Cotton Genomics and Genetics 2016, Vol.7, No.2, 1-23
6
(miR167) and 56.326 (miR14)]. It was further noticed that miRNA families, hme-bantam, miR2, miR14, miR156,
miR160, miR166, miR279, miR2111, miR3027, and miR3050 were immensely downregulated and one member
of miR169 family was significantly up-regulated in Suvin-WS (Supplementary Table 1).
1.3 Identification of novel miRNAs
In addition to conserved miRNAs, deep sequencing had also helped to identify new miRNAs in cotton that have
not yet reported in miRBase. Since, the sequencing of the
G. hirsutum
and
G. barbadense
genome is incomplete
and information on complete genome-wide cotton small RNA population is unknown, accurate identification of
non-conserved miRNA in cotton is a difficult task. In order to ensure the truthful identification of miRNA and
considering of the complicated nature of plant small RNA, a series of strict filtrations was used to enhance
reliability of the result presented here. ESTs from NCBI and the genome sequence of
G. raimondii
(Wang et al.,
2012) were used as reference sequences to predict several numbers of miRNA precursors. However, with the
availability of genomic sequencing data from
G. hirsutum
and
G. barbadense
in near future, there is a greater
potential to find several additional miRNAs of
Gossypium
with this same data sets.
Though several hundreds of new miRNA loci were identified as eligible candidates, following a BLASTn search
and hairpin structure prediction, only 59 putative unique miRNAs were detected in all the four small RNA
libraries (Table 3). The RNA structures were predicted by mFold software and manually checked according to the
criteria of Meyers et al. (2008). The lowest minimum free energy (MFE) among all hairpin structures of the novel
miRNAs precursors was −43.9 kcal/mol (Table 3). All precursors of novel miRNAs had regular hairpin structures
and two of these (Ghi-miR(contig-4796) and Ghi-miR(contig-13785) are presented in Figure 3. GC content of the
identified new miRNA precursor sequences was ranged from 23-71 percent (Table 3).
Table 3 Details on putative, novel, drought responsive miRNAs identified in this study
Contig ID
Small RNA sequence identified in this study
Length
MFE
*
GC(%)
#
Contig_5142
UUCUGAGAGGCUGCCACAUAACAUCA
26
-74.7
46
Contig_4796
GCCUCCAGAAGAUACAUUAGCACCAUGGGAUAU
33
-121.1
46
Contig_8004
UGCCAAAUCAGGGAAGCGAAAG
22
-66.0
50
Contig_16568
UUUCCAUCAUAUUAUUCGCCAUG
23
-104.7
35
Contig_13785
UUCACAAAGCGGAAGCACCUUG
22
-80.9
50
Contig_15645
UUUCCAUGUCACAGAGAUGUUG
22
-65.4
41
Contig_6913
AGAAUCAAGUUAUCAAUAUGAUGAGUACGC
30
-46.1
33
Contig_16002
UAGUGUCCUGCAAACUCACAAACC
24
-46.7
46
Contig_14218
AGAUGCAGUAUGGGUUGUGAUUGAUAAGCUAAC
33
-53.1
39
Contig_18822
GGGAUCUUUUAAGAACUAAGUCACC
25
-43.9
40
Contig_17043
AGUGUCAUCCUUCUGCGUCAUUAU
24
-73.4
42
Contig_16448
GAAGCGCUGGAGGGGAGAGUGG
22
-63.6
68
Contig_17400
UCGUCAGCGCUGCUCUCAAUCAUG
24
-71.3
54
Contig_15122
UGGAAGGUUUGGAGGAGAUUGA
22
-63.7
46
Contig_9778
GUAAGGGAGAACCUAGAUUCAUUACCCAGC
30
-77.2
47
Contig_16528
GUAAGGGAGAUCUAGAUUCAUAA
23
-67.9
35
Contig_15303
CCAACGACCGAAGUUAUUGUUCC
23
-91.3
48
Contig_14086
AUCAACCGUGUUACUCUGUCUAAUC
25
-63.6
40
Contig_20897
GGGCACCUCUCACUUAGGCAGG
22
-69.8
64
Contig_13516
UGAGGGUCCAAGACUGACCUCC
22
-94.4
59
Contig_22213
UAUAGGUCUUUCAUUUAAAGU
21
-79.4
24
Contig_20063
UCUGUCGCAGGGGAGAUGGCUG
22
-74.9
64
Contig_21500
UUCCAUGUCACAGAGAUGUUG
21
-61.2
43
Contig_19382
AUUCCAGUGAUUUCCAGAGGCU
22
-67.8
46
