Computational protocol: Elaboration of bilateral symmetry across Knautia macedonica capitula related to changes in ventral petal expression of CYCLOIDEA-like genes

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[…] Knautia macedonica Griseb. plants were grown to flower at St. John’s University, Queens, NY, USA. Internal and external buds from immature capitula were collected or dissected into dorsal, left lateral, right lateral, and ventral petal types (i.e., corolla tube tissue excluded from dissections) at various stages of development up to anthesis and prior to pigment production. Because of limited petal tissue per floret, dissections were pooled from multiple accessions and different capitula to obtain 20–25 mg of tissue for RNA extraction. Leaf RNA was also collected for comparison. Total RNA was extracted using the RNeasy Plant Mini Kit, including the RNase-free DNase (Qiagen) on-column step. Sample concentrations and purities were determined using a Thermo Scientific NanoDrop 2000.Total cDNA was generated using the Omniscript RT kit (Qiagen) following the standard protocol and including 4 µL of ~50 ng/µL concentration of each RNA sample. qPCR primers were designed in Geneious Pro v.6.1.8 (; []) based on available CYC-like genes and GAPDH sequences (see Additional files , ). Amplicons were confirmed via sequencing at the Yale DNA Analysis Facility. Six new CYC-like paralog sequences obtained of K. macedonica were combined with 159 CYC-like gene sequences downloaded from GenBank (Additional file ) representing all major lineages of the broadly circumscribed clade Caprifoliaceae (including Dipsacaceae; []) for phylogenetic analyses. Alignments from [, , , ] were obtained via personal communication from D.G. Howarth and S.E. Carlson, and new sequences were added in Geneious Pro v.6.1.8 (; []) using the Geneious Alignment tool (default parameters) based on nucleotide sequences, and non-Caprifoliaceae sequences were removed. Aligned nucleotide datasets were then manually adjusted based on amino acid codon sequence.The best-fitting model of sequence evolution (TIM3 + I + G) was determined using the Bayesian information criterion (BIC) in jModeltest v.2.1.6 [, ]. ML and BI analyses incorporating the best model of sequence evolution were both conducted on the CIPRES Science Gateway v.3.3 ( using RAxML v.8.1.24 [] and MrBayes v.3.2.3 [–] (Additional file ). ML analyses were conducted using default parameters to obtain the single best tree and 1000 BS replicates under the GTRGAMMA model (suggested by the developer of RAxML). A single CYC1 gene was initially used to root the tree in RAxML. Trees were read into PAUP* [], derooted, and rerooted with all CYC1 genes used as the monophyletic outgroup for the CYC2 and CYC3 copies. Two BI analyses were run in parallel for 20,000,000 generations with four chains sampling every 2000 generations. Stationarity was determined using Tracer v.1.6 {Rambaut:-M7jROaJ}. Trees were read into PAUP* and 20 % were discarded as burn-in prior to constructing a 50 % majority rule consensus tree.Primer efficiency was determined using a melting curve analysis [, ]. Samples were normalized to 20 ng/µL, and qPCR was performed using 20 µL reactions of the iTaq universal SYBR green one-step kit (BioRad) on a MyIQ (BioRad) machine. Relative expression levels were calculated using the 2−∆∆CT method []. GAPDH was used as the reference gene based on preliminary data (Additional file ) that revealed consistent expression levels regardless of the tissue type (i.e., leaf tissue vs. bud tissue vs. petal tissue). All samples were normalized to GAPDH expression, and three biological replicates were analyzed in duplicate. ANOVA and post hoc Tukey HSD tests were performed in R (R Development Core Team 2015; see Additional file ). Plots were made using the ggplot2 package v.1.0.1 [] as implemented in R. […]

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