Computational protocol: Duplication and diversification of the LEAFY HULL STERILE1 and Oryza sativa MADS5 SEPALLATA lineages in graminoid Poales

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Protocol publication

[…] LOFSEP genes were identified by Basic Local Alignment Search Tool (BLAST) searches at the National Center for Biotechnology Information (NCBI) ( and PlantGDB ( using full-length coding regions of the rice OsLHS1/MADS1 (Os03g11614), OsMADS5 (Os06g06750) and OsPAP2/MADS34 (Os03g54170) sequences as search seeds. Full-length sequences were assembled and translated into conceptual amino acids using Mesquite 2.74 [] and then aligned using multiple sequence comparison by log-expectation (MUSCLE) [] before being manually adjusted using MacClade 4.0 []. As in previous analyses of the SEP gene family [,,,], no regions were considered unalignable and excluded from subsequent analysis (See Additional File for the phylogenetic data set). Bayesian phylogenetic analyses of the full-length LOFSEP data set using MrBayes 3.1.2 [] were run on the Grethor parallel processing cluster at the University of Missouri - St. Louis and consisted of two separate searches of ten million generations using flat priors and the General Time Reversible (GTR) model of sequence evolution with invariant sites and gamma distributed rates partitioned according to codon position (GTR + I + SS). Trees were sampled every 1,000 generations and convergence between the two runs was determined by examining the average standard deviation of the split frequencies. After ten million generations the split frequencies between the two runs was 0.006531. After convergence had been assured the first 25% of trees were removed as burn-in and clade credibility values estimated using MrBayes.The Shimodaira-Hasegawa test [] for significance between the best maximum likelihood (ML) tree identified in the Bayesian search and the best tree consistent with the LHS1/OSM5 duplication mapping at the base of the combined Bambusoideae, Ehrhartoideae and Pooideae (BEP) and Panicoideae, Aristidoideae, Chloridoideae, Centothecoideae, Micrairoideae, Arundinoideae and Danthonioideae (PACCMAD) clade and after the divergence of Pharioideae was conducted in PAUP* 4.0 [].MP and ML character reconstruction analyses were conducted using Mesquite version 2.74 []. To facilitate comparisons among the different taxa we followed Whipple et al. [], Sajo and Rudall [] and Preston et al. [] and considered: 1) bract 6 of the early diverging grass Streptochaeta angustifolia as homologous to one of the outer tepals in other monocots, a sepal in eudicots and the lemma in spikelet grasses, 2) bracts 7 and 8 of S. angustifolia as homologous to the remaining two outer tepals in other monocots, sepals in eudicots, and the palea of spikelet grasses, and 3) bracts 9-11 of S. angustifolia as homologous to the inner tepals in other monocots, petals in eudicots, and lodicules in spikelet grasses.Tests for sites potentially under positive selection within the LOFSEP alignment utilized the best ML topology from the Bayesian search and the CODEML program within the Phylogenetic Analysis Using Maximum Likelihood (PAML) package [] on the Grethor parallel processing cluster at University of Missouri - St Louis. Evidence of positive selection at particular codons was tested using the nested codon models M0 and M3, M1a and M2a, and M7 and M8 [-] with significance determined using a standard likelihood ratio test (LRT) statistic against a χ2 distribution with two degrees of freedom.We also tested for sites potentially under positive selection on the branch subtending the LHS1 clade, the branch subtending the OSM5 clade, and the branch subtending the LHS1+OSM5 clades (Branches 1 to 3, Figure ) using the modified branch-site models A and B [,]. Model A was compared with model M1a (NearlyNeutral) and model B was compared with M3 (discrete) with two site classes in a LRT against a χ2 distribution with two degrees of freedom [,] […]

Pipeline specifications

Software tools BLASTN, Mesquite, MUSCLE, MacClade, MrBayes, PAML
Databases PlantGDB
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Oryza sativa, Zea mays, Avena sativa, Hordeum vulgare, Cenchrus americanus, Sorghum bicolor
Chemicals Amino Acids, Cytosine, Thymine, Glutamic Acid