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The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data.
Each primer pair was checked for their specificity using the Primer Blaster tool in OryGenesDB (http://orygenesdb.cirad.fr/tools.html), which were further confirmed by dissociation curve analysis obtained after the QPCR reaction.
After 30 days of incubation, the reproductive success of each pair was checked [55].
Reproducibility of each primer pair was checked by carrying out two times the whole AFLP protocol for 30% of individuals randomly chosen as recommended by Bonin et al. (2004) [55].
The amplification success for each primer pair was checked with secondary singleplex PCRs.
Amplification efficiency of each primer pair was checked with serial dilutions of cDNA (data not shown).
Similar(49)
In the last step, each potential pair is checked component-wise to see if the corresponding vector ( overrightarrow{varDelta {L}_{lj}} ) exceeds the allowable value 0.5 ΔL lim of the spatial corridor.
In an iterative process, each matched pair is checked in the global model, and the pairs which have the highest geometric error between the reference and the second image are removed one by one until achieving the root mean square error (RMSE) < T RMSE [6].
After reads were deconvoluted, the Ves reads for each sequence pair were checked for complementarity to the 5′ 34 bp of the consensus Ves3ML using fastx_clipper from the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/, last accessed June 8 , 2015.
The efficiency of each oligo pairs was checked by diluting a series of control cDNAs.
The uniqueness of the designed primer pairs was checked by a BLAST homology search to ensure that homologous genes were not cross-amplified by the same primer pair.
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