In silico microsatellite transferability from Psidium guajava to Eucalyptus globulus validated by PCR
BLAST is a genomic local alignment search tool used to identify homology between genotypes and possible orthologous genes. In vitro microsatellite transferability is a strategy to enable or increase species molecular fingerprinting, but it is dependent on PCR technique. An initial in silico step using BLAST for transferability can be helpful to save resources in pre-selecting markers more likely to amplify. We aligned and transfered SSR sequences from Psidium guajava to Eucalyptus globulus using BLAST. Twenty-three SSR clone sequences from P. guajava (query) were retrieved from the NCBI website and aligned against the whole genome of E. globulus (subject) using a cut-off e-value<1.00e−20. Another 140 loci retrieved from the GuavaMap project were analyzed using as parameters e-values<1.7 and a maximum distance of 300 nucleotides between forward and reverse sequences. All loci were analyzed using BLASTN with MEGABLAST optimization. DNA extraction of four eucalypt trees was performed with the 2x CTAB protocol containing a sorbitol initial step. Validation of the SSR selected via BLASTN was performed by PCR reactions with 12 loci (seven selected on Blast hits and five without hits) and posterior visualization on polyacrylamide gel. Nine out of 23 microsatellite loci were transferable in the in silico , with a mean identity of 87%. With regard to the GuavaMap microsatellite loci, only three showed significant alignments, among the 140 tested, with the forward and reverse mean identity of 100% and 95%, respectively. All seven SSR with e-values<1.00e−20 (mPgCIR001, mPgCIR005, mPgCIR007, mPgCIR009, mPgCIR018, mPgCIR020, and mPgCIR026) showed easy-to-score amplicons on the polyacrylamide gel when using the in silico transferability strategy. However, the other five, without significant e-values or hits, showed no amplification. These results highlight the effectiveness of in silico transferability for full-length SSR loci, constituting a valid alternative to save time and costs in transferability studies between species.