Computational protocol: Detection of Genome Donor Species of Neglected Tetraploid Crop Vigna reflexo-pilosa (Créole Bean), and Genetic Structure of Diploid Species Based on Newly Developed EST-SSR Markers from Azuki Bean (Vigna angularis)

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Protocol publication

[…] Total RNA was extracted from seedlings and young pods of Erimo-shouzu using Plant RNA Purification Reagent (Invitrogen, CA, USA). Purification of polyadenylated RNA and conversion to cDNA were performed as described by . Synthesized cDNA was resolved by 1% agarose gel electrophoresis, and fragments ranging from 1 to 3 kb were recovered. The recovered fragments were cloned into the Eco RI-Xho I site of the pBluescript II SK- plasmid vector (Stratagene, CA, USA) and introduced into the E. coli ElectroTen-Blue strain (Stratagene, CA, USA) by electroporation. To generate ESTs, plasmid DNAs were amplified from the colonies using TempliPhi (GE Healthcare UK Ltd, Buckinghamshire, England) and subjected to sequencing using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, CA, USA). The reaction mixtures were run on an automated ABI PRISM 3730 DNA Analyzer (Applied Biosystems).Sequencing chromatograms were converted into nucleotide bases with Phred , and the sequences derived from the vector and linkers were removed with CROSSMATCH . The EST reads were quality-trimmed with TRIM2 using the Phred quality score ≥20, and ambiguous regions including more than ten X or N bases were trimmed. Contiguous, high-quality reads ≥100 bp were submitted to the DDBJ/EMBL/GenBank databases under the accession numbers HX939204 to HX950377 (11,174 entries). The PHRAP program with default parameters was used to cluster and identify non-redundant azuki bean ESTs .Simple sequence repeats (SSRs) ≥15 nucleotides in length, which contained all possible combinations of di-nucleotide (NN), tri-nucleotide (NNN), and tetra-nucleotide (NNNN) repeats, were identified from the non-redundant azuki bean ESTs using fuzznuc program in EMBOSS for SSRs within two mismatches. Primer pairs for amplification of SSR-containing regions were designed based on the flanking sequences of each SSR with the aid of the Primer3 so that the amplified fragment sizes were between 90 bp and 300 bp in length. The newly developed markers were designated as VES (Vigna EST-derived SSR) markers. […]

Pipeline specifications

Software tools EMBOSS, Primer3
Application qPCR
Organisms Vigna angularis, Viscum minimum, Vigna radiata
Diseases Erythema Infectiosum