DEVELOPMENT OF MOLECULAR MARKER IN Pinus pinaster BASED ON PINE ESTs

¹ UMR 1202 BIOGECO, INRA, Equipe de Génétique, 69 route d’Arcachon, 33612 Cestas cedex, France

² INRA – UREFV, Service Bioinfo-Biotechno, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon cedex, France

³ CIFOR-INIA, Departamento de Mejora Genetica Forestal, Carretera de la Coruna Km 7, 28040 Madrid, Spain

⁴ CNR, Istituto di Genetica Vegetale, Sezione di Firenze, Via Madonna del Piano, Edificio D, 50019 Sesto Fiorentino, Firenze, Italy

Large scale Expressed Sequence Tag (EST) sequencing is considered as the most cost-efficient strategy of gene discovery for organisms whose genome sequence is not yet available, as it is the case for the megagenome of conifer species. ESTs also constitute a valuable resource for the development of molecular markers, accessible in large quantity with only little laboratory development. We used the EST databases of two pine species (Pinus pinaster and Pinus taeda) to uncover two types of markers: i) An automated method based on Polybayes* was first developed to detect SNPs in maritime pine ESTs. A set of true SNPs manually detected in 14 gene sequences was used to optimise the pipeline parameters for SNP discovery in pine genes. As a result, false positive rate was rather low (4%) and a total of 98% of SNPs with allele frequency > 0.01 could be retrieved. The 18,498 P. pinaster ESTs assembled into contigs were then searched for SNPs. 540 SNPs were detected in contig size ranging from 15 to 25 sequence reads, corresponding to an average of 1 SNP every 175 bp. ii) In parallel, we used Sputnik** to search SSRs within the loblolly and maritime pine unigene sets. A total of 579 SSRs were found resulting in a frequency of 1 SSR per 48 genes. We tested the amplification of 72 primer pairs in 7 pine species and evaluated their polymorphism in natural population and mapping pedigrees of maritime pine.
*http://www.genome.wustl.edu/groups/informatics/software/polybayes/
**http://espressosoftware.com/pages/sputnik.jsp