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To further investigate the evolutionary relationships among the WRKY domains from different species, we estimated the phylogeny by using the NJ program from MEGA 5 for the WRKY domains from O. sativa, A. thaliana, and B. distachyon.
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Phylogenetic analysis was carried out using the NJ method in MEGA 3.0 program [ 65] and Maximum Likelihood (ML) optimality criteria in PHYML algorithm [ 66].
Phylogenetic trees were estimated for each distance table using the NJ method implemented in the Neighbor program of the PHYLIP 3.69 package.
Finally, two unrooted phylogenetic trees were constructed using the NJ method and were displayed using the MEGA4 program.
Trees were generated using the NJ and ME methods.
Nei's standard genetic distance (Nei 1972) was calculated for each lineage, and distance was displayed in the topology of an un-rooted neighbor-joining (NJ) tree using the analysis program phylip version 3.68 (Felsenstein 2008).
Protoplasts were incubated with 30 µg/ml PI in culture medium for 5 10 minutes [44] and PI staining was quantified in the FL-2 channel of a FACSCalibur instrument (Becton, Dickinson and Company, Franklin Lakes, NJ) using the CELL Quest program (Becton, Dickinson and Company).
Phylogenetic trees were constructed by the neighbor-joining (NJ) method [ 68], using the computer program MEGA, Version 4.0 [ 69], and by Bayesian analysis (MrBayes v3.0B4 program).
Neighbor-joining (NJ) trees were constructed using the NEIGHBOR program in PHYLIP and were drawn with TreeView.
To estimate NJ trees, distance matrices were calculated using the protdist program with the JTT amino acid substitution matrix and submitted to the program neighbor.
Phylogenetic trees were constructed using the Neighbor-joining (NJ) and maximum-likelihood (ML) methods using the MEGA program (vers. 5.0) [ 19].
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