Computational protocol: The Maize (Zea mays L.) AUXIN/INDOLE-3-ACETIC ACID Gene Family: Phylogeny, Synteny, and Unique Root-Type and Tissue-Specific Expression Patterns during Development

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Protocol publication

[…] In the initial version of the maize reference genome sequence B73 RefGen_v1 [] 31 Aux/IAA genes were predicted among 32,540 protein-encoding genes []. To obtain a comprehensive overview of the maize Aux/IAA gene family, the 31 previously identified maize Aux/IAA genes were used as query sequences for blast ( searches of the maize filtered gene set based on genome assembly version AGPv2 (, containing 39,656 high confidence genes. The protein sequences of the maize Aux/IAA genes were retrieved from MaizeGDB ( Moreover, the previously identified rice Aux/IAA protein sequences [] were retrieved from the Rice Genome Annotation Project (, and the published sorghum sequences [] were extracted from Gramene ( The four conserved domains in the maize Aux/IAA gene family were determined by multiple alignments with ClustalW ( Synteny of the maize sequences was determined with Comparative Genomics software (CoGe,; []) and association with maize subgenomes 1 and 2 were based on []. Phylogenetic analyses comparing maize, rice, and sorghum Aux/IAA protein sequences were conducted using the neighbor-joining algorithm in MEGA5 [] considering 1,000 replications with bootstrap analyses. [...] Expression of maize Aux/IAA genes was determined by quantitative real-time-PCR in a Bio-Rad CFX 384™ Real-Time System ( using gene-specific oligonucleotides (). The oligonucleotides were designed by Primer3Plus ( and checked with NetPrimer (PREMIER Biosoft, software. Each reaction contained 4 µl MESA Blue qPCR™ Mastermix Plus for SYBR Assay no ROX (Eurogentec,, 1 µl cDNA sample and 250 nM gene-specific oligonucleotide primers to a final volume of 8 µl. The primer efficiency of each oligonucleotide was calculated using the following dilution series: 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128. The relative expression levels of the transcripts were calculated with reference to the housekeeping gene myosin (Genbank AC: 486090G09.x1). Differential gene expression was determined by a two sided Student´s t-test. […]

Pipeline specifications

Software tools Primer3, NetPrimer
Application qPCR
Organisms Zea mays