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Cite this: RSC Adv., 2015, 5, 85383

Identication of miRNAs and their targets in

transgenic Brassica napus and its acceptor (Westar)
by high-throughput sequencing and degradome
analysis
Cheng-Yi Tang,ab Min-Kai Yang,a Feng-Yao Wu,a Hua Zhao,a Yan-Jun Pang,a
Rong-Wu Yang,a Gui-Hua Lu*ab and Yong-Hua Yang*ab
MicroRNAs (miRNAs) are a class of noncoding small RNAs (sRNAs) that play many roles in plant growth,
development, and the stress response. Recently, the Brassica napus (B. napus) genome was reported,
which allows the genome-wide identication of miRNAs and their targets in B. napus cv. Westar and its
transgenic cultivar. Thus, two sRNAs libraries and two degradome libraries of the transgenic B. napus
(TG) and its acceptor (B. napus cv. Westar, WA) were constructed. Following high-throughput
sequencing and miRNA identication, 139 unique conserved and 305 unique novel miRNA sequences
were identied in the two sRNA libraries, and through degradome analysis, 540 targets corresponding to
167 unique miRNA sequences were identied in the two degradome libraries. 11 dierentially expressed

Received 24th July 2015

Accepted 2nd October 2015

unique miRNA sequences were veried by quantitative real-time PCR (qRT-PCR), and the results nearly
agreed with the high-throughput sequencing data. The present study increases the number of miRNAs

DOI: 10.1039/c5ra14672k

identied in B. napus and suggests that there are possible dierences in the miRNA expression between

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transgenic B. napus and its acceptor.

Introduction
In eukaryotes, gene expression is regulated and controlled at
the transcriptional and post-transcriptional levels, and one
class of regulators of post-transcriptional gene silencing
(PTGS) is small RNAs (sRNAs).1,2 MicroRNAs (miRNAs), which
constitute a major class of sRNAs that are approximately 2124
nucleotides (nt) in length, are endogenous, noncoding, regulatory sRNA molecules that have been widely identied in
animals, plants and viruses.25 Increasing lines of evidence
have shown that miRNAs play an important role in a wide
range of plant processes, including development, signal
transduction, and various biological and environmental stress
responses.5
In higher plants, miRNA genes are transcribed into primary
miRNA (pri-miRNA) transcripts by RNA polymerase II, and these
transcripts form stable hairpin structures. Dicer-like 1 (DCL 1),

State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of

Plant Molecular Biology, School of Life Sciences, Nanjing University, Nanjing
210093, China. E-mail: yangyh@nju.edu.cn; guihua.lu@nju.edu.cn; Fax: +86-02589686305; Tel: +86-025-89686305

Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry

University, Nanjing 210037, China
Electronic supplementary
10.1039/c5ra14672k

information

(ESI)

These authors contributed equally.

This journal is The Royal Society of Chemistry 2015

available.

See

DOI:

hyponastic leaves 1 (HYL 1) and serrate (SE) protein then

process the hairpin structures into stem-loop precursor miRNAs
(pre-miRNAs) and then cleaved these into miRNA duplexes
containing two stands (5p and 3p).1,6,7 The duplexes are then
loaded into the RNA-induced silencing complex (RISC) in the
cytoplasm. One strand, either the 5p or 3p mature miRNA,
binds the Argonaute (AGO) protein in the RISC complex, and
the other strand is degraded.1,7 The miRNARISC complex then
targets mRNAs and guides their cleavage, resulting in the
regulation of gene expression.
The recently proposed combination of high-throughput
sequencing with bioinformatics analysis has improved the
discovery of novel miRNAs in several plants due to the
conservation of miRNAs among related plant species. 811
Furthermore, degradome sequencing combined with gene
annotation has also been used for the large-scale
conrmation of several miRNA targets in many plants
because plant miRNAs completely or almost completely
bind their targets.1215 However, this strategy for
identifying miRNAs and their targets strongly depends on
the available genomic information and miRNA databases
(e.g., miRBase).
Brassica napus (B. napus) is one of the main edible oil crops
worldwide.11 Westar (Canola) coming from Canada is a cultivar
of B. napus, which was bred to reduce erucic acid that have
pathogenic potential and is implicated as a factor in

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hypercholesterolemia.16 Several previous studies of miRNAs

in B. napus have focused on miRNA identication, seed
development, the cadmium response and fungus infection.1725 Xie et al. (2007) detected 21 potential miRNAs only by
using computational analysis.17 Buhtz et al. (2008) rstly
identied 32 annotated miRNAs in the phloem of B. napus by
using high-throughput sequencing with bioinformatics analysis.18 Aer that, Xu et al. (2012) and K
orbes et al. (2012)
respectively used high-throughput sequencing to identify
miRNAs in the whole B. napus.19,20 In addition, Huang et al.
(2013) detected numerous potential miRNA sequences in B.
napus seed maturation;21 Zhao et al. (2012) identied
59 miRNAs involved in B. napus oil production;22 Zhou et al.
(2012) compared B. napus miRNA expression between
cadmium-treated and non-treated.23 Shortly aer, Verma et al.
(2014) and Shen et al. (2014) respectively researched miRNA
response to fungi infection in B. napus.24,25 Although highthroughput sequencing has widely been applied into the B.
napus miRNAs and their targets identication in these
previous studies, these miRNA identication approaches still
lacked the whole genome information of B. napus and were
mainly based on expressed sequence tags (ESTs), tentative
consensus sequences (TCs) and the genomes of related
species (e.g., Brassica rapa and Brassica oleracea). This strongly
limits B. napus miRNA researches. However, the B. napus
genome was reported recently,26 and miRBase has now been
updated to version 21.0 (current edition). Therefore,
a genome-wide the identication and comparison of miRNAs
and their targets in transgenic B. napus (TG) and its acceptor
(Westar, WA) can now be performed.

Materials and methods

Plant materials
The transgenic B. napus constructed by Dr Song Cheng was
transformed with a construct (a plasmid pCNFIRnos) containing ihpRNA targeted to the endogenous B. napus fad2 gene.27
Both transgenic B. napus and its acceptor (Westar) were
germinated in a glasshouse in the experimental eld at the
Jiangsu Academy of Agricultural Sciences, Nanjing, China; and
were placed under a light/dark (16 h/8 h) photo-period with
light intensity of $8000 lx at 23
1  C. Leaves and stalks from
three-month-old seedlings were collected. To minimize interindividual dierences, three samples from the same line were
mixed together and then frozen in liquid nitrogen and stored at
80  C.
RNA extraction
Samples from the WA and TG lines were individually subjected
to RNA extraction using the TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). The RNA concentrations were measured
with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was
assessed by agarose gel electrophoresis. The total RNA extracted
was used for miRNA identication, degradome analysis and
qRT-PCR verication.

85384 | RSC Adv., 2015, 5, 8538385394

Paper

High-throughput sequencing
Two sRNA libraries were constructed using the Illumina TruSeq
Small RNA Preparation Kit (LC Sciences, Hangzhou, China). The
total sRNA was ligated to 3p and 5p adapters (ADTs; 3p adapter:
50 -TGGAATTCTCGGGTGCCAAGG-30 ; 5p adapter: 50 -GUUCAGAGUUCUACAGUCCGACGAUC-30 ), and the corresponding cDNA
was obtained by reverse-transcription PCR. Following purication, the cDNA from the two sRNA libraries was sequenced
using an Illumina Genome Analyzer II (LC Sciences, Hangzhou,
China). Aer removing low-quality data and low-copy-number
reads (reads < 10), the raw reads were obtained using the
Illumina Pipeline v1.5 soware (LC Sciences, Hangzhou,
China). The ACGT101-miR program (LC Sciences, Hangzhou,
China) was then used for further data analysis.2833
miRNA identication
Aer ltering out ADTs data, sequences with lengths < 17 and >25
nt, junk data, RFam (http://rfam.janelia.org) and repeats (http://
www.girinst.org/repbase), the remaining reads (clean reads) were
subjected to miRNA identication using the Brassicaceae premiRNAs and miRNAs in miRBase 21.0 (http://www.mirbase.org)
and the B. napus genome database (http://www.genoscope.cns.fr/
brassicanapus). Three mismatches were allowed between the
candidate miRNAs and the known Brassicaceae pre-miRNA and
miRNA sequences.3136 The reads that mapped to known Brassicaceae pre-miRNAs and whose pre-miRNAs or candidate miRNAs
mapped to the B. napus genome were identied as conserved
miRNAs. In addition, the reads that did not map to known Brassicaceae pre-miRNAs but mapped to the B. napus genome were
considered novel miRNAs. Furthermore, the stem-loop structures
(secondary structures) of all of the identied and potential
pre-miRNAs from the B. napus genomic positions were predicted
using the UNAFold soware (http://www.mfold.rna.albany.edu/?
qmfold/RNA-Folding-Form),34,37 and only those with high
minimal folding energy indexes (MFEIs) were considered (MFEI $
0.7 for conserved miRNAs, MFEI $ 0.9 for novel miRNAs).38
Degradome sequencing
Two degradome libraries were constructed based on the
method described by Ma et al.39 poly(A)-enriched RNAs were
obtained and ligated to a 5p adapter harboring the EcoP15 I
recognition site. Through reverse-transcription PCR, the ligated
sequences were converted to cDNA. Following EcoP15 I digestion and purication, the cDNA was subjected to cluster analysis with an Illumina Cluster Station (LC Sciences, Hangzhou,
China) and sequenced using an Illumina Genome Analyzer II
(LC Sciences, Hangzhou, China). Lastly, aer removing lowquality data, the raw reads were obtained using the Illumina
Pipeline v1.5 soware (LC Sciences, Hangzhou, China).
Target identication and annotation
Aer ltering out ADTs data and reads with lengths <15 nt, the
remaining reads were compared with a cDNA library in the B.
napus genome database. Aerward, the mapped-cDNA reads
were compared with the identied miRNAs to perform an

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alignment analysis as described previously using CleaveLand

3.0 (LC Sciences, Hangzhou, China).14,40 Low alignment scores
(#4) were considered. Furthermore, based on the number of
degradome sequences and their abundance values, the miRNA
targets were classied into 5 categories (0, 1, 2, 3 and 4) as
previous studies:14,15,20,24,29,31,33 Category 0, the abundance of
degradome sequences (>1) was equal to the maximum value,
and there was only one maximum value; Category 1, the
abundance of degradome sequences (>1) was equal to the
maximum value, and there was more than one maximum value;
Category 2, the abundance of degradome sequences (>1) was
between the maximum and median values; Category 3: the
abundance of degradome sequences (>1) was below the median
value; and Category 4: only one degradome sequence was
detected. To further elucidate the functions of miRNAs and
their targets, these were annotated through Gene Ontology (GO,
http://www.geneontology.org) and Kyoto Encyclopedia of Genes
and Genomes (KEGG, http://www.genome.jp/kegg) pathway
analyses.
qRT-PCR
Total RNA was rstly carried out reverse transcription reaction.
Sequentially, by using SYBR Premix Ex Taq (Takara, Dalian,
China) system, quantitative real-time PCR (qRT-PCR) was conducted on a BIOER Line-Gene K RT-PCR machine (BIOER,
Hangzhou, China), whose primers were listed in Additional le
10: Table S10.1. U6 snRNA was used as the internal reference.30,32,33 In addition, all reactions were performed in triplicate, and relative expression levels were quantied by using the
2DDCt method.
Statistical analysis
log2 ratio, Chi-square 2  2 and Fisher's exact tests were performed to identify dierences in miRNA expression and in the
degradation of their targets between the WA and TG libraries. p
values both from Chi-square 2  2 test and Fisher's exact test
have been automatically adjusted to control False Discovery
Rate (FDR) as previous studies,2933,43 according to Benjamini
Hochberg procedure.41,42

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(Fig. 1, Additional le 2: Table S2), which is consistent

with previous reports in other species, particularly
plants.15,1825,2933,4346 Additionally, we also analyzed Rfam
data for conrming the stability of the other non-coding
sRNAs. The results roughly shown that rRNA, snRNA,
snoRNA should be stable in both libraries as a general
understanding (Additional le 2: Table S2.2).
Identication of conserved miRNAs
To identify conserved miRNAs in B. napus, the clean reads
were compared with the known B. napus pre-miRNAs in
miRBase 21.0, and these pre-miRNAs were mapped to the B.
napus genome. A total of 75 conserved unique miRNA
sequences (corresponding to 119 miRNAs and 74 pre-miRNAs)
were identied as conserved B. napus miRNAs (abbreviated as
Con-BN) (Additional le 3: Table S3.1). Similar to the
conserved miRNAs identied in B. napus, 56 unique miRNA
sequences (corresponding to 66 miRNAs and 47 pre-miRNAs)
were found to be conserved with Brassicaceae miRNAs
(abbreviated as Con-BA) (Additional le 3: Table S3.2).
Furthermore, the remaining reads, which mapped to Brassicaceae pre-miRNAs but whose pre-miRNAs did not map to the
B. napus genome, were compared with the B. napus genome. 8
conserved unique miRNA sequences (corresponding to 11
miRNAs and 9 predicted pre-miRNAs) were identied as
conserved-novel miRNAs (abbreviated as Con-N) (Additional
le 3: Table S3.3). Additionally, all of the identied and
predicted pre-miRNAs in the B. napus genome are capable of
forming stem-loop structures (secondary structures), and only
those with a minimal folding free index (MEFI) of at least
0.7 were considered (Additional le 3: Table S3). In summary,
139 conserved unique miRNA sequences corresponding to
196 miRNAs and 130 pre-miRNAs and belonging to 52 miRNA
families were identied in the WA and TG libraries (Fig. 2,
Additional le 3: Table S3, Additional le 4: Table S4), and
among these, 25 unique sequences, such as bra-miR158-3p,
bna-miR159, bna-miR166f, bna-miR396a_1ss21TG and bnamiR166a,b,c,d,e, presented high expression (normalized
reads $ 1000) (Table 1).

Results
Analysis of sRNA libraries
To identify conserved and novel miRNAs in transgenic B.
napus (TG) and its acceptor (B. napus cv. Westar, WA), two
sRNA libraries were constructed, rstly. Through highthroughput sequencing, a total of 15 287 111 (from the WA
library) and 13 084 245 (from the TG library) raw reads were
generated (Additional le 1: Table S1). Aer ltering out the
3ADT data, reads with lengths < 17 and >25 nt, junk data,
RFam and repeats, 11 285 339 and 9 574 008 clean reads
corresponding to 3 073 405 and 2 579 167 unique reads with
lengths of 1725 nt were obtained for the two libraries,
respectively (Additional le 1: Table S1, Additional le 2:
Table S2.1). The distribution of the lengths of the clean
reads was mainly between 21 and 24 nt in the two libraries

This journal is The Royal Society of Chemistry 2015

Fig. 1 Length distribution of the clean reads in the TG and WA libraries.

(WA total) total sRNA sequences from WA library; (TG total) total sRNA
sequences from TG library; (WA unique) unique sRNA sequences from
WA library; (TG unique) unique sRNA sequences from TG library.

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Fig. 2

Paper

Distribution of the families of the conserved unique miRNA sequences in the WA and TG libraries.

For comparison of the WA and TG libraries, the conserved

unique miRNA sequences were analyzed through Chi square
2  2, Fisher's exact and log2 ratio tests of the normalized
data. According to the standard of highly signicant variation (p < 0.01 from both Chi-square 2  2 and Fisher-exact
tests and |log2(WA/TG)| $ 1), 30 dierentially expressed
conserved unique sequences (12 upregulated and 18
downregulated in the TG library) were detected in the WA

Table 1

and TG libraries (Fig. 3, Additional le 4: Table S4).

Among these sequences, 5 unique sequences, e.g., athmiR399c-3p, presented high expression (normalized reads
$ 1000). In addition, 7 unique sequences exhibited highly
dierential expression (p < 0.01 and |log2 (WA/TG)| $ 2) with
above-average expression levels (normalized reads $ 100),
such as bna-miR156e_R+1 and bra-miR398-3p (Tables 1
and 2).

Conserved unique miRNA sequences that are highly expressed (normalized reads $ 1000) in the WA and TG libraries

Namea

Sequence

WA norm

TG norm

log2 ratiob

bna-miR156e_R+1
bra-miR158-3p
bra-miR158-5p
bna-miR159
bna-MIR159-p5
ath-MIR159a-p5, aly-MIR159a-p5
bra-miR162-3p
bna-miR166a,b,c,d,e
aly-miR166a-5p
bna-miR166f
aly-miR166b-3p_L+1
bna-MIR167c-p3
bna-miR168a
bra-miR168b-5p,c-5p
ath-miR390a-3p_R-1_1ss5AG
bna-miR396a_1ss21TG
bra-miR398-3p
ath-miR399c-3p
bna-miR403
bna-MIR824-p3
bna-miR1140
bna-MIR1140-p5
bra-miR1885b
bra-MIR5719-p3_1ss21AT
bra-MIR9563a-p5_1ss17CT

TGACAGAAGAGAGTGAGCACT
TTTCCAAATGTAGACAAAGCA
CTTTGTCTATCGTTTGGAAAAG
TTTGGATTGAAGGGAGCTCTA
AGCTGCTAAGCTATGAATCCC
AGCTGCTAAGCTATGGATCCC
TCGATAAACCTCTGCATCCAG
TCGGACCAGGCTTCATTCCCC
GGAATGTTGTCTGGCTCGAGG
TCGGACCAGGCTTCATCCCCC
TTCGGACCAGGCTTCATTCCCC
GATCATGTTCGTAGTTTCACC
TCGCTTGGTGCAGGTCGGGAA
TCGCTTGGTGCAGGTCGGGAC
CGCTGTCCATCCTGAGTTTC
TTCCACAGCTTTCTTGAACTG
TGTGTTCTCAGGTCACCCCTG
TGCCAAAGGAGAGTTGCCCTG
TTAGATTCACGCACAAACTCG
CCTTCTCATCGATGGTCTAGA
ACAGCCTAAACCAATCGGAGC
TCCGATTGGCTTTAGGCTGTT
TACATCTTCTCCGCGGAAGCTC
TCGGATTATCATCACAACACT
AGCGGAATATAAGAACTCGTCTCT

3496
18 164
11 274
29 676
1078
1476
2464
131 410
3809
36 772
1859
4435
2131
3475
546
22 401
3207
3183
8430
2537
6607
1526
8990
949
3901

687
26 437
14 289
18 564
472
867
2548
213 973
2522
59 919
2380
3639
1828
3766
1043
24 363
39 219
1257
10 001
4809
3771
671
8240
1608
2617

2.35**
0.54
0.34
0.68
1.19**
0.77
0.05
0.70
0.59
0.70
0.36
0.29
0.22
0.12
0.93
0.12
3.61**
1.34**
0.25
0.92
0.81
1.19**
0.13
0.76
0.58

a
Name regulation following Additional le 3: Table S3; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm); **
indicates highly signicant variation (p-value < 0.01 from both Chi-square 2  2 & Fisher's exact test and |log2(WA/TG)| $ 1) between the WA and TG
libraries.

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Fig. 3 Comparison of the WA and TG libraries. (Con) conserved

unique miRNA sequences; (Nov) novel unique miRNA sequences. (Up
expression) up expressed unique miRNA sequences in the TG library;
(down expression) down expressed unique miRNA sequences in the
TG library; (equal expression) equal expressed unique miRNA
sequences in the WA and TG libraries.

Identication of novel miRNAs

To identify novel miRNAs, the remaining clean reads (removed
conserved miRNAs) mapped to the B. napus genome. A total of
305 novel unique miRNA sequences corresponding to 339

miRNAs and 303 predicted pre-miRNAs were identied. In

addition, these predicted pre-miRNAs could form stem-loop
structures with a MEFI of at least 0.9 (Additional le 5: Table
S5). Compared with the conserved miRNAs, no highly
expressed novel unique miRNA sequences were found in the
two libraries; in contrast, most of the unique miRNA
sequences presented low expression (normalized reads < 100),
and only 22 unique sequences, e.g., PC-3p-9, PC-3p-88, PC-5p21 and PC-5p-50, exhibited above-average expression (Table 3,
Additional le 6: Table S6). The comparison revealed 105
dierentially expressed novel unique miRNA sequences
(29 upregulated and 76 downregulated in the TG library) (p <
0.01 and |log2(WA/TG)| $ 1), including 8 unique sequences
that were identied only in the WA library (Fig. 3, Additional
le 6: Table S6). Among these, 6 dierentially expressed
unique sequences, for example PC-3p-72, presented aboveaverage expression, and only 1 unique sequence (PC-5p-137)
exhibited highly dierential expression (p < 0.01 and |log2(WA/TG)| $ 2) (Table 3).
Identication and annotation of miRNA targets
To explore the potential miRNA targets and their biological
function, a degradome analysis, which is an ecient
platform for miRNA-cleaved sequencing combined with
bioinformatics-based prediction and annotation, was performed. A total of 31 003 060 and 24 766 218 raw reads were
generated from the WA and TG degradome libraries, respectively (Additional le 7: Table S7). Aer removing the 3ADT
data and reads with lengths <15 nt, the remaining reads were

Table 2 Dierentially expressed conserved unique miRNA sequences (p < 0.01 and |log2(WA/TG)| $ 1) with above-average expression levels
(normalized reads $ 100) in the WA and TG libraries

Namea

Sequence

WA norm

TG norm

log2 ratiob

bna-miR156d,e,f,a_R-1
bna-miR156e_R+1
bna-MIR159-p5
bna-miR160a,b,c,d
bna-MIR162a-p5
bra-miR168b-3p,c-3p
bna-MIR169m-p3
ath-MIR169e-p3_L+1
ath-miR171-5p, bna-MIR171g-p5
bna-miR390a,b,c
bra-miR391-3p
bna-miR393_R+1
bra-miR398-3p
bra-miR398-5p
bol-miR398a-3p_R-1
ath-miR399c-3p
bna-MIR1140-p5
bra-miR5654a
bra-MIR5719-p5_1ss16AC
bna-miR6028_R+2
bna-miR6030
bna-miR6031
bra-MIR9557-p3_1ss13GA

TGACAGAAGAGAGTGAGCAC
TGACAGAAGAGAGTGAGCACT
AGCTGCTAAGCTATGAATCCC
TGCCTGGCTCCCTGTATGCCA
GGAGGCAGCGGTTGATCGATC
CCCGCCTTGCATCAACTGAAT
GCAAGTCGACTTTGGCTCTG
GCAAGTTGACTTTGGCTC
TATTGGCCTGGTTCACTCAGA
AAGCTCAGGAGGGATAGCGCC
ACGGTATCTCTCCTACGTAGC
TCCAAAGGGATCGCATTGATCC
TGTGTTCTCAGGTCACCCCTG
GGGTCGACATGAGAACACATG
TGTGTTCTCAGGTCACCCCT
TGCCAAAGGAGAGTTGCCCTG
TCCGATTGGCTTTAGGCTGTT
ATAAATCCCAAGCATCATCCA
TGTTGTGATGATAATCCGACT
TGGAGAGTAAGGACATTCAGATC
TCCACCCATACCATACAGACCC
AAGAGGTTCGGAGCGGTTTGAAGC
AATGAGGGCTGAATTGGAACGC

291
3496
1078
104
241
87
139
116
124
815
77
56
3207
21
35
3183
1526
84
144
176
93
375
170

81
687
472
366
91
229
30
21
19
408
172
143
39 219
143
366
1257
671
192
330
72
370
118
62

1.85
2.35
1.19
1.82
1.41
1.40
2.21
2.47
2.71
1.00
1.16
1.35
3.61
2.77
3.39
1.34
1.19
1.19
1.20
1.29
1.99
1.67
1.46

Name regulation following Additional le 3: Table S3; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm).

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Table 3

Paper

Novel unique miRNA sequences with above-average expression (normalized reads $ 100) in the WA and TG libraries

Namea

Sequence

WA norm

TG norm

log2 ratiob

PC-3p-9
PC-3p-15
PC-3p-20
PC-3p-47
PC-3p-53
PC-3p-72
PC-3p-88
PC-3p-101
PC-3p-128
PC-3p-133
PC-3p-138
PC-5p-21
PC-5p-23
PC-5p-50
PC-5p-92
PC-5p-94
PC-5p-102
PC-5p-110
PC-5p-116
PC-5p-127
PC-5p-137
PC-5p-138

GCTTTCCCCGGACGACTTTAAATT
TATCCCGGACGACCTTAAATT
ATTCCGACAAGAACTCCGCCCT
AAAGATTGTTGTGGTCTTGTGCCT
ATCCATTAGTATCAGACGATC
AGAGTCGGGTTCTTATATTCTGCT
ATATTCCGTTAAGAACTCCACCCT
TGCTTTCCCCGGACGACTTTAAAT
AACTTCCGCTAAGAACTCCATCCT
GGTCATGTCTGACAGCTTCACT
TAAGAACCCACATTAATCATG
TGAACCCAAGATCTCACCCTT
AGCGGAATATAAGAACCTGACTCT
ATCGGAATATAAGAACTCGTCTCT
ATCGGAATATAAGAAACTGTCTCT
AACGGAATATAAGAATTTGACTCT
AGCGGAATATAAGAATTTGTCTCT
GTTTTGAGAGATTGGGAAGCT
ACCGGAATATAAGAACTCGTCTCT
AGCGGAAAATAAGAACTCGTGTCT
GAGTGAGAAATGGAGTGATGAACA
AGCGGAATATAAGAACTCGACTCT

183
209
102
153
156
288
215
46
73
101
53
333
238
353
173
200
137
225
121
139
339
121

473
237
212
169
164
131
360
114
125
66
105
377
130
180
97
122
79
69
72
93
51
64

1.37**
0.18
1.06**
0.14
0.07
1.14**
0.74
1.31**
0.78
0.61
0.99
0.18
0.87
0.97
0.83
0.71
0.79
1.71**
0.75
0.58
2.73**
0.92

a
Name regulation following Additional le 5: Table S5; red marked miRNAs were detected in degradome analysis. b log2(WA norm/TG norm); **
indicates highly signicant variation (p-value < 0.01 from both Chi-square 2  2 & Fisher's exact test and |log2(WA/TG)| $ 1) between the WA and TG
libraries.

compared with the B. napus cDNA library. A total of

23 597 707 and 18 863 658 mapped cDNA reads corresponding to 6 891 809 and 5 351 033 unique reads were obtained
from the two degradome libraries (Additional le 7: Table
S7). The mapped cDNA reads were then compared with the
identied miRNAs. Gene annotation [Gene Ontology (GO)
and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway analysis] revealed 741 miRNA targets corresponding
to 540 unique target transcripts, which are targeted by
167 unique miRNA sequences, including 81 conserved
(Additional le 8: Table S8.1) and 86 novel sequences
(Additional le 8: Table S8.2), in the two degradome
libraries. GO annotation included 3 ontologies: biological
process, cellular component, molecular function.47 According to GO annotation, targets corresponding to conserved
miRNAs classication revealed 26 categories in biological
process were classied, followed by 14 categories for cellular
component and 15 categories for molecular function,
respectively; while targets corresponding to novel miRNAs
classication revealed 12 categories in biological process
were classied, followed by 4 categories for cellular
component and 11 categories for molecular function,
respectively (Fig. 4).
To further compare the WA and TG degradome libraries,
the high-condence miRNA targets (categories 0, 1, 2 and 3)
were rst categorized by gene annotation and then analyzed
using the Chi-square 2  2 and Fisher's exact tests and log2
ratios based on their abundance values. Of the 441 highcondence miRNA targets (corresponding to 346 unique
target transcripts), 404 (312 target transcripts) were

85388 | RSC Adv., 2015, 5, 8538385394

predicted to bind 150 homologous genes, and 37 miRNA

targets (34 target transcripts) were absent (Additional le 9:
Table S9). Additionally, 227 dierentially expressed highcondence miRNA targets corresponding to 185 unique
target transcripts [97 downregulated miRNA targets
(78 downregulated target transcripts) and 130 upregulated
miRNA targets (107 upregulated target transcripts) in the TG
degradome library] were found in the two degradome
libraries (p < 0.01 and |log2(WA TPB/TG TPB)| $ 1). Of these
miRNA targets, 66 (55 target transcripts) were found only in
the WA degradome library, whereas 32 (28 target transcripts)
were identied only in the TG degradome library (Additional
le 9: Table S9).

Verication of miRNAs
To verify the miRNA expression prole identied by highthroughput sequencing, 11 dierentially expressed unique
miRNA sequences at above-average expression (normalized
reads $ 100) targeting high-condence transcripts (categories
0, 1, 2 and 3) were selected for verication by quantitative realtime PCR (qRT-PCR). The results nearly agreed with the highthroughput sequencing data: bra-miR168b-3p,c-3p, bnamiR390a,b,c, bna-miR393_R+1, bra-miR398-3p, ath-miR399c3p and PC-3p-72, were possible dierentially expressed in the
two libraries (Fig. 5, Additional le 10: Table S10.2). However,
some of them did not exhibit signicant dierences in two lines
in the qRT-PCR verication, such as bna-miR156e_R+1, bnamiR160a,b,c,d and bna-miR6030 (Fig. 5, Additional le 10:
Table S10.2).

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Fig. 4 Classication and count of Gene Ontology (GO) annotation of the miRNA targets in the WA and TG degradome libraries. There are three
ontologies in GO: biological process, cellular component, molecular function.47. (Con BP) conserved miRNAs' targets in biological process; (Nov
BP) novel miRNAs' targets in biological process; (Con CC) conserved miRNAs' targets in cellular component; (Nov CC) novel miRNAs' targets in
cellular component; (Con MF) conserved miRNAs' targets in molecular function; (Nov MF) novel miRNAs' targets in molecular function.

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qRT-PCR validation of 11 potential dierentially expressed unique miRNA sequences in the WA and TG lines from high-throughput
sequencing.

Fig. 5

Discussion
Conserved and novel miRNAs
Although high-throughput sequencing is an ecient platform
for miRNA identication, it remains incapable of distinguishing highly homologous miRNAs. For example, bna-miR160a,
bna-miR160b, bna-miR160c and bna-miR160d, which correspond to the same sequence, had identical raw data (Additional
le 3: Table S3.1). However, through pairwise comparisons,
some miRNAs could be recognized. For example, only bnaMIR160c-p3 and bna-MIR160d-p3 were detected, which
demonstrates that the sequence only represents bna-miR160c
and bna-miR160d because these are from dierent arms of
the same pre-miRNA (Additional le 3: Table S3.1).
In miRBase 21.0, there are 92 B. napus miRNAs corresponding to 90 pre-miRNAs belonging to 33 miRNA families. As
expected, 119 B. napus miRNAs corresponding to 74 premiRNAs belonging to 27 miRNA families were identied in
this study. Among these miRNAs, 52, including bna-MIR159-p5,
bna-MIR166d-p5, bna-MIR167c-p3 and bna-MIR824-p3, are
novel strand miRNAs that map to B. napus pre-miRNAs but not
B. napus miRNAs because they are generated from the opposite
pre-miRNA arm (Additional le 3: Table S3.1). Furthermore,
the analysis also revealed 66 miRNAs (including 24 novel strand
miRNAs) corresponding to 47 pre-miRNAs belonging to 33
miRNA families that are included in miRBase 21.0 as detected
in other Brassicaceae species, i.e., Brassica rapa, Brassica oleracea, Arabidopsis thaliana and Arabidopsis lyrata, and 24 of the
miRNA families are not found in the B. napus data included in

85390 | RSC Adv., 2015, 5, 8538385394

miRBase 21.0, particularly the highly expressed miRNAs

(normalized reads $ 1000) miR158 (e.g., bra-miR158-3p),
miR398 (e.g., bra-miR398-3p), miR1885 (e.g., bra-miR1885b),
miR5719 (e.g., bra-MIR5719-p3_1ss21AT) and miR9563 (e.g.,
bra-MIR9563a-p5_1ss17CT) (Additional le 3: Table S3.2).
Based on the analysis of miRNA conservation in related species
and the qRT-PCR validation, the results indicated that
bna-MIR171g-p5, bra-miR168b-3p,c-3p, bra-miR398-3p, athmiR399c-3p and bra-miR5654a might exist in B. napus.
However, most of the conserved-novel and novel miRNAs presented a low expression level, and most of their targets were in
Category 4 (low condence) (Additional le 3: Table S3.3,
Additional le 5: Table S5, Additional le 8: Table S8) as same
as previously reported in other plants.15,1825,2933,4346 Therefore,
only 1 novel miRNA (i.e., PC-3p-72) targeting high-condence
transcripts at above-average expression was veried by
qRT-PCR. These results suggest that PC-3p-72 might exist in
B. napus.
miRNA targets
To evaluate the accuracy of the predicted miRNAtarget interactions, the miRNA targets were analyzed by degradome
sequencing combined with bioinformatics analysis. In total,
540 targets, including 441 high-condence targets, were detected. The results demonstrate that many homologous and
heterogeneous targets are cleaved by the same unique miRNA
sequence, indicating that the same miRNA may be involved in
multiple biological activities, as previously reported. For
example, SPL2, SPL6, SPL9, SPL10, SPL11, SPL13A and SPL15

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(squamosa promoter-binding-like protein family) are targeted by

bna-miR156e_R+1, whereas AFB2, AFB3 (auxin signaling F-box
family), BH076, BH077 (transcription factor bHLH family),
GRH1 (GRR1-like protein family) and TIR1 (transport inhibitor
response family) are targeted by bna-miR393_R+1 (Additional
le 9: Table S9). In contrast, many homologous miRNAs
usually target homologous and similar targets. For example,
TOE2 and TOE3 (AP2-like ethylene-responsive transcription
factor TOE family) are cleaved by bna-miR172a, bna-miR172b,c
and bna-miR172a_R-1,d_R-1 (bna-miR172 family), and three
transcripts, namely BnaA02g06490D, BnaA10g12950D and
BnaC09g35430D, which correspond to TOE2, are cleaved by
bna-miR172a, bna-miR172b,c and bna-miR172a_R-1,d_R-1
(bna-miR172 family), respectively (Additional le 9: Table
S9). Interestingly, homologous and similar targets are occasionally cleaved by heterogenetic miRNAs; for example, the
BnaC09g37630D transcript corresponding to PPR37 (pentatricopeptide repeat-containing protein At1g12620) is targeted by
bna-miR161 and bra-miR5654a, and the BnaA03g45020D transcript corresponding to Y4117 (putative disease resistance
protein At4g11170) is targeted by bra-miR1885b and bnaMIR6028-p3 (Additional le 9: Table S9). Similar to miRNA
high-throughput sequencing, degradome sequencing is also
unable to distinguish highly homologous sequences
corresponding to identical cleavage sites, such as the
BnaAnng07000D transcript, which is cleaved by bna-miR164a
and bna-miR164b,c,d at position 817, in agreement with the
raw data (Additional le 9: Table S9).

Potential dierences in miRNA expression

In total, 135 unique miRNA sequences that are dierentially
expressed (p < 0.01 and |log2(WA/TG)| $ 1) between the WA and
TG libraries were identied in the present study. Of these
sequences, 41 (12 conserved and 29 novel) are signicantly
upregulated in the TG library, and 94 (18 conserved and
76 novel) are signicantly downregulated. Among these unique
sequences, 13 (12 conserved and 1 novel) that were predicted to
target transcripts and belonged to high-condence categories (0, 1, 2 and 3) presented above-average expression
(normalized reads $ 100). Therefore, 11 of these unique
miRNA sequences were veried by qRT-PCR, which suggested
that most of them, particularly bra-miR168b-3p,c-3p, bnamiR390a,b,c,
bna-miR393_R+1,
bra-miR398-3p,
athmiR399c-3p and PC-3p-72, were dierentially expressed in
the two libraries. Vaucheret (2006, 2009) reported that
miR168 family response to endogenous or environmental
uctuations through targeting AGO1. 48,49 Jiang et al. (2014)
suggested that bra-miR168a-3p and bra-miR168b-3p were upregulated in the pollen development of the male fertile
Brassica campestris line Bcajh97-01B (vs. line Bcajh9701A).31 In our study, the unique sequences of the bramiR168b-3p,c-3p detected by high-throughput sequencing
and qRT-PCR had possible dierentially expressed in the TG
and WA libraries. In addition, degradome analysis shown
that bra-miR168b-3p,c-3p might target NADH-cytochrome b5
reductase 1 (NB5R1) involved in amino sugar and nucleotide

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sugar metabolism (Additional le 8 and 9: Table S8 and S9).

Previous studies indicated that miR390 and miR393 family
take part in the leaf development in Arabidopsis thaliana (A.
thaliana) and other plants, particularly miR390 and miR393
family could regulate the auxin level, because miR390 family
targets auxin response factors family (ARF) and miR393
family targets auxin signaling F-box family (AFB) and transport inhibitor response 1 (TIR1).5058 In the present study, we
found that bna-miR390a,b,c and bna-miR393_R+1 also have
potential dierentially expression in the two libraries. In
addition, bna-miR390a,b,c might target PHD nger protein
ALFIN-LIKE 7 (ALFL7), and bna-miR393_R+1 might not only
target AFB, TIR1 but also GRR1-like protein 1 (GRH1) (Additional le 8 and 9: Table S8 and S9). According to previous
researches, miR398 family plays an important role in
response and resistance mechanism, especially thermotolerance, in A. thaliana.5962 Yu et al. (2012) discovered that
bra-miR398 might are responsive to heat stress in Brassica
rapa.63 The annotation of the bra-miR398-3p target
(BnaC09g42920D) in this study was not clear, although bramiR398-3p highly signicant dierentially expression
between the TG and WA libraries (Additional le 8 and 9:
Table S8 and S9). A potential novel miRNA PC-3p-72 also
dierentially expressed in two libraries. It might target
tonoplast dicarboxylate transporter (TDT) involved in intracellular pH regulation (Additional le 8 and 9: Table S8 and
S9). In short, the results of high-throughput sequencing and
qRT-PCR verication showed that the possible potential
dierences in miRNA expression between transgenic B. napus
and its acceptor (Westar).

Conclusions
In summary, through high-throughput sequencing and degradome analysis, the present study identied numerous
conserved and novel unique miRNA sequences and their corresponding targets in transgenic B. napus and its acceptor
(Westar). These results contribute valuable information for
further increases in the number of identied miRNAs and the
identication of their target transcripts in B. napus. Furthermore, our results also suggest that some miRNAs might be
dierentially expressed between transgenic B. napus and its
acceptor. Moreover, dierential miRNA expression might lead
to dierential target degradation.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
CT, MY, and YY contributed to the experiment design and
management. CT, HZ and GL performed the experiments. CT,
FW, YP and RY participated in the data analyses. CT written
the manuscript. All authors checked and approved the
manuscript.

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Abbreviations
WA
TG
sRNA
miRNAs
nt
PTGS
pri-miRNA
pre-miRNA
DCL 1
HYL 1
SE
RISC
AGO
ESTs
TCs
ADTs
MFEIs
GO
KEGG

Brassica napus cv. Westar

Transgenic B. napus
Small RNA
MicroRNA
Nucleotides
Post-transcriptional gene silencing
Primary miRNA
Precursor miRNA
Dicer-like 1
Hyponastic leaves 1
Serrate
RNA-induced silencing complex
Argonaute
Expressed sequence tags
Tentative consensus sequences
Adapters
Minimal folding energy indexes
Gene ontology
Kyoto encyclopedia of genes and genomes

Acknowledgements
This work was nancially supported by the Program for
Changjiang Scholars and Innovative Research Team in University (IRT_14R27) and the National Natural Science Foundation
of China (NSFC). We appreciated Dr Song Chen for providing
the seedlings of the transgenic B. napus and its acceptor (B.
napus cv. Westar).

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