Computational protocol: Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley

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Protocol publication

[…] The DNA coding sequences (Table ), protein sequences (Table ), and DNA genomic sequences (Table ) of HvSPLs were obtained from the Ensemble H. vulgare database (http://plants.ensembl.org/Hordeum_vulgare/Info/Index) database. The pHMMER search function was used, with the A. thaliana SBP domain (Pfam: PF03110) sequence as the query. The IPK Barley BLAST server (http://webblast.ipk-gatersleben.de/barley_ibsc/) and Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Hvulgare_er) databases were also searched by performing TBLASTN using SBP domain sequence as a query. The HvSPL gene accession numbers were extracted. The nomenclature of putative SPL genes in H. vulgare was based on rice and wheat orthologs. [...] The Gene Structure Display Server program (http://gsds.cbi.pku.edu.cn/index.php) was used to predict the exon/intron structure of each HvSPL gene by comparing their coding and genomic sequences. SPL sequences of A. thaliana were obtained from TAIR (http://www.arabidopsis.org/index.jsp). SPL sequences of rice were obtained from the rice genome annotation project database. Wheat SPL sequences were taken from. The amino acid sequences of conserved SBP domains were selected for phylogenetic analysis. The SBP domain sequences of SPL proteins from A. thaliana, rice, wheat, and barley were identified by the SMART tool and are presented in Table . The phylogenetic tree was constructed using the maximum likelihood method using JTT + G as a best model. SBP domain amino acid sequences were converted to the PHYLIP format and analysed with the PhyML3.0 software (http://www.atgc-montpellier.fr/phyml/), which uses the approximate likelihood-ratio test (aLRT) and depends on a non-parametric Shimodaira-Hasegawa-like (SH-like) approach. [...] A search for conserved motifs within HvSPL proteins was performed by using the MEME 4.11.0 tool (http://meme-suite.org/tools/meme), using default settings, except that the maximum width was 50, the minimum width was 6, and the maximum number of motifs to find was 10. The online WebLogo3 platform (http://weblogo.threeplusone.com/) was used to create the sequence logo of the barley SBP domain. The genomic and cDNA sequences of HvSPLs were analysed to predict the putative target sites of miR156 using psRNATarget tool (http://plantgrn.noble.org/psRNATarget/?function). Information on alternative splice events for each HvSPL gene was obtained from the Ensemble database (http://plants.ensembl.org/Hordeum_vulgare/Info/Index). Promoter regions, defined as the 1000-bp sequences upstream of start codons were searched for cis-regulatory elements using the PlantCARE database. [...] ‘Morex’ RNA-seq data was obtained from plant expression ATLAS (https://www.ebi.ac.uk/gxa/plant/experiments) which was generated by International Barley Sequencing Consortium (https://ics.hutton.ac.uk/morexGenes/), and the log2-transformed fragments per kilobase per million fragments measured (FPKM) values were used to study the expression of HvSPLs in eight tissues: 4-day-old embryos dissected from germinating grains (EMB); roots (Roo) and shoots (LEA) collected from seedlings (10-cm shoot stage); developing inflorescences (5 mm; INF1 (5 mm) and INF2 (1–1.5 cm); developing tillers at the six-leaf stage (3rd internode, NOD); developing grain 5 days post anthesis (DPA); spikelets with bracts removed at 5 DPA (CAR5) and 15 DPA (CAR15). A heat map of the expression of HvSPLs was generated by the average hierarchical clustering method using the MeV tool (http://www.tm4.org/mev.html). […]

Pipeline specifications

Software tools GSDS, PHYLIP, PhyML, MEME, MEME Suite, WebLogo, psRNATarget, TM4
Databases ATGC PlantCARE
Applications Phylogenetics, RNA-seq analysis, Genome data visualization
Organisms Hordeum vulgare, Arabidopsis thaliana, Triticum aestivum, Oryza sativa