Title: Transcriptome and Resequencing Analyses Provide Insight into Differences in Organic Acid Accumulation in Two Pear Varieties

題目：轉(zhuǎn)錄組和重測(cè)序揭示兩個(gè)梨品種有機(jī)酸積累的差異

通訊作者：張紹玲
機(jī)構(gòu)：南京農(nóng)業(yè)大學(xué)
雜志：Int. J. Mol. Sci.
IF/分區(qū)：5.9/Q2
發(fā)表時(shí)間：2021.9

研究意義：

了解兩個(gè)梨品種中有機(jī)酸的含量、表達(dá)和馴化的差異提供了依據(jù)，確定了幾個(gè)與有機(jī)酸相關(guān)的候選基因。

M&M：

材料：DSHS和AMT
性狀：蘋果酸（MA）、草酸（OA）、檸檬酸（CA）
方法：
1.RNA-seq Data De Novo （兩個(gè)品種，四個(gè)生長(zhǎng)時(shí)期，各三個(gè)生物學(xué)重復(fù)，共24個(gè)樣）
2.重測(cè)序：NCBI
3.top 1% of XP-CLR；top5% of Fst
4.WGCNA：R包
5.qRT-PCR

結(jié)論：

1.在兩個(gè)梨品種中，蘋果酸（MA）均是含量最的果酸。AMT中的三種果酸含量均高于DSHS，其中MA和檸檬酸（CA）的含量差異顯著，草酸（OA）差異不明顯。

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的參考基因組未知。通過(guò)系統(tǒng)進(jìn)化樹比較，AMT與DSHS的親緣關(guān)系比‘Bartlett’更加接近，因此選擇P. bretschneideri作為參考基因組。通過(guò)對(duì)轉(zhuǎn)錄組數(shù)據(jù)分析，表明AMT和DSHS在早期生長(zhǎng)階段（S1-S3）的基因表達(dá)情況類似，AMT比DSHS擁有更高的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(chǎn)’ 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.

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個(gè)基因進(jìn)行WGCNA，23944個(gè)gene被分為了22個(gè)model，其中三個(gè)model分別與MA、CA、OA具有極強(qiáng)的相關(guān)性，證明這些模塊中的基因主要與有機(jī)酸相關(guān)，且9個(gè)TCA中的差異基因均屬于這三個(gè)核心模塊。

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(chǎn)’ 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(chǎn)’. (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.通過(guò)WGCNA和差異基因分析，在AMT中找到12個(gè)候選基因，并在AMT和DSHS中構(gòu)建了兩個(gè)調(diào)控網(wǎng)絡(luò)。兩個(gè)調(diào)控網(wǎng)絡(luò)中只有一個(gè)相同的轉(zhuǎn)錄因子，證明兩個(gè)品種中具有不同的調(diào)控網(wǎng)絡(luò)。

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.對(duì)兩個(gè)品種進(jìn)行Fst和XP-CLR分析，有兩個(gè)基因同時(shí)被檢測(cè)到，一個(gè)是TCA途徑編碼基因，一個(gè)是ERF轉(zhuǎn)錄因子，且都是AMT在S3的上調(diào)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.對(duì)17個(gè)候選基因進(jìn)行qRT-PCR檢測(cè)，計(jì)算與FPKM的相關(guān)性，其中有9個(gè)基因的相關(guān)性達(dá)到0.8，為下游分析提供依據(jù)。

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.

亮點(diǎn)：

DEGs + WGCNA + sweep selection + qRT-PCR

原文鏈接：

https://www.mdpi.com/1422-0067/22/17/9622/htm#