Transition from vegetative growth
to reproductive growth is an extremely vital process in life cycle of higher
plants, and this is realized by flowering. The correct structure of a floral
organ determines the success of fruit formation and affects seed yield greatly
in crop plants.
The rapeseed (Brassica napus L.) is an important oil crop globally. Besides
edible oil, it also provides feed rich in protein for animals and raw materials
for biodiesel production. It is an allotetraploid
(AACC, 2n=38) derived from the natural corssing
between progenitors B. rapa (AA, 2n=20) and B. oleracea
(CC, 2n=18) (Cai et
al. 2012). The three most important yield-influencing factors in rapeseed
are seed weight, seed number per silique and silique number per plant (Xu et al. 2014). The seed weight, usually
indicated by 1000-seed weight (TSW), has been extensively studied and > 130
loci have been identified (Fu et al.
2015; Zhao et al. 2016; Sun et al. 2018). For instance, Geng et al.
(2016) crossed a large-seed line with a small-seed line and then used the
doubled haploid (DH) population to identify a hot region associated with the
TSW trait on Chromosome A09, followed by screening out four genes highly
related to seed weight based on annotations; Sun et al. (2018) detected 25 QTLs for TSW and 152 for seven seed-shape
traits by a recombinant inbred line (RIL). Additionally, the multi-locular Brassica juncea, presenting higher locule
number in siliuqe than normal, increased yield per
plant, which was considered due to the production of more seeds in a silique (Xiao et al.
2013, 2018). Tetra-locular trait in B. rapa
(subspecies trilocularis)
was also reported: Yadava et al. (2014) fine-mapped the locus and revealed it was due to a CLAVATA3 homologous gene. However,
reports about variation in silique number are
relatively rare.
We previously reported a multi-silique trait in
rapeseed (Chai et al. 2019). Unlike
the multi-locular trait mentioned above, our multi-silique rapeseed line “zws-ms” showed
three siliques on each carpopodium,
which was resulted from its aberrant floral structure: three pistils, including
two extra-outgrowth pistils, in one floral organ (Jiang et al. 1998; Chai et al.
2019). We located the underlying loci on chromosome A09 and C08, respectively;
and the differentially expressed genes (DEGs) between the two near-isogenic
lines (NILs) zws-217 (normal silique) and zws-ms at budding stage were preliminarily analyzed.
However, subsequent developmental stages were not investigated. Thus far, Zhu et al. (2019a) performed RNA-seq in a natural mutant of rapeseed exhibiting abnormal
differentiation of stems, finding that genes participating in the shoot apical
meristem (SAM) activity maintenance, cytokinin
biosynthesis, and signal transduction displayed greatly variation at
transcriptional level. Lee et al.
(2018) elucidated physiological and molecular mechanisms underlying seed
development of the tetralocular silique
in B. rapa
by using the dynamic transcriptome profiling, which
involved differentially expressed metabolic genes and changes in transcript
abundance of genes in different developing seeds (21, 28, 35, and 42 days after
flowering), as well as seed storage compounds. In addition, RNA-seq was also used for dynamic study in microRNAs (miRNAs) and their target genes grapevine (Vitis vinifera), by
investigating their variations systematically and spatiotemporally (Wang et al. 2014).
In order to further investigate the molecular mechanism underlying this
multi-silique trait in rapeseed, we dynamically
analyzed transcriptome data in multi-silique zws-ms and its
NILzws-217at both flowering stage and green podding
stage in this study. DEGs, including up-regulated genes and down-regulated
genes were identified. Particularly, DEGs sharing the same expression
tendencies at the two stages were analyzed. Annotations based on Gene Ontology
(GO) and the Kyoto Encyclopedia of Genes or Genomes (KEGG) were then further
analyzed. These results provide a deepened understanding for this multi-silique morphology in rapeseed.
The homozygous multi-silique rapeseed material “zws-ms”
and its near isogenic line (NIL) “zws-217” were developed at the Crop Research
Institute, Sichuan Academy of Agricultural Sciences. The only difference
between the two lines was the silique(s) morphology
(Fig. 1). They were grown in an experimental field in the Xindu
(altitude of 472 m, 30°47′10″N 104°12′12″E), located in the Sichuan Basin, with an annual
average temperature of 16.2°C in humid subtropical monsoon climate.
Three zws-ms
plants (samples T07, T08, and T09) and three zws-217 plants (T10, T11, and T12)
were selected respectively at flowering stage; while three individuals (T13,
T14, and T15) from zws-ms line and three individuals
(T16, T17, and T18) from zws-217 line were sampled, respectively, at green podding stage. The total RNA was isolated using an RNA
Isolation Kit (Tiangen, Beijing, China). The quality
control of RNA was then performed on NanoDrop 2000
(Thermo Fisher Scientific, Waltham, USA) by estimating concentration at
OD260/OD230. The sequencing libraries were generated using an RNA Library Prep
Kit for Illumina (New England Biolabs,
Ipswich, USA) according to the given instructions.
The RNA-seq was performed on the Illumina
HiSeq X-ten platform. The initially produced raw
reads were processed to generate clean reads by using in-house Perl scripts to
remove adapter sequences, reads containing poly-N, or low-quality reads.
Furthermore, the Q30, GC-content, and sequence duplicationlevels
of the clean reads were calculated. Clean reads were then aligned to the
reference genome of Brassica napus “Darmor-bzh” (www.genoscope.cns.fr/brassicanapus/data/) by using
Tophat2 tools, in order to screen out the reads with a perfect match or one
mismatch for consequent investigation.
DESeq R package (Wang et al. 2010) was used to detect DEGs. The P value was adjusted by controlling the false discovery rate (FDR)
described by Benjamini and Hochberg (1995). Genes
with an adjusted log2FC of 2 and a P value < 0.01 were
then identified as DEGs.
GO database was used to annotate
the genes. The GO enrichment of the DEGs was calculated by the GOseq R packages (Young et
al. 2010). The KEGG database (http://www.genome.jp/kegg/) was used to
explore the high-level functions and utilities of the biological system (Kanehisa et al.
2007). KOBAS software (Mao et al.
2005) was used to test the statistical enrichment of the DEGs in various KEGG
pathways. We also compared genes with their orthologs
from model plant Arabidopsis, based
on the TAIR database (www.arabidopsis.org/).
We performed the transcriptome
sequencing (RNA-seq) and the six samples at flowering
stage (T07, T08 and T09 from multi-silique line zws-ms and T10, T11 and T12 from NIL zws-217) totally
generated 62.44 Gb of raw data, with average Q30 value of 90.40% and GC content
of 47.61%; while the six samples at podding stage
(T13, T14 and T15 from zws-ms and T16, T17 and T18
from zws-217) produced 56.58 Gb raw data in total, with average Q30 value of 90.13% and GC content of 47.84% (Table 1).
For each sample generated
approximately 34.9 M and 31.6 M clean reads at average in T07~T12 group
(flower) and T13~T18 group (pod), respectively (Table 1). The proportion of
total reads mapped to the reference genome in T07~T12 group ranged from 72.91
to 75.09%; while it ranged from 71.30 to 74.57% (Table 2).
Table 1: Summary of the transcriptome sequencing data
Sample |
Number of clean reads |
Number of clean bases |
GC content (%) |
% ≥ Q30 |
T07 |
39,872,033 |
11,902,667,750 |
47.38 |
90.28 |
T08 |
29,835,132 |
8,894,586,194 |
47.66 |
91.44 |
T09 |
37,493,875 |
11,172,228,764 |
47.49 |
90.14 |
T10 |
37,185,677 |
11,091,447,160 |
47.37 |
90.28 |
T11 |
34,064,197 |
10,166,051,118 |
47.84 |
90.38 |
T12 |
30,883,341 |
9,214,076,026 |
47.91 |
89.86 |
T13 |
39,613,493 |
11,825,231,742 |
47.93 |
89.78 |
T14 |
26,988,155 |
8,030,791,800 |
47.80 |
90.05 |
T15 |
39,013,568 |
11,636,718,296 |
47.83 |
89.48 |
T16 |
29,122,944 |
8,688,230,392 |
47.41 |
91.36 |
T17 |
27,011,899 |
8,047,537,522 |
48.05 |
89.69 |
T18 |
28,023,293 |
8,348,109,052 |
48.01 |
90.43 |
Note: T07, T08, and T09: stamens and
pistils from three zws-ms individuals at the
flowering stage; T10, T11, and T12: stamens and pistils of three zws-217
individuals at the flowering stage; T13, T14 and T15: pods from three zws-ms individuals at the green podding
stage; T16, T17 and T18: pods of three zws-217 individuals at the green podding stage
Fig. 1: The multi-silique trait
in zws-ms compared with its near isogenic line,
normal zws-217
Fig. 2: The
number of differentially expressed genes (DEGs) in multi-silique
line zws-ms compared with zws-217, at flowering and
green podding stages
Genes with expression level fold
change > 4 (log2FC >2) and FDR <0.001 were identified as
DEGs. In the stamens and pistils at the flowering stage, 123 DEGs were
identified, among which 70 were up-regulated in zws-ms
and 53 were down-regulated (Fig. 2, Table S1). In the green pods, we found 268
DEGs totally. Compared with zws-217, 219 of the genes were up-regulated while
49 were down-regulated in zws-ms (Fig. 2, Table S2).
Further analysis found that 77 DEGs shared the same expression in the two
stages. Precisely, 46 of these genes were up-regulated in multi-silique zws-ms at both flowering
and podding stages, while 31 genes were
down-regulated at both stages. The expression level of these genes, including
12 new genes, represented stably line specific.
GO terms were generally divided
into three categories: biological processes (BP), cellular components (CC), and
molecular functions (MF). We analyzed the GO terms of all the DEGs between zws-ms and zws-217. At the flowering stage (Fig. 3a): the
BP terms with the highest levels of enrichment included “cellular process (GO:
0009987)” and “metabolic process (GO: 0008152)”, involved in 58 and 52 DEGs,
respectively; in the CC category, most enriched terms were “cell part (GO:
0044464)”, “cell (GO: 0005623)” and “organelle (GO: 0043226)”; in the MF
category, “catalytic activity (GO: 0003824)” and “binding (GO: 0005488)”
accounted the top enriched terms, involved in 46and 36 DEGs. While at the green
podding stage (Fig. 3b), which provided more DEGs:
the BP terms with the highest levels of enrichment included “cellular process
(GO: 0009987)”, “metabolic process (GO: 0008152)” and “single-organism process
(GO: 0044699)”, involved in 130, 137 and 126 DEGs, respectively; in the CC
category, the three most enriched terms were “cell part (GO: 0044464)”, “cell
(GO: 0005623)” and “organelle (GO: 0043226)”, the same as those at flowering
stage; the top two enriched terms were also the same to those in flowers, but
with larger amount in gene numbers, that was, 142 DEGs in “catalytic activity
(GO: 0003824)” term and 86 DEGs in “binding (GO: 0005488)” term. It could be
seen that at both flowering and podding stages, the
DEGs represented the highly similar top enriched GO terms.
Table 2: Mapped reads from the transcriptome sequencing
data
Sample |
Total reads |
Mapped reads |
Unique mapped reads |
Multiple mapped reads |
Reads mapped to '+' |
Reads mapped to '-' |
T07 |
79,744,066 |
58,732,384 |
52,322,869 |
6,409,515 |
27,498,178 |
27,527,387 |
T08 |
59,670,264 |
44,805,427 |
40,097,058 |
4,708,369 |
21,066,421 |
21,076,839 |
T09 |
74,987,750 |
54,766,420 |
48,293,165 |
6,473,255 |
25,431,146 |
25,461,399 |
T10 |
74,371,354 |
54,308,878 |
46,502,178 |
7,806,700 |
24,557,783 |
24,584,252 |
T11 |
68,128,394 |
50,765,850 |
42,596,102 |
8,169,748 |
22,570,858 |
22,584,713 |
T12 |
61,766,682 |
45,032,876 |
37,624,886 |
7,407,990 |
19,986,073 |
20,007,944 |
T13 |
79,226,986 |
57,391,532 |
47,797,521 |
9,594,011 |
25,507,679 |
25,527,520 |
T14 |
53,976,310 |
39,199,615 |
32,621,936 |
6,577,679 |
17,378,575 |
17,386,457 |
T15 |
78,027,136 |
55,632,827 |
48,605,556 |
7,027,271 |
25,698,426 |
25,704,707 |
T16 |
58,245,888 |
43,040,388 |
39,108,157 |
3,932,231 |
20,596,179 |
20,600,427 |
T17 |
54,023,798 |
39,689,203 |
33,286,563 |
6,402,640 |
17,732,478 |
17,738,347 |
T18 |
56,046,586 |
41,796,426 |
35,683,464 |
6,112,962 |
18,976,344 |
18,991,906 |
Note: T07, T08, and T09: stamens and
pistils from three zws-ms individuals at the
flowering stage; T10, T11, and T12: stamens and pistils of three zws-217
individuals at the flowering stage; T13, T14, and T15: pods from three zws-ms individuals at the green podding
stage; T16, T17, and T18: pods of three zws-217 individuals at the green podding stage
cKEGG pathway enrichment analysis revealed that at
flowering stage (Fig. 4a), the two enriched pathways with most genes were “phagosome (ko04145)” and “oxidative phosphorylation
(ko00190)”. “Phagosome (ko04145)” involved 6 DEGs:
BnaA09g55930D, BnaC08g29080D, BnaC08g35500D, BnaC08g35720D, BnaCnng75420D and
Cole_newGene_1984; “oxidative phosphorylation (ko00190)” enriched 5 DEGs:
BnaC08g29080D, BnaC08g35720D, BnaC08g36360D, BnaCnng75420D and
Cole_newGene_1984. But “vitamin B6 metabolism (ko00750)” got the highest value
of enrichment factor, at 12.8, involving only one gene: BnaC08g37550D. At green
podding stage (Fig. 4b), “starch and sucrose
metabolism (ko00500)”, “pentose and glucuronate interconversions (ko00040)” and “carbon metabolism
(ko01200)” had most genes enriched, six for each pathway respectively. The
“flavonoid biosynthesis (ko00941)”, involved 4 genes BnaA03g60670D,
BnaC04g18950D, BnaC07g37670D and BnaC09g17150D obtained the highest value of
enrichment factor, at 12.2.
Fig. 3: Gene ontology (GO) terms of the DEGs at flowering and podding
stages
a: GO terms at flowering stage; b:
GO terms at green podding stage
Overall, 60 of the stably line-specific 77 genes were annotated by GO or
KEGG (Table S3). When we aligned the above-mentioned 77 DEGs to model Arabidopsis, we found that 56 of these
genes had corresponding orthologs in Arabidopsis, 9 genes of them did not get corresponding genes
and 12 of them were new genes (Table 3). The chromosomes that harbored the most
stably line-specific DEGs were chromosome C08 and A09, where we had identified
the two associated regions according to our previous investigation (Chai et al. 2019). That was, 23 and 11 stably
line-specific DEGs on them (Table 3), respectively.
The present study performed RNA-seq at flowering and green podding
stage, dynamically analyzed the transcriptional level of genes in zws-ms, a rapeseed material with multi-silique
morphology. Similar trait (increased number of pistils) was reported in wheat (Triticum aestivum,
Yang et al. 2015; Wei 2017; Guo et al. 2019;
Zhu et al. 2019b), rye (Secale cereal, Malyshev
et al. 2001), alfalfa (Medicago sativa, Nair et al. 2008) and so on. However, there has been little report about
this trait in rapeseed. Multi-locular trait was
reported in Brassica plants B. juncea (Xu
Table 3: Ortholog information of the stably
line-specific DEGs
Gene in rapeseed |
Ortholog in Arabidopsis |
||
Gene ID |
Regulated in zws-ms |
Gene ID |
Description |
BnaA03g35870D |
up |
AT3G57550 |
guanylate kinase (AGK2) |
BnaA07g04500D |
up |
AT2G04900 |
unknown protein |
BnaA08g02930D |
up |
AT1G48920 |
nucleolin like 1 (NUC-L1) |
BnaA09g06740D |
up |
AT5G64180 |
unknown protein |
BnaA09g26320D |
up |
AT3G54620 |
basic leucine zipper 25
(BZIP25) |
BnaA09g44370D |
down |
AT1G19000 |
Homeodomain-like superfamily protein |
BnaA09g45300D |
down |
AT1G15000 |
serine carboxypeptidase-like
50 (scpl50) |
BnaA09g45310D |
up |
AT1G14990 |
unknown protein |
BnaA09g45320D |
down |
AT1G14980 |
chaperonin 10 (CPN10) |
BnaA09g45610D |
up |
AT1G14330 |
Galactose oxidase/kelch
repeat superfamily protein |
BnaA09g46080D |
down |
AT1G13780 |
F-box/RNI-like/FBD-like domains-containing protein |
BnaA09g46290D |
up |
AT5G03495 |
RNA-binding (RRM/RBD/RNP motifs) family protein |
BnaA09g48320D |
down |
AT1G09690 |
Translation protein SH3-like family protein |
BnaA09g49480D |
down |
AT1G07390 |
receptor like protein 1 (RLP1) |
BnaAnng30260D |
up |
AT3G54620 |
basic leucine zipper 25
(BZIP25) |
BnaC01g02500D |
up |
AT5G35640 |
Putative endonuclease or glycosyl
hydrolase |
BnaC01g43270D |
up |
AT5G57590 |
biotin auxotroph 1 (BIO1) |
BnaC02g06570D |
down |
AT5G16400 |
thioredoxin F2 (TRXF2) |
BnaC03g23820D |
up |
AT2G42670 |
Protein of unknown function (DUF1637) |
BnaC03g57080D |
up |
AT3G47630 |
CONTAINS InterPro DOMAIN/s:
Mitochondrial matrix Mmp37 |
BnaC04g29730D |
up |
AT1G76680 |
12-oxophytodienoate reductase
1 (OPR1) |
BnaC04g39120D |
down |
AT2G27450 |
nitrilase-like protein 1 (NLP1) |
BnaC05g26860D |
down |
AT1G14800 |
Nucleic acid-binding, OB-fold-like protein |
BnaC06g16950D |
up |
AT3G59000 |
F-box/RNI-like superfamily protein |
BnaC07g33980D |
up |
AT4G16900 |
Disease resistance protein (TIR-NBS-LRR class) family |
BnaC08g29060D |
down |
AT1G12820 |
auxin signaling F-box 3 (AFB3) |
BnaC08g35720D |
up |
AT2G21410 |
vacuolar proton ATPase A2 (VHA-A2) |
BnaC08g35850D |
up |
AT2G21300 |
ATP binding microtubule motor family protein |
BnaC08g36200D |
down |
AT2G20820 |
unknown protein |
BnaC08g37460D |
down |
AT1G17980 |
poly(A) polymerase 1 (PAPS1) |
BnaC08g38300D |
down |
AT1G79100 |
arginine/serine-rich protein-related |
BnaC08g39020D |
up |
AT1G15130 |
Endosomal targeting BRO1-like
domain-containing protein |
BnaC08g39120D |
up |
AT1G14990 |
unknown protein |
BnaC08g39130D |
up |
AT1G14980 |
chaperonin 10 (CPN10) |
BnaC08g39360D |
up |
AT1G14720 |
xyloglucan endotransglucosylase/hydrolase
28 (XTH28) |
BnaC08g39990D |
up |
AT1G14000 |
VH1-interacting kinase (VIK) |
BnaC08g40040D |
down |
AT1G13900 |
Purple acid phosphatases superfamily protein |
BnaC08g40320D |
up |
AT1G13450 |
Homeodomain-like superfamily protein |
BnaC08g40410D |
up |
AT5G19320 |
RAN GTPase activating protein
2 (RANGAP2) |
BnaC08g40810D |
up |
AT1G12970 |
plant intracellular ras
group-related LRR 3 (PIRL3) |
BnaC08g41390D |
down |
AT1G12240 |
ATBETAFRUCT4 |
BnaC08g41540D |
down |
AT1G12140 |
flavin-monooxygenase glucosinolate
S-oxygenase 5 (FMO GS-OX5) |
BnaC08g41720D |
down |
AT1G11910 |
aspartic proteinase A1 (APA1) |
BnaC08g42280D |
down |
AT1G10930 |
RECQ4A |
BnaC08g42450D |
down |
AT1G09970 |
LRR XI-23 |
BnaC08g49500D |
up |
AT1G18720 |
Protein of unknown function (DUF962) |
BnaC08g49610D |
down |
AT1G10760 |
STARCH EXCESS 1 (SEX1) |
BnaC08g49940D |
down |
AT1G10720 |
BSD domain-containing protein |
BnaC09g05960D |
up |
AT5G63550 |
DEK domain-containing chromatin associated protein |
BnaC09g06220D |
up |
AT5G64090 |
FUNCTIONS IN: molecular_function
unknown |
BnaC09g06260D |
up |
AT5G64180 |
unknown protein |
BnaCnng17490D |
up |
AT3G47680 |
DNA binding |
BnaCnng23190D |
down |
AT1G16880 |
uridylyltransferase-related |
BnaCnng24040D |
up |
AT1G53310 |
phosphoenolpyruvate carboxylase 1 (PPC1) |
BnaCnng68410D |
up |
AT3G57290 |
eukaryotic translation initiation factor 3E (EIF3E) |
BnaCnng75420D |
up |
AT2G21410 |
vacuolar proton ATPase A2 (VHA-A2) |
BnaA04g06410D |
up |
- |
- |
BnaAnng13790D |
up |
- |
- |
BnaC03g09190D |
down |
- |
- |
BnaC03g18930D |
up |
- |
- |
BnaC03g19830D |
up |
- |
- |
BnaC04g10370D |
down |
- |
- |
BnaC06g07110D |
up |
- |
- |
BnaC06g42000D |
up |
- |
- |
BnaC07g36960D |
up |
- |
- |
Cole_newGene_1983 |
down |
- |
- |
Cole_newGene_1984 |
down |
- |
- |
Cole_newGene_2071 |
down |
- |
- |
Cole_newGene_2073 |
up |
- |
- |
Cole_newGene_2243 |
down |
- |
- |
Cole_newGene_269 |
up |
- |
- |
Cole_newGene_3682 |
down |
- |
- |
Cole_newGene_4151 |
down |
- |
- |
Cole_newGene_4384 |
up |
- |
- |
Cole_newGene_4855 |
up |
- |
- |
Cole_newGene_6687 |
up |
- |
- |
Cole_newGene_8277 |
down |
- |
- |
et al.
2014; Xiao et al. 2018) and B. rapa
(Fan et al. 2014; Yadava et al.
2014; Lee et al. 2018). But that
trait increased the seed number per silique, rather
than silique number per plant, and tqas different from our zws-ms
line.
Fig. 4: Statistics of KEGG Pathway Enrichment
a: KEGG enrichment at flowering stage; b:
KEGG enrichment at green podding stage
This multi-silique trait was originally
discovered in hybridization progeny of B.
napus × B. rapa and in previous
researches (Jiang et al. 1998; Chai et al. 2019), we performed the
association analysis combined with bulked segregant
analysis (BSA) and detected two associated regions on chromosome A09 and C08,
respectively. After analyzing the transcriptional levels at budding stage and
annotations of the genes, we screened out some potential candidate genes. In
order to obtain a more comprehensive understanding of the variations in zws-ms, which might cause the difference to its NIL zws-27,
we further performed RNA-seq at flowering and green podding stages, dynamically displaying the transcriptional
level.
Transcriptome sequencing (RNA-seq) can rapidly detect all
transcripts from a specific tissue or in a particular state, and it provides a
general profile for transcriptional levels of all expressed genes, the survey
of alternative splicing events and even synteny
analysis between sub-genomes (Paritosh et al. 2014; Vitulo
et al. 2014; Xie
et al. 2015). In this study, RNA-seq generated approximately 9.9 Gb
for each sample (including both flowering and green podding
stage), this adequate data was then validated by the Q30 value and the
proportion of reads mapped to the reference genome.
Difference in expression level was indicated by log2FC and DEGs
were identified. At flowering stage, 70 DEGs were found up-regulated and 53
DEGs were down-regulated in multi-silique zws-ms; at the podding stage, the
number of total DEGs increased significantly, especially the up-regulated genes
in zws-ms. GO enrichment analysis showed that data
from the two stages were highly similar in the most enriched terms in each
category. Some genes were annotated to terms concerning flower/fruit structure
or development: BnaC08g29060D, showing no expression in zws-ms,
was annotated to “photoperiodism, flowering (GO:
0048573)”; BnaC08g39360D was annotated to “fruit development (GO:0010154)”; the term “ovule development (GO:0048481)”
involved three genes: BnaC08g41780D, BnaA09g45320D and BnaC08g39130D. Moreover,
these genes were all on chromosome A09 or C08, where we previously identified
two associated regions (Chai et al.
2019). As to the KEGG pathways, the enriched pathways at two stages were
different, but it also found that in the most enriched pathways, the genes
accounting highest proportion were also from chromosome A09 or C08. This
highlights the potential importance of these genes.
Arabidopsis and B. napus
have a common ancestor (Cai et al. 2012), and
Arabidopsis is a well-studied model
plants, of which most of the genes were investigated adequately. So orthologs from Arabidopsis
provide a reference for understanding the functions of their corresponding
rapeseed genes. Fifty six out of the 77 stably line-specific genes found orthologs in Arabidopsis.
After we further analyzed them and combined with our earlier report (Chai et al. 2019), we screened out following
genes: BnaC08g29060D, showing no expression in zws-ms,
was annotated to “photoperiodism, flowering (GO:
0048573)”. The ortholog in Arabidopsis is AT1G12820,
which is known as auxin signaling F-box 3 (AFB3). It is induced by nitrate in primary roots and the auxin receptor involved in primary and lateral root growth
inhibition (Dong et al. 2006). AT1G14720 encodes the xyloglucan
endotransglucosylase/hydrolase 28 (XTH28), a member
of Glycoside Hydrolase Family 16. In Arabidopsis
atxth28 mutant displayed abnormal floral structure: the lower elongation
ratio of the stamen at the pollination stage, the increased angle between the
stamen and pistil, and the aberrant orientation of anther dehiscence (Kurasawa et al. 2008). Thus, its ortholog
BnaC08g39360D in rapeseed, is supposed to cause some
unknown changes in flower potentially. The term “ovule development (GO:0048481)” involved three genes: BnaC08g41780D,
BnaA09g45320D and BnaC08g39130D. In fact, these three genes have been
highlighted in our earlier report (Chai et
al. 2019): AT1G11870, homologous to BnaC08g41780D, is identified as OVULE ABORTION 7 (OVA7), of which the disruption could result in ovule abortion in Arabidopsis (Berg et al. 2005);
thus the different expression of BnaC08g41780D between zws-ms
and zws-217 is also considered to result in some unknown process in fruit
development in rapeseed herein. In addition to “ovule development”,
BnaA09g45320D and BnaC08g39130D were also annotated to “response to cold (GO:0009409)”. It is notable that although they shared the same
ortholog (AT1G14980), the two genes represented
completely opposite expression model. That is, BnaA09g45320D was downregulated while BnaC08g39130D was unregulated in zws-ms in both flower and pod, which is consistent with our
earlier study in bud (Chai et al.
2019). Although how these hypothetic chaperonins, as
well as those genes mentioned above, causes abnormal flower development needs
further investigation, this study highlights their potential functions in this
multi-silique trait, especially BnaC08g39360D, BnaC08g41780D,
BnaA09g45320D and BnaC08g39130D.
We
performed the RNA-seq to analyze the DEGs between
multi-silique rapeseed zws-ms
and its NIL zws-217. 123 and 268 DEGs were identified at the flowering and
green podding stage, respectively. Further analysis
selected out 77 line-specific genes, which were expressed only in either zws-ms or zws-217. Combined with the information conferred
by GO annotations, KEGG pathways and ortholog
information from Arabidopsis, we
identified some potential candidate genes underlying the multi-silique trait, like BnaC08g39360D, BnaC08g41780D,
BnaA09g45320D and BnaC08g39130D. This deepens the understanding of molecular
mechanism for the multi-silique phenomenon in
rapeseed.
We thank the following funding for
supporting this study: the National Key Research and
Development Plan- International Cooperation Plan of Ministry of Science and
Technology (2018YFE0108200); the
International Cooperation Plan of SAAS (CGZH2019GH01); the Modern Agro-Industry
Technology Research System of China (CARS-12); Young Leading Talents Project of
Sichuan Academy of Agricultural Sciences (2019LJRC003 and 2019LJRC002).
Author Contributions
LC and JZ planned and carried out the experiments, performed
data analysis, and drafted the manuscript, HL, JJ, CC, and BZ revised the
manuscript and optimized the figures; PH and LZ assisted with DNA extraction
and data analysis; LJ designed and supervised the experiments.
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