2021-9-27【文献】转录组和重测序揭示两个梨品种有机酸积累
2021-10-18 本文已影响0人
Wei_Sun
Title: Transcriptome and Resequencing Analyses Provide Insight into Differences in Organic Acid Accumulation in Two Pear Varieties
题目：转录组和重测序揭示两个梨品种有机酸积累的差异
通讯作者：张绍玲
机构：南京农业大学
杂志：Int. J. Mol. Sci.
IF/分区：5.9/Q2
发表时间：2021.9
研究意义：
了解两个梨品种中有机酸的含量、表达和驯化的差异提供了依据，确定了几个与有机酸相关的候选基因。
M&M：
材料：DSHS和AMT
性状：苹果酸（MA）、草酸（OA）、柠檬酸（CA）
方法：
1.RNA-seq Data De Novo （两个品种，四个生长时期，各三个生物学重复，共24个样）
2.重测序：NCBI
3.top 1% of XP-CLR；top5% of Fst
4.WGCNA：R包
5.qRT-PCR
结论：
1.在两个梨品种中，苹果酸（MA）均是含量最的果酸。AMT中的三种果酸含量均高于DSHS，其中MA和柠檬酸（CA）的含量差异显著，草酸（OA）差异不明显。
Figure 1. The contents of three organic acids in four fruit developmental stages of AMT and DSHS pears. (A) The contents
of oxalic acid content. (B) The contents of malic acid content. (C) The contents of citric acid. The left boxplot displays overall
organic acid levels in AMT and DSHS, while the right line plot represents the organic acid levels in four developmental
stages in both AMT and DSHS. Blue and orange lines represent AMT and DSHS pears, respectively. S1/S2/S3/S4 on the X
axis represents 15/45/90/120 days after full bloom; mg/g FW on the Y axis represents mg/g fresh weight (FW); Student’s
t-test * p-value < 0.05; ** p-value < 0.01; ns: no significant.
2.AMT的参考基因组未知。通过系统进化树比较，AMT与DSHS的亲缘关系比‘Bartlett’更加接近，因此选择P. bretschneideri作为参考基因组。通过对转录组数据分析，表明AMT和DSHS在早期生长阶段（S1-S3）的基因表达情况类似，AMT比DSHS拥有更高的FPKM值。
Figure 2. Phylogenetic tree, PCA, and overall expression distributions in two pear varieties. (A) Phylogenetic tree generated by ASTRAL on the basis of all single-copy genes. Branch labels are bootstrap values. ‘Bartlett’ (P. communis), ‘Dangshansuli’ (P. bretschneideri) and ‘Amute’ (P. sinkiangensis) were using RNA-seq data from this study and others were using genome data obtained from GDR. (B) PCA of 24 samples. In aS1–aS4, ‘a’ represents AMT and S1–S4 represents 15–120 DAFB; that is, aS1–aS4 represents AMT at 15–120 DAFB. The dS1–dS4 represents DSHS at 15–120 DAFB. (B) Kernel plot of overall expression density of DSHS. The Y-axis represents gene density, and the X-axis represents Log10(FPKM). (C) Kernel plot of the overall expression density in DSHS; S1-S4 represents 15-120 DAFB in DSHS. (D) Kernel plot of the overall expression density in AMT; S1–S4 represents 15–120 DAFB in AMT.
3.在DSHS中，随着发育时间间隔的增加，差异基因数目也随之增加。但在AMT中的情况则相反。为确定差异基因，对12267个差异基因进行聚类分析，分组为0、5、6，其中有2236个gene在两个品种中均有表达。随后进行GO富集分析，分组0中，主要为转运蛋白，以及TCA循环路径中的基因。
Figure 3. DEGs between DSHS and AMT in four fruit developmental stages and cluster analysis (A) Pairwise comparisons within and between two species and the developmental stages. Red numbers represent up-regulation and green number represent down-regulation. ‘A’ and ‘D’ represent AMT and DSHS, respectively. S1–S4 indicate the four stages that occur from 15 to 120 DAFB. (B) Cluster profiles of clusters 0, 5, and 6. The X-axis represents 15 DAFB, 45, 90, and 120 DAFB. (C) GO enrichment analysis of cluster 0. Bubble size indicates the number of genes, and color indicates the corrected p-value. The enrichment (Rich) factor represents a ratio of genes enriched in this term to the background genes belonging to this term. (D) KEGG enrichment analysis of cluster 0. Ratio represents number of enriched genes divided by the number of background genes number of this term.
4.利用27239个基因进行WGCNA，23944个gene被分为了22个model，其中三个model分别与MA、CA、OA具有极强的相关性，证明这些模块中的基因主要与有机酸相关，且9个TCA中的差异基因均属于这三个核心模块。
Figure 4. The WGCNA network analysis of 23 samples and the expression profiles of three key modules. (A) Dendrogram of co-expression modules identified by WGCNA. The leaves of the tree represent genes, and their annotations are represented by colors (below). (B) Module–acid correlation heatmap. The color scale on the right shows correlations from −1 (red) to 1 (blue). (C) Module–sample correlation heatmap. The color scale on the right shows correlations from −1 (red) to 1 (blue). The sample name ‘a’ represents AMT and ‘d’ represents DSHS, 15, 45, 90, 120 represents 120/15/45/90 DAFB and 1/2/3 represents three replicates.
Figure 5. Expression heatmaps for all genes in three key modules. (A) The expression profile of 5988 genes in the turquoise module. Samples from aS1 and dS1 are marked by a red box. AMT is indicated by ‘a’. (B) The expression profile of 2191 genes in the yellow module. Samples from AMT are marked by a red box. (C) The expression profile of 99 genes in the light-yellow module. Samples from AMT are marked by a red box.
5.通过WGCNA和差异基因分析，在AMT中找到12个候选基因，并在AMT和DSHS中构建了两个调控网络。两个调控网络中只有一个相同的转录因子，证明两个品种中具有不同的调控网络。
Figure 6. Regulatory networks between two pear varieties and statistics of TFs in two networks. (A) Orange circle represents candidate genes. Red and green circles represent TFs in AMT and DSHS, respectively. Blue circles represent TFs shared in both regulatory networks. (B) Statistics of regarding TFs types in the AMT network. (C) Statistics of regarding TFs types in the DSHS network.
6.对两个品种进行Fst和XP-CLR分析，有两个基因同时被检测到，一个是TCA途径编码基因，一个是ERF转录因子，且都是AMT在S3的上调基因。
Figure 7. Selective signals and expression validations. (A) LD decay in different pear groups, including P. bretschneideri (red) groups, P. sinkiangensis (black) groups and Asian wild (blue) groups. (B) Distribution of XP-CLR values between P. bretschneideri groups and P. sinkiangensis groups across the whole genome of the pears. (C) XP-CLR signal of Pbr014487.1. (D) XP-CLR signal of Pbr038937.1. (E,F) Expression validation between ‘Yali’ and ‘Korla’. The upper boxplots represent the expression of Pbr014487.1 in S3 (including three biological replicates) between two varieties of pear. The middle heatmaps are gene profiles of two genes during S1−S4. The bottom boxplots represent the expression of Pbr038937.1 in S3 (including three biological replicates) between two varieties of pear. Wald test using DE-seq2 and Bonferroni−Holm method used to correct p-values, ** FDR < 0.01.
Figure 8. Expression profiles of DEGs related to the TCA and glyoxysome pathways. Schematic illustrations of TCA and glyoxysome pathways. DEG names and expression levels in AMT are presented in the pathway. The sky-blue frame represents the cell membrane, pink represents a mitochondrion, and the green circle represents the glyoxysome. The color scale from red to blue corresponds to expression levels from high to low, respectively, based on the log2(FPKM). SDH: succinate dehydrogenase, ACO: aconitase; FUM: fumarase; IDH: isocitrate dehydrogenase; MS: malate synthase; ICL: isocitrate lyase; NAD-MDH: NAD-Malate dehydrogenase; PEPC: phosphoenolpyruvate carboxylase; OGDH: ketoglutarate dehydrogenase; CS: citrate synthetase. Names of genes identified from WGCNA are marked in red.
7.对17个候选基因进行qRT-PCR检测，计算与FPKM的相关性，其中有9个基因的相关性达到0.8，为下游分析提供依据。
Figure 9. Validation of expression patterns by qRT-PCR of selected DEGs from RNA-seq analysis. The left Y-axis represents relative expression levels and their corresponding bar plot with standard deviations (denoted as an error bar in each plot). The right Y-axis represents expression levels calculated by fragments per kilobase per million reads (FPKM) method corresponds line plot in each plot. Additionally, the X-axis in each plot means successive developmental stages in two pear varieties. A/D15: 15 DAFB in ‘Amute’/ ‘Dangshansuli’ pear. A/D45: 45 DAFB in ‘Amute’/ ‘Dangshansuli’. A/D90: 90 DAFB in ‘Amute’/ ‘Dangshansuli’. A/D120: 120 DAFB in ‘Amute’/ ‘Dangshansuli’. R means Pearson Correlation Coefficient (PCC) between FPKM and relative expression, and P means p-value of PCC, * p < 0.05; ** p < 0.01.
亮点：
DEGs + WGCNA + sweep selection + qRT-PCR
