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

7. Echt CS, Saha S, Krutovsky KV, Wimalanathan K, Erpelding JE, Liang C, Nelson CD: An annotated genetic map of loblolly pine based on microsatellite and cDNA markers. *Bmc Genetics* 2011, 12.

P23

Effect of population structure and kinship relationships on the results of association mapping tests of growth and wood quality traits in four *Eucalyptus* populations

Eduardo Pablo Cappa¹*, Maria C Martínez², Martín N Garcia²,
Pamela V Villalba², Susana N Marcucci Poltri²

¹Instituto Nacional de Tecnología Agropecuaria. Instituto de Recursos Biológicos. Hurlingham, Buenos Aires, Argentina; ²Instituto Nacional de Tecnología Agropecuaria. Instituto de Biotecnología. Hurlingham, Buenos Aires, Argentina

E-mail: ecappa@cnia.inta.gov.ar

BMC Proceedings 2011, 5(Suppl 7):P23

**Background:** In recent years, association mapping studies have been reported for growth and wood quality traits in *Eucalyptus* (e.g. [1]). One problem with association studies is that they can be sensitive to the presence of population structure. The presence of population structure may generate spurious associations between markers and traits and leading to an elevated false-positive rate (e.g. [2]). Statistical approaches that account for population structure include model-based clustering [3], principal component analysis, genomic control and linear mixed model approach [4]. The mixed model of Yu et al. (2006) accounts for both major population structure, assigning individuals to subpopulations (the Q matrix), and the relatedness among individuals within and between subpopulations (the kinship (K) matrix). The mixed model approach generally performs best [2,4].

As part of the Biotech MERCOSUR project (Marcucci Poltri et al. this volume) molecular and phenotypic data from four *Eucalyptus* populations have been obtained: three open pollinated (OP, half-sib) progeny trials of *Eucalyptus grandis* from Argentina (EgrAr) and *Eucalyptus globulus* from Uruguay (EglUy) and Argentina (EglAr) and one clonal trial of *Eucalyptus grandis* from Paraguay (EgrPy). These populations differ in the underlying substructure and genetic relatedness among individuals. It is thus important to investigate the effects of population structure and kinship on the results of associations between markers and growth and wood quality traits from these *Eucalyptus* populations.

**Material and methods:** A total of 612 trees were sampled from the EgrAr (188), EgrPy (121), EglAr (134) and EglUy (169) populations. The number of OP families sampled in each OP progeny trials was 132 (EgrAr), 129 (EglAr) and 70 (EglUy) from different native stand sites in Australia (from 8 to 13) and land races (from 1 to 3). The number of trees per OP family varied from 1 to 3 (EgrAr), 1 to 8 (EglUy) and 1 to 2 (EglAr). One growth trait (diameter at breast height, DBH) and three quality wood traits (extractives in ethanol, Klason lignin and syringyl:guaiacyl ratio (S:G ratio)) were studied.

All the 612 trees were genotyped using Diversity Arrays Technology (DArT) molecular markers [5]. A subset of 2816 (EgrAr), 2693 (EgrPy), 2373 (EglAr) and 2300 (EglUy) DArT markers were used in the analysis after markers with frequency greater than 0.95 or less than 0.05 were excluded.

The association mapping tests were carried out at the DArTs level using two-steps. First, for the three OP trials, the overall mean and design effects or first order autoregressive residuals for rows and columns, were fitted to deal with environmental variation. Additionally, for the clonal trial, the best linear unbiased predictions (BLUP) of clonal values were predicted. Second, the markers effects were tested on the adjusted phenotypes (OP trials) or clonal BLUPs values (clonal trial) using four models [4]: 1) Simple model, in which Q and K matrices are ignored; 2) Q model, considers only Q matrix; 3) K model, considers only K matrix; and 4) Q+K model, considers both Q and K matrices. Except for the EglAr population, the Q matrix was calculated by the software STRUCTURE [3] on basis of 400 random DArT markers. The K matrix was calculated on basis of 800 random DArT markers using the software package SPAGEDI [6].

For each of the four populations, all the association tests were carried out using TASSEL software [http://www.maizegenetics.net/].

**Results:** All populations showed an optimum cluster number of 4. In general, for the three OP populations the compositions of the cluster coincide with the geographical native stand sites in Australia. More than 52% of the pair-wise kinship estimates were equal to 0, whereas about the 46% of the values were less than 0.25. Without taking into account the population structure and kinship (Simple model), from 0.9 to 8.5% of the DArT markers tested were associated with the growth and wood quality traits at *P* < 0.01. These preliminary results show a high number of associated markers that might suggest that several of them are likely to be false-positives due to population structure and/or kinship relationships among trees within each population. For all populations and most traits under consideration, the controlling only for population structure (Q model) reduced the number of significant DArTs (from 0.5 to 2.6%). This effect was more pronounced for the two OP *Eucalyptus globulus* populations. However, when the relative kinship coefficients between every pair of individuals were considered (K model), a more stringent reduction with respect to the Q model was observed (from 0.3 to 1.3%). This finding suggests that, when more complex interrelationship among individuals within and between subpopulations exists, the fitting Q model was not enough to reduce the number of spurious associations. It is no clear, across populations and traits, that the Q matrix should be added to the kinship effect. The reduction of number of significant DArT markers, excluding pedigree information (K matrix) in the model, appeared to be trait dependent.

**Conclusions:** Both population structure and kinship relatedness between individuals had effect on the association mapping tests. The kinship had a more stringent effect on the marker-trait associations. Effect on association tests depends on the populations and traits studied.

References

1. Thumma BR, et al: Polymorphisms in Cinnamoyl CoA Reductase (CCR) Are Associated With Variation in Microfibril Angle in *Eucalyptus* spp. *Genetics* 2005, 171:1257-1265.

2. Zhao K, et al: An *Arabidopsis* Example of Association Mapping in Structured Samples. *PLoS Genet* 2007, 3:e4, doi:10.1371/journal.pgen.0030004.

3. Pritchard JK, et al: Inference of Population Structure Using Multilocus Genotype Data. *Genetics* 2000, 155:945-959.

4. Yu J, et al: A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. *Nat. Genet* 2006, 38:203-208.

5. Sansaloni CP, et al: A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in *Eucalyptus*. *Plant Methods* 2010, doi:10.1186/1746-4811-6-16.

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

P24

Construction and analysis of a leaf cDNA library from cold stressed rubber tree clones

Carla Da Silva¹*, Tatiana Campos², Erivaldo Scaloppi Jr.³, Mírian Maluf⁴,
Paulo Gonçalves⁵, Anete Souza⁶

¹Molecular Genetics and Analysis Laboratory, Genetics and Evolution Department, Biology Institute, CBMEG/UNICAMP, Brazil; ²Agroforestry Research Center from Acre, Embrapa Acre, Brazil; ³São Paulo Northwest Regional Research Center, APTA, Brazil; ⁴Alcides Carvalho Coffee Center - IAC/Embrapa, Brazil; ⁵Rubber Tree Breeding Program - IAC/Embrapa, Brazil;

⁶Plant Biology Department, Biology Institute, UNICAMP, Brazil
E-mail: silvacbio@yahoo.com.br

BMC Proceedings 2011, 5(Suppl 7):P24

**Background:** Rubber tree [*Hevea braziliensis* (Willd. ex Adr. de Juss.) Muell-Arg.], a native species of the Amazon Rainforest in South America, belonging to the Euphorbiaceae family, is world's major source of natural latex. Brazil, despite being the center of origin and diversity of the species and latex main producer at the end of XIX century, nowadays imports around 70% of the rubber consumed in the country. Although the Amazonian basin provides optimal conditions for rubber plantation, the occurrence of South American leaf blight (SALB) disease, caused by the ascomycete *Microcyclus ulei*, limits the rubber production in
this region. Therefore, rubber plantation has been extended to sub-optimal areas that are prominently located in northeast India, Vietnam, southern China and southern plateau of Brazil. Besides the new conditions for crop development, these new areas present stress conditions like low temperatures, dry periods and wind. It was shown that low temperatures affect the development and latex production of rubber trees. Breeding programs have been searching for clones adapted to these suboptimal areas. A complete cycle of breeding and selection takes at least 20 years to succeed in obtaining a superior clone. As such, development of new methods for early evaluation is essential to reduce and optimize the breeding management. Aiming to help in the development of these methods, in this work, we have constructed a leaf cDNA library to study ESTs (Expressed Sequence Tags) from cold stressed rubber trees. The originated sequences from this library can be used to create an ESTs databank and will provide genetic knowledge about how rubber trees cope with chilling stress, being also a valuable source of polymorphic molecular markers, such as SSRs and SNPs. Some of these markers may be associated to chilling tolerance, and early evaluation would be possible in young rubber tree clones.

**Methods:** The young *Hevea Braziliensis* Muell. Arg. clones GT1, IAN873, PR255 and PB217 were obtained from Agronomic Institute of Campinas (IAC). GT1, PR255 and PB217 clones are genitors of mapping populations of ongoing projects at Molecular Genetics and Analysis Laboratory and IAN873 clone showed chilling tolerance in field. The 24h cold treatment was performed in a diurnal growth chamber (8°C, 12h photoperiod). The collected leaves were previously wrapped up in tinfoil to prevent transcript redundancy. Leaves were sampled in intervals of 6h, 10h and 24h and total RNA was extracted by LiCl precipitation. Total RNA extracted from clones were pooled in equal amounts, according to time of sampling. The In-fusion SMARTer cDNA Library Construction Kit (Clontech) was used to construct the cDNA library. A sequenced plaque

(96 clones) of each sub-library (6h, 10h and 24h) was chosen for a preliminary analysis of the library. Sequences were analyzed and clustered by the DNAStar package (DNAStar, Inc.) and Blast2Go software (http://www.blast2go.org) was used to annotate the contigs and singlets. The resulting ESTs were screened for microsatellite regions using Simple Sequence Repeat Identification Tool (SSRIT) (http://www.gramene.org/db/markers/ssrtool).

**Results and discussion:** The cold stressed leaf cDNA library has around 6,000 clones and is sub-divided into three libraries: "cold-6h", "cold-10h" and "cold-24h", having 2,000 clones each. The average fragment lengths from these libraries are 780bp, 625bp and 930bp, respectively. In order to perform a preliminary analysis of the libraries expression profiles, 96 sequenced clones from each library were chosen randomly. Among the 288 sequences analyzed, 87 were discarded due to sequencing reaction failure or sequencing slippage. Clustering of the remaining 201 sequences generated eight contigs and 180 singlets. All contigs and singlets were annotated and distributed into functional categories. "Hypothetical and Unknown Proteins" was the major functional category, containing 52 sequences (26%). Most of these sequences (25) belong to "cold-24h" library. Second larger functional category was "Metabolic Processes", constituted by 34 sequences (17%). It was also in that category that libraries showed a visible difference among them: while about 20% of the sequences of libraries "cold-6h" and "cold-10h" is involved in cellular metabolic processes, in the "cold-24h" library this number falls to 13%. Chilling stress mainly causes alterations in metabolic processes and decrease in enzymatic activities. An exposure of 24 hours to low temperatures seems to be enough to trigger such a change in stressed rubber trees, which maybe could explain the higher number of hypothetical proteins in that library; however analysis of more sequences is necessary to confirm the results. Sequences that presented no similarity to any sequence in GenBank database were grouped in the "No Match"

<figure><img src="image_26.png" /></figure>
category, which is third in number of sequences (22; 11%). These sequences may be specific to *Hevea Braziliensis* and several presented at least one ORF that could be translated. The relative distribution of all annotated ESTs into functional categories can be seen in figure 1. The presence of microsatellite regions in the sequences was also verified and 30 ESTs sequences bearing SSR regions (15%) were found: 13 dinucleotide (43%), 9 trinucleotide (30%) and 8 tetranucleotide motifs (27%). Microsatellite regions were present mostly in hypothetical protein sequences (13; 43%) and all of them were in the UTR regions. Primers for these sequences are being designed in order to evaluate their polymorphism. The sequencing process of the cold stressed leaf cDNA library is still ongoing. The comparison of a small number of sequences showed a difference in expression pattern between the sub-libraries that may be confirmed by a wide analysis of their sequences. A preliminary analysis for microsatellite regions suggested that around 15% of the sequences may have potential polymorphic SSR markers. Moreover the ESTs generated will be a rich source for polymorphic SNP markers. SNP and SSR molecular markers will be developed from these data and will be used in *H. Braziliensis* genetic mapping. These markers could provide tools for early evaluation of cold tolerant rubber tree clones, among other genetic studies.

P25

NextGen sequence analysis of two sex-linked *P. tremuloides* genomic regions on chromosome 19

Matthias Fladung<fnref n="1" />, Birgit Kersten

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):P25

**Background:** Sex determination in poplars is still an open question. In the male *P. tremuloides* parent we identified several sex-linked SSR markers and mapped them to a central position on the male map of linkage group XIX.

**Material and methods:** Using DNA probes derived from these SSR markers, two BAC clones were isolated from a BAC library of the *P. tremuloides* male clone (Pakull B. *et al.*, 2011, Can. J. For. Res. 41, 245-253) and further analyzed by 454 sequencing (GATC Biotech AG). The generated single end reads were assembled to contigs using Newbler and Mira. The set of combined contigs (Newbler and Mira) of each BAC was subjected to scaffolding to SeqMan Pro (DNASTAR Lasergene) together with the related BAC end sequences created by Sanger sequencing (Pakull B. *et al.*, 2011, Can. J. For. Res. 41, 245-253). The subsequent combination of the created scaffolds to BAC consensus sequences was assisted by Sanger sequencing of PCR amplified scaffold ends and connections.

**Results:** Mira created more contigs for both BACs than Newbler, where the N50-contig size was similar for both methods (~10,000 bp). The largest contig (38,518 bp) was assembled by Mira. Based on our strategy we already created a draft sequence of ~47,500 bp for one BAC clone. We expect a size of about 50 kb for the second BAC after finishing scaffolding.

**Conclusions:** Based on the final consensus sequences of both BACs, we will search for putative sex-determining regions or genes in the *P. tremuloides* genome and compare them with *P. trichocarpa* paralogs.

P26

A genetic linkage map for a Full sib population of *Eucalyptus grandis* using SSR, DArT, CG-SSR and EST-SSR markers

Martín García<fnref n="1" />*, Pamela Villalba<fnref n="1" />, Cintia Acuña<fnref n="1" />, Javier Oberschelp²,
Leonel Harrand², Mauro Surenciski², María Martínez<fnref n="1" />, César Petroli³,
Carolina Sansaloni³, Danielle Faria³, Dario Grattapaglia³,

Susana Marcucci Poltri<fnref n="1" />

E-mail: mgarcia@cnia.inta.gov.br

BMC Proceedings 2011, 5(Suppl 7):P26

**Background:** *Eucalyptus* are the most widely planted hardwood trees in the world, occupying globally more than 18 million hectares, as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Intensive planting programs of *Eucalyptus grandis* have been carried out in the Argentinian Mesopotamia.

Linkage maps are useful tools for quantitative trait loci (QTL) analyses and detection. Several maps for QTL analyses of growth and wood quality have been developed in this genus [1,2] and most of the *E. grandis* maps have been carried out in interspecific crosses. Improved marker density in genetic maps, high-throughput techniques and transferability across species are key aspects to increase resolution and speed for a variety of genomic applications in *Eucalyptus*. In this context, an important issue in association studies is the selection of appropriate mapped candidate genes that co-localize with QTL of interest.

As part of the Biotech MERCOSUR project (Marcucci et al., this journal), we here report the construction of a genetic linkage map for *E. grandis* in the context of a QTL study of this specie in an effort to understand the molecular basis for quantitative trait variation in wood quality. This map includes Diversity Arrays Technology (DArT) [3], microsatellite (SSR) markers [4], Candidate Genes-SSR (CG-SSR) for wood quality traits and stress responses functions and Expressed Sequence Tag-SSR (EST-SSR) for putative function related to stress responses and other functions (Acuña et al., this journal). These CG-SSR and EST-SSR were not mapped in *Eucalyptus* previously.

**Material and methods:** Plant material: *E. grandis* x *E. grandis* (EG-INTA-161 x EG-INTA-152) F1 population of 130 full-sib progeny cloned (3 ramets) and planted in 2007 in Entre Ríos, Argentina, was analyzed.

**Genotyping:** The parents of the mapping cross were initially screened with: 55 SSR, 12 CG-SSR and 37 EST-SSR markers; these last two classes of markers derive from a broad study (for details see Acuña et al., this journal). Capillary electrophoresis and fluorescent detection were carried out on an ABI 3130xL Genetic Analyzer. A DArT Microarray of 7,860 clones was screened for useful polymorphic markers.

**Linkage and bioinformatic analysis:** All loci were tested for goodness of fit to expected Mendelian segregation ratios using Chi-square goodness of fit tests. The assignment of DArT sequence function was performed using the Blast2GO software [http://www.blast2go.org/]. A consensus genetic linkage map was constructed with JoinMap v3.0 [5]. Linkage parameters were set as 10 minimum LOD and 0.4 maximum recombination fractions.

**Results and discussion:** In this intraspecific *E. grandis* population, 78% of the SSR markers tested could be mapped. Most mapped SSR loci were fully informative, segregating in approximate ratios of 1:1:1:1 (either heterozygous in both parents four alleles in total). Seven SSR loci that followed approximate segregation ratios of 1:1 (heterozygous in only one parent) were EMBRA 47,51,101,179,676,1244,2010.

From the DArT Microarray of 7,860 clones, 31% of the marker were selected because of their high call rate (>0.80) and polymorphism between parents.

A large proportion (1,503/2,381=63%) of the DArT markers displayed a Mendelian behavior indicating that they sample single copy regions and provide markers that can be used for genetic analyses (65% segregating in 1:1 ratio and 35% in 3:1 ratio).

The map was assembled with 1032 markers, including 976 DArT, 43 SSR loci (2-6 per linkage group), seven EST-SSR and six CG-SSR. The resulting integrated map featured the expected 11 major linkage groups, yielding a genome coverage of 1358.4 cM, and an average consecutive intermarker distance of 1.3 cM in accordance to other reports [1,4]. Linkage groups were numbered following the standardized nomenclature for Eucalyptus proposed by Brondani et al [4].

The six Candidate Genes and seven ESTs include enzymes involved in lignin and cell-wall polysaccharide biosynthesis and stress responses genes, while 267 DArT (29.7%) were assigned to a gene ontology (GO) categories and 296 loci (32.9%) had significant matches with the nonredundant protein database using BLASTX. Thus, 25 enzymes in 56 metabolic pathways were represented by at least one sequence with its corresponding EC number.

The inclusion of common previously mapped SSR markers in several different eucalypt species within the subgenus *Symphyomyrtus* (*E. globulus*, *E. camaldulensis*, *E. dunnii*, *E. tereticornis*, and predominantly *E. grandis* and *E. urophylla*) allowed comparison of linkage groups among this *E. grandis* population and other species of the genus. Linkage orders previously reported in *E. globulus*, *E. grandis* and *E. urophylla* were also observed in this intraspecific *E. grandis* population, supporting the developed map.

<footnote n="1">¹Instituto Nacional de Tecnología Agropecuaria. Instituto de Biotecnología. De Los Reseros y Dr. Nicolás Repetto s/nº, CP 1686, Hurlingham, Buenos Aires, Argentina; ²EEA INTA Concordia. Grupo de Mejoramiento Genético Forestal. Estación Yuquerí s/n, CP 3200, Concordia, Entre Ríos, Argentina; ³Embrapa Recursos Genéticos e Biotecnologia - Parque Estação Biológica - PqEB - Av. W5 Norte (final). Caixa Postal 02372, 70770-970 - Brasília, DF - Brazil</footnote>
**Conclusions:** In this work, 13 new functional SSR and 976 DArT (296 with assigned functions) markers are mapped in an intraspecific *E. grandis* F1 population. This map will be used to locate QTL for wood quality and growth traits in the specie. Also, this map can help to identify candidate genes and regions in the *Eucalyptus* genome useful for fine scale analysis with association studies that are being developed by our group (see Cappa et al., this volume). The putative functional approach combined with the genetic linkage mapping provides an advantage tool for future analysis in locating genes of interest in *Eucalyptus*.

References

1. Petroli CD, Sansaloni CP, Kilian A, Steane DA, Myburg AA, Pappas GJ, Faria DA, Vaillancourt RE, Grattapaglia D: A high-density sub-centiMorgan integrated DArT/microsatellite genetic linkage map for species of *Eucalyptus* based on 2,980 markers. *Resumos do 56º Congresso Brasileiro de Genética* 2010 [http://web2.sbg.org.br/congress/sbg2008/pdfs2010/GP159-34030.pdf].

2. Thumma BR, Southerton SG, Bell JC, Owen JV, Henery ML, Moran GF: Quantitative trait locus (QTL) analysis of wood quality traits in *Eucalyptus nitens*. *Tree Genet Genom* 2010, 6:305-317.

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

4. Brondani RP, Williams ER, Brondani C, Grattapaglia D: A microsatellite-consensus linkage map for species of *Eucalyptus* and a novel set of 230 microsatellite markers for the genus *BMC Plant Biology* 2006, 6:20.

5. Stam P: Construction of integrated genetic linkage maps by means of a new computer package: JoinMap. *Plant J* 1992, 3(5):739-744.

P27

QTL associated with resistance to defoliation (*Cylindrocladium pteridis* in *Eucalyptus spp*.

Talyta G Zarpelon¹, Lúcio MS Guimarães¹*, Marcelo M Coutinho²,
Braz Cápua Neto¹, Danielle A Faria³, Dario Grattapaglia³, Acelino C Alfenas¹
¹Universidade Federal de Viçosa, Brazil; ²Clonar Resistência a Doenças
Florestais, Brazil; ³Embrapa Cenargen, Brazil
E-mail: luciomsguimaraes@yahoo.com.br
BMC Proceedings 2011, 5(Suppl 7):P27

Leaf blight and defoliation caused by *Cylindrocladium pteridis* is one of the main leaf diseases found in Brazilian eucalypt plantations in warm and high rainfall regions. The inter and intraspecific variability in the intensity of defoliation indicate that your control can be achieved by planting resistant genotypes. In order to guide breeding programs and to predict the stability of resistance is essential to understand the genetic control of resistance in eucalypts to defoliation. Thus, the objectives of this paper were to map QTLs (Quantitative Trait Loci) associated with resistance to defoliation in a *Eucalyptus urophylla* x *E. camaldulensis* (EU11 x EC06) family and validate microsatellite markers linked to QTLs in four unrelated pedigrees involving different *Eucalyptus* species. Defoliation was evaluated on the branches of the lower third of the plants inoculated under controlled conditions. We identified five QTLs for resistance to defoliation (Rd), three on the map of the parental EU11, Rd-1 and Rd-2 located on linkage group (LG) 1 and Rd-4 located on LG6; and two QTLs located on the map of the parental EC06, present in LG2 (Rd-3) and LG8 (Rd-5). Markers linked to QTLRd-2 were validated in two unrelated families by simple linear regression analysis. This is the first genomic map of QTLs associated with resistance to defoliation caused by *C. pteridis* in *Eucalyptus*.

P28

Genomic of resistance for *Fusarium circinatum* in *Pinus radiata*

Rodrigo Hasbun¹*, Priscila Moraga², Pamela Wachtendorff³, Carolina Iturra³, Angela Carrasco², Claudio Balocchi⁴, Sofia Valenzuela²

¹Genomica Forestal S.A. Edificio Centro de Biotecnologia s/n. Universidad de Concepción. Concepción, Chile; ²Facultad de Ciencias Forestales. Universidad de Concepción. Concepción, Chile; ³Genomica Forestal S.A. Edificio Centro de Biotecnologia s/n. Universidad de Concepción. Concepción, Chile;

⁴Bioforest S.A. Concepción, Chile

E-mail: rodrigohasbun@udec.cl

BMC Proceedings 2011, 5(Suppl 7):P28

The pitch canker fungus *Fusarium circinatum* was first associated with mortality of *P. radiata* in California during the 1980s. Since then it has been dispersed to several countries of the Northern hemisphere together with South Africa and Chile in the southern hemisphere. In Chile the disease caused mortality of seedlings in *P. radiata* at nursery, and in South Africa the pathogen has already completed its life cycle in plantations. This situation could be replicated in Chile, in the short-term, becoming then a major problem due to that most of the commercial plantations correspond to radiata pine. Research and breeding programs to ensure the availability of resistant trees are essential.

Controlled inoculation trials using different pine species have shown the high susceptibility of *P. radiata* [1]. Different families of radiata pine in Australia, New Zealand and Chile have been screened, showing a considerable genetic variation for lesion length and a high heritability depending on the population, suggesting that selection for resistance should result in useful genetic gains [2]. Based on the above findings a molecular approach of pitch canker resistance in *P. radiata* was carried out. This study focuses on the discovery of genome regions associated with resistance and the genes involved in the response mechanism(s), to develop genomic tools for breeding resistance.

The resistance to *F. circinatum* was investigated using a Quantitative Trait Locus (QTL) mapping strategy in a full-sib family of *P. radiata*. This mapping project used a double pseudo testcross approach to map each of the parents with dominant markers. Microsatellite (SSR) and Amplified Fragment Length Polymorphism (AFLP) markers were used to genotype the two parents (XO and XP) and the progeny (86 clones). Both parental maps were joined using the co-dominant SSR and AFLP markers to obtain a saturated genetic map. For each clone (approximately 8 ramets/clone) inoculations were carried out at Bioforest S.A. and lesion lengths were measured 90 days later. Phenotypic data together with genetic map allowed identified and positioned QTLs that control part of the pitch canker resistance in the progeny. Using Windows QTL Cartographer three QTLs were detected for the trait of interest for both parents.

References

1. Hodge G, Dvorak W: Differential responses of Central American and Mexican pine species and Pinus radiata to infection by the pitch canker fungus. *New Forest* 2000, 19:241-258.

2. Matheson A, Devey M, Gordon T, Werner W, Vogler D, Balocchi C, Carson M: Heritability of response to inoculation by pine pitch canker of seedlings of radiata pine. *Australian Forestry Journal* 2006, 70:101-106.

P29

Genetic linkage map of willow (*Salix leucopithecia*×*S.erioclada* L) based on AFLP and SSR markers

Jian-Jun Hu*, Jin-Hui Lv, Meng-Zhu Lu
Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, PR China
E-mail: hujj@caf.ac.cn
BMC Proceedings 2011, 5(Suppl 7):P29

Due to its fast growth rate and little nursery requirements, willow is largely planted as short rotation coppice species for bioenergy resources in temperate regions. Willow coppices can be easily established by vegetative propagation of cuttings and can remain productive for up to 25 years. In China, diploid species *S. leucopithecia* and *S. erioclada* L. are two promising willow species that can be used for high biomass plantation. In order to further improve the biomass yield and resistance to drought and pests, we tried to create a genetic map to assist willow breeding. 560 F₁ individuals from a cross between *S. leucopithecia* x *S. erioclada* L were used to generate a willow genetic map using AFLP and SSR markers.

Both *Populus* and *Salix* are members of the Salicaceae. They share genomic homologues with high similarity and many common biological traits. As a model species for biological studies in trees, *Populus* has considerable genetic and genomic resources, which could provide cues for willow study. Five hundred pairs of SSR primers were selected from *Populus* database for willow analysis; 88 of them successfully generated polymorphic loci between *S. leucopithecia* x *S. erioclada* L. In addition, 24 pairs of AFLP primers also succeeded in detecting polymorphic fragments from F₁ individuals.
We selected 243 individuals to construct a framework linkage maps of parents using a two-way pseudo-testcross strategy. The linkage map of *S. erioclada* L contains 98 loci, organized in 15 linkage groups with total map distances between 76.7 cM and 209.5 cM. The average map distance between markers is 18.6 cM and the total genetic distance is about 1,810 cM, covering estimated 77.13% of the genome. The linkage map of *S. leucopithecia* contains 60 loci, arranged into 9 groups with map distances between 63.5 cM and 149.1 cM. The average map distance between markers is 19.57 cM, and the total genetic distance is about 1,037 cM, covering estimated 70.3% of the genome.

P30

Quantitative trait locus analysis of growth and wood density in an interspecific pseudo-backcross population of *Eucalyptus grandis* x *E. urophylla*

ARK Kullan¹*, MM Van Dyk¹, Nicky Jones², Arnulf Kanzler², Arlene Bayley³, AA Myburg¹

¹Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa; ²Sappi Forests Research, Shaw Research Centre, PO Box 473, Howick, 3290, South Africa;

³Sappi Technology Centre, PO Box 6, The Innovation Hub, Lynnwood, Pretoria 0087, South Africa

E-mail: s28583303@tuks.co.za

BMC Proceedings 2011, 5(Suppl 7):P30

**Background:** F1 hybrids of *E. grandis* and *E. urophylla* are commonly grown for pulp and paper production in clonal plantations in tropical and subtropical regions. Improving tree growth [1] and wood quality [2] are important objectives in eucalypt breeding programmes. The efficiency of selection for these traits can be enhanced by molecular breeding approaches enabled by high-throughput, genome-wide genotyping technologies and the recent completion of a reference genome sequence for *Eucalyptus* (*E. grandis* V1.0, JGI, http://www.phytozome.net). In this context, interspecific hybrid pedigrees are valuable for quantitative trait locus (QTL) dissection in *Eucalyptus* as there is abundant interspecific variation in such pedigrees. Breeding for wood property traits is mainly focussed on increasing pulp yield per hectare, reducing wood specific consumption (WSC) and increasing the efficiency of downstream processing by the pulp and paper industry. In this study, we are characterizing the genetic architecture of growth and wood quality traits in an interspecific pseudo-backcross mapping population of an F1 hybrid of *E. grandis* and *E. urophylla*.

**Methods:** A pseudo-backcross pedigree was developed by crossing an F1 hybrid (*E. grandis* x *E. urophylla*, GUSAP1, Sappi Forest Research) clone to non-parental individuals of the two parental species. Diameter (cm) at breast height (DBH) and wood basic density were measured at three years in a set of 305 and 319 progeny of the *E. grandis* and *E. urophylla* backcross families, respectively. The backcross progeny

were also genotyped with more than 1700 polymorphic Diversity Arrays Technology (DArT) markers. Framework genetic linkage maps were constructed for the *E. grandis*, *E. urophylla* backcross parents using 139 and 127 selected testcross (1:1) DArT markers distributed across the 11 *Eucalyptus* linkage groups spanning 926 cM and 1100 cM, respectively. The framework maps of the F1 hybrid contained 172 and 154 testcross DArT markers spanning 1061 cM and 1036 cM in the *E. grandis* and *E. urophylla* backcross families, respectively. The average marker interval of the parental framework maps ranged from 5.6 (F1 hybrid in the *E. grandis* backcross family) to 8.0 (*E. urophylla* backcross parent). DBH of the main stem was measured directly for three-year-old backcross progeny. For the assessment of basic density, a wood disk was taken at a height of 1.5 m. The wood disk from each tree was used to determine basic density by the water displacement method. QTL analysis was conducted using Windows QTL Cartographer using composite interval mapping (CIM) and a genome-wide significance threshold of α = 0.05.

**Results and conclusion:** A total of 9 QTLs were identified for DBH and 15 QTLs were identified for basic density (Table 1). These explained 3.1 to 9.6% and 3.6 to 13.1% of the phenotypic variation in the *E. grandis* and *E. urophylla* backcross families, respectively. Higher numbers of QTL were identified in the F1 hybrid parent (15) compared to the backcross parents (9) congruent with the expected interspecific and intraspecific genetic variation segregating in the backcross families.

Comparative QTL mapping in the F1 hybrid and backcross parents identified common and unique QTL regions in the parental maps. On LG6 and LG10, QTLs for DBH and basic density were detected in both F1 hybrid maps (*E. grandis* and *E. urophylla* backcross families). On LG4 and LG9, QTLs for density were shared between the *E. urophylla* and F1 hybrid parent, while a QTL for DBH on LG10 was shared between the *E. grandis* and F1 hybrid parents. Overall, six unique genomic regions with QTLs for DBH and eleven QTLs regions for basic density were identified in the two backcross families. The lower number of significant QTLs identified for DBH compared to that for wood basic density (Table 1) is consistent with previous QTL reports and genetic studies showing that wood quality traits have higher heritability than growth traits [2,3]. The genetic maps constructed and QTL regions identified in this study provide a resource for identifying markers for molecular breeding of volume growth and wood density in *E. grandis* x *E. urophylla* hybrids, as well as candidate gene identification based on the recently released *E. grandis* reference genome sequence.

References

1. Grattapaglia D, Plomion C, Kirst M, Sederoff RR: Genomics of growth traits in forest trees. *Current Opinion in Plant Biology* 2009, 12:148-156.

2. Raymond CA: Genetics of *Eucalyptus* wood properties. *Annals of Forest Science* 2002, 59:525-531.

3. Freeman JS, Whittock SP, Potts BM, Vaillancourt RE: QTL influencing growth and wood properties in *Eucalyptus globules*. *Tree Genetics & Genomes* 2009, 5:713-722.

<table><caption>**Table 1(abstract P30) Putative QTL for DBH and basic density identified by CIM in *E. grandis* and *E. urophylla* BC mapping population**</caption><thead><tr><th>Parent tree</th><th>Total number of QTLs identified</th><th>Linkage group (LG) number for DBH</th><th>Linkage group (LG) number for density</th><th>Percentage of variation explained by the DBH QTLs</th><th>Percentage of variation explained by the density of QTLs</th></tr></thead><tbody><tr><td><em>E. grandis</em> backcross parent</td><td>3</td><td>LG9, LG10</td><td>LG2</td><td>3.6 to 4.6%</td><td>4.8%</td></tr><tr><td><em>E. grandis</em> F1 hybrid parent</td><td>7</td><td>LG4, LG6, LG10</td><td>LG1, LG3, LG4, LG10</td><td>3.7 to 9.6%</td><td>3.1 to 5.5%</td></tr><tr><td><em>E. urophylla</em> F1 hybrid parent</td><td>8</td><td>LG6</td><td>LG2, LG4, LG6, LG8, LG9, LG10a, LG10b</td><td>7.0%</td><td>3.6 to 13.1%</td></tr><tr><td><em>E. urophylla</em> backcross parent</td><td>6</td><td>LG4, LG9, LG10</td><td>LG4, LG8, LG9</td><td>4.5-5.3%</td><td>4.6-7.8%</td></tr><tr><td>Total</td><td>24</td><td>9</td><td>15</td><td></td><td></td></tr></tbody></table>

QTL, Quantitative trait loci; DBH, Diameter at breast height; CIM, Composite interval mapping.
P31

Assessment of SNPs for linkage mapping in *Eucalyptus*: construction of a consensus SNP/microsatellite map from two unrelated pedigrees

Bruno Marco Lima¹*, Orzenil B Silva-Junior², Danielle A Faria²,
Eva MC Mamani², Georgios J Pappas³, Dario Grattapaglia³

¹EMBRAPA Genetic Resources and Biotechnology, EPqB 70770-910, Brazilia,
DF, Brazil / Department of Genetics, University of São Paulo - ESALQ/USP,
Brazil; ²EMBRAPA Genetic Resources and Biotechnology, EPqB 70770-910,
Brazilia, DF, Brazil; ³EMBRAPA Genetic Resources and Biotechnology, EPqB
70770-910, Brazilia, DF, Brazil /Genomic Sciences Program, Catholic University
of Brazilia, SGAN 916 Brazilia, DF, Brazil

E-mail: bmlima@esalq.usp.br

BMC Proceedings 2011, 5(Suppl 7):P31

**Background:** High throughput genotyping of SNP (Single Nucleotide Polymorphisms) based markers has been developed for an increasing number of plant and animal species. In forest trees large scale SNP development has been approached mainly by amplicon resequencing targeting specific genes for association genetics studies. This approach, although successfully employed in conifers aided by the use of haploid tissue, is technically laborious in diploids because of the very high levels of nucleotide and indel diversity in highly heterozygous tree genomes. Direct SNP development from large *in silico* sequence resources developed by next-generation sequencing is now a very efficient approach for SNP development in forest trees. We have recently developed a first set of 768 SNPs assayed by the Golden Gate Genotyping Technology for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database [1]. We saw that a careful sequence quality assessment and the application of stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. With the exception of 72 SNPs specifically selected in 20 candidate genes putatively associated with relevant wood phenotypes, all remaining validated SNPs were randomly picked based solely on in silico quality. In this study we wanted to position these SNPs relative to microsatellites and assess their information content for linkage map construction. To enhance our ability of mapping SNPs we employed two eucalyptus full-sib families involving four different Eucalyptus species.

**Material and methods:** Two inter-specific segregating populations of *Eucalyptus*, population IP (*E. grandis* x *E. urophylla*) and DGUGL [[*E. dunnii* x *E. grandis*] x (*E. urophylla* x *E. globulus*)] were used for linkage analysis and map construction. DNA was extracted through CTAB and picogreen quantified. The DNA was used for SNP genotyping on an Illumina BeadStation 500 GX. SNP data were analyzed using GenomeStudio V2009.1 and a GeneTrain score cutoff of 0.25 and call rate ≥0.95 were initially applied to the whole dataset. After that, every single SNP was manually checked for genotyping failures and potential calling errors that would bias the overall analysis and segregation ratios. Linkage analysis, individual and consensus map construction for both segregating populations was performed using Joinmap, v. 3.0 [2].

**Results:** The proportion of informative segregating SNPs out of the 768 assayed were similar in the two mapping populations: 215 SNPs in DGUGL (28%) and 239 in IP (31%). SNPs were mapped on top of existing microsatellite maps. The linkage map of the IP population with 409 markers on the 11 expected linkage groups had 215 microsatellites and 194 SNPs, with an observed length of 1,581.3 cM and average distance between markers of 3.9 cM. The DGUGL map also had 11 linkage groups and 430 markers being 236 microsatellites and 194 SNPs with an estimated length of 1,252.4 cM and average distance of 2.9 cM. The consensus map, constructed using both segregating populations included 624 unique markers, of which 320 were microsatellites and 304 SNPs with 1,451.4 cM and average distance of 2.3 cM. The proportion of segregating SNPs in each population individually (28 to 31%) was consistent with a within-species level observed heterozygosity of ~50% for this set of 768 SNPs (Grattapaglia et al. 2011), reminding that none of these parents were used in the generation of the EST database wherefrom these SNP were derived. The rate of mappable SNPs was enhanced to almost 40% (304 SNPs mapped out of 768) by using two mapping populations suggesting that by sampling more full-sib families it should be possible to map most if not all SNPs in this panel. No evident clustering of SNPs was observed suggesting that these SNPs are randomly distributed in the Eucalyptus genome.

**Conclusions:** This study shows that large numbers of informative SNPs can be developed directly from in silico sequence databases involving unrelated individuals to the parents of mapping populations. Evidently, by being biallelic, SNPs will be less efficient than multiallelic microsatellites for linkage mapping purposes. This drawback however is clearly compensated by the much higher throughput, automation and lower cost of SNP genotyping. While this is the first reported sizeable scale SNP mapping effort in Eucalyptus, a larger number of informative SNPs mapped at regular intervals will be necessary for broader applications. For example, to implement Genomic Selection in most Eucalyptus breeding programs some 4,500 to 6,000 informative SNPs will be necessary to provide a marker density of 3 to 5 markers/centiMorgan and reach selection accuracies above 70% [3]. This would require a SNP panel of some 9,000 to 12,000 SNPs. While this goal is fully achievable, per sample genotyping cost issues, however, will have to be considered before larger scale SNP developments are undertaken in Eucalyptus. Benchmarking the Golden Gate or Infinium genotyping technologies against other high throughput systems such as DArT and emerging genotyping-by-sequencing methods will eventually define how SNP variants will be assayed in Eucalyptus and for that matter in several other highly heterozygous forest tree species.

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

**References**

1. Grattapaglia D, Junior OBS, Kirst M, Lima BM, Faria DA, Jr GJP: High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species. *BMC Plant Biology* 2011, 11.

2. Stam P: Construction of Integrated Genetic-Linkage Maps by Means of a New Computer Package - Joinmap. *Plant Journal* 1993, 3:739-744.

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

P32

Identification of a novel QTL contributing to rust resistance in *Eucalyptus*

Bruno M Lima¹*, Juliana EC Teixeira², Rodrigo Gazaff¹, Antonio AF Garcia¹,
Dario Grattapaglia³, Raphaelle KD Valle⁴, Luis EA Camargo⁴

¹Departamento de Genética, Universidade de São Paulo - ESALQ/USP,
Piracicaba, SP, Brazil; ²Fibria, Jacarei, SP, Brazil; ³EMBRAPA Recursos Genéticos e Biotecnologia, Brazilia, DF, Brazil; ⁴Departamento de Fitopatologia e Nematologia, Universidade de São Paulo - ESALQ/USP, Piracicaba, SP, Brazil
E-mail: bmlima@esalq.usp.br

BMC Proceedings 2011, 5(Suppl 7):P32

**Background:** The genus *Eucalyptus* has many species that are well adapted to a wide range of environmental conditions in Brazil. However, in some areas diseases are a limiting factor, among which the Eucalyptus rust caused by *Puccinia psidii* Winter stands out as a destructive pathogen of the Myrtacea. The growth of plants with high levels of infection is severely compromised and these plants end up being dominated by adjacent plants which ultimately leads to their death. The most efficient method to control the disease is through the use of resistant genotypes. A major effect rust resistance QTL has already been identified [1,2], however a residual variance stays unexplained. In this context, our study aimed to identify genomic regions containing quantitative resistance loci to rust based on segregation data of a S<sub>1</sub> progeny from an inter-specific (*Eucalyptus grandis* x *E. urophylla*) resistant clone.

**Material and methods:** The progeny used in this study was generated by the breeding program of Fibria and is composed of 90 S<sub>1</sub> trees from the self-pollination of an *E. grandis* x *E. urophylla* hybrid tree. This hybrid (VCP-R) is heterozygous for a rust resistance locus [3] and highly resistant to rust. The plants were evaluated at two different sites with high incidence of rust starting in November 2005. Four disease severity evaluations were made at 90, 120, 150 and 180 days after planting by scoring the levels of disease using a four-score severity scale. Scores were used to calculate the area under the disease progress curve (AUDPC) for each plant and for BLUPs estimation. Genomic DNA was CTAB extracted from health young leaves and used in PCR amplifications of 121 microsatellite, 20 TRAP and 38 AFLP markers (totalizing 179 markers).
The Onemap software [4] was used in linkage analysis. The estimated BLUPs were used in QTL mapping [5]. The QTLs were mapped by interval mapping (IM) and composite interval mapping (CIM).

**Results:** Linkage analysis resulted in 11 linkage groups with 160 linked markers. The identity of the groups were as in [6] based on common microsatellite loci as references. The length of the map was 1075 cM with an average distance of 6.7 cM. IM analysis identified one QTL in linkage group 3 (LOD=7.7) with a large phenotypic effect that accounted for 28.5% of the variation (fig. 1). The negative additive effect was significant (p = 0.05), without dominance effects. QTL was located 1.6 cM from SSR EMBRA049 and 5.8 cM from AFLP E08M18_250. The CIM analysis mapped two QTLs, both located in linkage group 3. The peak LOD score (LOD=10.3) of QTL₁, which explained 39.5% of the phenotypic variation, mapped in the same interval of the QTL identified in the IM analysis. It was located close to Embra049 (1.6 cM) and presented significant negative additive effects and no significant dominance effect. The second QTL (QTL₂), with LOD=3.4, had a relative lower phenotypic effect (6.9%), mapped 3.1 cM close to the AFLP marker E08M18_300 AFLP. However, differently than QTL₁, QTL₂ presented both additive and dominance effects.

**Conclusions:** The use of the AUDPC permitted to transform discrete variables (symptoms scores) into a continuous variable, which gives greater power in QTL detection. The effect of the reduced size of the S₁

progeny in QTL detection was compensated by the BLUPs analysis, which considers only the genetic variation in QTL localization. QTL mapping was done by two different approaches (IM and CIM) that presented contrasting results. The non detection of QTL₂ in the IM analysis was probably due to the limitations of the method (e.g., non-independence in different intervals when there are more than one QTL and low precision of the position by the presence of linked QTLs). Previous studies on the inheritance of resistance to rust in eucalyptus identified a locus with large phenotypic effect, but also pointed to the existence of minor effects [1,2]. Our results confirm the hypothesis of the presence of at least one minor effect locus, by data analysis in quantitative approach and CIM, as never used before in Eucalyptus rust resistance mapping. This work revealed the existence of a second rust resistance locus in addition to one with large effect locus previously mapped in linkage group 3. The identification of markers linked to resistance loci gives perspectives of MAS of resistant genotypes, which is desirable in the case of this disease due to the technical and economical difficulties of selecting resistant genotypes based solely on phenotypes since this biotrophic requires large areas and skilled labor. In addition this study opens possibilities for the positional cloning of the resistance genes together with the availability of the genome sequence of eucalyptus.

**Financial support.** Fibria, CNPq, CAPES, ESALQ-USP.

<figure><img src="image_27.png" /><figcaption>**Figure 1 (abstract P32)** IM and CIM QTL mapping for Eucalyptus rust on linkage group 3.</figcaption></figure>
## References

1. Junghans DT, Alfenas AC, Brommonchenkel SH, Oda S, Mello EJ, Grattapaglia D: Resistance to rust (Puccinia psidii Winter) in Eucalyptus: mode of inheritance and mapping of a major gene with RAPD markers. *Theoretical and Applied Genetics* 2003, **108**:175-180.

2. Mamani EMC, Bueno NW, Faria DA, Guimaraes LMS, Lau D, Alfenas AC, Grattapaglia D: Positioning of the major locus for Puccinia psidii rust resistance (Ppr1) on the Eucalyptus reference map and its validation across unrelated pedigrees. *Tree Genetics & Genomes* 2010, **6**:953-962.

3. Teixeira JEC, Guedes FTP, Dias DC, Bonine CAV, Camargo LEA: Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection. *Tropical Plant Pathology* 2009, **34**:203-210.

4. Margarido GRA, Souza AP, García AAF: OneMap: software for genetic mapping in outcrossing species. *Hereditas* 2007, **144**:78-79.

5. Gazaffi R: Desenvolvimento de modelo genético-estatístico para mapeamento de QTLs em progênie de irmãos completos, com aplicação em cana-de-açúcar. *PhD Thesis* Universidade de São Paulo, Departamento de Genética 2009.

6. Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. *BMC Plant Biology* 2006, **6**.

P33

## Biotech MERCOSUR project: an integrated genotyping and phenotyping platform of *Eucalyptus* germplasm for mapping purposes

Susana N Marcucci Poltrini<fnref n="1" />, Nuno Borralho², José Carlos Rodrigues³, José Elizaur⁴, Mirtha Vera de Ortiz⁵, César Cardozo⁶, Guillermo Salvatierra⁷, Dario Grattapaglia⁸

E-mail: smarcucci@cnia.inta.gov.ar
BMC Proceedings 2011, 5(Suppl 7):P33

**Background:** The Biotech MERCOSUR project (85% founded by the European Union) launched in January 2009 and finished in December 2010. The general objective was to establish a regional scientific-technological network to develop a genotyping and phenotyping platform useful for the improvement of *Eucalyptus* in the region. Two advanced genomic strategies (genetic and association mapping) were applied to investigate the genetic basis of the industrial wood formation and energy production of *Eucalyptus* plantations. Up to date, no projects were carried out involving the four MERCOSUR countries altogether. The project brought together scientists from 3 MERCOSUR public research institutes and 2 industrial partners from 4 countries.

The project was possible thanks to the availability of five evaluated field trials of *Eucalyptus*, four segregating populations, a high-density Diversity Arrays Technology (DART) resource of wide genome coverage and high transferability technology for the genera [1], as well as phenotypic assessment methods of physical and chemical properties of wood using Near Infrared Reflectance (NIR) [2] together with the classical measurements of growth and wood quality tree properties.

The aim of this work is to present the main inputs and results achieved in the project.

**Materials and methods:** Plant material: Open pollinated (OP) progeny trial of *Eucalyptus grandis* planted in Argentina: 188 trees from 132 OP families (1 to 3 trees per OP family) from 13 native stand sites in Australia and 3 Argentinean land races.

Clonal trial of *E. grandis* planted in Paraguay: 123 clonal trees most of them with unknown provenances.

OP progeny trials of *E. globulus* planted in Uruguay: 169 trees from 132 OP families (1 to 8 trees per OP family) from 8 Australian geographic races and one Chilean land race.

OP progeny trial of *E. globulus* planted in Argentina: 134 trees from 129 OP families (1 or 2 trees per OP family) from 8 Australian geographic races and 2 landraces from Chile and Portugal.

Full-sib progeny trial of *E. grandis* planted in Argentina: 130 full-sib trees (see García et al. in this journal).

*E. grandisx E. urophylla*: described in [3].

**Genotyping:** Over 7,600 clones of DArT *Eucalyptus* microarray were screened.

From 12 to 19 polymorphic simple sequence repeat (SSR) markers [4] were used to estimate diversity parameters as well as inbreeding coefficients for the *E. grandis* and *E. globulus* populations. For map development, more than 300 SSR markers were tested.

**Growth, shape and wood quality tree measurements:** Standard methods were used to measure diameter at breast height, total height, stem straightness, pilodyn penetration and basic density for all population analyzed.

**Chemical wood properties for NIR spectroscopy predictions:** Lignin contents was measured by Klason method, extractives by an adjusted laboratory protocol, Syringyl:Guaiacyl ratio by analytical pyrolysis.

Pulp yields were calculated based on the oven-dry weight of wood chips charged to the reactor at Kappa number 18.

The NIR spectra were obtained by diffuse reflectance on a Bruker model MPA.

**Data analysis:** Several tools for data analysis were applied according to the objectives of the different approaches (preliminary association mapping, see Cappa et al.; genetic mapping, see García et al., this journal and [3]).

**Results:** 1. Germplasm characterization with DArT markers in two *E. grandis* and two *E. globulus* populations: between 2,300 and 2,816 DArT (call rate higher than 0.80, and polymorphic for more than 95% of individuals) were included in a preliminary association analysis (Cappa et al. in this volume).

2. Linkage Mapping for QTL detection with DArT (between 1500 and 2000 segregating markers per population), SSR, EST-SSR and candidate genes in *E. grandis* population (García et al., this volume) and in *E. grandis* x *E. urophylla* [3].

3. NIR-PLSR models were developed to assess extractives (ethanol and water) and lignin content (Klason and total) as well as lignin composition (S/G ratio) and pulping yield for *Eucalyptus globulus* and *E. grandis*. All models obtained were at least good enough for screening with RPD above 2 [2]. The RPD obtained varied with trait and species (*E. globulus*: extractives-2.3; Klason lignin-3.9; total lignin-3.8; S/G-3.8; pulping yield (K18)-3.5. *E. grandis*: extractives-5.9; Klason lignin-6.5; total lignin-3.2; S/G-4.2).

4. Phenotyping analyses: between 2,000 and 16,000 trees were characterized for 2 to 5 traits (diameter at breast height, total height, stem straightness, pilodyn penetration and basic density). This data for all population is available as a product of the project. The phenotypic distribution of all continuous traits was studied. Then, to deal with environmental variation, the data were analysed using a mixed linear model with separable first-order autoregressive residuals for rows and columns (i.e., with a standard spatial analysis).

5. Generation of clonal populations in Argentina and Uruguay for future association mapping studies: two *E. globulus* and one *E. grandis* (*n* > 200 each) for a more precise and thorough evaluation of phenotypes are being planted.

6. Origin assign for clone trees of unknown provenances. This was possible due to the combination of DArT markers analysis with population structure approaches. More than 50 clones were assigned to defined clusters.

7. Training of the participants involved: 4 lab training and 2 courses were developed for different participants of the project.

**Conclusions:** The genotyping and phenotyping platform was successfully established and encourage the collaboration between the participants of the different countries. Likewise different strategies have established the basis for developing new works in the near future using the resources and tools generated in the context of the Biotech MERCOSUR project.

**Acknowledgments:** C.Acuña, M.García, C.Martínez, P.Villalba, J.Oberschelp, L.Harrand, M.Surecinsky, P.Pathauer, E.P.Cappa, J.Diez, M.Marcó, N.Zelener, S.Torales, E.Hopp, G.Salvatierra, V.Escalada; C.Sansaloni, C.Petroli, D.Faría, J.Elizaul,M.Enciso, M.L.García, L.Monges. H.Nakayama, I.Peralta. BiotecSur UE 127118

<footnote n="1">¹Instituto Nacional de Tecnología Agropecuaria. Instituto de Biotecnología (INTA). De Los Reseros y Dr. Nicolás Repetto s/nº, CP 1686, Hurlingham, Buenos Aires, Argentina; ²Forestacion S.A. Oficina en Florida. Battle 843. Esq. Suarez, Montevideo, Uruguay; ³Instituto de Investigação Científica Tropical (IICT), Centro de Florestas e Produtos Florestais, Tapada Ajuda, 1349-017 Lisboa, Portugal; ⁴Desarrollos Madereros S.A. Supercarretera Itaipú Km. 32; Norte - Hernandarias, Paraguay; ⁵Facultad de Ciencias Agrarias. Universidad Nacional de Asunción, Paraguay; ⁶Centro Multidisciplinario de Investigaciones Tecnológicas / Dirección General de Investigación Científica y Tecnológica, Universidad Nacional de Asunción, Parguay; ⁷Garruchos S.A.Estancia Puerto Valle. Ruta Nacional N° 12 - KM 1.282, Corrientes, Argentina; ⁸Empresa Brasileira de Pesquisa Agropecuaria, Unidad Recursos Genéticos e Biotecnologia. Parque Estação Biológica - PQEB - Av. W5 Norte (final). Caixa Postal 02372 - Brasília, DF, Brazil</footnote>
## References

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

2. Williams PC, Sobering DC: Comparison of commercial near infrared transmittance and reflectance instruments for analysis of whole grains and seeds. *J. Near Infrared Spectrosc* 1993, 25-32.

3. Petroli CD, Sansaloni CP, Kilian A, Steane DA, Myburg AA, Pappas GJ, Faria DA, Vaillancourt RE, Grattapaglia D: A high-density sub-centiMorgan integrated DART/microsatellite genetic linkage map for species of *Eucalyptus* based on 2,980 markers. *Resumos do 56° Congresso Brazileiro de Genética* 2010 [http://web2.sbg.org.br/congress/sbg2008/pdfs2010/GP159-34030.pdf].

4. Brondani RP, Williams ER, Brondani C, Grattapaglia D: A microsatellite-consensus linkage map for species of *Eucalyptus* and a novel set of 230 microsatellite markers for the genus. *BMC plant biology* 2006, 6:20.

## P34

### A new set of 182 microsatellites for *Eucalyptus*: characterization and mapping in a four-species consensus linkage map

Daniela Faria¹*, Eva Mamani¹, Juliana Sena¹, Alexandre Alves¹, Clarissa Falcao¹, Rodrigo Lourenço¹, Georgios Pappas², Dario Grattapaglia²

¹Plant Genetics Laboratory, EMBRAPA Genetic Resources and Biotechnology, PgEB, CEP 70770-970, DF, Brazilia, Brazil; ²Plant Genetics Laboratory, EMBRAPA Genetic Resources and Biotechnology, CEP 70770-970, DF, Brazilia, Brazil, and Graduate Program in Genomic Sciences Biotechnology and Universidade Católica de Brasília, SGAN Qd 916, CEP 70790-160, DF, Brasília, Brazil

E-mail: danyafp@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P34

**Background:** *Eucalyptus* is the most widely planted hardwood crop in the tropical and subtropical world. Plantations of *Eucalyptus* species supply high-quality wood for industrial applications and are important sources of carbon neutral renewable energy in Brazil. *E. grandis* and *E. urophylla* and their hybrids are the most widely planted species in fast growing commercial forests in Brazil. *E. globulus* is the preferred raw material by the mills generating a pulp that is considered superior by the market. However as a pure species it does not grow adequately in Brazil but performs well in hybrid combinations. Breeding programs have increasingly incorporated *E. globulus* germplasm in fast moving elite populations. Molecular breeding in such populations will require information on markers, comparative mapping and QTL validation across pedigrees involving these different species. Highly multiallelic and transferable microsatellites not only are excellent tools for individual identification, but also provide robust and efficient framework genetic maps that serve well for mapping thousands of biallelic higher throughput markers such as Single Nucleotide Polymorphisms (SNPs) and Diversity Array Technology (DART). Furthermore microsatellites provide a powerful way for QTL validation across species. We describe the development and characterization of 182 new microsatellites, most of them derived from ESTs and some from a genomic shotgun library. These markers, together with other previously developed ones were used to build a consensus map involving three different pedigrees derived from intercrossing four species of *Eucalyptus*.

**Methods:** The source of ESTs used to mine microsatellites and the methods used to select loci and design primers were described earlier [1,2]. Data from a sample sequencing experiment, carried out back in 2003 using a Sanger sequenced shotgun genomic library, was used for microsatellite discovery (R.T. Lourenço, unpublished). Microsatellite, primer pairs were initially screened for assay success, polymorphism and inter-specific transferability by colorimetric detection on polyacrylamide gels. Selected loci had their forward primer re-synthesized with a fluorescence label and used in downstream genotyping. Loci were characterized using a set of 32 unrelated individuals each of *E. grandis* and *E. globulus*. Map construction was carried out for three different pedigrees of *Eucalyptus* one of them being the pedigree used earlier for a reference map construction [3]. Mapping was conducted using JoinMap 3.0 [4] with a minimum LOD score of 10. and marker order determined with the default mapping module parameters. Parameters of genetic information content (PIC polymorphism information content; PE = probability of paternity exclusion and PI = probability of identity) were estimated for all newly developed microsatellite markers for each species separately.

**Results:** Data mining of 22,298 unigenes provided 1,261 microsatellite loci. Most of them were (54.71%) trinucleotide repeats. After all screening steps, 494 microsatellites amplified discrete and polymorphic fragments and 210 were selected for fluorescence based genotyping. From the single-pass genomic shotgun library 41 microsatellites were also selected following then same screening steps. Genetic maps were initially built for each pedigree separately. A consensus map was then built by combining separately the homologous linkage groups of the three maps. The total number of microsatellite markers eventually mapped was 448 including the 234 previously mapped [3] and part of those published but not yet mapped earlier [1,2]. The map had an estimated total recombination distance of 1,297 cM and an average distance between adjacent markers of 3.48 cM. The linear order of markers was conserved on most linkage groups across the individual pedigree maps and the consensus map with no evidence of rearrangement of chromosomal blocks. For the 182 newly developed microsatellites characterized the allele size range did not vary between *E. grandis* and *E. globulus*. Only 15 loci for *E. grandis* and 17 loci for *E. globulus* were monomorphic in the set of individuals genotyped. The average number of alleles was very close in the two species, 4.6 for *E. grandis* and 4.8 for *E. globulus*. A significant estimate of null allele frequency was found for only seven loci and these coincided for the two species Average PIC, PE and PI for the set of loci were not substantially different between the two species. Furthermore the overall performance of the EST derived and genomic shotgun derived sets of microsatellite did not show any appreciable difference, somehow unexpectedly due to the generally lower polymorphism of genic microsatellites. The ascertainment bias introduced by the much larger set of EST derived microsatellites and rigorous screening applied might have contributed to this.

**Conclusion:** This work summarizes the development and characterization of a new set of 182 new microsatellites markers and presents a relatively dense microsatellite marker consensus map involving four different species of *Eucalyptus*. The new 182 microsatellites developed are robust and polymorphic enough to be used for applications in breeding programs that involve individual identification as well as for comparative QTL mapping and marker assisted selection.

**Acknowledgments:** This work was supported by the Brazilian Ministry of Science and Technology through CNPq grant 577047/2008-6 and FAP-DF Grant NEXTREE 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

## References

1. Faria DA, Mamani EMC, Pappas GJ, Grattapaglia D: Genotyping systems for *Eucalyptus* based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests. *Tree Genetics & Genomes* 2011, 7(1):63-77.

2. Faria DA, Mamani EMC, Pappas MR, Pappas GJ, Grattapaglia D: A Selected Set of EST-Derived Microsatellites, Polymorphic and Transferable across 6 Species of *Eucalyptus*. *Journal of Heredity* 2010, 101(4):512-520.

3. Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of *Eucalyptus* and a novel set of 230 microsatellite markers for the genus. *Bmc Plant Biology* 2006, 6:1-16.

4. Van Ooijen JW, R E: JoinMap 3.0, Software for calculation of genetic linkage maps. Wageningen, The Netherlands: Plant Research International 2001.

## P35

### Genomic characterization, high-density mapping and anchoring of DART markers to the reference genome of *Eucalyptus*

César Petroli¹*, Carolina Sansaloni¹, Jason Carling², Eva M C Mamani¹, Dorothy A Steane³, Alexander A Myburg⁴, René E Vaillancourt³, Andrzej Kilian², Georgios J Pappas⁵, Orzenil Bonfim da Silva⁵, Dario Grattapaglia⁵

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; ³School of Plant Science and CRC for Forestry, University of Tasmania, Private Bag 55. Hobart, Tasmania 7001. Australia; ⁴Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI).

University of Pretoria. Pretoria. 0002. South Africa; ⁵EMBRAPA Genetic Resources and Biotechnology - EPqB Final W5 Norte 70770-917 Brazilia DF, Brazil

E-mail: cesar@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P35
**Background:** Genetic linkage maps have been essential tools to examine the inheritance of qualitative and quantitative traits, to carry out comparative mapping and to provide markers for molecular breeding applications. Linkage maps for species of Eucalyptus have been reported for several pedigrees using different molecular marker technologies [1]. However improved marker density, throughput and transferability across species are necessary to increase resolution of current maps for a variety of genomic applications. We report the development of a high density linkage map for Eucalyptus based on microsatellites and DArT (Diversity Arrays Technology) markers generated by a standardized genotyping microarray [2]. DNA probes that constitute the DArT microarray were sequenced and positioned on the reference Eucalyptus genome providing information about their sequence content, their distribution relative to annotated genes as well as the relationship between physical and recombination distance in the Eucalyptus genome.

**Methods:** Map construction was carried out using an F1 progeny of 177 individuals derived from a cross between *E.grandis* and *E.europhylla*. Genomic representations of both parents and their F1 offspring were produced with the same complexity reduction method used to prepare the library (PstI/TaqI) generating the 'targets' for hybridizing to the arrays. Microarray imaging, data extraction polymorphism detection and marker scoring were carried out using DArTSoft v.7.44 (http://www.diversityarrays.com/). Polymorphic markers were filtered according to reproducibility, quality parameter (Q) and marker call rate as described earlier [2]. An integrated linkage map was constructed with JoinMap v3.0 [3] using a framework map of previously mapped fully informative anchor microsatellites. All 7,680 DNA probes that constitute the current DArT genotyping microarray were Sanger sequenced. Redundancy analysis was carried out at the sequence level using a 50bp minimum overlap, 98% identity, and allowing for 10% mismatch and gap size of one bp. These DArT probes were mapped on the annotated Eucalyptus reference genome as available in Phytozome. Distribution of DArT probes and mapped DArT markers relative to the predicted gene models was carried out by dividing the genome in 500 kbp bins corresponding approximately to a 1 cM recombination fraction.

**Results:** A segregation ratio filtering (both for 1:1 and 3:1 markers) together with a relaxed call rate threshold > 50% were initially applied to include the largest number of potentially mappable markers. A total of 4,271 DArT markers segregated 1:1 and 1,572 segregated 3:1. The complete dataset with 6,065 markers (5,843 DArT and 222 microsatellites) was submitted to a linkage analysis resulting in eleven groups at LOD ≥ 15 with a total of 2,484 markers. A subset of 1,032 markers positioned at high likelihood for ordering (864 DArTs and 168 microsatellites) provided a framework map with 1,176 cM and an average recombination distance between consecutive markers of 1.15 cM. When all the 2,484 mapped markers were fitted, the total recombining genome length increased to 1,303 cM with a resolution of 0.6 cM. A redundancy analysis of the 6,918 DArT probes for which sequences could be obtained resulted in 4,583 unique sequences (66%). The estimated redundancy of 34% represents a useful resource by providing alternative probes for detecting polymorphism in the same genomic region in different individuals. A total of 6,480 probes (93.7%) were confidently mapped onto the reference genome; 4,189 of them (65%) mapped to single positions while the remaining 2,291 probes mapped to a second position with a relatively high suboptimal alignment score and were therefore considered mapped at lower confidence. About 50% of these low confidence mapping (1,026) actually mapped to a sequence position containing a repeat element which might explain the mapping result. A total of 438 DArT probes could not be mapped to the current assembly. These DArT probes might further improve the current genome version by including some yet unassembled genome contigs. A total of 1,987 linkage mapped DArT markers for which sequence was available were positioned relative to the 41,208 gene models in the current genome, distributed in 500 kbp bins providing an average of 1.6 ± 2.4 DArT markers for the 34 ± 15 gene models present in each genome bin. Interestingly a total of 4,663 DArT probes (67%) mapped at less than ten basepairs from the nearest gene model and only 76 probes mapped at more than 10kbp. The largest distance from a DArT probe to the next gene was only 156 kbp and a modest although highly significant coincidence across the whole genome was seen between the number of DArT markers and the number of gene models (Spearman rank correlation R = 0.33 p<0.00000).

**Conclusions:** The linkage map presented in this work was used to aid the ordering of contigs during the assembly of the *Eucalyptus grandis* genome currently available through the JGI Phytozome website.

This map, together with other parallel mapping efforts that used this same genotyping platform have provided between 2,000 and 3,000 segregating markers irrespective of species of Eucalyptus. The mapping density achieved with the Eucalyptus DArT microarray provides unprecedented opportunities for comparative mapping across pedigrees, high resolution QTL analysis and molecular breeding applications across species of the genus. We have shown that not only the DArT marker sequences are highly enriched for genes, but that their distribution relative to the predicted gene models provides an extremely efficient tool to specifically tag genes at distances within the extent of LD in typical breeding populations. The use of this standardized high-throughput genotyping platform will therefore be instrumental to implement genome-wide selection strategies and positional cloning efforts in multiple Eucalyptus species.

**Acknowledgments:** This work was supported by the Brazilian Ministry of Science and Technology through CNPq grant 577047/2008-6 and FAP-DF Grant NEXTREE 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

**References**

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

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

3. Van Oojen J, Voorrips R: JoinMap 3.0 software for the calculation of genetic linkage maps. Wageningen, the Netherlands: Plant Research International 2001.

P36

**Development of highly-multiplexed SNP arrays in maritime pine for multi-objective genetic applications**

C Plomion¹*, E Chancerel¹, C Lepoittevin¹, I Lesur¹, SC González-Martínez², GG Vendramin³, MT Cervera²

¹INRA, UMR1202 BIOGECO, France; ²INIA, Center of Forest Research (CIFOR), Indonesia; ³Italian National Research Council, Institute of Plant Genetics, Italy
E-mail: plomion@pierroton.inra.fr

BMC Proceedings 2011, **5**(Suppl 7):P36

Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genetic variation in the genome. In this poster, we reviewed what has been achieved in terms of highly-multiplexed SNP genotyping assay construction in maritime pine (*Pinus pinaster* Ait.), the main conifer used for commercial plantation in southwestern Europe. Seven custom SNP-assays (384 and 1536-plex), oriented towards broad applications, have been designed. We illustrated here the usefulness of this genotyping technology to address specific questions related to i/ genetic diversity and population structure analysis, ii/ linkage and QTL mapping and iii/ association mapping.

With respect to genetic diversity and differentiation, a custom VeraCode assay for 384 SNPs mostly based on a larger array designed for linkage mapping (see below) allowed to obtain less blurred, albeit similar, breeding zone boundaries than a set of 12 nuSSRs screened on the same individuals. Levels of diversity were also more accurately estimated, showing clear differences among gene pools. Interestingly, a relatively small subset of SNPs would be enough to develop an application tool for origin certification, which could have a notable impact on current operational practices.

In terms of linkage mapping, a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (in vitro SNPs) and from Sanger-derived Expressed Sequenced Tags (in silico SNPs) was established. Offspring from two mapping pedigrees were genotyped. A consensus map comprising 357 SNPs from 292 different loci was constructed and the analysis of sequence homology between mapped markers and their orthologs in a *Pinus taeda* linkage map, made it possible to align the 12 linkage groups of both species. Moreover, QTL detection for different traits is underway.

In terms of association mapping, a custom GoldenGate assay with 384 SNPs was built and used to genotype 160 unrelated plus-trees from a half-sib experimental design for which breeding values (for height growth, circumference, stem straightness at 8 years, lignin content and extractives at 31 years) were available. Taking into account multiple
testing, one single SNP in a gene encoding a putative fasciclin-like arabinogalactan protein was found to be associated with growth traits. We conclude that the VeraCode/GoldenGate assays can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This first generation of SNP-arrays has been recently upgraded to an Infinium-array (containing 10.5k SNPs) thanks to deep sequencing based on new generation sequencing technologies. The Infinium-array also includes SNPs from comparative orthologous sequences with other major conifer species, providing a wider collection of anchor points for comparative genomics among these major groups of forest trees.

## P37

### How many genes might underlie QTLs for growth and wood quality traits in *Eucalyptus*?

Carolina Sansaloni¹, César Petroli¹, Georgios Pappas², Orzenil Da Silva², Dario Grattapaglia³

¹EMBRAPA Genetic Resources and Biotechnology - EPqB Final W5 Norte 70770-917 Brazilia DF and Dep. Cell Biology, Universidade de Brazil – UnB, Brazil; ²EMBRAPA Genetic Resources and Biotechnology - EPqB Final W5 Norte 70770-917 Brazilia DF, Brazil; ³EMBRAPA Genetic Resources and Biotechnology - Estação Parque Biológico, 70770-910, Brazil, DF and Genomic Sciences Program - Universidade Católica de Brasília - Brazil, Brazil E-mail: carols@cenargen.embrapa.br
*BMC Proceedings 2011, 5(Suppl 7):P37*

**Background:** QTL mapping is an unbiased approach where the phenotype reveals the location of regulatory genes or genomic regions affecting the trait of interest. The development of transferable molecular markers and the increased use of multiple pedigrees for QTL mapping have allowed comparative analysis of QTLs across independent studies thus providing validation data. Such QTL positional information, together with the availability of annotated genome sequences, now promises to identify strong candidate genes for a number of traits [1]. As large pedigrees become available and higher resolution mapping with SNPs, DArT and genotype-by-sequencing technologies becomes routine in forest trees, QTL positional information could be an alternative to the current approaches that rely on tentative candidate genes for association genetics studies. Among the several traits for which QTLs have been mapped in forest trees those that display higher heritability, such as wood chemical composition, are more likely to involve candidate genes of stronger effect although recent association studies show that even such genes explain very small proportion of the variation [2]. In this study we used a high-resolution map with over 2,000 Diversity Arrays Technology (DArT) markers to carry out an initial assessment of the number of annotated gene models in the reference genome sequence of Eucalyptus that putatively co-locate with QTLs for growth and wood quality traits.

**Methods:** A QTL mapping study was carried out with a clonally replicated segregating population of 171 F1 individuals derived from an *E. grandis* x *E. urophylla* cross. Individuals were genotyped with the Eucalyptus DArT microarray described earlier [3]. The DArT marker data were combined with 222 microsatellites and a linkage map for each parent was constructed using JoinMap 3.0 [4]. Six traits were measured: height growth (HG), circumference at breast height (CBH); wood specific gravity (WSG); cellulose pulp yield (%PULP); Total Lignin (TL); syringyl/guaiacyl ratio (S/G). QTL mapping was carried out using QTL Cartographer [5] on the two parental maps separately at 1 cM intervals. Empirical threshold significance levels for QTL detection were determined by 1,000 permutations considering a significance level of 5%. All the segregating DArT and microsatellite markers were mapped onto the 11 pseudo-molecules of the *Eucalyptus grandis* draft genome sequence covering 609 Mbp.

**Results:** QTL analyses were carried out using framework genetic linkage maps with high likelihood support for order. The maternal map had 825 markers (684 DArTs + 141 SSR) and the paternal map 511 markers (410 DArTs + 101 SSR). A total of 16 QTLs in *E. grandis* and 14 in *E. urophylla* were detected influencing growth and wood quality traits. High and significant positive phenotypic correlations were found between CBH and HG, TL and S/G, and S/G and %PULP. In the maternal *E. grandis* map, five QTLs were identified for TL (Linkage groups (LG) 1, 3, 4, 5 and 8), two QTLs for %PULP (LG 4 and 5), three for S/G (LG 1, 5 and 8), WSG (LG 6, 8 and 10) and HG (LG 1, 2 and 6). In the *E. urophylla* paternal map we detected three QTLs for %PULP (LG 1, 4 and 9), three for HG (LG7, 8 and 10), three for TL

(LG3, 4 and 8), two for CBH (LG7 and 10), two for S/G (LG 8 and 9), and one for WSG (LG 8). More than two QTLs were clustered on LG 4, 5 and 8 in *E. grandis* and on LG 4, 8 and 9 in *E. urophylla* suggesting interesting genomic regions to look for candidate genes to be tested in association mapping. Several of these QTLs were syntenic to QTLs found in other studies [6,7] providing some indirect support for their validity. The sequences of DArT and microsatellite markers bracketing QTLs were used to extract the gene models from the Eucalyptus reference genome. A total of 7,125 predicted gene models are found across all maternal QTLs, with an average of 445 genes per QTL. For the paternal QTLs 5,076 gene models exist, with an average of 362 genes per QTL.

**Conclusions:** As in many other QTL mapping studies in Eucalyptus [6-8] we have identified several QTLs that control a modest proportion of the phenotypic variation for a number of economically important traits. In this first assessment of putative candidate genes co-locating with these QTLs we found thousands of annotated gene models, hundreds of which could be tentatively suggested as being involved in trait variation. Notwithstanding the low mapping resolution provided by the small progeny, this preliminary study shows that tens or hundreds of genes will likely be always found underlying QTLs for such complex traits. Testing and validation of such large numbers of genes will require a gigantic effort. Furthermore a large proportion of the phenotypic variation remains unexplained by the few QTLs mapped. It is therefore questionable from the applied stand point how much useful information this approach will effectively provide for the advancement of association genetics and, for that matter, of breeding practice.

**Acknowledgments:** This work was supported by the Brazilian Ministry of Science and Technology through CNPq grant 577047/2008-6 and FAP-DF Grant NEXTREE 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

### References

1. Price AH: Believe it or not, QTLs are accurate! *Trends Plant Sci* 2006, 11(5):213-216.

2. Wegrzyn JL, Eckert AJ, Choi M, Lee JM, Stanton BJ, Sykes R, Davis MF, Tsai CJ, Neale DB: Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem. *New Phytol* 2010, 188(2):515-532.

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

4. Stam P: Construction of Integrated Genetic-Linkage Maps by Means of a New Computer Package - Joinmap. *Plant Journal* 1993, 3(5):739-744.

5. Wang S, Basten CJ, Zeng Z-B: Windows QTL Cartographer 2.5. North Carolina State University, Raleigh, NC; Department of Statistics 2011.

6. Thumma BR, Southerton SG, Bell JC, Owen JV, Henery ML, Moran GF: Quantitative trait locus (QTL) analysis of wood quality traits in. *Eucalyptus nitens*. *Tree Genetics & Genomes* 2010, 6(2):305-317.

7. Freeman JS, Whittock SP, Potts BM, Vaillancourt RE: QTL influencing growth and wood properties in Eucalyptus globulus. *Tree Genetics & Genomes* 2009, 5(4):713-722.

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

## P38

### Association mapping of local adaptation traits of Scots pine in a European wide population sample

Timo Knürr¹, Sonja Kujala², Mikko J Sillanpää³, Komlan Avia⁴, Aleksia Vaattovaara⁴, Katri Kärkkäinen⁵, Outi Savolainen⁴⁶
¹Dept. Mathematics and Stastistics, University of Helsinki, Finland; ²Dept. biology, Unviersity of Oulu, Finland; ³Dept. Mathematics and Statistics, University of Helsinki, Finland; ⁴Dept. Biology, University of Oulu, Finland; ⁵Finnish Forest Research Institute, Finland
E-mail: outi.savolainen@oulu.fi
*BMC Proceedings 2011, 5(Suppl 7):P38*

Traits related to local adaptation by definition show high phenotypic differentiation. The underlying genetic patterns could be clines at individual loci or small effects and extensive linkage disequilibrium at the underlying loci. In any case, including many populations in an analysis provides more information, but may simultaneously induce problems due to genetic structure. Even if the neutral loci have little genetic structure, loci related to other clinically selected traits could show more structure.
Here we have developed an approach to efficiently use the information along a latitudinal environmental gradient. Scots pine populations from central Europe to the species' northern range were sampled and patterns of phenotypic variation of both timing of budset and frost tolerance were measured in common garden experiments, (10 populations, a total of 270 halfsib families, 25 trees per family). By hierarchical modelling of the phenotype's clinal variation and accounting for varying allele frequencies across the 10 populations, the statistical approach simultaneously exploits the genetic variation between and within populations to detect association signals. We apply shrinkage-based Bayesian variable selection to detect genetic associations between timing of bud set and ~450 SNPs in Scots pine.

P39

Development of a genetic linkage map of rubber tree (Hevea braziliensis) based on microsatellite markers

Livia Moura Souza¹*, Camila Campos Mantello¹, Fernando Suzuki¹,
Rodrigo Gazaffi², Dominique Garcia³, Vincent Le Guen³,
Antonio Augusto Franco Garcia⁴, Anete Pereira Souza⁴
¹Molecular Biology Center and Genetic Engeniering, UNICAMP, Campinas,
SP,13083-875, Brazil; ²Department of Genetics “Luiz de Queiroz” College of
Agriculture University of São Paulo, Piracicaba, SP,13400-970, Brazil; ³CIRAD-BIOS,
UMR AGAP, Montpellier, Cedex 5, France; ⁴Department of Plant Biology –
Biology Institute and Molecular Biology Center and Genetic Engeniering,
UNICAMP, Campinas, SP,13083-875, Brazil
E-mail: liviamoura31@gmail.com
BMC Proceedings 2011, 5(Suppl 7):P39

**Background:** Rubber tree (*Hevea spp.*) is the main natural rubber producing crop, and it is cultivated in numerous equatorial, tropical and subtropical areas. *Hevea spp.* is an outcrossing perennial species and belongs to the botanical family Euphorbiaceae. Breeding of rubber tree, like for many other tree crops, is a long term process. Large scale cultivation of a new cultivar can only be reached after 20–25 years of field experiments on large areas. Breeders are thus interested in developing alternative strategies to improve and speed up the breeding scheme, using genetic markers. Molecular markers are being used for more than 15 years in *Hevea* breeding, mostly for diversity studies, assessment of genetic distance among cultivars, genetic mapping and identification of genetic loci implied in the expression of agronomical traits.

**Material and methods:** The mapping population was a full-sib progeny (F1 progeny) derived from a controlled cross between the cultivars PB217 and PR255. The PB217 has a high yield potential, which is expressed throughout the lifetime of its rubber production, low metabolic activity and a high level of sucrose in the latex vessels. Genotype PR255 showed tolerance to injury and cold. A set of 603 microsatellite primer pairs was tested for polymorphism the two parents and six F1 progenies genotypes. Among these, 200 genomic microsatellites have been developed by our laboratory (part published in [7]), 296 were developed by CIRAD [3] and 178 EST-SSRs were obtained from a published work [1]. The polymorphic microsatellites between the parents were selected for mapping in a F1 population. The PCR conditions were detailed by [7]. PCR products were separated using a DNA analyzer 4300 (LI-COR) through 6.5% polyacrylamide gels. Linkage map was obtained using OneMap [5]. This software generates integrated genetic maps from molecular markers with different segregation patterns; it considers multipoint technology based on Hidden Markov Models, as presented by [8]. LOD Score 4.5 and recombination fraction of 0.40 was considered to determine linkage between markers. The algorithms “compare” (until six markers) and “order” (more than six) were applied to obtain best order for each linkage groups, as presented in mapmaker/EXP [6].

**Results and discussion:** Out of 603 microsatellites markers evaluated, 309 (51%) showed polymorphism between the parents of the mapping population. Until now, 225 marks were genotyped (59 SSR genomic loci and 166 EST-SSR). Chi-square test was carried out on the genotyping polymorphic loci showed that 110 loci followed a segregation ratio of 1:1; 28 followed a ratio of 1:2:1 and 87 (38.7%) followed a ratio of 1:1:1:1. The map consists of 225 markers, distributed in 23 linkage groups (LG) and 2,471.2 cM in length with an average genetic distance of 11 cM between adjacent markers. The largest group has 215.9 cM (18 markers) and the smallest has 2.71 cM (2 markers). This reflects in a more realistic way the polymorphism of the full-sib cross.

In a previous work [4] a linkage map was constructed from a double pseudo testcross, and two maps were constructed separately for each parent. In the present work, instead of using only single dose markers (1:1) like [4], markers that segregate in both parents in the ratio 1:2:1 and 1:1:1:1 were also used enabling the construction of an integrated genetic map and facilitating the location of quantitative trait loci (QTL) [2]. Seven linkage groups are small, composed of two or three markers and covering a few cM. These small linkage groups probably occur because the map is not saturated enough and some chromosome regions could not be linked. The chromosome number accepted today, for most *Hevea* species, is 18 (2n = 36), as observed by [4].

**Conclusions:** This map is still under development and we expect to achieve a saturated map consisting of 18 linkage groups. The present map will be used for yield rubber QTL mapping and other important economical characteristics.

**Acknowledgements:** FAPESP and Michelin.

**References**

1. Feng SP, Li EWG, Huang EH, Wang JY, Wu EYT: Development, characterization and cross-species/genera transferability of EST-SSR markers for rubber tree (*Hevea Braziliensis*). *Molecular Breeding* 2009, 23:85-97.

2. Garcia AAF, Kido EA, Meza AN, Souza HMB, Pinto LR, Pastina MM, Leite CS, Silva JAG, Ulian EC, Figueira AVQ, Souza AP: Development of an Integrated Genetic Map of a Sugarcane (Saccharum spp.) Commercial Cross, based on a Maximum-Likelihood Approach for Estimation of Linkage and Linkage Phases. *Theoretical and Applied Genetics* 2006, 112:298-314.

3. Le Guen V, Gay C, Xiong TC, Souza LM, Rodier-Goud M, Seguin M: Development and characterization of 296 new polymorphic microsatellite markers for rubber tree (*Hevea Braziliensis*). *Plant Breeding* 2011, 130:294-296.

4. Lespinasse D, Rodier-Godier M, Grivet L, Leconte A, Legnate H, Seguin M: A saturated genetic linkage map of rubber tree (*Hevea spp.*) based on RFLP, AFLP, microsatellite, and isozyme markers. *Theoretical and Applied Genetics* 2006, 100:127-138.

5. Margarido GRA, Souza AP, Garcia AAF: OneMap: software for genetic mapping in outcrossing species. *Hereditas* 2007, 144:78-79.

6. Mollinari M, Margarido GRA, Vencovsky R, Garcia AAF: Evaluation of algorithms used to order markers on genetic maps. *Heredity* 2009, 103:494-502.

7. Souza LM, Mantello CC, Santos MO, Gonçalves PS, Souza AP: Microsatellites from rubber tree (*Hevea Braziliensis*) for genetic diversity analysis and cross-amplification in six *Hevea* wild species. *Conservation Genetics Resource* 2009, DOI10.1007/s12686-009-9018-7.

8. Wu R, Ma CX, Wu SS, Zeng ZB: Linkage mapping of sex-specific differences. *Genetical Research* 2002, 79:85-96.

P40

QTL analyses of drought tolerance in *Eucalyptus* under two contrasting water regimes

Juliana Teixeira¹*, Alexandre Missiaggia¹*, Donizete Dias¹, Edimar Scarpinati¹,
Juliana Viana¹, Nadia Paula², Rinaldo Paula³, César Bonine¹

¹Fibria Celulose S.A., Brazil; ²Faculdade de Tecnologia de Jaboticabal – FATEC-JB, Brazil; ³Universidade Estadual de São Paulo – UNESP, Brazil
E-mail: amissiaggia@fibria.com.br

BMC Proceedings 2011, 5(Suppl 7):P40

**Background:** Drought stress is one of the most important abiotic factors in *Eucalyptus* sp. plantations which influences the growth and limits productivity in cultivated areas, mainly in central, northern and northeastern areas in Brazil, where large parts of these areas have limitations on water supply. The breeders are now looking for tolerant genotypes to overcome this challenge and the QTL mapping approach will help to understand the genetic control of drought tolerance. The objective of this study was to identify genetic loci controlling the phenotypic variation in drought tolerance in a *Eucalyptus* segregant progeny grown under drought and irrigation conditions.

**Material and methods:** The progeny used in this study was generated by the breeding program of Fibria Celulose and is composed of 184 F1 genotypes from a cross between a tolerant and a susceptible clone to water stress, both *E. grandis* x *E. urophylla* hybrid tree. In a greenhouse condition, the progeny (seedlings with 70 days) was evaluated under two irrigation conditions (1 - control: assigned to a well-watered regime with watering equal to transpiration loss and 2: submitted to water deficit until the onset of initial drought symptoms) in 4 different experiments. Growth (the relative increase in height and stem diameter; leaf number;
leaf area; leaf, steam and root dry weight; root-shoot ratio) and physiological traits (net assimilation rate, stomatal conductance, transpiration, instantaneous and intrinsic water-use efficiency, relative water content in leaves, chlorophyll content index, photochemical efficiency and leaf water potential) were measured and BLUPs analysis were performed.Genomic DNA was extracted with CTAB protocol from young leaves and used in PCR amplifications of 121 microsatellite markers. The Onemap software [1] was used in linkage analysis. The estimated BLUPs were used in QTL mapping [2]. The QTLs were mapped by composite interval mapping (CIM).

**Results and discussion:** Linkage analysis resulted in 11 linkage groups with 101 markers. The groups were identified using a reference eucalyptus genetic map [3], through common microsatellite loci. The length of the map was 770 cM with an average distance of 7.2 cM between markers. The experimental design allowed analysis of genotype by drought tolerance interaction for the first time in a eucalyptus QTL mapping population, resulting in the identification of 66 loci that control traits under water restriction and 70 loci under irrigation condition, for all of 16 traits evaluated (considering LOD>3.0). Both additive and dominance effects were detected. Around 4 – 7 QTLs were identified for each trait and, in general, the QTLs identified explained from 11% to 30% of phenotypic variation, except by photochemical efficiency, where 59% of phenotypic variation was explained, mainly by 2 QTLs (25.16% and 25.17% and peak LOD score 3.46 and 18.73) mapped in linkage group 6. Most of QTLs identified were specific for each treatment and for just two traits (root and leaf dry weight) QTLs were co-localized for both irrigation conditions and they were mapped in linkage groups 2, 8, 10 and 11. Clusters of QTL for different traits were mapped close to each other at several linkage groups, indicating either a common genetic base or tightly linked QTL. The results are consistent with Ronnberg's and Yue's results [4,5],where many QTLs with minor effects are controlling drought tolerance.

**Conclusions:** This work revealed the existence of several QTLs that control drought tolerance in Eucalyptus and these QTLs identified may be involved in many tolerance mechanisms that plants can use to avoid this stress. Since clusters of QTL for different traits were identified, potential pleiotropic regulators could be coordinating these traits and these genomic regions can be used to identify key genes for these traits.

**Financial support.** FINEP and Fibria Celulose.

## References

1. Margarido GRA, Souza AP, Garcia AAF: OneMap: software for genetic mapping in outcrossing species. Hereditas 2007, 144:78-79.

2. Gazaffi R: Desenvolvimento de modelo genético-estatístico para mapeamento de QTLs em progênie de irmãos completos, com aplicação em cana-de-açúcar. PhD Thesis Universidade de São Paulo, Departamento de Genética; 2009.

3. Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. BMC Plant Biology 2006, 6.

4. Rönnberg-Wästljung AC, Glynn C, Weih M: QTL analyses of drought tolerance and growth for a *Salix dasyclados* *xSalix viminalis* hybridin contrasting water regimes. Theor and Appl Genet 2005, 110:537-549.

5. Yue B, Xue W, Xiong L, Yu X, Luo L, Cui K, Jin D, Xing Y, Zhang Q: Genetic Basis of Drought Resistance at Reproductive Stage in Rice. Genetics 2006, 172:1213-1228.

# S3. GENOMICS ASSISTED BREEDINGS

## P41

### Comparative assessment of SNPs and microsatellites for fingerprinting, parentage and assignment testing in species of *Eucalyptus*

Leonardo Correia¹*, Danielle Faria², Dario Grattapaglia¹

¹EMBRAPA Genetic Resources and Biotechnology – Estação Parque Biológico, 70770-910, Brazilia, DF and Genomic Sciences Program – Universidade Católica de Brasília - Brazilia, Brazil; ²EMBRAPA Genetic Resources and Biotechnology – Estação Parque Biológico, 70770-910, Brazilia, DF, Brazil

E-mail: leoqcorreia@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P41

**Background:** Brazil stands on the international scenario by having one of the largest natural forest heritage and extensive sustainable planted forests. Brazilian planted forestry is based mainly on fast growing

eucalyptus and pines, with the pulp, paper and steel industries as the major consumers. Introduced commercially in Brazil in the early twentieth century, the eucalypts have experienced increasing levels of genetic improvement over the years. Along with the classical breeding, the use of genetic markers has positively influenced breeding programs, contributing to quality control processes of clonal forestry and advanced breeding. Microsatellite markers have been the main tool used to date. They are multiallelic, highly polymorphic and thus very efficient for several applications that require identification and discrimination of elite clones and determination of parentage. While di and tri-nucleotide repeat microsatellites tend to be more polymorphic, tetra and higher order repeats allow a more robust allele calling [1]. SNP markers genotyped by a high-throughput system have been recently developed for Eucalyptus [2] but not yet routinely incorporated in Eucalyptus breeding programs. In spite of their lower genetic information content, many more SNPs can be simultaneously typed using automated systems, an appealing feature to operational breeding programs. The aim of this study was to comparatively evaluate the resolving power and precision of different sets of molecular markers (microsatellites and SNPs assayed by the Golden Gate technology) for the most common operational applications in *Eucalyptus* breeding programs.

**Methods:** Samples belonging to six species of Eucalyptus (*E. grandis*, *E. urophylla*, *E. globulus*, *E. nitens*, *E. camaldulensis* and *E. dunnii*) were genotyped with three different sets of markers: (1) 24 di- and trinucleotides microsatellites (2) 17 tetra-, penta- and hexanucleotides microsatellites and (3) selected subsets of SNPs from the 768 developed earlier [2]. Microsatellites were genotyped by capillary electrophoresis in an ABI3100 genetic analyser and data collected under dye set D using Genescan/Genotyper.SNP genotyping was performed using an Illumina BeadStation 500 GX at the Genome facility of the University of Florida and the data analyzed using the software GenomeStudio. Multilocus genotypes were obtained with the two microsatellite panels and a set of the 96 most informative SNPs for individual identification based on an algorithm that takes into account minimum allele frequency and observed heterozygosity within and across species. The genotype data were used to estimate the combined probability of paternity exclusion (PE) and probability of identity (PI) for the marker panels. Additionally, markers were assessed for their ability to estimate genomic ancestry of individuals, a particularly useful application for determining the origin of spontaneous Eucalyptus hybrids. All typed individuals were assigned probabilistically to a given number of populations inferred with a Bayesian approach without any prior population information using the STRUCTURE package [3]. The analyses were performed with each set of microsatellites and with a specific subset of 96 ancestry informative SNPs, selected with an algorithm that focused on SNPs that display contrasting allele frequencies among species.

**Results:** The panel of di- and trinucleotide microsatellites showed the fastest increase in the combined paternity exclusion with the addition of new markers. For example, a PE > 99% was achieved with only five markers in *E. grandis*. The panel of tetra-, penta- and hexanucleotide microsatellite was unable to reach a PE of 99% even with all 17 markers were used. The panel of 96 SNPs showed a slower increase in the combined paternity exclusion with the addition of new markers, but needed only 35 markers to reach a PE > 99%. The same trend was observed with the estimate of probability of identity. In the analysis of genetic structure, all three sets of markers correctly inferred the six genetically distinct populations, corresponding to the six species studied. Furthermore, both panels assigned correctly all individuals to their respective species. The SNP panel showed the highest average proportion of correct assignment for all six species, followed by the di- and trinucleotides and at last the tetra-, penta- and hexanucleotide panel, although these estimates were not statistically different between the three marker panels tested, (p= 0.327).

**Conclusions:** Results show that di- and trinucleotide microsatellites tested are around three times more informative than the tetra-, penta- and hexanucleotide microsatellites used and about seven times more informative than the specific SNPs employed for assessment of parentage and individual identification. Although more informative, the di- and trinucleotide microsatellites are subject to a higher inaccuracy in allele calling, which can result in inconsistencies across analyses carried out in different labs.All three marker panels showed high resolution power to detect genetic structure and carry out assignment tests in species of
**Eucalyptus.** We are now extending this analysis to controlled and spontaneous hybrids to compare the estimates of genomic ancestry provided by the three marker panels. Additionally we are developing much larger sets of SNPs by re-sequencing reduced genomic representations of pooled individuals from all six species. This should allow a targeted selection of more ancestral SNPs that are homogeneously polymorphic across species to be used for fingerprinting and parentage or historically more recent SNPs that display contrasting allele frequencies among species and thus be more useful for genomic ancestry determination. We anticipate that despite the lower individual genetic information content, careful selection of SNPs panels for targeted applications coupled to their accurate genotype calling and high throughput will become an increasingly attractive alternative for operational application in breeding programs.

**Acknowledgments:** This work was supported by the Brazilian Ministry of Science and Technology through CNPq grant 577047/2008-6 and FAP-DF Grant NEXTREE 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

## References

1. Faria DA, Mamani EMC, Pappas GJ, Grattapaglia D: Genotyping systems for Eucalyptus based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests. *Tree Genetics and Genomes* 2010, doi:10.1007/s11295-010-0315-9.

2. Grattapaglia D, Silva-Junior O, Kirst M, Lima BM, Faria DA, Pappas GJ: High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species. *BMC Plant Biology* 2011, 11:65.

3. Pritchard JK, Stephens M, Donnelly P: Inference of population structure using multilocus genotype data. *Genetics* 2000, 155(2):945-959.

## P42

### SNP discovery in apple cultivars using next generation sequencing

Sérgio Alencar¹*, Orzenil Silva-Junior¹, Roberto Togawa¹, Marcos Costa¹, Luís Fernando Revers², Georgios Pappas¹

¹Embrapa Recursos Genéticos e Biotecnologia - Parque Estação Biológica - Av. W5 Norte (final) Caixa Postal 02372 - Brasília, DF - 70770-917, Brazil;

²EMBRAPA Uva e Vinho - Rua Livramento, 515 - Caixa Postal 130 - Bento Gonçalves, RS - 95700-000, Brazil

E-mail: salencar@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P42

**Background:** Knowledge about single nucleotide polymorphism (SNP) markers is extremely important in the development of genotyping assays, allowing improvements in plant breeding through marker-assisted selection. With the emergence of next generation sequencing platforms, high-density SNP discovery in the genome of plant crops becomes more achievable. In this project, we carried out whole genome resequencing of two apple cultivars (M13/91 and Fred Hough, developed by Epagri [1,2]) using the recently released draft genome sequence of the domesticated apple genome (*Malus x domestica* Borkh.) [3] as a reference for SNP discovery.

**Materials and methods:** We sequenced four DNA samples (two from M13/91 and two from FH cultivars) on four lanes of the Illumina GA II platform (single-end sequencing), with the Illumina PhiX sample used as a control. Image analysis and base calling were performed using on-instrument real time analysis, after which the Off-Line Basecaller was used to convert per-cycle base call files (.bcl) into per read base call files (.qseq). The resulting short reads were aligned to the reference genome with the BWA software [4], with the maximum edit distance set to 3, quality threshold for read trimming set to 15, and no gap opens allowed. Next, SAMtools [5] utilities were used to convert SAM into BAM format, to remove read duplicates and indels, and also to sort reads by coordinates. SNP discovery was then carried out using the GATK Unified Genotyper SNP caller [6].

**Results:** Alignment of the M13/91 and FH reads to the reference genome resulted in a total of 37,757,897 and 60,400,252 reads mapped, respectively. Considering only reads with root-mean-square mapping quality greater than 20, and occurring at raw read depths (DP) greater than 6 and lower than 20, a total of 143,468 and 474,483 heterozygous putative SNPs were identified when comparing the reference genome with the M13/91 and FH cultivars, respectively. A total of 80,554

heterozygous putative SNPs are shared by both M13/91 and FH cultivars. When considering only homozygous putative SNPs, a total of 20,296 (M13/91) and 70,659 (FH) SNPs were identified. A search was also made between the M13/91 and FH cultivar genomes, resulting in a total of 2,631 SNPs which are homozygous in FH and heterozygous in M13/91, and 4,768 SNPs which are homozygous in M13/91 and heterozygous in FH. In order to determine whether the differences in SNP frequencies between these cultivars are due to differences in read coverage obtained from sequencing, we set up a cut-off value above which all SNP calls in both cultivars had the same coverage, and it showed that their SNP frequency is similar.

**Conclusions:** We have used next generation sequencing data combined with high-density SNP detection methods to discover large numbers of putative SNPs in apple cultivars, which can be used in the development of genotyping assays.

## References

1. Denardi F, Hough LF: Apple breeding in Brazil. *Hortscience* 1987, 22:1231-1233.

2. Denardi F, Camilo AP: Novas cultivares de macieira: proposta de nova composição de pomares com polinizadoras/produtoras. *Agropecudria Catarinense* 1997, 10:25-30.

3. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R: The genome of the domesticated apple (*Malus x domestica* Borkh.). *Nature Genetics* 2010, 42:833-839.

4. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler Transform. *Bioinformatics* 2009, 25:1754-60.

5. Li H, Handsaker B, Wyosoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup: The Sequence Alignment/Map format and SAMtools. *Bioinformatics* 2009, 25(16):2078-79.

6. Deplisto MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, Del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ: A framework for variation discovery and genotyping using next-generation DNA sequencing data. *Nature Genetics* 2011, 43(5):491-498.

## P43

### Improvement of *Eucalyptus* sp for biomass and bioenergy production in the north of Spain

Carmen Diaz-Sala¹†, María Dolores Velez²†, Elena Carneros¹†, Dolores Abarca¹†, Carmen Barbero²†, Alicia Del Amo¹†, José Antonio Cabezas³†, Claudia Escudero⁴†, Libertad Juez⁴†, Carlos Tejedor⁴†, María Teresa Cervera²†+

¹Department of Plant Biology, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain; ²Departamento de Ecología y Genética Forestal INIA-CIFOR, Ctra. de La Coruña Km 7,5, 28040 Madrid, Spain; ³Departamento de Investigación Agroalimentaria, Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario, Finca El Encín. Ctra A-2, Km38200, 28800 Alcalá de Henares, Spain; ⁴SNIACE. Ctra. Ganzo, s/n, 39300 Torrelavega, Cantabria, Spain

E-mail: cervera@inia.es

BMC Proceedings 2011, 5(Suppl 7):P43

*Eucalyptus globulus* Labill. has been used in Spain for decades as a cellulose source for the paper and textile industry. Since 1997, Sniace group has tested new provenances and families from 29 different *Eucalyptus* species to explore their capacity for biomass and bioenergy production.

Plus trees for growth, wood quality, rooting capacity and tolerance to *Mycosphaerella* sp have been identified by mass selection, and more than 300 controlled crosses among those trees have been carried out. However, the restrictions caused by the high susceptibility to *Mycosphaerella* leaf
disease and the low rooting capacity of the species *Eucalyptus globulus*
*Labill.* delay the application of the gains to a commercial scale.

The objective of this project is to improve the production of *Eucalyptus* in
the North of Spain focused on the improvement of two traits of economic
importance: clonal/rooting capacity and tolerance to *Mycosphaerella* sp. To
achieve this goal three partial objectives have been approached:

**Mass propagation of selected adult clones and rooting improvement by in vitro tissue culture:** Clonal propagation by sequential subcultures of axillary buds in proliferation and elongation media and further rooting of elongated shoots will be developed. This will further optimize a simple *in vitro* protocol for the micropropagation of identified elite mature trees for raising plantations. Micropropagation can maintain selection gains, developed in improvement programmes, to be transmitted directly to plantations or to seed orchards, adding value to the subsequent products and reducing production costs in the long term. For this purpose, it is required to test differences between *in vitro* produced trees and seed-derived trees, regarding some important traits such as juvenility, growth, productivity, uniformity and morphological traits.

**Certification of clones and varieties by the use of specific molecular markers:** A collection of 97 elite clones have been genotyped using 22 SSRs [1,2], and four multiplex PCR reactions. This analysis has allowed the genetic characterization of each individual clone, the analysis of genetic similarity and inferring tentative relatedness among clones. This information will be used to improve crossing designs as well as to certify clonal material.

**Genetic dissection of rooting capacity and tolerance to Mycosphaerella sp:** A strategy combining transcriptomics, genetic mapping, phenotypic characterization of the targeted traits and QTL analysis has been established. For this purpose a mapping progeny was obtained using two contrasting progenitors: A tree with high rooting capacity, tolerant to *Mycosphaerella* sp, and a tree with low rooting capacity, susceptible to *Mycosphaerella* sp. The steps to reach this goal include: 1) Construction of a cDNA library made of pooled RNAs, from different tissues collected from the progeny plants grown under different conditions. This cDNA library is used as template for Roche GS-FLX Titanium high throughput sequencing. Once the unigene is identified, SNPs from selected genes are chosen to design a 1536 Golden Gate array which is used to genotype both progeny individuals and progenitors. Segregating SNPs are used to construct genetic maps. 2) Evaluation of rooting capacity and response to infection during two consecutive years. 3) Identification of association between SNPs and trait parameters using QTL analysis followed by identification of favourable alleles (SNP variant).

**Acknowledgements:** Finannical support for this investigation has been
provided by the Ministry of Science and Innovation "Investment fund for
employment.-Government of Spain" by the CDTI in the project BioSos-
CEN-2009-1040 and by the project Eucaful.

References

1. Steane DA, Vaillancourt RE, Russell J, Powell W, Marshall D, Potts BM: Development and characterization of microsatellite loci in *Eucalyptus globules* (Myrtaceae). *Silvae Genet* 2001, 50:89-91.

2. Assis Faria D, Mamani EMC, Pappas GJ, Grattapaglia D: Genotyping systems for *Eucalyptus* based on tetra-, penta-, and hexanucleotide repeat EST microsatellites and their use for individual fingerprinting and assignment tests. *Tree Genetics and Genomes* 2011, 7:63-77.

P44

Genome wide selection for *Eucalyptus* improvement at international
paper in Brazil

Carla Garcia¹, Bruno Lima², Adriano Almeida³, Wesley Marques⁴,

Marcos Resende⁵, Roland Vencovsky⁶, Dario Grattapaglia⁷

¹International Paper in Brazil, Mogi Guaçu, SP, Brazil; ²Department of Genetics, University of São Paulo - ESALQ/USP, Piracicaba, SP, Brazil;

³International Paper in Brazil; ⁴University of Campinas, Campinas, SP, Brazil;

⁵EMBRAPA Forestry/UFVicosa, Brazil; ⁶Department of Genetics, University of São Paulo - ESALQ/USP, Piracicaba, Brazil; ⁷EMBRAPA Genetic Resources and Biotechnology, EPqB 70770-910, Brazilia, DF, Brazil

E-mail: carla.garcia@ipaperbr.com

BMC Proceedings 2011, 5(Suppl 7):P44

**Background:** The efficiency of plant breeding depends mainly on two actions of the breeder: creation and subsequent identification of superior

genotypes. In both actions, selection plays a fundamental role in the
definition of crosses to be performed, with interest in creating new
genotypes and identifying superior trees to be used commercially. The
great attraction of molecular tools for plant breeding is the direct use of
DNA information in selection, allowing higher efficiency, quickness in
obtaining genetic gains with relatively low costs, when compared to the
traditional selection based on phenotypic data. All these objectives can
be reached through a new approach: genome-wide selection (GWS) or
just genomic selection (GS). Genomic selection can be applied in
breeding programs of any species. Results obtained in simulated data
indicated that GWS can be very profitable in eucalyptus breeding [1].
GWS is based on selection exclusively by molecular markers, after having
their genetic effects estimated based on phenotypic data, in a breeding
population sample [2]. The present work aims to characterize and
estimate genetic parameters of a hybrid progeny test, the population that
will compose the genome-wide selection study at International Paper in
Brazil.

**Material and methods:** The International Paper Brazil population chosen for genome-wide selection purposes is installed in a Hybrid Progeny Test, comprising 58 crosses from controlled pollination of *Eucalyptus grandis* and *Eucalyptus urophylla* and five common checks (commercial clones of the company), totaling 63 treatments. These 58 families are derived from 56 different parents crossed. The test was installed in July 2006 in Brotas (São Paulo State, Brazil) in randomized complete block design, with six plants per plot and eight blocks, corresponding to a total of 3,024 plants. In 2011 the test was evaluated, performing the measurement of diameter at breast high (DBH) and plant height of the progenies. The obtained data were analyzed in Selegen-REML/BLUP software for the estimation of genetic parameters. The analysis, will allow the identification of the elite individual trees, which comprised the GWS population, ranking the best living trees of the test by estimating the annual average increment and morphological aspects (removing dead, forked and broken trees). Following the selection of the GWS 1,000 trees population, xylem samples were collected and sent for DNA extraction and genotyping with DArT and SNP markers.

**Results:** Three variables were analyzed to characterize the test population: DBH, plant height and wood volume (calculated as function of plant diameter and height). DBH had general mean of 13.8 cm and phenotypic variance between progenies of 1.61 cmÅ². The individual narrow-sense heritability of the character was 0.255 ± 0.039, very similar to the plant height heritability of 0.236 ± 0.037. Plant height presented general mean of 21.3 meters and genotypic variance of 2.17 mA². The estimated wood volume had general mean of 0.168 m3 per plant and genotypic variance of 0.0012 (m3)Å². The individual narrow-sense heritability was slightly higher than for DBH and plant height, namely 0.297 ± 0.042. All estimates were typical of quantitative traits controlled by many loci of small effects. Based on the analysis of the experiment, the best trees were selected, as potential clones and parents for crossing. This analysis revealed 44 crosses with elite clones, to be used as training/discovery population for GWS purposes. The superior individuals of these crosses were selected to complete 1,000 trees, dispersed through the entire test. The number of crosses (44) selected and the different parents identified (45) are important informations for estimating easily the effective population size (Ne), an important factor in GWS [1].

**Conclusions:** The information and data generated will lead the studies of the next steps into GWS approach. With the deep understanding of the training population that we have, the genomic breeding values (GBV) can be estimated and the GWS applied using phenotypic and marker data simultaneously. A correct choice of a breeding population is essential for traditional plant breeding and is not different in integrated molecular approaches. Subsequently, the wood phenotyping will be performed and the GBV also estimated for wood quality traits. These data will start up the implementation of GWS in International Paper breeding program in Brazil, as operational procedure, looking at quick selection and, consequently, cost decreases.

**Financial support.** International Paper Brazil, FAPESP, Embrapa-Cenargen
References

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

2. Resende MDV, Lopes PS, Silva RL, Pires IE: Seleção Genômica Ampla e maximização da eficiência do melhoramento genético. *Pesquisa Florestal* Brazileira 2008, 56:63-77.
P45

Genome-wide genotyping and SNP discovery by ultra-deep Restriction-
Associated DNA (RAD) tag sequencing of pooled samples of *E. grandis*
and *E. globulus*

Dario Grattapaglia¹*, Sergio de Alencar¹, Georgios Pappas²

¹EMBRAPA Genetic Resources and Biotechnology – Estação Parque
Biológico, 70770-910 and Genomic Sciences Program - Universidade Católica
de Brazilia, 70790-160 Brazilia, DF, Brazil; ²EMBRAPA Genetic Resources and
Biotechnology – Estação Parque Biológico, 70770-910, Brazilia, DF, Brazil

E-mail: dario@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P45

**Background:** The availability of next generation sequencing (NGS) technologies has opened the door to new strategies of SNP discovery and genotyping. Rapid genome-wide SNP detection via deep resequencing of reduced representation libraries of restriction digested pools of genomic DNA combined with a reference genome has been successfully used for SNP discovery in microorganisms [1], plants [2]and domestic animals [3]. Taking a step further from using NGS for SNP discovery, Baird et al [1]showed that NGS of short tags derived from barcoded multiplexed genomic representations generated with restriction enzymes could be used for direct genotyping of individuals, calling this method RAD (Restriction-site associated DNA) sequencing. RAD sequencing involves cutting a genome with at least one restriction enzyme and NGS the ends of the resulting fragments. We have recently developed a first set of SNPs for high-throughput genotyping of species of *Eucalyptus*. Although SNP assay success was high, the proportion of polymorphic SNPs declined as phylogenetic distance between species increased, down to <20% when contrasting *E. grandis* and *E. globulus*, the two main worldwide commercially planted species were considered [4]. In this work we used RAD sequencing to discover polymorphic SNPs across these two species. Additionally we were interested in assessing the potential of RAD for direct genotyping-by-sequencing in *Eucalyptus*.

**Methods:** DNA was extracted separately from 18 unrelated individual trees of *E. grandis* and 18 of *E. globulus*. For each species three bulks of six individuals were prepared with equimolar amounts of picogreen quantified DNA. DNA samples were delivered to Floragenex who carried out the RAD reduced representation library preparation using PstI and Illumina 75 bp single-end sequencing on a GAIIx. Raw sequence data was filtered for quality and mapped onto the 11 chromosomes of the *E. grandis* reference genome available in Phytozome. SNPs in the short sequence tags were called for nucleotides with quality Q> 30 at the position and a minimum of 6X coverage.

**Results and conclusions:** The average sequencing depth exceeded 28X for all bulked samples, providing a minimum estimated ~5X coverage for each individual present in each bulk providing a 93.75% probability of detecting a heterozygous SNP position. With 18 individuals per species (36 chromosomes), the probability of detecting a SNP allele with a Minimum Allele Frequency (MAF) > 0.1 is > 95% [5] therefore providing good power to select informative SNPs in each species separately and even more so in both species simultaneously. RAD sequence tags may be present or absent in specific individuals depending on the presence of the PstI restriction site providing large numbers of dominant markers; SNPs detected within the aligned tags provide additional co-dominant markers (Figure 1).

Out of a total of 200,712 SNPs declared with high confidence, 42,300 were simultaneously polymorphic in the two species while the remaining were fixed in one or the other. These 42,300 SNPs provide an average density of one SNP every 14 kbp in the *Eucalyptus* genome. These SNPs could be immediately used to select an evenly spaced set of SNPs for the development of a high density SNP genotyping platform.

Taken together, the RAD tags plus the SNPs into them provide excellent marker density for applications such as Genomic Selection [6]. Besides the RAD method, Elshire et al. [7] recently described a straightforward method of genotyping-by-sequencing. Additionally the DArT complexity reduction protocol has also been streamlined based on NGS for a number of plant genomes including *Eucalyptus* (see Sansaloni et al. this meeting). All these NGS based genotyping methods will cause a paradigm shift in our ability to carry out high density, high throughput and low-cost genotyping of large numbers of samples, unlocking incredible opportunities in forest tree genetics and breeding in the years to come.

**References**

1. 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).

2. Myles S, Chia JM, Hurwitz B, Simon C, Zhong GY, Buckler E, Ware D: Rapid Genomic Characterization of the Genus Vitis. *Plos One* 2010, 5(1).

3. Van Tassell CP, Smith TPL, Matukumalli LK, Taylor JF, Schnabel RD, Lawley CT, Haudenschild CD, Moore SS, Warren WC, Sonstegard TS: SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. *Nature Methods* 2008, 5(3):247-252.

4. Grattapaglia D, Silva-Junior O, Kirst M, Lima BM, Faria DA, Pappas GJ: High-throughput SNP genotyping in the highly heterozygous genome of *Eucalyptus*: assay success, polymorphism and transferability across species. *BMC Plant Biology* 2011, 11:65.

5. Ott J: Strategies for Characterizing Highly Polymorphic Markers in Human Gene-Mapping. *American Journal of Human Genetics* 1992, 51(2):283-290.

<figure><img src="image_28.png" /></figure>
<figure><img src="image_29.png" /><figcaption>**Figure 2(abstract P45)** (A) Sampling of the PstI sites in the 11 chromosomes of *Eucalyptus* provided by RAD sequencing. Counts (into bars) and percent (Y axis) of unsampled PstI sites, RAD sequence tags generated in both directions out of the PstI site or in single directions (fwd or rev); (B) Distribution of the distances between adjacent RAD sequencing tags across the 605 Mbp of the *Eucalyptus* genome.</figcaption></figure>

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

7. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE: *A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species*. *PLoS One* 2011, 6(5):e19379.

P46

Identification of five commercial *Eucalyptus* species by SCAR markers
development

Karine Kettener¹*, Aletéa Madacki¹, Tânia Mara Bortoloto¹, Shinitiro Oda²,
Celso Luis Marino¹

¹Genetics Department, Biosciences Institute of Botucatu-SP, Brazil; ²Suzano
Pulp and Paper, Brazil

E-mail: karinekettener@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P46

The genus *Eucalyptus* native to Australia, had some species introduced in Brazil between the nineteenth and early twentieth century and is currently planted in tropical and subtropical regions of the world. Currently, species of the genus have been highlighted as some of the most important in reforestation projects to provide wood for pulp, paper, energy and solid wood products. *Eucalyptus* breeders frequently face difficulties to identify closely related species and their hybrids that share common morphological traits, some of them expressed only later in the breeding cycle [1]. Molecular markers could be useful for early

identification of species and hybrids. A strategy to identify species specific molecular markers is the use RAPD (Random Amplified Polymorphic DNA) combined with bulked segregant analysis [2]. We used RAPD markers and BSA to find putative species specific markers for five pure species of *Eucalyptus*: E. tereticornis, E. saligna, E. grandis, E. brassiana and E. urophylla. We tested 112 RAPD primers which generated 187 candidates polymorphic bands, of which 44 have proved reliable after validation carried out by genotyping individually all plants that composed the bulks. A good species-specific candidate RAPD marker was defined as being exclusive to one bulk, have a size between 300 bp and 2000 bp; provide easy visualization in agarose gel and display repeatability in independent assays. The best candidate bands were provided by the Operon primers: AD01, H03, H19, H20, K10, X06, W03, W05 and W07. As expected, although polymorphic between species, none of the RAPD markers were totally exclusive to one species and absent in the others. However by combining sets of two or more RAPD markers with contrasting frequencies among species it was possible to discriminate all species with high confidence. The most informative polymorphic RAPD amplicons were isolated, purified and cloned into pGEM*-T Easy Vector System I (Promega) and cloned into competent cells of strain DH5α-UltraMAXTM FTTM (Life Technologies, GibcoBRL) for subsequent sequencing and primers design. Based on the sequences obtained a set of SCAR markers was derived after carrying out a temperature gradient test ranging between 52°C and 69°C. The SCAR marker CXT1 allows the identification of E. tereticornis individuals with 80% efficiency. A combination of markers CAS1 and CAS2 or CAS 1 and
CWB 1 or CAS2 and CWB 1 allows identifying 90% of the E. maligna individuals. SCAR markers CAG1 combined with CHG1 can identify 100% of the E. grandis individuals; markers CXG1 and CWB1 combined provide 90% identification of E. urophylla and CWB1 is a strong candidate to uniquely identify E. brassiana. These results indicate that the development of SCAR markers from intensive RAPD marker screening can be an inexpensive way to discriminate different species and possibly hybrids.

### References

1. Domingues DS, Farr APC, Coscrato VE, Mello EJ, Oda S, Marino CL: Identificação de marcador RAPD e SCAR relacionado ao caráter florescimento precoce em Eucalyptus grandis. Ciência Florestal 2006, 16:251-260.

2. Michelmore RW, Paran I, Kesseli RV: Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating animal populations. PNAS 1991, 88:9828-9832.

## P47

### QTL tools applied to forest breeding

Luciano Medina-Macedo¹*, Alexandre Coelho²

¹Biotechnological Processes, Federal University of Parana, Curitiba, Parana, 81531-990, Brazil; ²Department of Agronomy, Federal University of Goias, Goiania, Goias, 74001-970, Brazil

E-mail: medinacwb@hotmail.com

BMC Proceedings 2011, 5(Suppl 7):P47

**Background:** In last decades, the progress of molecular techniques, bioinformatics tools and genome analysis equipment allowed genomes to be sequenced and analyzed for several plants. For Eucalyptus, the combination of quantitative trait loci (QTL) mapping with genomic analyses, has allowed the identification and validation of a few genomic regions involved in growth and wood quality characters. However, several forest companies still have difficulties to apply Marker Assisted Selection (MAS) at the operational level in their genetic improvement programs. Using as case study of a Eucalyptus hybrid progeny, which had QTLs mapped for growth and wood quality characters, the present study compared the possible outcomes of traditional selection used in forest breeding programs, where individuals are classified by genetic values (eBLUP), with selection based only on superior QTL genotypes (MAS).

**Methods:** Genotype selection included 30 trees as the maximum number selected by both selection methods, an amount that corresponds to a 15% selection intensity in relation to the mapping population size used in this study. About 200 individuals of an interespecific hybrid progeny had 3 growth characters evaluated in the field and 5 wood quality characters predicted by Near Infra-Red Spectroscopy (NIRS). This progeny was genotyped with 76 microsatellite markers (SSR). An integrated genetic map was built on LOD 3 and 0.40 recombination fraction, composed by 65 SSR markers distributed in 12 linkage groups with total length of 1,365 cM and average distance among markers of 21 cM [1,2]. The genetic map built covered approximately 83% of the Eucalyptus genome estimated by Brondani et al. [3]. Due to the low density and large distance among markers obtained, QTL mapping was only carried out by single marker analysis using R, at p-values smaller than 0.01.

**Results:** Out of the 65 mapped loci, 10 of them (~15%) showed association with a total of 12 QTLs; 10 loci were associated with only one character and 2 loci with two different growth characters. Three characters showed association to only one QTL. The average volume growth of the 30 individuals selected by MAS was 55% lower than the average of the 30 best individuals selected by phenotypic eBLUP values. One QTL was found for syringil/guayacil lignin ratio and the average of the 30 selected trees by MAS was 17% lower than the average of the best individuals selected by eBLUP. Two QTLs were found for wood density, diameter and lignin content. For density and lignin content, multiple selection for superior alleles at two QTL didn't restrict the number of selected trees, and the average values of individuals selected by MAS were respectively 45% and 83% lower than the average of the best individuals selected by eBLUP. The simultaneous selection for superior alleles at two QTL for diameter restricted from 30 to 23 the

number of selected trees. The simultaneous selection for these two QTLs reduced the average value of the 23 individuals selected by MAS to almost half of that of the 23 best individuals selected by eBLUP. For cellulose yield, the simultaneous selection for superior alleles at three QTL reduced the number of selected trees from 30 to 29. The average value of MAS selected trees was 78% lower than the one of the 29 trees selected by eBLUP. In conclusion, for all traits evaluated, selection based on eBLUP was significantly more efficient than MAS. This result was expected, due firstly to the low marker density of our experiment and hence low power to detect all QTLs involved in the trait. Secondly due to the overestimated QTL effects that bias the real value of each QTL used in selection and finally to the fact that eBLUP based selection captures the final effect of all genes involved in trait expression, while MAS based on a few QTLs captures only a small portion of the variation. These results indicate that MAS for a few QTLs will hardly be useful as the sole selection criteria in forest breeding programs.

**Acknowledgements:** Post-Graduate Program in Agronomy UFG, CAPES and GENOLYPTUS consortium.

### References

1. Medina-Macedo L: Mapeamento de QTL para características de interesse industrial da madeira de *Eucalyptus* em progênie de híbridos interespecíficos. *143 f. Dissertação (Mestrado) – Universidade Federal de Goiás, Escola de Agronomia e Engenharia de Alimentos 2009.*

2. RITTER E, GEBHARDT C, SALAMINI F: Estimation of recombination frequencies and construction of RFLP linkage maps in plants from crosses between heterozygous parents. *Genetics* 1990, 125:645-654.

3. BRONDANI RPV, WILLIAMS ER, BRONDANI C, GRATTAPAGLIA D: A microsatellite-based consensus linkage map for species of *Eucalyptus* and a novel set of 230 microsatellite markers for the genus. *BMC Plant Biology* 2006, 6:20-36.

## P48

### Eucalypt pulp yield QTL from Raiz as compared to the literature

Cristina MP Marques¹*, Víctor J Carocha²,³, Carla Ribeiro³, Fátima Cunha¹, António Mendes de Sousa¹, José A Araújo¹, João Costa e Silva⁵, José Carlos Rodrigues⁵, Ana Freitas⁶, Ana M Pires⁶, Gabriel Dehon Rezende¹
¹RAIZ, Portugal; ²ITQB, Portugal; ³CBA/DBA, FCUL, Portugal; ⁴CEF, ISA/UTL, Portugal; ⁵IICT, Portugal; ⁶DM-CEMAT, IST/UTL, Portugal

E-mail: cristina.marques@portucelsoporcel.com

BMC Proceedings 2011, 5(Suppl 7):P48

**Background:** RAIZ is a Portuguese private non-profit research institute owned by the Pulp & Paper Portucel Soporcel Group (http://www.raiz-iifp.pt). RAIZ *E. globulus* genetic improvement program is managed in order to generate trees with increased economic value, through gains in forest productivity and wood properties. Molecular markers have been used for clonal identification and genetic diversity management in the program since 1990. Moreover, RAIZ has been engaged in a longer term genomics project aiming to identify quantitative trait *loci* (QTL) for wood properties. The ability to detect QTL depends on sample size, genetic background, environment and genetic interactions. Most importantly, the ability to use detected QTL depends on their adequate map location and the identification of the molecular variation behind them. This in turn is determined by linkage map quality, the choice of phenotypes and the precision of phenotyping. There are many reports in the literature on QTL detection for economically important traits in *Eucalyptus*, but very few present data on QTL verification (at the statistical and/or biological levels). We illustrate the importance of this issue for pulp yield related traits by comparing available results from QTL mapping studies in the literature with those obtained from a QTL detection and verification experiment pursued by RAIZ.

**Methods:** An F₁ full-sib family with 361 progeny from an intraspecific *E. globulus* cross was planted in a field trial and phenotyped (at age 4½ years) for pulp yield using near infrared spectroscopy. Phenotypic data was adjusted for spatial variation in the field trial using a first-order separable autoregressive model. The NIR-PLSR spectra were recorded using a Bruker equipment (vector 22N model), and the calibration model to estimate pulp yield (RTIK16) was constructed using partial least-squares regression as implemented in the *QUANT2* software. DNA extraction and microsatellite genotyping were carried out as described in [1,2]. Linkage analysis and map construction were performed on independent
male and female datasets using MAPMAKER/EXP® 3.0[3] under the F₂ backcross model. Framework maps with evenly distributed selected SSR and gene markers were used for QTL detection [4-8]. The selection of SSR markers took into consideration the possibility to establish syntenity between available eucalypt maps in the literature. Interval Mapping and Multiple Interval Mapping QTL detection results were compared with the MultiQTL vs 2.6 software. QTL detection was repeated in data from 100 different simulations (using the R software), after adding to the original phenotypes randomly selected values from a normal distribution with mean zero and a standard deviation that accounted for the reference essay error and the prediction error of the NIR calibration model.

**Results:** Many QTL for pulp yield related traits have been reported for every eucalypt chromosome, in the literature. There is insufficient mapping information to infer if these QTL where detected in similar genomic locations. In 6 of the 11 eucalypt linkage groups (LG2, LG4, LG5, LG6, LG9 and LG10), QTL for pulp yield related traits were detected in different species (E. globulus, E. grandis x E. urophylla and/or E. nitens) (Figure 1). RAIZ reports two verified QTL for pulp yield, in an E. globulus intraspecific cross, in linkage groups 3 and 11, detected in all simulations. Although these results can reflect the polygenic nature of the trait (as suggested by [9]) we cannot exclude the putative biases resulting from current limitations of existing studies in terms of experimental designs, phenotyping and/or data analysis.

**Conclusions:** QTL studies in some plant species have proved useful to target genomic regions for subsequent genomics investigation [10]. Forest trees experience a variety of environmental conditions and it is expected that some QTL will be age/environment specific and some will be consistently detected over multiple growing seasons. In order to raise the scope of inferences that can be drawn from QTL research in *Eucalyptus* and the prospects of delivering breeding tools from gene sequences, future QTL detection studies should account for experimental phenotyping error, in order to reduce putative false positive results, as we have done in RAIZ QTL detection experiment. Existing QTL mapping studies from different institutions could be upgraded in this perspective, in order to allow comparative mapping and the identification (or not) of

specific genomic regions, detected in multiple *pedigrees* and environments, that could be explored further.

**References**

1. Marques C, Vasquez-Kool J, Carocha V, Ferreira J, O'Malley D, Liu B, Sederoff R: Genetic dissection of vegetative propagation traits in *Eucalyptus* tereticornis and *E. globulus*. *Theor Appl Genet* 1999, 99:936-946.

2. Marques M, Brondani V, Grattapaglia D, Sederoff R: Conservation and syntenity of SSR loci and QTLs for vegetative propagation in four *Eucalyptus* species. *Theor Appl Genet* 2002, 105(2-3):474-478.

3. Lander E, Green P, Abrahamson J, Barlow A, Daly M, Lincoln S, Newburg L: MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. *Genomics* 1987, 1:174-181.

4. Brondani R, Williams E, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of *Eucalyptus* and a novel set of 230 microsatellite markers for the genus *Bmc Plant Biology* 2006, 6(1):20.

5. Foucart C, Paux E, Ladouce N, San-Clemente H, Grima-Pettenati J, Sivadon P: Transcript profiling of a xylem vs phloem cDNA subtractive library identifies new genes expressed during xylogenesis in *Eucalyptus*. *New Phytologist* 2006, 170(4):739-752.

6. Paux E, Tamasloukht M, Ladouce N, Sivadon P, Grima-Pettenati J: Identification of genes preferentially expressed during wood formation in *Eucalyptus*. *Plant Molecular Biology* 2004, 55(2):263-280.

7. Rengel D, Clemente HS, Servant F, Ladouce N, Paux E, Wincker P, Couloux A, Sivadon P, Grima-Pettenati J: A new genomic resource dedicated to wood formation in *Eucalyptus*. *Bmc Plant Biology* 2009, 9.

8. Steane DA, Vaillancourt RE, Russell J, Powell W, Marshall D, Potts BM: Development and characterisation of microsatellite loci in *Eucalyptus globulus* (Myrtaceae). *Silvae Genetica* 2001, 50(2):89-91.

9. Thumma BR, Southerton SG, Bell JC, Owen JV, Henery ML, Moran GF: Quantitative trait locus (QTL) analysis of wood quality traits in *Eucalyptus nitens*. *Tree Genetics & Genomes* 2010, 6(2):305-317.

10. Price AH: Believe it or not, QTLs are accurate! *Trends Plant Sci* 2006, 11(5):213-216.

<figure><img src="image_30.png" /><figcaption>**Figure 1(abstract P48)** Raiz and literature QTL studies for pulp yield related traits in *Eucalyptus*, compared at the linkage group (chromosome) level.</figcaption></figure>
<table><caption>Table 1(abstract P49) Heritability, BLUP and GS accuracy for models 1, 2, 3 and 4</caption><thead><tr><td>ModelNumber</td><td>Heritability<sup>1</sup></td><td>BLUP<sup>2</sup>Accuracy<sup>2</sup></td><td>Deregressed</td><td>GS<sup>3</sup>Accuracy<sup>3</sup></td></tr></thead><tbody><tr><td>1</td><td>0.26</td><td>0.80</td><td>Yes</td><td>0.64</td></tr><tr><td>2</td><td>0.31</td><td>0.85</td><td>Yes</td><td>0.77</td></tr><tr><td>3</td><td>0.34</td><td>0.87</td><td>Yes</td><td>0.58</td></tr><tr><td>3</td><td>0.34</td><td>0.87</td><td>No</td><td>0.87</td></tr><tr><td>4</td><td>0.36</td><td>0.89</td><td>Yes</td><td>0.60</td></tr><tr><td>4</td><td>0.36</td><td>0.89</td><td>No</td><td>0.88</td></tr></tbody></table>

3) deregress BVs prior to use as input for construction of GS prediction models.

### References

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

2. Sander K, Bennewitz J, Kalm E: Wrong and missing sire information affects genetics gain in the angeln dairy cattle population. *J Dairy Sci* 2006, 89:315-321.

3. Powell J, Visscher P, Goddard M: Reconciling the analysis of IBD and IBS in complex trait studies. *Nat Rev Genet* 2010, 11:800-805.

4. Lee S, Goddard M, Visscher P, van der Werf J: Using the realized relationship matrix to disentangle confounding factors for the estimation of genetic variance components of complex traits. *Genet Sel Evol* 2010, 42:22.

5. Habier D, Fernando R, Dekkers J: The impact of genetic relationship information on genomic-assisted breeding values. *Genetics* 2007, 142:5.

6. Yang J, Benyamin B, McEvoy B, Gordon S, Henders A, Nyholt D, Madden P, Heath A, Martin N, Montgomery G, Goddard M, Visscher P: Common SNPs explain a large proportion of the heritability for human height. *Nat Genet* 2010, 42:565-571.

### P50

#### Recent application developments in genomic research using the AdvanCE™ FS capillary electrophoresis platform

Julio Petersen*, Dylan Baker, Deepak Dibya, Pierre Varineau, Jeremy Kenseth
Advanced Analytical Technologies, INC
E-mail: jpetersen@aati-us.com

BMC Proceedings 2011, 5(Suppl 7):P50

Central to genomic research is the analysis of nucleic acids, which requires versatile, sensitive and high throughput technologies that can deliver accurate results in a short time. Reported herein are recent developments for sizing and/or quantifying nucleic acids (dsDNA and RNA) on a parallel capillary electrophoresis platform with LED-based fluorescence detection (AdvanCE™ FS system). This flexible multi-capillary platform has been employed for applications such as SSR, gDNA analysis, RNA analysis, quantification of next generation sequencing (NGS) libraries, and the separation and sizing of large cDNA from *Populus spp.*, This poster will discuss recent validation of the platform for a subset of these applications in detail. The AdvanCE™ FS system successfully scored all SSR samples with resolution as low as 2 bp. Assessment of the quality/concentration of gDNA is also demonstrated with a new separation matrix, for separation of large DNA fragments. Separation and sizing of large cDNA was also demonstrated with good resolution and accuracy. A method for rapid and sensitive detection of total RNA concentration and quality will be discussed, as well as the sizing and quantification of NGS libraries. The AdvanCE™ FS platform offers rapid separation and ample resolution with excellent sensitivity and dynamic range, to benefit a variety of applications in genomic research.

### P51

#### Novel design and deployment of orthologous genic SSR markers in *Eucalyptus camaldulensis* Dehn

Kengavanar Nagabhushana*, Prasad Suresh Hendre, Navin Sharma, Rajkumar Rathinavelu*
Plant Molecular Biology, ITC R&D Centre, Bangalore-58, India
E-mail: rajkumar.rathinavelu@itc.in

BMC Proceedings 2011, 5(Suppl 7):P51

**Background:** *Eucalyptus camaldulensis* is a widely planted tree species in India, because of its rapid growth and adaptability to dry regions. Genetic improvement through informed breeding of *E. camaldulensis* largely depends on availability of molecular markers, linkage maps and genome information. Microsatellite markers, also called as simple sequence repeats (SSRs) have wide application due to their unique advantages over other marker systems. In Eucalyptus, SSR markers have been used in various breeding applications from DNA fingerprinting to QTL mapping [1,4]. Despite of their advantages, the major drawback is the time required for their development [6]. There are less than twenty five *E. camaldulensis* specific SSR markers available in the public database [5]. Although, there are large number of SSRs available in other *Eucalyptus* species [2,3], their species transferability in *camaldulensis* is questionable for practical use. Nevertheless, the existing *camaldulensis* specific SSRs are insufficient for developing linkage maps, QTL and comparative mapping studies. Besides, EST based markers are handicapped due to exclusion of introns, which sometimes lead to compromise on product sizing. Therefore we have modeled a novel strategy that targets highly conserved domain in the genic region using both publicly available ESTs as well as genome sequences.

**Methods:** Targeting conserved genic region: A set of publicly available *E. globulus* ESTs were assembled as unigenes using CAP3 algorithm and mined for repeat motifs using MISA program [http://pgrc.ipk-gatersleben.de/misa/]. The repeat rich unigenes were marked and mapped on the whole genome scaffolds of *E. grandis*. The sequence of mapped coordinates were extracted from EUCAGEN database [http://web.up.ac.za/eucagen/] and further validated for repeat motifs and the presence of exons using MISA and GENSCAN (http://genes.mit.edu/genscan.html). A total of 300 ± base pairs were marked in the genic region of scaffolds flanking the repeat motifs and further primers were designed on the flanking region of repeat motifs using the Primer3 tool [http://primer3.sourceforge.net/], following the appropriate parameters (Figure 1). The entire pipeline was automated and multi-threaded using a set of in-house PERL programs.

**PCR, multiplexing and genotyping:** We adopted an innovative two-tier polymerase chain reaction (PCR) system to reduce the cost 12 times on labeled oligo synthesis. The first PCR was performed with M13 tagged forward primer at 5' end whereas the second PCR was performed with fluorescently labeled M13 as forward primer and SSR reverse primer. Multiplexing was carried out at post PCR stage. The first PCR consisted of template DNA (5 ng), primers (2 pM), 10X PCR buffer, dNTPs (1 µM), MgCl₂ (1.5mM) and AmpItaq Gold Taq DNA polymerase (0.25 U). The PCR profile consisted of denaturing the template DNA at 94°C for 5 min, followed by 35 cycles, each at 94°C for 30 sec, 50-65°C for 30 sec and 72°C for 1 min, followed by 72°C for 8 min. The first PCR products were resolved on 2% high resolution agarose. The second PCR consisted of template DNA (2 ng), 2 pM each of forward (labeled M13) and reverse (SSR) primer, 10X PCR buffer, dNTPs (1 µM), and 0.5U of Taq DNA polymerase. The PCR conditions remained same except reduced number of cycles to 20. The amplicons were resolved using ABI 3730 sequencer and each amplicon was manually validated for their allele size.

**Results and conclusion:** About 13,441 *E. globulus* ESTs and 13,380 *camaldulensis* EST/genomic sequences were collected from public database and mined for repeat motifs. A total of 2,330 repeat motifs were identified on 1873 ESTs of which, 1159 were di; 1128 tri and 43 were tetra repeats. There were more than 290 repeats found to be in compound form. The repeat containing ESTs were assembled to a total of 301 unigenes. These ESTs were mapped on *E. grandis* genome scaffolds.

<footnote n="1">¹ Narrow sense heritability, ² Average of accuracy among all clones, ³ correlations between BLUP BVs and GS-BVs prediction.</footnote>
<figure><img src="image_31.png" /><figcaption>**Figure 1(abstract P51)** Strategy summarizing the in silico development of orthologus SSRs.</figcaption></figure>

A total of 124 SSR positive scaffolds were identified and used for designing 230 primer pairs. Primers were standardized using gradient PCR at appropriate annealing temperatures. Of the 230 primers, 179 were successfully amplified and validated in *E. camaldulensis* resulting in 77.82% success. About 95% of the primers were amplified as single and clean product, indicating their locus specificity (Figure 2). These markers

were validated in 4 *Eucalyptus* and 2 *Corymbia* species with multiple alleles ranging from 4 to 12. Surprisingly, 92% of cross species amplification was observed within the genus *Eucalyptus*, while only 21% in *Corymbia*. Higher species transferability in *Eucalyptus* genus shows the power of design as they originate from conserved domain. Further to validate these markers in *E. camaldulensis*, some of the selected primers

<figure><img src="image_32.png" /><figcaption>**Figure 2(abstract P51)** Electropherogram of multiplexed PCR products of primers IME3126 (black), IME3130 (blue) and IME3285 (green) in eight clones of *E. camaldulensis*.</figcaption></figure>
were successfully utilized for parentage analysis, confirmation of interspecific hybrid and genotyping of seedling seed orchard in *E. camaldulensis*. Unlike SSR markers developed from conventional *in silico* methods, orthologous SSRs resulted in very high success rate in *Eucalyptus* species due to targeted repeats in conserved domain. Our present strategy successfully demonstrated the power of orthologous SSR markers and its application in informed breeding in *E. camaldulensis*.

## References

1. Brondani RPV, Brondani C, Grattapaglia D: Towards a genus-wide reference linkage map for eucalyptus based exclusively on highly informative microsatellite markers. *Mol Genet Genomics* 2002, 267:338-347.

2. Brondani RPV, Williams ER, Brondani C, Grattapaglia D: A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus. *BMC Plant Biol* 2006, 6:20.

3. Faria DA, Mamani EMC, Pappas MR, Pappas GJ Jr, Grattapaglia D: A selected set of EST-derived microsatellites, polymorphic and transferable across 6 species of Eucalyptus. *J Hered* 2010, 101:512-520.

4. Kirst M, Cordeiro GD, Rezende SP, Grattapaglia D: Power of microsatellite markers for fingerprinting and parentage analysis in *Eucalyptus grandis* breeding populations. *J Hered* 2005, 96:1-6.

5. Silva JM, Souza ACB, Souza AP, Mori ES, Freitas MLM, Sebbenn AM, Moraes MLT: Development and characterization of 14 microsatellite loci from enriched genomic library of Eucalyptus camaldulensis Dehnh. *Conservation Genet Resour* 2009, 1:465-469.

6. Zane L, Bargelloni L, Patarnello T: Strategies for microsatellite isolation: a review. *Mol Ecol* 2002, 11:1-16.

## P52

### Identification of SNPs in candidate genes related to water stress in *Eucalyptus*

Cintia Helena Duarte Sagawa¹*, Leonardo Curi Martin¹, Andréia Santoro¹, Julio Cezar Santos Otto¹, Juliana Erika de Carvalho Teixeira², Rinal do César de Paula³, Celso Luis Marino¹

¹Departamento de Genética, UNESP, Botucatu, São Paulo, 18618-970, Brazil;
²FIBRIA, São Paulo, 12340-010, Brazil; ³Departamento de Produção Vegetal, UNESP, Jaboticabal, São Paulo, 14884-900, Brazil

E-mail: cintiasagawa@ibb.unesp.br

BMC Proceedings 2011, 5(Suppl 7):P52

**Background:** Forestry companies have occupied Northern and Northeastern of Brazil with plantations of *Eucalyptus* species; however, limitations on water supply are affecting the biomass production and reducing the yield significantly [1]. Tolerant plants develop defense mechanisms like hormone production of abscisic acid (ABA) and osmoprotector glycine betain (GB) when submitted to drought conditions [2]. Identifying and studying genomic regions related to water stress tolerance are important for tree improvement programs. In this work, SNPs in candidate genes, 9-cis-epoxy-carotenoid dioxygenase (NCED) and choline monooxygenase (CMO), of ABA and GB biosynthetic pathways related to drought tolerance in a population of *Eucalyptus* were identified, as well as, a possible molecular marker associated to genomic regions related to plant response to water stress through AFLP technique [3] combined with bulk segregant analysis method [4].

**Materials and methods:** Contrasting plants of *E. grandis*, *E. urophylla*, and their hybrids were selected according to their physiology. Specific primers were designed from homology sequences from *Eucalyptus* ESTs databank and amplification products submitted to sequencing which allowed the identification of SNPs and the genotyping of these plants. For the identification of the AFLP marker, contrasting DNA bulks were digested with restriction enzymes combination and routine protocols were followed. The selective amplification products were separated on 6% denaturing polyacrylamide gel and seen in the silver nitrate staining [5].

**Results and conclusions:** Seven SNPs were identified in a region of 1230 bp on NCED gene from which five were in codified regions and generated synonymous mutations. For the CMO gene, 49 SNP were identified in a region of 3885 bp, which 12 were in codified regions and 37 in UTRs and intron regions. Especially for these codified regions; 83,3% of the mutations were synonymous and 16,7% were non-synonymous. Through the genotyping of the SNPs, the NCED and CMO genes presented respectively, seven haplotypes with 15 different genotypes and 18 haplotypes with 16 diverse combinations. Nevertheless, CMO gene showed some unique

genotypes for some species. Then, the genotyping of individuals by the allele-specific extension technique demonstrated to be efficient; moreover the SNPs primers designed can decrease costs and permit the genotyping of these mutations in large scale of contrasting populations to water deficit and in population studies. From the analysis of the DNA bulks, 50 AFLP primer combinations were tested from which 27 generated polymorphic bands between the bulks and just one primer combination was confirmed in all susceptible plants of the bulk by now. This fragment sequence will be compared and converted into PCR-based markers.

## References

1. Whitehead D, Beadle CL: Physiological regulation of productivity and water use in Eucalyptus: a review. *For Ecol Manag* 2004, 193:113-140.

2. Chen THH, Murata N: Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. *Curr Opin Plant Biol* 2002, 5:250-257.

3. Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M: AFLP: a new technique for DNA fingerprinting. *Nucleic Acids Res* 1995, 23:4407-4414.

4. Michelmore RW, Paran I, Kesseli RV: Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. *Proc. Natl. Acad. Sci. USA* 1991, 88:9828-9832.

5. Creste S, Tulmann Neto A, Figueira A: Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. *Plant Mol Biol Rep* 2001, 19:299-306.

## P53

### Identification of genomic regions related to early flowering in *Eucalyptus*

Vanusa Socorro Leite¹, Julio César Santos Otto¹, Cintia Helena Duarte Sagawa¹*, Esteban Roberto Gonzalez², Maria Giziane Fagundes², Shinitiro Oda², Celso Luis Marino¹

¹Departamento de Genética, UNESP, Botucatu, São Paulo, 18618-970, Brazil;
²Suzano Papel e Celulose, Suzano, São Paulo, 08613-000, Brazil

E-mail: cintiasagawa@ibb.unesp.br

BMC Proceedings 2011, 5(Suppl 7):P53

**Background:** The genus *Eucalyptus* is the most important planted tree to the Brazilian economy. Besides being a source of timber, its wood pulp is mostly used for papermaking. There is a considerable interest in its genetic improvement due to the economic importance of the gender to forestry companies. The time required to obtain ideal populations, after selective breeding, is a limiting factor of classical breeding in these species. However, the breeding program of Suzano Papel & Celulose company obtained a plant of *E. grandis* with the early flowering character, whose first flowering took place between 60 and 90 days. This plant was grown and its seeds were collected and germinated. Progeny segregated for early flowering after approximately 60 to 90 days.

**Materials and methods:** AFLP markers [1] combined with bulk segregant analysis method (BSA) [2] were used to search for specific DNA polymorphisms in the genome to produce markers which could identify the character at any stage of development. AFLP markers showed polymorphisms based on the distribution of restriction sites and differential amplification of the fragments. The methodology followed the routine protocols for the AFLP technique and BSA method. Contrasting bulks were prepared, one bulk with ten normal flowering plants and another with ten early flowering plants. DNA was extracted and digested using two restriction enzymes, a frequent cutting (*MseI*) and a rare cutting (*EcoRI*), followed by connection of the adapters.

**Results and conclusion:** The pre-amplification and selective amplification were performed to test 32 primer combinations, which were seen in 6% polyacrylamide gel stained with silver nitrate [3]. As a result, 13 primers amplified and showed polymorphic bands. These amplified fragments are candidates to be specific molecular markers but still need confirmation. Hence, new analyses are being conducted using DNA from each individual separately in order to verify its co-segregation with the early flowering phenotype. Moreover, confirming its effectiveness as a marker in these plants, new strategies will be adopted for validation, such as cloning, sequencing and the development of primers for testing in other populations of *Eucalyptus*. These results indicate the feasibility of using AFLP technique to detect polymorphisms, which might contribute to the improvement of the species.
## References

1. Vos P, Hogers R, Bleeker M., Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M: AFLP: a new technique for DNA fingerprinting. *Nucleic Acids Res* 1995, 23:4407-4414.

2. Michelmore RW, Paran I, Kesseli RV: Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. *Proc. Natl. Acad. Sci. USA* 1991, 88:9828-9832.

3. Creste S, Tulmann Neto A, Figueira A: Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. *Plant Mol Biol Rep* 2001, 19:299-306.

## P54

### Diversity Arrays Technology (DART) and next-generation sequencing combined: genome-wide, high throughput, highly informative genotyping for molecular breeding of *Eucalyptus*

Carolina Sansaloni¹*, Cesar Petroli¹, Damian Jaccoud², Jason Carling², Frank Detering², Dario Grattapaglia³, Andrzej Kilian²

¹EMBRAPA Genetic Resources and Biotechnology - EPqB Final W5 Norte 70770-917 Brazilia DF and Dep. Cell Biology, Universidade de Brazil - UnB, Brazil; ²Diversity Arrays Technology Pty Ltd - PO Box 7141, Yarralumla, ACT 2600, Australia; ³EMBRAPA Genetic Resources and Biotechnology - Estação Parque Biológico, 70770-910, Brazilia, DF and Genomic Sciences Program - Universidade Católica de Brasília - Brazil, Brazil

E-mail: carols@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P54

**Background:** Wider genome coverage and higher throughput genotyping methods have become increasingly important to meet the resolution and speed necessary for a variety of applications in genomics and molecular breeding of forest trees. Developed more than 10 years ago [1], the Diversity Arrays Technology (DART) has experienced an increasing interest worldwide for it has efficiently satisfied the requirements of throughput, genome coverage and inter-specific transferability for over 40 different plant species to date, including *Eucalyptus* [2] and recently *Pinus* (Dione Alves-Freitas, this meeting). DART is based on genome complexity reduction using restriction enzymes, followed by hybridization to microarrays to simultaneously assay hundreds to thousands of markers across a genome. Genome complexity reduction for genotyping has now been taken to another level when combined to next generation sequencing (NGS) technologies. Such a strategy has been used for rapid SNP discovery in different organisms [3], and proposed as a way to genotype with RAD (Restriction-associated DNA) sequencing [4]and recently by a similar method generally termed GB5 (Genotyping-by-Sequencing)[5]. In this work we assessed the power of the now well established DART marker platform in combination with Illumina short read sequencing to generate a linkage map for a segregating outcrossed F1 population derived from *E. grandis* BRASUZ1, the donor of the *Eucalyptus* reference genome.

**Methods:** A segregating population of 89 individuals derived from the intra-specific cross BRASUZ1 x M4D31 was provided by Suzano company. Correct parentage of all individuals was certified by microsatellite genotyping. DNA samples of parents and progeny were processed for the conventional array-based DART genotyping as described earlier [2] to generate marker data for comparative analysis with the NGS based DART data. For the sequencing based DART genotyping two complexity reduction methods optimized for several other plant species at DART PL were used: PstI_ad/TaqI/Hpall_ad and PstI_ad/TaqI/Hhal_ad with TaqI restriction enzyme used to eliminate a subset of PstI -Hpall and PstI-Hhal fragments, respectively. PstI-site specific adapter was tagged with 92 different barcodes enabling encoding a plate of DNA samples to run within a single lane on an Illumina GAIIx. PstI adapter included also a sequencing primer, so that the tags generated were always reading into the genomic fragments from the PstI sites. After the sequencing run the FASTQ files (full reads of 77 bp) were quality filtered using the threshold of 90% confidence for at least 50% of the bases and in addition filtered more stringently for barcode sequences. The filtered data were split into their respective target (individual) data using barcode splitting script. After producing various QC statistics and trimming of the barcode the sequences were aligned against the reference *Eucalyptus grandis* genome available in Phytozome. The output files from alignment (generated using Bowtie software) were processed using an analytical pipeline developed by DART PL to produce "DART score" tables and "SNP" tables. A linkage maps was constructed with JoinMap 3.0 [6]using the

microarray-based DART markers, DART NGS markers, and 40 microsatellites of known map position as anchors. A parallel analysis exclusively meant to estimate the total number of potential SNPs within the short read tags was carried out using CLC genomic workbench v4.6 software [7] with a minimum read coverage of 6 and minimum variant frequency of 25%.

**Results:** The microarray-based DART platform yielded 1,088 high quality markers of which 505 (46.4%) segregated in a 1:1 pseudo-testcross while the remaining 583 (53.6%) segregated 3:1. This relatively lower number of markers when compared to other Eucalyptus mapping populations was expected. Not only it is an intra-specific cross but also involves BRASUZ1, a know self (S1) individual with a lower level of sequence heterozygosity. DART genotyping using NGS technology yielded 2,835 polymorphic presence/ absence markers, almost three-fold the number produced by the microarray platform. Of these, 2,449 markers mapped to the 11 chromosome scaffolds with an average of 222 markers per scaffold, while the remaining 386 markers fall out of the 11 scaffolds, potentially allowing the localization of a fraction of the still unassembled smaller genome scaffolds. In total, an integrated linkage map with 564 DART markers, 1,930 DART-NGS and 29 microsatellites was preliminarily built. Furthermore, from the 148 million reads generated (~10.5 Gb), 83.6 million (6.1 Gb) were successfully mapped on the Eucalyptus reference genome. Although a very large number of SNPs can be identified when all reads combined are mapped, only a fraction that displays sufficient coverage allows robust scoring at the individual level. Still, over 1,500 SNPs could be confidently genotyped providing a further advantage of adding co-dominant markers to the already large number of dominant markers obtained.

**Conclusion:** These initial results show that the combined use of DART as a robust genome complexity reduction method with optimized barcoded NG sequencing protocol provides at least three fold more dominant markers than the conventional microarray-based DART method and an additional set of co-dominant SNPs. We are now genotyping a much larger set of distantly related individuals of a training population to be used for Genomic Selection (GS). The possibility of delivering large numbers of both dominant and co-dominant markers with the same platform will enable fitting dominance effect in predictive models therefore increasing the selection accuracy.

## References

1. Jaccoud D, Peng K, Feinstein D, Kilian A: Diversity arrays: a solid state technology for sequence information independent genotyping. *Nucleic Acids Res* 2001, 29(4):e25.

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

3. Van Tassell CP, Smith TPL, Matukumalli LK, Taylor JF, Schnabel RD, Lawley CT, Haudenschild CD, Moore SS, Warren WC, Sonstegard TS: SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. *Nature Methods* 2008, 5(3):247-252.

4. 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).

5. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE: A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species. *PLoS One* 2011, 6(5):e19379.

6. Van Ooijen J, Voorrips R: JoinMap 3.0 software for the calculation of genetic linkage maps. Wageningen, the Netherlands: Plant Research Internation 2001.

7. Knudsen B, Knudsen T, Flensborg M, Sandmann H, Heltzen M, Andersen A, Dickenson M, Bardram K, Steffensen PJ, Mønsted S, et al: CLC Genomics Workbench.bio C, 4.6.1.

## P55

### Evaluation of the genetic diversity of a set of parents of *Eucalyptus* spp. by using microsatellite markers to direct matings

Lucas Santos¹*, Eullaysa Sabóia¹, Douglas Almeida², Jupiter Abad³, Alexandre Missiaggia³, Norma Pereira³, Dário Ahnert¹, Fernanda Gaiotto¹, Ronan Corrêa¹

¹Universidade Estadual de Santa Cruz, Brazil; ²Universidade Estadual do Sudoeste da Bahia, Brazil; ³Centro de Tecnologia – Fibria Celulose S.A., Brazil E-mail: luftspeter@gmail.com

BMC Proceedings 2011, 5(Suppl 7):P55

**Background:** To obtain genetically superior cultivars in a breeding program, methods and procedures are necessary to allow the identification
of selected individuals over several cycles of selection while at the same time maintain broad genetic base of the breeding populations. This is crucial to guarantee continuous genetic gains along the program. The establishment of efficient breeding strategies depends on methods and analytical tools. The assessment of genetic diversity with molecular markers of parents used in mating designs could aid optimizing the recombination phase. Microsatellites provide good information content and require small amounts of DNA and may be transferable between species of the same genus. In this study we evaluated the genetic diversity in a set of *Eucalyptus* parent trees and indicated those to be preferentially crossed in a recombination process to potentially maximize variation in the offspring for individual selection of clones.

**Materials and methods:** A total of 20 genotypes were selected from 44 families obtained by crossing 24 parents in different types of hybridizations of populations of *Eucalyptus grandis* and *E. urophylla*. Genomic DNA was extracted from leaves and amplified with microsatellites developed during the Genolyptus project and previously used for *Eucalyptus spp*[1]. The amplifications were performed using the GeneAmp PCR System 9600 (Applied Biosystems) thermal cycler using the following amplification cycle: 96 °C for 2 min, 30 cycles at 94 °C for 1 min, specific primer annealing temperature for 1 min, 72 °C for 1 min and a final extension step at 72 °C for 7 min. Loci were amplified in single and duplex system. Tocher clustering were developed using the genetic distance matrix with the software Genes.

**Results and conclusions:** This study showed wide genetic diversity among the 20 genotypes under evaluation; all nine SSR loci analyzed were polymorphic, with a total of 77 alleles. The number of alleles per locus ranged from 5 (EMBRA 646) to 10 (Embra 645) averaging 9.1. The lowest value of polymorphism was obtained for marker EMBRA 915 (PIC = 0.59) and higher for the marker EMBRA 645 (PIC = 0.89). The expected heterozygosity ranged from 0.66 to 0.89, averaging 0.80. Based on genetic distances, the 20 genotypes were clustered into six distinct groups. The greatest genetic distance was observed between genotypes C022H (GIV) and M4320G (GVI), and the smallest among genotypes P023H (GI) and C407 (GII). Group II clustered the largest number of genotypes (C386H, C407H, C053H, C051H, TC10H, P044H, VR3748H, VR3709, P4295H), consistent with the fact that these genotypes are considered to belong to the same "heterotic" group by the breeders. Based on the genetic distances, the following trees were selected to be preferentially crossed: M4320H, P4295H, P082G, VR3709H, C053H, C219H, C041H and C022H. It remains to be seen if the progenies derived form these crosses will in fact display a wider segregation and allow, as expected, effective selection of extreme phenotypes for growth and wood quality.

**Reference**

1. Cupertino F, Leal J, Corrêa R, Gaiotto F: Comparative study between genomic and EST microsatellite markers to estimate genetic diversity of *Eucalyptus* hybrids. *Biologia Plantarum* 2010, 55:379-382.

**P56**
Chloroplast SNP-marker as powerful tool for differentiation of *Populus* species in reliable poplar breeding and barcoding approaches

Hilke Schroeder¹, Aki Höltken², Matthias Fladung¹

¹Johann Heinrich von Thünen Institut (vTI), Institute for Forest Genetics, 22927 Grosshansdorf, Germany; ²University of Hamburg, Department of Wood Science, Wood Biology, 21031 Hamburg, Germany

E-mail: hilke.schroeder@vti.bund.de

BMC Proceedings 2011, 5(Suppl 7):P56

**Background:** The genus *Populus* is one of the world's most important tree genera. High growth rates, particularly of some interspecies hybrids, and a broad range of applicability from wood and paper to energy production, led to their widespread cultivation in Europe and North America. For the use in SRCs (short rotation coppices), in particular interspecies-hybrids are well suited because of their superior growth and advanced resistance traits. However, *Populus* hybrids are often morphologically extreme variable and can show either more criteria of the one or the other parental species. Thus, species identification within the genus *Populus* using morphological characters can sometimes be difficult. Furthermore, systematically performed records during breeding or vegetative propagation of poplar hybrids and/or clones are not available to date. For breeding activities with the priority of

registration of new high-efficiency clones, clear species identification is inevitably necessary. Therefore, we evaluate the usability of already published plant barcoding regions ("barcoding"; Barcode of Life [http://www.barcodeoflife.org/]) for their efficacy to differentiate seven often used poplar species. We present data on amplification success of the barcoding regions by using the already published primers. Moreover, novel primers were established in promising chloroplast regions to differentiate *Populus* species.

**Methods:** Twenty three published barcoding primer combinations were used for PCR amplification of coding and non-coding (intergenic spacers) regions. Additionally, 17 primer combinations have been newly designed taken advantage of the sequence of the *Populus trichocarpa* chloroplast genome. Obtained sequences were aligned and screened for presence of SNPs by using either the software SeqMan 7.1.0 from DNASTar (Lasergene, GATC Biotech, Konstanz, Germany) or Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, USA). The sequences around the SNPs were checked for restriction sites using the software NEBcutter V2.0 from New England BioLabs Inc (Ipswich, USA).

**Results:** Twelve of the 23 used barcoding primer combinations and fifteen of the seventeen newly designed primer combinations resulted in PCR amplification products in all individuals for the seven species tested. Twenty four of these amplification products have been sequenced and checked for species-specific SNPs or indels. Three chloroplast regions revealed species-specific SNPs using the original barcoding primers and within seven chloroplast regions species-specific SNPs or indels were identified when using the newly designed primers. A ranking, taking into account the percentage of variable sites for the ten chloroplast regions, reveals the first five places being occupied by intergenic spacers: trnH-psbA, psbK-psbI, trnG-psbK, ndhE-ndhG and rbcL-accD, followed by two coding regions: rpoC and rbcL, again one intergenic spacer, rps2-rpoC2, and finally two coding regions rpoB and matK. Besides the usability of the identified SNPs and indels as contribution to the "barcoding of life" project, they should also be used to identify *Populus* species for breeding purposes. For an efficient application, rapid test methods without the need for sequencing or capillary electrophoresis facilities are required. Thus, all sequences with SNPs were checked for restriction sites for use as PCR-RFLPs. For five of the 21 identified SNPs suitable restriction enzymes were found. Thus, we can identify five of the seven used species by using PCR-RFLPs.

**Discussion and conclusions:** Altogether, the use of intergenic spacers seems to be more successful to differentiate closely related species as given within the genus *Populus*, because of the higher overall variability. Thus, the choice of suitable barcoding regions is obviously different for different groups of land plants. Our results support the recommendation of some authors of focussing the examinations of the plastid genome on the barcoding potential of the trnH-psbA spacer. We recommend focusing even more on intergenic spacers in general (for example trnG-psbK). And, therefore, our results also follow the idea of several authors in using multi-locus combinations, because differentiation of only seven species within the genus *Populus* requires three chloroplast regions.

Our study clearly show that SNP markers combined with methods as PCR-RFLPs and length polymorphisms are convenient, easy to use, and - necessary in most breeding programs - fast and of low cost for application in breeding projects.

**P57**
Genome-wide SNP discovery from a pooled sample of accessions of the biofuel plant *Jatropha curcas* based on whole-transcriptome Illumina resequencing

Orzenil B Silva-Junior¹*, Tatiana B Rosado², Bruno G Laviola², Marilia R Pappas¹, Georgios J Pappas Jr³, Dario Grattapaglia³

¹EMBRAPA Genetic Resources and Biotechnology, Brazil; ²Embrapa Agroenergy, Brazil; ³EMBRAPA Genetic Resources and Biotechnology - Estação Parque Biológico, 70770-910, Brazilia, DF and Genomic Sciences Program - Universidade Católica de Brasília - Brazilia, Brazil

E-mail: orzenil@cenargen.embrapa.br

BMC Proceedings 2011, 5(Suppl 7):P57

**Background:** *Jatropha curcas* (JC) is an oil-rich, drought-tolerant perennial shrub of the *Euphorbiaceae* family widely dispersed throughout the world. Thought to be native to Central America, it has been the object of an increasing number of studies in recent years for it exhibits a number of
appealing attributes as a promising source of biodiesel. Although its
undomesticated nature and preferential outcrossed mating system would
suggest a high degree of genetic variation to be exploited in breeding,
studies have shown limited genetic diversity in the existing germplasm
collections [1]. In spite of the increased interest in this bioenergy plant,
challenges still exist to turn this species into a genuine crop and
improved varieties that consolidate desirable traits are not yet available,
making JC large scale plantation an uncertain business [2]. Genomic
studies to potentially assist JC breeding efforts have started in the last
few years. JC is diploid (2n=22), with a haploid genome size estimated at
416 Mbp [3]. EST databases focusing on gene discovery were constructed
[4]and a draft genome sequence was recently published covering 285
Mbp (~68%) of the genome in 120,586 contigs with 40,929 predicted
gene models [5]. The focus of our work with JC is to provide effective
tools to accelerate breeding through Genomic Selection (GS) [6] and to
help assess the levels, organization and enrichment strategies of genetic
diversity in germplasm banks and breeding populations. To this end we
have started the development of SNP markers. Available EST databases
built from single individual plants do not provide the necessary sequence
diversity for SNP discovery.In this work we report on the discovery of a
set of SNPs for JC derived from a pool of genetically diverse accessions
using Illumina sequencing and a SNP selection pipeline recently
described [7].

**Methods:** Genetic diversity data was used to select twelve JC accessions that maximized genetic diversity out of a germplasm collection currently serving as the foundation of a breeding program [1]. Total RNA of young expanding leaves was extracted from each individual plant and a pool of equimolar quantities of RNA was prepared. Two Illumina GAIIx single end lanes were sequenced following standard protocols. Raw reads were processed and aligned on the mapped reference genome using GSNAP [9]. GATK Unified Genotyper [10] was used to estimate the allele

frequency in the pooled samples and to provide an accurate posterior
probability of there being a segregating variant allele at each locus using
a Bayesian genotype likelihood model. SNPs were then specifically
selected to design Illumina Golden Gate Genotyping Technology (GGGT)
assays based on an *in silico* estimated minor allele frequency MAF >0.10
and at least 60 bases available on each SNP flank with no additional
SNPs following a procedure described earlier [7].

**Results and discussion:** The two lanes yielded a total of 74 million reads from which 66.5 million were filtered providing 11.8 Giga bases of high quality sequence. Upon mapping on the JC draft genome sequence 28,110 unigenes were sampled covering 22.1 Mbp of the 39.7 Mbp total unigene length, i.e. 56% of the transcribed portion currently predicted in the draft genome. From the 66.5 million reads, 60.8 were aligned on the genome with an average coverage of 152X of the unigene sampled. A large percentage of these reads (73%) were identical, derived either from abundant transcripts or more likely from amplification bias introduced by the PCR enrichment step during library preparation a standard occurrence in NGS [8]. After removing read amplification bias a total of 16.4 million de-replicated reads were aligned providing an average coverage of 26X of the sampled unigenes and a much more reliable substrate for SNP discovery. The distribution of percent coverage levels attained for each sampled gene was estimated (Figure 1). If a gene was completely covered by reads from the first base to the last at a depth >1X, then this gene was given a value of 100%. With no MAF and flanking sequences filtering a total of 18,225 SNPs were detected.When a MAF>0.10 was applied, 9,164 SNPs in 2,907 genes survived. When 60 bases with no SNPs on both flanks were required 1,574 high quality SNPs were recovered sampling 895 genes while 561 SNPs had coordinates not falling into any predicted gene model. These results corroborate the low level of sequence polymorphism in the breeding material and further highlight the need to widen the current germplasm base for successful breeding [1]. While a set of 768 high quality

<figure><img src="image_33.png" /><figcaption>**Figure 1(abstract P57) Distribution of percent gene coverage.** Distribution of percent gene coverage attained by the transcriptome sequencing at various coverage depth levels using dereplicated reads.</figcaption></figure>
SNPs likely to show high conversion rate with the GGGT can now be developed, alternative genotype-by-sequencing technologies might provide wider genome coverage and thus assay a larger number of sequence polymorphisms.

## References

1. Rosado TB, Laviola BG, Faria DA, Pappas MR, Bhering LL, Quirino B, Grattapaglia D: Molecular Markers Reveal Limited Genetic Diversity in a Large Germplasm Collection of the Biofuel Crop Jatropha curcas L. in Brazil. Crop Science 2010, 50(6):2372-2382.

2. Divakara BN, Upadhyaya HD, Wani SP, Gowda CLL: Biology and genetic improvement of Jatropha curcas L.: A review. Applied Energy 2010, 87(3):732-742.

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

4. Natarajan P, Parani M: De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing. BMC Genomics 2011, 12(1):191.

5. Sato S, Hirakawa H, Isobe S, Fukai E, Watanabe A, Kato M, Kawashima K, Minami C, Muraki A, Nakazaki N, et al: Sequence Analysis of the Genome of an Oil-Bearing Tree, Jatropha curcas L. DNA Research 2011, 18(1):65-76.

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

7. Grattapaglia D, Silva-Junior O, Kirst M, Lima BM, Faria DA, Pappas GJ: High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species. BMC Plant Biology 2011, 11:65.

8. Smith DR, Quinlan AR, Peckham HE, Makowsky K, Tao W, Woolf B, Shen L, Donahue WF, Tusneem N, Stromberg MP, et al: Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res 2008, 18(10):1638-1642.

9. Wu Thomas D, Nacu S: Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 2010, 26:873-881.

10. DePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics 2011, 43(5):491-498.

## P58

**Use of SSR-Tools for clone certification in Uruguayan *Eucalyptus grandis* and *Eucalyptus dunnii* breeding programs**

Diego Torres-Dini<fnref n="1" />*, Zohra Bennadi², Milton Cabrera³, Carmelo Centurion³, Fernando Resquin², Gustavo Balmelli²

E-mail: dtorres@tb.inia.org.uy

BMC Proceedings 2011, 5(Suppl 7):P58

**Background:** Over the last two decades, commercial plantations in Uruguay have increased exponentially, particularly those destined for pulp and paper industry. Although the about 700,000 ha of commercial plantations of Eucalyptus are a continuous source for the selection of elite genotypes, there is no national registration system for Eucalyptus clones in this country. The traditional procedure of clonal identification accepted by the UPOV (International Union for the Protection of New Varieties of Plants) involves the evaluation of morphological descriptors such as leaf shape, bark texture, fruit shape, etc. [1]. The assessment of these characters varies from one observer to another, potentially leading to ambiguous results [2,3]. This restriction has led countries such as Brazil to include the use of microsatellite markers as additional descriptors, in their legislation. The hypervariability and single inheritance of microsatellite markers provide a powerful clonal characterization system through fingerprinting. The identification of elite clones is nowadays one of the most widely used applications of molecular markers. This could generate a labeling system to follow the material traceability in companies with large-scale production of clonal nurseries. Proper identification makes new clones releasing easier and improves the management of seed orchards and controlled pollination breeding programs [4-6]. The aim of this study was to characterise 24 elite clones

of several breeding programs in Uruguay, by the use of microsatellites. This was achieved by verifying the potential for discrimination of these molecular tools by assigning a specific molecular pattern of fingerprinting to each tested clone.

**Methodology:** Plant material was collected from 24 elite clones of Eucalyptus, provided by INIA Uruguay and Forestal Oriental. They were subdivided in 14 samples of E. grandis and 10 of E. dunnii. The tested clones were analyzed by PCR with 5 microsatellite loci using EMBRA 4, EMBRA 5, EMBRA 10, EMBRA 11 and EMBRA 16 markers under the same conditions as previously reported [7,8]. The molecular weight of the alleles was estimated with an automatic sequencer Macrogen Koerea [http://www.macrogen.com/eng/macrogen/macrogen_main.jsp] that employed Peak Scanner software V1.0 [https://products.appliedbiosystems.com]. The data obtained for each microsatellite was subjected to statistical assay to determinate the following parameters: the probability of identity (PI), the paternity exclusion probability (Pe), the heterozygosity (He) and the observed heterozygosity (Ho). This analysis was performed with the software IDENTITY 1.0 [9].

**Results and discussion:** The generated data was sufficient to assign a specific profile for each tested clone. The five EMBRA markers amplified a total of 90 alleles and a maximum of 23 alleles for the EMBRA 11 marker. The minimum was found for the EMBRA 16 marker with 14 alleles. The remaining three markers showed 20, 18 and 15 alleles per locus. The probability of identity (PI) was 5.43 x10⁻⁹ and the paternity exclusion (Pe) 0.999. The expected heterozygosity (He) for all loci ranged from 0.894 to 0.958 while the observed heterozygosity (Ho) showed values ranging from a minimum of 0.471 to a maximum of 0.936 for every loci. A likelihood value of complete identity of 5.43 x10⁻⁹, guarantees the individual identity of each clone, as the probability of two being equal by chance is 1 in 184 million. A precise molecular pattern for each of the 24 genotypes could be assigned and the information produced is sufficiently robust to ensure the molecular traceability for each of these materials at the different stages of nursery production and/or field trials. However, the five markers derived from genomic sequence containing dinucleotide repeats. These markers, even when making a great discrimination, have reduced precision of genotyping, necessary for comparative multilocus profiling across laboratories or even at different times in the same equipment [10]. Considering a National System of Clones Registration, it proves necessary to increase the number of markers employed by including tetra and pentanucleotide markers to strengthen the precision and reproducibility among different laboratories. In addition, special emphasis should be laid on their selection, focusing on those microsatellites with high interspecies transferability. The testing of different species of Eucalyptus with the same molecular methodology and standardized procedures, will contribute to the registration of elite material and the protection of intellectual property.

**Conclusions:** The results show that using five microsatellite markers it is possible to discriminate among 24 clones and assigning a molecular fingerprint characteristic for each of them. The transferability of the five markers used is also verified, since they amplified both *E. grandis* and *E. dunnii*.

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<footnote n="1">¹Forestry Biotechnology Laboratory, National Institute for Agriculture Research. INIA Tacuarembó, Uruguay; ²Forestry Breeding Program, INIA Tacuarembó, Uruguay; ³Forestal Oriental, UPM, Paysandú, Uruguay</footnote>