Computational protocol: Diversification of the plant-specific hybrid glycine-rich protein (HyGRP) genes in cereals

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Protocol publication

[…] To identify the HyP/GRP gene family in the rice genome, we employed BlastP searches of RAP-DB (http://rapdb.dna.affrc.go.jp/) and MSU-RGAP (http://rice.plantbiology.msu.edu/) using the protein sequence of qLTG3-1 as a query. The HyP/GRP gene family in monocots was searched by BlastP programs. The protein sequences in maize, sorghum, and Brachypodium were retrieved from Phytozome version 10 (http://phytozome.jgi.doe.gov/pz/portal.html). Barley gene sequences were retrieved from the IPT Barley blast server (http://webblast.ipk-gatersleben.de/barley/). The 1 kb upstream region from the transcription start site on each gene was also retrieved from databases as the promoter sequence.Multiple sequence alignment of the HyP/GRP protein sequences was performed using the ClustalW method with MEGA version 6 built in with a default setting (http://www.megasoftware.net/). A phylogenic tree was constructed by the neighbor-joining method considering 1000 replications with bootstrap analyses. The similarity of the amino acid sequences and identities of the promoter sequences among the HyP/GRP gene family were calculated using alignment data by the SIAS program with a default setting (http://imed.med.ucm.es/Tools/sias.html). The cis-acting elements in the promoter region were analyzed using the MEME suite (http://meme.nbcr.net/meme/) with the following parameters. The optimum width of each motif was between 6 and 20 bp. The number of differential motifs was 20, while that of the minimum motif site was 5. E = 1.1e+4 was used as a threshold for shuffle sequences. [...] Syntenic dotplots for the chromosomal regions around qLTG3-1 orthologous genes were generated using PipMaker (http://pipmaker.bx.psu.edu/pipmaker/). The orthologous genomic regions were identified through comparative genomics analysis of the putative highly conserved gene pairs in monocots. The 163 kb in rice chromosome 3 including 22 genes, the 117 kb in Brachypodium chromosome 1 including 21 genes, the 200 kb in sorghum chromosome 1 including 15 genes, the 383 kb in maize chromosome 1 including 17 genes, and the 209 kb in barley chromosome 4 including 12 genes were used for the initial analysis. The CDSs in these orthologous genomic regions were then used to analyze collinearity among the monocots. […]

Pipeline specifications

Software tools BLASTP, Clustal W, MEGA, SIAS, MEME Suite, PipMaker
Databases Phytozome RAP-DB RGAP
Applications Phylogenetics, Genome data visualization
Organisms Oryza sativa, Hordeum vulgare, Zea mays, Sorghum bicolor
Chemicals Glycine