Computational protocol: Molecular evidence of RNA polymerase II gene reveals the origin of worldwide cultivated barley

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Protocol publication

[…] Multiple sequence alignments were performed using ClustalX. Nucleotide diversity was estimated by Tajima′s π and Watterson’s statistics. Tests of neutral evolution were performed as described by Tajima, and Fu and Li. The above calculations were conducted using the software program DnaSP version 5.0. Each insertion/deletion (indel) was considered as a single mutation event, and all indels were therefore coded as single positions. Identical sequences were grouped into haplotypes (Hap). Phylogenetic analysis was performed with the computer program MEGA 6 using the maximum likelihood (ML) method under the Kimura 2-parameter model, the minimum-evolution (ME) and neighbor-joining (NJ) methods with the model of Tajima-Nei. The confidence of each clade was calculated based on the bootstrap values with 1,000 replications.The population structure was analyzed using STRUCTURE software (version 2.3.4). Haplotypes were recoded as unique alleles. Multistep approach (after several trial runs) was applied to infer the genetic structure in our wild, cultivated as well as all barley samples, respectively. The first step of the analysis consisted of estimating K-value (the putative number of genetic groups). Twenty independent runs of K from 1 to 10 were performed, with 100,000 MCMC (Markov Chain Monte Carlo) iterations and a burn-in period of 50,000 replicates under the ‘admixture model’. The most likely K-value was estimated by the log probability of data [LnP(D)] and an ad hoc statistic ΔK based on the rate of change of LnP(D) between successive K values as described by Evanno et al.. To infer the appropriate number of K, STRUCTURE HARVESTER ( was used. In a second step, after the inference of K, the STRUCTURE procedure was repeated with a fixed K and 10 independent runs with 50,000 MCMC iterations and a burn-in period of 25,000. An individual was assigned to a certain cluster if its q value was higher than 0.75. […]

Pipeline specifications

Software tools Clustal W, DnaSP, MEGA, Structure Harvester
Applications Phylogenetics, Population genetic analysis
Organisms Hordeum vulgare