POSTER PRESENTATION Open Access

Comparative assessment of SNPs andmicrosatellites for fingerprinting, parentage andassignment testing in species of EucalyptusLeonardo Correia1*, Danielle Faria2, Dario Grattapaglia1

From IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and DeliveryArraial d’Ajuda, Bahia, Brazil. 26 June - 2 July 2011

BackgroundBrazil stands on the international scenario by havingone of the largest natural forest heritage and extensivesustainable planted forests. Brazilian planted forestry isbased mainly on fast growing eucalyptus and pines, withthe pulp, paper and steel industries as the major consu-mers. Introduced commercially in Brazil in the earlytwentieth century, the eucalypts have experiencedincreasing levels of genetic improvement over the years.Along with the classical breeding, the use of geneticmarkers has positively influenced breeding programs,contributing to quality control processes of clonal for-estry and advanced breeding. Microsatellite markershave been the main tool used to date. They are multial-lelic, highly polymorphic and thus very efficient forseveral applications that require identification and dis-crimination of elite clones and determination of paren-tage. While di and tri-nucleotide repeat microsatellitestend to be more polymorphic, tetra and higher orderrepeats allow a more robust allele calling [1]. SNP mar-kers genotyped by a high-throughput system have beenrecently developed for Eucalyptus [2] but not yet routi-nely incorporated in Eucalyptus breeding programs. Inspite of their lower genetic information content, manymore SNPs can be simultaneously typed using auto-mated systems, an appealing feature to operationalbreeding programs. The aim of this study was to com-paratively evaluate the resolving power and precision ofdifferent sets of molecular markers (microsatellites andSNPs assayed by the Golden Gate technology) for the

most common operational applications in Eucalyptusbreeding programs.

MethodsSamples belonging to six species of Eucalyptus(E. grandis, E. urophylla, E. globulus, E. nitens, E. camal-dulensis and E. dunnii) were genotyped with three dif-ferent sets of markers: (1) 24 di- and trinucleotidesmicrosatellites (2) 17 tetra-, penta- and hexanucleotidesmicrosatellites and (3) selected subsets of SNPs fromthe 768 developed earlier [2]. Microsatellites were geno-typed by capillary electrophoresis in an ABI3100 geneticanalyser and data collected under dye set D using Gen-escan/Genotyper.SNP genotyping was performed usingan Illumina BeadStation 500 GX at the Genome facilityof the University of Florida and the data analyzed usingthe software GenomeStudio. Multilocus genotypes wereobtained with the two microsatellite panels and a set ofthe 96 most informative SNPs for individual identifica-tion based on an algorithm that takes into accountminimum allele frequency and observed heterozygositywithin and across species. The genotype data were usedto estimate the combined probability of paternity exclu-sion (PE) and probability of identity (PI) for the markerpanels. Additionally, markers were assessed for theirability to estimate genomic ancestry of individuals, aparticularly useful application for determining the originof spontaneous Eucalyptus hybrids. All typed individualswere assigned probabilistically to a given number ofpopulations inferred with a Bayesian approach withoutany prior population information using the STRUC-TURE package [3]. The analyses were performed witheach set of microsatellites and with a specific subsetof 96 ancestry informative SNPs, selected with an

* Correspondence: leoqcorreia@gmail.com1EMBRAPA Genetic Resources and Biotechnology – Estação ParqueBiológico, 70770-910, Brazilia, DF and Genomic Sciences Program -Universidade Católica de Brasília - Brazilia, BrazilFull list of author information is available at the end of the article

Correia et al. BMC Proceedings 2011, 5(Suppl 7):P41http://www.biomedcentral.com/1753-6561/5/S7/P41

© 2011 Correia et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

algorithm that focused on SNPs that display contrastingallele frequencies among species.

ResultsThe panel of di- and trinucleotide microsatellitesshowed the fastest increase in the combined paternityexclusion with the addition of new markers. For exam-ple, a PE > 99% was achieved with only five markers inE. grandis. The panel of tetra-, penta- and hexanucleo-tide microsatellite was unable to reach a PE of 99% evenwith all 17 markers were used. The panel of 96 SNPsshowed a slower increase in the combined paternityexclusion with the addition of new markers, but neededonly 35 markers to reach a PE > 99%. The same trendwas observed with the estimate of probability of identity.In the analysis of genetic structure, all three sets of mar-kers correctly inferred the six genetically distinct popu-lations, corresponding to the six species studied.Furthermore, both panels assigned correctly all indivi-duals to their respective species. The SNP panel showedthe highest average proportion of correct assignment forall six species, followed by the di- and trinucleotides andat last the tetra-, penta- and hexanucleotide panel,although these estimates were not statistically differentbetween the three marker panels tested, (p= 0.327).

ConclusionsResults show that di- and trinucleotide microsatellitestested are around three times more informative than thetetra-, penta- and hexanucleotide microsatellites usedand about seven times more informative than the speci-fic SNPs employed for assessment of parentage andindividual identification. Although more informative, thedi- and trinucleotide microsatellites are subject to ahigher inaccuracy in allele calling, which can result ininconsistencies across analyses carried out in differentlabs.All three marker panels showed high resolutionpower to detect genetic structure and carry out assign-ment tests in species of Eucalyptus. We are now extend-ing this analysis to controlled and spontaneous hybridsto compare the estimates of genomic ancestry providedby the three marker panels. Additionally we are develop-ing much larger sets of SNPs by re-sequencing reducedgenomic representations of pooled individuals from allsix species. This should allow a targeted selection ofmore ancestral SNPs that are homogeneously poly-morphic across species to be used for fingerprinting andparentage or historically more recent SNPs that displaycontrasting allele frequencies among species and thus bemore useful for genomic ancestry determination. Weanticipate that despite the lower individual genetic infor-mation content, careful selection of SNPs panels for tar-geted applications coupled to their accurate genotypecalling and high throughput will become an increasingly

attractive alternative for operational application inbreeding programs.

AcknowledgmentsThis work was supported by the Brazilian Ministry of Science andTechnology through CNPq grant 577047/2008-6 and FAP-DF Grant NEXTREE193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004

Author details1EMBRAPA Genetic Resources and Biotechnology – Estação ParqueBiológico, 70770-910, Brazilia, DF and Genomic Sciences Program -Universidade Católica de Brasília - Brazilia, Brazil. 2EMBRAPA GeneticResources and Biotechnology – Estação Parque Biológico, 70770-910, Brazilia,DF, Brazil.

Published: 13 September 2011

References1. Faria DA, Mamani EMC, Pappas GJ, Grattapaglia D: Genotyping systems for

Eucalyptus based on tetra-, penta-, and hexanucleotide repeat ESTmicrosatellites and their use for individual fingerprinting andassignment 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 ofEucalyptus: assay success, polymorphism and transferability acrossspecies. BMC Plant Biology 2011, 11:65.

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

doi:10.1186/1753-6561-5-S7-P41Cite this article as: Correia et al.: Comparative assessment of SNPs andmicrosatellites for fingerprinting, parentage and assignment testing inspecies of Eucalyptus. BMC Proceedings 2011 5(Suppl 7):P41.

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Correia et al. BMC Proceedings 2011, 5(Suppl 7):P41http://www.biomedcentral.com/1753-6561/5/S7/P41

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