Abstract
Through agronomic traits and sequencing data, the cultivated and wild
varieties of grapes and peaches were analyzed and compared in terms of
fruit size, fruit flavor, fruit resistance, and fruit color. Cultivated
grapes and peaches have advantages in fruit size, soluble sugar
content, sugar and acid ratio, etc. Wild grapes and peaches have
utility value in resistance. The results showed that there were 878 and
301 differentially expressed genes in cultivated and wild grapes and
peaches in the three growth stages, respectively based on the
next-generation sequencing study. Ten and twelve genes related to the
differences between cultivated and wild grapes and peaches were found
respectively. Among them, three genes, namely chalcone synthase (CHS),
glutathione S-transferase (GST) and malate dehydrogenase (MDH1) were
present in both cultivated and wild grapes and peaches.
Subject terms: Plant domestication, Transcriptomics
Introduction
Grapes (Vitis vinifera L.) are woody vines of the Vitaceae family
(Vitis L.)^[34]1. There are currently approximately 70 different grape
types grown throughout the world, with the majority found in temperate
or subtropical regions of East Asia and North America^[35]2. In 2021,
the world’s grape production exceeded 82 million tons (2021, FAOSTAT).
The peach tree (Prunus persica L.) is a perennial tree in the rose
family^[36]3. In 2020, China’s peach cultivation area and output both
ranked first in the world, with an output exceeding 77,600 tons.
Through the improvement of fruit tree varieties and the continuous
optimization of cultivation methods, from 2000 to 2021, the production
of grapes and peaches in China increased exceeding fourfold (2021,
FAOSTAT).
Artificial selection and natural selection have resulted in cultivated
and wild grapes and peaches having great differences in fruit traits
such as quality, peel color, and flavor. The present study was designed
to reveal the associated differences between cultivated and wild grape
and peach fruit traits, and to conduct transcriptome analysis to
identify differences in gene expression^[37]4. Such sequencing
technology can accurately quantify the expression level of specific
genes, and their alleles; this is helpful for in-depth analysis of
biological problems^[38]5.
Much research has been conducted on transcriptome sequencing of grapes
and peaches^[39]6–[40]11. For example, QTL analysis revealed candidate
genes (Ppa004358m、Ppa010376m、Ppa018828m and Ppb018041m) related to
fruit swelling, and discussed the significance of fruit swelling speed
and during fruit growth and development and the potential gene
regulatory network related to peach fruit. Ye^[41]12 selected the peel
of peach variety ‘Jinxiu’ at three time points (105, 120 and 135 days
after flowering) as samples, performed transcriptome sequencing and
annotated 25,694 unigenes, 31 genes were found significantly related to
anthocyanin content, including 8 enzyme genes
(CHS、CHI、F3H、DFR、UFGT、4CL、FLS and F3'H). This study providesd an
important reference for further understanding of peach skin color
formation mechanism. Most previous transcriptome studies have focused
on the differences under different treatment conditions and there are
few studies conducted on the differences between cultivated and wild
fruit trees. The regulatory mechanism of domestication research is
required because many great genes in wild germplasm resources can be
exploited for the domestication and cultivation of cultivated kinds.
In this study, cultivated grapes ‘Pinot Noir’ (Vitis vinifera L.) and
wild grapes ‘Changbai No. 9’ (Vitis amurensis Rur.), cultivated peaches
‘Okubo’ (Prunus persica L.) and wild peach ‘Lianyungang Maotao’
(Amygdalus persica L.) were selected as the experimental material to
study the difference in the fruit traits:- single fruit weight,
horizontal diameter, vertical diameter, soluble sugar content and
organic acid content. Gene expression of grapes and peaches from
typical cultivated and wild fruit trees, and their influence on fruit
quality during domestication were investigated. The differences in
fruit traits such as size, resistance and peel color were discussed.
These findings are of great significance for the utilization of
excellent characters in wild fruit tree resources and the optimized
breeding of cultivated fruit trees.
Results
Transcript sequencing data set
This study included 16 libraries of samples from grapes and peaches at
three growth stages (Table [42]1). The comparative results of grape
sample sequencing data are shown in Table [43]2. A total of 186,132,112
reads were generated, of which C_G_t2 had the largest number of reads
with 29,858,769 reads. The least is C_G_t1, with 15,933,645 reads.
After comparing the reads to the grape reference genome, the highest
similarity frequency is 77.60% (C_G_t1), the average frequency of four
samples of cultivated grapes reached 73.15%, and the average similarity
frequency of four samples of wild grapes was 59.5%. After assembly, a
total of 27,264 genes and 66,713 transcripts were obtained. The length
of contig N50 reached 2715 kb, and the average length of contigs
reached 1881 kb. The annotation results showed that 44,007 transcripts
were completely matched to the reference genome.
Table 1.
Sample abbreviation specifications.
Species Sample abbreviation Sample description
Grape C_G_t1 Fruit samples of cultivated grape ‘Pinot Noir’ in young
fruit stage
C_G_t2 Fruit samples of cultivated grape ‘Pinot Noir’ in swelling stage
C_G_t3 Fruit samples of cultivated grape ‘Pinot Noir’ in maturity
C_G_leaf Leaf samples of cultivated grape ‘Pinot Noir’ in maturity
W_G_t1 Fruit samples of wild grape ‘Changbai No. 9’ in young fruit
stage
W_G_t2 Fruit samples of wild grape ‘Changbai No. 9’ in swelling stage
W_G_t3 Fruit samples of wild grape ‘Changbai No. 9’ in maturity
W_G_leaf Leaf samples of wild grape ‘Changbai No. 9’ in maturity
Peach C_P_t1 Fruit samples of cultivated peach ‘Okubo’ in young fruit
stage
C_P_t2 Fruit samples of cultivated peach ‘Okubo’ in swelling stage
C_P_t3 Fruit samples of cultivated peach ‘Okubo’ in maturity
C_P_leaf Leaf samples of cultivated peach ‘Okubo’ in maturity
W_P_t1 Fruit samples of wild peach ‘Lianyungang Maotao’ in young fruit
stage
W_P_t2 Fruit samples of wild peach ‘Lianyungang Maotao’ in swelling
stage
W_P_t3 Fruit samples of wild peach ‘Lianyungang Maotao’ in maturity
W_P_leaf Leaf samples of wild peach ‘Lianyungang Maotao’ in maturity
[44]Open in a new tab
Table 2.
Comparison results of transcript sequencing data.
Species Sample abbreviation Total pairs Mapped reads Concordant pairs
Grape C_G_t1 15 933 645 82.80% 77.60%
C_G_t2 29 858 769 79.30% 73.80%
C_G_t3 22 781 342 76.60% 70.60%
C_G_leaf 25 566 026 75.60% 70.60%
W_G_t1 17 098 859 73.00% 63.40%
W_G_t2 26 403 616 68.90% 59.50%
W_G_t3 23 370 214 69.90% 60.60%
W_G_leaf 25 119 641 62.40% 54.70%
Peach C_P_t1 24 512 137 85.20% 81.00%
C_P_t2 17 326 896 88.20% 84.20%
C_P_t3 30 382 911 88.10% 84.40%
C_P_leaf 16 055 962 87.60% 83.60%
W_P_t1 15 770 694 86.50% 82.20%
W_P_t2 17 409 126 88.20% 84.20%
W_P_t3 29 320 743 87.10% 84.30%
W_P_leaf 26 376 610 85.80% 81.50%
[45]Open in a new tab
The comparative results of the sequencing data of the peach samples are
shown in Table [46]2. A total of 177,155,079 reads were generated, of
which C_P_t1 had the largest number of reads with 30,382,911 reads, and
the smallest was W_P_t1 with 15,770,694 reads. After comparing reads to
the peach reference genome, it was found that the highest similarity
frequency was 84.40% (C_P_t1), and the lowest was 81.00% (C_P_t3). The
average similarity frequency of the four samples of ‘Okubo’ reached
83.3%, ‘the average similarity frequency of the four samples of
‘Lianyungang Maotao’ is 83.1%. A total of 27,999 genes were obtained
after assembly. With 56,876 transcripts, the length of contig N50
reached 2475 kb, and the average length of contigs reached 1635 kb. The
annotation results showed that 29,542 transcripts were perfectly
matched to the reference genome.
Differences in transverse diameter, longitudinal diameter and single fruit
weight
In the grape fruits (Fig. [47]1, Table [48]3), the horizontal and
vertical diameters of cultivated grape ‘Pinot Noir’ have relatively
large changes during the three development periods. In the horizontal
diameter, the expansion stage of ‘Pinot Noir’ increased by 2.28 mm,
which was 1.25 times, compared with the young fruit stage; and the
mature stage increased by 0.24 mm, which was 1.02 times, compared with
the expansion stage. Also, it increased by 2.5 mm from the young fruit
stage to the mature stage, which was 1.28 times. In the longitudinal
diameter, the expansion stage of 'Pinot Noir' increased by 1.82 mm,
which was 1.18 times compared with the young fruit stage; and the
mature stage increased by 0.8 mm, which was 1.07 times, compared with
the expansion stage, and it increased by 2.6 mm, which was 1.26 times,
from the young fruit stage to the mature stage.
Figure 1.
[49]Figure 1
[50]Open in a new tab
Comparison of fruit diameter and fruit weight between cultivated and
wild grape and peach. Changes in the horizontal and vertical diameters
and individual fruit weight of cultivated and wild grapes and peaches
in the three periods. The top abscissa represents cultivated and wild
samples in three periods. The left axis is the horizontal and vertical
diameter (mm), and the right axis is the weight of a single fruit (g).
The grid column represents the changing trend of the fruit’s horizontal
diameter, and the diagonal column represents the fruit’s vertical
diameter. The broken line represents the change trend of fruit weight.
(A) refers to cultivated grape ‘Pinot Noir’, (B) refers to wild grape
‘Changbai No. 9’, (C) refers to cultivated peach ‘Okubo’, D refers to
wild peach ‘Lianyungang Maotao’.
Table 3.
Sugar and acid content of cultivated and wild grapes and peach fruits
in three periods.
Sample Soluble sugar (
[MATH: mg·g :MATH]
^−1) Organic acid (
[MATH: mg·g :MATH]
^−1)
Sorbitol Fructose Glucose Sucrose Quinic Citric Malic Oxalic Shikimic
Grape C_G_t1 – 1.98 1.80 – 10.64 – 10.02 0.04 0.03
C_G_t2 – 56.00 51.02 2.50 1.84 – 1.27 0.05 –
C_G_t3 – 50.65 45.79 3.11 3.67 0.11 1.34 0.08 –
W_G_t1 – 1.04 0.11 0.05 11.51 – 4.87 0.01 –
W_G_t2 – 12.06 9.61 1.01 4.81 0.16 5.54 0.01 0.01
W_G_t3 – 21.75 18.99 2.80 0.60 0.26 3.54 0.01 0.01
Peach C_P_t1 5.64 16.38 15.90 4.83 3.01 0.01 3.58 0.22 –
C_P_t2 6.27 7.38 7.01 20.30 0.77 0.35 1.24 0.10 0.02
C_P_t3 0.86 9.63 9.56 28.15 1.23 – 2.95 0.31 0.04
W_P_t1 5.21 3.54 24.03 6.98 6.49 – 8.70 0.73 0.26
W_P_t2 3.00 2.45 4.67 4.87 3.19 2.28 4.38 0.57 0.13
W_P_t3 0.92 0.95 3.78 9.45 0.30 – 0.56 0.13 0.01
[51]Open in a new tab
The horizontal and vertical diameters of the wild grape ‘Changbai No.
9’ had relatively small changes in the three periods. For the
horizontal diameter, the expansion stage and young fruit stage of
‘Changbai No. 9’ were basically the same, and the mature stage is
0.61 mm longer than the expansion stage, which was 1.02 times. From the
young fruit stage to the mature stage, the increase was 0.54 mm, which
was 1.06 times. For the longitudinal diameter, the swelling stage and
the young fruit stage of ‘Changbai No. 9’ are basically the same. The
mature stage is 0.85 mm, which was 1.09 times longer than the swelling
stage, and 0.68 mm, which was 1.07 times from the young fruit stage to
the mature stage.
The cultivated and wild grapes maintain the same trend in fruit weight
per fruit. During the three periods, the weight gradually increased
with the growth and development of the fruit. The change of fruit
weight of the cultivated grape ‘Pinot Noir’ in the three periods is
more significant than that of the wild grape ‘Changbai No. 9’
(Fig. [52]2). The single fruit weight of ‘Pinot Noir’ increased by
0.41 g at swelling period, which was 1.75 times compared with the young
fruit period; and the mature period increased by 0.05 g, which was 1.06
and 1.85 times when compared with the swelling period and the young
fruit stage respectively. The single fruit weight of the wild grape
‘Changbai No. 9’ had a small change compared with the cultivated grape
‘Pinot Noir’. The single fruit weight of ‘Changbai No. 9’ was basically
the same in the swelling stage and the young fruit stage, and the
ripening stage was increased by 0.1 g compared with the swelling stage,
which was 1.18 times. Also, the mature stage was 0.12 g more than the
young fruit stage, which was 1.22 times. The growth rate of the
cultivated grape ‘Pinot Noir’ increased significantly compared with the
wild grape ‘Changbai No. 9’, especially from the young fruit stage to
the expansion stage, while the growth rate of ‘Changbai No. 9’ was more
obvious from the expansion stage to the mature stage.
Figure 2.
[53]Figure 2
[54]Open in a new tab
Wild and cultivated fruits in different periods. Comparison of fruit
size changes between cultivated and wild grapes and peaches in three
periods T1 represents young fruit period, T2 represents swelling
period, and T3 represents maturity period.
The horizontal and vertical diameters of cultivated peach 'Okubo'
varied greatly in the three periods (Fig. [55]1, Table [56]3). For the
horizontal diameter, the expansion period of wild peach ‘Okubo’
increased by 33.15 mm compared with the young fruit stage, which was
2.28 times. Compared with the swelling stage, the mature period
increased by 18.18 mm, which was 1.31 times. Also, the total increase
from the young fruit stage to the mature stage was 51.3 mm, which was
2.98 times of the young fruit stage. For the longitudinal diameter, the
swelling stage of cultivated peach ‘Okubo’ increased by 24.46 mm, which
was 1.77 times, compared with the young fruit stage, while the mature
stage increased by 10.15 mm, which was 1.18 times, compared with the
swollen stage. Also, the total increase from the young fruit stage to
the mature stage increased by 34.61 mm, which was 2.09 times.
In terms of the single fruit weight of peach fruit, the weight of
cultivated peach ‘Okubo’ changed significantly in the three stages. The
expansion stage increased by 96.85 g, which was 9.79 times, compared
with the young fruit stage, and the mature stage increased by 126.97 g,
which was 2.18 times, compared with the expansion stage. Compared with
the young fruit stage, the mature stage increased by 223.82 g, which
was 21.32 times.
The horizontal and vertical diameter changes of the wild peach
‘Lianyungang Maotao’ in the three stages were smaller than that of the
‘Okubo’. For the horizontal diameter, the expansion period of the wild
peach ‘Lianyungang Maotao’ increased by 5.8 mm compared with the young
fruit stage, which was 2.28 times at the mature stage. Compared with
the swelling period, it increased by 17.21 mm, which was 1.31 times.
From the young fruit stage to the mature stage, total increase was
23.01 mm, which was 2.99 times. For the longitudinal diameter, the
swelling stage of the wild peach ‘Lianyungang Maotao’ increased by
4.24 mm, which was 1.77 times compared with the young fruit stage, and
the mature stage increased by 11.62 mm, which was 1.18 times compared
with the swollen stage, and the total increase from the young fruit
stage to the mature stage increased by 15.85 mm, which was 2.09 times.
The individual fruit weight of the wild peach ‘Lianyungang Peach’ had
relatively little change. The mature stage increased by 34.91 g, which
was 4.27 times compared with the expansion stage, and the total
increased by 40.6 g, which was 9.12 times compared with the young fruit
stage.
Combining the above results, it can be seen that the cultivated peach
‘Okubo’ had a higher growth rate than the wild peach ‘Lianyungang
Maotao’, and it is dominant in the transverse diameter, longitudinal
diameter and single fruit weight, and the skin color of fruit is more
red.
Difference in soluble sugar and organic acid content of fruit
Soluble sugars in grape and peach fruits include sorbitol, fructose,
glucose, and sucrose, and organic acids include quinic acid, citric
acid, malic acid, oxalic acid, and shikimic acid. As shown in Table
[57]3, the total sugar content of cultivated grape ‘Pinot Noir’
increased from 3.78 mg·g^−1 to 99.55 mg·g^−1, with an increase of
95.77 mg·g^−1 from the first stage to the third stage, while the sugar
content of ‘Changbai No.9’ increased from 1.2 mg·g^−1 to 43.54 mg·g^−1,
with an increase of 42.34 mg·g^−1. In terms of sugar content, the
increase of ‘Pinot Noir’ was 53.43 mg·g^−1 more than that of ‘Changbai
No.9’, which was 2.26 times.
The average sugar-acid ratio of ‘Pinot Noir’ was 4.27 times that of
‘Changbai No.9’, which was 18.13. The average sugar-acid ratio of
‘Changbai No.9’ was 4.25. At maturity stage, the sugar-to-acid ratio of
'Pinot Noir' was 1.86 times that of ‘Changbai No. 9’, reached 19.56,
and that of ‘Changbai No. 9’ was 10.49. It can be seen that the sugar
content of the cultivated grape ‘Pinot Noir’ in the three periods was
higher than that of the wild grape ‘Changbai No.9’. Also, the
sugar-acid ratio was higher, which means an advantage in fruit
sweetness. For the cultivated grape ‘Pinot Noir’, the content of
fructose and glucose changed significantly. From the young fruit stage
to the mature stage, the fructose content increased by 48.67 mg·g^−1
and the glucose content increased by 43.99 mg·g^−1. From the expansion
stage to the mature stage, the sucrose increased to 3.11 mg·g^−1.
Compared with the cultivated grape ‘Pinot Noir’, the wild grape
‘Changbai No.9’ had a small increase in soluble sugar content. From the
young fruit stage to the swelling stage, the fructose content increased
by 11.02 mg·g^−1, the glucose increased by 9.5 mg·g^−1, and the sucrose
only increased by 0.96 mg·g^−1. From the expansion stage to the mature
stage, the fructose content increased by 9.69 mg·g^−1, glucose
increased by 9.38 mg·g^−1, and sucrose increased by 2.8 mg·g^−1. The
content of quinic acid in the cultivated grape ‘Pinot Noir’ decreased
from 6.97 mg·g^−1 to 3.67 mg·g^−1 from the young fruit stage to the
mature stage, and the wild cultivar ‘Changbai No.9’ decreased by
10.91 mg·g^−1 to 0.6 mg·g^−1in the same period. The content of malic
acid in ‘Pinot Noir’ decreased from 8.68 mg·g^−1 from the young fruit
stage to the mature stage to 1.34 mg·g^−1. Also, in the same period,
the content of ‘Changbai No.9’ decreased from 1.33 mg·g^−1 to
3.54 mg·g^−1. The content of oxalic acid in ‘Pinot Noir’ ranged from
0.04 mg·g^−1 in the young fruit stage to 0.08 mg·g^−1 in the mature
stage, while the content of ‘Changbai No.9’ was maintained at
0.01 mg·g^−1. In terms of citric acid content, ‘Pinot Noir’ maintained
a low level, with a content of 0.11 mg·g^−1 in the mature stage, and a
citric acid content of 0.26 mg·g^−1 in ‘Changbai No.9’.
The soluble sugar content of cultivated peach 'Okubo' was higher than
that of wild peach ‘Lianyungang Maotao’ at every stage, and the organic
acid content was lower. The total soluble sugar content of cultivated
peach 'Okubo' increased from 42.75 mg·g^−1 to 48.2 mg·g^−1, while the
total soluble sugar content of wild peach ‘Lianyungang Maotao’
decreased from 39.76 mg·g^−1 to 15.1 mg·g^−1. In terms of total soluble
sugar content, cultivated peach ‘Okubo’ increased 30.11 mg·g^−1 more
than wild peach ‘Lianyungang Maotao’. The total organic acid content of
the cultivated peach ‘Okubo’ increased from 6.82 mg·g^−1 to
4.53 mg·g^−1, which increased 2.29 mg·g^−1, while the total organic
acid content of the wild peach ‘Lianyungang Maotao’ decreased from
16.18 mg·g^−1 to 1 mg·g^−1. In terms of total organic acid content, the
reduction of wild peach ‘Lianyungang Maotao’ is 12.89 mg·g^−1 more than
that of cultivated peach ‘Okubo’. The sucrose content of cultivated
peach ‘Okubo’ and wild peach ‘Lianyungang Maotao’ maintained an
increasing trend during the three periods. The sucrose content of
cultivated peach ‘Okubo’ ranged from 4.83 mg·g^−1 to 28.18 mg·g^−1,
accounting for 58% of the total soluble sugar content. The sucrose
content of wild peach ‘Lianyungang Maotao’ ranges from 6.98 mg·g^−1 to
9.45 mg·g^−1, accounting for 62% of the total soluble sugar content,
and the content of all other soluble sugars showed a downward trend.
The content of quinic acid and malic acid in the cultivated peach
‘Okubo’ and the wild peach ‘Lianyungang Maotao’ decreased significantly
in the three periods.
Analysis of gene differential expression in cultivated and wild grapes and
peach
The volcano diagram of the relative changes in expression level of
grapes in each period (Fig. [58]3A), which compares the transcripts of
‘Changbai No. 9’ to the transcripts of ‘Pinot Noir’. The results showed
that in the young fruit stage, the number of up-regulated transcripts
was 1,552 and the number of down-regulated transcripts was 737; in the
swelling stage, the number of up-regulated transcripts was 747, and the
number of down-regulated transcripts was 1284. In the mature stage, the
number of up-regulated transcripts was 733, and the number of
down-regulated transcripts was 1036. It can be seen that the
transcripts of 'Pinot Noir' were more abundant in the young fruit
stage, and more transcripts were up-regulated. In the expansion and
maturity stages, number of upward adjustments is more. A total of
55,956 transcripts were expressed in ‘Pinot Noir’, and a total of
53,446 transcripts were expressed in ‘Changbai No. 9’, with 49,232
co-expressed transcripts. After the differential expression screening
of the transcript expression level, it was found that the differential
transcripts in the young fruit stage, the swelling stage, and the
mature stage gradually decreased. There were 3505, 3077 and 2707
differential transcripts respectively (Fig. [59]4A), and a total of 878
common transcripts in the three periods. There were 6086 common
differential transcripts and a total of 6086 non-repetitive
differential transcripts.
Figure 3.
[60]Figure 3
[61]Open in a new tab
Volcano diagram of genes expression of grape and peach in three
periods. (A) Grape ‘Pinot Noir’ and ‘Changbai No. 9’. (B) ‘Okubo’ and
‘Lianyungang Maotao’ From left to right are the Volcano diagrams of the
gene expression of grape and peach in the young fruit stage, swelling
stage, and mature stage; the abscissa is log[2] (Fold Change), and the
ordinate is -log[10](P-value); up: significantly up-regulated gene,
down: significantly down-regulated gene, no_diff: no significant change
in gene.
Figure 4.
[62]Figure 4
[63]Open in a new tab
Venn diagram of differential expression of genes of ‘Pinot Noir’ and
‘Changbai No. 9’ in three periods of grapes (A) and peaches (B)
respectively.
A differential expression analysis of peach transcripts was performed
(Fig. [64]3B). Compared with 'Lianyungang Maotao', ‘Okubo’ contained
636、989 and 1,890 up-regulated transcripts at three stages
respectively, and 1082、915 and 2,716 down-regulated transcripts
respectively. In the young fruit stage and the mature stage, the
transcripts of ‘Okubo’ were more abundant, and there were more
transcripts up-regulated. In ‘Okubo’, a total of 46,172 transcripts
were expressed, and in ‘Lianyungang Maotao’, a total of 46,372
transcripts were expressed. According to the selection criteria of
P-value < 0.05 and |Fold Change|> 1, significantly differentially
expressed transcripts were screened (Fig. [65]4B). The results showed
that the number of differentially expressed transcripts in the young
fruit stage and the swelling stage were similar, 907 and 957
respectively. The number of differentially expressed transcripts in the
mature stage was larger, which was 3540. There were 301 common
differentially expressed transcripts in the young fruit stage, swelling
stage and mature stage.
STEM analysis of differentially expressed genes
Time series analysis of 878 and 301 co-differentially expressed genes
in grape and peach was performed by STEM software. As shown in
Fig. [66]5, the differentially expressed genes of 'Pinot Noir' are
divided into 16 modules, of which there are five modules with
significant differential expression, namely modules 0, 4, 11, 14, and
15, of which module 4 contains the most genes, reached 259, and module
15 is the least, with only 42. There are 3 significant modules in
‘Changbai No. 9’, namely module 3, 4 and 14, of which module4 has the
most genes (113). Module3 has 34 genes. The expression trends of the
two grape species in the module are same, and the difference is mainly
reflected in the expression level of the genes. In the two different
significant expression modules 0, 3, 11, and 15, the expression level
of 'Pinot Noir' is higher than that of ‘Changbai No. 9’. In the same
significant expression module14, it was first up-regulated and then
down-regulated, and the expression level of ‘Changbai No. 9’ in this
module was higher.
Figure 5.
[67]Figure 5
[68]Open in a new tab
Short time-series expression miner analysis of gene expression in three
periods of cultivar and wild grape and peach. Expression trends
analysis of 878 differential expression genes in cultivar and wild
grape species and 301 differentially expressed genes in cultivar and
wild peach species. Each box represents a transcript expression
pattern. The colored box indicates that there is a significant
distribution of transcripts, the number in the upper left corner is the
model number; the black line represents the total expression trend of
the box, and the red line represents the expression trend of each gene
in the box.
It can be seen from Fig. [69]5 that the genes of ‘Okubo’ are
significantly clustered in four modules (12, 13, 14, 15), and the genes
of ‘Lianyungang Maotao’ are significantly clustered in five modules
(11, 12, 13, 14, 15). Module14 and Module15 in ‘Okubo’ are the most
representative modules. The expected distribution of genes are 14.6 and
13.3, respectively, and the actual distribution of genes are 49.5 and
45. The most representative modules in ‘Lianyungang Maotao’ are
module11 and module14, the expected distribution of genes are 19.8 and
15.4, respectively, and the actual distribution are 33 and 56.5.
Similar expression patterns are maintained in the common modules (12,
13, 14, 15) of ‘Okubo’ and ‘Lianyungang Maotao’. The difference between
the two modules is mainly reflected in the amount of expression. In
module12, the average expression level of ‘Lianyungang Maotao’ is
higher, reaching 167.4, and that of ‘Okubo’ is 113.9. With module13,
the expression of ‘Okubo’ was more active, reaching 52.5, and the
expression of ‘Lianyungang Maotao’ was 33.8. With module14, the
expression levels of ‘Okubo’ and ‘Lianyungang Maotao’ were
significantly different, which was 68.9 and 37.9, respectively. With
module 15, the expression levels of ‘Okubo’ were lower than that of the
‘Lianyungang Maotao’, which was 26.6 and 42.8 respectively. The
significant expression module, No. 11 of ‘Lianyungang Maotao’ is not
significant in ‘Okubo’. Therefore, after analyzing the function of the
transcript in this module, it is found that its function is mainly
concentrated in the overall composition of the membrane, chloroplast,
and chloroplast thylakoid membrane, iron ion binding, chlorophyll
binding, flavonoid biosynthesis process, anti-stress response and
photosynthesis, etc.
Analysis of co-expression network of differential expressed genes
The 878 and 301 common differentially expressed genes of grapes and
peaches were analyzed by weighted gene co-expression network analysis
(WGCNA), and their subsequent co-expression modules were studied to
reveal their differences. According to the expression level of the
transcript, a cluster dendrogram was made for the grapes in the three
periods. The expression level of ‘Changbai No. 9’ in the young fruit
stage is quite different from that of other samples (Fig. [70]6A),
while the samples in the expansion stage and mature period appeared
clustered. Six modules are finally obtained with the 878 genes of
grapes, of which module Turquoise, Blue, Brown, Yellow and Green
contains 302, 241, 156, 104 and 70 genes respectively (Fig. [71]6C).
The Grey module includes genes not belonging to any modules and with no
function.
Figure 6.
[72]Figure 6
[73]Open in a new tab
Analysis of weighted gene co-expression network of common
differentially expressed genes of grape and peach. (A) Gene clustering
dendrogram for the three periods of ‘Pinot Noir’ and ‘Changbai No. 9’.
(B) Gene clustering dendrogram for the three periods of ‘Okubo’ and
‘Lianyungang Maotao’. (C) Module construction for the three periods of
‘Pinot Noir’ and ‘Changbai No. 9’. (D) TModule construction for the
three periods of ‘Okubo’ and ‘Lianyungang Maotao’.
The function of the genes in the Turquoise module is mainly related to
the overall composition of the membrane and the response to low
temperature stress. The genes in the Blue module are related to the
nucleus and plasma membrane. The genes in the Brown module are related
to the heat stress response and cytoplasm. The genes in the Yellow
module are related to the binding of metal ions and the transcript DNA
template, and the Green module is related to the overall composition of
the nucleus and membrane.
Analysis of the 301 common differentially expressed genes of ‘Okubo’
and ‘Lianyungang Maotao’ found that the expression level of
‘Lianyungang Maotao’ at the maturity stage (W_P_t3) was significantly
different from other samples (Fig. [74]6B). As shown in Fig. [75]6D,
four modules are finally formed, of which the Turquoise module contains
183 genes, the Blue module contains 56 genes, the Brown module contains
35 genes, and the Grey module with no function. After annotating the
genes function in each module, it is found that the genes in the
Turquoise module are mainly enriched in the cytoplasm, ATP binding and
stress resistance; the genes in the Blue module are mainly concentrated
in the nucleus, DNA binding and stress resistance; the genes in the
Brown module are mainly enriched in the overall composition of the
membrane, chlorophyll binding and photosynthesis.
Differentially expressed genes GO function enrichment analysis
878 and 301 co-expressed genes of grape and peach were analyzed by GO
enrichment. The 878 common differentially expressed genes of ‘Pinot
Noir’ and ‘Changbai No. 9’ have six annotations at the Cellular
component, 12 annotations at the Molecule function, and 16 annotations
at the Biological process (Table [76]4). In terms of cell components,
annotations are mainly focused on integral component of membrane,
nucleus, plasma membrane, mitochondrion, golgi membrane and
nucleoplasm. In terms of molecular functions, annotations mainly focus
on ATP binding, zinc ion binding, DNA binding, sequence-specific DNA
binding transcription factor activity, RNA binding, and protein
serine/threonine kinase activity. In the biological process,
annotations mainly focus on transcription, DNA-templated, regulation of
transcription, defense response, protein transport, embryo development
ending in seed dormancy and mRNA processing.
Table 4.
GO function enrichment analysis of common differentially expressed
genes of cultivated and wild grape species.
GO GO term Number P-value
Cellular component Integral component of membrane 119 2.19E − 04
Nucleus 96 3.44E − 09
Plasma membrane 79 2.32E − 02
Mitochondrion 27 5.91E − 03
Golgi membrane 9 2.77E − 02
Nucleoplasm 2 1.59E − 02
Molecular function ATP binding 58 3.11E − 26
Zinc ion binding 36 1.63E − 02
DNA binding 30 1.14E − 08
Sequence-specific DNA binding transcription factor activity 20
2.09E − 06
RNA binding 19 8.57E − 03
Protein serine/threonine kinase activity 11 9.39E − 15
Ligase activity 9 1.83E − 02
Nucleic acid binding 3 4.76E − 03
Phosphoprotein phosphatase activity 2 4.90E − 02
Calmodulin binding 2 4.38E − 03
Serine-type endopeptidase activity 1 2.13E − 02
ADP binding 1 3.36E − 23
Biological process Transcription, DNA-templated 38 2.00E − 08
Regulation of transcription, DNA-templated 25 1.41E − 06
Defense response 17 2.54E − 10
Protein transport 8 4.18E − 02
Embryo development ending in seed dormancy 6 2.04E − 02
mRNA processing 4 2.47E − 03
Plant-type hypersensitive response 3 2.26E − 04
Metabolic process 2 3.80E − 02
Flower development 2 4.05E − 03
DNA replication 1 1.08E − 02
DNA repair 1 8.21E − 05
DNA recombination 1 2.13E − 02
Intracellular protein transport 1 1.96E − 02
Mitotic nuclear division 1 1.81E − 02
Chromatin modification 1 1.87E − 02
[77]Open in a new tab
The GO function enrichment analysis of 301 common differentially
expressed genes of peaches found that there are 10 annotations at the
Cellular component, 8 annotations at the Molecule function, and 4
annotations at the Biological process (Table [78]5). In terms of cell
components, annotations are focused on integral component of membrane,
nucleus,、chloroplast, plasma membrane, cytosol, mitochondrion,
endoplasmic reticulum membrane, golgi apparatus, nucleolus and
chloroplast stroma. In terms of molecular functions, annotations are
concentrated on ATP binding, metal ion binding, DNA binding, protein
serine/threonine kinase activity, zinc ion binding, RNA binding,
sequence-specific DNA binding transcription factor activity and GTP
binding. In terms of biological processes, annotations focus on
regulation of transcription, DNA-templated, transcription, mRNA
processing and protein ubiquitination.
Table 5.
GO function enrichment analysis of 301 common differentially expressed
genes of cultivated and wild peach species.
GO GO term Number P-value
Cellular component Integral component of membrane 28 7.89E − 06
Nucleus 21 6.28E − 09
Chloroplast 14 4.63E − 02
Plasma membrane 10 7.02E − 07
Cytosol 7 1.14E − 03
Mitochondrion 5 1.09E − 03
Endoplasmic reticulum membrane 3 4.55E − 02
Golgi apparatus 3 1.80E − 02
Nucleolus 2 2.94E − 02
Chloroplast stroma 1 1.00E − 02
Molecular function ATP binding 26 1.64E − 05
Metal ion binding 11 1.05E − 03
DNA binding 9 6.90E − 05
Protein serine/threonine kinase activity 6 4.32E − 04
Zinc ion binding 5 1.86E − 04
RNA binding 4 4.88E − 03
Sequence-specific DNA binding transcription factor activity 2
6.51E − 06
GTP binding 1 4.67E − 02
Biological process Regulation of transcription, DNA-templated 8
2.99E − 03
Transcription, DNA-templated 7 7.85E − 07
mRNA processing 1 3.08E − 02
Protein ubiquitination 1 4.45E − 03
[79]Open in a new tab
The differentially expressed genes of cultivated and wild materials in
our study have similar items in GO functional enrichment analysis. Cell
component included integral component of membrane, nucleus, plasma
membrane, and mitochondrion; molecular functions included ATP binding
and zinc ion binding, DNA binding, sequence-specific DNA binding
transcription factor activity, RNA binding, and protein
serine/threonine kinase activity; biological processes included
regulation of transcription, DNA-templated, transcription, and mRNA
processing.
Differentially expressed genes KEGG pathway enrichment analysis
KEGG pathway enrichment analysis was performed on 878 and 301 common
differentially expressed genes of cultivated and wild materials to
explore the function of genes in the plant growth process from multiple
angles. The 878 common differentially expressed genes of ‘Pinot Noir’
and ‘Changbai No. 9’ are concentrated in 103 pathways. The functions
are mainly concentrated in metabolic pathways, biosynthesis of
secondary metabolites, protein processing in the endoplasmic reticulum,
carbon metabolism, glutathione metabolism, amino acid biosynthesis,
phenylpropane biosynthesis, peroxisomes, glycolysis/glycolysis raw and
pyruvate metabolism etc. According to the results of KEGG and GO
analysis, a total of ten genes related to grape resistance, fruit
color, peel color and fruit flavor were screened. Among them, there are
five genes related to grape fruit resistance: superoxide dismutase
(SOD2), glutathione S-transferase (GST), serine/threonine-protein
kinase (PBS1), basic endochitinase B (CHIB) and calreticulin (CALR);
fruit size is related to two genes: auxin-responsive protein (IAA) and
ABA responsive element binding factor (ABF); two fruit flavor-related
genes: malate dehydrogenase (MDH1) and pyruvate kinase (PK); one peel
color-related gene: chalcone synthase (CHS).
The 301 common genes of ‘Okubo’ and ‘Lianyungang Maotao’ clustered in
51 pathways. The functions of the differentially expressed genes are
mainly concentrated in metabolic pathways, flavonoid biosynthesis,
tyrosine metabolism, pyrimidine metabolism, alpha-linolenic acid
metabolism, photosynthesis, pyruvate metabolism, photosynthesis-antenna
proteins, pantothenate and CoA biosynthesis. These pathways may be
closely related to the differences in the traits of ‘Okubo’ and
‘Lianyungang Maotao’ in peel color, fruit weight, sugar and acid
content. Combining the results of KEGG pathway enrichment analysis and
GO function annotation results, it was found that 12 genes were related
to peach fruit resistance, peel color and fruit growth. Among them,
eight genes are related to fruit resistance: polyphenol oxidase,
peroxidase, glutathione S-transferase (GST), chitinase,
12-oxophytodienoic acid reductase (OPR), light-harvesting complex II
chlorophyll a/b binding protein 4 (LHCB4), 9-cis-epoxycarotenoid
dioxygenase (NCED) and alcohol dehydrogenase class-P (ADH1); there are
three genes related to fruit color: chalcone synthase (CHS),
phenylalanine ammonia-lyase (PAL) and bifunctional dihydroflavonol
4-reductase/flavanone 4-reductase (DFR), and one gene related to fruit
growth: SAUR family protein (SAUR). Considering KEGG annotations in
grapes and peaches: metabolic pathways and pyruvate metabolism are the
common pathways, which may explain the differences between cultivated
and wild fruit trees.
Discussion
‘Pinot Noir’ is a world-renowned grape wine variety, and ‘Changbai No.
9’ is widely planted in production as a wild grape strain with strong
cold resistance. The cultivated and wild species of grapes and peaches
have similar differences in fruit horizontal and vertical diameter and
single fruit weight: the horizontal diameter, vertical diameter and
single fruit weight of the cultivated species are greater than those of
the wild species. The horizontal diameter of ‘Pinot Noir’ in the mature
stage is 1.14 times that of ‘Changbai No. 9’, and the longitudinal
diameter is 1.24 times. The difference in single fruit weight is the
most significant, reaching 1.51 times. The horizontal diameter of
‘Okubo’ in the mature period is 1.78 times that of ‘Lianyungang
Maotao’; the longitudinal diameter is 1.65 times, and the difference in
single fruit weight is 5.15 times.
In this study, an auxin-responsive gene related to the difference in
agronomic traits of cultivated and wild fruits was identified in
peaches: AUR family protein (SAUR). Auxin plays a key regulatory role
in plant cell division, elongation and plant growth and
development^[80]13–[81]15. The SAUR gene family is a plant-specific
gene family and it can interact with calmodulin to affect the
relationship between the calmodulin secondary signaling system and the
auxin signaling pathway^[82]16. Previous studies have shown that during
plant growth and development, auxin-responsive genes have a close
regulatory effect on fruit development by regulating the balance of
auxin in the plant body. In this study, two genes related to fruit size
were found: auxin-responsive protein (IAA) and ABA responsive element
binding factor (ABF). AUX_IAA and ARF can recognize the auxin response
element (Aux REs), thereby regulating auxin-responsive genes^[83]17.
The GO annotation results of the differential transcripts of ‘Pinot
Noir’ and ‘Changbai No. 9’ contain ATP binding, zinc ion binding,
protein transport and other functions, which can promote the
transportation and absorption of nutrient elements in plants, thereby
affecting plant growth and fruit swelling and development.
Fruit flavor is affected by plant genetic factors, environmental
factors, and cultivation techniques^[84]18. ‘Pinot Noir’ and ‘Okubo’
are grown in artificial greenhouses, while ‘Changbai No. 9’ and
‘Lianyungang peach’ are grown in nature, therefore cultivated and wild
grapes and peaches have big differences in fruit flavors, which are
reflected in the content of soluble sugars and organic acids. In this
study, the sugar content of cultivated varieties was higher than that
of wild materials (Fig. [85]1); this was attributed to the differences
caused by different growth conditions between cultivated and wild
varieties. These differences were also reflected at the gene level,
which also proved that in the process of cultivation, domestication and
utilization of grapes, the direction of increasing the ratio of sugar
and acid has been developed.
The flavor quality of peach fruit is affected by the soluble sugar,
organic acid, sugar-acid ratio and volatile aromatic substances in the
fruit. The sweetness of the fruit is most affected by fructose,
followed by sucrose, glucose and sorbitol^[86]19. In mature peach
fruit, sucrose is the main component of its sugar component, accounting
for 40–85% of the total sugar content^[87]20, which is consistent with
the results of this study, ‘Okubo’ and ‘Lianyungang Maotao’ sucrose
content accounted for 58.4% and 62.6% of the total soluble sugars,
respectively. There was a big difference in sugar acid content and
sugar acid ratio between ‘Okubo’ and ‘Lianyungang Maotao’. Through the
identification of 878 common differentially expressed transcripts of
'Pinot Noir' and ‘Changbai No. 9’ in three periods, it was found that
two genes were related to fruit flavor: malate dehydrogenase (MDH1) and
pyruvate kinase (PK), and participated in glycolysis/gluconeogenesis
and MAPK signaling pathways. Studies have shown that pyruvate kinase
can determine the final product of glycolysis, so as to cultivate
plants that are more in line with needs^[88]21.
The color of grape peel has an important impact on its economic value.
At present, many studies have shown that the content and type of
anthocyanins play a decisive role in the color of grape peel.
Anthocyanins are glycoside derivatives of anthocyanins. The content of
anthocyanins in grape fruits is higher and contains many kinds of
anthocyanins^[89]22. Anthocyanidins exist in the vacuoles of plant
cells and are synthesized in the cytoplasm from flavonoids through the
shikimate pathway^[90]23,[91]24. Chalcone synthase is a key enzyme in
the synthesis of flavonoids, and its expression has been shown to be
closely related to the accumulation of anthocyanins^[92]25. After
studying the color changes of the fruits of ‘Pinot Noir’ and ‘Changbai
No. 9’, it was found that chalcone synthase (CHS), a gene related to
anthocyanins, was enriched in pathways such as flavonoid biosynthesis
and monoterpene biosynthesis. The fruit colors of ‘Pinot Noir’ and
‘Changbai No. 9’ in the young fruit stage are both green, and there is
a big difference in the swelling stage. The fruit of ‘Pinot Noir’
remains green, while the color of ‘Changbai No. 9’ is purple and black.
In terms of peel color, there is no obvious difference between the
young fruit stage and the swelling stage of the ‘Lianyungang Maotao’
fruit peel, but the peel color of the 'Okubo' is significantly redder
at the mature stage. There are 301 common differentially expressed
genes between ‘Okubo’ and ‘Lianyungang Maotao’. After analysis, three
genes related to peel color were found: chalcone synthase (CHS),
phenylalanine ammonia-lyase (PAL) and bifunctional dihydroflavonol
4-reductase/flavanone 4-reductase (DFR). Research shows that CHS in
peaches found that inhibiting the expression of CHS gene caused the
anthocyanin metabolic pathway to shift to the direction of chlorogenic
acid and complexes, proving that CHS gene is closely related to the
metabolism of flavonoids^[93]26. The expression levels of CHS
gene-related transcripts of ‘Okubo’ and ‘Lianyungang Maotao’ also
showed a significant difference of 5 times, and the expression levels
at the mature stage were 349 and 69.3. Research shows that the
anthocyanin biosynthesis of peaches of different colors and showed that
PAL gene may affect the anthocyanin metabolism pathway in peach
fruits^[94]27. The DFR gene plays a key role in the formation of
anthocyanins^[95]28. DFR can use three different substrates (DHK, DHQ
and DHM) to synthesize different colors^[96]29.
Grapes are susceptible to infection by pathogens such as anthracnose,
downy mildew, white rot and black pox. They also face abiotic stresses
such as drought and ultraviolet radiation, which greatly affect the
yield of grapes. Studies by Carvolho^[97]30 and Daldoul^[98]31 have
shown that wild grapes have important research significance in
biological/abiotic resistance. Salicylic acid accumulates in a stress
environment, and its signaling pathway is related to plant
resistance^[99]32. Under abiotic stress, salicylic acid still has an
effect on resistance^[100]33,[101]34. In the process of plants
resisting stress, antioxidant systems play a key role, such as:
superoxide dismutase (SOD), peroxidase (POD), catalase (CAT)^[102]35.
The KEGG enrichment pathway analysis of 878 common differential genes
found that the metabolic pathways related to biotic/abiotic stress are
mainly concentrated in: peroxisomes, glutathione metabolism,
phagosomes, interactions between plants and pathogens and other
pathways. Five genes related to resistance of grape berries were
screened: superoxide dismutase (SOD), glutathione S-transferase (GST),
serine/threonine-protein kinase (PBS1), basic endochitinase B (CHIB)
and calreticulin (CALR). SOD has the ability to eliminate superoxide
anion free radicals and participates in response pathways under various
stress environments of plants. Studies have shown that glutathione
S-transferase (GST), glutathione peroxidase (GPX), and glutathione
reductase (GR) are the key enzymes that glutathione participates in
plant defense mechanisms^[103]36. Known studies have shown that PBS1
participates in Pep-induced plant defense response signal transmission.
After analyzing the expression of resistance-related transcripts, it
was found that the average TPM value of the resistance-related
transcripts of ‘Changbai No. 9’ was 798.9, and the average TPM value of
the resistance-related transcripts of ‘Pinot Noir’ was 156.9. This
result is consistent with previous studies and proves that the
anti-stress ability of ‘Pinot Noir’ still has a large scope for
domestication and improvement, and there are resistance genes worth
studying in ‘Changbai No. 9’.
Peach has become the most widely used rootstock species in China due to
its excellent resistance^[104]37. This study screened the common
differential genes of ‘Okubo’ and ‘Lianyungang Maotao’ and found eight
fruit resistance-related genes: polyphenol oxidase, peroxidase,
glutathione S-transferase (GST), chitinase, 12-oxophytodienoic acid
reductase (OPR), light-harvesting complex II chlorophyll a/b binding
protein 4 (LHCB4), 9-cis-epoxycarotenoid dioxygenase (NCED) and alcohol
dehydrogenase class-P (ADH1). The GO function annotation contains
multiple resistance-related annotations: response to wounding
(GO:0009611) response to nematode (GO:0009624) and response to insect
(GO:0009625). The polyphenol oxidase gene has been shown to have an
effect on plant resistance^[105]38. The chitinase
enzyme^[106]39,[107]40, affects and degrades chitin in the fungal cell
wall, and acts as an elicitor to regulate the defense response of the
plant. After analyzing the expression levels of resistance genes in
‘Okubo’ and ‘Lianyungang Maotao’, it was found that their expression
levels were quite different: the average expression level of chitinase
gene-related transcripts in ‘Okubo’ in the three periods was 30.53,
while the expression level of the more resistant ‘Lianyungang Maotao’
was 102.93, indicating that ‘Okubo’ still has a great potential for
improvement and needs further cultivation and domestication.
It has been proved that chalcone synthase (CHS), glutamate S-transfer
(GST) and malate dehydrogenase (MDH1) genes play an important role in
the growth of grapes and peaches. The gene expression pattern of
infected grapes was detected by qRT-PCR. It was found that the highly
expressed CHS gene could enhance the ability of grape leaves to resist
gray mold and downy mildew, which was consistent with the results of
this study (the average expression amount of related genes in
cultivated grapes was 16.35, while the average expression amount of
related genes in wild grapes was 63.07)^[108]41. In peaches, the CHS
gene regulates the metabolism of flavonoids, affects the synthesis of
anthocyanin glycosides, quercetin glycosides, and chlorodermin, and has
an important impact on peel color^[109]42. The correlation between GST
gene function deletion allele and white peel has been verified by
genetic markers in peach^[110]43. The correlation between MDH1 gene in
grape and malic acid content in fruit has been proved by RNA seq and
qRT PCR^[111]44.
Methods
Experimental material collection
The cultivated plants ‘Lianyungang Maotao’ and ‘Okubo’ were healthy
plants cultivated for 5 years by Jiangsu Academy of Agricultural
Sciences (Nanjing Jiangsu Province, China) and ‘Changbai No. 9.’ and
‘Pinot Noir’ for 8 years by Zhengzhou Fruit Tree Institute (Zhengzhou
City, Henan Province, China) respectively.
Grape fruit materials are collected in the young fruit period (June 6),
swelling period (July 17) and ripening period (July 30). Peach fruit
materials are collected in the young fruit period (May 6), the swelling
period (June 24) and maturity period (cultivated peach: July 17; wild
peach: August 15) (Table [112]1). Selected normal fruits and leaves
from branches with similar height from the ground and free of disease
and insect marks. At least 10 fruits of each variety were mixed in each
period as a sample. The sample was frozen in liquid nitrogen
immediately after collection, and then stored in ultra-low temperature
refrigerator at minus 80 degrees Celsius.
The plant materials involved in this experiment had been approved by
the unit before collection, and the research was in compliance with
local policies and regulations.
Determination of fruit transverse and longitudinal diameter and single fruit
weight
The horizontal diameter, vertical diameter and single fruit weight of
grapes were measured by vernier calipers and electronic balance. And
average of the horizontal diameter, vertical diameter and single fruit
weight of the fruit at different growth stages were made according to
the measured data.
Measurement of fruit soluble sugar and organic acid
Took two grams of fruits stored in a liquid nitrogen environment and
fully grind them in the mortar, then add the extract, heat treatment,
ultrasonic extraction and centrifugation in a 37 °C water bath, and
collect the supernatant in a volumetric flask, performed three
repetitions. After constant volume, used a rotary evaporator to process
and add 1 mL of ultrapure water. The extracted liquid was filtered
through a chromatographic column SEP-C18 column (Waters, WAT021515) and
Sep-Pak filter.
The soluble sugar indicators included sorbitol, sucrose, fructose and
glucose. The soluble sugar content in the fruit was determined by high
performance liquid chromatography. The equipment and parameters were
set as followed: Carbohydrate column (Transgenomic COREGET-87C) and
guard column (Transgenomic CARB Sep Coregel 87C cartridge);column
setting temperature: 85 °C, reference cell temperature: 35 °C; flow
rate setting: 0.8 mL/min; ultrapure water; injection volume 5 μm.
Organic acid indicators include quinic acid、citric acid、malic
acid、oxalic acid and shikimic acid. The equipment and parameter
settings are as follows: Zobar SB-Aq column; flow rate setting:
0.7 mL/min; 20 mmol/l disodium hydrogen phosphate buffer; 2% methanol
98%; column temperature setting: 35 °C; water UV detector: wavelength
210 nm; injection volume 5 μm. Calculate the soluble sugar and organic
acid content based on the output data.
RNA extraction and cDNA library preparation
RNA samples were extracted by the cetyltrimethyl ammonium bromide
method (CTAB)^[113]45. The pulp tissue samples of grapes and peaches
were added to CTAB solution and liquid nitrogen for grinding, and
approximately 1 mg was extracted respectively. According to the
Illumina^®TruSeq™RNA sample preparation process, the extracted RNA
sample was used to prepare a cDNA library, first strand cDNA was
synthesized using random hexamer primer and second strand cDNA
synthesis was subsequently performed using DNA Polymerase I and RNase
H.
Transcriptome sequencing, assembly, annotation and quantification process
Library quality was assessed on the Agilent Bioanalyzer 2100 system and
then sequenced with Illumina platform and paired-end reads were
generated. The adaptor sequences and low-quality sequence reads were
removed from the raw sequences. Download grape and peach genome data
and annotations from NCBI (National center for biotechnology
information) database. These clean reads were then mapped to reference
genome by Tophat tool^[114]46. TransDecoder was used first to identify
potential coding regions (CDS) in the sequences, Diamond software was
then used for sequence similarity comparison. After the comparison was
completed, Hmmer was used to search for protein domains, and finally
the above annotation informations were integrated through the Trinotate
annotation tool. In this study, Kallisto^[115]47 data quantification
software was used to calculate gene expression by TPM (Transcripts per
kilobase million) standardized method
([116]https://pachterlab.github.io/kallisto/).
Analysis and identification of differentially expressed genes
The identification of differentially expressed genes were identified by
the R program platform DEGseq package^[117]48. DEGseq was used to
analyze the fruit transcript expression levels of samples. The
threshold of significantly differentially expressed transcripts and
false positive rates of grape and peach were | Fold Change |> 1 and
P-value < 0.01, | Fold Change |> 2 and P-value < 0.05, respectively.
Short time-series expression miner (STEM)
([118]http://www.cs.cmu.edu/~jernst/stem/) was a JAVA-based gene
expression trend analysis program, which could be logarithmic perform
cluster analysis and visualization of gene expression data at each time
point, and used to perform Gene ontology (GO) annotations on genes with
the same expression pattern^[119]49. The differential transcript
expression levels of wild grapes ‘Changbai No. 9’, cultivated grapes
‘Pinot Noir’, cultivated peaches ‘Okubo’ and wild peaches ‘Lianyungang
Maotao’ at the same stage were input into expression profile data
analysis. The software analyzes the expression trends of the three
periods.
Functional analysis of differentially expressed genes
Weighted gene correlation network analysis (WGCNA) can organize genes
with the same expression pattern into modules, and association analysis
between the modules and phenotypic data were performed to mine
potential key genes^[120]50. In this study, 878 and 301 differential
genes between wild grape ‘Changbai No. 9’ and cultivated grape ‘Pinot
Noir’, cultivated peach ‘Okubo’ and wild peach ‘Lianyungang Maotao’ at
three periods as input data respectively. The soft threshold power was
screened and the genes were divided into modules.
Gene ontology^[121]51 annotation analysis includes three parts:
Cellular component, Molecular function and Biological process. Gene
ontology describes the characteristics of genes and gene products
through these three aspects, and GO annotation analysis was widely used
in gene function annotation as a unified tool in biology. The Kyoto
Encyclopedia of Genes and Genomes (KEGG) can assign functional meanings
to genes and genomes at the molecular level and understand the
functions of biological systems^[122]52. This study used GO and KEGG to
annotate the differential expression transcripts of cultivated grape
‘Pinot Noir’ and wild grape ‘Changbai No.9’, cultivated peach ‘Okubo’
and wild peach ‘Lianyungang Maotao’ to further understand the function
and pathway differences between cultivated and wild fruit trees.
Author contributions
W.J.H. analyzed the data and drafted the manuscripts. M.L. designed
experiment, collected samples and revised the manuscript. H.W.Z.
revised the manuscript and provided guidance for article writing.
J.Y.W. and S.L.Z. supported for experimental design and revised the
manuscript. All authors have read and approved the manuscript.
Data availability
Raw reads of the experiment are submitted to NCBI SRA database, the
accession number of the project is PRJNA792624,
[123]https://www.ncbi.nlm.nih.gov/bioproject/PRJNA792624.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
These authors contributed equally: Weijian Huang and Meng Li.
Contributor Information
Meng Li, Email: mli@njau.edu.cn.
Shaoling Zhang, Email: slzhang@njau.edu.cn.
References