Computational protocol: Mutation of SELF-PRUNING homologs in cotton promotes short-branching plant architecture

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Protocol publication

[…] Paired-end reads were aligned against the cotton reference genome (G. hirsutum acc. TM-1) using BWA-MEM (version 0.7.12) (). SAMtools (version 0.1.19) () was used to convert mapping results to the bam format, and duplicated reads were filtered with the help of the Picard package (version 1.128). Only the uniquely aligned reads that were mapped to unique locations in the reference genome were retained (SAMtools flags −F 256 −f 3 −q 20). The threshold of single nucleotide polymorphism (SNP) calling was set to 20 for both base quality and mapping quality. SNP identification and genotype calling were performed based on the outputs from mpileup of the SAMtools software package. Only the non-singleton SNPs, defined as those where more than two accessions demonstrate the presence of the alternative alleles, were retained. Unreliable SNP sites were then filtered out, the candidate SNP loci were required to be bi-allelic, and all the singleton SNPs were excluded. We required that the common SNPs had a minor allele frequency (MAF) of more than 5%. We only analysed the SNPs that were located in the 26 pseudomolecules of the TM-1 assembly, and the SNPs in the small scaffolds () were removed. The EMMAX (Efficient Mixed-Model Association eXpedited) program was used to perform a large-scale genome-wide association study (GWAS). Association analysis was performed using the EMMAX software package. The significance threshold was determined by modified Bonferroni correction (Genetic type 1 Error Calculator (GEC) version 0.2) (). [...] Alignment of the amino acid sequences of the CENTRORADIALIS families in cotton and other species was performed using the CLUSTALX program (). MEGA5.1 () was used for a phylogeny reconstruction analysis using the Neighbor-Joining method and Poisson correction distance model. Bootstrap analysis was performed to estimate nodal support on the basis of 1000 resamplings. [...] The structure of the cotton SP proteins were predicted against the Antirrhinum CEN protein structure (PDB ID: 1QOU) using Deep View/Swiss-pdb viewer 4.0.1 as described previously (). The 3D structures of GoSP proteins were derived from the pre-structured proteins using SWISS-MODEL and compared with each other. Alignment diversity color was applied. […]

Pipeline specifications

Software tools Clustal W, MEGA, Swiss-PdbViewer, SWISS-MODEL
Applications Phylogenetics, Protein structure analysis
Organisms Gossypium hirsutum, Gossypium barbadense, Solanum lycopersicum
Diseases Plant Poisoning