Validação in silico de marcadores ssr de eugenia uniflora e transferibilidade para sete espécies de eucalyptus

Abstract

The progress of next generation sequencing techniques (NGS) has enabled the genome sequencing of economically important species not only with relatively low costs but also developing primers with efficiency in amplifying microsatellite rich regions. The validation of markers is of major importance since it allow us to obtain reliable and suitable results. Transferability assays in silico requires a database containing the gene sequences of the species under study that may be found through the Expressed Sequence Tag (EST) database. ESTs are markers of interest as they control phenotypic traits with economic importance and to assess the distribution of microsatellite molecular markers in transcribed and non-transcribed sites. In this regard, economically important species from the Myrtaceae family were chosen for the present study: Eugenia uniflora and Eucalyptus spp. This study aimed to validate E. uniflora primers and test its transferability to seven species of Eucalyptus. For this purpose, data from the sequencing, draft genome assembly of the E. uniflora, prospection of microsatellites and primer design were used. The validation of the primers was held through a gel electrophoresis simulation. After that, data from ESTs were obtained from the Taxonomy browser on the NCBI website for seven Eucalyptus species, requiring the removal of redundancy since this database has no curation which implies the possibility of many repeated sequences. These repeated sequences were excluded and, lastly, the PCR simulation was fulfilled. For the primers validation through virtual gels, from an amount of 111 primers tested, 80 primers amplified only one locus in the evaluated area, proving to be viable for amplification in laboratory. Besides, eighteen primers showed amplification of two loci in the assessed region and because of that, they need to be evaluated in laboratory for adjustments in reagent concentrations and annealing temperatures. Moreover, thirteen primers showed amplification of overlapped loci or above three loci in the assessed region, thus making their evaluation unfeasible. For transferability by comparing the SSR Eugenia uniflora primers (111) with the Eucalyptus EST database by virtual PCR, it was possible to obtain the number of transferable primers for each species. The largest number of transferable primers was E. gunni (95), followed by E. camaldulensis (94), E. globulus (89), E. pellita (86) E. grandis (84), E. urophylla (76) and E. tereticornis (35). The transferability of primers was relatively high in comparison to the data presented in literature meaning, therefore, success. Yet, the fact that some species show a low level of transferability may also occur for the low number of sequences available in the database. These markers have a high development cost and, for this reason, a cheaper alternative is of the utmost interest. As a conclusion, it was possible to evidence a reduction in laboratory costs through the validation of the primers in addition to reveal the possibility of transferability Eugenia uniflora SSR primers for the seven species of Eucalyptus.