Discovery, validation, and in silico functional characterization of EST-SSR markers in Eucalyptus globulus

ACUÑA, C.; FERNANDEZ , P.; VILLALBA, P.; GARCIA, M.; HOPP, H,E.; S.N. MARCUCCI POLTRI

TREE GENETICS & GENOMES

SPRINGER HEIDELBERG

Año: 2012 vol. 8 p. 288 - 301

1614-2942

Eucalyptus globulus Labill. (Tasmanian blue gum) is a forest tree native to south-eastern Australia and is the most worldwide planted hardwood species for pulpwood in temperate regions. Recently, genomic research in Eucalyptus have made significant progress, leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. In the present study these sequences were utilized to identify non redundant sequences, to in silico predict putative gene functions and to discover simple sequence repeats (SSRs). These new functional molecular markers (SSR) from ESTs published in public databases from Eucalyptus globulus were also validated for their utility in analysis of functional genetic diversity. Results From 12,690 updated E. globulus EST database published in National Center for Biotechnology Information (NCBI) (December 2008), a total of 4,924 non-redundant sequences were identified. From these ones, 952 unigenes (approximately 23%) contained 1140 SSRs, being SSR motifs mainly trimeric (58.6%) as expected but also dimeric ones (22.8%). A new set of 1,017 primers were designed for SSR-markers after bioinformatic analysis. The putative functions of these SSR-ESTs were adjudged, including biological process, molecular function and cellular component Gene Ontology (GO) categories. Most of SSR-ESTs (78.7%) had significant matches (E¡Ü10-5) to the non-redundant (nr) protein database using BLASTX. Gene Ontology (GO) categories were assigned to 226 SSR-ESTs (30.2%). A random set of 57 primer pairs designed were validated in eight genotypes coming from six geographical races, being seventeen SSRs polymorphic (2-8 alleles per locus) and 16 showed in silico cross-transferability between species through other Eucalyptus spp. EST databases. Conclusion Bioinformatics tools and genomic public databases provide knowledge to look for new functional SSR markers in ESTs, showing an easy alternative to quantify functional diversity. The set described here allowed the characterization of new markers and promise to be useful in genetic studies such as: genetic diversity, taxonomic studies, linkage mapping, mapped based cloning of genes, and facilitate marker-assisted breeding in Eucalyptus in an easy way of application.