td><td>n.d.</td><td>[9]</td></tr><tr><td>Phaseolus</td><td>PAL2</td><td>AC-II</td><td>CCACCAACCC...</td><td>n.d.</td><td>[9]</td></tr><tr><td>Petroselinum</td><td>4CL1</td><td>AC-II</td><td>CTCACCAACCC</td><td>n.d.</td><td>[10]</td></tr><tr><td>Populus</td><td>CCoAOMT</td><td>AC-II</td><td>CTCACCAACCC...</td><td>n.d.</td><td>[7]</td></tr><tr><td>Eucalyptus</td><td>EgCCR</td><td>MBSIIG</td><td>ccACCTACC</td><td>EgMYB2<sup>§</sup></td><td>[3]</td></tr><tr><td>Eucalyptus</td><td>EgCCR</td><td>MBSIIG</td><td>ccACCTACC</td><td>EgMYB1</td><td>[11]</td></tr><tr><td>Arabidopsis</td><td>4CL1</td><td>AC-II</td><td>tCACCAAC</td><td>MYB58/63</td><td>[6]</td></tr><tr><td>Zea</td><td>ZmCOMT</td><td>AC-II</td><td>tcACCAAC</td><td>ZmMYB31</td><td>[12]</td></tr><tr><td>Populus</td><td>GT43A</td><td>AC-type</td><td>CCACCAAC</td><td>PttMYB021<sup>§</sup></td><td>[2]</td></tr></tbody></table>

In motif sequences, lowercase indicates ases outside the defined motif, and asterisks highlight invariant bases. Similarity to the GT43A AC-type elements is underlined. †Indicates which MYB protein binds the motif. §Ortholog of MYB46.

by us (Table 1), so it remains to be established whether they are all representatives of one identical core sequence motif, as previously proposed [7]. Establishing the exact sequence requirements for AC-element recognition by distinct MYBs will facilitate elucidation of lignocellulose-related regulatory networks in different species, through bioinformatic analysis [8].

**Conclusions:** The AC regulatory element, here preliminarily redefined as the sequence CCACCAAC, is a true "lignocellulose synthesis response element", mediating MYB46-dependent transactivation of the whole secondary cell wall gene program. This knowledge will enable inference of MYB46-centered regulatory networks in different species, through bioinformatic analysis.

**Acknowledgements:** We thank David Caparrós-Ruiz for helpful comments and discussions.

**References**

1. Ohashi-Ito K, Fukuda H: Transcriptional regulation of vascular cell fates. *Curr Opin Plant Biol* 2010, 13:670-676.

2. Winzell A, Aspeborg H, Wang Y, Ezcurra I: Conserved CA-rich motifs in gene promoters of PtxtMYB021-responsive secondary cell wall carbohydrate-active enzymes in Populus. *Biochem Biophys Res Commun* 2010, 394:848-853.

3. Goicoechea M, Lacombe E, Legay S, Mihaljevic S, Rech P, et al: EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. *Plant J* 2005, 43:553-567.

4. Zhong R, Lee C, Ye ZH: Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. *Mol Plant* 2010, 3:1087-1103.

5. Yamaguchi M, Mitsuda N, Ohtani M, Ohme-Takagi M, Kato K, Demura T: VASCULAR-RELATED NAC-DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation. *Plant J* 2011 in press.

6. Zhou J, Lee C, Zhong R, Ye ZH: MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. *Plant Cell* 2009, 21:248-266.

7. Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W: Genome-wide characterization of the lignification toolbox in Arabidopsis. *Plant Physiol* 2003, 133:1051-1071.

8. Pilpel Y, Sudarsanam P, Church GM: Identifying regulatory networks by combinatorial analysis of promoter elements. *Nat Genet* 2001, 29:153-159.

9. Hatton D, Sablowski R, Yung MH, Smith C, Schuch W, Bevan M: Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. *Plant J* 1995, 7:859-876.

10. Hauffe KD, Lee SP, Subramaniam R, Douglas CJ: Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco. *Plant J* 1993, 4:235-253.

11. Legay S, Sivadon P, Blervacq AS, Pavy N, Baghdady A, et al: EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar. *New Phytol* 2010, 188:774-786.

12. Fornalé S, Shi X, Chai C, Encina A, Irar S, et al: ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. *Plant J* 2010, 64:633-644.

# S7. PROPAGATION AND IN VITRO TECHNOLOGIES

## O41

### Analysis of cellulose synthase (CesA) promoter function in trees using Induced Somatic Sector Analysis (ISSA)

Nicky Creux¹, Alexander Myburg¹, Gerd Bossinger², Antanas Spokevicius²*
¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; ²The University of Melbourne, Department of Forest and Ecosystem Science, Creswick, Victoria 3363, Australia

E-mail: avjs@unimelb.edu.au

BMC Proceedings 2011, **5**(Suppl 7):O41

Detailed knowledge of the tissue specificity of gene expression is of central importance not only for our understanding of developmental processes during wood formation, but also is a prerequisite for the deliberate manipulation of xylogenesis candidate genes. Today, much of our knowledge about specific gene expression is based on annual model plants in part because perennial tree systems are often seen as too cumbersome for detailed promoter studies. Here we address this issue in a novel way with the investigation of a number of well characterised cellulose synthase (CesA) genes in the stems of perennial tree species using the *in vivo* Induced Somatic Sector Analysis (ISSA) system. Three primary and four secondary cell wall associated CesA gene promoters from *Eucalyptus grandis* and *Arabidopsis thaliana* were previously isolated and cloned in front of the GUS reporter gene then, using *Agrobacterium*-mediated transformation, transformed into cambial initials in stems of eucalypt and poplar plants. A CAMV35S promoter::GUS vector was also used as a control. After a period of growth, stems were harvested and GUS-staining patterns were analysed and interpreted. Results from this work show that in eucalypts the staining patterns are consistent with observations in other species whereas in poplar conflicting results were observed. These findings and the general utility of ISSA for these studies are discussed.

## O42

### Variation of telomeric repeats in Scots pine (Pinus sylvestris) – is there a connection to ageing and loss of regeneration ability?

Tuija Aronen
Finnish Forest Research Institute, Punkaharju Unit, FI-58450 Punkaharju, Finland

E-mail: tuija.aronen@metla.fi

BMC Proceedings 2011, **5**(Suppl 7):O42

The shortening of telomeres, the specific structures of repeated DNA sequence at the end of the eukaryotic chromosomes, has been connected with ageing and loss of cell replication or regeneration capacity. There is, however, only limited information available on telomeres of long-living trees. Physiological ageing, on the other hand, represents a serious
problem for vegetative propagation of conifers, while in deciduous tree
species ageing has no such a strict influence on regeneration ability. The
aim of this study was, for the first time, to determine the variation in
telomeric repeats in an economically important Nordic conifer, Scots pine
(Pinus sylvestris L.), and to study potential connections to tree ageing.

Scots pine individuals ranging from immature embryos to 200-year-old
trees were examined. In 1- and 5 year-old seedlings and 50-, 100- and
200-year old trees, cambium, bud and needled samples were collected
from different positions within a tree. The length of telomeric repeats in
the extracted total genomic DNA was then determined by Southern
hybridisation. In addition, telomerase enzyme activity was measured from
the protein extracts prepared from the same tissues using TRAP
(Telomeric Repeat Amplification Protocol).

In Scots pine samples, the size of telomeric repeats detected was variable, ranging from 0.9 kb up to 26 kb. Based on exonuclease treatment, high molecular weight repeats seem to be genuine telomeres at the ends of the chromosomes, while the low molecular weight signals probably originate at interstitial or centromeric positions. The Southern hybridisation was thus adjusted to show only high-molecular weight signals, and analyses were performed based on them.

A decline in the average length of the telomeric repeats was observed with increasing level of tissue differentiation: immature and germinating embryos had the longest repeats, followed by cambium, elongating buds with tiny meristems and, finally, full-sized needles without meristems having the shortest repeats. When embryo samples were excluded from the data, the age of the tree had no significant effect on the length of telomeric repeats, although there was a tendency for telomere shortening with increasing age. In cambium samples, also the position of the tissue within tree (stem base versus top) affected the length of telomeric repeats, the telomeres shortening towards top of tree. The telomeric repeats also varied remarkably between tree individuals. Potential connections to regeneration ability are discussed.

## O43
Agrobacterium-mediated co-transformation of American Chestnut
(Castanea dentata) somatic embryos with a wheat oxalate oxidase gene

Bo Zhang*, Andrew Newhouse, Linda McGuigan, Charles Maynard,
William Powell

State University of New York-College of Environmental Science and Forestry
(SUNY-ESF), USA

E-mail: bozhang@syr.edu

BMC Proceedings 2011, 5(Suppl 7):O43

**Background:** American chestnut is a tree of great historical, ecological, and economical importance. It once dominated forests in eastern United States until the introduction of chestnut blight fungus (Cryphonectria parasitica) in the late 19th century. Within 50 years, C. parasitica killed almost all of the 4 billion American chestnut trees in the eastern United States. The fungus first infects wounded stem, secretes oxalic acid to decrease the pH of the infected tissue to toxic levels for the tree, but optimum for fungal enzymes, and then mycelia fans spread forming a canker which when it girdles a branch prevents water and nutrient transport, eventually killing the tree above the canker. The fungus does not infect the roots, thus allowing the growth of adventitious shoots to keep the tree alive. However, this survival is only temporary because these spouts will again get infected by the fungus and die back to the ground. It is this continuing circle that made American chestnut, once a great canopy tree, to no more than an early-succession-stage shrub today. Efforts to restore American chestnut back to its native range are currently being made. In our labs, we use an *Agrobacterium*-mediated co-transformation system to deliver potential resistant genes into somatic embryos of American chestnut together with a selectable marker gene. The gene of interest we used is an oxalate oxidase gene from wheat (*Triticum aestivum*). Oxalate oxidase (OxO) can degrade the fungus secreted oxalic acid to carbon dioxide and hydrogen peroxide. This action has a dual-effect: bringing up the pH of the infected sites thus neutralizing the virulent effect of oxalic acid, and increasing the expression of defense related genes induced by hydrogen peroxide byproduct. The selectable marker we used is Green Florescent Protein (GFP) from *Aequorea victoria*,which allows us to have a direct visual selection of successful transformants.

**Methods:** *Agrobacterium tumefaciens* strain EHA105 was used for the co-transformation. Vectors containing OxO and GFP were introduced to EHA105 separately via electroporation so each *Agrobacterium* had only one vector. Transformed EHA105 were grown separately under the selection of kanamycin and rifampicin until an O.D. of 0.8-1.2 was reached. *Agrobacterium* with optimum O.D. were harvested and re-suspended in *vir* induction medium containing acetosyringone. To approximately equalize the concentration of bacteria samples with different vectors, the amount of *vir* induction medium added was calculated by multiplying the O.D. value with 50ml. Bacteria samples were incubated in *vir* induction medium for three to four hours under room temperature with slow agitation. After *vir* induction, bacteria samples containing different vectors were mixed together in two different volume ratios: 2 (OxO):1 (GFP) and 4 (OxO):1 (GFP). The mixture was then added to embryo clusters to bring a total volume of 6ml followed by co-cultivation on a turning machine for an hour at room temperature. After the co-cultivation, embryo clusters were taken out to put in desiccation plates (plates with 200ul ddH₂O saturated filter paper) and kept in dark for two days under room temperature. After desiccation, embryos were transferred to *Agro*-kill medium containing carbenicillin and cefotaxime and kept in dark under room temperature. After a week, embryos were transferred to selection medium containing carbenicillin, cefotaxime, and appropriate selection agents (herbicide PPT and paromomycin in our case). Embryos were transferred to fresh selection medium every two weeks and were examined for GFP expression under a UV microscope (Figure 1).

GFP positive embryos were separated and multiplied in selection medium followed by genomic DNA extraction and PCR. Positive transformants with both GFP and OxO together with six GFP-only events were selected for regeneration as previously developed in our labs. The expression and copy number of OxO were tested by RT-PCR (Real Time-PCR).

**Results and discussion:** Thirteen experiments with a total of 208 embryo clusters were done using the 2 (OxO):1 (GFP) ratio resulting in 50 GFP positive events. 10 out of the 50 events were also OxO positive, giving a co-transformation efficiency of 20%, or 4.8% per embryo cluster. Three experiments with a total of 150 embryo clusters were done using the 4 (OxO): 1 (GFP) ratio resulting in 45 GFP positive events. 18 out of the 45 events were also OxO positive, giving a co-transformation efficiency of 40%, or 12% per embryo cluster. Doubling the ratio of vector containing gene of interest to vector containing the selectable marker from 2:1 to 4:1 gave a 2.5-fold increase in co-transformation efficiency. All of the 28 events went through the regeneration process together with 6 GFP-only events. 4 out of the 10 events produced by 2:1 ratio regenerated into whole plants. 17 out of the 18 events produced by 4:1 ratio regenerated into whole plants. All 6 GFP-only events regenerated into whole plants. These regenerated American chestnut plants seem to exhibit normal morphology. A gradient of OxO expression levels were found with the highest being more than 200 fold higher than the lowest expressed event.

<figure><img src="image_16.png" /><figcaption>**Figure 1(abstract O43)** GFP expression in transformed American chestnut embryo cluster under UV light.</figcaption></figure>
Copy numbers of the transgene *oxo* were also tested via RT-PCR showing most events with 2 copies, few with single copy, and a couple with higher copy numbers. The difference in regeneration ability and expression levels among these events was likely a result of position effect, which the expression of foreign gene is affected by the insertion site and nearby regulatory factors in the genome.

**Conclusions:** The efficiency of *Agrobacterium*-mediated co-transformation of American chestnut somatic embryos improved significantly when increasing the ratio of vector containing the gene of interest to vector containing the selectable marker in the co-cultivation mixture. Other factors of co-transformation including *Agrobacterium* strain, co-cultivation time, and desiccation time, etc. can also be investigated to improve the overall efficiency of this process. Co-transformation proved to be an effective way to introduce foreign genes into American chestnut to test their effectiveness on chestnut blight resistance. Having GFP on a separate plasmid provided a useful tool for removing of the marker gene in future. However, further tests need to be done to determine whether the GFP gene is linked to *oxo* in the genome.

## O44

### Evaluation of reference genes for quantitative PCR analysis during somatic embryogenesis in conifers

José Javier de Vega-Bartol¹, Raissa Raquel Santos, Marta Simões, Celia Miguel Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa (IBET/ITQB-UNL), Portugal E-mail: devegabartol@itqb.unl.pt

BMC Proceedings 2011, 5(Suppl 7):O44

Clonal propagation by somatic embryogenesis (SE) has a great application potential in conifers, but its efficiency still needs further improvement. Quantitative PCR (qPCR) is widely used for gene expression analysis during SE as part of studies to characterize gene function. In qPCR, data is normalized against a reference gene with a uniform expression during the tested conditions. Studies have shown that no single housekeeping gene is universal for all experiments, so a systematic characterization of several endogenous genes for concrete conditions is fundamental for biological accuracy.

We evaluated 10 genes present in *Pinus pinaster* selected among those commonly used as endogenous reference in conifers (*actin*, *α-tubulin*, *elongation factor 1 α*, *adenosine kinase* and *ubiquitin*), previously tested in model plants (*Histone 3*, *SAND* protein and *clathrim adaptor complex* -CAC- subunit), and with a lower variation of expression in five microarray samples at different embryogenesis stages according to mRNA hybridization results (homologous to a heat shock protein -HEATSK- and to an ether reductase -REDUC-) (Simões *et al.*, unpublished results). Stability of the expression in three developmental stages (embryogenic tissue in proliferation, mature embryos and germinated embryos) was evaluated with two different algorithms (implemented by programs geNorm and Normfinder), both in *Pinus pinaster* and *Picea abies*, so results could be expanded to other conifers. Previously, efficiency was calculated with programs Linreg and qPCR miner and the distribution of Cts by statistical boxplot analysis, for each amplicon.

Results evidenced that the geometric averaging of two-to-four of these genes is accurate enough. Using genes *elongation factor 1 α*, *SAND* and *α-tubulin* as reference is the better generic combination, and so probably the preferable option to consider in other conifers. Among them, *SAND* and *α-tubulin* had a reduced range of Cts, while it was higher in *elongation factor 1 α*, especially in *Pinus pinaster*. Some discrepancies were detected in the ranking base on stability for the other genes, so this generic combination can be improved in certain stages or species, like *adenosine kinase* in *Pinus pinaster* or CAC when analysing embryogenic tissue in proliferation. *Adenosine kinase* and *ubiquitin* are also acceptable options, as they showed small ranges of Cts and medium stability values in the three stages in both species. By contrast, several genes showed a higher variation in our experiments, as both selected from the microarray analysis (REDUC and HEATSK), and one commonly used reference gene (*actin*), which evidence the importance of these kind of studies.

**Acknowledgements:** This work has been supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through projects PTDC/AGR-GPL/102877/2008, P-KBBE/AGR-GPL/0001/2009 and grant SFRH/BD/32037/2006 (MS).

## O45

### Optimisation of methods for *Agrobacterium rhizogenes* mediated generation of composite plants in *Eucalyptus camaldulensis*

Aiyar Balasubramanian¹, Rathinavel Venkatachalam¹, K R Selvakesavan¹, Abraham Sanu Mary¹, Hassen Gherbi², Sergio Svistoonoff², Claudine Franche², Didier Bogusz², N Krishna Kumar¹, Mathish Nambiar-Veeti¹*

¹Institute of Forest Genetics and Tree Breeding, Coimbatore, Tamil Nadu-641002, India; ²Institut de Recherche pour le Développement (IRD), Montpellier-34394, France

E-mail: nvmathish@yahoo.com

BMC Proceedings 2011, 5(Suppl 7):O45

**Background:** The wide adaptability, fast growth rate, good form, excellent wood and fiber properties make *Eucalyptus* the most widely planted genus of plantation forest trees. With the availability of *Eucalyptus* genome sequence information, assignment of function to genes contributing to desired traits require functional validation via overexpression or silencing in the context of the plant environment. However, plant regeneration from transformed tissues has been the most time consuming and rate-limiting step for rapid gene function analysis in tree species. The composite plants with transgenic hairy roots derived via *Agrobacterium rhizogenes* mediated transformation are therefore increasingly being used in species with such limitations to study root development and root-biotic interactions [1]. In this paper we describe a protocol developed for generation of composite plants using *A. rhizogenes* mediated transformation of *Eucalyptus camaldulensis*.

**Methods:** Transformation experiments were carried out using an agropine mannopine-type strain A4RS, harbouring a green fluorescent protein (sGFP) based binary vector pHKN29, constructed from pCAMBIA 1300 by replacing hygromycin phosphotransferase (hpt) with the sGFP(S65T) [2]. Infection was carried out using a sterile needle swabbed with *A. rhizogenes* grown for 48 hours in YEB plates containing 100 µM Acetosyringone. The infected plants of *E. camaldulensis* were then placed under dim light on modified MS [3] medium comprising half strength MS macro elements, full strength MS minor elements and organic supplements. The petridishes were placed slantingly with the plants in an upside-down position. Following cocultivation, the cultures were transferred to fresh medium with plants in an upright position. Hairy roots were made bacteria-free by making transfers every 15 days to fresh medium containing 500mg/l cefotaxime. The hairy roots generated were examined under a stereo fluorescence microscope (Nikon, Japan) for visualization of GFP following excitation by blue light (Excitation filter 495nm, Barrier filter 520nm). The nontransformed roots were removed using sterile blades. The composite plants were placed in test tubes, and different media including full MS, modified MS, Knop's, BS and Hoagland's solutions were evaluated for growth under hydroponic conditions. The composite plants were shifted to the transgenic greenhouse, where in the cotton plugs were loosened gradually and finally removed after a period of 2 weeks. The plants were then placed so that roots were immersed in the liquid media and shoots were above the test tube rim covered with aluminium foil. Few of these plants were subjected to stepwise increasing concentration of 50mM NaCl to determine their tolerance to salt stress.

**Results and discussion:** Inoculation with A4RS resulted in roots, arising mainly from the injured surface of the explants. Root initiation occurred within first 7 days after inoculation of explants with *A. rhizogenes*. The roots reached a length of 8-10cm in 45 days. The results were similar to the report of MacRae and Van Staden (1993), where in genetic transformation using the *A. rhizogenes* strain LBA 9402 was assessed by the detection of opines in the root generated from *in vitro* propagated seedling explants of *E. grandis*, *E. dunnii* and *E. nitens*. In our study, the bright green fluorescence due to co-transformation of the pHKN29 vector harbouring the sGFP(S65T) was used for screening of transgenic roots (Figure 1). No fluorescence was observed in untransformed control roots of *Eucalyptus*.

**Effect of explant type:** Seedlings showed root induction in 68% of the explants at an average of 1.08 roots per plant of which 36% were cotransformed with the pHKN29 as confirmed by the GFP expression. The *in vitro* propagated clonal plantlets showed root induction in 37.5% of the explants at an average of 0.45 roots per plant of which 4.1% showed GFP expression.

**Effect of cocultivation conditions:** In a separate experiment, out of the four different basal media used for cocultivation, modified MS media gave the highest average number of hairy roots at 1.3 per plant, when
<figure><img src="image_17.png" /><figcaption>**Figure 1 (abstract O45)** GFP expressing hairy root of *E. camaldulensis*.</figcaption></figure>

compared to 0.8, 0.7 and 0.6 in case of 0.1 MS, Knop's and Hoagland's media (Figure 2).

Different supplements like phosphate buffer and calcium chloride were included in the cocultivation media to evaluate their influence on hairy root induction. Addition of calcium chloride to the cocultivation media reduced the average number of hairy roots induced per plant by 30%. Similarly, phosphate buffer also did not significantly influence the hairy root generation.

However, the inclusion of 100 µM acetosyringone in the cocultivation medium increased the average number of hairy roots to 1.6 per plant as

compared to 1.3 for media without acetosyringone (Figure 2). Acetosyringone has been known to enhance transformation efficiency through activation of *vir* genes in *Agrobacterium*.

Preliminary experiments indicated that, cotransformation efficiency was higher when cocultivation was carried out at 16.5°C for 14 days when compared to 22°C for 7 days.

**Effect of different liquid media on hardening of composite plants:**

Different liquid media were evaluated for hardening of composite plantlets. Hoagland's solution showed the highest survival (73.3%) when compared to MS (20%), modified MS (16%), B5 (0%) and Knop's (3.3%).

<figure><img src="image_18.png" /><figcaption>**Figure 2 (abstract O45)** Effect of different media / supplements on the average number of roots generated per plant post cocultivation.</figcaption></figure>
To determine the tolerance of these composite plants under salt stress, they were subjected to weekly increase in the concentration of NaCl by 50mM. Fifty percent of the Eucalyptus composite plants were able to tolerate up to 300 mM NaCl stress.

**Conclusion:** A composite plant strategy was developed utilising GFP based screening of transgenic hairy roots. Different parameters were evaluated to increase transformation efficiency using *A. rhizogenes*. Our studies showed that, modified MS media containing 100 µm Acetosyringone was optimum for enhancing the cotransformation efficiency. Hoagland's solution was identified as an appropriate media for hardening of Eucalyptus composite plants under transgenic green house conditions. Roots being the major portal of entry of ions to plants, the composite plant strategy will provide a rapid tool for the functional analysis of the genes vital for ion transport and root development. The composite plant strategy will be used to validate the function of *EchKT1/EthKT1*, the homologue of which have been reported to contribute to Na⁺ uptake and xylem unloading of Na⁺ in model plant and crop species.

### References

1. Gherbi H, Nambiar-Veetil M, Zhong C, Félix J, Autran D, Girardin R, Vaissaye V, Auguy F, Bogusz D, Franche C: Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata. *Mol Plant Microbe In* 2008, 21:518-524.

2. Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H: Non-invasive detection and applications of non-toxic, S65T-type green fluorescent protein in living plant. *Plant J* 1999, 18:455-463.

3. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. *Plant Physiol* 1962, 15:473-479.

4. MacRae S, Van Staden J: *Agrobacterium rhizogenes*-mediated transformation to improve rooting ability of eucalypts. *J of Tree Physiol* 1993, 12:411-418.

## O46

### New cytokinin derivatives – their discovery, development and use for micropropagation of endangered tree species

Karel Dolezal<fnref n="1" />, Jana Malá<fnref n="2" />, Pavlina Máchová<fnref n="2" />, Helena Cvrcková<fnref n="2" />, Michal Karady<fnref n="1" />, Ondřej Novák<fnref n="1" />, Lucie Szucova³, Jaromir Mikulík<fnref n="1" />, Lukas Spichal³, Miroslav Strnad³

BMC Proceedings 2011, 5(Suppl 7):O46

Elms (*Ulmus* sp.) are highly valuable trees for their great strength, tightly twisted grain, and durability, as well as for their cold and salt tolerance [1]. Wych elm (*Ulmus glabra*, Huds.) is a native species in Europe. In the Czech Republic, it is spread especially in mountain regions. During the years 1970–80, the second epidemic of Dutch elm disease destroyed most elm populations in Europe. The elm trees, which have survived the disease, are capable to maintain a high degree of resistance against the repeated infection. Micropropagation represents promising alternative of their vegetative reproduction.

The wild service tree (Sorbus torminalis (L.) Crantz) is a slow-growing tree reaching maximum height of 20–25 m at around 80–100 years. According to the latest data, the species is also disappearing from forests of the Czech Republic and it is considered to be endangered. On the other hand, the wild service tree is rated as one of the most valuable hardwoods [2] with a great potential for wider use in forestry and forest ecology, and also for its importance in the timber industry.

The *in vitro* induction of organogenesis, rooting and acclimatization of above mentioned elm species as well as wild service tree were optimized and standartized. Widely used BAP is an important and affordable cytokinin routinely utilized for its effective stimulatory properties in micropropagation practice. On the other hand, BAP may negatively influence the growth, rooting and acclimatization processes in some crops [3]. It is known that hydroxylated aromatic cytokinins (topolins) are more resistant to cytokinin oxidase (CKX), more stable and in some biological systems active at lower concentrations than the isoprenoid CKs. In addition, *meta* - topolin does not

inhibit root formation which is a typical inconvenient effect of high concentrations of BAP. These properties might help to enhance the future productivity of plant tissue culture industry crops [3,4]. The development of other new CK derivatives exhibiting high morphogenetic activity might consequently be of a great practical importance in plant biotechnology. During our recent search for naturally occurring aromatic cytokinins (ARCKs) in plants, another new group of endogenous BAP derivatives – methoxytopolins – exhibiting high biological, especially anti-senescence (and surprisingly also anti-cancer) activity, was discovered. Based on these results, we synthesized several groups of their synthetic analogues that exhibited high activity in three different CK bioassays and showed the ability to activate cytokinin receptors and/or to inhibit CKX. Best compounds so far (6-(3-hydroxybenzylamino)purine (mT) and the 6-(3-methoxybenzylamino) purine-9-ribofuranoside (MeomTR)) were used for retardation of senescence during multiplication stage of micropropagation of above mentioned tree species as well as for rooting support. Subsequently, wide range of endogenous plant hormones (isoprenoid cytokinins, IAA, ethylene) were quantified (using UPLC-MS/MS and/or GC/FID, respectively) and compared in relation to cytokinin exogenously used.

The results about optimal endogenous plant hormone concentrations and their dependence on different exogenous cytokinin used in the cultivation media, may improve *in-vitro* micropropagation efficiency as well as quality of *ex vitro* acclimatized plants of above mentioned elm species, wild service tree and possibly also of other tree species.

This work was supported by the Ministry of Agriculture of the Czech Republic (Mze 0002070202, QI 92A247), Czech Ministry of Education (MSM 6198959216), ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research.

### References

1. Heybroek HM, Elgersma DM, Scheffer RJ: Dutch elm disease - an ecological accident. *Outlook Agric* 1982, 11:1-9.

2. Demesure B, Leguerroué B, Lucchi G, Prat D, Petit RJ: Genetic variability of a scattered temperate forest tree Sorbus torminalis L-(Crantz). *Ann. For. Sci* 2000, 57:63-71.

3. Strnad M: The aromatic cytokinins. *Physiol. Plant* 1997, 101:674-688.

4. Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC: *Meta-topolin*, an alternative to benzyladenine in tissue culture? *Physiol. Plant* 1996, 98:291-298.

# S8. GENOMIC, PROTEOMIC AND METABOLOMIC TECHNOLOGIES

## O47

### Haploid transcriptome analysis reveals allelic gene expression variants, co-expressed gene groups, and linkages between expression and copy number variation

Jukka-Pekka Verta<fnref n="1" />*, Sebastien Caron<fnref n="1" />, Isabelle Giguère<fnref n="1" />, Brian Boyle<fnref n="1" />, Christian R Landry<fnref n="2" />, John MacKay<fnref n="1" />

BMC Proceedings 2011, 5(Suppl 7):O47

**Background:** Genetic variation can cause changes in gene expression (mRNA abundance) among individuals. This so-called heritable variation in gene expression is affected by genetic variants that are co-segregating with the gene locus (local/cis effects) and/or segregating independently from it (distant/trans effects). Genetic variation in gene expression can be measured to estimate the extant of variation in gene expression within a population, and to determine to what degree expression alleles of different genes are connected within regulatory networks. Furthermore, determining whether variation in the expression of a gene is linked to local or distant effects allows us to make inferences about how heritable variation may change depending on gene function, the number of interacting partners, genetic architecture and evolutionary history [1,2].

Detecting heritable variation in gene expression can be a challenging task in diploid organisms, mainly because of tissue-specificity and dominance effects of allelic expression. For example, up to 70% of gene expression alleles in *Drosophila* may be masked by dominance [3]. We developed an experimental system to overcome these obstacles by utilizing the conifer

<footnote n="1">¹Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany AS CR, 783 71 Olomouc-Holice, The Czech Republic;</footnote>

<footnote n="2">²Institute of Forest and Game Management, Strnad 136, 252 02 Jilovišt?, The Czech Republic; ³Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitel? 11, CZ 783 71 Olomouc, Czech Republic</footnote>

<footnote>E-mail: karel.dolezal@upol.cz</footnote>

<footnote n="1">¹Centre d'étude de la forêt, Université Laval, Québec QC, Canada;</footnote>

<footnote n="2">²Department of Biology, Université Laval, Québec QC, Canada</footnote>

<footnote>E-mail: jukka-pekka verta.1@ulaval.ca</footnote>
seed's maternally derived haploid tissue, the megagametophyte. Analyzing a set of sibling megagametophytes allows us to first, measure separately the expression each of the two alleles in the maternal genome in the absence of dominance and second, identify genes whose expression levels are co-segregating. In addition, the megagametophyte allows us to categorize the underlying genetic variants into local or distant with a simple co-segregation assay.

**Methods:** We set out to characterize segregating variation in gene expression in white spruce (*Picea glauca* [Moench] Voss). We analyzed the transcriptomes of germinating sibling megagametophytes from two controlled-crossed families (C9412516: male 2388 x female 77111 & C9612856: male 80109 x female 80112) with a custom microarray comprised of 32,000 spotted oligonucleotides, which represent over 25,000 unique white spruce genes. Each megagametophyte was split into two halves to provide technical replicates that were analyzed separately. A separate comparison of microarray results and RNA-Seq data has been carried out to validate the quality of the microarray.

The single-color microarray data was background-corrected and quantile-normalized with the R package Limma [4]. We used the R package Mclust [5] to test for unimodal vs. bimodal expression distributions of each gene across sibling megagametophytes. Genes that exhibited at least two expression alleles, which segregated within the 95% IC of their expected proportions, were selected. The segregation had to be repeatable with a replicate sample set to be considered valid.

**Results and discussion:** Analysis of two families of sibling megagametophytes (n=18) identified close to a thousand genes with segregating gene expression patterns in both of the two families. Approximately 10% of these genes were shared. Zero replicates are expected to have the same clustering pattern by chance alone (binomial p=3.810-6 n=18). The number of variable genes is comparable to that found between 50 nearly isogenic *Drosophila* lines [6].

We have discovered a large number of genes with gene expression patterns segregating in a Mendelian way in white spruce. We are presently analyzing the relationships between gene function and paralog number with the variation in its expression in order to determine whether heritable variation in gene expression is associated with same genetic attributes in white spruce as what has been reported in model organisms. We have also begun investigating the contribution of local vs. distant effects on the expression alleles identified in the megagametophytes, and their nature, by studying the co-segregation of gene expression, SNPs and copy number variations (CNVs). Preliminary comparative genomic hybridization data suggests a significant portion of genes which show segregating gene expression alleles also exhibit CNVs. In follow-up experiments, we will address the amount of dominance between the gene expression alleles by comparing gene expression in megagametophytes versus self-fertilized embryos.

**Acknowledgments:** The seed material was provided by J. Beaulieu (Canadian wood fibre centre).

**References**

1. Landry CR, Lemos B, Rifkin SA, Dickinson WJ, Hartl DL: Genetic properties influencing the evolvability of gene expression. *Science* 2007, 317:118-121.

2. Kliebenstein D: A role for gene duplication and natural variation of gene expression in the evolution of metabolism. *PLoS One* 2008, 3:e1838.

3. Lemos B, Araripe LO, Fontanillas P, Hartl DL: Dominance and the evolutionary accumulation of cis-and trans-effects on gene expression. *PNAS* 2008, 105:14471-14476.

4. Smyth GK, Speed T: Normalization of cDNA microarray data. *Methods* 2003, 31:265-273.

5. Fraley C, Raftery AE: MCLUST version 3 for R: Normal mixture modeling and model-based clustering. *University of Washington Tech Report* 2009, 504.

6. Hsieh W, Passador-Gurgel G, Stone EA, Gibson G: Mixture modeling of transcript abundance classes in natural populations. *Genome Biology* 2007, 8:R98.

O48

**Targeted sequencing in the loblolly pine (Pinus taeda) megagenome by exome capture**

Leandro Neves¹*, John Davis², Brad Barbazuk³, Matias Kirst²

¹Plant Molecular and Cellular Biology Program, University of Florida,
Gainesville, FL, USA; ²Plant Molecular and Cellular Biology Program and
School of Forest Resources and Conservation, University of Florida,

Gainesville, FL, USA; ³Plant Molecular and Cellular Biology Program and
Department of Biology, University of Florida, Gainesville, FL, USA
E-mail: gomide@ufl.edu

BMC Proceedings 2011, 5(Suppl 7):O48

**Background:** An essential use of genomics is in the discovery of genes controlling complex, quantitative traits. In forestry, attempts to identify genes that regulate quantitative variation are still limited to a few Association Studies (AS) focused largely on candidate genes [1]. In most studies, few markers have been identified in association with quantitative traits.

Recent advances in DNA sequencing create the potential for high-throughput SNP genotyping at low cost, by re-sequencing genomes of interest [2]. For the particular case of conifers, two obstacles remain: (i) the lack of a reference genome to align the DNA sequences and identify SNPs, and (ii) the size and complexity of the genome that hinders the de novo assembly of reads. Whereas whole-genome sequencing of a large number of conifer genotypes is still unfeasible, concentrating the sequencing on gene-rich regions is an alternative to generate markers that are more likely to capture variation associated to complex traits. Here we report our approaches to develop methods of genotyping based on whole-exome capture using in-solution target enrichment (Agilent's SureSelect) followed by high-throughput sequencing (Illumina's GAllx).

**Methods: Probes for target enrichment:** To capture a defined genic portion of the *Pinus taeda* genome we designed 54,773, unique, 120-mer RNA probes that tilled across a collection of 14,729 EST assemblies (unigenes). For 3,615 genes we could predict gene models and avoid probes on known exon-intron junctions.

**Genetic material, library preparation and sequencing:** A total of 72 seeds collected from a female tree (10-5 genitor) were used for library preparation. Haploid megagametophyte tissue was dissected and DNA was extracted using standard protocols. Libraries were prepared as recommended by Illumina, with some custom modification of concentration, adapter sequence and fragment size selection. Custom barcoded adapters were used to pool up to eight genotypes in a single hybridization, which followed Agilent's SureSelect protocol. Captured-libraries were sequenced using GAllx with 115nt single-end run.

**Results and discussion: Preliminary validation:** To test the potential of target enrichment in pine we prepared sequencing libraries from three different genetic complexities from single genotypes/haplotypes: haploid cDNA from megagametophyte (n=12), haploid DNA from megagametophyte (n=12) and diploid DNA extracted from needles (2n=24). After hybridization of these libraries to SureSelect probes, we cloned each captured library into vectors and sequenced 96 fragments using Sanger sequencing. After reads were aligned to the reference sequences used for probe design (BLAST e-value=1x10⁻¹⁰), 70, 84 and 80% of the quality-filtered reads from haploid cDNA, haploid DNA and diploid DNA aligned uniquely to a unigene, respectively. Therefore, sequence capture is highly specific, even when the complexity of the material increases.

**Pipeline development for mapping population:** We modified the sequence capture protocol to increase throughput by constructing libraries from haploid DNA of 72 megagametophytes in 96-well plates. An average of 25.9 million single-end reads were obtained from flow-cell channel containing 8 barcoded megagametophytes, for a total of 56 megagametophytes sampled across 7 channels lanes. The remaining two pools (16 individuals) have not been sequenced. Despite multiplexing a large number of megagametophytes, we detected low variation in read numbers between haplotypes within a multiplex reaction (Figure 1). Due to the lack of a reference genome, bioinformatics experiments were designed to test whether the best approach to identify polymorphisms would result from aligning the sequences directly to the unigene sequence using Mosaik (http://bioinformatics.bc.edu/marthlab/Mosaik), or by alignment of reads to a new reference sequence defined by de novo assembly of the captured reads with the ABySS [3] short read sequence assembler.

The analysis using Mosaik compared different levels of polymorphism between reads and the unigene sequences resulting from alignments over a range of mismatch tolerances (from 1% to 50%). Figure 2A shows that the number of reads that aligned to a single region of the reference (i.e. uniquely aligned) increases linearly until 10% of mismatches in the alignment are accepted, and stabilizes after that. Interestingly, the