Computational protocol: Comparative Systems Biology Reveals Allelic Variation Modulating Tocochromanol Profiles in Barley (Hordeum vulgare L.)

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Protocol publication

[…] Genotyping and linkage analysis were performed according to Islamovic et al. . Analysis of variance (ANOVA), means, and broad sense heritability estimates were calculated for each tocochromanol form using the JMP 9.0 software (SAS Institute, Cary, NC). Heritability within the RIL population was calculated by dividing genotypic variance by total variance, calculated from variance components estimates for genotype, location, and year in a full-factorial random effects model. Based on significant interactions within the model, each genotype, location, and year were treated as separate traits for QTL identification ().QTL were detected with WinQTL Cartographer , using marker locus positions from an existing Falcon x Azhul SNP map . Experiment-wise thresholds, determined through 1000 permutations of the data, were used to determine significant QTL . Peaks were initially detected using single marker analysis, and QTL positions, LOD scores, and phenotypic variance were determined with composite interval mapping (CIM), using stepwise regression, a window size of 10 cM, walk speed of 2 cM, and significance threshold of P = 0.05. Clustered peaks were counted as separate QTL if individual peaks spanned more than one LOD score. [...] Primers for gene cloning were designed based on coding sequences of locus AK355075 for VTE4, locus AK366699 for VTE2, and locus AY222860 for HGGT (available in NCBI http://www.ncbi.nlm.nih.gov). Primers for promoter regions (contigs 254212 and 2207823 for VTE4, contig 1025503 for HGGT) were designed from sequences obtained from the morex_rcba database in ViroBLAST, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) (http://webblast.ipk-gatersleben.de/barley/viroblast.php). Primer sequences are listed in . PCR amplification was performed using the Phusion High-Fidelity PCR Kit (Finnzymes, #F-553S, distributed by New England BioLabs, Inc.). Reaction preparation and thermocycling were generally as recommended by the manufacturer, with 40 cycles of 10 s denaturation, 63°C annealing, and 30 s extension. Gel bands corresponding to target regions were purified using a QIAquick PCR Purification Kit (Qiagen, #28104), and purified DNA was cloned using a StrataClone Blunt PCR Cloning Kit (Stratagene, #240207) and plated on LB-Ampicillin agar with 40 µl 5-bromo-4-chloro-3-indolyl-β-D galactopyranoside (20 mg/ml). White colonies were cultured in 5 ml LB-Ampicillin broth and plasmids were purified with a QuickClean II Plasmid Miniprep Kit (GenScript, #L00420). Sequencing was performed at the Idaho State University Molecular Research Core Facility, using an Applied Biosystems 3130xl instrument and plasmid reactions purified with a BigDye XTerminator Purification Kit (Applied Biosystems, #4376485). Sequences were assembled and analyzed using Geneious Pro v5.5.6 software . Intron regions were identified through comparative alignment of full-length genes with coding sequences. Promoter motif sequences, including transcription start sites, were identified using the Neural Network Promoter Prediction v2.2 software, Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/promoter.html). Additional promoter motifs were identified using the PlantCARE database of cis-acting regulatory elements (http://bioinformatics.psb.ugent.be/webtools/plantcare/html). [...] Gene-specific, PCR-based markers were developed for HGGT and VTE4 based on sequence differences between Falcon and Azhul. Primers were designed to amplify a 50–80-bp region containing the polymorphism, and genotyping of the FA population was performed using high-resolution melt analysis, as described . Markers were appended to the existing map using MultiPoint software (http://www.multiqtl.com) and QTL analysis was repeated separately for addition of each marker, using the same conditions as previously. […]

Pipeline specifications

Software tools FALCON-X, ViroBLAST, Geneious, NNPP, MultiQTL
Databases PlantCARE BDGP
Applications Genome annotation, WGS analysis, Nucleotide sequence alignment
Organisms Hordeum vulgare, Oryza sativa