Computational protocol: Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria

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Protocol publication

[…] Total genomic DNA was extracted from fresh leaves using the CTAB procedure []. The 5S rDNA gene fragments from each sample were amplified using primers designed on conserved regions of the 5S rDNA sequences from barley and wheat [] with FP: 5′-GGACCTCCTGCGAAGTCCT-3′ and RP: 5′-CCCATCCGTGTACTACTCTC-3′. The PCR conditions were 95°C for 5 min, 32 cycles of 94°C for 55 s, 62°C for 25 s and 72°C for 35 s, followed by a final extension of 72°C for 10 min. The PCR mixture (20 μl) contained 100 ng template DNA, 250 μM of each dNTP (Takara), 0.5 μM of each primer, 2.0 μl of reaction buffer, and 1 unit of Ex Taq DNA polymerase (Takara). The knotted1 (kn1) gene fragments including part of the first intron, the whole sequences of the second intron and the second exon as described by Doust et al [] were amplified using the primers pair of kn211-402F: 5′-TCAGAACTTTTGGCCGTGGGT-3′ and kn612-402R: 5′-GAGATGGACAGCGAGTTGAGC-3′. PCR reactions were denatured for 5 min at 95°C and then followed a step-down procedure where annealing temperature was stepped down from 65°C to 59°C, and then 35 amplification cycles were performed, each cycle including denaturation at 95°C for 30 s, annealing at 57°C for 1 min, primer extension at 72°C for 1 min, and a final extension of 72°C for 10 min. Products were separated by electrophoresis on 1.5% agrose gels, and the major band of each sample was isolated from the gel, cleaned using Qiagen columns, cloned into the PUCm-T or PMD18-T vector system, and then transformed into the DH5a strain of E. coli. After identification of recombinant clones, for diploid species, a minimum of two clones were sequenced, for tetraploid species, at least six clones were picked and sequenced. Kn1 and 5S rDNA fragments obtained were confirmed by comparison with Genbank. Sequences from the same accession were aligned using DNAMAN, and redundant sequences were deleted. Accession number for each clone has been deposited in Gene Bank and listed in Additional file : Table S1. Outgroup sequences [AB023618, DQ351339, JQ947589, X61308] were obtained from Genbank.Multiple alignments of unique sequences from each sample were carried out using T-Coffee [-]. The phylogenetic trees based on 5S rDNA and kn1 sequences were analyzed under neighbor joining (Mega version 5.1) [] and Bayesian approaches (Mr Bayes 3.1.2) [,]. Evolutionary models were chosen using jModeltest version 0.1.1 [,], and in both cases were a general time reversible model with a 4 category gamma rate model. Neighbor-joining analyses were run under the Maximum-composite likelihood model, with gamma values estimated in jModeltest, and tree support estimated with 1,000 bootstrapped sample sets. Mr Bayes was run using a GTR plus gamma model, with branch length unconstrained, branch length priors set to an exponential distribution with a parameter of 10, and shape parameter priors set to an exponential distribution with a parameter of 10. Bayesian analyses were run for 5,000,000 generations, with 4 chains on each of two nodes. Chains were compared every generation and nodes compared every 1,000 generations. Tracer version 1.5 [] was used to estimate burnin, and a conservative 1,000,000 generations from the beginning of each run were removed. The remaining trees from each run were combined and a majority rule consensus tree used computed to summarize the data. […]

Pipeline specifications

Software tools DNAMAN, T-Coffee, MEGA, jModelTest
Application Phylogenetics
Organisms Setaria viridis, Setaria italica, Sciadopitys verticillata, Sarocalamus faberi, Cenchrus americanus, Styela plicata