Cotton Genomics and Genetics 2016, Vol.7, No.2, 1-23
14
deep-sequencing data provides several additional signals to help distinguish miRNAs from other sequences
(Kozomara and Griffiths-Jones, 2011).
The lowest minimum free energy (MFE) among all hairpin structures of the novel miRNAs precursors was −43.9
kcal/mol (Table 4), which is significantly lower than the threshold of −30 kcal/mol reported in a previous study
[25]. Among the 36 novel miRNAs identified in this report, none of the novel miRNA had homologs in miRBase
Release 19. Hence, we assumed that they were Gossypium-specific, or restricted to closely related species.
Further, there is a chance to get more novel or conserved miRNAs if new genomic resources are developed in near
future as discussed above.
In conclusion, our previous computation study (Boopathi and Pathmanaban, 2012) has shown that conserved
miRNAs were shown to be linked with abiotic stress resistance. Here we provided experimental proof for those
and other miRNAs for their roles in drought tolerance in cotton. During water stress responsive transcript
profiling, it is important to target the different tissues and their precise stage besides the dynamics of the stress
(such as time, duration and intensity), for isolation of RNA. Thus, it will be more useful if the ultimate
economically important tissues, the flower, boll and fiber, are used during sampling instead of leaves that was
used in this study. Further, instead of using genotypes with different genetic backgrounds, near-isogenic lines,
which differ only in the target trait, would be ideal genetic material which ensures that differentially expressed
genes are linked to the drought resistance and not to the genetic background. Our laboratory has already started to
work on these lines. The number of new miRNAs identified in this study is relatively low when compared with
other crops. This is mainly due to lack of genome sequence in
G. hirsutum
and
G. barbadense
. Release of their
genome sequences in the near future may help to identify more number of novel miRNAs in cotton in response to
drought stress using this data.
3 Materials and Methods
3.1 Plant materials
Two contrasting accessions of
Gossypium,
which are considered as ideal genetic materials for studying drought
tolerance mechanism in cotton, were used in this study. One was short duration upland cotton,
G. hirsutum
L. var.
KC 3 (hereafter referred as KC3) that has good performance under rainfed tract of Tamil Nadu, India and provide
assured minimum yield with relatively good quality fiber even under severe water stress. Another line was the
long duration
G. barbadense
L. var. Suvin (hereafter referred as Suvin) which provide poor yield with inferior
fiber qualities under drought stress (Boopathi et al., 2011). These two lines have also shown significant
dissimilarity at molecular level (Thiagu et al., 2011) and hence they were selected for development of different
types of mapping population at this laboratory. Therefore, the differentially expressed miRNAs identified in this
study could also be genetically mapped, since there is no genome sequence currently available in
G. hirsutum
or
G.
Barbadense.
3.2 Experimental conditions in the field
Field trial was conducted under upland condition at TNAU, Coimbatore, India during the regular cotton cultivating
season,
kharif
(July - December, 2012). Both the cotton lines were grown in a single row by following 75 x 40 cm
spacing between and within rows, respectively with two replications and evaluated under two water regimes: irrigated
(control or non-stress) and water limited (stress). A buffer area of 3.0 m wide and 0.75 m deep along the length of the
experimental plot divided the control and stress plots. All the standard agronomic practices were equally applied to
both the treatments. Both the lines were surface irrigated as per the regular cultivation practices, except for water
limited regime. Water stress was imposed by withholding irrigation when the plants were in peak vegetative phase
to stress plots i.e., from 30 days after sowing (DAS) and there were continuous rain free period of 20 days. The
leaf samples were collected from both the accessions and treatments on 50
th
DAS with two replications and stored
in Shrimpex lysis buffer (Shrimpex Biotech Services Private Limited, Chennai, India).
3.3 Small RNA library construction and sequencing
