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 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the maize filtered gene set based on genome assembly version AGPv2 (http://www.maizegdb.org/), containing 39,656 high confidence genes. The protein sequences of the maize Aux/IAA genes were retrieved from MaizeGDB (http://maizegdb.org/). Moreover, the previously identified rice Aux/IAA protein sequences [] were retrieved from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml), and the published sorghum sequences [] were extracted from Gramene (http://www.gramene.org/). The four conserved domains in the maize Aux/IAA gene family were determined by multiple alignments with ClustalW (http://www.clustal.org/). Synteny of the maize sequences was determined with Comparative Genomics software (CoGe, http://genomevolution.org/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 (http://www.bio-rad.de) using gene-specific oligonucleotides (). The oligonucleotides were designed by Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and checked with NetPrimer (PREMIER Biosoft, http://www.premierbiosoft.com) software. Each reaction contained 4 µl MESA Blue qPCR™ Mastermix Plus for SYBR Assay no ROX (Eurogentec, http://www.eurogentec.com), 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