Cotton Genomics and Genetics 2016, Vol.7, No.2, 1-23
3
are remain to be recognized. Further, among the reported miRNAs, only very few that were specific to drought
and other abiotic stress has been reported in cotton using computational (Boopathi and Pathmanaban, 2012) or
experimental (Yin et al., 2011; Wang et al., 2013) approaches, even though such abiotic stress specific miRNAs
have been expressively established in Arabidopsis, rice and other crops (Sunkar et al., 2008). Identification of
miRNAs in cotton, in response to water stress, certainly have important implications for gene regulation under
drought and such knowledge can impressively enhance the drought tolerance breeding efficiency besides
contributing significantly to the goal of having a complete profile of miRNAs in cotton. Besides, knowledge on
miRNA-guided stress regulatory networks would provide new tools for the genetic improvement of other abiotic
stress tolerances. In addition, all the miRNA studies employed
G. hirsutum, G. arboreum, G. herbaceum
and
G.
raimondii
. However, miRNA expressions in
G. barbadense
, which has superior fiber quality traits that suit to
modern textile mills, remain to be explored. Hence, in view of the importance of miRNAs in regulation of water
stress resistance in cotton, this study was conducted with the following objective: to capture and characterize the
differentially expressed miRNAs in drought tolerant and susceptible cotton cultivars using next generation
sequencing platform.
1 Results
1.1 Small RNA libraries
Totally four different small RNA libraries were constructed from the leaf tissues of KC3 and Suvin collected from
two extreme water regimes and intensely sequenced using ion proton
TM
technology. After the deletion of low
quality reads and several kinds of contaminant tags 9 826 785 clean reads from leaf tissues of KC3-C were
obtained (Table 1). Similarly, clean reads of 8 672 895 from KC3-WS; 8 627 325 from Suvin-C and 8 017 500
from Suvin-WS were obtained (Table 1) and they were used for analysis of differentially expressed small RNAs.
Altogether, there were 39 407 089 reads found in all the four small RNA libraries with a mean read length of 22 nt.
Table 1 Output details of the small RNA reads of four libraries
Name of the library
Bases
Reads
Mean read length (nt)
KC3-C
192,062,293
9,826,785
19
KC3-WS
192,360,514
8,672,895
22
Suvin-C
179,836,027
8,627,325
20
Suvin-WS
181,349,766
8,017,500
22
Total number of reads
39,407,089
22
The sequence reads of the each library was normalized to RPKM and each reads that were differentially expressed
were recorded in RPKM (Table 2 and Figure 1). Small RNA reads with a size of 27 nt were found to be present
in significantly greater number in all the four comparisons followed by 28 or 29 nt reads (Figure 1). Small RNAs
in the size range of 24-33 nt that were differentially expressed in drought susceptible cotton cultivar, Suvin, under
two different water regimes were significantly higher than the other three comparisons. However, predominantly
the size range of 20-23 nt were differentially expressed between KC3-WS and Suvin-WS (Figure 1). Totally 8 469
differentially expressed distinct reads were found between Suvin-C and Suvin-WS followed by 7 494
differentially expressed distinct reads between KC3-WS and Suvin-WS (Table 2). ESTs from NCBI and the
genome sequence of
G. raimondii
(Wang et al., 2012), were used as reference sequences to predict the miRNA
precursors since no allopolyploid cotton genome sequence was available at the time of this analysis. Nevertheless,
to a maximum of 15.35% of the small RNA reads obtained from this study
were mapped with
G. raimondii
genome sequences (Table 2). The distinct reads that were identified as rRNA, snRNA, snoRNA, tRNA, tmRNA
during the analysis (number of reads in each small RNA class is given in Table 2) were separated and the
remaining reads (which constitutes the majority of the reads; Table 2) were used further.
1.2Differentially expressed conserved miRNA families
RPKM reads of each miRNA were used to notify their abundance and differential expression in two different
contrasting cotton cultivars under two different water regimes. Simple blast analysis with matured miRNA
