Abstract
Background
Vaccinium uliginosum (Ericaceae) is an important wild berry having high
economic value. The white-fruited V. uliginosum variety found in the
wild lacks anthocyanin and bears silvery white fruits. Hence, it is a
good resource for investigating the mechanism of fruit color
development. This study aimed to verify the differences in the
expression levels of some structural genes and transcription factors
affecting the anthocyanin biosynthesis pathway by conducting
high-throughput transcriptome sequencing and real-time PCR analysis by
using the ripening fruits of V. uliginosum and the white-fruited
variety.
Results
We annotated 42,837 unigenes. Of the 325 differentially expressed
genes, 41 were up-regulated and 284 were down-regulated. Further, 11
structural genes of the flavonoid pathway were up-regulated, whereas
two were down-regulated. Of the seven genes encoding transcription
factors, five were up-regulated and two were down-regulated. The
structural genes VuCHS, VuF3’H, VuFHT, VuDFR, VuANS, VuANR, and VuUFGT
and the transcription factors VubHLH92, VuMYB6, VuMYBPA1, VuMYB11, and
VuMYB12 were significantly down-regulated. However, the expression of
only VuMYB6 and VuMYBPA1 rapidly increased during the last two stages
of V. uliginosum when the fruit was ripening, consistent with
anthocyanin accumulation.
Conclusions
VuMYB6 was annotated as MYB1 by the BLAST tool. Thus, the white fruit
color in the V. uliginosum variant can be attributed to the
down-regulation of transcription factors VuMYB1 and VuMYBPA1, which
leads to the down-regulation of structural genes associated with the
anthocyanin synthesis pathway.
Electronic supplementary material
The online version of this article (10.1186/s12864-018-5351-0) contains
supplementary material, which is available to authorized users.
Keywords: Vaccinium uliginosum, White-fruited variant, RNA-seq, qRT-PCR
Background
Vaccinium uliginosum is a perennial deciduous shrub of the Ericaceae
family. It is distributed in the northeast regions of China, including
Xiao Xing’an Mountains, Da Xing’an Mountains, Inner Mongolia, and
Changbai Mountain forest area, it also occurs in Sphagnum swamp meadow
at an elevation of more than 700 m, along with a large community of
other plant species such as Carex, Larix gmelinii, Betula ovalifolia,
and Ledum palustre. This species can be found at temperatures as low as
− 40 °C to 50 °C [[35]1]. The berries are fragrant, delicious, and
nutritious and can be consumed either raw or after processing [[36]2].
Although V. uliginosum is a very valuable species, it has not been
intensively investigated. V. uliginosum berries contain abundant amino
acids, trace elements, anthocyanins, procyanidins and other polyphenols
[[37]3]. They have various benifical health effects such as blood
vessels softening, disease prevention and health care.
A V. uliginosum variety with white berries has been reported, but no
further studies on this species were performed [[38]4, [39]5]. During
resource investigation, we found a white-fruited V. uliginosum variety
in Wangqing Country of Jilin Province in the Lanjia forest farm. They
were sporadically distributed in the wild community. The berries were
round or oblong, and the fruit weight was approximately 1–1.5 g, the
ripening berries were silvery white and translucent. Preliminary
investigation revealed that the berries lacked anthocyanins, and their
total phenolic and flavonoid content was lower [[40]6], whereas the
vitamin C and titratable acid contents were higher than those of the
wild-type V. uliginosum. During the survey, we also found a chimera
with blue and white berries on the same plant during the survey
(Fig. [41]1). This phenotypic variation might have been caused by
somatic mutation. This resource has a potential value in theoretical
research and application for elucidating the mechanism of the synthesis
and regulation of anthocyanidin. The white-fruited V. uliginosum
variety produces tastier and sweeter berries with low tannin content
than those of the wild type. This might reduce the processing and
marketing cost of the berries [[42]5].
Fig. 1.
Fig. 1
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The ripening white Vaccinium uliginosum variety and the chimera at the
experimental site. The site is in Wangqing County of Jilin Province
Lanjia forest farm
Anthocyanin is a flavonoid produced via the flavonoid biosynthesis
pathway during plant metabolism. It is an important water-soluble,
nature, edible pigment, which exists widely in the vacuoles of plant
epidermal cells and imparts orange and red to blue color to flowers,
fruits, stems, leaves, and toots [[44]7]. It is safe, non-toxic, and
abundant, with important nutritional and pharmacological effects, such
as antioxidant capacity, and ability to scavenge free radicals, prevent
cardiovascular diseases, tumors, mutation, and radiation, regulate the
activity of platelets, prevent platelet condensation, and induce
immunomodulatory activity. In addition, it helps in improving the cold
and drought resistance ability of plants [[45]8].
The anthocyanin biosynthetic pathway is most extensively studied
secondary metabolic pathway in plants studied most extensively,
particularly in Arabidopsis, Zea and Petunia. Anthocyanin biosynthesis
can be divided into two stages. First, phenylalanine transforms into
4-coumaryl: CoA referred to as the phenylpropanoid metabolic pathway.
Subsequently, 4-coumaryl: CoA transforms into all kinds of flavonoid
compounds, which is referred to as the anthocyanin biosynthetic
pathway. Anthocyanin biosynthesis involves various structural and
regulatory genes [[46]9] .
The genes involved in anthocyanin biosynthesis can be classified into
two major groups. The first group consists of structural genes that
directly encode the key enzymes in anthocyanin biosynthesis. The other
group is transcriptional factors. Transcriptional regulation is an
important aspect of the regulation of gene expression in the
anthocyanin biosynthetic pathway of plant. Its mechanism is very
complicated. At present, three main types of regulatory factors have
been identified: MYB, bHLH and WD40 transcription factors [[47]10].
Anthocyanin biosynthesis in most of the species is regulated by the
transcription factors that form a protein complex that binds to the
promoter of the structural genes. The MADS-box transcription factor
VmTDR4 in V. myrtillus was suggested to play an important role in the
synthesis of anthocyanins by the direct or indirect regulation of MYB
transcription factors [[48]11]. Moreover, the microRNA miR156 and its
target gene SPL3 decrease anthocyanin biosynthesis in Arabidopsis
thaliana [[49]12].
Fruit color is an important factor affecting the appearance and quality
of the fruit. The study of the mechanism and regulation of fruit
coloration is very important for elucidating somatic cell mutations in
the peel. Mutations affecting fruit coloration, have been investigated
in the model plants such as grape [[50]13, [51]14], V. myrtillus
[[52]15], Duchesnea indica [[53]16], and Syzygium malaccense [[54]17].
The variations in the regulatory or structural genes in the anthocyanin
biosynthesis pathway of these species are responsible for the
mutations.
However, few studies have investigated the variations in the molecular
mechanism of anthocyanin biosynthesis in V. uliginosum. This study
aimed to analyze the differences among wild V. uliginosum and fruit
color varieties at the molecular level by using transcriptome
sequencing analysis. In addition we conducted a bioinformatics analysis
and quantitative polymerase chain reaction (q-PCR) to identify the
genes related to anthocyanin biosynthesis pathway and glucose
metabolism. Our findings might reveal the main reason for the lack of
anthocyanins in the V. uliginosum mutant.
Results
Transcriptome sequencing and assembly
In order to investigate the mutation mechanism of the white-fruited
variant, we repeated the transcriptome sequencing analysis thrice. We
obtained 34.62Gb of Clean Data, which reached the 5.3Gb threshold for
each sample and Q30 base percentage was ≥88.73%. The Read Number was
more than 21,044,015, and the base number for each sample was not less
than 5,303,019,780. The V. uliginosum GC content was in the range of
46.69—47.17%, whereas that of the white-fruited variant was in the
range of 47.17—47.46% (Table [55]1).
Table 1.
Statistical evaluation of sequencing data
Samples Read Number Base Number GC Content Q30 (≥%)
A1 21,137,847 5,326,737,444 46.78% 88.73%
A2 23,116,662 5,825,398,824 46.69% 90.89%
A3 25,635,645 6,460,182,540 47.17% 90.61%
B1 24,328,728 6,130,839,456 47.36% 90.85%
B2 22,137,140 5,578,559,280 47.46% 90.61%
B3 21,044,015 5,303,091,780 47.17% 90.97%
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Note: A1, A2, A3 is the sample of Vaccinium uliginosum. B1, B2, B3 is
the sample of its white-fruited variant
The assembled sequences showed 225,777,919 transcripts items and 89,725
unigenes. The N50 value of the transcripts and unigenes were 1058.35
and 1094, respectively. This showed that the assembly had high
integrity (Table [57]2). The number of contigs with 200—300 nt was the
highest (27,838,434; 99.62%) and that of contigs with length of
> 2000 nt was the lowest (6441; 0.02%). In addition, the highest number
of transcripts had length in the range of 200—300 nt, and the lowest
number of transcripts had 1000—2000 nt (27,320; 12.81%). However, no
unigenes had length in the range of 200—300 nt and the highest number
of unigenes had length of 300—500 nt (42,605 accounting for 47.48% of
the total).
Table 2.
Assembly results
Length Range Contig Transcript Unigene
200–300 27,732,934(99.62%)* 68,779(32.24%) 0%
300–500 52,388(0.19%) 65,549(30.73%) 42,605(47.48%)
500–1000 32,032(0.12%) 51,682(24.23%) 26,647(29.70%)
1000–2000 14,639(0.05%) 27,320(12.81%) 13,722(15.29%)
2000+ 6441(0.02%) 213,330 6751(7.52%)
Total Number 27,838,434 225,777,919 89,725
Total Length 1,139,232,020 1516 73,630,818
N50 Length 43 1058.35 1094
Mean Length 40.92 820.63
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Analysis by sequence alignment revealed approximately 18,355,782 mapped
reads in V. uliginosum, accounting for 78.77% of the total clean reads.
The number of mapped reads of the white-fruited variant was
approximately 17,688,233 (78.58%). The mapped reads were not
significantly different between the two samples (Table [59]3).
Table 3.
Comparison of the results of sequencing and assembly
Number Clean Reads Mapped Reads Mapped Ratio
A1 21,137,847 16,607,615 78.57%
A2 23,116,662 18,181,365 78.65%
A3 25,635,645 20,278,365 79.10%
B1 24,328,728 19,199,505 78.92%
B2 22,137,140 17,420,598 78.69%
B3 21,044,015 16,444,595 78.14%
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Functional annotation of unigenes
A total of 89,725 unigenes were annotated from different databases,
with a total of 42,837 unigenes with functional annotations. Unigenes
with a length of 300—1000 nt and ≥ 1000 nt were 26,734 (62.41%) and
16,103 (37.59%), respectively. Analysis by sequence alignment revealed
that the maximum number of unigenes were annotated in NR (40,121,
accounting for 93.66% of all the annotations). Whereas, the least
number were annotated in COG (12,991, accounting for 30.33% of the
total). The remaining unigenes were annotated in GO, KEGG, KOG, Pfam,
and Swiss-prot databases (Table [61]4).
Table 4.
Unigene annotation in different databases
Anno_Database Annotated_Number 300 ≤ length ≤ 1000 Length ≥ 1000
COG_Annotation 12,991 6985 6006
GO_Annotation 24,469 15,061 9408
KEGG_Annotation 17,732 11,297 6435
KOG_Annotation 25,080 15,162 9918
Pfam_Annotation 29,299 15,609 13,690
Swissprot_Annotation 27,560 16,170 11,390
nr_Annotation 40,121 25,211 14,910
All_Annotated 42,837 26,734 16,103
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Screening of differentially expressed genes
For differential expression analysis, we corrected the significance
value of the original hypothesis of the accepted effective
Benjamini—Hochberg method. Finally, we adopted the corrected P value
named false discovery rate (FDR) as the key indicator of differential
gene expression, which can help to reduce the false positive rate by
considering the expression levels of numerous of genes in an
independent statistical hypothesis test.
A total of 325 differentially expressed genes (DEGs) were obtained,
among which 41 were up-regulated and 284 genes were down-regulated
(Fig. [63]2). A total of 118,804 genes showed no significant
differences in expression level.
Fig. 2.
Fig. 2
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Differential expression gene expression pattern clustering map. A1, A2,
A3: sample of V. uliginosum ripening fruits. B1, B2, B3: sample of
white-fruited variant ripening fruits
Of the 41 genes of up-regulated genes, 28 and 13 DEGs annotated and
unannotated, respectively. Unannotated genes might be the new
transcripts of V. uliginosum. The expression level of F3’5’H (c101538)
and DFR (c130571) genes of the flavonoid biosynthesis pathway was high
(6.1665 and 2.5403, respectively). The c74288 gene expressed in the
white-fruited variant, but not in V. uliginosum; this could be
attributed to the difference in aromatic compound metabolism
(Additional file [65]1: Table S1).
Of the 284 down-regulated genes, 148 and 136 DEGs were annotated and
unannotated, respectively. Moreover, 71 genes were expressed in V.
uliginosum, but not in the white-fruited variant. The c17593 gene
related to transcription factor B3, c114838 gene related to MADs-box
transcription factor, and c86331 gene related to transcription factor
MYB3 were down-regulated in the white-fruited variant, with expression
fold values of − 5.1825, − 4.7228, and − 6.6772, respectively. The gene
c89580 related to sugar metabolism was not expressed in the
white-fruited variant, but was expressed in V. uliginosum. In addition,
the c116690 gene was down-regulated in the white-fruited variant and
its expression fold value was − 2.9947 (Additional file [66]2: Table
[67]S2).
Functional annotation of DEGs
In all, 176 annotated DEGs were different between V. uliginosum and its
white-fruit variant among the different databases. In the NR database,
a maximum of 159 DEGs were annotated. In addition, a minimum of 39 DEGs
were annotated in the COG database (Table [68]5).
Table 5.
The number of annotated differential expression genes
DEG Set Annotated COG GO KEGG KOG Pfam Swiss-Prot nr
A vs B 176 39 82 50 94 145 114 159
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GO function enrichment of DEGs
GO function enrichment revealed 82 annotated DEGs (Fig. [70]3). They
participate in three major aspects including the biological process,
molecular function and cellular components. In all, 88 GO terms were
enriched. Of them, 47 were significantly enriched (P ≤ 0. 05). Moreover
GO: 0009773 was the most significantly associated (P = 0.0002) with the
light system I photoelectron transfer. The GO: 0009058 node was related
to the biosynthesis process, and two DEGs were associated with this
node one is sucrose synthase (c122231), the FPKM value of which was
four fold lower in V. uliginosum and the other is c134490 of the
glycosyl transferase group which was not expressed in the white-fruited
variant. However, its FPKM value was 10.7933 in V. uliginosum
(Additional file [71]3: Table S3).
Fig. 3.
[72]Fig. 3
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The secondary node of GO annotation statistical figure of
differentially expressed genes
Enrichment analysis of DEGs in the KEGG pathway
In living organisms, different gene products coordinate to perform
different biological functions. Pathway annotation analysis of DEGs is
helpful for further understanding the function of genes. KEGG database
is the main public database on pathways. We analyzed of whether the
appearance of DEGs in a pathway reflected pathway enrichment. KEGG
pathway enrichment analysis revealed 42 annotated DEGs and 15 pathways.
These pathways involve five aspects, including cellular processes,
genetic information processing, human diseases, metabolism, and
organismal systems.
Among them, the number of unigenes and DEGs involved in the
phenylpropanoid biosynthesis pathway was 240 and 5, respectively. The
flavonoid biosynthesis pathway had 80 unigenes, and three DEGs. In
addition, the numbers of unigenes and DEGs in starch and sucrose
metabolism pathways were 390 and two, respectively
(Additional file [74]4: Table S4).
Anthocyanin synthesis-related genes expression
The color of fruit peel is known to be correlated with the expression
of anthocyanin synthesis related genes. Our expression analysis results
showed that the expression of structural genes was mostly
down-regulated in the white-fruited variant. However the expression of
transcription factors in the anthocyanin biosynthesis pathway and genes
in the sugar metabolism pathway were mostly up-regulated (Fig. [75]4).
The structural genes peroxidase (VuPOD, c132702), cinnamoyl-CoA
reductase (VuCCR, c104371), and VuCHI (c126113) related to anthocyanin
biosynthesis were significantly up-regulated in the white-fruited
variety. In contrast, the genes VuCHS (c127976),
flavanone-3β-hydroxylase (VuFHT), flavonoid 3′-hydroxylase (VuF3’H,
c123712), VuDFR (c130571), VuANS (c122374), anthocyanidin reductase
(VuANR, c128846), and VuUFGT (c127140) were significantly
down-regulated. However, the expression of flavonoid3’, 5’hydroxylase
(VuF3’5’H, c132630) and leucoanthocyanins reductase (VuLAR, c124332)
was not significantly different between the white-fruited variant and
wild-type V. uliginosum. The transcription factors were in the most
up-regulated in the white-fruited variant. The genes VubHLH63 (c80718),
and VuTDR4 and transcription factors VubHLH130 (c113885), VuMADS-box
(c114838) and transcription factor B3 (c117593) were significantly
up-regulated in the white-fruited variant. However the expression of
VubHLH93 (c90489) was not significantly different from that of V.
uliginosum. In the white-fruited variant, the VubHLH92 (c112979) gene
was significantly down-regulated. Among the MYB transcription factors,
VuMYB2 (c119481), VuMYB4 (c47872), VuMYB7 (c111166), VuMYB8 (c86331),
and VuMYB10 (c113018) were significantly up-regulated. The
transcription factors VuMYB6 (c99078) and VuMYBPA1 (c115051) were
significantly down-regulated. Moreover, the VuMYB12 (c117353)
transcription factor was significantly down-regulated. In the
white-fruited variant sugar metabolism, beta-glucosidase (c112037) and
glycosyl transferases group 1 (c134490) were significantly
up-regulated, and the sugar (and other) transporter (c130693) was
significantly down-regulated. The sucrose synthase (c122231) expression
was not significantly different from that of V. uliginosum.
Fig. 4.
[76]Fig. 4
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Relative expression of the genes of V. uliginosum and its white-fruited
variant. Values represent mean ± SD of three replicates. *P ≤ 0. 05,
**P ≤ 0. 01, ***P ≤ 0. 001, and “n” is not statistically significant
difference (independent samples t-test) Note: CK: V. uliginosum
ripening fruits related genes expression. The other is related genes
are expressed in white-fruited variant ripening fruits
The expression of differentially expressed transcription factors at different
developmental stages in V. uliginosum
Four transcription factors, VubHLH92 (c112979), VuMYB6 (c99078),
VuMYBPA1 (c115051), and VuMYB12 (c117353), were expressed in the
white-fruited variant, but significantly down-regulated in V.
uliginosum.
Analysis of the expression of these four transcription factors at
different developmental stages of V. uliginosum showed that the
expression of VubHLH92 was first decreased and then increased
(Fig. [78]5). When the fruit was about to ripen, almost no VubHLH92
expression was noded. The expression of the VuMYB6 was almost the same
during the first four stages, and then the expression suddenly and
rapidly increased when the fruit was ripening, to approximately 400
times of that during the first period, consistent with the accumulation
patten of anthocyanin. Evidently, this transcription factor is related
to the synthesis of anthocyanin in V. uliginosum. The VuMYB12
transcription factor expression was first decreased and then increased,
followed by a decline. The expression began to increase when the fruit
was ripening, but its expression was lower than that during the first
and third periods. The expression of VuMYBPA1 transcription factor
first increased gradually and decreased in the fourth period, and
increased rapidly in the last two periods. The expression ratio of the
last period was approximately 16 times higher than that for the first
period, consistent with the accumulation of anthocyanin.
Fig. 5.
[79]Fig. 5
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The differential expression of transcription factors in V. uliginosum
at the six developmental stages. The column represent means ± SD from
three independent biological replicates. Statistical analysis was
performed using Duncan test at the level of P ≤ 0. 05
Discussion
As early as 1986, Zhang et al. [[81]5] identified a variant of V.
uliginosum in Heihe City, Heilongjiang Province. They found that the
sugar content of the white-fruited variety was higher than that of the
blue-fruited wild-type one. Moreover, Ma et al. [[82]4] reported that
the white-fruited variant was slightly sweeter than the original
variety from the Da Xing’an Mountains of China. These findings might
explain why most of the genes related to sugar metabolism were
up-regulated in the white-fruited variant. The expression of
beta-glucosidase (c112037) was significantly up-regulated, probably
because glucose was the principle sugar in the fruits of the
white-fruited variant. Glycosyltransferase is the key enzyme associated
with the catalytic synthesis of sugar chains; thus, the extremely
significant up-regulation of glycosyl transferases group 1 (c134490)
was reasonable. Moreover the sucrose is not primarily responsible for
the sweetness of the white-fruited variety; thus, sucrose synthase
(c122231) expression was not significantly different from that in V.
uliginosum. After sugar formation in ripe fruits, the expression of
sugar (and other) transporter (c130693) was significantly
down-regulated in the white-fruited variant.
According to our survey, the morphological characteristics of the
white-fruited variant did not differ from those of V. uliginosum,
except that the ripe fruit was silver-white and lacked anthocyanin.
This is not caused by the external environment, as the silver-white
coloration does not change even in winter [[83]4]. The formation of
fruit peel color is closely related to the synthesis of anthocyanins,
and its mechanism is complex. The expression of structural genes in the
anthocyanin biosynthetic pathway is generally required for upstream
transcription factor binding to initiate gene transcription and
expression, to finally synthesize anthocyanins and impart color to the
fruit and peel [[84]18].
The expression of structural genes VuF3’ 5’ H (c132630) and VuLAR
(c124332) of the anthocyanin biosynthesis pathway in the white-fruited
variant did not differ significantly from that in V. uliginosum. These
genes do not direct effects on anthocyanin synthesis. The expression of
VuCHS (c127976), VuFHT, VuF3’H (c123712), VuDFR (c130571), VuANS
(c122374), VuANR (c128846) and VuUFGT (c127140) genes was extremely
significantly down-regulated in the white-fruited variety their
expression is closely linked to anthocyanin synthesis. This is probably
because of the inhibition of transcription factor expression, which can
affect the accumulation of anthocyanin.
Transcription factor B3 is unique to plants; it is a super-family
existing mainly in gymnosperms, mosses, and algae and other plants. Luo
et al. found that this transcription factor B3 plays an important role
in the plant stress response and in plant growth and development
[[85]19]. Zhang et al. [[86]20] found that transcription factor B3
plays a key role in the development and maturation of the embryo in
Theobroma cacao. In Arabidopsis, the excessive expression of the
transcription factor B3 resulted in the loss of petals [[87]21]. At
present, no studies have shown a relationship between transcription
factor B3 and coloring. The expression of transcription factor B3 in
the white-fruited variant was extremely significantly up-regulated;
therefore, it might play a role in other aspects of development.
The MADS-box and TDR4 genes encode transcription factors, and the TDR4
gene is a member of the MADS gene family [[88]11]. Previous studies
have showed that MADS and TDR4 genes were involved in plant anthocyanin
synthesis. Another study showed that IbMADS10 was involved in the
synthesis of anthocyanin in Ipomoea batatas [[89]22]. In the V.
myrtillus mutant, down-regulated expression of the VmTDR4 gene directly
or indirectly inhibited the expression of the VmMYB2 gene, resulting in
the down-regulation of the structural genes of the anthocyanin
biosynthesis pathway, inhibiting the synthesis of anthocyanin [[90]11].
The MADS super family has many members, and each member has its own
function. We verified that the expression of the VuMADS-box (c114838)
gene was extremely significantly up-regulated and that of the VuTDR4
gene was significantly up-regulated in the white-fruited variety.
Therefore, they might have no relation with anthocyanin synthesis.
At present, studies on anthocyanin synthesis in plants suggest that the
known transcription factors can be mainly divided into three
categories, namely the R2R3-MYB type, bHLH and WD40 complex protein
transcription factors [[91]10]. Some of these are single gene
regulatory structural genes, some are two genes whose interaction
controls anthocyanin synthesis, and some are MBW (MYB bHLH WD40)
complexes regulating anthocyanin accumulation. Park et al. [[92]23]
revealed that the bHLH2 gene contained a DNA transposon resulting in a
change in the color of the petals of Pharbitis purpurea flowers. A
single gene mutation can also affect anthocyanin accumulation. Qian et
al. [[93]24] found that PyMYB10 regulated the synthesis and expression
of anthocyanin in Pyrus pyrifolia. Azuma et al. [[94]25] confirmed that
ViMYBA1–3 is a key gene that influences anthocyanin biosynthesis in
grape peel. Aharoni et al. [[95]26] repored that yellow strawberry
FaMYB1 inhibited the accumulation of anthocyanin and flavonoids in
transgenic tobacco. Primetta et al. [[96]27] showed that the homologous
MYBPA1 and MYB2 are the members of the R2R3MYB family, and their
down-regulation inhibited the expression of CHS, DFR, LAR, ANR, ANS and
UFGT structural genes, affecting the accumulation of anthocyanin.
Single-gene transcription factor can also directly regulate anthocyanin
synthesis. Wada et al. [[97]28] reported that Arabidopsis MYB (CPC) and
bHLH (GL3) interact with each other to influence bHLH anthocyanin
pigment synthesis in Lycopersicon esculentum. Schwinn et al. [[98]29]
reported that MYB and bHLH transcription factors increased the
intensity of the color of Petunia and Platycodon, and strongly enhanced
the phenotype of Petunia. Liu et al. [[99]30] reported that MrbHLH1 and
MrMYB1 affected anthocyanin synthesis in tobacco and Myrica rubra. Two
transcription factors can also interact to regulate the accumulation of
anthocyanin. Xie et al. [[100]31] showed that MdbHLH3 regulated MdMYB1
expression to mediate the low-temperature-induced anthocyanin
accumulation and coloration of apples. A transcription factor can also
be regulated by another transcription factor to affect the biosynthesis
of anthocyanin. Albert et al. [[101]32] confirmed that the MBW complex
transcription factor activated a single transcription factor and then
the R2R3-MYB type transcription factors TrMYB133 and RrMYB134 regulated
the biosynthesis of anthocyanin and procyanidin in Trifolium repens.
Thus, a single transcription factor can thus be regulated by a complex
of transcription factors to induce the synthesis of anthocyanin.
Primetta et al. [[102]27] used V. uliginosum and the white-fruited
variant from Finland as study material and indicated that the
expression of structural genes CHS, DFR, LAR, ANR, ANS and UFGT,
related to the anthocyanin synthesis pathway, was significantly
down-regulated, and the expression of CHI and F3’5’H genes did not
differ significantly from those in V. uliginosum. However, in our
study, the expression of VuCHS, VuFHT, VuF3’H, VuDFR, VuANS, and VuUFGT
was significantly down-regulated in the white-fruited variety, and the
expression of the VuF3’ 5’ H and VuLAR genes did not differ
significantly from that in V. uliginosum. Nevertheless, the expression
of VuCHI was significantly up-regulated. The results of our study and
those of Primetta et al. differ to some extent owing to the fact that
mutants are formed differently because of the difference geographical
regions.
In this study, high-throughput transcriptome sequencing was used to
identify DEGs, the expression of which was verified to differ
significantly between wild-type V. uliginosum and the white-fruited
variant using qRT-PCR. Moreover, the expression of four transcription
factors related to anthocyanin biosynthesis was significantly
down-regulated, namely VubHLH92 (c112979), VuMYB6 (c99078), VuMYBPA1
(c115051), and VuMYB12 (c117353). The expression of only VuMYB6 and
VuMYBPA1 increased rapidly in ripening fruit, among the six different
developmental stages of V. uliginosum, consistent with the accumulation
of anthocyanin, whereas the expression of other transcription factors
did not increase during the anthocyanin accumulation period, indicating
they had no direct relationship with anthocyanin synthesis. VuMYBB6 was
annotated to MYB1 by BLAST sequence alignment analysis, and MYB1 and
MYBPA1 were found to both be R2R3-MYB type transcription factors. The
significant down-regulation of the expression of VuMYB1 and VuMYBPA1
inhibited the expression of structural genes VuCHS, VuFHT, VuF3’H,
VuDFR, VuANS, and VuUFGT, affecting the anthocyanin accumulation in the
white-fruited variant. This study provided another possibility for the
mechanism of mutation in V. uliginosum. We intend to verify the
specific regulatory mechanism in the future.
Conclusions
According to the transcriptome analysis of DEGs related to anthocyanin
synthesis pahway in the ripening fruits of V. uliginosum and
white-fruited variant, the down-regulated expression of transcription
factors VubHLH92, VuMYB6, VuMYB12, and VuMYBPA1 might lead to the
down-regulation of the structural genes related to the anthocyanin
synthesis pathway.
The results of the expression of these four transcription factors in
different developmental stages ofV. uliginosum showed that only the
expression of VuMYB6 and VuMYBPA1 is related to the accumulation of
anthocyanins.
VuMYB6 was annotated as MYB1 by the BLAST tool. Therefore, the
down-regulation of the transcription factors VuMYB1 and VuMYBPA1 leads
to the down-regulation of the structural genes associated with the
anthocyanin synthesis pathway, which is the main reason for the lack of
anthocyanins in the white berries.
Materials and methods
Plant materials
The ripening fruits (60 days after full-bloom stage) of V. uliginosum
and its variant for RNA-seq were collected from Lanjia forest farm,
Wangqing Contry, Jilin Province, China, and stored at − 80 °C.
Different developmental stages of V. uliginosum materials for q-PCR
were also collected: blooming flowers (LS1), fruits with prominent
ovarian enlargement 15 days after full-bloom stage (LS2), 0.5—0.7 cm
fruits 30 days after full-bloom stage (LS3), 0.7—1.0 cm fruits 40 days
after full-bloom stage (LS4), fruits at the stage when color changes
50 days after full-bloom stage (LS5), and the ripening fruits 60 days
after full-bloom stage (LS6).
Methods
Extraction and detection of total RNA
The total RNA was isolated from the ripening fruits of V. uliginosum
and its variant by using the RNA rapid extraction kit (Hua Yueyang,
Beijing, China). Spectrophotometry method was used to detect the
purity, concentration and integrity of RNA samples to ensure that the
high-quality samples were used for RNA-seq.
RNA was also isolated from the materials collected at different
developmental stages of V. uliginosum by using the RNA rapid extraction
kit. The first chain of cDNA was synthesized using a reverse
transcription Kit (Takara, Dalian, China) for q-PCR.
cDNA library, deep transcriptome sequencing and assembly
Enrichment of V. uliginosum and its variant mRNA with magnetic beads
with Oligo (dT), randomly interrupt mRNA by adding Fragmentation
Buffer. With the mRNA from the fruits V. uliginosum and its variant as
template, the first cDNA strand was synthesized using random hexamers,
then a second cDNA strand was synthesized by adding buffer, dNTPs,
RNase H and DNA polymerase I, and cDNA was purified using AMPure XP
beads. The end was trimmed and a poly (A) tail was added at the
sequencing joint. The AMPure XP beads were used to select the fragment
size, and the cDNA library was obtained using PCR enrichment.
After the library was constructed, the concentration and insert size of
the library were detected using Qubit2.0 and Agilent 2100 respectively,
and the effective concentration of the library was accurately
quantified by q-PCR method to ensure the library quality. Based on
Sequencing By Synthesis technology, deep transcriptome sequencing was
performed using Illumina HiSeq 2500 (Illumina, USA) and the read length
was PE125. The Raw Data were filtered by removing the connector
sequence and low-quality reads to achieve high-quality clean Data.
Sequence alignment was conducted between the clean data for each sample
and assembled transcript or unigene library. The obtained transcript
and unigene reads were called Mapped Reads and were used for subsequent
analysis. The two samples of V. uliginosum and its white-fruited
variant were sequenced three times in order to ensure the accuracy of
the experiment.
Functional annotation of unigenes
The BLAST tool [[103]33] was used to compare the unigene sequences with
those in NR [[104]34], Swiss-Prot [[105]35], GO [[106]36], COG
[[107]37], KOG [[108]38], and KEGG [[109]39] databases. The results of
unigene orthology in KEGG were obtained using KOBAS 2.0 [[110]40].
After the amino acid sequences of the unigenes were predicted, the
unigenes were annotated by comparing with HMMER [[111]41] by using the
Pfam [[112]42] database.
Expression calculation of unigenes
The reads of each sequenced sample were compared with the unigene
database by using Bowtie [[113]43]. Based on this comparison we
estimated the expression levels by using RSEM [[114]44]. We used the
value of FPKM to indicate the expression abundance of unigenes.
FPKM can eliminate the effect of gene length and sequencing on the
calculation of gene expression. The calculated gene expression can be
directly used to compare the differences in gene expression between
different samples. The formula is as follows:
[MATH:
FPKM=cDNA Fragment<
mtext>Mapped FragmentMollionsxTranscript Lengthkb :MATH]
Differential gene expression and anthocyanin synthesis-related gene analysis
The FDR of less than 0.01 and the difference in expression level (fold
change; FC) of ≥2 were used as a screening criterion. FC represents the
ratio of the expression levels for two samples (groups). Therefore,
FC > 2 indicates differences in the expression level of the two
samples.
The DEGs and anthocyanin synthesis pathway and glucose
metabolism-related genes expression in the ripening fruits of V.
uliginosum and its white-fruited variant were analyzed using
quantitative real-time polymerase chain reaction (qRT-PCR). Specific
primers were designed and synthesized according to the gene sequence
obtained using high throughput sequencing. Futher, specific primers of
anthocyanin biosynthesis-related genes designed by Michael Zifkin
[[115]45] for Vaccinium corymbosum were used to verify the expression
levels of the genes by using q-PCR (Additional file [116]5: Table S5).
The VuGAPDH gene [[117]27] (GenBank Accession No. [118]KP218509) was as
reference gene. The SYBR FAST qPCR Kit Master Mix (2×) and the
universal dye method (KAPA Biosystems, USA) were used to validate the
expression of the genes by using ABI7900HT real time quantitative PCR
instrument (ABI Company, USA). The PCR condition was as follows:
pre-incμbation at 95 °C for 5 min; amplification at 95 °C for 3 s,
60°C for 20 s and 95 °C for 15 s. The melting cμrves were measured at
60 °C for 15 s, 95 °C for 15 s. The genes expression levels of the
ripening fruits of the white-fruited variant were compared with those
of the blue ripening fruits of V. uliginosum. Data analysis was
performed using the 2 ^-△△CT method. Statistical analysis was performed
using t-test at the level of P ≤ 0. 05.
Differential expression of transcription factors at the different
developmental stages of V. uliginosum
The expression of significantly down-regulated transcription factors at
the different developmental stages of the white-fruited variety of V.
uliginosum was detected by performing q-PCR by using the 2 ^-△△CT
method. The PCR condition was the same as mentioned abrove. Moreover,
the gene expression level and anthocyanin accumulation in the samples
were assessed using VuGAPDH as the reference gene. The expression of
transcription factors at the different developmental stages of V.
uliginosum was detected and compared with that at the full-bloom stage
(LS1). Statistical analysis is performed using Duncan test at the level
of P ≤ 0. 05. Each gene from each sample was analyzed three times to
ensure the accuracy of the experimental results.
Additional files
[119]Additional file 1:^ (48.5KB, xls)
Table S1. The up-regulated expression genes from the annotated DEGs
(XLS 48 kb)
[120]Additional file 2:^ (182.5KB, xls)
Table S2. The down-regulated expression genes from the annotated DEGs
(XLS 182 kb)
[121]Additional file 3:^ (37KB, xls)
Table S3. Differentially expressed genes identified using GO function
enrichment (XLS 37 kb)
[122]Additional file 4:^ (13.1KB, xlsx)
Table S4. Differentially expressed genes from KEGG enrichment (XLSX 13
kb)
[123]Additional file 5:^ (13.1KB, xlsx)
Table S5. Specific primers used for anthocyanin biosynthesis pathway
related genes (XLSX 13 kb)
Acknowledgements