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However, we note that shuffling of the seed sequence is not an ideal random model and statistical testing of the tRF seed regions is complicated by the fact that a tRNA sequence is under multiple selective constraints for its structure and function related to translation (and furthermore different from the constraints of a miRNA).
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This might be caused by strong selective constraints (negative selection) at high temperature [ 19, 20].
In the models using any physico-chemical evaluation of selective constraints, the significance of multiple nucleotide changes has been indicated; see Tables 2 and 3.
If a codon substitution matrix is used for model fitting with the assumption of multiple nucleotide changes, all 190 parameters of selective constraints <img src="http://journals.plos.org/plosone/article/asset?id=info?doi/10.1371/journal.pone.0017244.e392.PNG" class= inline-graphic"/> will be able to be optimized.
This comparison would distinguish between relaxed selective constraints or increased positive selection on male genes.
A GERP score is determined using multiple sequence alignments of orthologous genes and the level of selective constraints on each position.
Positive selection will be taken into account if selective constraints are variable over sites.
Thus, a broad breadth of expression imposes selective constraints limiting the possible action of positive selection.
On the basis of the multiple sequence alignment, we estimated the rate of evolution for each CP sequence in order to infer the selective constraints.
Multiple studies have observed the hourglass pattern across diverse biological processes, including transcriptome divergence, transcriptome age, molecular interaction, and evolutionary selective constraints.
We infer that the weak positive selection and multiple branches are considered as foreground branch may explain this phenomenon, because power will be reduced unless the same sites and selective constraints are occurring along all foreground branches [ 46].
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