Cotton Genomics and Genetics 2016, Vol.7, No.2, 1-23
16
of this study sat at one arm of the stem and asymmetric bulges was minimal in size (one base at most) and
infrequent (typically one or less) within the miRNA/miRNA* duplex) and ii) the minimum free energy should be
lower than the threshold of−30 kcal/mol
(Bonnet et al., 2004).
3.6 Differentially expressed miRNAs
Differential expression analysis of both conserved and novel miRNAs between the two contrasting genotypes
under two different water regimes were identified as described below. The miRNA reads of the each libraries
were assembled
de nova
into contigs and normalized to reads per kilobase per million (RPKM; Mortazavi et al.,
2008) and RPKM was used as a statistical value for the analysis of differences in expression among the samples.
The increased or decreased level of expression (up- or down- regulation) of miRNA in each sample under given
treatment were represented in RPKM fold change (as described in Rada-Iglesias et al., 2011) and minimum of two
fold change in RPKM were considered further. Such analysis was helped to identify major (thus insignificant
miRNAs were not included in this report) and differentially expressed miRNAs in leaf samples between i) KC3
irrigated control (hereafter mentioned as KC3-C) and KC3 water stressed (hereafter mentioned as KC3-WS) ii)
KC3-WS and Suvin water stressed (hereafter mentioned as Suvin-WS) iii) Suvin irrigated control (hereafter
mentioned as Suvin-C) and Suvin-WS and iv) KC3-C and Suvin-C.
3.7 Identification of miRNA’s targets
The targets of miRNAs was predicted by the web tool psRNATarget (Dai and Zhao, 2011) using the
Gossypium
(cotton) DFCI Gene Index (CGI) Release 11
) as the sequence library
for the targets search. No more than three mismatches between miRNAs and targets were allowed, especially no
mismatches were allowed within the maximum expectation region.
Acknowledgements
The authors gratefully acknowledge DBT, Government of India for the financial assistance.
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