Analysis of small RNA library data sets and the small RNA profile
To identify miRNAs related to DGMS and RGMS during pollen development, the flower buds were collected from the sterile (6251A, 6284A) and fertile (6251B and 6284B) lines of the RGMS lines. Meanwhile, the flower buds were also respectively collected from the sterile (4001A, 4006A) and fertile (4001B and 4006B) lines of the DGMS lines. Three biological replicates were conducted for each of the eight kinds of samples. Thus, total of 24 sRNA libraries were constructed and deep-sequenced. The raw reads of the 24 sRNA libraries ranged from 20.58 to 42.68 million (Table 1). The raw reads of the 24 sRNA libraries were uploaded to SRA database of NCBI and 24 accession numbers were obtained, including SRX11350295, SRX11350296, SRX11350307, SRX11350312, SRX11350313, SRX11350315, and SRX11350316 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA743414?reviewer=t674c02cj415380e8oldre4s5a). After removing the low-quality reads and contaminated adapter sequences, the clean reads of the 24 sRNA libraries ranged from 19.77 to 41.61 million. The mapped reads were further annotated against the Pfam database and subsequently divided into rRNAs, tRNAs, snRNAs, snoRNAs, ta-siRNA, and others. The endogenous sRNAs were identified as known and novel miRNAs. The average sRNA lengths of the three biological replicates for each sample were calculated, which showed the length distribution patterns of the sRNAs being similar to one another. In general, the majority of the small RNAs ranged from 21 nt to 24 nt in size. The 24 nt small RNAs were the most dominant, followed by 21 nt small RNAs (Figure. 1).
Table 1 Overview of sRNA sequencing reads in Brassica napus
Sample
|
Raw reads
|
N% > 10%
|
Low quality
|
5, adapter contamine
|
3, adapter null or insert null
|
With ployA/T/G/C
|
Clean reads
|
4001B-1
|
24481718
|
41
|
16955
|
8590
|
761698
|
14436
|
23679998
|
4001B-2
|
27695084
|
70
|
36718
|
12896
|
698693
|
16785
|
26929922
|
4001B-3
|
20576690
|
38
|
16651
|
10078
|
768766
|
9577
|
19771580
|
4006B-1
|
28457208
|
131
|
44382
|
14481
|
721967
|
14232
|
27662015
|
4006B-2
|
27355185
|
45
|
7473
|
9466
|
895573
|
11463
|
26431165
|
4006B-3
|
35841966
|
57
|
25238
|
16152
|
1597289
|
18301
|
34184929
|
6251B-1
|
22307540
|
36
|
18341
|
18038
|
533864
|
14517
|
21722744
|
6251B-2
|
24409491
|
46
|
15603
|
27336
|
689062
|
7177
|
23670267
|
6251B-3
|
24485022
|
34
|
10518
|
24084
|
477366
|
16924
|
23956096
|
6284B-1
|
25552984
|
64
|
12764
|
21674
|
567970
|
12962
|
24937550
|
6284B-2
|
24763779
|
34
|
16161
|
23880
|
431298
|
15913
|
24276493
|
6284B-3
|
25378477
|
40
|
16388
|
25065
|
1546184
|
6044
|
23784756
|
4001A-1
|
21968633
|
56
|
16757
|
10355
|
723740
|
8680
|
21209045
|
4001A-2
|
26445702
|
23
|
5505
|
11592
|
1192819
|
7631
|
25228132
|
4001A-3
|
27397803
|
96
|
35289
|
12605
|
1483390
|
12457
|
25853966
|
4006A-1
|
23437578
|
357
|
10961
|
9403
|
612982
|
14712
|
22789163
|
4006A-2
|
42675389
|
50
|
10345
|
14879
|
1018244
|
21106
|
41610765
|
4006A-3
|
27452096
|
93
|
40026
|
12496
|
749916
|
14657
|
26634908
|
6251A-1
|
21484268
|
19
|
17038
|
20165
|
462483
|
17972
|
20966591
|
6251A-2
|
24127081
|
56
|
12533
|
20730
|
464608
|
24999
|
23604155
|
6251A-3
|
21608183
|
357
|
7763
|
13126
|
393936
|
10376
|
21182625
|
6284A-2
|
23525443
|
27
|
11236
|
20073
|
746946
|
12136
|
22735025
|
6284A-2
|
27605112
|
37
|
14315
|
23792
|
1211985
|
12779
|
26342204
|
6284A-3
|
29689151
|
56
|
30879
|
21998
|
753037
|
16533
|
28866648
|
Identification of known and novel miRNAs in B. napus
To identify known miRNAs in B. napus, all mapped small RNA sequences were compared with the known mature bna-miRNA sequences deposited in the miRBase database 22.1. Forty-six small RNAs that have the same sequences with the known bna-miRNAs in miRBase were identified. The numbers of reads of the 46 known miRNAs in 24 libraries were listed in Additional Table S1. Among the 46 known miRNAs, bna-miR159a, bna-miR166a, and bna-miR167c showed very high expression levels.
To predict novel miRNAs in B. napus, all the mapped small RNAs were blasted to the B. napus genome sequence in Brassica database and known plant miRNAs in miRBase. The small RNAs that exactly map to the genome sequence but not the known plant miRNAs were classified as candidate novel miRNAs. Five criteria described in the Materials and Methods were used to search for novel miRNAs. As a result, 35 pairs of novel miRNA-3p/miRNA-5p were identified. The mature sequences, reads numbers, positions in chromosomes, precursor sequences and minimum free energy are listed in Table 2. The length distribution of the novel miRNAs was between 18 nt to 26 nt. The length of the novel miRNA precursors ranged from 51 nt to 300 nt with an average length of 154 nt. The minimum free energy ranged from − 240.26 to -9.9 kcal mol− 1 with an average of -70.14 kcal mol− 1. The precursor sequences of the novel miRNA are shown in Additional Tables S2.
Table 2 Novel miRNAs identified in the fertile and sterile lines of Brassica napus by high-throughput sequencing
miR_ name
|
Sequence
|
Len
|
Read
|
pre-position
|
pre-len
|
MFE
|
bna-novel_1-3p
|
CUUCCUCCUAACACCAAUUGAUU
|
23
|
67
|
chrA09:27889716..27889818
|
102
|
-34.6
|
bna-novel_1-5p
|
AUCAAUUGGUUUUAGGUUAAGAAGCC
|
26
|
124
|
|
|
|
bna-novel_2-3p
|
UGGCAUUGGUAGUAAUGAGUGU
|
22
|
190
|
chrC04:42905032..42905108
|
76
|
-26.9
|
bna-novel_2-5p
|
ACUCAUUACCAUCAGAGCCAC
|
21
|
7
|
|
|
|
bna-novel_3-3p
|
UCAAUGUUGGCUCAAUUAUGU
|
21
|
120
|
chrC02:22515980..22516065
|
85
|
-29.5
|
bna-novel_3-5p
|
UCAUUGAGUGCAGCGUUGAUGU
|
22
|
12
|
|
|
|
bna-novel_4-3p
|
AUUAUCGACACUGAUCUCAUC
|
21
|
106
|
chrC08:2975781..2975915
|
134
|
-80.8
|
bna-novel_4-5p
|
UAAGGUCACUGUGGUAAUCC
|
20
|
52
|
|
|
|
bna-novel_5-3p
|
UCAAUGUUGGCUCAAUUAUG
|
20
|
49
|
chrC09:40959623..40959707
|
84
|
-31.6
|
bna-novel_5-5p
|
UCAUUGAGUGCAGCGUUGAUGU
|
22
|
12
|
|
|
|
bna-novel_6-5p
|
AAGGACUCUAAUCAGAAAUAUUGG
|
24
|
143
|
chrC06:35249139..35249190
|
51
|
-9.9
|
bna-novel_6-3p
|
AAUGGUCUUAUCUGGAAUCCUUAA
|
24
|
11
|
|
|
|
bna-novel_7-5p
|
UGCCUGGCUCCCUGUAUACCA
|
21
|
83
|
chrA08:8293061..8293144
|
83
|
-32.4
|
bna-novel_7-3p
|
GUGUAUAGAGUAGUCAAGCAUG
|
22
|
2
|
|
|
|
bna-novel_8-5p
|
AUCUCUAAUGUAUAACUCCAUUUU
|
24
|
24
|
chrA03:20065617..20065865
|
248
|
-95.7
|
bna-novel_8-3p
|
AAUGGAGUAGAUAUGGAGAUGCCC
|
24
|
1
|
|
|
|
bna-novel_9-3p
|
UUGGACUGAAGGGAACUCCCU
|
21
|
1527
|
chrA09:14645700..14645869
|
169
|
-64.4
|
bna-novel_9-5p
|
AGAGUUUCCUUAAGUCCAUUC
|
21
|
17
|
|
|
|
bna-novel_10-5p
|
UAAGAUCUUUGUACUUUCGGG
|
21
|
67
|
chrA10:15442817..15442916
|
99
|
-39.8
|
bna-novel_10-3p
|
CGAAAGUACAAAGAUCUGAAA
|
21
|
3
|
|
|
|
bna-novel_11-5p
|
AACAGUUGGAUUGGCUCUACGUGG
|
24
|
27
|
chrA09_random:3088720..3089020
|
300
|
-65.5
|
bna-novel_11-3p
|
ACGAUGGAGGACAAAACUGAUGCA
|
24
|
2
|
|
|
|
bna-novel_12-5p
|
UUUUCAGCAAUCUCUUUUCCAUU
|
23
|
44
|
chrA05_random:416209..416319
|
110
|
-29.5
|
bna-novel_12-3p
|
AUGGGAAAGAUUGUUGAUCAGA
|
22
|
6
|
|
|
|
bna-novel_13-5p
|
UAAAGUAGAGCUCGGUGACGG
|
21
|
1163
|
chrC03:20992442..20992727
|
285
|
-240.3
|
bna-novel_13-3p
|
GUCACCGAGCUCUACUUUAUA
|
21
|
1058
|
|
|
|
bna-novel_14-5p
|
UCGCUUCUGUUGAAUAAUUUUGAC
|
24
|
22
|
chrC04:45707769..45708016
|
247
|
-148
|
bna-novel_14-3p
|
CAAAAUUAUUCAACAGAAGCGAAU
|
24
|
23
|
|
|
|
bna-novel_15-5p
|
AUAUGAGGGUACAAUAGGAAG
|
21
|
137
|
chrAnn_random:33598332..33598540
|
208
|
-139.1
|
bna-novel_15-3p
|
UAUUGUACCCUCAUAUAUAGC
|
21
|
89
|
|
|
|
bna-novel_16-3p
|
CUAAGAGAUCUGUAAUAAACAUGC
|
24
|
30
|
chrC04:7124159..7124378
|
219
|
-117.5
|
bna-novel_16-5p
|
AUGUUUAUUGUAGGUCUUUUAGGUU
|
25
|
7
|
|
|
|
bna-novel_17-5p
|
ACGAACACUGAGUAAUAUCUG
|
21
|
15
|
chrC01_random:3928762..3929012
|
250
|
-164.8
|
bna-novel_17-3p
|
GAUAUUACUCAGUGUUCGUUG
|
21
|
13
|
|
|
|
bna-novel_18-3p
|
ACACUGCAGUGCACUGUACAUUGC
|
24
|
17
|
chrCnn_random:79954335..79954585
|
250
|
-113.8
|
bna-novel_18-5p
|
GUUGUACAUUGUACACAGCGGUGUAC
|
26
|
3
|
|
|
|
bna-novel_19-5p
|
UUGCAAACUGAAUUAUGAGUC
|
21
|
20
|
chrA09:30966144..30966235
|
91
|
-45.4
|
bna-novel_19-3p
|
CUCAUAAUUCAGUUUGCAAUC
|
21
|
20
|
|
|
|
bna-novel_20-5p
|
AAGAUACGGUCUCUUAACUUUUAG
|
24
|
259
|
chrC04_random:3818028..3818149
|
121
|
-67.8
|
bna-novel_20-3p
|
GUUAAUAGACCGUAUCUUAUA
|
21
|
13
|
|
|
|
bna-novel_21-5p
|
AACGAUCUUGUUUGGUUUUGAAGA
|
24
|
18
|
chrA05:21620032..21620188
|
156
|
-82.1
|
bna-novel_21-3p
|
UUCAAAACCAUACAAGAUCGUUUU
|
24
|
24
|
|
|
|
bna-novel_22-3p
|
GAUCAUGUUCGUAGUUUCACC
|
21
|
445
|
chrCnn_random:35712007..35712108
|
101
|
-47.3
|
bna-novel_22-5p
|
UGAAGCUGCCAGCAUGAUCU
|
20
|
3
|
|
|
|
bna-novel_23-3p
|
UUCUUGUGCGUUUAUAGGUAG
|
21
|
55
|
chrA06:23040126..23040236
|
110
|
-52.8
|
bna-novel_23-5p
|
ACCUCUAAAACACACAAGAAGA
|
22
|
3
|
|
|
|
bna-novel_24-5p
|
UGUUUCGCUGUUACUCAUGC
|
20
|
40
|
chrC02:8973302..8973545
|
243
|
-93.1
|
bna-novel_24-3p
|
AUGAGUAACAGCGAAACAAA
|
20
|
26
|
|
|
|
bna-novel_25-3p
|
AAACUGUGUGAACUCUCCAUGGAG
|
24
|
389
|
chrC02_random:2214651..2214881
|
230
|
-73.8
|
bna-novel_25-5p
|
CCAUAAAAAGGGUUCACAAAGUAUUU
|
26
|
1
|
|
|
|
bna-novel_26-3p
|
UUGAUACAUGUAGCUCUUUG
|
20
|
2089
|
chrA03:669098..669269
|
171
|
-83.8
|
bna-novel_26-5p
|
AAGUGCUACCGGUAUCCACGUG
|
22
|
840
|
|
|
|
bna-novel_27-5p
|
UUAAUCGUUUUGUGACUCUU
|
20
|
244
|
chrA07:19146929..19147018
|
89
|
-34.7
|
bna-novel_27-3p
|
UAGUUACAAAACGAUUAGUGC
|
21
|
24
|
|
|
|
bna-novel_28-3p
|
AUCAACGUUGGCUCAAUUAUG
|
21
|
453
|
chrA10_random:1861693..1861781
|
88
|
-33.2
|
bna-novel_28-5p
|
UCAUUGAGUGCAGCGUUGAUGU
|
22
|
12
|
|
|
|
bna-novel_29-3p
|
UCUUGUUACUGAGCUCGACG
|
20
|
308
|
chrA02:7209993..7210285
|
292
|
-99.9
|
bna-novel_29-5p
|
UUCAGCUGGGUACGAGCCACC
|
21
|
710
|
|
|
|
bna-novel_30-5p
|
AUCUGCAUCGAGUGAACUCUAUGG
|
24
|
426
|
chrCnn_random:65177977..65178226
|
249
|
-72.9
|
bna-novel_30-3p
|
AUGGAAUUCACUGAUGCAGAUGCU
|
24
|
7
|
|
|
|
bna-novel_31-5p
|
UUCUUGUGGUUGUAGAGUCUUG
|
22
|
367
|
chrA06:4069612..4069740
|
128
|
-56.1
|
bna-novel_31-3p
|
AGACUCUACAACAUCAGAAAC
|
21
|
47
|
|
|
|
bna-novel_32-5p
|
CGGAUUUUAGCUGCGUAGCUA
|
21
|
322
|
chrAnn_random:44030328..44030409
|
81
|
-42.5
|
bna-novel_32-3p
|
GGCUACGCUGCUGAAUCCGC
|
20
|
2
|
|
|
|
bna-novel_33-3p
|
UUGUAGAAUUUUGGGAAGGGC
|
21
|
289
|
chrC05_random:138762..138826
|
64
|
-32.6
|
bna-novel_33-5p
|
CCUUCCCAAAAUUCUACAAUU
|
21
|
39
|
|
|
|
bna-novel_34-5p
|
ACUUUGAAACUUUGAUCUAGA
|
21
|
5292
|
chrC06:5179422..5179524
|
102
|
-42.4
|
bna-novel_34-3p
|
UAGAUCAAAGCUUUAAUGU
|
19
|
20
|
|
|
|
bna-novel_35-3p
|
UUUUCGAUCUGUAAAUUU
|
18
|
4
|
chrA03:11978303..11978381
|
78
|
-30.5
|
bna-novel_35-5p
|
CAUUUACAGAUCGAAGACAUU
|
21
|
3
|
|
|
|
miR_name, miRNA name; Len, length of mature miRNA; pre-position, the position of miRNA precursor sequences in chromosomes of Brassica napus, pre-len, length of miRNA precursor sequences; MFE, minimum folding free energy.
Novel miRNA on the other arm of known pre-miRNA
Through sRNA high-throughput sequencing, miRNA-3p and miRNA-5p were found to always be simultaneously present on the 5′ arm and 3′ arm of pre-miRNA secondary structures. To identify novel miRNAs on the other arm of known pre-miRNAs, all mapped small RNA sequences were compared to the known precursor sequences of bna-miRNAs in the miRBase database 22.1. Finally, 27 novel miRNAs on the other arm of known B. napus pre-miRNAs were identified. The miRNA sequences and the number of reads in 24 libraries are listed in Table 3.
Table 3 Identification of novel miRNAs on the other arm of known pre-miRNAs in Brassica napus
bna-miRNA name
|
mature miRNA sequence
|
Length
|
Read count
|
bna-miR156f-3p
|
GCUCACUGCUCUUUCUGUCAGA
|
22
|
749
|
bna-miR156e-3p
|
UGCUCACCUCUCUUUCUGUCAGU
|
23
|
344
|
bna-miR160a-3p
|
GCGUAUGAGGAGCCAUGCAUA
|
21
|
32
|
bna-miR160c-3p
|
GCGUACAGAGUAGUCAAGCAUG
|
22
|
24
|
bna-miR160d-3p
|
GCGUACAGAGUAGUCAAGCAUG
|
22
|
24
|
bna-miR161-3p
|
GUCACUUUCAAUGCGUUGAUC
|
21
|
7
|
bna-miR164b/c/d-3p
|
CACGUGUUCUACUACUCCAAC
|
21
|
21
|
bna-miR166d-5p
|
GGACUGUUGUCUGGCUCGAGG
|
21
|
135
|
bna-miR166e-5p
|
GGAAUGUUGUCUGGCACGAGG
|
21
|
10
|
bna-miR166f-5p
|
GGAAUGUUGUCUGGAUCGAGG
|
21
|
202
|
bna-miR167a/b-3p
|
GAUCAUGUUCGCAGUUUCACC
|
21
|
750
|
bna-miR167a/b-3p
|
GAUCAUGUUCGCAGUUUCACC
|
21
|
750
|
bna-miR168a-3p
|
CCCGCCUUGUAUCAAGUGAAU
|
21
|
104
|
bna-miR171a/b/c-5p
|
AGAUAUUAGUGCGGUUCAAUC
|
21
|
51
|
bna-miR171d-5p
|
AGAUAUUGGUGCGGUUCAAUC
|
21
|
12
|
bna-miR172a-5p
|
GCAGCACCAUCAAGAUUCAC
|
20
|
48
|
bna-miR172b-5p
|
GCAGCAUCAUUAAGAUUCACA
|
21
|
3
|
bna-miR172c-5p
|
GCAGCAUCAUCAAGAUUCACA
|
21
|
9
|
bna-miR172d-5p
|
GCAUCAUCAUCAAGAUUCAGA
|
21
|
218
|
bna-miR2111d-3p
|
AUCCUCGGGAUACGGAUUACC
|
21
|
25
|
bna-miR390b-3p
|
CGCUGUCCAUCCUGAGUUUCA
|
21
|
1109
|
bna-miR390c-3p
|
CGCUAUCCAUCCUGAGUUCC
|
20
|
19
|
bna-miR395a/b/c-5p
|
GUUCCUCUGAGCACUUCAUUG
|
21
|
61
|
bna-miR395d/f-5p
|
GUUCCCUUUAACGCUUCAUUG
|
21
|
13
|
bna-miR399b-5p
|
GGGCAAGAUCUCUAUUGGCAGG
|
22
|
12
|
bna-miR403-5p
|
UGUUUUGUGCGUGAAUCUAAUU
|
22
|
287
|
bna-miR824-3p
|
CCUUCUCAUCGAUGGUCUAGA
|
21
|
1640
|
Identification of new conserved miRNA families and new miRNA members
To identify new conserved miRNAs in B. napus, all mapped small RNAs were mapped to known plant miRNAs in miRBase and B. napus genome sequences. If the small RNAs can match known plant miRNAs with no more than three mismatches and can exactly map to B. napus genome sequences, then these small RNAs were initially classified as candidate new conserved miRNAs. Five criteria described in the Materials and Methods were used to strictly screen the candidate conserved miRNAs. As a result, 44 miRNAs (22 pairs of miRNAs) belonging to 15 miRNA families were identified (Table 4). Among them, bna-miR159b was a new miRNA member of bna-miR159 family. The rest of the 36 miRNAs (14 pairs of miRNAs) have not been previously reported as bna-miRNAs in miRBase; they show high sequence similarity to some of the known plant miRNAs. The bna-miR158a.1 and bna-miR158a.2 were identified for bna-miR158a member. The two pairs of bna-miR158a shared the same mature sequences. Their precursor sequences were highly similar with each other, and these sequences were from different loci of the B. napus genome. These two pairs of miRNAs were called sub-members. This type of sub-member was also observed for bna-miR159b and bna-miR408a. Four sub-members (bna-miR159b.1, bna-miR159b.2, bna-miR159b.3, and bna-miR159b.4) were identified for bna-miR159b, and two sub-members (bna-miR408a.1 and bna-miR408a.2) were identified for bna-miR408a. This phenomenon suggests that some MIRNA genes might be produced through a replication event from one origin to another one, which results in more copies of the miRNA group. Two members were identified for bna-miR319 and bna-miR398 families. Except the above mentioned five miRNA families, the rest of 10 miRNA families had only one miRNA member (Table 4).
Table 4 Identification of new conserved miRNA families in Brassica napus
bna-miRNA
|
Sequence
|
Len
|
Read
|
pre-position
|
bna-miR158a.1-5p
|
CUUUGUCUAUCGUUUGGAAAAG
|
22
|
3884
|
chrA08:2748114..2748220
|
bna-miR158a.1-3p
|
UUUCCAAAUGUAGACAAAGCA
|
21
|
32292
|
|
bna-miR158a.2-5p
|
CUUUGUCUAUCGUUUGGAAAAG
|
22
|
3884
|
chrC08:3581242..3581348
|
bna-miR158a.2-3p
|
UUUCCAAAUGUAGACAAAGCA
|
21
|
32292
|
|
bna-miR159b.1-5p
|
AGCUGCUAAGCUAUGGAUCCC
|
21
|
258
|
chrA02:9865184..9865001
|
bna-miR159b.1-3p
|
UUUGGAUUGAAGGGAGCUCUA
|
21
|
46073
|
|
bna-miR159b.2-5p
|
AGCUGCUAAGCUAUGGAUCCC
|
21
|
258
|
chrA07_random:1944377..1944191
|
bna-miR159b.2-3p
|
UUUGGAUUGAAGGGAGCUCUA
|
21
|
46073
|
|
bna-miR159b.3-5p
|
AGCUGCUAAGCUAUGGAUCCC
|
21
|
258
|
chrC02:19215807..19215624
|
bna-miR159b.3-3p
|
UUUGGAUUGAAGGGAGCUCUA
|
21
|
46073
|
|
bna-miR159b.4-5p
|
AGCUGCUAAGCUAUGGAUCCC
|
21
|
258
|
chrC06:33954934..33954749
|
bna-miR159b.4-3p
|
UUUGGAUUGAAGGGAGCUCUA
|
21
|
46073
|
|
bna-miR319a-5p
|
AGAGCUUCCUUGAGUCCAUUC
|
21
|
27
|
chrC01:10651723.. 10651921
|
bna-miR319a-3p
|
UUGGACUGAAGGGAGCUCCCU
|
21
|
4848
|
|
bna-miR319b-5p
|
GGAGAUUCUUUCAGUCCAGUC
|
21
|
4
|
chrC04:46407584.. 46407846
|
bna-miR319b-3p
|
UUGGACUGAAGGGAGCUCCUU
|
21
|
27901
|
|
bna-miR391-5p
|
UUCGCAGGAGAGAUAGCGCCA
|
21
|
110
|
chrA10:10707678..10707812
|
bna-miR391-3p
|
ACGGUAUCUCUCCUACGUAGC
|
21
|
237
|
|
bna-miR398a-5p
|
GGGUCGACAUGAGAACACAUG
|
21
|
141
|
chrA03:2288822..2288945
|
bna-miR398a-3p
|
UGUGUUCUCAGGUCACCCCUG
|
21
|
9870
|
|
bna-miR398b-5p
|
GGAGUGUCAUGAGAACACGGA
|
21
|
25
|
chrC02:37793584..37793689
|
bna-miR398b-3p
|
UGUGUUCUCAGGUCACCCCUU
|
21
|
145
|
|
bna-miR400-5p
|
UAUGAGAGUAUUAUAAGUCAC
|
22
|
78
|
chrAnn_random:40582790..40582930
|
bna-miR400-3p
|
GACUUAUAAUGAUCUCAUGAA
|
22
|
237
|
|
bna-miR408a.1-5p
|
GGGAGCCAGGGAAGAGGCAGU
|
22
|
1232
|
chrA05:478954..479121
|
bna-miR408a.1-3p
|
UGCUUGUUCCCUGUCUCUCUC
|
22
|
1002
|
|
bna-miR408a.2-5p
|
GGGAGCCAGGGAAGAGGCAGU
|
22
|
1232
|
chrCnn_random:8448205..8448064
|
bna-miR408a.2-3p
|
UGCUUGUUCCCUGUCUCUCUC
|
22
|
1002
|
|
bna-miR9554-5p
|
GAAUGAUACUUGGAUAUAAUC
|
21
|
5
|
chrA06:19718101..19718250
|
bna-miR9554-3p
|
UCAUAUCCAAGUAUCAUUCCU
|
21
|
81
|
|
bna-miR9558-5p
|
AGAGAUGUCUGGCUUGCAACA
|
21
|
3
|
chrC03_random:1702602..1702746
|
bna-miR9558-3p
|
UUGCAAGCCAGACAUUUCCUUU
|
22
|
8
|
|
bna-miR9559-5p
|
UUUGGAUUUUGGUCAUUGUUG
|
21
|
5
|
chrAnn_random:36404086.. 36404194
|
bna-miR9559-3p
|
ACAAUGAACGAAAUCCAAAUC
|
21
|
3
|
|
bna-miR9560a-5p
|
ACAGGUGGUGGAACAAAUAUGAGU
|
25
|
30
|
chrA06:19552830..19552965
|
bna-miR9560a-3p
|
UCAUAUUAGUUCUACCUCCUGCUG
|
25
|
2
|
|
bna-miR9562-5p
|
ACUAUGCAAUUGUGAACAAAC
|
21
|
4
|
chrA02_random:1408210..1408358
|
bna-miR9562-3p
|
UUAUUCACAACUGCAUAAUUC
|
21
|
3
|
|
bna-miR9563a-5p
|
ACCCGUCUCUUAACUUUUAAC
|
22
|
15
|
chrAnn_random:9932700..9932850
|
bna-miR9563a-3p
|
UAAAAGUUAAGAGACAAGUUA
|
22
|
17
|
|
bna-miR9568-5p
|
UGCGGAUAUCUUAGGAUGAGGU
|
22
|
13
|
chrA03:13274664..13274813
|
bna-miR9568-3p
|
UCAUCGUAAGAGAUCUGCAUU
|
21
|
2
|
|
bna-miR9569-5p
|
UGAGUUAUCAUUGGUCUUGUG
|
21
|
1198
|
chrAnn_random:21855323..21855514
|
bna-miR9569-3p
|
ACACAGGAACAAUACUAACUCAUU
|
24
|
3509
|
|
Len, length of mature miRNA; pre-position, the miRNA precursor sequences in chromosomes of Brassica napus
Expression profiling of differentially expressed miRNAs in sterile and fertile lines
The normalized expression levels of miRNAs were used for identifying differentially expressed miRNAs between the sterile line and the corresponding fertile line, such as “4001A” and “4001B”, “4006A” and “4006B”, “6251A” and “6251B”, and “6284A” and “6284B”. The known, identified conserved and novel miRNAs were followed to differential expression analysis criteria (qvalue < 0.01 and |log2 (fold change)| > 0.73). As a result, 6, 2, 6, and 14 differentially expressed miRNAs were obtained between the flower buds of “4001A” and “4001B”, “4006A” and “4006B”, “6251A” and “6251B”, and “6284A” and “6284B”, respectively (Fig. 2, Tables 5). To further explore the miRNAs involved in the two DGMS lines, a Venn diagram analysis was conducted. The results indicated that two differentially expressed miRNAs were shared between the DGMS lines “4001AB” and “4006AB” (Fig. 3A). In addition, four differentially expressed miRNAs were shared between the RGMS lines “6251AB” and “6284AB” (Fig. 3B). The novel miRNA “bna-novel_34-5p” was the only miRNA that was simultaneously differentially expressed in the DGMS and RGMS lines.
Table 5 Differentially expressed miRNAs in “4001AB”, “4006AB”, “6251AB” and “6284AB” libraries and their candidate targets by sRNA sequencing and transcriptome analysis in Brassica napus
Sample
|
miR_name
|
A
|
B
|
log2(FoldChange)
|
Candidate Targets
|
4001
|
bna-novel_4-3p
|
8.2
|
0.4
|
1.90
|
BnaC01g31810D,BnaA07g24350D,BnaC02g41520D,BnaA08g22150D,BnaA04g13020D,BnaA07g18960D,BnaA09g38810D,BnaC03g26470D,BnaC02g03200D,BnaA04g00990D,BnaA03g41600D,BnaA05g07140D,BnaA08g19190D
|
bna-miR390a/b/c
|
26.8
|
11.1
|
1.32
|
BnaC03g02760D,BnaC01g03570D,BnaA05g28390D,BnaC03g53360D,BnaC01g22410D
|
bna-novel_31-5p
|
10.7
|
55.2
|
-2.16
|
BnaC01g21190D,BnaA01g17940D,BnaC03g13660D,BnaA03g10950D,BnaA09g00170D,BnaA10g02710D,BnaC01g22250D,BnaA03g38770D,BnaA02g29970D,BnaA02g18470D
|
bna-novel_34-5p
|
0.6
|
624.0
|
-8.16
|
BnaA09g14240D,BnaA05g30840D,BnaA01g24360D,BnaA07g34690D,BnaA03g14400D,BnaA05g09000D,BnaC01g09790D,BnaA08g26410D
|
bna-novel_1-3p
|
0.1
|
8.1
|
-2.64
|
--
|
|
bna-miR158-3p
|
554.7
|
935.8
|
-0.74
|
BnaA05g08940D,BnaA04g29200D
|
4006
|
bna-novel_34-5p
|
0.2
|
125.1
|
-5.82
|
BnaA05g29360D,BnaA09g14240D,BnaA05g30840D,BnaA01g24360D,BnaA07g34690D,BnaC03g41430D,BnaA03g35600D
|
|
bna-novel_31-5p
|
4.7
|
53.3
|
-2.81
|
BnaC01g21190D,BnaC03g13660D,BnaA09g19450D,BnaA05g09930D
|
6251
|
bna-novel_28-3p
|
5.2
|
38.3
|
-2.48
|
BnaA09g36810D,BnaA10g16750D
|
|
bna-novel_73-5p
|
30.6
|
64.2
|
-1.19
|
BnaAnng19370D,BnaA06g16700D,BnaA05g05990D,BnaA10g17750D,BnaC03g50150D,BnaA10g19150D,BnaA03g58190D,BnaA05g28260D,BnaA07g15220D,BnaA01g18450D,BnaA09g50980D,BnaA10g21470D,BnaA09g13370D
|
|
bna-miR408-5p
|
23.8
|
60.6
|
-1.15
|
BnaA02g05230D,BnaA10g18650D,BnaA08g27620D,BnaAnng21260D,BnaA10g02870D,BnaA08g00080D
|
|
bna-novel_88-3p
|
214.1
|
511.8
|
-1.16
|
BnaA04g16310D,BnaA09g36970D,BnaA05g24870D,BnaC01g43200D
|
|
bna-miR398a-3p
|
0
|
5.1
|
-2.10
|
BnaA05g02320D,BnaA06g14440D,BnaA08g08840D
|
|
bna-novel_34-5p
|
23.2
|
57.5
|
-1.11
|
BnaA02g05230D,BnaA10g18650D,BnaC01g01790D,BnaA10g10960D,BnaAnng21260D,BnaA10g14890D,BnaA08g27620D,BnaA07g15260D,BnaAnng36200D,BnaA10g15730D,BnaA02g11840D,BnaA10g25360D,BnaA10g02870D,BnaA08g00080D,BnaA10g17820D,BnaA09g25870D
|
6284
|
bna-miR394a/b
|
195.2
|
87.5
|
1.04
|
BnaA05g16640D,BnaA01g24160D,BnaA05g11890D,BnaC02g17150D,BnaAnng09250D,BnaA08g10740D,BnaA07g36430D,BnaC02g29160D,BnaA06g08380D,BnaC01g07190D,BnaA05g00820D,BnaA05g13120D,BnaC01g39490D,BnaC01g16400D,BnaA02g34270D,BnaC02g43190D,BnaA01g33370D,BnaC01g39860D,BnaA08g27810D
|
|
bna-novel_36-3p
|
1594.6
|
900.7
|
0.82
|
BnaA03g08820D,BnaA01g00910D,BnaC01g40540D,BnaA04g27250D,BnaA02g08190D,BnaC02g11600D,BnaA10g30150D,BnaA02g24120D,BnaC02g31870D,BnaA04g05090D,BnaA02g02340D
|
|
bna-novel_67-3p
|
129.3
|
50.0
|
1.23
|
BnaA03g02150D,BnaC01g00150D,BnaA07g02160D,BnaC03g01120D,BnaA03g00810D,BnaC01g01370D,BnaA07g25630D,BnaA03g55940D,BnaA04g24770D,BnaC01g25070D,BnaA01g20170D,BnaA03g48520D,BnaA09g17190D,BnaA09g39530D,BnaAnng28310D,BnaA01g35930D,BnaAnng08400D,BnaC02g23160D,BnaC03g60340D,BnaA04g00950D,BnaA03g33460D,BnaA04g22260D,BnaA03g27270D,BnaA07g37210D,BnaA06g37910D,BnaC03g21760D,BnaA03g18230D,BnaA09g05960D,BnaA03g34840D,BnaA09g05960D
|
|
bna-novel_33-3p
|
30.2
|
8.4
|
1.76
|
BnaA02g26940D,BnaA09g44810D,BnaA06g12040D,BnaA06g14250D,BnaA02g23840D,BnaA10g26020D,BnaA03g12030D,BnaA09g06400D,BnaC01g11370D,BnaA10g03030D,BnaA03g05700D,BnaA10g26690D,BnaA05g01020D,BnaA02g00710D
|
|
bna-miR159a
|
2320.1
|
1295.7
|
0.82
|
BnaAnng27960D,BnaA04g18810D,BnaA03g15690D,BnaA07g18670D,BnaA07g12970D,BnaA03g22590D,BnaA07g25350D,BnaA06g20460D,BnaA01g18450D,BnaC01g19500D,BnaA01g16350D,BnaA09g27090D,BnaA06g13170D,BnaA09g42230D,BnaA09g30160D,BnaAnng05670D,BnaA06g18020D,BnaA08g20300D,BnaA02g05410D,BnaAnng21510D,BnaA09g08360D,BnaAnng14630D,BnaA09g02220D,BnaA04g25320D,BnaA09g10390D,BnaA02g30030D,BnaA09g44380D,BnaC01g19800D,BnaA01g35420D,BnaA08g27930D
|
|
bna-novel_9-3p
|
114.4
|
68.3
|
0.75
|
BnaA03g33680D,BnaA08g01260D,BnaA01g05980D,BnaAnng27960D,BnaA03g22590D,BnaA07g12970D,BnaA04g09220D,BnaA07g12970D,BnaA02g05410D,BnaA09g55500D,BnaAnng13060D,BnaA01g23170D,BnaA09g13960D,BnaA09g47880D,BnaA04g01370D,BnaA08g17490D,BnaC02g08160D,BnaA02g33550D,BnaC02g42310D,BnaA10g14420D,BnaA05g16460D,BnaA03g42760D
|
|
bna-miR398a-3p
|
436.2
|
972.6
|
-1.06
|
BnaA08g19040D,BnaC02g28060D,BnaC01g27860D,BnaA10g26450D,BnaC03g13330D,BnaA03g10640D,BnaC02g12070D,BnaC01g18280D,BnaA01g15390D,BnaC02g42090D,BnaAnng31090D,BnaC01g43200D,BnaA05g30940D,BnaA01g29240D,BnaC01g36670D
|
|
bna-novel_3-3p
|
3.1
|
23.4
|
-2.43
|
BnaA05g24640D,BnaA09g38650D,BnaA06g39700D,BnaA09g39360D,BnaA09g54170D,BnaA06g10130D,BnaA10g14600D,BnaC01g22370D,BnaA10g24950D,BnaA10g23300D,BnaA04g14120D,BnaA09g41640D,BnaC01g09540D
|
|
bna-novel_5-3p
|
1.5
|
13.5
|
-1.77
|
BnaA05g24640D,BnaA06g39700D,BnaC02g14840D,BnaA06g10130D,BnaA09g38650D,BnaC01g22370D,BnaA09g39360D,BnaA10g04390D,BnaA09g54170D,BnaA10g14600D,BnaA01g28170D,BnaA06g28940D,BnaA10g23460D,BnaA06g07690D,BnaA10g24950D,BnaA09g01710D,BnaA10g16750D,BnaA10g23300D,BnaC01g09540D
|
|
bna-novel_73-5p
|
44.5
|
80.8
|
-0.84
|
BnaAnng19370D,BnaC02g44840D,BnaA06g16700D,BnaA05g05990D,BnaC03g50150D,BnaA10g19150D,BnaA05g06360D,BnaA03g26400D,BnaA09g49880D,BnaA10g04230D,BnaA03g28580D,BnaA06g06040D,BnaA03g29130D,BnaA07g15220D,BnaA10g27030D,BnaA04g10410D,BnaAnng07200D,BnaC01g14980D,BnaA04g10200D,BnaA08g03230D,BnaA10g23200D,BnaA10g21470D,BnaA03g07290D,BnaA07g21850D,BnaC02g16740D,BnaA10g09600D,BnaA03g21270D,BnaA08g29090D,BnaA09g13370D
|
|
bna-novel_68-3p
|
0
|
7.1
|
-2.20
|
BnaA04g25570D,BnaA02g31820D,BnaC02g40450D,BnaAnng04800D,BnaA06g27210D,BnaA03g20670D,BnaC01g31630D,BnaA03g20660D,BnaA08g29130D,BnaA09g12560D,BnaA09g14580D,BnaA09g06270D,BnaA10g26450D,BnaA02g05300D,BnaA03g26340D,BnaA09g32850D
|
|
bna-miR408-5p
|
41.3
|
94.4
|
-1.13
|
BnaA03g22490D,BnaA10g18290D,BnaA07g26450D,BnaA06g37530D,BnaC02g29240D,BnaA05g29880D,BnaA10g21900D,BnaC01g16210D,BnaA02g09770D,BnaA10g23140D,BnaA02g34310D,BnaC02g43230D,BnaA01g08900D,BnaA06g10230D,BnaA10g15210D,BnaA05g14430D,BnaC02g46010D,BnaA03g04360D,BnaA04g08040D,BnaAnng32010D,BnaA03g30060D
|
|
bna-novel_1-3p
|
0.9
|
7.5
|
-1.72
|
BnaA02g30100D,BnaC02g38520D,BnaA10g21900D,BnaAnng29380D
|
|
bna-novel_34-5p
|
1.0
|
456.9
|
-6.87
|
BnaA02g05300D,BnaA05g27100D,BnaA07g28780D,BnaA06g28070D,BnaA09g14240D,BnaA05g30840D,BnaA08g29690D,BnaC03g48340D,BnaA04g22150D,BnaC01g31340D,BnaA01g24360D,BnaA03g23950D,BnaC03g53270D,BnaA06g01440D,BnaA02g14960D,BnaA09g03120D,BnaA09g31030D,BnaC02g03920D,BnaA07g34690D,BnaA03g35600D,BnaA04g02140D,BnaC02g14950D,BnaA03g10460D,BnaAnng21410D
|
A:fertile, B:sterile lines.
qRT-PCR was conducted to verify the expression profiles of these differentially expressed miRNAs in deep sequencing. Five differentially expressed miRNAs were chosen for qRT-PCR analysis. The results were consistent with those of deep sequencing. In qRT-PCR, miR158 was up-regulated in “4001B” flower buds compared with that in “4001A”. Novel34 was greatly up-regulated in “4006B” flower buds (730-fold) compared with that in “4006A”. miR159 and miR827 were both up-regulated in “6284A” flower buds compared with that in “6284B”. miR398 was up-regulated in “6284B” flower buds compared with that in “6284A” (Fig. 4).
Target prediction and identification of differentially expressed miRNAs in sterile and fertile lines
To make clear the potential regulatory roles of differentially expressed miRNAs, a plant small RNA target analysis server psRNATarget (http://plantgrn.noble.org/psRNATarget/) was performed to predict their target genes. A total of 707 transcripts were predicted to be targets of the 20 miRNAs (Additional Table S3). In addition, transcriptome sequencing was conducted using the same samples as sRNA sequencing (unpublished data). The differentially expressed and up-regulated mRNAs were predicted as the candidate targets for the differentially expressed and down-regulated miRNAs. At the same time, the differentially expressed and down-regulated mRNAs were predicted as the candidate targets for the differentially expressed and up-regulated miRNAs. As shown in Table 5. Thirty eight candidate target genes were predicted for the six differentially expressed miRNAs between “4001A” and “4001B”. Eleven candidate genes were predicted for the two differentially expressed miRNAs between “4006A” and “4006B”. Forty-four candidate genes were predicted for the six differentially expressed miRNAs between “6251A” and “6251B”. Two hundred and sixty-seven candidate genes were predicted for the 14 differentially expressed miRNAs between “6284A” and “6284B”.
To further demonstrate the potential target genes, 5’ modified RACE was performed using mixed samples from flower buds of the fertile lines (“6284B” and “4001B”). Three target genes were validated using 5’ modified RACE (Fig. 5). Bn.A09.CSD1 (BnaA09g48720D) was cleaved by bna-miR398a-3p. Bn.A09.PPR (BnaA09g11120D) was cleaved by bna-miR158-3p. Bn.Cnn.MYB (BnaCnng51960D) was cleaved by bna-miR159a.
Overexpression of bna-miR159a affected seeds and siliques develpoment in Arabidopsis
Among all the differentially expressed miRNAs in the two DGMS and RGMS lines, bna-miR159a had the highest expression level. Thus, bna-miR159a was chosen to analyze its function. Two vectors, namely, p35S::MIR159a-C6 and p35S::MIR159a-A7, which contained the precursor sequences of MIR159a located in C6 and A7 chromosomes of B. napus, were transformed into Arabidopsis, respectively. The corresponding Arabidopsis transgenic plants were named as MIR159OE-1 and MIR159OE-2. The expression levels of bna-miR159 and its target genes were analyzed in wild-type, MIR159OE-1, and MIR159OE-2.
To reveal miR159 potential function in Arabidopsis, the expression level of miR159a was analyzed by qRT-PCR. In wild-type plants of Arabidopsis, the transcript of mature miR159a and its targets (AtMYB33 and AtMYB65) were detected in root, stem, rosette leaf, stem leaf, flower, and silique through qRT-PCR. The expression level of mature miR159a was the highest in silique (5875-fold), relatively lower in stem, stem leaf, and flower compared with that in the root. The expression levels of AtMYB33 and AtMYB65 were very low and almost undetectable in silique, whereas they were relatively high in stem, stem leaf, and flower compared with that in the root (Fig. 6). In MIR159OE-1 and MIR159OE-2, the transcripts of mature miR159a and its targets were detected in stem leaf through qRT-PCR. The mature miR159a was overexpressed in MIR159OE-1 (4.04-fold) and MIR159OE-2 (13.6-fold) compared with that in WT. Meanwhile, the transcripts of AtMYB33 and AtMYB65 were suppressed in MIR159OE-1 and MIR159OE-2, especially in MIR159OE-2, compared with that in WT (Fig. 7).
The morphological characters of MIR159OE-1, MIR159OE-2, and WT were observed along with their development processes, especially in the flowering and fruiting periods. No significant difference was observed between transgenic and WT plants in the vegetative growth phase. However, during the reproductive growth period, in the MIR159OE-1 and MIR159OE-2 transgenic plants, the seed setting rate decreased, and siliques became shorter compared with that in the WT (Fig. 8). The length of siliques from WT, MIR159OE-1, and MIR159OE-2 transgenic plants were measured. The results showed that the silique lengths of WT were approximately 13.4 mm, whereas the average silique lengths of MIR159OE-1 and MIR159OE-2 were 5.9 and 5.8 mm, respectively. The results indicated that the overexpression of MIR159 led to significantly shorter siliques and reduced seed setting rate (Fig. 8E).