Computational protocol: Gene-based microsatellite development for mapping and phylogeny studies in eggplant

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Protocol publication

[…] In all, 3,357 eggplant sequences were retrieved from the SGN and EMBL nucleotide databases, using the Sequence Retrieval System (SRS6, ). A stand-alone nucleotide database was built for local BLAST2 searches []. PolyA and polyT tracts were removed, by applying the criterion that no 50 bp window contain a run of ten A's or ten T's. ClustalW [] alignment was used to eliminate redundancy, by setting the following two criteria: (i) where a cluster contained two or more identical sequences, the longest was retained, and (ii) where the members of a cluster fell into recognisable sub-groups, only one member of each sub-group was retained. Sequences composed entirely of SSR motif (i.e., lacking any flanking sequence) were discarded, since their uniqueness could not be established, and in any case, primer design is not possible. SSR-containing sequences were identified using MISA software [], a Perl script which allows both perfect and compound SSRs to be detected. A sequence was considered an SSR where a motif was repeated at least 12 times (1 nt motif), seven times (2 nt) or five times (3–6 nt), allowing for only one mismatch. For compound repeats, the maximum default interruption (spacer) length was set at 100 bp.Primer pairs were designed from the flanking sequences, using PRIMER3 software [] in batch mode via the p3_in.pl and p3_out.pl Perl5 scripts within the MISA package. The target amplicon size was set as 100–300 bp, the optimal annealing temperature as 60°C, and the optimal primer length as 20 bp. The resulting markers were each assigned the prefix EEMS (EST Eggplant MicroSatellite). Local BLASTN analyses were carried out using all EEMS sequences as queries. The target database contained 1,524,584 entries derived from a variety of solanaceous species, retrieved from the EMBL sequence database (Release 93) […]

Pipeline specifications

Software tools Clustal W, MISA, Primer3, BLASTN
Applications WGS analysis, qPCR
Organisms Solanum melongena, Solanum tuberosum, Solanum lycopersicum