percentage of reads non-uniquely aligned remains lower than 2%, regardless of the mismatch rate. Next we tested if accepting higher mismatch rates adds more reads to the same genes, or if genes previously not captured are now represented. Again, at a 10% mismatch rate (Figure 2B) the total number of genes with at least one read aligned reaches its maximum, with 22% of the reads aligning to more than 10,000 genes.

Analyzing the data using a *de novo* assembly approach does not appear to be more suitable. ABYSS assemblies were performed utilizing a range of k-mer values (50 to 95, increments=5) and the contigs formed were analyzed. As expected, the number of contigs drops considerably as a function of the k-mer size (Figure 2C). To evaluate the quality of contigs generated at each k-mer, we compared them to the unigene sequences (BLAST identity≥85%) and counted the number of genes represented by at least one contig. This number decreased as a function of the k-mer size (Figure 2D); and, within the same genotype the sequence assemblies generated using a high k-mer were already presented in the assembly using a lower k-mer. However, when comparing two genotypes assembled with the same k-mer value about 30% of the genes are unique to each genotype. This suggests that additional sequencing is necessary for an adequate analysis of the efficiency of sequence capture. We are currently sequencing the same libraries from both ends (i.e. paired-end sequencing) to increase coverage and depth. This sequencing data is expect to identify additional SNP markers for segregation analysis, and to help define the sequencing requirements to confidentially perform target-enrichment resequencing and genotyping in complex genomes.

### References

1. Grattapaglia D, Plomion C, Kirst M, Sederoff RR: *Genomics of growth traits in forest trees.* *Curr Opin Plant Biol* 2009, 12(2):148-156.

2. Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Lewis ZA, Selker EU, Cresko WA, Johnson EA: *Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers.* *PLoS ONE* 2008, 3(10):e3376.

3. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I: ABYSS: a parallel assembler for short read sequence data. *Genome Res* 2009, 19(6):1117-1123.

## O49

**The *Eucalyptus* genome integrative explorer (EucGenIE): a resource for *Eucalyptus* genomics and transcriptomics**

Charles Hefer¹*, Eshchar Mizrachi², Fourie Joubert¹, Alexander Myburg²

¹Bioinformatics and Computational Biology Unit, Department of Biochemistry, University of Pretoria, Pretoria, South Africa; ²Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa

E-mail: charles.hefer@up.ac.za

BMC Proceedings 2011, 5(Suppl 7):O49

**Background:** The fast growth and good wood properties of *Eucalyptus* tree species and hybrids make them excellent renewable sources of fiber for pulp and paper production, and woody biomass for bioenergy production. Our research is aimed at understanding the genetic regulation of wood formation in eucalypts, with a focus on transcriptomes, regulatory sequences and gene families involved in secondary cell wall biosynthesis.

**Methods:** We have performed deep mRNA sequencing (using Illumina RNA-Seq technology) of several primary and secondary tissues of three *Eucalyptus grandis* trees with the aim to investigate the transcriptional control of cellulose biosynthesis and wood formation. The transcriptome datasets range from nearly mature xylogenic tissues to immature shoot tips, and consists on average 35 million paired-end 80bp short reads. The Illumina short reads were mapped to the latest *Eucalyptus grandis* genome sequence (DOE-JGI v. 1.0, http://www.phytozome.net), and the set of predicted gene models provided by the Joint Genome Institute (JGI). We calculated tissue specific gene expression (FPKM) profiles for each of the ~44 000 predicted genes across the sequenced transcriptome datasets. The results were stored in a relational database for further analysis and analysis of co-expression patterns in the expression profiles. We ultimately aim to identify genes that are differentially expressed and co-regulated during different stages of wood formation.

**Results:** We describe the initial development of an Integrative *Eucalyptus* Genome Explorer (EucGenIE), modeled after the poplar resource, PopGenIE (http://www.popgenie.org, Sjödin *et al*, 2009). EucGenIE relies on a

relational database system that allows for the efficient storage and retrieval of gene models and expression values from the database, which is then presented to the user in novel and intuitive ways. The web-based front-end makes use of tools available in the Generic Model Organism Database (GMOD, http://gmod.org) toolkit to enrich the query interface as well as result visualization. Links to the Phytozome GBrowse instance provides genomic context to the expressed gene sets. Custom queries allows the user to find genes with similar gene expression profiles across the various datasets, as well as perform bulk searches on sequence features annotated on the gene set. EucGenIE also provides access to common analyses tools, such as homology searching and online clustering tools. The first version of the EucGenIE database and online portal is available at http://eucgenie.bi.up.ac.za with restricted access.

### Reference

1. Sjödin A, Street NR, Sandberg G, Gustafsson P, Jansson S: *The Populus Genome Integrative Explorer (PopGenIE): a new resource for exploring the Populus genome.* *New Phytologist* 2009, 182:1013-1025.

## O50

**Investigation of genetic variation in *Jatropha curcas* by Ecotilling and ISSR**

Fatemeh Maghuly¹*, Joanna Jankowicz-Cieslak², Alberto Calari¹, Rose Ramkat¹, Bradley Till², Margit Laimer¹

¹Plant Biotechnology Unit (PBU), Dept. Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria; ²Plant Breeding Unit, FAO/IAEA Agricultural and Biotechnology Laboratory, International Atomic Energy Agency, P.O. Box 100, Vienna International Centre, 1400 Vienna, Austria

E-mail: fatemeh.maghuly@boku.ac.at

BMC Proceedings 2011, 5(Suppl 7):O50

**Background:** The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and population histories that have played a role in the species' evolution. A number of different techniques for identifying SNPs have been developed, all having their limitations.

Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variations in natural or mutant populations. Ecotilling, a variant of TILLING (Targeting Induced Local Lesions IN Genomes) technique, allows high-throughput analyses of natural genetic diversity in plants [1], particularly in species with limited genetic diversity.

*Jatropha curcas* L. is a perennial, monoecious shrub of the *Euphorbiaceae* family, native to America but distributed widely in the tropical and subtropical areas [2]. Wild or semi-cultivated types of *J. curcas* can grow well under unfavourable climatic and soil conditions [3]. *J. curcas* has attracted a great deal of attention worldwide, regarding its potential as a new energy plant. The seeds of *J. curcas* contain 30-45% oil [4] with a high percentage of monounsaturated oleic and polyunsaturated linoleic acid [5]. For genomic analyses, *J. curcas* is an interesting model species, since it has a relatively small genome (2C DNA content of 0.850 ± 0.006 pg or C DNA content of 0.416 × 109 bp) [6].

However, to achieve specific breeding goals in *Jatropha* for wider ecological adaptation, disease resistance and novel seed quality, the use of germplasm from different group and regions is necessary. Understanding the population structure of the alternative bioenergy plant *Jatropha curcas* is challenging due to limited genetic variability and information on phylogenetic relationships between accessions and related species. The development of cultivars of *Jatropha curcas* by conventional breeding will profit largely from biotechnological support (pathogen-free accessions with specific traits, non-toxic, high yielding varieties).

The knowledge about *J. curcas* remains limited and little genomic research has been done so far [7]. In fact, the genetic map of *J. curcas* is not well-developed and only few molecular markers exist that could be used to clearly distinguish world wide accessions. Therefore, a resource database of SNPs in *J. curcas* would provide researchers with a tool for answering questions concerning population structure or adaptation and allow comparison of this species with related species.

**Methods:** The identification of novel SNPs that account for natural variation was used to study genetic diversity and the relationships between and within *Jatropha* species. ISSRs (Inter Simple Sequence
Repeats) also were considered as a tool in selecting germplasm for
breeding purposes.

An *in vitro* germplasm collection of 1300 accessions from 12 countries was established. This collection will serve different purposes: a) conserve valuable genetic resources, b) survey genetic variation, and c) serve as starting material for genetic improvement with different breeding goals. Ecotilling was applied to 12 different genes of interest related to stress tolerance, toxin and oil metabolism. 50 ISSR primers were used to assess the genetic diversity of *Jatropha curcas* and related species. Four different pooling strategies were used to identify homozygous and heterozygous SNP variations. In fact, variation was analyzed both within a single tree (heterozygous) as well as between individual trees and a reference samples. Due to the reported low variations between *Jatropha* accessions [8,9] and large size of our collection, the 8 x 8 pooling strategy was chosen to estimate the level of variations among 12 selected genes.

**Results and conclusions:** To elucidate genetic relationship among *Jatropha* accessions from different regions and related species, a dendrogram was produced using NJ analysis of Nei's genetic distance for 5 ISSR markers. The dendrogram is divided into two groups, one containing all *Jatropha* accessions and the other containing the related species. The main *Jatropha curcas* cluster is divided into two subclusters, one containing samples from Kenya and the other containing the remaining *Jatropha* accessions. The data showed clear variations not only among individuals but also between different regions.

Ecotilling was found to be more efficient for large-scale studies of genetic variation in *Jatropha*, compared to RAPD, SSR and AFLP. Ecotilling is a low cost, high-throughput reverse genetic method for haplotyping and SNPs discovery. The level of differentiation observed was based on the geographic distribution pattern, i.e. it was higher in the centre of origin. ISSR analysis yielded highly reproducible patterns with 5/50 primers.

**Acknowledgements:** Financial support of FFG project 820132 and Biplant R & D is acknowledged.

**References**

1. Comai L, Young K, Till B, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S: Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. *Plant J* 2004, 37:778-786.

2. Cano-Asseleih L M, Plumbly RA, Hylands PJ: Purification and partial characterization of the hemagglutination from seeds of *Jatropha curcas* L. *Jour Food Biochem* 1989, 13:1-20.

3. Katwal RPS, Soni PL: Biofuels: an opportunity for socioeconomic development and cleaner environment. *Indian Forester* 2003, 129:939-949.

4. Openshaw K: A review of *Jatropha curcas*: an oil plant of unfulfilled promise. *Biomass Bioenerg* 2000, 19:1-15.

5. Akintayo ET: Characteristics and composition of Parkia biglobossa and *Jatropha curcas* L. oils and cakes. *Bioresour Technol* 2004, 92:307-310.

6. Carvalho CR, Clarindo WR, Praca MM, Arauja FS, Carels N: Genome size, base composition and karyotype of *Jatropha curcas* L., an important biofuel plant. *Plant Science* 2008, 174:613-617.

7. Wen M, Wang H, Xia, Zou M, Lu C, Wang W: Development of EST-SSR and genomic-SSR markers to assess genetic diversity in *Jatropha curcas* L. *BMC. Res Notes* 2010, 3:42.

8. Achten MJW, Nielsen LR, Aerts R, Lengkeek AG, Kjaer ED, Trabucco A, Hansen JK, Maes WH, Graudal L, Akinnifesi FK, Muys B: Towards domestication of *Jatropha curcas*: a review. *Biofuels* 2010, 1:91-107.

9. Vollmann J, Laimer M: Novel and traditional oil crops and their biorefinery potential. *Bioprocessing Technologies in Integrated Biorefinery for Production of Biofuels, Biochemicals, and Biopolymers from Biomass* John Wiley & Sons, Inc. (USA): Yang S-T, El Enshasy HA, Thongchul N, Martin Lo Y 2010.

information on putative genes, however, often without any idea on their functions. To understand how a cell works one needs to know the function of almost every gene in its genome. Once the whole-genome information is available for an organism, the challenge turns from identifying the parts to understanding their function as well as to improving genome structure. In the short term, the first goal is to assign some element of function to each of the genes in an organism also referred to as 'functional genomics', and to do this with high-throughput, systematic approaches.

**Material and methods:** Gene technology is a very powerful tool in forest tree breeding programs as well as for functional genomics in order to unraveling gene functions. Various reverse and forward genetics strategies are ongoing to determine the functions of genes and regulatory sequences. A significant progress has been made by using T-DNA as a vehicle to induce either "Knock-out" or "Knock-in" ("gain-of-function" or Activation tagging") in different plant species including poplar [1].

The application of a transposon-based activation tagging system for poplar has been proposed early by [2]. It could be shown that the maize transposable element Ac is functional in the *Populus* genome [3], and re-integrations occur in high frequencies in or near coding regions [4]. Further, the majority of the re-integrations were found scattered over many unlinked sites on other scaffolds than the one carrying the original integration locus, confirming that Ac does in fact cross chromosome boundaries in poplar [5].

**Results:** In this paper, we describe for the first time the development of an efficient activation tagging system for poplar based on a non-autonomous "Activation Tagging Ds" (ATDs) system [6] in combination with a heat-inducible Ac-transposase. First, seven independent transgenic lines were obtained transformed with HSP:transposase construct. From these, two transgenic lines were selected for super-transformation with the ATDs-rolC construct based on the construct by [6]. The ATDs original integration could be determined from 18 double transgenic lines. Induction of the transposase gene and mobility of the ATDs element could be confirmed. Four activation-tagged populations comprising in total 12,083 individuals from 22 different ATDs transgenic lines have been produced and phenotyped. So far, from 18 different putatively tagged variants the new ATDs genomic position was successfully determined. Sequences obtained were blasted against the publicly available genome sequence of *P. trichocarpa* v2.0 (Phytozome v5.0; http://www.phytozome.net/poplar). E-values of hits ranged from -35 down to zero. Annotation of the sequences obtained against *P. trichocarpa* revealed for 13 variants possible transcripts. For five putative proteins either no functional annotation or unknown function was found.

In a second approach, 300 randomly selected individuals revealing no obvious phenotype from the forth activation-tagged population were screened for ATDs excision. In approximately one third of the investigated individuals transposition of ATDs was confirmed, and analyses of the new genomic positions of ATDs reveal a very high percentage of tagged genes.

**Conclusions:** Tagging approaches based on T-DNA insertion are effective only for plant species (like Arabidopsis and poplar), whose are easily transformable in combination with high frequencies of tagged lines obtained. An advantage of T-DNA based activation tagging could be that even T-DNA insertion sites are not randomly distributed in the genome but do show some insertion site preferences to the 5'UTR of a gene coding region. For transposable elements, however, new insertion sites were found scattered throughout the genome at many unlinked sites. But similar to [5] results for poplar, also other reports describe preferential transposon insertion around transposon donor sites [7].

The fact that a transposon is able to jump to other chromosomes, thus passing chromosomal boundaries, leads to the convenient situation that only a few primary transposon transgenic lines are sufficient for the establishment of large transposon tagging populations in order to tag at least theoretically every gene in a tree genome. This is obviously difficult for T-DNA tagging as plant transformation is time consuming and, therefore, the genome can't easily be saturated with T-DNA tags.

Taken together, so far the strategy for both T-DNA and transposon activation tagging is first to phenotype an existing tagging population and then to determine the new genomic insertion locus of the tag. A novel strategy of activation tagging can be suggested based on the described power of the ATDs transposon approach and the simplicity to induce ATDs transposition *in vitro*. The ATDs-based strategy allows first the production of a very high number of independent ATDs-transposed plants whose can be screened for new ATDs flanking genomic loci.

O51

Activation tagging in poplar by using an inducible Ac/Ds transposon system

Matthias Fladung*, Olaf Polak
Johann Heinrich von Thünen Institute (vTI), Institute for Forest Genetics,
Sieker Landstr. 2, D-22927 Grosshansdorf, Germany
E-mail: matthias.fladung@vti.bund.de
BMC Proceedings 2011, 5(Suppl 7):O51

**Background:** The sequence of the whole genome of black cottonwood (*Populus trichocarpa*) was made available in public domain (http://www.phytozome.net/poplar) few years ago. Sequencing and annotation of the genome of an organism yields a tremendous amount of data and
## References

1. Busov V, Brunner A, Meilan R, Filichkin S, Ganio L, Gandhi S, Strauss SH: Genetic transformation: A powerful tool for dissection of adaptive traits in trees. *New Phytol* 2005, 167:9-18.

2. Fladung M, Deutsch F, Hönicka H, Kumar S: DNA and transposon tagging in aspen. *Plant Biol* 2004, 6:5-11.

3. Fladung M, Ahuja MR: Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus. *Plant Mol Biol* 1997, 33:1097-1103.

4. Kumar S, Fladung M: Somatic mobility of the maize element Ac and its usability for gene tagging in aspen. *Plant Mol Biol* 2003, 51:643-650.

5. Fladung M: Analysis of re-integrated Ac element positions in the genome of Populus provides a basis for Ac/Ds transposon activation tagging in trees. *Trees* 2011, 25:551-557.

6. Suzuki Y, Uemura S, Saito Y, Murofushi N, Schmitz G, Theres K, Yamaguchi I: A novel transposon tagging element for obtaining gain-of-function mutants based on a self-stabilizing Ac derivative. *Plant Mol Biol* 2001, 45:123-131.

7. Raina S, Mahalingam R, Chen FQ, Fedoroff N: A collection of sequenced and mapped Ds transposon insertion sites in *Arabidopsis thaliana*. *Plant Mol Biol* 2002, 50:93-110.

## O52

### Building up resources and knowledge to unravel transcriptomics dynamics underlying *Eucalyptus globulus* xylogenesis

Jorge AP Paiva¹*, José Carlos Rodrigues², Pedro Fevereiro³, Lucinda Neves⁴, Clara Araújo⁴, Cristina Marques⁵, Ana Teresa Freitas⁶, Hélène Bergès⁷, Jacqueline Grima-Pettenati⁸

¹Instituto de Investigação Científica e Tropical (IICT/MCTES) Palácio Burnay - Rua da Junqueira, 30, 1349-007 Lisboa, Portugal Instituto de Biologia Experimental e Tecnológica (IBET) Av. da República, Quinta do Marquês, 2781-901 Oeiras, Portugal; ²Instituto de Investigação Científica e Tropical (IICT/MCTES) Palácio Burnay - Rua da Junqueira, 30, 1349-007 Lisboa, Portugal; ³Instituto de Biologia Experimental e Tecnológica (IBET) Av. da República, Quinta do Marquês, 2781-901 Oeiras, Portugal; ⁴ALTRI FLORESTAL SA, Quinta do Furadouro 2250-582 Olho Marinho, Portugal; ⁵RAIZ, Herdade de Espíra, 2985-270 Pegões, Portugal; ⁶INEC-ID - Instituto de Engenharia de Sistemas e Computadores, R. Alves Redol 9, 1000 Lisbon, Portugal; ⁷INRA-CNRGV - chemin de Borde Rouge 31326 Castanet-Tolosan, France; ⁸UMR CNRS/Université Toulouse III 5546, Pôle de Biotechnologies Végétales, 24 chemin de Borde Rouge, BP42617 Auzeville, 31326 Castanet, Tolosan, France E-mail: jorge@itqun.pt

BMC Proceedings 2011, 5(Suppl 7):O52

The economic importance of some *Eucalyptus* species, including interspecific hybrids, has been extended from the traditional interest of pulp and paper production to the emergent areas of bio-fuels and bio-materials. New genomic resources and high throughput technologies have provided the *Eucalyptus* research international community with the opportunities to identify genomic regions of interest in order to comprehensively dissect, catalogue and characterize genes involved in the determination of wood formation and quality. Similar strategies can be now applied to identify key regulator genes and better understand the cellular mechanisms by which they modulate the complex molecular events occurring in xylogenesis.

The *Geneglob*<sup>wq</sup> project (2006-2010) produced and used a set of genomic tools which, associated with Next Generation Sequencing (NGS) technologies, allowed us to expand the knowledge of the *Eucalyptus* genome by focusing on regions potentially involved in the determination of wood properties, namely pulp yield and lignin content. We first start to characterize two *E. grandis* BAC libraries [1] constructed by the Arizona Genome Institute (with DNA from the clone Brasuz S1 whose genome was sequenced recently by the DOE [http://www.jgi.doe.gov/sequencing/why/99176.html], and two *E. globulus* BAC libraries made available by RAIZ [http://www.raiz-ifp.pt/]. We than used 3D-pools of BAC libraries and BAC macroarrays to characterize genomic environment of several lignin and lignin-regulator genes (e.g. *EguCCR*, *EguCAD2* and *EguRAC1*) both in *E. grandis* and *E. globulus*. The shotgun sequencing of selected BAC clones containing those genes generated a high amount of sequencing data that made it possible to map the *E. globulus* BAC sequenced clones against the *E. grandis* genome (8X coverage). These comparative analyses showed extended microlinearity between both genomes, at least in the studied regions. Additionally, we have sequenced and annotated the chloroplast genome of *E. grandis* (GeneBank Accession NC_014570) [1].

A global approach to unravel *E. globulus* transcriptome dynamics has also been included and structured in the *Geneglob*<sup>wq</sup> project, aiming at the identification of genomic hotspots of transcription activity. Various *E. globulus* xylogenesis "models" have been considered comprising several paired, contrasting wood forming tissues (provided by RAIZ): i) xylem samples collected along the year (season variation); ii) juvenile and adult individuals of a single genotype; iii) contrasting genotypes for pulp yield. Samples from these tissues were used for transcriptome sequencing using *IlluminaHi-Seq* technology (*mRNA-SEQ*). The same *E. globulus* genotype used for both *E. globulus* BAC libraries (a parent tree used in controlled crosses by RAIZ) has been re-sequenced (pair-end 100bp), and provided the first draft of an *E. globulus* genome. This resequencing data was mapped against the *E. grandis* Brasuz S1 reference genome. Transcriptomic data were also blatted against the gene models annotated in *E. grandis* genome, to evaluate *in silico* the expression of each gene.

More recently, the *microEGo* project (2010-2012) started the identification and characterization of *Eucalyptus globulus* microRNAs and their target genes, involved in the regulation of wood formation. The *E. globulus* season variation xylogenesis "model" was used considered for this project as well as an *E. globulus* reaction wood "model". The latter comprises reaction wood tissues (tension / opposite wood) formed in bent trees at different kinetic times of gravitropic stimulation and control wood (non-bent trees). Small RNA libraries have been generated from those tissues and sequenced using *IlluminaHi-Seq* technology (*Small RNAs-SEQ*). The sequencing data from both *microEGo* and *Geneglob*<sup>wq</sup> projects together with a genome wide bioinformatics analysis of *E. grandis* reference genome and *E. globulus* genome are being used for identification of miRNA gene and putative their putative target-genes.

These projects will hopefully constitute an important piece in the assemblage of whole new categories of knowledge and genomic resources for the *Eucalyptus* community, providing insights into the nature of the molecular machinery involved in wood formation and most importantly in the identification of key players determining the variability of wood characteristics and its end-uses.

**Acknowledgments:** This work has been partially supported by Fundação para a Ciência e Tecnologia (Portugal): http://PTDC/AGR-GPL/098179/2008, "microEGO - Did you ask for something small? The microRNAs power in a *Eucalyptus* tension world! (https://sites.google.com/site/microeogovernment/); PTDC/AGR-GPL/66564/2006, "GenEglob<sup>wq</sup> - Scanning for candidate genes underlying a pulp yield QTL in *Eucalyptus globulus*" (http://geneglob.inesc-id.pt/). We would like to acknowledge ALTRI FLORESTAL SA (Portugal) and RAIZ (Portugal) for providing the plant material used in these studies.

The authors would like to thank the valuable contribution of actual and former members of the involved groups and institutions: Susana Araújo<sup>1,2</sup>, Teresa Quilho<sup>1</sup>, Nuno Borralho<sup>1</sup>, Ricardo Barrela<sup>1</sup>, Douglas Freitas<sup>1</sup>, Victor Carocha<sup>1,2</sup>, Clara Graça<sup>1</sup>, Joana Amado<sup>1</sup>, Filipa Cardeano<sup>1</sup>, Paulo Fonseca<sup>5</sup>, Nuno Mendo<sup>5</sup>, Elisa Prat<sup>6</sup>, Sonia Vautrin<sup>6</sup>, Joelle Fourment<sup>6</sup>, Nathalie Ladouce<sup>7</sup>, Hélène San-Clemente<sup>7</sup>, Hua Wang<sup>7</sup> and Andreia Fonseca (IMM-UL, Lisboa).

## Reference

1. Paiva JAP, Prat E, Vautrin S, Santos MD, San-Clemente H, Brommonschenkel S, Fonseca PG, Grattapaglia D, Song X, Ammiraju JSS, Kudrna D, Wing RA, Freitas AT, Berges H, Grima-Pettenati J: Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries. BMC Genomics 2011, 12:137, doi:10.1186/1471-2164-12-137.

## S9. BIOSAFETY, CERTIFICATION AND ECONOMICS OF TREE BIOTECHNOLOGY

### O53

#### Highly regulated but can't be certified as sustainable. Responsible use principles are bridging the gap for biotech trees

Adam Costanza
Institute of Forest Biotechnology
E-mail: adam costanza@forestbiotech.org
BMC Proceedings 2011, 5(Suppl 7):O53

Biotech trees are some of the most heavily regulated plants in the world, yet no forest management system certifies the use of them as sustainable. The Responsible Use: Biotech Tree Principles are the first, and
only, set of practices designed specifically for the long-term stewardship of biotech trees. These Principles may serve as a framework that certification systems can use when considering whether biotech trees meet their certification standards.

Responsible use principles were developed to guide the long-term stewardship of biotech trees. As forest biotechnology has taken root and genetically modified trees are being field-tested for biofuels, species restoration, and disease resistance, the need for a set of global guiding principles for responsible use of these trees became apparent. Since this need was not being met by the international forestry certification schemes, the Institute of Forest Biotechnology (IFB) developed the Responsible Use: Biotech Tree Principles in a transparent, stakeholder driven process over nearly three years.

A broad set of stakeholders have set aside the issues of whether biotech trees should be used and created these stewardship principles. The Principles are in recognition that responsibly used biotech trees have the potential to benefit society, economies, and the environment in ways that other trees cannot. Central to these Principles are core beliefs that:

* Biotech trees should benefit people, the environment, or both

* Risks and benefits of biotech trees must be assessed

* Transparency is vital and stakeholders must be engaged

* Social equity and indigenous rights are important and must be respected

* Biotech tree use must follow regulations of the appropriate country Regulations for biotech trees are stringent in most parts of the world. From product inception in the lab, to seedlings grown by tree farmers, it can take years and cost millions of dollars – and the biotech tree may never get deregulated. Long growth cycles make gathering scientific information necessary for deregulation difficult in many countries. However, regulations vary widely in practice from one country to another. We will discuss the regulatory frameworks of biotech trees in Brazil, Chile, U.S., Canada, South Africa, China, and New Zealand.

Certification systems have yet to address biotech tree use in a holistic, scientific manner. We will discuss how the top certification systems in Brazil, Chile, U.S., Canada, South Africa, China, and New Zealand address biotech trees. Along with the regulatory review of these countries, a framework will be developed that shows where regulations and certification systems overlap, diverge, or have significant gaps. This framework will be a tool to begin a dialogue with certification systems on how to best bridge these gaps using the Responsible Use: Biotech Tree Principles.

People have highly disparate opinions about using biotech trees. The reality is that biotech trees are already in use today, and they will be put to use even more in the future. It is important that we engage society, assess environmental risks and benefits, and follow best practices for the stewardship of biotech trees and their products. The IFB, through the Responsible Use Initiative, will work with relevant forest certification systems to provide science-based, stakeholder driven assessments that will help meet the requirements of their own sustainable forestry mechanisms. The IFB is the only organization to address the sustainability of forest biotechnology on a global scale. The IFB has been in operation for 10 years as a non-profit organization. In December 2010 the IFB published the Responsible Use: Biotech Tree Principles. The Principles along with a comprehensive Biotech Tree Primer are available at http://www.responsibleuse.org. Learn more at IFB's main website, http://www.forestbiotech.org.

### O54
#### Assessing ectomycorrhizal associations and transgene expression in transgenic *Castanea dentata*

Katherine D'Amico*, Thomas Horton, Charles Maynard, William Powell
State University of New York College of Environmental Science and Forestry,
USA

E-mail: kmdamico@syr.edu

BMC Proceedings 2011, 5(Suppl 7):O54

**Background:** American chestnut (*Castanea dentata*) once dominated the forests of the eastern United States until the introduction of a Chinese fungal pathogen *Cryphonectria parasitica* (causal agent of the chestnut blight) decimated the species. This tree was important not only ecologically, but also played a significant role economically. Restoration programs are implementing a variety of techniques, including genetic

transformation, to develop a blight resistant tree that can be reintroduced into the native chestnut range (S.A. Merkle, 2007, *Tree Genetics and Genomics*, 3, 111-18).

Specific genes involved in plant defense responses are being introduced into American chestnut via *Agrobacterium*-mediated transformation, with the hope that one gene or a combination of genes will aid in chestnut defense against blight (L.D. Polin, 2006, *Plant Cell, Tissue, and Organ Culture*, 84, 69-79). One gene of particular interest to the transgenic project is oxalate oxidase, a defense related gene from wheat that has also been shown to enhance the defense response of other plants when introduced through genetic transformation (H. Liang, 2001, *Plant Molecular Biology*, 45, 619-29). American chestnut transformation with a binary vector containing oxalate oxidase has produced a number of transgenic events, many of which have already been regenerated plants (unpublished data).

Before releasing any transgenic plant it is necessary to assess any non-target impacts the introduced genes may have on associated microbial communities. *C. parasiticais* a fungal pathogen, so we are particularly interested in studying the effects of any transgene on the symbiotic relationship between the host plant and mycorrhizae-forming fungi. Prior studies have examined the potential impact of transgenic plants on ectomycorrhizal fungi (K. L. Oliver, 2008, *Applied and Environmental Microbiology*, 74, 5340-48).

**Objectives:** 1. Analyze and compare transgene expression in root, stem, and leaf tissue in American chestnut trees transformed with a gene for oxalate oxidase.

2. Assess potential non-target impacts on mycorrhizal fungi by comparing ectomycorrhizal associations with wildtype American chestnut and a transgenic American chestnut expressing a gene for oxalate oxidase.

**Methods:** Wildtype American chestnut seeds and transgenic American chestnut ('Darling4') trees were grown in a potting mix containing field soil in a soil bioassay to bait for mycorrhizal fungi. All trees were grown in a greenhouse for at least one year before mycorrhizal root tips were harvested. The root system of each tree was rinsed over a fine sieve, and segments of root were collected. Mycorrhizal root tips were quantified and grouped based on morphotype. Fungi were identified using RFLP and sequence analysis of the fungal ITS region.

RNA was extracted for gene expression studies from all tissues using a CTAB extraction method. RNA samples were then DNase treated and used to synthesize cDNA, which was used as a template in all subsequent quantitative RT-PCR experiments. qRT-PCR studies were conducted to determine the relative level of transgene expression in leaf, stem, and root tissue in 'Darling4' trees.

**Results:** Gene expression study results based on qRT-PCR indicate that the oxalate oxidase gene is expressed in root tissue of 'Darling4' American chestnut and not in the wildtype control (Fig. 1). Transgene expression results for both stem and leaf tissue are comparable to that found in root tissues.

Based on mycorrhizal tip quantification for transgenic and wildtype American chestnut, it appears that there is not a significant difference in total percent fungal colonization between these tree types (Fig. 2).

**Discussion and conclusions:** The results of the gene expression portion of this study show oxalate oxidase is expressed in the root tissue of 'Darling4' transgenic American chestnut trees. As expected, there is no oxalate oxidase expression in root tissue of wildtype American chestnut trees. Oxalate oxidase expression in root tissue has the potential to influence associated microbial communities, specifically mycorrhizal fungi that are in direct contact with chestnut roots. It is therefore necessary to assess mycorrhizal associations in transgenic chestnut to determine if there are any non-target impacts as a result of this expression. Mycorrhizal root tips were quantified for both the Darling 4 transgenic chestnut and the wildtype. The results of the total percent ectomycorrhizal fungal colonization portion of the study show that there is no significant difference in overall levels of mycorrhizal abundance between the two tree types. It can be inferred from this that American chestnut trees expressing oxalate oxidase are able to form mycorrhizal associations in a similar manner to the wildtype.

As with any other transgenic plant, 'Darling4' and other transgenic American chestnut trees are required to go through extensive deregulation procedures before they can be released back to the native chestnut range. The results from this study will be used in conjunction with other ecological impact studies to inform the deregulation process in
an effort to restore the American chestnut. This study will also contribute to a body of existing work examining the potential environmental impacts of transgenic forest trees that can inform future policy and procedures relating to the genetic modification of important species.

## O55
Mycorrhization of transgenic apple trees with increased resistance against fungal pathogens

Tina Schäfer¹, Henryk Flachowsky², Stephan König³, Stefan Hempel⁴,
Tesfaye Wubet³, Andreas Peil², Michael Kaldorf⁵, Magda-Viola Hanke²,
François Buscot¹

¹UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany & University Leipzig, Faculty of Biological Science, Pharmacy and Psychology, Institute of Biology I, Department of Terrestrial Ecology, Leipzig, Germany; ²Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Horticultural and Fruit Crops, Dresden, Germany; ³UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany; ⁴Freie Universität Berlin, Institute of Biology, Dahlem Center of Plant Sciences, Department of Plant Ecology, Berlin, Germany; ⁵Gesellschaft für Umwelt- und Innenraumanalytik mbH, Mönchengladbach, Germany & University Leipzig, Faculty of Biological Science, Pharmacy and Psychology, Institute of Biology I, Department of Terrestrial Ecology, Leipzig, Germany

E-mail: tina.schaefer@ufz.de

BMC Proceedings 2011, 5(Suppl 7):O55

The project 'Mycorrhizal symbioses of transgenic apple trees with increased resistance against fungal pathogens' (Subproject of the framework 'Biosafety of transgenic woody plants', supported by the German Federal Ministry for Education and Research) investigates whether the transformation of apple trees with fungal chitinase encoding genes - besides increasing resistance against fungal pathogens - also interferes with mycorrhizal fungi. Pathogenic fungi such as the ascomycete species *Venturia inaequalis*, the pathogenic agent of apple scab, cause severe damage in apple cultivation. In commercial apple production, pesticides are employed frequently and in high quantities. Alternative methods such as the use of mycorporasitic *Trichoderma* fungi to control fungal plant pathogens have been explored [1]. Genetic engineering of apple cultivars with *Trichoderma* chitinases has lead to an increase of the trees' resistance against apple scab [2]. However, like many fruit tree species, apple trees form symbiotic associations with arbuscular mycorrhizal fungi (AMF) at their roots. AMF receive fixed carbon compounds, while the plants benefit from the symbiosis by enhanced uptake of nutrients and water, promoted growth, and increased resistance against biotic and abiotic stress [3]. Like in most higher fungi, the cell walls of AMF contain chitin as a major skeletal component [4]. Consequently, the symbiosis between apple and AMF may be negatively affected by the expression of *Trichoderma* chitinase genes within the plant. At the Julius Kühn-Institute in Dresden, the apple cultivar 'Pinova' was transformed by introducing the construct pBIN (Endo + Nag) into the transgenic lines. With the help of promoter CaMV 35S the transgenic lines constitutively express endochitinase gene *ech42* and exochitinase gene *nag70* from the biocontrol fungus *Trichoderma atroviride*. In order to study whether the constitutive expression of *ech42* and *nag70* has an effect on mycorrhization of plants with transgenic roots (local effect) and - as apple cultivars are propagated vegetatively by grafting- whether expression of *ech42* and *nag70* in a transgenic scion grafted on a non-transgenic rootstock has an effect on mycorrhization (systemic effect), a series of greenhouse experiments was conducted. In a first experiment, non-grafted clones of wildtype apple cultivar 'Pinova' and two transgenic lines were cultivated in artificial substrate and inoculated with two AMF species, *Glomus intraradices* and *G. mosseae*. After four months of cultivation, root colonization rates were significantly lower in the transgenic lines compared to the non-transformed cultivar 'Pinova' [5]. Samples were taken again after another four months of cultivation under enhanced inoculation pressure (increased inoculum mass, decreased nutrient availability in the substrate). The experimental approach was broadened by using soil from different field sites in order to supply the sample trees with complex soil matter, AMF adapted to the very soil conditions, and the appropriate soil bacteria which also play a role in the formation of mycorrhizal associations. Two sets of non-grafted sample trees (clones of apple cultivar 'Pinova' and two

transgenic lines) were cultivated in the greenhouse; one set in pots with soil from an intensively managed apple plantation, the other set in pots with soil from an extensively managed apple orchard. Root sampling was conducted three and six months after transferring the plants to soil. To assess the systemic effect (expression of *ech42* and *nag70* in a transgenic scion grafted on a non-transgenic rootstock) on mycorrhization, two further experiments were carried out. Grafted sample trees (clones of apple cultivar 'Pinova' and two transgenic lines, all grafted on commonly grown rootstock M9) were cultivated in the greenhouse in pots with artificial substrate and inoculum of *Glomus intraradices* and *G. mosseae*. Root samples were taken after four and eight months. A second set of grafted sample trees was cultivated in pots with soil from an intensively managed plantation in the 'semi-natural' environment of a gossamer cage. Root samples were taken after 12 and 24 months. For all experiments, root colonisation rates were determined microscopically. Molecular methods - PCR with taxa specific primer sets [6], cloning of PCR products, Sanger sequencing - were applied to study the impact of the transformation on the composition of the fungal communities associated with the roots of the sample trees cultivated in soil. Chitinase activity in leaves and roots of sample trees was determined using fluorescence spectrometry and substrates 4-methylumbelliferyl N-acetyl-β-D-glucosaminide for exochitinase and 4-methylumbelliferyl β-D-N,N',N''-triacetylchitotrioside for endochitinase activity. The results of this biosafety research concerned with potential adverse effects on beneficial soil fungi will be presented and discussed.

## References

1. Howell : Mechanisms employed by *Trichoderma* species in the biological control of plant diseases: The history and evolution of current concepts. *Plant Dis* 2003, 87:4-10.

2. Bolar, Norelli, Harman, Brown, Aldwinckle : Synergistic activity of endochitinase and exochitinase from *Trichoderma atroviride* (*T. harzianum*) against the pathogenic fungus (*Venturia inaequalis*) in transgenic apple plants. *Transgenic Res* 2001, 10:533-543.

3. Smith, Read : Arbuscular mycorrhizas. *Mycorrhizal Symbiosis* London: Academic Press: Smith and Read, 3 2008, 11-145.

4. Bonfante-Fasolo, Faccio, Perotto, Schubert : Correlation between chitin distribution and cell wall morphology in the mycorrhizal fungus *Glomus versiforme*. *Mycol. Res* 1990, 94:157-165.

5. Schäfer, Flachowsky, König, Kaldorf, Hanke, Buscot : First results on the effect of increased chitinase expression in transgenic apple trees on mycorrhization with *Glomus intraradices* and *G. mosseae*. *Acta Hort* 2009, 839:719-724.

6. Wubet, Weiss, Kottke, Teketay, Oberwinkler : Phylogenetic analysis of nuclear small subunit rDNA sequences suggests that the endangered African Pencil Cedar, *Juniperus procera*, is associated with distinct members of *Glomeraceae*. *Mycol. Res* 2006, 110:1059-1069.

## O56
Introduction of alkali-labile units into lignin in transgenic plants by genetic engineering

Yasuyuki Ishikawa¹, Yukiko Tsuji¹, Kanna Sato¹, Amiu Shino²,
Yoshihiro Katayama³, Jun Kikuchi², Hirofumi Hara⁴, Shoji Hishiyama⁵,
Eiji Masai⁶, Shinya Kajita¹²

¹Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan;

²Metabolomics Research Group, RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan; ³Department of Forest Science and Resource, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan; ⁴Department of Biomedical Engineering, Okayama University of Science, Okayama, Okayama 700-0005, Japan; ⁵Department of Biomass Chemistry, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan; ⁶Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan

E-mail: kajita@cc.tuat.ac.jp

BMC Proceedings 2011, 5(Suppl 7):O56

**Background:** Lignin is one of major components of plant secondary cell wall. In plant cell wall, it is synthesized via radical coupling of precursors such as *p*-coumaryl, coniferyl, and sinapyl alcohols. In early stage of the lignification, 8-O-4', 8-8' and 8-5' dimers are thought to be synthesized mainly from the precursors in the wall. A gram-negative bacterium,
<figure><img src="image_19.png" /><figcaption>**Figure 1 (abstract O56)** The reaction catalyzed by LigD. This bacterial enzyme catalyzes oxidation of benzyl position of 8-O-4' lignin dimers. Both of guaiacyl-guaiacyl and syringyl-syringyl dimers can be oxidized by the enzyme.</figcaption></figure>

*Shingobium* sp. strain SYK-6 (hereafter refer to as SYK-6) is able to catabolize a wide variety of phenolic compounds including the lignin precursors by its unique enzymatic system. One of catabolic enzymes, LigD, catalyzes oxidation at alpha (benzyl) position of 8-O-4' dimers and forms carbonyl group at the position (Figure 1). This oxidation is the first step of catabolic pathway of 8-O-4' dimers in SYK-6. When we express LigD polypeptide in the cell wall of transgenic plants, the oxidative dimers will be expected to be generated and then incorporated into lignin polymer. In some past studies, it has been shown that the presence of carbonyl groups at the alpha position of aryl propane units in lignin greatly speeds up the rate of cleavage of beta-aryl ether linkages during kraft pulping condition [1,2]. In order to contribute to efficient and sustainable production of kraft pulp and the other biomass-derived products such as bioethanol, we introduced the *ligD* gene into *Arabidopsis* and hybrid aspen and tried to generate transgenic plants whose lignin can be easy to remove from holocellulose fraction under alkaline conditions.

**Method:** Because of codon usage is significantly different between genes in plants and SYK-6, we chemically synthesized open reading frame (ORF) of the *ligD* gene for improving its expression in the transgenic plants. After addition of nucleotide sequence for apoplast-targeting signal peptide to the synthesized *ligD* ORF, it was introduced into *Arabidopsis thaliana*, tobacco BY-2 and hybrid aspen under the control of cauliflower mosaic virus 35S promoter. LigD expression in the transgenic plants was monitored by Western blot analysis and enzymatic activity with crude extract prepared from each transgenic line. Preliminary analysis of lignin structure by 2D-NMR and nitrobenzene oxidation was also performed.

**Results and discussion:** At first we confirmed expression of the *ligD* transgene in *Arabidopsis* by Western blot analysis with antiserum against LigD polypeptide. Positive expressions of the *ligD* were detected in some of the transgenic plants analyzed. Enzymatic activities of LigD in crude extracts prepared from both cytosolic and apoplastic fractions of the transgenic *Arabidopsis* plants were also detected, but it was relatively higher in the latter case. As expected, 2D NMR (¹H-¹³C HMQC) analysis suggests that the abundance of the alpha-keto (alpha carbonyl) structure in 8-O-4' units of lignin in the transgenic plants is relatively higher than that in the wild-type plant. Chemical compositions of lignin (syringyl/guaiacyl ratio) and neutral sugars could not be distinguishable between the transgenic and wild-type plants. Generation of transgenic hybrid aspen and its analysis are now in progress.

**Acknowledgements:** This research project was supported partly by the New Energy and Industrial Technology Development Organization (NEDO).

References

1. Gierer J, Ljunggren S, Ljungquist P, Norén I: Reactions of lignin during sulfate pulping.18. The significance of α-carbonyl groups for the cleavage of β-aryl ether structures. *Sven Papperstidn* 1980, 83:75-82.

2. Gierer J, Norén I: Oxidative pretreatment of pine wood to facilitate delignification during kraft pulping. *Holzforschung* 1982, 36:123-130.

O57

Environmental and health risk assessments of genetically modified
eucalypts in Brazil

Giancarlo Pasquali¹*, Leandro V Astarita², Eliane R Santarém³,
Rogério Margis⁴, Rafael Roesler⁵, Eduardo Cassel³, Aline M Lucas³,
Juliana Gerhardt³, Maria N M Lima⁶, Joseane B Carvalho⁷, Karen A Freitas³,
Rochele P Kirch⁸

¹UFRGS, Porto Alegre, RS, Brazil; ²PUCRS, Porto Alegre, RS, Brazil; ³PUCRS, Brazil; ⁴UFRGS & Vitatec, Porto Alegre, RS, Brazil; ⁵UFRGS & NeuroAssay, Porto Alegre, RS, Brazil; ⁶NeuroAssay, Brazil; ⁷Vitatec, Brazil; ⁸UFRGS, Brazil

E-mail: pasquali@cbiot.ufrgs.br

BMC Proceedings 2011, 5(Suppl 7):O57

Motivated by the Brazilian Ministry of Agriculture, Farming and Supply (MAPA), we created in 2009 the "Collaborating Center in Agriculture Defense Relative to the Biosafety of Genetically Modified Eucalypts" (Project "CDA Eucalyptus") in order to collect information and conduct research to assess the biosafety of GM eucalypts in the Brazilian context. The Normative Resolution Nr. 5 of the National Biosafety Technical Commission (CTNBio) is the official document presenting all information needed to propose the commercial release of GMOs in Brazil. Based on this document and along with the personnel of the Suzano Paper & Cellulose Co., we conducted a series of experiments with GM and non-GM eucalypts planted in a test field in the state of São Paulo to start collecting the necessary information. Two independent groups of transgenic plants, harboring two different transgene constructions along with non-GM control plants are being assayed. The genetic traits, the identity or names of the transgenes as well as the identity of each tree individual will not be revealed due to intellectual property request still pending. Each group of plants was represented by four independent events in triplicates (2 groups x 4 events x 3 clonal trees + 3 non-GM clonal trees), therefore totaling 27 individuals under analysis. Samples were identified by random numbers and all assays were conducted in a simple-blind or a double-blind fashion. Tests concluded until now included (i) the detection of transgene regulatory sequences in purified DNA samples by conventional PCR and RT-qPCR, confirming the expected sampling conducted; (ii) extraction, chemical characterization and analysis of the antifungal effects of essential (volatile) oils extracted from leaves; (iii) pollen germination in vitro; (iv) flower morphology; (v) seed production; (vi) initial seedling development; (vii) leaf allelopathy; (viii) measurements of total phenolic compounds in leaves and roots; and (ix) effects of leaf extracts on the viability of human colon cells. All results obtained from experiments (ii) to (ix) revealed no statistical differences between GM- and non-GM-derived samples. A second round of experiments will be conducted to confirm these results. Proteomic and transcriptomic profiling of GM and non-GM trees are under analysis, as well as a series of experiments that include the chemical, nutritional and biological analysis of honey samples derived from bee hives located in fields of GM versus non-GM plants; and bee (*Apis mellifera*) population dynamics.
## IURO WORKSHOP COST ACTION FP0905

### O58
#### Biological characterization of genetically modified trees (GMTs)

Gilles Pilate¹*, Hely Haggman², Isabel Allona², Lucian Curtu⁴, Terezia Salaj⁵, Ove Nilsson⁶, Matthias Fladung⁷, Cristina Vettori⁸

¹INRA, UR588 Amélioration, Génétique, et Physiologie Forestières, F-45075

Orléans cedex 2, France; ²University of Oulu, Department of Biology, P.O. Box 3000, FI-90014 Oulu, Finland; ³Centro di Biotecnologia y Genomica de Plantas (CBGP-UPM/INIA), Campus de Montegancedo, UPM, 28223 Madrid, Spain; ⁴Transilvania University Brasov, Faculty of Forest Sciences, 1 Sirul Beethoven, Brasov, Romania; ⁵Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P. O. Box 39A, 950 07 Nitra, Slovakia; ⁶Department of Forest Genetics and Plant Physiology, SLU; ⁷Johann Heinrich von Thünen Institute (vTI) Institute for Forest Genetics Sieker Landstr. 2, D-22927 Großhansdorf, Germany; ⁸Plant Genetics Institute, National Research Council, Via Madonna del Piano 10, 50019 Sesto, Fiorentino (FI), Italia

E-mail: pilate@orleans.inra.fr

BMC Proceedings 2011, 5(Suppl 7):O58

In the last 20 years, a large number of GMTs have been generated, characterized and field tested to evaluate their agronomic behaviour and their potential performance for technological applications.

Therefore, important knowledge has been gained from these studies on the potential of GMTs whereas biosafety concerns have been raised on the plantation of transgenic trees in the field. The European Cooperation in Science and Technology (COST) action FP0905 on the biosafety of forest transgenic trees aims to help forthe building and implementation of EU policy directives to ensure a safe development and practical use of GMTs in the future. This Action is expected to generate important benefits thanks to the experience gained by the research community, to provide general information on these studies as well as to address public concerns about biosafety issues raised by transgenic tree plantations, and finally to help for the establishment and implementation of EU policy directives in the view of a future deployment of GMTs cultivation in Europe. Within this action, the objective of the working group 1 (WG1) is to gather the existing knowledge on genetically modified forest trees (GMTs) including both field trials and greenhouse experiments. More specifically, the action of WG1 is focused on the compilation of:

- Data from literature and methods developed on gene flow and containment strategies

- Data available on the traits targeted and the function of the gene introduced, on their intended and eventually unintended effects

- Data on host species characteristics especially related to biosafety issues such as their biological cycle, their way of pollination and the characteristics of pollen and seed dispersal.

This information will be collected from published papers in books and reviews as well as from reports from different agencies available on the web. Moreover, additional data will hopefully be gained through the responses of expert scientists from public or private institutions to an ongoing survey accessible at the website of the COST Action FP0905: [http://www cost-action-fp0905 eu/index.php](http://www cost-action-fp0905 eu/index.php)

option=com_kunena&Itemid=74&func=view&catid=4&id=11)

The information collected will be used to:

- 1) establish a list of protocols for the control of transgene flow and gene containment.

- 2) establish the present state of the art in gene targeting strategies for site specific integration of transgenes.

- 3) get an appraisal on the types of construct and genes inserted in GMTs in the past; in the present and in the future.

This information will be compiled in a database that will be made available to the scientific community as well as to the European policy makers.

The results of this work will enable the participants and end-users to get a clear factual overview of the global status of GMTs and especially the status of Europe compared to non-European countries. Furthermore, the information gathered will also be useful for assessment of environmental impacts of GMTs that is the object addressed in the WG2 of the COST Action FP0905.

### O59
#### Environmental impact assessment and monitoring of genetically modified trees

Fernando Gallardo¹*, Lucia Ionita², Marja Ruohonen-Lehto³,

Antoine Harfouche⁴, Stefano Bircolti⁵, Wout Boerjan⁶, Boet Glandorf⁷,

Lise Jouanin⁸, Matthias Fladung⁹, Cristina Vettori¹⁰

¹Departamento de Biología Molecular y Bioquímica, University of Málaga, 29071

Málaga, Spain; ²Department Genetics, Forest Research and Management

Institute, 077190 Ilfov, Romania; ³Finnish Environment Institute, 00251 Helsinki,

Finland; ⁴Department of Forest Environment and Resources, University of

Tuscia, 01100 Viterbo, Italy; ⁵Dipartimento di Ortoflorofrutticoltura, University of

Florence, 50019 Sesto Fiorentino, Italy; ⁶VIB Department of Plant Systems

Biology and, University of Gent, 9052 Gent, Belgium; ⁷National Institute for Public Health and the Environment, 3720BA Bilthoven, Netherlands; ⁸UMR1318

INRA-AgroParisTech Versailles, Institute Jean-Pierre Bourgin, 78026 Versailles,

France; ⁹Institute of Forest Genetics, Johann Heinrich von Thünen Institute, D-22927 Grosshansdorf, Germany; ¹⁰Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, 50019 Sesto, Fiorentino, Italy

E-mail: fgallardo@uma.es

BMC Proceedings 2011, 5(Suppl 7):O59

Transgenic biotechnology can assist forest tree improvement programs but it may also raise environmental safety concerns. The environmental effects of genetically modified transgenic trees (GMTs) have been studied in many countries during the last 15 years. Today there is an urgent need of putting together this scattered knowledge to build-up a European knowledge platform for addressing GMTs in plantations. The main aims of Working Group 2 (WG2) of the European Cooperation in Science and Technology (COST) Action FP0905 "Biosafety of transgenic forest trees" (http://www cost-action-fp0905 eu/) are (1) to discuss, based on scientific facts, whether current containment strategies are appropriate or need to be improved for GMTs, (2) to define a common protocol to track the transgene from the laboratory to the final product, and (3) to assess the possible impacts of GMTs on the environment. The potential risks of GMTs, the fate of recombinant material and the potential relevance of recombinant genes on plant's omics are other main aspects to be considered and compared to similar processes with endogenous genes in conventional breeding. The group involves experts from public research, government and independent regulatory sectors across COST and non-COST member countries. The activities of this group have been organized into three Task Groups focusing on (1) risk assessment studies and guidance documents, (2) the monitoring of the transgenes and recombinant plant material, and (3) the impact of GMTs on exposed ecosystems. As a first step of WG2 activity, a database of guidance documents from national and transnational sources dealing with impacts and risk assessments of GMTs is being created to identify common and case-specific issues on biosafety. It is expected that the information gained will facilitate (1) a science-based understanding of the impacts of GMTs on the environment in comparison with that of traditionally tree breeding, and (2) future socio-economic and cost/benefits analyses of GMTs in plantations.

### O60
#### Dissemination of Cost Action FP0905 information by website

Fabio Migliacci¹*, Matthias Fladung², Cristina Vettori³, Jean-Charles Leplé⁴, Klaus Minol⁵, Francesca Donnarumma³

¹Organo Metallic Chemistry Compound Institute, CNR, Via Madonna del

Piano 10, I-50019 Sesto Fiorentino (FI), Italy; ²Johann Heinrich von Thünen Institute (vTI), Institute of Forest Genetics, Sieker Landstr. 2, D-22927

Grosshansdorf, Germany; ³Plant Genetics Institute, CNR, Via Madonna del

Piano 10, I-50019 Sesto Fiorentino (FI), Italy; ⁴INRA, UR588 AGPF, Centre de

recherche d'Orléans, 2163 Avenue de la Pomme de Pin, CS 40001 Ardon, 45075 Orléans Cedex 2, France; ⁵Genius GmbH, Robert-Bosch-Str. 7, 64293 Darmstadt, Germany

E-mail: Fabio.migliacci@cnr.it

BMC Proceedings 2011, 5(Suppl 7):O60

**Background:** The Cost Action FP0905 aims to evaluate the scientific knowledge of GMT biosafety protocols coordinating the existing information generated in various European countries as basis for future EU policy and regulation for the environmental impact assessment.
For spreading the information relevant to the Action, the website http://www_cost-action-fp0905.eu/has been set up, educating the general population of the technical, social, environmental and economic aspects of this issue. The principal aims of the website are: to provide a database with the main information on forest GMTs, that should be available to the scientific community and Europe organisations; to update the website with science-based information for public interest in the utilization of GMTs in forest plantation and at the same time safeguarding the environment, using all the information collected to identify particular topics which could be useful to develop research projects collaborating with the other working groups.

**Material and methods:** The Website was built using the Open Source Software Joomla, a program for web site design and content management. This software has not been written for profit, but to benefit the user community. The difference between Joomla and other web design software systems is that there are thousands of fully functional, complete web site templates available in the Internet, so that all what is to do is to choose the layout you like and to add information in the templates.

However, Joomla as a "Content Management System" is not only a "simple" web site construction program, furthermore it can also be used as a database. It is used to build/design a "php/mysql database" to

<figure><img src="image_20.png" /><figcaption>**Figure 1(abstract O60)** image of the COST Action FP0905 website.</figcaption></figure>
collect the information. But the possibilities of the program on how to use it as a database are endless. In this way it will be simple to create the database tables and fields. The software generates the scripts to upload them into the website, to setup the MySQL tables and the scripts and to manage the tables once uploaded.

The most important features of the Joomla program are:

1. Instantly publish PHP MySQL database applications ready for uploading on the website of Action.

2. Manage easily the database.

3. Automatic generation of php forms to display and browse data content of tables, with the ability to edit, delete and add records.

4. Data navigation, sorting, searching, search filtering, modification, addition, deletion, file uploads. The website is also a very friendly searching engine. Google will regularly visit this website site, due to the frequent content changes that result from the news feeds. They are usually updated on a daily basis, even if the web site is not updated on a regular basis.

**Results:** Working group 4 is developing and managing the web site of the Action enhancing communication and information among the participants and with those organisations interested in the data provided. The website has provided a forum for the organizations working within and outside the consortium; discussing for example, any effect of a transgenic plantation needs in the context of already existing and accepted forestry practise. The Action has also been introduced in facebook.

## O61

### Field trials of GM trees in the USA: activity and regulatory developments

Steven H Strauss*, Venkatesh Viswanath
Department of Forest Ecosystems and Society, Oregon State University, 321 Richardson Hall, Corvallis, OR 97331, USA
E-mail: Steve.Strauss@oregonstate.edu
BMC Proceedings 2011, 5(Suppl 7):O61

Field trials are extremely important for all facets of research, breeding, and biotechnology. Tree physiology is distinctively different in greenhouses or growth chambers vs. field environments. Thus, results from non-field environments can be very misleading with respect to identification of promising biotechnologies and elite varieties. Large regulatory impediments to conduct of field tests of genetically modified (GM) trees will therefore tend to stifle scientific and technological development. This appears to be the case for genetically modified trees,

where regulatory burdens to conducting field tests have grown in stringency in the USA and elsewhere in recent years [1,2]. Here, we present the record of recent field trials and some new regulatory developments in the USA.

**Overview of field trials in the USA:** Information Systems for Biotechnology (ISB) [3] maintains an easily used database of GM crop data in the United States. There have been nearly 600 field trials (including both "acknowledgments" and "permits") conducted since 1989, with a five-fold increase occurring in 2000-2009 compared with 1990-1999 (Figure 1).

Fifteen different genera have been tested, including *Populus*, *Pinus*, *Eucalyptus*, and *Malus*, with the first two accounting for approximately 60% of all field trials (Figure 2). Marker genes and tolerance to biotic stresses were the two most common research objectives. Private companies carried out 60% of field trials, and their activity has grown dominant in recent years.

Currently, there are more than sixty field trials covering eleven different genera, with *Populus* accounting for 35% of all field trials. *Eucalyptus*, *Liquidambar*, and *Malus* together account for another 37% of trials. Other genera being tested include *Castanea* (American chestnut), *Pinus*, *Ulmus* (American elm), *Prunus*, *Musa*, *Citrus*, and *Juglans*. Forty field trials are being conducted by private companies, with ArborGen alone accounting for 36 of the 40 active field trials. Cornell University, North Carolina State University, Oregon State University, Purdue University, the United States Department of Agriculture (ARS) and the University of California, Davis are among the public institutions currently conducting field trials. One of ArborGen's field trials with cold tolerant *Eucalyptus* covers 197.2 acres. This is the largest current field trial in terms of acres and is being conducted in six different states. In terms of research objectives of current trials, marker genes dominate in terms of frequency of trials, with modification of wood quality the next most common objective.

**Regulation of field trials in the USA:** In the USA, transgenic trees are regulated in the same manner as agricultural crops [4], using regulatory laws created prior to the development of transgenic biotechnology. The three agencies involved are the United States Department of Agriculture (USDA), which considers agricultural safety and economics; the Food and Drug Administration (FDA), which considers human and animal feed safety; and the Environmental Protection Agency (EPA), which considers pesticidal properties of transgenic organisms [5]. Regulations in the United States are perceived to be less stringent than those in Europe and Asia, however, a recent survey in the USA found that forest scientists—including breeders, biotechnologists, and ecologists—see regulations as major obstacles to field research and commercial development of GM trees [6].

<figure><img src="image_21.png" /><figcaption>**Figure 1(abstract O61)** Total number of field trials with transgenic trees in the USA between 1990 and 2009.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>Figure 2(abstract O61) Major genera of transgenic trees in field trials in the USA, 1990 to present.</figcaption></figure>

**Recent regulatory developments:** A company known as Okanagan Specialty Fruits has taken the lead in the production of transgenic tree varieties produced by the insertion of cisgenes and intragenes [7]. Non-browning versions of established apple varieties were developed by the company by the silencing of the polyphenol oxidase gene. These apples have been named Arctic™ apples because of the color of their skin. The company has petitioned the Animal and Plant Health Inspection Service (APHIS) for deregulation of the product in the USA [8].

Cold tolerant eucalyptus varieties were developed by ArborGen [9]. The company believes that these varieties would permit the planting of highly productive eucalypt hybrids for bioenergy and pup north of Florida in the USA. In May 2010, the USDA authorized a large scale flowering field trial of these trees, thus permitting the planting of nearly 260,000 trees over ~300 acres in 7 states [10]. However, several environmental organizations filed a lawsuit against APHIS because they believed that APHIS performed an inadequate environmental analysis, thus violating the US National Environmental Policy Act (NEPA).

A new two year pilot project has recently been announced by APHIS [11]. Under the provisions of the new project—whose aim is to speed up the review process before deregulation of biotech crops—environmental assessments of transgenic crops will be conducted by companies themselves, or by contractors authorized by the USDA. Although USDA would still need to review and approve the analyses, critics of the new policy believe that it might lead to less stringent environmental analyses.

## References

1. Strauss SH, Tan H, Boerjan W, Sedjo R: Strangled at birth? Forest biotech and the Convention on Biological Diversity. *Nature Biotechnology* 2009, 27:519-527.

2. Strauss SH, Kershen DL, Bouton JH, Redick TP, Tan H, Sedjo R: Far-reaching deleterious impacts of regulations on research and environmental studies of recombinant-DNA modified perennial biofuel crops in the USA. *BioScience* 2010, 60:729-741.

3. Information Systems for Biotechnology: USDA field tests of GM crops-Search the release data. 2011 [http://www.isb.vt.edu/search-release-data.aspx].

4. Harry DE, Strauss SH: Biotechnology and genetic engineering in forest trees. 2010 [http://www.agribiotech.info/details/Strauss%20and%20Harry%20Draft%20Final%2002%20print.pdf].

5. McHughen A, Smyth S: US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars. *Plant Biotechnol J* 2007, 6:2-12.

6. Strauss SH, Schmitt A, Sedjo R: Forest scientist views of regulatory obstacles to research and development of transgenic forest biotechnology. *Journal of Forestry* 2009, 107:350-357.

7. Okanagan Specialty Fruits: Non-browning apple. 2011 [http://www.okspecialtyfruits.com/ps-non-browning-apple.php].

8. Animal and Plant Health Inspection Service (APHIS): Petitions for nonregulated status pending. 2011 [http://www.aphis.usda.gov/biotechnology/not_reg.html].

9. ArborGen: Freeze tolerant eucalyptus. 2011 [http://www.arborgen.us/index.php/products/product-pipeline/freeze-tolerant-eucalyptus].

10. Vaidyanathan G: Government approves GM trees; company plans for biofuel use. 2010 [http://www.eenews.net/climatewire/2010/05/17/3/].

11. Perkowski M: USDA plans to outsource biotech studies. 2011 [http://www.capitalpress.com/content/mp-biotech-process-041511].

## O62

### Research in tree genetic engineering; the Canadian context for field trial of GM trees

Armand Séguin

Natural Resources Canada, Canadian Forest Service, Laurentian Forestry

Centre, Canada

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

BMC Proceedings 2011, **5**(Suppl 7):O62

Canada is in a unique position for the regulation of GM (genetically modified) plants since it is based on the novelty of the resulting traits and not on the process to obtain it. For this reasons the term "plant with novel traits" (or PNTs) is used and these traits can be introduced into plants using biotechnology, mutagenesis or conventional breeding techniques.

In Canada, less than a handful of GE tree field trials have been conducted. In all cases, poplars or spruces were used. Consistent with international guidance provided for living modified organisms, particular attention is given to a specific trait by considering both its novelty for the Canadian environment and its potential effect on human health and the environment. Our research is in line with the development of a consolidated research approach and knowledge needed for international guidance (e.g. Cartagena Protocol on Biosafety under the Convention on Biological Diversity) developed for genetically modified organisms. Important losses due to insects and fungal pathogens result in a considerable reduction of yield in Canadian forest productivity. Compounding these problems is the increased movement of pests that has been facilitated by increased international trade of agricultural and forest products as well as global climate change. More recently the increased need for biomass production and specific bioproducts to sustain crop-based biofuel production has emerged. Forest biotechnology
has the potential to maintain forest sustainability by contributing to the transformation of our forest industry so as to improve productivity in the context of tree plantation. In this presentation, we will show how genetic engineering of forest trees has been used in improving tree adaptation and pest protection. We will also describe the work being done on environmental assessment of field-grown trees and results on long-term expression of engineered genes.

**O63**

**A brief overview of field testing and commercial application of transgenic trees in China**

Meng-Zhu Lu<sup>*</sup>, Jian-Jun Hu
State Key Laboratory of Forest Genetics and Tree Breeding, Chinese Academy of Forestry, Beijing 100091, China
E-mail: lumz@caf.ac.cn
*BMC Proceedings* 2011, **5**(Suppl 7):O63

Since the first report on transformation of *Bt* gene into *Populus nigra* was published in 1989, transgenic approaches have been widely used in breeding trees for high tolerance to insects and environmental stresses in China. Notably, field testing of the transgenic poplar with *Bt* was performed in 1994 and permitted to be commercialized as the first commercialized transgenic tree in the world. Up to now, nearly 50 species have been used as recipients for genetic transformation including poplar, birch, locust tree, walnut, aimed to improve their tolerance to insects and diseases, environmental stresses, prolonged storage lifetime, wood property, flowering time control, etc.

On May 5, 2006, the State Forest Administration (SFA) launched its first regulatory framework on "Management measures on the inspection and permit of forest genetic engineering" taken effect on July 1, 2006. It states that small-scale field testing, environmental release and pilot production test of each transgenic line are required before its permit of commercialization, with each phase must be evaluated by the experts. "A technical guideline on safety evaluation of the transgenic forest plants and their products" was published by SFA on June 4, 2007, which provides a standard procedure for molecular breeders to follow in order to commercialize their transgenic trees.

To the year 2010, 128 field trials have been granted by SFA, 84 of which belong to tree species while 44 to grass species. Thirty three of them were granted to transgenic poplars, with tolerance to insects and diseases, drought and salt stresses, altered wood property, etc., and 25 to locust tree. Transgenic *Populus tomentosa* with antisense *CCoAOMT* (coding for a key enzyme involved in lignin monomer biosynthesis), transgenic *Robinia pseudoacacia* with *BADH*, *Sophora japonica* with *RD29A* and *P. nigra* with *Bt* are under environmental release testing.

The transgenic poplar plantation has increased to 450 hm² this year since two *Bt* transgenic poplars were commercialized in 2001 in China. The transgenic poplar plantations have effectively inhibited the fast-spread of target insects and significantly reduced the times of insecticide application on poplar plantation. The transgenic *Populus nigra* was also used in hybridization with non-transgenic *P. deltoides* as an insect-resistant source for breeding new hybrid clones.

# S1. POPULATION GENOMICS, CONSERVATION AND ADAPTATION

**P1**

**Identification of microsatellite loci in Pinus tecunumanii**

Valderês Aparecida de Sousa¹, Camila C Mantello²,
Ananda Virginia de Aguiar¹*, Daiane Rigoni Kresting¹, Anete Pereira de Souza²,
Laila Toniol Cardin³

¹Embrapa Forestry, Brazilian Agricultural Research Corporation, Colombo,
Paraná, 83411-000, Brazil; ²UNICAMP, Campinas, São Paulo, 13083-970, Brazil;
³UNESP, Ilha Solteira, São Paulo, 15385-000, Brazil

E-mail: ananda@cnpf.embrapa.br
*BMC Proceedings* 2011, **5**(Suppl 7):P1

**Background:** *Pinus tecunumanii* has displayed good performance in tropical regions of Brazil and showed high potential for commercial

exploitation. Embrapa Forestry and its partners own many of the species seed production areas. In spite of its importance, the majority of P. tecunumanii germplasm collections remain still genetically uncharacterized. Thus identifying genetic markers is an important tool to genetically characterize these collections. We describe the initial steps to develop microsatellites for *Pinus tecunumanii* by enriched library construction with the ultimate goal of characterizing accessions of the germplasm collections of EMBRAPA.

**Methods:** The genomic-enriched library was constructed following the protocol described by [1]. The genomic DNA of *P. tecunumanii* was digested with AFAI and enriched in (CT)₈ and (GT)₈ repeats. Enriched fragments were amplified by polymerase chain reaction (PCR), connected to a pGEM T-easy vector and transformed into competent XL1- blue *Escherichia coli* cells. The positive clones were selected using the B-galactosidase gene and then grown overnight in an HM/F medium with ampicillin. After PCR 95 positive clones were sequenced in both directions using the T7 and SP6 primers as well as the Big Dye terminator Kit. The sequences were assembled and edited in Seqman (DNAStar), the repetitive regions were found using the Simple Sequence Repeat Identification Tool [2]. Primer select (DNAStar) and Primers Plus were used to design primer pairs flanking the microsatellite regions.

**Results and conclusion:** Of the ninety five sequences cloned only eleven contained microsatellite sequences and five showed repeats and adequate flanking regions for primer design. The observed proportion of dinucleotide was 5.26% (5), while trinucleotide and tetranucleotide proportions were 1.05% (1) and 5.26% (5), respectively. Ninety one percent of nucleotides were simply perfect and 9% were compost perfect. The explanation for this low yield (11.6%) can be attributed to the genomic-enriched procedure. To overcome this problem this procedure will be repeated. The obtained sequences will be used for validation of *P. tecunumanii* microsatellite primers and used to estimate the genetic diversity from the germplasm collection located in various regions of Brazil.

**Acknowledgments:** The authors would like to thank Selma Buzzetti de Moraes and Mario Luiz Teixeira de Moraes for their assistance with DNA extraction procedures and the Valor Florestal Company for collecting material used in this research.

**References**

1. Billote N, Risterucci AM, Baurens FC: Microsatellite enriched libraries: applied methodology for the development of SSR markers in tropical crops. *Fruits* 1999, 54:277-288.

2. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S: Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. *Genome Research* 2011, 11:1441-1452.

**P2**

**Development of microsatellite markers for Pinus maximinoi derived from microsatellite-enriched libraries**

Ananda Virginia Aguiar¹*, Camila C Mantello², Daiane Rigoni Kesting¹,
Valderês Aparecida Sousa¹, Anete Pereira Souza², Laila Toniol Cardin³

¹Embrapa Forestry, Brazilian Agricultural Research Corporation, Colombo,
Paraná, 83411-000, Brazil; ²UNICAMP, Campinas, São Paulo, 13083-970, Brazil;
³UNESP, Ilha Solteira, São Paulo, 15385-000, Brazil

E-mail: ananda@cnpf.embrapa.br
*BMC Proceedings* 2011, **5**(Suppl 7):P2

**Background:** Integrating the use of molecular techniques in ongoing breeding can help identifying divergent genotypes to be used in controlled crosses for the development of hybrids, estimating parentage and monitoring the efficient use of genetic variation. Breeding programs carried out at EMBRAPA have established a broad population genetic base for various species of pine, from which trees with high productivity and wood quality have been selected. *P. maximinoi* was included in this program as its timber has specific characteristics of great economic importance. One of the main features of *P. maximinoi* is its rapid growth, reaching 20 to 40 meters in height with a diameter at breast height of 100 cm. The species' straight trunk with few branches is advantageous to the sawmill, and the timber's thick fiber is necessary for quality paper production. IN this work we report on the initial steps of the development of microsatellite markers for *P. maximinoi* derived from
microsatellite-enriched libraries. These markers will be used to characterize the breeding populations and germplasm collections of EMBRAPA.

**Methods:** A microsatellite-enriched genomic library was constructed following the protocol described by [1]. The genomic DNA of *P. maximinoi* was digested with AFAI and enriched for two microsatellite motifs (CT)₈ and (GT)₈. Enriched fragments were amplified by PCR, ligated to a pGEM T-easy vector and transformed into competent XL1- blue *Escherichia coli* cells. The positive clones were selected using the B-galactosidase gene and then grown overnight in an HM/F medium with ampicillin. After PCR, 88 positive clones were sequenced in both directions. The sequences were assembled and edited in Seqman (DNAStar), and the repetitive regions were found using the Simple Sequence Repeat Identification Tool [2] Primer select (DNAStar) was used to design primer pairs flanking the microsatellite regions.

**Results and conclusion:** Of the eighty sequences cloned only eight contained microsatellite sequences showing repeats and adequate flanking regions for primer design. The observed proportion of dinucleotide was 6.81%, while the tetranucleotide proportion was 2.27%. Seventy five percent of nucleotides were simple perfect repeats and 25% were compost perfect. The explanation for this low yield (9.09%) can be attributed to the genomic-enriching procedure. To overcome this problem the procedure will be repeated. The obtained sequences will be used for validation of *P. maximinoi* microsatellite primers. Eight microsatellite sequences of simply perfect and compost perfect nucleotides were observed for *P. maximinoi*. These markers will be validated and used to estimate the genetic diversity from the germoplasm collection of the Brazilian Agricultural Research Corporation (EMBRAPA).

**Acknowledgments:** The authors would like to thank Selma Buzzetti de Moraes and Mario Luiz Teixeira de Moraes for their assistance with DNA extraction procedures and the Valor Florestal Company for collecting material used in this research.

## References

1. Billote N, Risterucci AM, Baurens FC: Microsatellite enriched libraries: applied methodology for the development of SSR markers in tropical crops. *Fruits* 1999, 54:277-288.

2. Temnykh S, DeClерck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S: Computational and experimental analysis of microsatellites in rice (*Oryza sativa* L.): frequency, length variation, transposon associations, and genetic marker potential. *Genome Research* 2001, 11:1441-1452.

## P3

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

Komlan Avia¹*, Kärkkäinen Katri², Outi Savolainen¹

¹Department of Biology, University of Oulu, PO Box 3000, FI-90014 Oulu, Finland; ²Finnish Forest Research Institute, FI-91500 Muhos, Finland

E-mail: komlan.avia@oulu.fi

BMC Proceedings 2011, 5(Suppl 7):P3

Conifers, like other perennial plants adapt their growth rhythm to seasonal changes in the environment. Growth cessation and bud set are among the main adaptations used by the plants to prepare for winter. These adaptive traits are mainly controlled by photoperiod, temperature and light quality and show a clinal variation in different species including Scots pine. In conifers, efforts have been made to understand the genetic basis of these traits. In Norway spruce for instance, a *Flowering Locus T* homolog (*PaFT4*) has been shown to be implicated in the control of growth rhythm.

We previously evaluated genetic variation for timing of bud set and cold tolerance in a large association population of Scots pine (*Pinus sylvestris* L.) from Punkaharju, Southern Finland, along with five other populations used as control for clinal variation. To correlate this genetic variation with the expression pattern of some candidate genes already described in other species including conifers as involved in the control of bud set (e.g. FT4, PRR1, Gi, Myb) and cold tolerance (e.g. Aba responsive, DHN3, ERD, LEA), the Punkaharju population was used together with two others from Kolari (Northern Finland) and Poland. For photoperiod treatments, seedlings of eight families from each population were grown in a greenhouse under natural photoperiod conditions and in growth chambers under constant light for three months before transfer to three different photoperiod

conditions (20 h/4 h, 17 h/7 h and 8 h/16 h light/dark). For cold treatments, the same families were grown in growth chambers under constant light for two and a half months and divided into two parts. The first part was directly submitted to the cold treatments (+4°C for one week followed by -5°C for 48 hours) while for the second part, seedlings were allowed to set buds under short day conditions (8 h light/16 h dark) before treatments. The results of this expression study will be described in relation to the phenotypic variance already observed.

## P4

### Construction of Brazil nut (*Bertholletia excelsa* Lecythidaceae) cambium normalized cDNA libraries for 454 next generation sequencing

Peter Inglis, Ana Ciampi, Vânia Azevedo¹

Laboratório de Genética Vegetal, Embrapa Genetic Resources and Biotechnology, Brazilia, Brazil

E-mail: vania.azevedo@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P4

**Background:** The Brazil nut, *Bertholletia excelsa* (Lecythidaceae), is the most important non-timber forest resource in the Amazonian biome, where nuts for the international trade are exclusively wild-collected. The monotypic genus possesses a discontinuous distribution and is thought to have rapidly and recently radiated south and westward from a northern/eastern Amazonian origin, largely as a result of ancient human activity [1]. It's genetic variability is currently thought to be low and conserved in one or very few populations [2], and in order to better understand the population genetics of this tree, there is a need for additional, or more informative, molecular markers than is currently available.

**Methods:** Trunk cambium is an attractive source of material for cDNA library construction, in contrast to foliar material, where photosynthetic genes are grossly overrepresented. Furthermore, samples can be taken from mature trees at the forest floor level. Cambium samples were collected from both Acre and Amapá states of Brazil, and from both highly, and poorly-productive trees from the respective states, with the aim of detecting microsatellites (SSRs) and single nucleotide polymorphisms (SNPs) for genetic analysis. The samples were frozen in the field on dry ice and later kept at -80 °C in the laboratory. Total RNA was extracted from cambium ground with a pestle and mortar under liquid nitrogen, using either the RNeasy kit (QIAGEN) or a combination of hot CTAB pre-treatment with repeated chloroform extraction and the RNeasy kit. Messenger RNA was purified using oligo dT-magnetic bead separation (Dynabeads; Invitrogen). The Clontech SMART kit and Superscript III (Invitrogen) reverse transcriptase were used for cDNA synthesis and four normalized, PCR-amplified libraries constructed using the Trimmer Direct kit (Evrogen). The four cDNA libraries were multiplex tagged and submitted to a single 454 pyrosequencing production run, using a GS FLX-Titanium instrument.

**Results and conclusions:** Cambium RNA extracted with the RNeasy kit alone or with the CTAB pretreatment possessed A260/280 and A260/230 ratios above 2.0, when measured by Nanodrop spectrometry, indicating high purity, and appeared to be of high quality by agarose electrophoresis. However, the RNA extracted by RNeasy alone was not successfully reverse-transcribed and gave no significant products in RT-PCR, even after mRNA purification by magnetic bead separation, indicating the presence of recalcitrant and bound enzyme inhibitory substances. In contrast, the CTAB pretreatment yielded RNA that could be readily reverse transcribed, which was subsequently used for amplified and normalized cDNA library construction.

Data yield for the four normalized libraries averaged 73 million total bases. 270 thousand validated sequences with a mean read length of 265 bp, respectively, per library. Sequences were assembled into contigs using Newbler version 2.5 and microsatellites were searched for using Microsatellite Commander v. 0.8.2. The mean numbers of microsatellites identified per repeat class per library were: 507 dinucleotide, 897 trinucleotide, 29 tetranucleotide, 9 pentanucleotide, 41 hexanucleotide, and 138 compound/ interrupted repeats, with little variation in microsatellite yield or repeat class present between libraries. These libraries appear to be a rich source of new markers, which will greatly enhance our efforts to better understand the population genetics of *B. excelsa*, to determine appropriate conservation strategies, and to work towards rational selection of productive lines.
## References

1. Shepard GH, Ramirez H: "Made in Brazil": Human Dispersal of the Brazil Nut (Bertholletia excelsa, Lecythidaceae) in Ancient Amazonia. *Econ Bot* 2011, 65:44-65.

2. Buckley DP, O'Malley DM, Apsit V, Prance GT, Bawa KS: Genetics of Brazil nut (Bertholletia excelsa Humb. & Bonpl.: Lecythidaceae). *Theor Appl Genet* 1988, 76:923-928.

## P5

### Genetic diversity of *Bertholletia excelsa*, an Amazonian species of wide distribution

Patrícia Suji¹, Karina Martins², Lucia Wadt³, Vânia Azevedo⁴*, Vera Solferini¹
¹Laboratório de Diversidade Genética, Universidade Estadual de Campinas, Campinas, Brazil; ²Universidade Federal de São Carlos, Brazil; ³Embrapa Acre, Brazil; ⁴Laboratório de Genética Vegetal, Embrapa Recursos Genéticos e Biotecnologia, Brazilia, Brazil

E-mail: vania.azevedo@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P5

**Background:** Amazonian upland forests are expansive and can comprise large continuous tracts. There have been several studies on the population genetic structure of species in this kind of forest, but there are few studies that aim to understand genetic structure throughout the Amazon [1]. The Brazil-nut tree, *Bertholletia excelsa*, is a monotypic genus, endemic to upland forests and distributed along almost the entire expanse of the Amazon [2-5].

Genetic diversity distribution through Amazon forest is an understudied issue, specially in plants. Quantifying and understanding population genetic structure, associated to gene flow and mating system studies are recognized as important tools for the development of strategies for conservation and management. Such information can also be helpful in identifying effects of habitat fragmentation [6,7].

This study aimed to evaluate genetic structure of *B. excelsa* populations in the Amazon and to verify if the structuring is influenced by distance between them.

**Methods:** Seven polymorphic microsatellite markers were developed from a dinucleotide-enriched genomic library. Material from 379 individuals was collected from nine Amazonian subpopulations distributed in five Brazilian states (AC: Acre, AM: Amazonas, AP: Amapá, PA: Pará, RR: Roraima). All seven microsatellite markers developed for the species were used with four others previously published markers [8], for genotyping.

Analyses within and among populations were performed to evaluate genetic diversity and population structure. All nine populations were characterized with 11 microsatellite loci for number of alleles per locus, allele frequency and observed and expected heterozygosity under Hardy-Weinberg expectation. Wright fixation indices were also estimated. Genetic distance estimates were correlated with different potential factors to find possible causes of genetic structure.

**Results and conclusions:** A few alleles were found in each subpopulation and considerable variation was observed in alleles found in each subpopulation and in their allele frequencies, especially when compared with very distant subpopulations. Heterozygote excess was observed in five subpopulations while in the other four subpopulations, estimates of *F*<sub>IS</sub>, non-significantly different from zero, were found. The observed high heterozygote proportions in most subpopulations and absence of inbreeding support the existence of self-incompatibility mechanisms and selection in favor of heterozygotes. Maintenance of genetic variability is favored by the alogamous breeding system, which is an important feature to be considered in conservation and management strategies, in order to avoid fertility deficits.

Fine-scale structure, when present, was small. Estimates of inter population genetic structure varied from low (*q*= 0,02) to high (*q*= 0,244). *B. excelsa* genetic structure can be analyzed on three different scales: (i) within population; (ii) among moderately distant populations (<500km); and (iii) among very distant subpopulations. At all scales, significant correlations were found between genetic structure and geographic distance between pairs of individuals or populations. This may indicate that distance is an important factor in this population's genetic structure, but probably there are other factors acting in conjunction, especially on a large geographical scale.

## References

1. Sampaio AJ: A flora amazônica. *Revista Brazileira de Geografia* 1942, 4(2):57-72.

2. Maués MM: Reproductive phenology and pollination of the Brazil nut tree (Bertholletia excelsa Humb. & Bonpl., Lecythidaceae) in Eastern Amazonia. *Pollinating Bees The Conservation Link Between Agriculture and Nature - Ministry of Environment / Brazilia*. P. KEVAN & V.L. Imperatriz Fonseca 2002, 245-254.

3. Mori SA, Prance GT: Taxonomy, ecology and economic botany of the Brazil nut (Bertholletia excelsa Humb. & Bonpl. Lecythidaceae). *Advances in Economic Botany* 1990, 8:130-150.

4. Moritz A: Estudos biológicos da floração e frutificação da castanha-do-Brazil. *DocumentosEmbrapa/Cpatu, Belém* 1984, 29:1-78.

5. Salomão R: Densidade, estrutura e distribuição especial de castanheira-do-Brazil (Bertholletia excelsa H. & B.) em dois platôs de floresta ombrófila densa na Amazônia setentrional Brazileira. *Boletim do Museu Paraense Emilio Goeldi* 2009, 4:11-25.

6. Azevedo VCR, Kanashiro M, Ciampi AY, Grattapaglia : Genetic structure and mating system of Manilkara huberi (Ducke) A. Chev., a heavily logged Amazonian timber species. *Journal of Heredity* 2007, 98:646-654.

7. Erickson DL, Hamrick JL, Kochert GD: Ecological determinants of genetic diversity in an expanding population of the shrub *Myrica cerifera*. *Molecular Ecology* 2004, 13:1655-1664.

8. Reis AMM, Braga AC, Lemes MM, Gribel R, Collevatti RG: Development and characterization of microsatellite markers for the Brazil nut tree Bertholletia excels Humb & Bonpl. (Lecythidaceae). *Molecular Ecology Resources* 2009, 9:920-923.

## P6

### Molecular Phylogenetics of the genus *Hexachlamys* (Myrtaceae) using chloroplast and nuclear markers

Fernanda Cruz¹*, Andreia Turchetto-Zolet¹, Nicole Veto², Cláudio Mondin³, Mário Maurício Almerão², Rogério Margis⁴

¹Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil; ²Centro de Biotecnologia UFRGS, Brazil; ³Pontifícia Universidade Católica do Rio Grande do Sul, PUCRS, Brazil; ⁴Centro de Biotecnologia UFRGS, Pesquisador CNPq, Brazil

E-mail: fernandacruz86@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):P6

**Background:** Myrtaceae family includes more than 3,800 species of trees and shrubs, distributed mainly in tropical and subtropical regions of the world. Representatives of this family have great ecological significance for forest ecosystems, and are economically important species in the pharmaceutical, food, cosmetic and perfumery industry [1]. The genus *Hexachlamys* Berg. (Myrtaceae) has about 10 species distributed from the Southern to the Southeast of Brazil, and it also occurs in Paraguay, Argentina, Bolivia and Uruguay [2,3]. Since 1968 it has been considered an independent genus. It is distinguished morphologically from the genus *Eugenia* by pentameric or hexameric flowers and exserta radicle, and as many genera and species of the Myrtaceae family, it has a complex taxonomic classification [2,4,5]. The goal of this study was to conduct a molecular phylogenetic analysis among species of the genus *Hexachlamys* (Myrtaceae) using chloroplast (cpDNA) and nuclear (nrDNA) markers, and to verify its phylogenetic relationships with the genus *Eugenia* in order to contribute to the systematics and taxonomy of it.

**Methods:** The samples were collected as leaf material (from herbaria species and samples collected in the field) of the genus *Hexachlamys* representing all described species, as indicated in herbariums and species of the *Eugenia* genus that occur in the Rio Grande do Sul state. Total genomic DNA was extracted using the CTAB method based on the protocol already described [6] and used for the PCR reactions. PCR products were sequenced on ABI PRISM 3100 sequencer (Applied Biosystem). Nucleotide sequences were aligned using CLUSTALW [7] implemented in MEGA5 (Molecular Evolutionary Genetics Analysis) version 5.0 [8], then checked visually and carefully improved manually before analysis. The phylogenetic analysis was reconstructed after nucleotide sequence alignments using three different approaches: the neighbor-joining (NJ), the Bayesian and the Maximum-Likelihood (ML) methods. The NJ, Bayesian and ML analysis were performed in MEGA 5.0, MrBayes [9] and PhyML [10], respectively.
**Results and conclusions:** To date we obtained DNA sequences of 21 species of the genus Eugenia and seven species of the genus *Hexachlamys*. Universal primers that amplify chloroplast region (trnL-trnF, trnL-intron, ycf5, accD, rbcLa, psbA-trnH, rpoB, rpoC1, ndhJ, matK and rps16) and nuclear (ITS) were tested. The regions used for the phylogenetic analysis were chloroplast *accD, rpoB, rpoC1*genes and the nuclear ITS, since the other chloroplast regions were not possible to amplify and sequence in all sampled species. These cpDNA and nrDNA regions presented polymorphisms among species studied. Results from Bayesian, NJ and ML tree analysis produced similar topologies and revealed that *Hexachlamys* species could not form a monophyletic clade. The *Hexachlamys* species have grouped together *Eugenia* species with high bootstrap values, indicating that *Hexachlamys* can be a synonymous of genus *Eugenia*. These results corroborate with morphological data [11]. To confirm these results, other cpDNA and nrDNA markers will be tested and *Hexachlamys* species will be collected to substitute the species obtained in herbarium, since we found difficulties in the amplification of these species with all markers previously tested. This study can contribute to taxonomic classification of this group, as well as the field of conservation, since these species have an important economically and ecologically role.

Financial support: CNPq and FAPERGS

## References

1. Barroso GM, Peron MV: Myrtaceae Aspectos florísticos das espécies vasculares.J. Botânico 1994, 261-302.

2. McVaugh R: The genera of american myrtaceae, a interim report. Taxon 1968, 17:354-418.

3. Legram CD, Klein RM: Mirtáceas. Flora ilustrada catarinense 1969, 172.

4. McVaugh R: Tropical american myrtaceae. Chicago Natural History Museum 1956, 29.

5. Schmid R: Resolution of eugenia-syzygium controversy (myrtaceae). Am J Bot 1972, 59:423.

6. Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. Focus 1987, 12:13-15.

7. Thompson JD: Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22(22):4673-4680.

8. Tamura K, Dudley J, Nei M, Kumar S: Mega4: Molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol 2007, 24:1596-9.

9. Ronquist F, Huelsenbeck JP: Mrbayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19(12):1572-1574.

10. Guindon S, J F: Estimating maximum likelihood phylogenies with PhyML. Mol Biol 2009, 9(3):384-385.

11. Sobral M: A Família das Myrtaceae no Rio Grande do Sul. Edição Unisinos 2003, 216.

# P7

## Twenty four microsatellite markers for *Aspidosperma polyneuron* (*Apocynaceae*), an endangered tree species

Ronai Ferreira-Ramos¹,¹, Mariza Monteiro², Maria Imaculada Zucchi³, José Baldin Pinheiro², Carlos Alberto Martinez⁴, Moacyr Antonio Mestriner⁵, Ana Lilia Alzate-Marin⁵

¹Programa de Pós-Graduação em Biologia Comparada, FFCLRP, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil; ²Departamento de Genética, ESALQ, USP, Piracicaba, SP, Brazil; ³APTA Pólo Centro-Sul, Piracicaba, SP, Brazil; ⁴Departamento de Biologia, FFCLRP, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil; ⁵Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil

E-mail: ronai1977@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P7

**Background:** *Aspidosperma polyneuron* (Apocynaceae), commonly known as peroba-rosa, is a native and perennial species characteristic of semideciduous forest in submontane formation of Atlantic rain forest [1]. This species is recommended for ecosystems recovery and restoration of riparian forests in areas without flooding. But, the crescent logging, agriculture and urban expansion caused the reduction of native population of *A. polyneuron*. It is now included in the Red List of

Threatened Species [http://www.iucnredlist.org/apps/redlist/details/32023/0]. Little is known about the population structure of *A. polyneuron* and the effects of the spatial isolation on the levels of genetic diversity and gene flow. In this study, we developed microsatellite loci for *A. polyneuron* aiming future studies of genetic structure, gene flow and mating system.

**Material and methods:** Genomic DNA was extracted from leaf tissue [2] from one individual located in Cravinhos Municipality, São Paulo state, southeastern Brazil (21°17'41.8"S; 47°42'08.5"W). The Microsatellite-enriched library was constructed using the protocols described earlier [3]. The DNA was digested with *Rsa*1, bound to the adapters (*Rsa*21 and *Rsa*25) and amplified through polymerase chain reaction (PCR). The products were purified by Quiaquick PCR purification kit (Qiagen). Fragments containing microsatellite sequences were selected by hybridization with biotinylated oligo-probes (CT)₈, (GT)₈ and (CTT)₈ and recovered by streptavidin-coated paramagnetic beads. After that, it was carried out a second PCR in order to increase the enriched fragments number and then an aliquot of the PCR product was bound to the pGEM-T vector (Promega) and transformed into *Escherichia coli* XL-1 Blue strains. The clones were sequenced using the Big Dye Terminator Kit and ABI 377 sequencer (Applied Biosystems) and the primers were designed using PRIMER3 [http://frodo.wi.mit.edu/primer3/].

Thirty *A. polyneuron* individuals were used for screening and the amplification were carried out in 10 µL reactions containing 2.5 ng of template DNA, 0.3 µM of each primer, 0.25 mM of each dNTP, 1x PCR buffer [75 mM Tris-HCl pH 9.0, 50 mM KCl and 20 mM (NH₄)₂SO₄], 1.5 mM MgCl₂ and 1U TaqDNA polymerase (Bitools); and performed using a MasterCycler® (Eppendorf); 96°C for 4 min followed by 30 cycles (94°C for 40 s, 50-54°C for 1 min and 72°C for 1 min) and 72°C for 7 min. PCR products were denatured and separated on 8% denaturing polyacrylamide gels stained with silver nitrate. Allele sizes were estimated by comparison by 10 bp DNA ladder standard (Invitrogen) and the original DNA used for library development. FSTAT software [http://www2.unil.ch/popgen/softwares/fstat.htm] was used to calculate the genetic parameters per locus, alleles number, the genetic diversity (*H*<sub>e</sub>) and the linkage disequilibrium. The cumulative exclusion probabilities were calculated by CERVUS 3.0 software [4].

**Results and conclusions:** A total of 384 clones were obtained and sequenced. Thirty-nine clones (10.2%) presented microsatellite repeats and 25 were suitable for primers design. The most common repeat motif was GA/CT as expected due the employing technique. Among the loci analyzed, one did not amplify, eight were monomorphic and 16 loci showed polymorphism in this sample. Twenty loci were dinucleotide (83.3%), one was tetra-, other one penta- and two were rare compounds loci: di/tri- (Apn8) and di/tetra- (Apn9). Mendelian inheritance analyses were carried out for each polymorphic locus using one mother tree and their open-pollinated family (26 progeny). All sibs displayed at least one of the maternal alleles, confirming Mendelian inheritance. From a total of 121 alleles presented in the sample, the genetic diversity was high (*H*<sub>e</sub> = 0.65) and the linkage disequilibrium, applying Bonferroni correction for multiple comparisons, was not significant. The total paternity exclusion probabilities over 16 loci were very high for one (*P*<sub>exclu(1)</sub> = 0.9995) or both parents available (*P*<sub>exclu(2)</sub> = 0.9999) indicating that the set is suitable for paternity analysis. Our study provides a new set of variable microsatellite loci for *A. polyneuron* that may be used to estimate genetic parameters such as genetic diversity, population structure, gene flow, and mating system. These markers might also prove transferable for genetic analyses in related taxa.

Financial Support: FAPESP, CNPq, CAPES-PROEX, FAEPA

## References

1. Lorenzi H: Árvores Brazileiras. Plantarum: Nova Odessa, 4 2002, 1:41.

2. Alzate-Marin AL, Guidugli MC, Soriani HH, Martinez CAH, Mestriner MA: An efficient and rapid DNA min preparation suitable for PCR/SSR and RAPD analysis in tropical forrest tree species. Braz Arch Biol Technol 2009, 52:1217-1224.

3. Billotte N, Lagoda PJL, Risterucci AM, Baurens FC: Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits 1999, 54:277-288.

4. Kalinowski ST, Taper ML, Marshall TC: Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 2007, 16:1099-1106.
P8

**Genetic diversity assessed in individuals of *Aspidosperma polyneuron* and *Cariniana estrellensis* used as seed donors in an forest gene bank**

Ana Lilia Alzte-Marin¹, Ronai Ferreira-Ramos²*, Marcela Guidugli¹, Carlos Alberto Martinez², Moacyr Antonio Mestriner¹

¹Departamento de Genética, FMRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil; ²Programa de Pós-Graduação em Biologia Comparada, Departamento de Biologia, FFCLRP/USP, Brazil; ³Departamento de Biologia, FFCLRP/USP, Brazil

E-mail: ronai1977@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P8

**Background:** The neotropical tree species *Aspidosperma polyneuron* and *Cariniana estrellensis*, known as *peroba-rosa* and *jequitibá-branco* respectively, are characteristic of late secondary semideciduous Atlantic forest in submontane formation. Their wood is widely used for carpentry and construction. Increasing logging, intensive agriculture and urban expansion placed *A. polyneuron* in the Red List of Threatened Species [http://www. iucnredlist.org/apps/redlist/details/32023/0] while *C. estrellensis* is considered an endangered species. So, *in situ* conservation of these forest species is critical while *ex situ* collections as gene banks is an important complementary approach. Our study assessed and compared the genetic diversity of *A. polyneuron* and *C. estrellensis* in two stands aiming to provide subsidies for the *ex situ* conservation of these important genetic resources.

**Material and methods:** Leaf tissues of mature trees of two disturbed stands (SI and SII) of *A. polyneuron* and *C. estrellensis* were sampled and stored at -20°C; SI was composed by isolated individuals of an extensive area located among Pardo river and Mogi-Guaçu river basins used as seed donor of a Forest Genetic Bank, at the University of São Paulo in Ribeirão Preto campus (BG-USP/RP), SP-Brazil. SII was one of the last natural populations around Ribeirão Preto region of an isolated fragment of 7.5 ha located on Águas Claras farm in Cravinhos Municipality (21°17'47"S; 47°40'29"W).

DNA extraction was performed according to [1]. SSR markers development for *C. estrellensis*[2] and for *A. polyneuron* (Ferreira-Ramos R., unpublished)) were used for this study (Tables 1,2). Microsatellite loci were amplified individually according [3]. FSTAT software [http://www2.unil.ch/popgen/softwares/fstat.htm] was used to calculate the genetic parameters per locus and sample: mean number of alleles (A), the observed ($H_o$) and expected heterozygosities ($H_e$). Wright's fixation index was calculated as $1 - H_o/H_e$. Deviation from Hardy-Weinberg equilibrium (HWE) was measured using GDA software[http://hydrodictyon.eeb.uconn.edu/people/plewis/software.php].

**Results and discussion:** For both species, the genetic diversity was higher in SI than SII ($H_e$ $_{SI/SII}$= 0.65/0.47, $H_e$ $_{SI/SII}$= 0.72/0.61 for *A. polyneuron* and *C. estrellensis*, respectively). For *A. polyneuron*, the expected heterozygosity ($H_e$) was higher than observed heterozygosity ($H_o$) in SI and SII. For *C. estrellensis* $H_e$ was higher than $H_o$ in SI and the opposite was found in SII. Fixation index was higher in SI than SII for both species ($F_{SI/SII}$= 0.30/0.11, $F_{SI/SII}$= 0.29/-0.05 for *A. polyneuron* and *C. estrellensis*, respectively), suggesting inbreeding. Significant departures from HWE were observed for most loci in both species mainly in SI, which might be due to population substructure (Wahlund effect).

In summary, our study revealed a high diversity for the seed donor trees of *A. polyneuron* and *C. estrellensis* (SI), suggesting that this diversity was incorporated into BG-USP/RP. Indeed future progeny studies will be able to confirm these results. The low diversity found in SII for *A. polyneuron* highlights its threatened situation in this stand, on of the last natural populations in Ribeirão Preto (SP-Brazil). For *C. estrellensis*, a higher genetic diversity was observed which may be due to greater number of trees in the population, preferentially outcrossed mating system and probably gene flow from outside the fragment studied.

**Financial Support:** FAPESP, CNPq, CAPES-PROEX, FAΕΑ

**References**

1. Alzte-Marin AL, Guidugli MC, Soriani HH, Martinez CAH, Mestriner MA: An efficient and rapid DNA min preparation suitable for PCR/SSR and RAPD analysis in tropical forest tree species. *Braz Arch Biol Technol* 2009, 52:1217-1224.

2. Guidugli MC, Campos T, Sousa ACB, Feres JM, Sebbenn AM, Mestriner MA, Contel EPB, Alzte-Marin AL: Development and characterization of 15

microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical tree species. *Conserv Genet* 2009, 10:1001-1004.

P9

**Genetic diversity and divergence applied to Enviromental services for *Araucaria angustifolia* (Brazil)*

Luciano Medina-Macedo¹*, Juliana Bittencourt², Carlos Soccol¹, Andre Lacerda³

¹Biotechnological Processes, Federal University of Parana, Curitiba, Parana, 81531-990, Brazil; ²Federal Technological University of Parana, Ponta Grossa, Parana, 84016-210, Brazil; ³EMBRAPA FLORESTAS, Colombo, Parana, 83411-000, Brazil

E-mail: medinacwb@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):P9

Humans derive many utilitarian benefits from the environmental services of biotas and ecosystems.Yet ecosystem, species and genetic levels is increasingly lost from agricultural landscapes mainly due to maximisation of production [1,2]. The genetic variability is a result of several forces including mutation, recombination and gene flow. Allele frequencies are altered by natural selection and by genetic drift. Whilst mutation, recombination and genetic drift are random and independent processes, natural selection is a directional process towards evolutionary change [3]. The Araucaria Rainforest is one of the most important biomes occurring naturally in southern Brazil. The extensive logging and agricultural expansion became this forest extremely fragmented. Particularly important is the factor of ecosystem resilience, which appears to underpin many of the services. While biodiversity often plays a key role, the services can also derive from biomass and other attributes of biotas, for example, genetic diversity of a particular species. Landscape genetics are being used in this project to improve the understanding of fragmentation impact to assist an environmental services scheme. The objective of this study was to assess by microsatellites markers, the genetic diversity and dynamics in remnant patches of *Araucaria angustifolia* rainforest, with different levels of human modification. The project compared two different forest conditions: one is 1.157,48 ha of continuous forest, and the other is a fragmented forest remnant, with size from 8 ha, appraised 5 km from the continuous forest area. Genetic diversity and divergence of seedlings and adult individuals present in forest fragment was compared with the genetic composition of samples appraised in continuous forest. Cambium and seed material were collected from each recorded tree, and genomic DNA was extracted using the method described in Mazza & Bittencourt (2000). The seeds for DNA extraction had undergone a slight adjustment – Proteinase K was added to megagametophyte (maternal origin) and embryo extraction. Polymerase chain reaction (PCR) conditions used were establish by Qiagen Multiplex Master Mix protocol (1022830) and use fluorescents dye labelling. Two multiplexed systems of microsatellites were applied with the 8 primers. The eight loci used were CRCAc2, Ag23, Ag62, Ag45, CRCAC1, Ag20, Ag56 and As90. Following PCR this dilution was running in ABI sequencer 3100. Gene Scan and Genotyper software were used for data collection and alleles analysis. The average number of alleles per locus among adult trees in the continuous forest was 8.10, compared to 5.15 appraised in forest fragment. The means of expected heterozygosity were 0.623 for continuous forest and 0.579 for forest fragment, while observed heterozygosity range was respectively, 0.571 and 0.723. Subpopulations of forest fragments were more distinguished than subpopulations of continuous, due the average fixation index was 0.082 for subpopulations in the continuous forest and 0.210 in fragment. It is worth noting that the levels of genetic differentiation among all subpopulations can be considered to be high. Paternity analysis, within the continuous forest indicated that 48% of offspring were fertilized by pollen from trees outside the plot site. The average pollination distance within the continuous was 95 m. In the trees from forest fragment, the analysis showed that 42% to 65% of the offspring was fertilized by pollen from trees outside fragment. The effective number of pollen donors in the continuous forest ranged among seed-trees from 2 to 10, and in the fragment from 2 to 6. The results suggest high pollen
dispersal distance in both conditions and an absence of reproductive isolation. They also show high pollen immigration and dispersal distance among the tree groups. The results suggest that fragmentation increases divergence in *Araucaria angustifolia*. The population in continuous forest showed higher genetic diversity in the adult population than the population of trees in fragment. The reducing the heterozygosity were low, may be due the recent forest fragmentation history. There are more inbreeding in fragmented population than in continuous population. Fragmentation increased the genetic divergence among the fragmented population. However, the genetic results indicated the presence of long-distance dispersal leading to functional connectivity between isolated forest fragment. The survival of remnants of *Araucaria angustifolia* patches as well as single trees in the agricultural landscape is key factors for species conservation and could be applied to environmental payment services, these strategy for *A. angustifolia* is an integration of conservation strategies across reserves and the surrounding matrix, including productive agricultural areas, to assist gene flow movement between temporally suitable habitats.

**Acknowledgements:** EMBRAPA Florestas, UTFPR Campus Ponta Grossa and Post Graduate Program in Biotechnological Processes UFPR

## References

1. Millennium Ecosystem Assessment. *Ecosystems and human well-being: General synthesis* Washington, DC: Island Press 2005.

2. Swinton SM, Lupi F, Robertson GP, Landis DA: Ecosystem services from agriculture: looking beyond the usual suspects. *American Journal of Agricultural Economics* 2006, 5:1160-1166.

3. Lindenmayer DB, Franklin JF: Conservation forest biodiversity: comprehensive multiscale approach. Washington: Island Press 2002.

## P10

### Microsatellite variability between apricot and related Prunus species

Fatemeh Maghuly*, Margit Laimer*

Plant Biotechnology Unit (PBU), Dept. Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria

E-mail: m.laimer@iam.boku.ac.at

BMC Proceedings 2011, 5(Suppl 7):P10

**Background:** Apricots, family *Rosaceae*, are economically important representatives of the genus *Prunus*. The number of apricot species ranges from three to ten, depending on the classification system adopted. The major obstacles to expansion of apricot production are irregular yields and low resistance to diseases. Compared with the high genetic variability in related species, apricots are not so variable and thus interspecific hybrids were proposed as a means to overcome deficiencies inherent in the common apricot. To estimate the extent of variation in apricot germplasm, between ecogeographical groups and related species,

is very useful for planning breeding programmes, through diversity analysis, cultivar identification or marker-assisted selection.

**Methods:** One hundred accessions of *P. armeniaca* representing the European, Irano-Caucasian, Central Asian and North American genepool, three accessions of *P. mandshurica* and one accession each of *P. ansu*, *P. brigantiaca*, the interspecific hybrid Plumcot, *P. x dasycarpa*, *P. mume*, *P. siberica* were analysed with 10 apricot SSR loci on an ABI 3100 capillary sequencer [1]. A total of 196 alleles were detected varying from 11 to 27 with an average of 16.9. Out of them, 59 alleles occurred only once (so-called private alleles). 34 (58%) private alleles were also found in related species. Non-amplified alleles were observed in four samples of the related species (*P. siberica* and *P. brigantina*). *FST* values ranged from 0.5121 to 0.3503, with an average of 0.4459 (Table 1). It is known, that *FST* values up to 0.05 indicate negligible genetic differentiation whereas >0.25 means very great genetic differentiation within the population analyzed. In *Prunus*, high levels of genetic differentiation could be explained by the mating system. The data indicate a low gene flow (as described by Nm; Table 1). These results could be attributed to different geographic origins of the species or the influence of the breeding strategy.

**Results and conclusions:** Genetic similarity among common apricots and related species was quantified using Nei's [2] genetic distance and genetic identity based on allele frequencies (data not shown). The lowest genetic identity (0.000) was found among *P. mandshurica*, *P. brigantina*, *P. mume* and *P. dasycarpa*, and between Plumcot and *P. mume*. The highest genetic identities (0.81) were found between Western European and North American accessions and among Irano Caucasian and Eastern European groups. To demonstrate the genetic relationship between common apricots from different ecogeographical regions and related species, a neighbor joining dendrogram based on genetic distance was produced (Figure 1). The accessions are divided into two groups, one containing all common apricots and hybrids thereof, and the other containing all related species. In general, results show that the common apricots are remote from related species. The tree supports that the *P. x dasycarpa* and Plumcot, known to be a *P. armeniaca* hybrid, are intermediates between common apricot and other related species. *P. ansu* and *P. siberica* appear distant from the others accessions, and in fact they are the most distantly related species. It is interesting to know that some authors even consider *P. ansu* as a separate species [3]. In this study *P. siberica*, the species having the largest distribution area of all apricot species, and *P. mandshurica*, being present in the very cold area, cluster together. In the analyses they appear far from the common apricot species, which confirms them as being markedly different from *P. armeniaca*. Maghuly et al. [1] described that *P. x dasycarpa*, *P. brigantiaca* and Plumcot were distant from the common apricot cluster. In fact, *P. brigantiaca* is the most distantly related species, while *P. x dasycarpa*, a hybrid between *P. armeniacax* *P. cerasifera*, was found intermediate between the apricot groups and Plumcot, which is a hybrid between *P. armeniaca* and *P. salicinia*.

<table><caption>**Table 1(abstract P10) Variability parameters calculated for 10 SSR markers in 100 apricot cultivars origin of five ecogeographical region and related species using POPGENE**</caption><thead><tr><th>Locus</th><th>Number of putative alleles</th><th>Effective alleles per locus(Ne)</th><th>Observed heterozygosity (Ho)</th><th>Expected heterozygosity (He)</th><th>Inbreeding coefficient (F<sub>ST</sub>)</th><th>Gene flow (Nm)</th></tr></thead><tbody><tr><td>SsrPaCITA7</td><td>18</td><td>4.6280</td><td>0.8091</td><td>0.7875</td><td>0.4252</td><td>0.3380</td></tr><tr><td>ssrPaCITA10</td><td>21</td><td>4.4906</td><td>0.5545</td><td>0.7809</td><td>0.4461</td><td>0.3105</td></tr><tr><td>ssrPaCITA19</td><td>14</td><td>4.1052</td><td>0.7636</td><td>0.7599</td><td>0.5121</td><td>0.2382</td></tr><tr><td>ssrPaCITA23</td><td>11</td><td>4.5846</td><td>0.5872</td><td>0.7855</td><td>0.4951</td><td>0.2550</td></tr><tr><td>ssrPaCITA27</td><td>12</td><td>2.9979</td><td>0.3738</td><td>0.6696</td><td>0.6130</td><td>0.1578</td></tr><tr><td>UDAp-407</td><td>27</td><td>7.2979</td><td>0.7182</td><td>0.8669</td><td>0.3823</td><td>0.4039</td></tr><tr><td>UDAp-410</td><td>15</td><td>5.5415</td><td>0.8440</td><td>0.8233</td><td>0.4493</td><td>0.3064</td></tr><tr><td>UDAp-414</td><td>20</td><td>5.3862</td><td>0.5000</td><td>0.8181</td><td>0.3503</td><td>0.4636</td></tr><tr><td>UDAp-415</td><td>15</td><td>3.7162</td><td>0.6273</td><td>0.7342</td><td>0.4072</td><td>0.3640</td></tr><tr><td>UDAp-420</td><td>16</td><td>3.5567</td><td>0.5909</td><td>0.7221</td><td>0.4102</td><td>0.3594</td></tr><tr><td>Mean</td><td>16.9</td><td>4.6305</td><td>0.6369</td><td>0.7748</td><td>0.4459</td><td>0.3107</td></tr><tr><td>St.Dev</td><td>4.77</td><td>1.2233</td><td>0.1471</td><td>0.0565</td><td>-</td><td>-</td></tr></tbody></table>
<figure><img src="image_23.png" /><figcaption>**Figure 1 (abstract P10)** Neighbor joining dendrogram for apricots from five ecogeographical groups and related species.</figcaption></figure>

Results to date indicate that crosses between apricot and apricot related species are successful, when made in either direction and the resulting hybrids are viable [3]. In this manner, *P. mandshurica* and *P. siberica* were used in common apricot breeding as a source of cold hardiness [4]. Likewise, adaptation to humid climates should be easy to transmit through hybridization with *P. mume* and *P. armeniaca* var. *ansu*. In addition, Rubio et al. [5] suggested *P. manchurica* as the possible origin of the apricot cultivars resistance to Sharka. It should be emphasized that in the interest of still further extending the genetic diversity available for posterity, additional efforts should be committed to the systematic exploration for unique phenotypes of apricots, and also of related species.

## References

1. Maghuly F, Borroto Fernandez E, Ruther S, Bistray G, Pedryc A, Laimer M: Microsatellite variability in apricots (Prunus armeniaca L.) reflects their geographic origin and breeding history. *Tree Genetics and Genomes* 2005, 1:151-165.

2. Nei M: Estimation of average heterozygosity and genetic distance from a small number of individuals. *Genetics* 1978, 89:583-590.

3. Mehlenbacher SA, Cociu V, Hough F: *Apricot* (*Prunus*). *Genetic resource of temperate fruit and nut crops* ISHS Wageningen: Moore JN and Ballington RJ 1991, 65-107.

4. Paunovic SA: Apricot germplasm, breeding, selection, cultivar, rootstock and environment. *Acta Hort* 1988, 209:13-28.

5. Rubio M, Dicenta F, Martínez-Gómez P: Susceptibility to sharka (*Plum pox virus*) in*Prunus mandshurica* x *Prunus armeniaca* seedlings. *Plant Breeding* 2003, 122:465-466.

P11

# Development of microsatellite molecular markers and genetic diversity in *Hevea Braziliensis*

Camila Campos Mantello¹*, Fernando Issamu Suzuki¹, Livia Moura Souza¹, Paulo de Souza Gonçalves², Anete Pereira de Souza³,¹

¹Molecular Genetic Analysis Laboratory - Molecular Biology Center and Genetic Engineering (CBMEG), UNICAMP, Campinas, São Paulo, 13083-875, Brazil; ²Agronomy Institute of Campinas (IAC), Campinas, São Paulo, 13012-970, Brazil; ³Molecular Genetic Analysis Laboratory - Molecular Biology Center and Genetic Engineering (CBMEG), UNICAMP, Campinas, São Paulo, 13083-875, Brazil and Department of Botany, Biology Institute, UNICAMP, Campinas São Paulo, 13083-970, Brazil

E-mail: camila.mantello@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P11

**Background:** The rubber tree [*Hevea Braziliensis* (Willd. ex Adr. De Juss.) Muell-Arg.] is native from the Amazon region which has great economic importance for being the largest source of natural rubber in the world. Although Brazil is the center of origin and genetic diversity of this culture, the country is currently responsible for only 1% of the world production. Besides offering optimal weather conditions for crop development, the Amazon rainforest region is also favorable to the development of the fungus known as SALB (South American leaf blight), which causes the disease-of-leaves. Leaves fall sharply reducing the production of latex, limiting the production of rubber in this region. Thus breeding programs are seeking for clones which are resistant to this fungus and with high production in escape regions, which provide stress conditions such as low temperatures, high altitude, wind and other diseases [1]. The rubber tree is perennial and requires about 30 years to obtain an improved variety, starting from the controlled pollination of a clone to the final recommendation. Molecular markers such as microsatellites (Simple Sequence Repeats, SSRs) are an important tool for diversity studies and potentially to assist breeding programs. This study aimed to develop an enriched microsatellite library for *H. Braziliensis*, characterize these developed microsatellite markers and test the transferability of these markers to six other species of the genus *Hevea*.

**Material and methods:** For this study we used 36 accessions of *H. Braziliensis* donated by the Agronomy Institute of Campinas and one accession of each of six other species of genus *Hevea* (*H.nitida*, *H. pauciflora* (2), *H.camargoana*, *H.guianensis*, *H.rigidifolia* and *H.benthamiana*) provided by Embrapa. Genomic DNA samples were extracted from lyophilized leaf tissues using a modified CTAB method [2]. Trinucleotide and dinucleotide enriched genomic libraries for *H. Braziliensis* were constructed. The DNA samples were digested with AFAI and enriched using (CT)8 and (GT)8 biotinylated microsatellite probes for the dinucleotide library and (ATC)8 and (CCT)8 for the trinucleotide library. The clones obtained were sequenced and the sequences were evaluated with the Microsat program, which removes parts of the vector and the adapters and verifies the presence of restriction site within the sequence. After this step, the sequences were aligned and edited using the program SeqMan (DNAStar Inc.), which also allows analyzing the redundancy of the library. The identification of microsatellites was performed using a research tool SSRs SSRIT – "The Simple Sequence Repeat Identification Tool" avaialable at Gramene [http://www.gramene.org]and primers complementary to sequences flaking the microsatellites were designed by Primer Select Program (DNAStar Inc) and Primer 3. Amplification tests were made from a temperature gradient to know the annealing temperature and the products were evaluated and resolved on 3% agarose gels stained with ethidium bromide and in denaturing 6% polyacrylamide and silver stained [3]. The loci were characterized on the number of alleles per locus, allele frequency and the Polymorph Information Content (PIC). It was also made analysis of ancestry for de accessions of *H. Braziliensis* using the program Structure v 2.3.3 [4].

**Results and discussion:** A total of 384 clones from the dinucleotide library were sequenced while 288 from the trinucleotide library resulting in 133 and 62 microsatellites in each library respectively. It was possible to design 55 and 32 primer pairs for the dinucleotide and trinucleotide libraries respectively. Of the 87 microsatellite loci designed 69 were characterized. The maximum number of alleles per locus was 17 and eight loci were monomorphic. The PIC values ranged from 0.83 to 0.06, the observed and expected heterozygosity ranged from 0.86 to 0.06 to 0.90 and 0.06 respectively. A population analysis divided the 36 accessions into two groups. The cross-amplification of these microsatellites loci was tested in six other species of the genus *Hevea*. The percentage of cross-amplification in others species of *Hevea* ranged from 94.2% to 82.6%, with *H. nitida* with the highest percentage of transferability and *H. carmagoana* with the lowest one. This high percentage indicates that the regions flaking the microsatellites are well conserved between species of *Hevea*. These markers will be useful to breeding and conservation studies of *H. Braziliensis* and related species.

**Acknowledgments:** Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Agronomy Institute of Campinas (IAC).

## References

1. Pushparajah E: Problems and potentials for establishing Hevea under difficult environmental conditions. *The Planter* 1983, 59:242-251.

2. Doyle JJ, Doyle JL: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. *Phytochem Bull* 1987, 19:11-15.
3. Creste S, Tulmann A, Figueira A: Detection of Single Sequence Repeat Polymorphism in denaturing Polyacrylamide Sequencing Gels by Silver Staining. *Plant Molecular Biology Reporter* 2001, **19**:299-306.

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

## P12
### Phylogeography of the disjunct *Schizolobium parahyba* (*Fabaceae-Caesalpinioideae*)

Rogerio Margis¹*, Andreira Turchetto-Zolet¹, Fernanda Cruz¹,
Fabiano Salgueiro², Giovanni Vendramin³, Marcelo Simon⁴, Stephen Cavers⁵,
Marcia Margis-Pinheiro¹

¹Universidade Federal do Rio Grande do Sul, Brazil; ²Universidade Federal do Estado do Rio de Janeiro, Brazil; ³Plant Genetics Institute – NRC, Brazil;

⁴EMBRAPA, Brazil; ⁵Center Ecology and Hidrology, Brazil

E-mail: rogerio.margis@ufrgs.br

BMC Proceedings 2011, 5(Suppl 7):P12

This work aims to analyze the phylogeography of *Schizolobium parahyba* (*Fabaceae*), which includes two varieties with a disjunct distribution, from Southern Brazil to Central America. Neotropical rain forests, focus on four largest wet forests: Atlantic forest, Amazonian forests, Andean forest and Central America forest. The genetic diversity and differentiation of populations among *S. parahyba* populations using sequences of three cpDNA regions (*psbA-trnH*, *trnL-trnFandmatK*) and one nrDNA region (ITS) were analyzed. The presence of the significant phylogeographic structure was inferred by testing if $G_{ST}$ and $N_{ST}$ were significantly different and a spatial analysis of molecular variance was made with both markers. Using cpDNA (*matK*) sequences of the *S. parahyba* and other Fabaceae species and fossils, we estimated de divergent time for *Schizolobium* clade and using the average ITS substitution rate reported for a range of woody plants, we estimated the divergence time between the two varieties. The high levels of genetic diversity in some populations of *S. parahyba* and two centres of genetic diversity that correlate with the two known varieties: one in the southeast Atlantic forest, and the other in the Amazonian basin. In contrast, the populations from Northeast Atlantic forest and Andean-Central America forests showed low level of genetic diversity and divergent haplotypes, probably because the founder effect after dispersion. The results suggest dispersion from southeast Atlantic forest to Amazonian, Andean and Central America forests. We verify high level of genetic structure, with 68% (nrDNA) and 82% (cpDNA) of the total genetic diversity due to differences among populations. Twenty-one haplotypes were found with cpDNA and four with nrDNA and no haplotypes were shared between varieties. The age for *Schizolobium* clade using *matK* sequences was estimated ranging from 8.4-23.0 My and the divergence between two varieties using ITS sequence variation was of the 6.5 My. In conclusion, the variation pattern of cpDNA (maternally inherited) and nrDNA (biparentally inherited) markers provides different insights into the phylogeographic structure and gene flow in *S. parahyba*. This comparative analysis of cpDNA and nrDNA markers can help a deeper understanding of the dynamics responsible for both ancient and more recent events that have shaped the current distribution of genetic variability in Neotropical plants. The results are relevant to conservation efforts and ongoing work on the genetics of population divergence and speciation in these Neotropical rainforests. Also, for the long-term conservation of the genetic diversity of *S. parahyba*, including the divergent lineages of the two varieties, it would be important to design strategies that aim to preserve most of its lineages.

## P13
### Genetic divergence in Cork Oak based on cpDNA sequence data

Joana Costa¹, Célia Miguel²*, Helena Almeida³, Margarida M Oliveira²,
José A Matos⁴, Fernanda Simões⁴, Manuela Veloso⁵, Pinto C Ricardo²,
Octávio S Paulo¹, Dora Batista⁶

¹Faculdade de Ciências da Universidade de Lisboa/Centro de Biologia Ambiental (CBA), Computational Biology and Population Genomics Group (http://cobig2.fc.ul.pt/), Edificio C2, Campus da Faculdade de Ciências, 1759-016 Lisboa, Portugal; ²Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL)/Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, 2780-501 Oeiras, Portugal; ³Universidade Técnica de Lisboa, Instituto Superior de Agronomia (ISA), Centro de Estudos

Florestais, Tapada da Ajuda, 1349-017 Lisboa, Portugal; ⁴Instituto Nacional de Recursos Biológicos, I.P. (INRB), L-INIA - Pólo do Lumiar, Unidade Investigação Recursos Genéticos, Ecofisiologia e Melhoramento de Plantas, Grupo Biologia Molecular, Estrada Paço Lumiar 22, Ed.S 1º, 1649-038 Lisboa, Portugal; ⁵INRB, I.P., L-INIA - Pólo de Oeiras, Unidade Recursos Genéticos, Ecofisiologia e Melhoramento de Plantas, Quinta do Marquês, 2784-505 Oeiras, Portugal; ⁶Instituto de Investigação Científica Tropical IICT/CIFC, Quinta do Marquês, 2784-505 Oeiras, Portugal

E-mail: cmiguel@itqb.unl.pt

BMC Proceedings 2011, 5(Suppl 7):P13

**Background and objectives:** Cork oak (*Quercus suber* L.) is one of the dominant broadleaved woody species in the western Mediterranean Basin, defining unique open woods. These woodlands have an outstanding economical and ecological value in this region, particularly in Portugal, where they sustain a strong cork industry. In the context of a prospective management of these sustainable ecosystems and renowned reservoirs of biodiversity, it is vital to better understand how the genetic variation of *Q. suber* natural populations is spatially organized so reasonable guidelines for conservation can be provided. On the other hand, knowledge of how past climate fluctuations influenced the patterns and dynamics of *Q. suber*, shaping the ranges of the species in the Mediterranean peninsulas, is of the utmost importance for our perception of what can happen in the future. Although a great deal of details on the genetic divergence of the Mediterranean cork oak populations has been uncovered and several hypotheses have been advanced concerning its evolutionary history, there is still much to unravel. For instance, Portuguese natural population sampling included so far in previous studies has been very deficient. To achieve this goal a different and complementary analysis of cork oak's genetic diversity was initiated under a phylogeographical framework based on chloroplastidial DNA sequences. This study is the starting up of a project aiming at assessing the genetic diversity and differentiation of natural cork oak populations from the entire Mediterranean distribution, with the intent of understanding patterns of biodiversity, gene flow and population admixture, as well as to infer possible evolutionary events.

**Materials and methods:** We used 3-5 samples from 25 populations collected across the range of distribution of cork oak, in a total of 115 individuals. Three chloroplastidial DNA (cpDNA) intergenic spacer regions (*TrnL-F* [1], *TrnS-PsbC* [2] and *TrnH-psbA* [3]) were amplified and sequenced. Fourteen individuals of *Q. rotundifolia*, five of *Q. coccifera*, two of *Q. ilex*, and one of *Q. robur*, *Q. faginea*, *Q. pyrenaica*, *Q. lusitanicus*, *Q. rubra* and *Q. canariensis* were also analyzed as comparative references. *Castanea crenata* was used as outgroup. The alignment was developed in Clustal X 2.0.12 [4] and manually refined. The phylogenetic trees were made with PAUP 4.0d99 [5], using a Maximum Parsimony analysis method.

**Results:** Regarding the cpDNA regions under study, two main lineages of cork oak haplotypes were found: one lineage closely related to *Quercus rotundifolia* and *Q.coccifera*, ("introgressed lineage"), mainly present in Iberia and Morocco; and a second lineage that contrastingly seems "pure", which is the most common and does not appear to be shared with any other *Quercus* species ("pure lineage"). Three distinct sublineages are shown in the "pure" lineage, corresponding to the Eastern populations, Sicily and the Western populations, although separated by minor differences. Some populations present cpDNA haplotypes belonging to both lineages, while others are specific for one of them. An absence of introgression in the easternmost populations of cork oak seems also apparent.

**Conclusions:** Although these preliminary results seem to confirm previous ones, some major differences are suggested, such as too few differences between the western and eastern groups to be explained by a Tertiary divergence pattern, as previously suggested [6], but rather more consistent with a more recent expansion from few refugia; and widespread and multiple introgression events from *Q. rotundifolia* and *Q. coccifera*, common in peripheral western populations.

**Acknowledgements:** Fundação para a Ciência e a Tecnologia (FCT) is acknowledged for financial support, through project PTDC/AGR-GPL/104966/2008.

### References

1. Taberlet P, Gielly L, Pautou G, Bouvet J: Universal primers for amplification of three non-coding regions of chloroplast DNA. *Plant Mol Biol* 1991, **17**:1105-1109.
2. Nishizawa T, Watano Y: Primer pairs suitable for PCR-SSCP analysis of chloroplast DNA in angiosperms. *J Phytogeo Taxon* 2000, **48**(1):67-70.

3. Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DA: Use of DNA barcodes to identify flowering plants. *PNAS* 2005, **102**(23):8369-8374.

4. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. *Nucl Acids Res* 1997, **25**(24):4876-4882.

5. Swofford DL: PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods*). Version 4. Sunderland, Massachusetts: Sinauer Associates 2002.

6. Magri D, Fineschi S, Bellarosa R, Buonami A, Sebastiani F, Schirone B, Simeone MC, Vendramin GG: The distribution of *Quercus suber* chloroplast haplotypes matches the palaeogeographical history of the western Mediterranean. *Mol Ecol* 2007, **16**:5259-5266.

P14

Tracking down worldwide *Puccinia psidii* dispersal

Rodrigo Neves Graca¹*, Amy Ross-Davis², Ned Klopfenstein², Mee Sook³,

Tobin Peever⁴, Phil Cannon², Janice Uchida⁵, Acelino Couto Alfenas¹

¹Universidade Federal de Vicos, Brazil; ²USDA Forest Service, USA; ³Kookmin

University, South Korea; ⁴Washington State University, USA; ⁵University of

Hawaii at Manoa, USA

E-mail: mevesgs@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P14

**Background:** *Puccinia psidii* causes rust disease on many host species in the Myrtaceae [1]. First reported in 1884 on guava in Southern Brazil [2], the rust has since been detected on several myrtaceous in South America, Central America, Caribbean, Mexico, USA: in Florida, California, and Hawaii. More recently, *P. psidii* was reported in Japan infecting *M. polymorpha* [3]. Of special note is that a rust was found infecting Myrtaceae species in Australia, where the fungus was reported as *Uredo rangelii*, based on the tonsure found on the urediniospores surface. However, DNA sequence data did not differentiate that rust from *P. psidii* [4], and the same tonsure patch, was also observed on rust urediniospores collected from several host species in Brazil [unpublished data]. We have hypothesised that *P. psidii* was introduced into Hawaii through California by trade of rust infected myrtaceous plants, and that *P. psidii* populations from South America are distinct from the rust populations that became established in California and Hawaii.

**Material and methods:** Fast-evolving microsatellite markers were used to determine genetic relationships among rust populations from South America, California and Hawaii. Such genetic markers allow inferences about the potential sources of pathogen introduction.

**Results:** The eight microsatellite loci analyzed revealed 14 multilocus genotypes (MGs) within the 221 *P. psidii* isolates. Isolates collected on seven different hosts in South America presented distinct MGs. In contrast, all rust isolates collected on nine myrtaceous hosts in the Hawaiian Islands were composed of only a single, unique MG. The same unique MG observed in Hawaii, was also detected on isolates from two different hosts in California, indicating a common origin of the rust genotype found in Hawaii and California. The isolates were grouped using a principal coordinates analysis (PCA). The first two axes of the PCA plot explained 89% of the total observed variation, with the first axis explaining 50% and the second 39%. According to the PCA, the *S. cumini* group is the South American group that is most closely related to the Hawaiian and Californian groups.

**Conclusions:** The MG comprising all isolates from Hawaii and California is distinct from the MGs found in South America so far, suggesting that the Hawaiian and Californian isolates did not come directly from South America. Isolates from Florida, Central America, and the Caribbean must be analyzed to better understand potential relationships with pathogen dispersion to Hawaii.

References

1. Farr DF, Rossman AY: Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. 2010 [http://nt.ars-grin.gov/fungaldatabases/].

2. Winter G: Repertorium. Rabenhorstii fungi europaei et extraeuraopaei. *Hedwigia* 1884, 23:164-175, Centuria XXXI et XXXII.

3. Kawanishi T, Uematsu S, Kagiwada S, Hamamoto H, Horie H, Namba S: First report of rust disease on ohia and the causal fungus, *Puccinia psidii*, in Japan. *J Gen Plant Pathol* 2009, **75**:428-431.

4. Carnegie AJ, Lidbetter JR, Walker J, Horwood MA, Tesoriero L, Glen M, Priest MJ: *Uredo rangelii*, a taxon in the guava rust complex, newly recorded on Myrtaceae in Australia. *Australasian Plant Pathology* 2010, **39**:463-466.

P15

Genetic diversity of *Salix koreensis* based on inter-simple sequence repeat (ISSR) in South Korea

Jae In Park¹*, Go Eun Choi¹, Jae Ik Nam¹, Young Mi Kim²

¹Chungbuk National University, Korea; ²Chungbuk National University, Korea

Forest Research Institute, Korea

E-mail: jipark@chungbuk.ac.kr

BMC Proceedings 2011, 5(Suppl 7):P15

**Background:** *Salix koreensis* native to Korea, Japan and China, is a deciduous broadleaved tall tree growing throughout Korea. This species is dioecius and planted along the streets and for landscaping. Along the rivers the species grows well reaching up to 20 meters in height. Trees bloom in April and fruits ripe in May. This study was carried out to investigate the genetic diversity of *S. koreensis* within and among 10 populations throughout South Korea using ISSR marker analysis.

**Materials and methods:** From 10 populations identified throughout the country 8 to 11 individual trees per population were sampled, totalling 84 individual trees. Twigs with leaves were collected and used as material for genetic analysis. DNA extraction was performed using GENE ALL plant SV DNA purification Kit (Gean all, Korea) and amplified by PCR using 5 UBC (University of British Columbia) primers (#807, #834, #842, #856, #881) which showed good results in previous experiments. PCR products were electrophoresed and gels photographed. Amplified fragments were visually scored as present (1) or absent (0). Observed and effective numbers of alleles, Shannon's information index, number of polymorphic loci, Nei's genetic distance were estimated using POPGENE 3. and genetic distance was analysed using UPGMA.

**Results and conclusions:** The overall number of polymorphic ISSR amplicons was 80 and mean per primer was 16. The percentage of polymorphic loci was as follows: Namwon population had the highest level (61.25%), Seomjin river population had the lowest level (37.50%) and the overall mean was 48%. Shannon's Information Index indicating genetic diversity ranged from 0.17 (Seomjin river population) to 0.28 (Hanam population) with a mean of 0.24. Shannon's information index of this species was lower than the one found in other species. This result is likely due to the fact that *S. koreensis* is frequently propagated by asexual reproduction. The UPGMA dendrogram based on Nei's genetic distance did not show any clear geographic pattern. The Seomjin river population with low genetic diversity was genetically more distant from the other populations and the Upo population was the next more distant one. Hanam and Yangchon populations were the ones more genetically related.

P16

Development and characterization of microsatellite loci in *Grevillea robusta*

Camila Mantello¹, Daiane Rigoni Kestrings²*, Valderês Aparecida Sousa²,

Ananda Virginia Aguiar², Anete Pereira Souza¹

¹UNICAMP, Brazil; ²Embrapa Florestas, Brazil

E-mail: drigoni@cnpf.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P16

**Background:** *Grevillea robusta* is a native tree to the subtropical coastal regions of northern New South Wales and southern Queensland in Australia. In Brazil, Embrapa Forestry and its partners have established many provenance/progeny tests to increase the species genetic basis and aim to implement a breeding program to improve timber production. Genetic variability of these tests has been monitored through the assessment of quantitative traits. However, the genetic evaluation of materials based on phenotypic traits is influenced by many environmental factors. Estimates of some genetic parameters such as gene flow and
parentage are possible using molecular tools as most common molecular
marker, microsatellite (Simple Sequence Repeats, SSRs) which are
codominant and highly polymorphic. Genetic markers have intensively
applied for the main strategies in breeding programs, especially when
economically important traits are difficult measure because of low
heritability. The aim of this study was develop microsatellite markers for
*Grevillea robusta* through enriched library in order to estimate the genetic
diversity and structure of the species, and direct efforts for the
conservation and management of its active germplasm banks.

**Methods:** The genomic-enriched library was constructed following the protocol described by [1]. The genomic DNA was digested with AFAI and enriched in (CT)₈ and (GT)₈ repeats. Enriched fragments were amplified by polymerase chain reaction (PCR), connected to a pGEM T-easy vector and transformed into competent XL1- blue *Escherichia coli* cells. The positive clones were selected using the B-galactosidase gene and then grown overnight in an HM/F medium with ampicillin. After PCR, 95 positive clones were sequenced in both directions using the T7 and SP6 primers as well as the Big Dye terminator Kit. The sequences were assembled and edited in Seqman (DNAStar) and the repetitive regions were found using the Simple Sequence Repeat Identification Tool [2]. Primer Select (DNAStar) and Primer 3 plus were softwares used to design primer pairs flanking the microsatellite regions.

**Results and conclusions:** Of the ninety five sequences cloned, seven contained microsatellite sequences. Only five of them showed repeats and adequate flanking regions for primer design. The observed proportion of trinucleotide was 4.21% (4), while tetranucleotide and pentanucleotide were 2.11% (2) and 1.05% (1), respectively. Of the total of nucleotides found 100% are simply perfect. The explanation for this low yield sequences (7,4%) can be attributed to the genomic-enriched

procedure. These sequences will be validated and promptly used to
estimate the genetic diversity, gene flow and parentage of the Brazilian
germoplasm collections.

**References**

1. Billote N, Risterucci AM, Baurens FC: Microsatellite enriched libraries: applied methodology for the development of SSR markers in tropical crops. *Fruits* 1999, 54:277-288.

2. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S: Computational and experimental analysis of microsatellites in rice (*Oryza sativa* L.): frequency, length variation, transposon associations, and genetic marker potential. *Genome Research* 11:1441-1452.

P17

Phylogeographic patterns of *Calophyllum Braziliense* Camb.
(*Calophyllaceae*) based on the psbA-trnH cpDNA locus

Fabiano Salgueiro¹, Jordana Neri², Marcio Alves-Ferreira³, Fabio Scarano⁴

¹Departamento de Botânica, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; ²Programa de Pós-Graduação em Botânica, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, Brazil; ³Departamento de Genética, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; ⁴Departamento de Ecologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

E-mail: fabiano.salgueiro@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P17

**Background:** Past climate changes have severely influenced the current distribution of species and their genetic diversity. Phylogeography is the study of the principles and processes governing the geographic

<figure><img src="image_24.png" /></figure>
distributions of genealogical lineages [1]. South America has the world's largest area of swamps, floodplains and wetlands in general [2]. Brazil's major wetlands cover 2% of the country's huge territory [3]. However, from a plant ecology standpoint, Brazilian freshwater wetlands are largely unknown and the scarce data available refer mostly to flooded forests of the Amazon [4]. *Calophyllum Braziliense* Camb. (Calophyllaceae), also known as guanandi, jacareúba or landim, is a canopy tree species typical of waterlogged areas from South and Central America. This species occurs in the humid tropical forests of Central America, Amazon Forests, Atlantic Forest (including restingas); and in the riverines forests of the Cerrado biome (brazilian savannah) [5,6]. However, unlike other species typical of flooded areas, *C. Braziliense* shows none of the morphological features common to flood-adapted plants. *Calophyllum Braziliense* is a hermaphroditic tree pollinated by bees. Its seeds are animal (mainly by bats) or water dispersed. The timber has excellent characteristics and is widely used. *Calophyllum Braziliense* is also used in vegetation restoration programs and its leaves extract presents anti-inflammatory activity [7]. This study examines the phylogeographic patterns of *C. Braziliense* based on the cpDNA intergenic region *psbA-trnH*.

**Methods:** Twenty four populations of *C. Braziliense* were sampled from Costa Rica (10° 12'N, 83° 47'W) to the Paraná State in Brazil (25° 34'S, 48° 27'W). Samples were collected from about 5-10 adult trees in each population, totaling 192 individuals. Total DNA was extracted from leaves or cambium using the CTAB procedure described by Doyle & Doyle [8]. After a screening for cpDNA amplification and polymorphism in *C. Braziliense*, the *psbA-trnH* intergenic regions was selected [9]. Sequences were aligned using CLUSTAL-W implemented in the MEGA 4 software. The cpDNA haplotypes were defined by analyzing the sequences with DNASP 4.01. The genetic diversity indexes were estimated in ARLEQUIN 3.01. The phylogenetic relationships among the haplotypes were estimated using the median-joining algorithm implemented in NETWORK 4.1. An analysis of molecular variance (AMOVA) was performed in ARLEQUIN. A spatial analysis of molecular variance (SAMOVA) was conducted using the SAMOVA 1.0 software. To evaluate the hypothesis of population expansion, neutrality tests were computed in DNASP and ARLEQUIN.

**Results:** A total of 263 aligned positions were obtained for the *psbA-trnH* locus. Twenty-eight variable characters were analyzed resulting in seven cpDNA haplotypes (Figure 1). The haplotype diversity (*h*) for each population ranged from 0.0 to 0.533 and the nucleotide diversity (*π*) from 0.0 to 0.01882. Similar levels of genetic diversity were observed for other tropical species [10,11]. Most of the Atlantic rain forest populations (13/14) are monomorphic and present the same haplotype (H1). Generally, the remaining populations present different private haplotypes. Spatial analysis of molecular variance (SAMOVA) identified seven phylogroups (*k*=7, *Fct* = 0,926), one consisting of 13 monomorphic populations from the Brazilian Atlantic rain forest and more three populations from de Cerrado biome. Most of the others populations constitute different phylogroups. Neutrality tests suggest expansion for the Atlantic rain forest populations. The AMOVA analysis reveals that most of the variation was found between populations (0.8788, *p*<0.00005). A great genetic distance was observed between Central America (Costa Rica) and the others populations, even from the Brazilian Amazon forest. Similar results were observed when *Swietenia macrophylla* (mahogany) populations from Central and South America were compared [10].

**Conclusions:** The genetic data obtained here for *C. Braziliense* based on chloroplast DNA diversity indicate a recent expansion for the Atlantic rain forest populations. Our results suggest that the Northeast of Brazil maintained large populations during the last glacial period and that the Southeast and South populations may have undergone a pronounced retraction process, followed by a recolonization process with a strong founder effect. Thus, the recolonization of the South and Southeast region of the Atlantic rain forest probably occurred from these more stable areas in the Northeast.

**Acknowledgements:** This work was funded by FAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro. Jordana Neri received a fellowships from CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

## References

1. Avise J: Phylogeography: the history and formation of species. Harvard University Press 2000.

2. Aselmann J, Crutzen P: Global distribution of natural freshwater wetlands and rice paddies, their net primary productivity, seasonality and possible methane emissions. *J Atmos Chem* 1989, 8:307-358.

3. World Conservation Monitoring Centre. Chapman & Hall, London 1992, 594.

4. Scarano F, Ribeiro K, Moraes L, Lima H: Plant establishment on flooded and unflooded patches of a freshwater swamp forest in Southeastern Brazil. *J Trop Ecol* 1997, 14:793-803.

5. Reitz R, Klein R, Reis A: Projeto madeira de Santa Catarina. Sellowia 1978, 28-30:218-224.

6. Oliveira-Filho A, Ratter J: A study of the origin of central Brazilian forests by the analysis of plant species distribution patterns. *Edin B J Bot* 1995, 52(2):141-194.

7. Carvalho P: Espécies florestais Brasileiras recomendações silviculturais, potencialidades e uso da madeira. *Colombo, Embrapa Florestas* 1994, 640.

8. Doyle J, Doyle J: Isolation of plant DNA from fresh tissue. *Focus* 1987, 12:13-15.

9. Hamilton M: Four primers pairs for the amplification of chloroplast intergenic regions with intraspecific variation. *Mol Ecol* 1999, 8:521-523.

10. Lemes M, Dick C, Navarro C, Lowe A, Cavers S, Gribel R: Chloroplast DNA microsatellites reveal contrasting phylogeographic structure in mahogany (*Swietenia macrophylla* King, Meliaceae) from Amazonia and Central America. *Trop Plant Biol* 2010, 3:40-49.

11. Novaes R, Lemos-Filho J, Ribeiro R, Lovato M: Phylogeography of *Plathymenia reticulate* (Leguminosae) reveals patterns of recent range expansion towards northeastern Brazil and southern Cerrados in Eastern Tropical South America. *Mol Ecol* 2010, 19:985-998.

## P18

### Optimizing sampling efforts for ex situ conservation of genetic variability of *Dipteryx alata* Vogel

Dayane B Melo¹, José Alexandre F Diniz-Filho², Guilherme Oliveira³, Ludmilla L Santana⁴, Thannya N Soares⁴, Lázaro J Chaves⁵, Ronaldo V Naves⁵, Rosane G Collevatti⁴, Mariana P de C Telles⁴

¹Programa de Pós-Graduação em Agronomia (UFG), Brazil; ²Depto. Ecologia, Universidade Federal de Goiás (UFG), Brazil; ³Programa de Pós-Graduação em Ecologia & Evolução, ICB, UFG, Brazil; ⁴Laboratório de Genética & Biodiversidade, Depto. Biologia Geral, ICB, UFG, Brazil; ⁵Escola de Agronomia & Engenharia de Alimentos, UFG, Brazil

E-mail: tellesmpc@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P18

**Background:** The "Baru" tree (*Dipteryx alata* Vogel) is an endemic species from the Cerrado biome, but widely distributed within the biome and abundant in several of its habitats [1]. The species is also important in commercial terms for recover of degraded areas, and seeds are consumed *in natura* and are source of raw material for small and middle-sized food industries [2]. Thus, a more detailed knowledge of its population variability and structure is needed for better establishing both *in situ* and *ex situ* conservation efforts. Data for such analyses can be provided by new molecular markers, such as microsatellites, whereas spatial analyses and optimization procedures can be applied to analyze these data and thus improve conservation efforts. Here we investigate the genetic variability within the germplasm bank of *D. alata* based on microsatellite data and compared it with a large sample of 25 widely distributed natural populations of the species. We then used a simulated annealing algorithm to find the smallest number of these natural populations that should be sampled to conserve the full genetic variability of the species.

**Methods:** We described the genetic variability of 816 individuals of *D. alata* from 25 natural population widely distributed in the Cerrado biome, and from 180 individuals preserved in an active germplasm bank, situated in the "Escola de Agronomia e Engenharia de Alimentos" Universidade Federal de Goiás, totalizing 996 individual plants genotyped for 9 microsatellite loci. The origin of the individuals in the bank is not known in detail, but it is certain that they came from several localities from Goiás State.

Overall description of genetic variability in the natural populations and in the germplasm bank was done by percentage of polymorphic loci (P), mean number of alleles per loci (A), expected heterozigosity under H-W equilibrium (He) and observed heterosigosity (Ho). Population structure
and divergence among natural populations were analyzed using $F_{ST}$ statistics.

A simulated annealing algorithm, implemented in the software SITES [3] was used to establish the minimum number of local populations necessary to complement the germoplasm bank so that all alleles (expressed as present or absent in each local population) are represented at least once (i.e., representation goal). The search was performed using two hundred runs and 10,000,000 iterations for each run. The problem is analogous to the ecological problem of finding a minimum number of new conservation priority areas that represent all species, after fixing a few already established natural reserves (see [4]). However, there may be frequently multiple combinations of local populations that satisfy the representation goal, so the first 100 solutions of SITES were also retained. The relative frequency of each local population in the alternative optimized solutions is an estimate of the "irreplaceability", or relative importance, of the populations to the overall goal of representing the entire genetic variability (alleles).

**Results and discussion:** The genetic variability of *D. alata* for the microsatellite loci analyzed here was low compared with other forest species in the same region (e.g., [5]). Out of the 9 loci, on average 78% were polymorphic within natural population, and 67% were polymorphic in the germplasm bank. The mean number of alleles per loci were 2.7 and 4.4 for the natural populations and for the germplasm bank, respectively. The *He* and *Ho* were equal to 0.36 and 0.30 for natural populations, and 0.36 and 0.23 for the germplasm bank. The $F_{ST}$ was significant and equal to 0.259, indicating a relatively high differentiation among the local populations.

The simulated annealing revealed that only 4 local populations, located in Mato Grosso and Mato Grosso do Sul (Figure 1; 1-CMT. 5-AMS. 19-RAMT e 25-CAMT), are necessary to represent all alleles that appeared in the species, complementing in an optimum way the genetic variability already present in the germplasm bank. The solution is unique so that all these 4 local populations have 100% of irreplaceability.

Our analyses confirm that the germplasm bank currently established in the Universidade Federal de Goiás preserves the genetic variability of a relatively large proportion of species' range in the Cerrado biome. To improve such representation it is important, however, to sample more local populations in the west portion of the species' distribution, in Mato Grosso region. It is important to note that these populations possess unique alleles that are not found elsewhere, so they are irreplaceable for achieving the conservation goal. If these populations are lost (i.e., extinct), there will be genetic erosion reducing total genetic variability in this

species. So, an effort to sample and grow up individuals or progenies from the 4 populations, preferentially coupled with in situ conservation programs in these natural populations from Mato Grosso region, must be urgently done. Our optimization analyses reveals that only a few samples are necessary to achieve a more complete representation for the species, and this is important to reduce sampling and maintenance costs without losing efficiency in terms of establishing conservation and future breeding programs for the species.

**References**

1. Almeida SP, Proença CEB, Sano SM, Ribeiro JF: Cerrado: espécies vegetais úteis. Planaltina: Embrapa-CPAC 1998.

2. Sano SM, Ribeiro JF, Brito MA: Baru: biologia e uso. Planaltina: Embrapa Cerrados 2004.

3. Possingham H, Ball I, Andelman S: Mathematical methods for identifying representative reserve networks. Quantitative methods for conservation biology New York: Springer-Verlag; Ferson S, Burgman M 2000, 291-305.

4. Margules C, Pressey RL: Systematic conservation planning. Nature 2000, 405:243-253.

5. Collevatti RG, Grattapaglia D, Hay JD: Population genetic structure of the endangered tropical tree species *Caryocar braziliense*, based on variability at microsatellite loci. Molecular Ecology 2001, 10:349-356.

P19

Chloroplast DNA variation and phylogeography of *Eugenia uniflora* L. (Myrtaceae) in the Brazilian Atlantic forest

Andreia Carina Turchetto-Zolet¹, Fabiano Salgueiro², Fernanda Cruz¹, Nicole Veto³, Rogerio Margis¹

¹Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil; ²Departamento de Botânica, Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Brazil; ³Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil

E-mail: aturchetto@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P19

**Introduction and objectives:** The Atlantic Forest (AF) is considered the second largest tropical forest in South America with high species richness and endemisms, harboring a large diversity of animals, plants and habitats types [1]. This biome covers an area of more than one million square kilometres along the Brazilian coast and extending to eastern Paraguay and northeastern Argentina [2]. AF is considered one of the

<figure><img src="image_25.png" /></figure>
most threatened ecosystems on Earth due to intense disturbance, having been reduced to only 7.5% of its original area [1]. Despite an increase in research efforts in the past few years, studies of AF species diversification and knowledge about its evolutionary history is still scarce. Vegetation changes in the Atlantic Forest related to climatic changes during the Pleistocene have been registered in paleopalinological studies, with the replacement of large areas of forests by subtropical grasslands and savannas during cooler and drier conditions [3,4]. Also, studies of paleoclimatic models, predicted the presence of historically stable areas (refugia) in the Atlantic Forest during the Late Quaternary [5]. *Eugenia uniflora* L. (Myrtaceae), a shrubby tree with edible cherry-like fruits which is locally known as pitanga or Brazilian cherry. This species is one of the key species in the Atlantic rain forest geomorphological domain, which includes the Atlantic forest and the adjacent Restinga ecosystem [6]. *E. uniflora* occurs in areas of medium and large levels of rainfall and can also be found in different vegetation types and ecosystems. This species present economic and folk medicinal applications and is an important pioneer species in the Restinga ecosystem and has been used to recover and manage disturbed and fragmented areas. Our aim in this study was to investigate the phylogeography and genetic diversity of *Eugenia uniflora* to help elucidate the evolutionary history of this species as a model for gain insights into past vegetation patterns in the Brazilian Atlantic Forest.

**Methods:** Forty-six populations of *E. uniflora* were sampled across the Brazilian Atlantic forest. The samples were collected as leaf material (silica gel dried) from natural populations. Total genomic DNA was isolated using the CTAB method [7]. Two cpDNA regions (*psbA/trnH* and *trnC/ycF6*) were using for population analysis based on sequence quality and degree of variation. These regions were amplified using universal primers. The individual consensus sequences were aligned through CLUSTALW [8] implemented in MEGA4 [9], then carefully improved manually. The genealogical relationships among haplotypes were estimated by using the median-joining method implemented in Network 4.2.0.1 (Fluxus Technology Ltd. at http://www.Fluxus-engineering.com). Molecular diversity estimates were calculated using Arlequin 3.1[10] and DNAsp 5.0 [11]. Genetic structure was further examined by the analysis of molecular variance (AMOVA), as implemented in Arlequin version 3.1.

**Results and conclusions:** The total combined cpDNA matrix presented 1224 sites, which eight were variable. Eight haplotypes were found and the haplotype diversity (h) ranged from 0 to 0.733 and the nucleotide diversity (π) from 0 to 0.00140. Total haplotype and nucleotide diversities were 0.433 and 0.00088, respectively. The highest haplotype diversity was found in populations ITAP, PALM, CAPI and GRAV from the Atlantic forest South. Populations from Northeast were monomorphic. The most common haplotype was H1, present in 39 of 46 populations. The AMOVA analysis showed a very strong differentiation among all *E. uniflora* populations (FST =0.771, P<0.0001). These results can help a deeper understanding of the dynamics responsible for both ancient and more recent events that have shaped the current distribution of genetic variability in Atlantic Forest and also have implications for conservation efforts. For the long-term conservation of the genetic diversity of *E. uniflora*, it would be important to design strategies that aim to preserve most of its lineages. For such, the South region is a key piece, as it houses many divergent and endemic haplotypes and lineages.

**Financial support:** FAPERGS and CNPq

**References**

1. Myers N: Biodiversity hotspots for conservation priorities. *Nature* 2000, 403(6772):853-858.

2. Oliveira AT, Fontes MAL: Patterns of floristic differentiation among Atlantic forests in southeastern Brazil and the influence of climate. *Biotropica* 2000, 32:793-810.

3. Behling H: South and southeast Brazilian grasslands during Late Quaternary times: a synthesis. *Palaeogeogr Palaeocl* 2002, 177:19-27.

4. Behling H: Late Quaternary environmental changes in the Lagoa da Curuca region (eastern Amazonia, Brazil) and evidence of Podocarpus in the Amazon lowland. *Quaternary Res* 2001, 56:383-389.

5. Carnaval AC, Moritz C: Historical climate modelling predicts patterns of current biodiversity in the Brazilian Atlantic forest. *J. Biogeography* 2008, 35:1187-1201.

6. Scarano FR: Structure, function and floristic relationships of plant communities in stressful habitats marginal to the Brazilian Atlantic rainforest. *Ann Bot* 2002, 90:517-524.

7. Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. *Focus* 1987, 12:13-15.

8. Thompson JD, Higgins DG, Gibson TJ: Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. *Nucleic Acids Res* 1994, 22:4673-4680.

9. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. *Mol Biol Evol* 2007, 24:1596-1599.

10. Excoffier L, Laval G, Schneider S: Arlequin ver. 3.0: An integrated software package for population genetics data analysis. *Evol Bioinformatics Online* 2005, 131:479-491.

11. Librado P, Rozas J: DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. *Bioinformatics* 2009, 25:1451-1452.

P20

**Detection of genes involved in bud phenology in sessile oak (Quercus petraea Matt. Liebl) combining digital expression analysis and Q-PCR**

Saneyoshi Ueno¹, Christophe Köpp², Céline Noirot², Valérie Léger³, Elodie Prince³, Antoine Kremer³, Christophe Plomion³, Grégoire Le Provost³

¹Forestry and Forest Products Research Institute, Brazil; ²Plateforme bioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA, Brazil; ³INRA, UMR 1202, BIOGECO, Brazil

E-mail: saueno@ffpri.affrc.go.jp

BMC Proceedings 2011, 5(Suppl 7):P20

**Background:** In temperate zones, the seasonal cycling of perennials comprises two main steps: a growing period when environmental conditions are favourable and a non-growing period in winter. Winter dormancy consists of two phases: endodormancy and ecodormancy [1]. The former is the deepest state of the dormancy controlled by the meristem itself, while the latter is controlled by environmental factors, such as accumulated temperatures. This phenological cycle has been shown to be strongly affected by climatic change: higher temperature having a positive effect on ecodormancy phase by accelerating meristem cell growth and early bud break is particularly crucial to consider not only in respect to increasing length of the growing season, but also regarding the risk of frost damage. For these reasons, understanding the molecular mechanism controlling the shift from endo- to ecodormancy is of main importance to optimize the management of forest tree plantations to their most likely environmental conditions.

The objective of this study was to characterize the transcriptome of endo- and eco-dormant sessile oaks (a major broadleaf species in Europe) by RNA-seq and to identify differentially expressed genes between these two dormancy stages. Four cDNA libraries were generated using total RNA extracted from apical buds harvested during endo- or ecodormancy of two contrasted populations. Transcriptome characterization was performed on a Roche 454 FLX platform. Digital expression analysis leads us to identify a set of 48 genes differentially regulated between these two developmental phases (there was no differences between populations). Their function was identified by sequence homology and this result contributes to the characterization of the molecular network underlying vegetative bud dormancy, an important life history traits of these long lived organisms.

**Methods:** Acorns of sessile oak (*Quertus petreae* (Matt) Lieb.) were collected in 2006 on two populations from Northern France: Longchamp (LC) and Saint Jean (SJ). These populations are contrasted for their bud burst date (early vs. late flushing). We obtained 560 and 468 seedlings for the SJ and the LC populations, respectively. A forcing test experiment was performed in a greenhouse from July 30th 2007 to February 25th 2008 located at the INRA forestry research station (southwestern France). For each population, approximately 25 seedlings were selected each week. Ten apical buds from 10 seedlings were used for RNA extraction. The 15 remaining seedlings were transferred in a greenhouse with the following parameters: a 16-hours photoperiod and a day/night temperature of 25/18 °C. For each batch of seedlings, bud burst date was evaluated three times a week allowing us to determine the number of days necessary to induce bud burst. We, thus, identified periods corresponding to eco- and endodormancy.

Total RNA from apical buds harvested on September 17th, 24th and October 1st for endo-dormancy as well as on January 14th, 28th and February 11th for eco-dormancy were extracted independently, and
mixed equimolarly to obtain a composite cDNA library for each population and developmental stage. The libraries were sequenced on a Roche 454 FLX to produce 495,915 reads in total. After cleaning and removal of duplicated reads, reads were mapped onto oak unigenes [2]. A digital gene expression analysis was performed using both *r* statistics [3] and R BioConductor packages (edgeR, DEGseq and goseq). Homology searches were carried out by BlastX against SWISS-PROT and TAIR9 databases with e-value cutoff of 1e-5. Gene ontology (GO) annotations were based on the top blast hit against SWISS-PROT database. To validate bioinformatic analysis, Q-PCR was performed for 13 selected genes. One microgram of total RNA was reverse transcribed using Improm-II™ reverse transcription system (Promega) according to the manufacturer's instructions. Q-PCR reaction and quantification were performed on a Chromo4 Multicolor Real-Time PCR Detection System (Bio-Rad)

**Results and conclusions:** We identified 48 common contigs that showed differential expression by three bioinformatic programs (R statistics, edgeR and DEGseq) and selected 13 genes for Q-PCR analysis with the main goal to validate the result obtained in silico. One gene (heat shock protein) showed multi-banding pattern and discarded from the analysis. Among the 12 remaining genes, only one gene (cold shock protein) showed contradictory expression pattern between in silico and Q-PCR analysis. The high success rate of Q-PCR analysis (11/13 = 84.6%) indicates that the verified genes are likely to represent endo- and ecodormancy stages. Two genes (Globulin and XET) were confirmed as up-regulated in endodormancy, while nine genes (GASA, GST, DRM1, XERO2, PIP, AWPM, GID, LEA and ELP) were found to be up-regulated in ecodormancy. Analysis in terms of GO showed genes responsible to abiotic and endogenous stimulus as well as stress related were enriched in the set of 48 genes. Similar trend was observed in expression analysis for raspberry bud dormancy, where high percentage of stress-response/defense/detoxification-related genes were identified [4]. In grape also, most of genes expressed in buds at the end of dormancy were attributed to the oxidative processes and stress responses [5]. Although the number of genes identified is small and further verification through time course change in their expression will be needed, these candidates will serve as stepping stone to dissect the molecular mechanisms involved in bud dormancy in forest trees.

## References

1. Lang GA, Early JD, Martin GC, Darnell RL: Endo-, Para-, and Ecodormancy: physiological terminology and classification for dormancy research. *HortScience* 1987, 22:371-377.

2. Ueno S, Le Provost G, Leger V, Klopp C, Noirot C, Frigerio JM, Salin F, Salse J, Abrouk M, Murat F, et al: Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak. *BMC Genomics* 2010, 11:650.

3. Stekel DJ, Git Y, Falciani F: The comparison of gene expression from multiple cDNA libraries. *Genome Res* 2000, 10:2055-2061.

4. Mazzitelli L, Hancock RD, Haupt S, Walker PG, Pont SD, McNicol J, Cardle L, Morris J, Viola R, Brennan R, et al: Co-ordinated gene expression during phases of dormancy release in raspberry (*Rubus idaeus*) L. buds. *J Exp Bot* 2007, 58:1035-1045.

5. Pacey-Miller T, Scott K, Ablett E, Tingey S, Ching A, Henry R: Genes associated with the end of dormancy in grapes. *Funct Integr Genomics* 2003, 3:144-152.

# P21
## Spatial genetic structure of *Stryphnodendron adstringens* (MART.)

### COVILLE (Leguminosae-Mimosoideae) using SSR markers

Léo Zimback¹, Edson Mori², Eliete Branco³, Adriano Lima⁴, Eliseu Baena¹

¹Instituto Florestal, Avaré, SP, 18701-180, Brazil; ²Unesp, Botucatu, SP, 18610-307, Brazil; ³University Sagrado Coração, Bauru, SP, 17011-160, Brazil

E-mail: Izimback@terra.com.br

BMC Proceedings 2011, 5(Suppl 7):P21

**Background:** The forest fragments of *Stryphnodendron adstringens* have disappeared because of the high rate of Brazilian Savannah deforestation for commercial purposes, such as eucalypt, citrus, soy bean, and sugar cane crops that have been intensely cultivated on those areas [1]. Economically, *S. adstringens* is important for manufacture industries of paints and tanneries, but also is utilized for therapy against ulcer, scurvy, and for antiseptic and healing purposes [2]. Considering the forestry

sector, the species is potential for building uses because of their wood have high density and durability [3]. Aiming for germplasm preservation and for breeding programs we studied the spatial genetic structure of *S. adstringens* based on SSR markers.

**Methods:** Juvenile leaves of 38 individuals of *S. adstringens* were collected in natural population, of approximately 200 individuals, in Botucatu State Forest belonging to Forestry Institute, São Paulo State, Brazil. The population is located from 22° 55' 55" S to 22° 56' 39" S latitude and from 48° 27' 19" W to 48° 27' 33" W longitude, through 860 m altitude, totaling 33 hectares of savannah. The lab procedure protocol based on Ferreira and Grattapaglia [4], and adapted to *S. adstringens*, for DNA extraction was used. We assessed the transferability of 98 primer pairs developed for different tree species of several families and genera. The amplification was performed at 92° C for 2 minutes by initial denaturation, 45 cycles of 1 minute at 92° C, 1 minute at annealing temperature, and 1 min at 72° C, and ending to 10 minutes at 72° C. The analysis of spatial autocorrelation of sampled population, with local coordinates, and variable alleles of individuals was estimated. The "Spatial Genetic Software (SGS) version 1.0 program for distance classes per each allele, and considering 1000 bootstraps to get the Moran's I Index [5] were used. The expression was:

$$I = \left( \frac{n}{W} \right) \cdot \left[ \frac{\sum_{i} \sum_{j} (p_i - p)(p_j - p)W_{ij}}{\sum_{i} (p_i - p)^2} \right]$$

where:

* I = Moran's I index, which can take values between -1.0 (negative autocorrelation) and + 1.0 (positive autocorrelation);

* n = number of individuals;

* pi and pj = allele frequency for i and j individuals;

* p = average of p;

* Wij = element of symmetric matrix square and W were nxn dimensions, which is given the value 1 for individual neighbors and 0 otherwise;

* W = matrix that expresses the spatial correlation between individuals and the sum over i and j is the value equal W.

**Results and discussion:** Forty eight out of 98 pairs were amplified showing 23 polymorphic primers, but only 10 of them were conserved, and presenting no null alleles: EMBRA 03, EMBRA 06 (*Eucalyptus grandis*), EMBRA 72, EMBRA 210 (*Eugenia dysenterica*) Empas 02 (*Prunus avium*) LMCH 12, LMCH 14 (*Annona cherimoya*), SCU 056, SCU 062 (*Melaleuca alternifolia*), and SP 06 (*Schizolobium parahyba*). O LMCH 12 locus was eliminated to present low accuracy. Table 1 shows the correlogram of *S. adstringens* indicating that the distances, among individuals of Botucatu State Forest, from 0 to 33 and from 99 to 132 meters have

<table><caption>**Table 1 Correlogram using Moran's Index (D) with confidence interval (CI), probability of exclusion (P), number of comparisons (CN), and value indicating absence of spatial autocorrelation D = -0.0270 for *Stryphnodendron adstringens***</caption><thead><tr><th>Distance (m)</th><th>D(-CI)</th><th>D(obs.)</th><th>D(+CI)</th><th>P(D)&lt; (-CI)</th><th>P(D)&gt; (+CI)</th><th>CN</th></tr></thead><tbody><tr><td>0-33</td><td>-0.080841</td><td>0.043351</td><td>0.034583</td><td>0.015</td><td>0.985</td><td>40</td></tr><tr><td>33-66</td><td>-0.078096</td><td>-0.005722</td><td>0.023789</td><td>0.190</td><td>0.810</td><td>45</td></tr><tr><td>66-99</td><td>-0.079640</td><td>-0.010610</td><td>0.031820</td><td>0.291</td><td>0.709</td><td>41</td></tr><tr><td>99-132</td><td>-0.081936</td><td>-0.096614</td><td>0.032199</td><td>0.995</td><td>0.005</td><td>35</td></tr><tr><td>132-165</td><td>-0.079713</td><td>-0.015566</td><td>0.027000</td><td>0.355</td><td>0.645</td><td>38</td></tr><tr><td>165-198</td><td>-0.090142</td><td>0.025456</td><td>0.033938</td><td>0.046</td><td>0.954</td><td>37</td></tr><tr><td>198-231</td><td>-0.064909</td><td>-0.059062</td><td>0.007197</td><td>0.954</td><td>0.046</td><td>75</td></tr><tr><td>231-264</td><td>-0.086119</td><td>-0.063560</td><td>0.031723</td><td>0.867</td><td>0.133</td><td>25</td></tr><tr><td>264-297</td><td>-0.114183</td><td>-0.054740</td><td>0.074730</td><td>0.715</td><td>0.285</td><td>7</td></tr><tr><td>297-330</td><td>-0.103925</td><td>-0.058399</td><td>0.052559</td><td>0.800</td><td>0.200</td><td>10</td></tr></tbody></table>
presented spatial structure. According to Sebbenn [6] is common in
tropical species, similarities until 100 meters; therefore to collect high
variability of *S. adstringens* it is important obeyed distance up to 132
meters.

**Conclusions:** Significant results were observed for the class distances from 0 to 33 meters and 99 to 132 meters, showing there is spatial autocorrelation for *S. adstringens*.

**Acknowledgements:** The authors thank FAPESP to provide financial funds to develop the study, CNPq and CAPES for scholarships.

References

1. Machado SR: Pesquisa aponta degradação do Cerrado. *Jornal Diário da Serra* 2006, A3.

2. Jacobson, Garcia J, Santos SC, Duarte JB, Farias JG, Kliemann HJ: Influência de fatores edáficos na produção de fenóis totais e taninos de duas espécies de barbatimão (Stryphnodendron sp.). Pesquisa Agropecuária Tropical 2005, 35:163-169.

3. Almeida SP, Sano SM: Cerrado: espécies vegetais úteis. *Brasília: EMPRAPA Cerrado* 1998, 347-351.

4. Ferreira ME, Grattapaglia D: Introdução ao Uso de Marcadores Moleculares em Análise Genética. 1998, 121.

5. Sokal RR, Oden NL: Spatial autocorrelation in biology. 2. Some biological implications and four applications of evolutionary and ecological interest. *Biological Journal of the Linnaean Society* 1978, 10:229-249.

6. Sebbenn AM: Sistemas de reprodução em espécies tropicais e suas implicações para a seleção de árvores matrizes para reflorestamentos ambientais. *Pomar de sementes de espécies florestais nativas* Higa AR, Silva LD 2006, 93-138.

## S2. LINKAGE AND ASSOCIATION MAPPING

P22

Development of DArT (Diversity Arrays Technology) for high-throughput genotyping of *Pinus taeda* and closely related species

Dione MT Alves-Freitas¹*, Andrzej Kilian², Dario Grattapaglia³

¹EMBRAPA Genetic Resources and Biotechnology - EPqB Final W5 Norte 70770-917 Brazilia DF and Dep. Cell Biology, Universidade de Brazilia – UnB, Brazil; ²Diversity Arrays Technology Pty Ltd, 1 Wilf Crane Crescent, Yarralumla, ACT 2600, Australia; ³EMBRAPA Genetic Resources and Biotechnology - EPqB, 70770-910 Brazilia, Brazil; Dep. Cell Biology, Universidade de Brazilia – 70910-900 Brasilia, Brazil; Genomic Sciences Program - Universidade Católica de Brasília - Brazilia, Brazil

E-mail: dionebio@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P22

**Background:** *Pinus (Pinaceae)* is the largest existing genus of conifers, with over 100 widely recognized species. Pines are an important source of wood, paper and resins, among others. Genetic improvement of *Pinus* species is a challenging endeavor. Breeding cycles typically last for decades, significant changes take place in wood properties and growth patterns with the transition from the juvenile to the adult phase and most traits are multi-factorial with low heritability [1]. Genomic Selection (GS) could radically reduce the time required for completion of a cycle of genetic improvement by precluding the progeny testing phase, significantly increasing the selection efficiency relative to conventional breeding [2,3]. To put GS into practice, genome-wide, high-throughput and cost-efficient marker are needed to advance this genomics assisted breeding approach focusing purely on prediction of performance, precluding gene-trait association discovery. To this end we are developing a high-throughput DNA genotyping platform for *Pinus taeda* based on the genome complexity reduction DArT (Diversity Arrays Technology) technology that has been successfully developed for many plant species including some with very large and complex genomes such as wheat [4] and sugarcane [5].

**Methods:** For DArT array probe development population samples of 16 species of *Pinus*, 24 provenances of *Pinus taeda* L. and two mapping populations were used as starting material organized in pooled samples. For microsatellite and DArT mapping a set of 288 individually extracted haploid megagametophyte DNA samples of clone 7-56 were used. DArT development involved reduction of genome complexity of pooled samples

by digestion with *Pst*I as the primary cutter and *BstNI* as a frequently and secondary cutter followed byligation of adaptors to *Pst*I overhangs and PCR amplification of intact *Pst*I fragments following a protocol described earlier [6]. Genomic representations (targets) generated by PCR amplification were cloned into six separate libraries. A total of 7,680 clones were randomly picked being 2,304 clones from the 16 species libraries and 4,608 clones from *P. taeda* only libraries. A first test panel of 96 *Pinus* individuals was used to screen the 7,680 probes for polymorphic markers. Markers were declared reliable and polymorphic at a cutoff of 80% call rate, 95% reproducibility, and polymorphism information content > 0.1. Parallel to DArT marker development a linkage map of microsatellites was generated to provide a framework onto which the DArT markers will eventually be mapped. DNA from haploid megagametophyte samples of 288 seeds of Loblolly pine tree 7-56 was extracted. To immortalize the limited amount of haploid DNA of this mapping population, whole genome amplification was used. Haploid samples were genotyped with a selected set of 65 microsatellites. Genotyping was performed in an ABI PRISM 3100XL using 4-color fluorescent detection in multiplexed sets of 2 co-amplified microsatellites. Linkage analyses and map construction was carried out using JOINMAP 3.0.

**Results:** A total of 856 and 930 markers were found polymorphic across samples of 24 individuals of each one of two mapping populations, while 1,776 DArT markers were polymorphic in a panel of 16 species, two individuals per species and 16 *Pinus taeda* US provenances, one individual per provenance. A significant number of probes could not be adequately scored due to background signal resulting from the presence of highly repetitive DNA that the *PstI/BstNI* complexity reduction could not remove from the 23 pg *Pinus taeda* genome. Cot-1 DNA, i.e. genomic DNA highly enriched for repetitive elements was therefore isolated from *P. taeda* DNA by shearing genomic DNA in autoclave, followed by DNA denaturing and re-annealing and S1 nuclease treatment. Using the Cot-1-DNA to quench repetitive probes, the recovery of assayable DArT markers was increased by 40%. When mapping microsatellites extensive locus duplication of the 65 microsatellite primer pairs resulted in a total of 182 individually "loci" scored for presence/absence in the haploid gametes. Based on the map position 96 of these "loci" actually corresponded to 48 effective loci and the other 86 are actual individual loci totaling 134. From the 134 segregating loci 110 were confidently mapped in the 12 expected linkage groups leaving two unlinked groups of 5 and 3 markers. Locus order was essentially the same for the 50 loci derived from 31 microsatellites in common with those recently mapped [7]. Interestingly, microsatellite PTTX3021 amplified 17 individually scored "loci". Eight were allelic pairs and were consolidated in four loci while the remaining 9 were unique. Additional duplications were seen for 15 microsatellites in different linkage groups suggesting paralogous loci.

**Conclusion:** In spite of the complex and repetitive nature of the *Pinus taeda* genome several hundred DArT markers could be confidently genotyped indicating that DArT is a robust technology to be used for conifers. A new round of DArT probe picking and marker screening coupled to Cot-1 DNA use is now underway and should provide the target number of 7,680 selected probes for an operational array. The haploid mapping population will be genotyped with this array to provide information on distribution of the DArT markers in terms of recombination fraction and genome coverage relative to the framework of microsatellites. Additionally, probes detecting mapped markers will be sequenced to eventually aid the assembly of the 7-56 genome sequence.

**Acknowledgments:** PRONEX FAP-DF grant 193.000/570/2009, CNPq Grant 474645/2007-9 and CNPq doctoral fellowship 560831/2008-0 to D.M.T.A.F.; we thank Arborgen for donating 7-56 seeds.

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