Computational protocol: Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids

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Protocol publication

[…] Gene-based markers were designed from wheat expressed sequence tags (ESTs) mapping to the chromosome 2DS1-0.33 deletion bin (; http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi), from selected rice unspliced gene sequences (Rice MSU Rice Genome Annotation Project Database v6.1, ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_6.1/all.dir/; RAP database v5, http://rapdb.dna.affrc.go.jp/download/index.html), and from selected B. distachyon unspliced gene sequences (release 1.0, www.modelcrop.org; ftp://ftp.ensemblgenomes.org/pub/plants/release-3/fasta/brachypodium_distachyon/cdna/). Wheat assembled ESTs were downloaded from the TIGR Plant Transcript Assemblies database (ftp://ftp.tigr.org/pub/data/plantta/Triticum_aestivum/). For all data sets, a local BLAST database was produced using the command formatdb. A local similarity search (blastall –p blastn) () was performed on wheat ESTs against the unspliced gene databases to identify the corresponding unspliced gene. Wheat EST sequences with a first-hit similarity score of >150 were aligned to the genomic sequence of the identified gene using the est2genome algorithm (EMBOSS). To reduce false positives, results were filtered for repetitive elements (Triticeae Repeat database: TREP http://wheat.pw.usda.gov/ITMI/Repeats), and for wheat ESTs that hit genes outside the desired genomic interval when re-BLASTed. Primers amplifying products of ~500–600 bp were designed on EST sequence-spanning predicted introns, and when possible on 3'-untranslated regions (UTRs), with Primer3 (http://frodo.wi.mit.edu/, ). Wheat ESTs from which markers were designed were also BLASTed against the SWISSPROT database for predicting the putative function (ftp://ftp.uniprot.org/pub/ databases/uniprot/knowledgebase/uniprot_sprot.fasta.gz).Polymorphic markers () between parents of the coarse mapping population (89 RILs; ) were amplified from all RILs. Genomic DNA was extracted from 2-week-old seedlings with an adapted method of ). Amplification was conducted in a 20 μl volume containing 50 ng of genomic DNA, 1 μM of each primer, 1.25 mM dNTPs, 1× PCR buffer (Invitrogen), and 0.4 U of Taq DNA polymerase (Invitrogen). After an initial denaturation at 94 °C for 1 min, 35 amplification cycles were performed: 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min. Single-stranded amplification products of newly developed gene-based markers were separated according to their conformation by single strand conformation polymorphism (SSCP) analysis as described by ). Linkage of molecular markers on chromosome 2DS was analysed with JoinMap version 3.0, with a logarithm (base 10) of odds (LOD) score >5.0, and the Haldane mapping function. […]

Pipeline specifications

Software tools BLASTN, EMBOSS, Primer3
Databases UniProtKB PlantTA RGAP
Application qPCR
Organisms Triticum aestivum, Homo sapiens, Oryza sativa, Brachypodium distachyon
Chemicals Gibberellins