From 2b055261db0348e8319025cda6c27fc40a686907 Mon Sep 17 00:00:00 2001
From: MWA_AG_Fernie_MPIMP <Wijesingha@mpimp-golm.mpg.de>
Date: Tue, 9 May 2023 12:20:31 +0200
Subject: [PATCH] updated LC isa.assay.xlsx files

---
 .../cmQTL_val1_GH_2020_GC_MS/isa.assay.xlsx   |  Bin 315209 -> 344387 bytes
 .../isa.assay.xlsx                            |  Bin 2759 -> 280177 bytes
 assays/cmQTL_val1_GH_2020_phenotype/README.md |    0
 .../dataset/.gitkeep                          |    0
 .../dataset/Phenotype_data.xlsx               |    3 +
 .../isa.assay.xlsx                            |  Bin 0 -> 2696 bytes
 .../protocols/.gitkeep                        |    0
 .../isa.assay.xlsx                            |  Bin 2753 -> 276680 bytes
 isa.investigation.xlsx                        |  Bin 4117 -> 4767 bytes
 .../cmQTL_val1_GH_2020_phenotype/README.md    |    0
 .../isa.study.xlsx                            |  Bin 0 -> 3098 bytes
 .../protocols/.gitkeep                        |    0
 .../resources/.gitkeep                        |    0
 .../220531_correlation_matrices.R             |  281 ++
 .../GC_MS_figures/Figure maker_cmQTL1_paper.R | 2362 +++++++++++++++++
 .../210114_cmQTL_val_phenotype_analysis.R     |  826 ++++++
 .../Figure maker_phenotypes.R                 | 1448 ++++++++++
 17 files changed, 4920 insertions(+)
 create mode 100644 assays/cmQTL_val1_GH_2020_phenotype/README.md
 create mode 100644 assays/cmQTL_val1_GH_2020_phenotype/dataset/.gitkeep
 create mode 100644 assays/cmQTL_val1_GH_2020_phenotype/dataset/Phenotype_data.xlsx
 create mode 100644 assays/cmQTL_val1_GH_2020_phenotype/isa.assay.xlsx
 create mode 100644 assays/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep
 create mode 100644 studies/cmQTL_val1_GH_2020_phenotype/README.md
 create mode 100644 studies/cmQTL_val1_GH_2020_phenotype/isa.study.xlsx
 create mode 100644 studies/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep
 create mode 100644 studies/cmQTL_val1_GH_2020_phenotype/resources/.gitkeep
 create mode 100644 workflows/GC_MS_correlation/220531_correlation_matrices.R
 create mode 100644 workflows/GC_MS_figures/Figure maker_cmQTL1_paper.R
 create mode 100644 workflows/Phenotype_analysis/210114_cmQTL_val_phenotype_analysis.R
 create mode 100644 workflows/Phenotype_figures/Figure maker_phenotypes.R

diff --git a/assays/cmQTL_val1_GH_2020_GC_MS/isa.assay.xlsx b/assays/cmQTL_val1_GH_2020_GC_MS/isa.assay.xlsx
index b8164b2d3765b2022354f54487473f2f9c124569..fec9ae80414f7a938612073d165363cbe9515ac4 100644
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diff --git a/assays/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep b/assays/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep
new file mode 100644
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diff --git a/assays/cmQTL_val1_GH_2020_polar_LC_MS/isa.assay.xlsx b/assays/cmQTL_val1_GH_2020_polar_LC_MS/isa.assay.xlsx
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diff --git a/studies/cmQTL_val1_GH_2020_phenotype/README.md b/studies/cmQTL_val1_GH_2020_phenotype/README.md
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diff --git a/studies/cmQTL_val1_GH_2020_phenotype/isa.study.xlsx b/studies/cmQTL_val1_GH_2020_phenotype/isa.study.xlsx
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HcmV?d00001

diff --git a/studies/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep b/studies/cmQTL_val1_GH_2020_phenotype/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/cmQTL_val1_GH_2020_phenotype/resources/.gitkeep b/studies/cmQTL_val1_GH_2020_phenotype/resources/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/workflows/GC_MS_correlation/220531_correlation_matrices.R b/workflows/GC_MS_correlation/220531_correlation_matrices.R
new file mode 100644
index 0000000..1f44b87
--- /dev/null
+++ b/workflows/GC_MS_correlation/220531_correlation_matrices.R
@@ -0,0 +1,281 @@
+library(tidyverse)
+library(glue)
+library(here)
+
+
+# Directory setting -------------------------------------------------------
+
+current <- getwd()
+source <- str_c(current,"/..")
+
+cur_date <- str_c(str_replace_all(Sys.Date(),"^.{2}|-",""))
+
+out <- str_c(cur_date, "correlation", sep = "_")
+
+if (file.exists(out)) {
+  cat("The folder already exists")
+} else {
+  dir.create(out)
+}
+
+out_dir <- str_c(current, out, sep = "/")
+
+latest <- str_sort(str_extract(list.files(pattern = "^\\d{6}_analysis$"),
+                               pattern = "^\\d{6}_analysis"),
+                   decreasing = T)[[1]]
+
+latest_analysis <- str_c(current, "/", latest)
+
+setwd(latest_analysis)
+
+latest_analysis_date <- str_extract(latest, pattern = "^\\d{6}")
+
+# File loading ------------------------------------------------------------
+
+fc_1 <- read_csv("mean_values_se_n.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+genotypes <- fc_1 %>% 
+  distinct(alias, genotype) %>% 
+  mutate(genotype_label = str_remove_all(genotype, "\\*"))
+
+fc_1_ind <- read_csv("individual_values.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC <- read_csv("p_values.csv")
+
+fc_1_lt <- read_csv("mean_values_se_n_levene.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+fc_1_ind_lt <- read_csv("individual_values_levene.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC_lt <- read_csv("p_values_levene.csv")
+
+fc_1_cv <- read_csv("mean_values_se_n_cv.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+fc_1_ind_cv <- read_csv("individual_values_cv.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC_cv <- read_csv("p_values_cv.csv") %>% 
+  mutate(group1 = as_factor(if_else(group1 == "MoneyMaker", glue("{group1}"),glue("*{group1}*"))),
+         group1 = fct_relevel(group1, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")),
+         group2 = as_factor(if_else(group2 == "MoneyMaker", glue("{group2}"),glue("*{group2}*"))),
+         group2 = fct_relevel(group2, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+setwd(out_dir)
+
+
+# Data combination --------------------------------------------------------
+
+
+met_dat <- fc_1 %>% 
+  distinct(met, Compound_Name, Compound_Class)
+
+
+per_comp_y <- fc_1 %>%
+  group_by(tissue, treatment, met) %>% 
+  summarise(tot_val = max(mean_fc + se))
+
+cb_scale <- c("#E69F00", "#56B4E9", "#009E73", "#F0E442",
+              "#0072B2", "#D55E00","#000000")
+bw_scale <- c("black", "black", "black", "black", "black", "black", "black")
+
+
+# Correlation network -------------------------------------------------------------
+library(jmuOutlier)
+
+g_vec <- genotypes$genotype %>%
+  sort() %>%
+  as.character()
+
+g_vec_lab <- genotypes %>%
+  arrange(genotype) %>% 
+  select(genotype_label) %>% 
+  as_vector()
+
+
+## Loop start --------------------------------------------------------------
+
+
+for(g in seq_along(genotypes$genotype)) {
+  print(str_c("starting with", g_vec_lab[[g]], sep = " "))
+  
+cor_base <- fc_1_ind %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c(g_vec[[g]]),
+    tissue == "fruits")
+
+cor.GC <- cor_base %>% 
+  group_by(met) %>% 
+  mutate(log_norm = log2(fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(genotype, treatment, LIMS_ID),
+              names_from = met,
+              values_from = fc) %>% 
+  #arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+cor.GC_numeric <- cor.GC %>% 
+  select(starts_with("m_"))
+
+corr_mat <- cor(cor.GC_numeric, method = "spearman")
+
+x <- vector(mode = "list", length = length(colnames(cor.GC_numeric)))
+names(x) <- colnames(cor.GC_numeric)
+
+start <- Sys.time()
+for (i in seq_along(colnames(cor.GC_numeric))) {
+  #names(x[[i]]) <- colnames(cor.GC_numeric)
+  for (n in seq_along(colnames(cor.GC_numeric))) {
+    if(i > n) {
+      x[[i]][[n]] <- NA
+    } else {
+    x[[i]][[n]] <- perm.cor.test(cor.GC_numeric[[i]], cor.GC_numeric[[n]],
+                                 method = "spearman",
+                                 num.sim = 20000
+                                 )$p.value
+    }
+  } 
+  print(i)
+}
+
+stop <- Sys.time()
+stop-start
+
+for (i in seq_along(colnames(cor.GC_numeric))) {
+  names(x[[i]]) <- colnames(cor.GC_numeric)
+}
+
+corr_p <- x %>% 
+  reduce(bind_rows)
+
+corr_p_long <- corr_p %>% 
+  mutate(met1 = colnames(cor.GC_numeric)) %>% 
+  pivot_longer(cols = starts_with("m_"),
+               values_to = "cor_p_val",
+               names_to = "met2") %>% 
+  mutate(met1_num = as.numeric(str_extract(met1,"(?<=m_)\\d+")),
+         met2_num = as.numeric(str_extract(met2,"(?<=m_)\\d+"))) %>% 
+  mutate(small = if_else(met1_num < met2_num, met1_num, met2_num),
+         big = if_else(met1_num > met2_num, met1_num, met2_num),
+         sorted = str_c(small, big, sep = "_")) %>% 
+  filter(!is.na(cor_p_val)) %>% 
+  mutate(p_adj = p.adjust(cor_p_val, method = "fdr")) %>% 
+  filter(!met1 == met2)
+
+corr_mat_long <- corr_mat %>% 
+  as_tibble(rownames = "met1") %>% 
+  pivot_longer(cols = starts_with("m_"),
+               values_to = "cor_val",
+               names_to = "met2") %>% 
+  mutate(met1_num = as.numeric(str_extract(met1,"(?<=m_)\\d+")),
+         met2_num = as.numeric(str_extract(met2,"(?<=m_)\\d+"))) %>% 
+  mutate(small = if_else(met1_num < met2_num, met1_num, met2_num),
+         big = if_else(met1_num > met2_num, met1_num, met2_num),
+         sorted = str_c(small, big, sep = "_")) %>% 
+  distinct(sorted, .keep_all = T) %>% 
+  filter(!met1 == met2) %>% 
+  mutate(pos_neg = as_factor(if_else(cor_val > 0, "pos", "neg")),
+         weight = cor_val)
+
+corr_mat_comb <- corr_mat_long %>% 
+  left_join(corr_p_long)
+
+write_csv(corr_mat_comb,
+          str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                "_corr_mat_",
+                g_vec_lab[[g]],
+                ".csv"))
+
+}
+
+## Loop end ----------------------------------------------------------------
+
+stop()
+
+library(viridis)
+library(igraph)
+library(ggnetwork)
+
+library(ggraph)
+
+classes_tidy <- met_dat %>% 
+  mutate(`ChEBI simplified` = if_else(str_detect(Compound_Class,"carbohydrate"),"carbohydrate or derivative",
+                                      if_else(str_detect(Compound_Class,"amino_acid"), "amino acid or derivative",
+                                              if_else(str_detect(Compound_Class,"carboxylic_acid"), "carboxylic acid",
+                                                      "other"))))
+
+annotation_col_corr <- classes_tidy %>% 
+  select(`ChEBI simplified`) %>% 
+  as.data.frame()
+
+rownames(annotation_col_corr) <- classes_tidy$met
+
+vir_scale <- plasma(4, begin = 0.2, end = 1)
+ann_colors_corr = list(
+  `ChEBI simplified`= c("amino acid or derivative"= vir_scale[1],
+                        "carbohydrate or derivative" = vir_scale[2],
+                        "carboxylic acid" = vir_scale[3],
+                        "other" = vir_scale[4])
+)
+
+
+
+ann_colors_graph = tibble(`ChEBI simplified`= names(ann_colors_corr$`ChEBI simplified`),
+                          color = ann_colors_corr$`ChEBI simplified`)
+
+node_cols <- tibble(met = colnames(cor.GC_numeric)) %>% 
+  left_join(classes_tidy) %>% 
+  left_join(ann_colors_graph)
+
+node_col_vec <- node_cols$color
+
+
+net_raw <- graph_from_data_frame(corr_mat_comb, vertices = node_cols, directed = F)
+
+
+set.seed(200)
+plot.igraph(net_raw, vertex.color = node_col_vec, vertex.label = "")
+
+set.seed(121)
+layout <- layout_with_graphopt(net_raw , charge = 0.2,
+                               spring.length = 1,
+                               spring.constant = 200,
+                               mass = 50)
+
+ggraph(net_raw, layout = layout) +
+  geom_edge_arc0(aes(color = pos_neg), strength = 0.1) +
+  geom_node_point(aes(color = `ChEBI simplified`), size = 3)
+#  geom_node_text(aes(label = name))
+
+
+ggnetwork(net_raw, layout = layout) %>% 
+    ggplot(aes(x = x, y = y, xend = xend, yend = yend)) + 
+    geom_edges(aes(color = pos_neg),curvature = 0.1) +
+    geom_nodes(aes(fill = `ChEBI simplified`), color = "black",  shape = 21, size = 3) +
+    theme_blank() +
+    scale_fill_manual(values = vir_scale, aesthetics = "fill")
+  
+#other methods
+  
+  corr_mat_plot <- corr_mat_long %>% 
+    filter(abs(cor_val) >= 0.75) %>% 
+    select(met1, met2) %>% 
+    as.matrix()
+  
+  corr_mat_plot <- corr_mat
+  corr_mat_plot[abs(corr_mat_plot)<0.5] <- 0
+  
+  net_raw <- graph_from_edgelist(corr_mat_plot, directed = F)
+  net_raw <- graph_from_adjacency_matrix(corr_mat_plot,
+                                         weighted=T,
+                                         mode="undirected",
+                                         diag=F)
diff --git a/workflows/GC_MS_figures/Figure maker_cmQTL1_paper.R b/workflows/GC_MS_figures/Figure maker_cmQTL1_paper.R
new file mode 100644
index 0000000..4a3cd4c
--- /dev/null
+++ b/workflows/GC_MS_figures/Figure maker_cmQTL1_paper.R	
@@ -0,0 +1,2362 @@
+rm(list = ls())
+library(tidyverse)
+library(ggpubr)
+library(glue)
+library(ggtext)
+library(cowplot)
+library(ggbeeswarm)
+library(extrafont)
+library(viridis)
+
+# Directory setting -------------------------------------------------------
+
+
+setwd(dirname(rstudioapi::getActiveDocumentContext()$path))
+getwd()
+
+current <- getwd()
+source <- str_c(current,"/..")
+
+cur_date <- str_c(str_replace_all(Sys.Date(),"^.{2}|-",""))
+
+out <- str_c(cur_date, "Figures", sep = "_")
+
+if (file.exists(out)) {
+  cat("The folder already exists")
+} else {
+  dir.create(out)
+}
+
+out_dir <- str_c(current, out, sep = "/")
+
+latest <- str_sort(str_extract(list.files(pattern = "^\\d{6}_analysis$"),
+                               pattern = "^\\d{6}_analysis"),
+                   decreasing = T)[[1]]
+
+latest_analysis <- str_c(current, "/", latest)
+
+setwd(latest_analysis)
+
+latest_analysis_date <- str_extract(latest, pattern = "^\\d{6}")
+
+# File loading ------------------------------------------------------------
+
+fc_1 <- read_csv("mean_values_se_n.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+genotypes <- fc_1 %>% 
+  distinct(alias, genotype) %>% 
+  mutate(genotype_label = str_remove_all(genotype, "\\*"))
+
+fc_1_ind <- read_csv("individual_values.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC <- read_csv("p_values.csv")
+
+fc_1_lt <- read_csv("mean_values_se_n_levene.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+fc_1_ind_lt <- read_csv("individual_values_levene.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC_lt <- read_csv("p_values_levene.csv")
+
+fc_1_cv <- read_csv("mean_values_se_n_cv.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+fc_1_ind_cv <- read_csv("individual_values_cv.csv") %>% 
+  select(-genotype) %>% 
+  left_join(genotypes)
+
+sig_GC_cv <- read_csv("p_values_cv.csv") %>% 
+  mutate(group1 = as_factor(if_else(group1 == "MoneyMaker", glue("{group1}"),glue("*{group1}*"))),
+         group1 = fct_relevel(group1, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")),
+         group2 = as_factor(if_else(group2 == "MoneyMaker", glue("{group2}"),glue("*{group2}*"))),
+         group2 = fct_relevel(group2, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+
+setwd(out_dir)
+
+
+# Data combination --------------------------------------------------------
+
+
+met_dat <- fc_1 %>% 
+  distinct(met, Compound_Name, Compound_Class)
+
+classes_tidy <- met_dat %>% 
+  mutate(`ChEBI simplified` = if_else(str_detect(Compound_Class,"carbohydrate"),"carbohydrate or derivative",
+                                      if_else(str_detect(Compound_Class,"amino_acid"), "amino acid or derivative",
+                                              if_else(str_detect(Compound_Class,"carboxylic_acid"), "carboxylic acid",
+                                                      "other")))) %>% 
+  mutate(ChEBI = if_else(str_detect(Compound_Class,"carbohydrate"),"CH",
+                                      if_else(str_detect(Compound_Class,"amino_acid"), "AA",
+                                              if_else(str_detect(Compound_Class,"carboxylic_acid"), "CA",
+                                                      "other"))),
+         ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other")))
+
+genotypes <- fc_1 %>% 
+  distinct(alias, genotype) %>% 
+  mutate(genotype_label = str_remove_all(genotype, "\\*"))
+
+per_comp_y <- fc_1 %>%
+  group_by(tissue, treatment, met) %>% 
+  summarise(tot_val = max(mean_fc + se))
+
+cb_scale <- c("#E69F00", "#56B4E9", "#009E73", "#F0E442",
+              "#0072B2", "#D55E00", "#CC79A7","#000000")
+bw_scale <- c("black", "black", "black", "black", "black", "black", "black")
+vir_scale <- plasma(4, begin = 0.2, end = 1)
+
+
+# Used plots --------------------------------------------------------------
+
+com_theme <- theme(axis.text.x = element_markdown(angle = 45, hjust = 1, size = 6, family = "sans"),
+                   axis.text.y = element_text(size = 6, family = "sans"),
+                   axis.title.x = element_blank(),
+                   axis.title.y = element_text(size = 6, family = "sans"),
+                   panel.background = element_rect(fill = "white"),
+                   panel.border = element_rect(color = "black",fill = NA),
+                   strip.text = element_text(size = 8, family = "sans", margin = margin(t = 1, r = 1, b = 1, l = 1 , unit = "pt")),
+                   text = element_text(size = 6, family = "sans"),
+                   legend.title = element_blank(),
+                   legend.text = element_markdown(size = 6),
+                   plot.margin = unit(c(1,1,1,1), "mm"),
+                   legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm"))
+
+make_box_dot_plot <- function(plot_met, plot_tissue, plot_label, plot_legend, plot_genotypes, plot_fill){
+  
+  per_comp_y <- fc_1_ind %>%
+    filter(genotype %in% plot_genotypes) %>% 
+    group_by(tissue, treatment, met) %>% 
+    summarise(min_y = min(fc),
+              max_y = max(fc)) %>% 
+    mutate(tot_val = 1.1*max_y) %>% 
+    ungroup()
+  
+  sig_bar <- sig_GC %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    group_by(tissue, treatment, met) %>% 
+    mutate(y.position = 1.1 * tot_val,
+           p.signif = if_else(p.value <= 0.05, "*", "ns")) %>% 
+    ungroup() %>% 
+    filter(met == plot_met, tissue == plot_tissue,
+           group1 %in% plot_genotypes, group2 %in% plot_genotypes) 
+  
+  binwidth <- sig_bar %>% 
+    summarise(min_y_comp = min(min_y),
+              max_y_comp = max(max_y)) %>% 
+    mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+    select(binwidth) %>% 
+    as_vector() %>%
+    as.numeric() 
+    
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue, genotype %in% plot_genotypes) %>% 
+    ggplot(aes(x = genotype, y = fc)) +
+    geom_boxplot(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_dotplot(aes(fill = genotype, color = genotype), binaxis = "y", stackdir = "center", binwidth = binwidth, dotsize = 1.5) +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(legend.position = plot_legend) +
+    com_theme +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+   # ggtitle(label = "") +
+    scale_fill_manual(values = cb_scale[plot_fill], aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+make_box_dot_plot_levene <- function(plot_met, plot_tissue, plot_label, plot_legend, plot_genotypes, plot_fill){
+  
+  per_comp_y <- fc_1_ind_lt %>%
+    filter(genotype %in% plot_genotypes) %>% 
+    group_by(tissue, treatment, met) %>% 
+    summarise(min_y = min(lev_t),
+              max_y = max(lev_t)) %>% 
+    mutate(tot_val = 1.1*max_y) %>% 
+    ungroup()
+  
+  sig_bar <- sig_GC_lt %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    group_by(tissue, treatment, met) %>% 
+    mutate(y.position = 1.1 * tot_val,
+           p.signif = if_else(p.value <= 0.05, "*", "ns")) %>% 
+    ungroup() %>% 
+    filter(met == plot_met, tissue == plot_tissue,
+           group1 %in% plot_genotypes, group2 %in% plot_genotypes) 
+  
+  binwidth <- sig_bar %>% 
+    summarise(min_y_comp = min(min_y),
+              max_y_comp = max(max_y)) %>% 
+    mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+    select(binwidth) %>% 
+    as_vector() %>%
+    as.numeric() 
+   
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_1_ind_lt %>% 
+    filter(met == plot_met, tissue == plot_tissue, genotype %in% plot_genotypes) %>% 
+    ggplot(aes(x = genotype, y = lev_t)) +
+    geom_boxplot(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_dotplot(aes(fill = genotype, color = genotype), binaxis = "y", stackdir = "center", binwidth = binwidth, dotsize = 2) +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(legend.position = plot_legend) +
+    com_theme +
+    ylab("Levene's transformed value")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    #ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale[plot_fill], aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+
+make_box_dot_plot_cv <- function(plot_met, plot_tissue, plot_label, plot_legend, plot_genotypes, plot_fill){
+  
+  per_comp_y_cv <- fc_1_ind_cv %>%
+    filter(genotype %in% plot_genotypes) %>% 
+    group_by(tissue, met) %>% 
+    summarise(min_y = min(cv),
+              max_y = max(cv)) %>% 
+    mutate(tot_val = 1.1*max_y) %>% 
+    ungroup()
+  
+  sig_bar <- sig_GC_cv %>%  
+    left_join(per_comp_y_cv) %>% 
+    group_by(tissue, met) %>% 
+    mutate(y.position = 1.1 * tot_val,
+           p.signif = if_else(p.value <= 0.05, "*", "ns"))  %>% 
+    ungroup() %>% 
+    filter(met == plot_met, tissue == plot_tissue,
+           group1 %in% plot_genotypes, group2 %in% plot_genotypes)
+  
+  binwidth <- sig_bar %>% 
+    summarise(min_y_comp = min(min_y),
+              max_y_comp = max(max_y)) %>% 
+    mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+    select(binwidth) %>% 
+    as_vector() %>%
+    as.numeric()
+  
+  plot_out <- fc_1_ind_cv %>% 
+    left_join(met_dat) %>% 
+    filter(met == plot_met, tissue == plot_tissue, genotype %in% plot_genotypes) %>% 
+    ggplot(aes(x = genotype, y = cv)) +
+    geom_boxplot(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_dotplot(aes(fill = genotype, color = genotype), binaxis = "y", stackdir = "center", binwidth = binwidth, dotsize = 2) +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    #facet_grid(cols = vars(treatment)) +
+    com_theme +
+    theme(legend.position = plot_legend) +
+    ylab("CV")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale[plot_fill], aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+
+# Malic acid fruits PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_70", "fruits", "Malic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+saveRDS(last_plot(), "Malic_acid_nominal_pank4-1_fruits.RDS")
+
+p2 <- make_box_dot_plot_cv("m_70", "fruits", "Malic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+saveRDS(last_plot(), "Malic_acid_cv_pank4-1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_70", "fruits", "Malic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+saveRDS(leg, "Malic_acid_legend_pank4-1_fruits.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Malic_acid_fruits_pank4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p2 <- make_box_dot_plot_levene("m_70", "fruits", "Malic acid levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Malic_acid_fruits_pank4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Malic_acid_lt_pank4-1_fruits.RDS")
+
+# Malic acid leaves PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_70", "leaves", "Malic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+saveRDS(last_plot(), "Malic_acid_nominal_pank4-1_leaves.RDS")
+
+p2 <- make_box_dot_plot_cv("m_70", "leaves", "Malic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+saveRDS(last_plot(), "Malic_acid_cv_pank4-1_leaves.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_70", "leaves", "Malic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+saveRDS(leg, "Malic_acid_legend_pank4-1_leaves.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Malic_acid_leaves_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p2 <- make_box_dot_plot_levene("m_70", "leaves", "Malic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Malic_acid_leaves-panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Malic_acid_lt_pank4-1_leaves.RDS")
+
+
+# Malic acid fruits and leaves pank4-1 ------------------------------------
+
+p1 <- make_box_dot_plot("m_70", "fruits", "Malic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+p2 <- make_box_dot_plot_cv("m_70", "fruits", "Malic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+p3 <- make_box_dot_plot("m_70", "leaves", "Malic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p3
+
+p4 <- make_box_dot_plot_cv("m_70", "leaves", "Malic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_70", "leaves", "Malic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Malic_acid_fruits_leaves_panK4-1_combined.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+# Phenylalanine fruits log2-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_81", "fruits", "Phenylalanine nominal", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p1
+
+saveRDS(last_plot(), "Phenylalanine_nominal_log2-1_fruits.RDS")
+
+p2 <- make_box_dot_plot_cv("m_81", "fruits", "Phenylalanine CV", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p2
+
+saveRDS(last_plot(), "Phenylalanine_CV_log2-1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_81", "fruits", "Phenylalanine", "bottom", c("MoneyMaker","*log2-1*"), c(1,3)))
+leg
+
+saveRDS(leg, "Phenylalanine_leg_log2-1_fruits.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Phenylalanine_fruits_log2-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p2 <- make_box_dot_plot_levene("m_81", "fruits", "Phenylalanine levene transformed", "bottom", c("MoneyMaker","*log2-1*"), c(1,3))
+p2
+ggsave("Phenylalanine_fruits_log2-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Phenylalanine_lt_log2-1_fruits.RDS")
+
+
+# Phenylalanine leaves log2-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_81", "leaves", "Phenylalanine nominal", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p1
+saveRDS(last_plot(), "Phenylalanine_nominal_log2-1_leaves.RDS")
+
+p2 <- make_box_dot_plot_cv("m_81", "leaves", "Phenylalanine CV", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p2
+saveRDS(last_plot(), "Phenylalanine_cv_log2-1_leaves.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_81", "leaves", "Phenylalanine", "bottom", c("MoneyMaker","*log2-1*"), c(1,3)))
+leg
+saveRDS(leg, "Phenylalanine_leg_log2-1_leaves.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Phenylalanine_leaves_log2-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p2 <- make_box_dot_plot_levene("m_81", "leaves", "Phenylalanine levene transformed", "bottom", c("MoneyMaker","*log2-1*"), c(1,3))
+p2
+ggsave("Phenylalanine_leaves_log-2-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Phenylalanine_lt_log2-1_leaves.RDS")
+# Phenylalanine fruits  and leaves log2-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_81", "fruits", "Phenylalanine nominal", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p1
+
+p2 <- make_box_dot_plot_cv("m_81", "fruits", "Phenylalanine CV", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p2
+
+p3 <- make_box_dot_plot("m_81", "leaves", "Phenylalanine nominal", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p3
+
+p4 <- make_box_dot_plot_cv("m_81", "leaves", "Phenylalanine CV", "none", c("MoneyMaker","*log2-1*"), c(1,3))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_81", "leaves", "Phenylalanine", "bottom", c("MoneyMaker","*log2-1*"), c(1,3)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Phenylalanine_fruits_leaves_log2-1_combined.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+# Erythritol fruits PanK4-1-------------------------------------------------------
+
+
+p1 <- make_box_dot_plot("m_24", "fruits", "Erythritol nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+saveRDS(last_plot(), "Erythritol_nominal_pank4-1_fruits.RDS")
+
+p2 <- make_box_dot_plot_cv("m_24", "fruits", "Erythritol CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+saveRDS(last_plot(), "Erythritol_cv_pank4-1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_24", "fruits", "Erythritol", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+saveRDS(leg, "Erythritol_leg_pank4-1_fruits.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Erythritol_fruits_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p2 <- make_box_dot_plot_levene("m_24", "fruits", "Erythritol levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Erythritol_fruits_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Erythritol_lt_pank4-1_fruits.RDS")
+
+# Erythritol leaves PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_24", "leaves", "Erythritol nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+saveRDS(last_plot(), "Erythritol_nominal_pank4-1_leaves.RDS")
+
+p2 <- make_box_dot_plot_cv("m_24", "leaves", "Erythritol CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+saveRDS(last_plot(), "Erythritol_cv_pank4-1_leaves.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_24", "leaves", "Erythritol", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+saveRDS(leg, "Erythritol_leg_pank4-1_leaves.RDS")
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Erythritol_leaves_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p2 <- make_box_dot_plot_levene("m_24", "leaves", "Erythritol levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Erythritol_leaves_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Erythritol_lt_pank4-1_leaves.RDS")
+
+
+# Isocitrate fruits PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_63", "fruits", "Isocitrate nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+saveRDS(last_plot(), "Isocit_nominal_pank4-1_fruits.RDS")
+
+p2 <- make_box_dot_plot_cv("m_63", "fruits", "Isocitrate CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+saveRDS(last_plot(), "Isocit_cv_pank4-1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_63", "fruits", "Isocitrate", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+saveRDS(leg, "Isocit_leg_pank4-1_fruits.RDS")
+
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Isocitrate_fruits_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p2 <- make_box_dot_plot_levene("m_63", "fruits", "Isocitrate levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Isocitrate_fruits_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Isocit_lt_pank4-1_fruits.RDS")
+
+# Isocitrate leaves PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_63", "leaves", "Isocitrate nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+p2 <- make_box_dot_plot_cv("m_63", "leaves", "Isocitrate CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_63", "leaves", "Isocitrate", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Isocitrate_leaves_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p3 <- make_box_dot_plot_levene("m_63", "leaves", "Isocitrate levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Isocitrate_leaves_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Isocit_nominal_pank4-1_leaves.RDS")
+saveRDS(p2, "Isocit_cv_pank4-1_leaves.RDS")
+saveRDS(p3, "Isocit_lt_pank4-1_leaves.RDS")
+saveRDS(leg, "Isocit_leg_pank4-1_leaves.RDS")
+
+# Isocitrate fruits and leaves pank4-1 ------------------------------------
+
+p1 <- make_box_dot_plot("m_70", "fruits", "Isocitrate nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+p2 <- make_box_dot_plot_cv("m_70", "fruits", "Isocitrate CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+p3 <- make_box_dot_plot("m_70", "leaves", "Isocitrate nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p3
+
+p4 <- make_box_dot_plot_cv("m_70", "leaves", "Isocitrate CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_70", "leaves", "Isocitrate", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Isocitrate_fruits_leaves_panK4-1_combined.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+
+# GABA CV -----------------------------------------------------------------
+
+p4 <- make_box_dot_plot_cv("m_17", "fruits", "GABA", "bottom",  c("MoneyMaker","*panK4-1*"), c(1,2))
+p4
+
+saveRDS(last_plot(), "GABA_cv_pank4-1_fruits.RDS")
+
+# beta-alanine CV -----------------------------------------------------------------
+
+p4 <- make_box_dot_plot_cv("m_8", "fruits", "beta-alanine", "bottom",  c("MoneyMaker","*panK4-1*"), c(1,2))
+p4
+
+saveRDS(last_plot(), "beta-alanine_cv_pank4-1_fruits.RDS")
+
+# Valine CV -----------------------------------------------------------------
+
+p4 <- make_box_dot_plot_cv("m_122", "fruits", "Valine", "bottom",  c("MoneyMaker","*panK4-1*"), c(1,2))
+p4
+
+saveRDS(last_plot(), "Valine_cv_pank4-1_fruits.RDS")
+
+# Succinic acid fruits PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_102", "fruits", "Succinic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+
+p2 <- make_box_dot_plot_cv("m_102", "fruits", "Succinic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_102", "fruits", "Succinic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Succinic acid_fruits_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p3 <- make_box_dot_plot_levene("m_102", "fruits", "Succinic acid levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Succinic acid_fruits_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Succinic acid_nominal_pank4-1_fruits.RDS")
+saveRDS(p2, "Succinic acid_cv_pank4-1_fruits.RDS")
+saveRDS(p3, "Succinic acid_lt_pank4-1_fruits.RDS")
+saveRDS(leg, "Succinic acid_leg_pank4-1_fruits.RDS")
+
+# Succinic acid leaves PanK4-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_102", "leaves", "Succinic acid nominal", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p1
+
+p2 <- make_box_dot_plot_cv("m_102", "leaves", "Succinic acid CV", "none", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_102", "leaves", "Succinic acid", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Succinic acid_leaves_panK4-1_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p3 <- make_box_dot_plot_levene("m_102", "leaves", "Succinic acid levene transformed", "bottom", c("MoneyMaker","*panK4-1*"), c(1,2))
+p2
+ggsave("Succinic acid_leaves_panK4-1_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Succinic acid_nominal_pank4-1_leaves.RDS")
+saveRDS(p2, "Succinic acid_cv_pank4-1_leaves.RDS")
+saveRDS(p3, "Succinic acid_lt_pank4-1_leaves.RDS")
+saveRDS(leg, "Succinic acid_leg_pank4-1_leaves.RDS")
+
+# Glucose fruits transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_44", "fruits", "Glucose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_44", "fruits", "Glucose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_44", "fruits", "Glucose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Glucose_fruits_combined_transp1-1.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p3 <- make_box_dot_plot_levene("m_44", "fruits", "Glucose levene transformed", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Glucose_fruits_box_dot_plot_levene_transp1-1.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Glucose_nominal_transp1-1_fruits.RDS")
+saveRDS(p2, "Glucose_cv_transp1-1_fruits.RDS")
+saveRDS(p3, "Glucose_lt_transp1-1_fruits.RDS")
+saveRDS(leg, "Glucose_leg_transp1-1_fruits.RDS")
+# Glucose leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_44", "leaves", "Glucose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_44", "leaves", "Glucose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_44", "leaves", "Glucose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Glucose_leaves_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p3 <- make_box_dot_plot_levene("m_44", "leaves", "Glucose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Glucose_leaves_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Glucose_nominal_transp1-1_leaves.RDS")
+saveRDS(p2, "Glucose_cv_transp1-1_leaves.RDS")
+saveRDS(p3, "Glucose_lt_transp1-1_leaves.RDS")
+saveRDS(leg, "Glucose_leg_transp1-1_leaves.RDS")
+
+
+# Glucose fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_44", "leaves", "Glucose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_44", "leaves", "Glucose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_44", "fruits", "Glucose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_44", "fruits", "Glucose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_44", "fruits", "Glucose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Glucose_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+# Maltose fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_72", "leaves", "Maltose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_72", "leaves", "Maltose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_72", "fruits", "Maltose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_72", "fruits", "Maltose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+saveRDS(last_plot(), "Maltose_cv_transp1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_72", "fruits", "Maltose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Maltose_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+# Fructose-6-phosphate fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_27", "leaves", "Fructose-6-phosphate nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_27", "leaves", "Fructose-6-phosphate CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_27", "fruits", "Fructose-6-phosphate nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_27", "fruits", "Fructose-6-phosphate CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+
+saveRDS(last_plot(), "Fructose_6_phosphate_cv_transp1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_27", "fruits", "Fructose-6-phosphate", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Fructose-6-phosphate_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+p3 <- make_box_dot_plot_levene("m_72", "fruits", "Fructose-6-phosphate levene transformed", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Fructose_fruits_box_dot_plot_levene_transp1-1.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+
+# Glucose-6-phosphate fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_41", "leaves", "Glucose-6-phosphate nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_41", "leaves", "Glucose-6-phosphate CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_41", "fruits", "Glucose-6-phosphate nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_41", "fruits", "Glucose-6-phosphate CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+
+saveRDS(last_plot(), "Glucose_6_phosphate_cv_transp1_fruits.RDS")
+
+leg <- get_legend(make_box_dot_plot("m_41", "fruits", "Glucose-6-phosphate", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Glucose-6-phosphate_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+
+# Fructose fruits transp1-1-------------------------------------------------------
+
+
+p1 <- make_box_dot_plot("m_29", "fruits", "Fructose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+
+p2 <- make_box_dot_plot_cv("m_29", "fruits", "Fructose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_29", "fruits", "Fructose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Fructose_fruits_combined_transp1-1.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p3 <- make_box_dot_plot_levene("m_29", "fruits", "Fructose levene transformed", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Fructose_fruits_box_dot_plot_levene_transp1-1.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Fructose_nominal_pank4-1_fruits.RDS")
+saveRDS(p2, "Fructose_cv_pank4-1_fruits.RDS")
+saveRDS(p3, "Fructose_lt_pank4-1_fruits.RDS")
+saveRDS(leg, "Fructose_leg_pank4-1_fruits.RDS")
+# Fructose leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_29", "leaves", "Fructose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_29", "leaves", "Fructose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_29", "leaves", "Fructose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Fructose_leaves_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p3 <- make_box_dot_plot_levene("m_29", "leaves", "Fructose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Fructose_leaves_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Fructose_nominal_pank4-1_leaves.RDS")
+saveRDS(p2, "Fructose_cv_pank4-1_leaves.RDS")
+saveRDS(p3, "Fructose_lt_pank4-1_leaves.RDS")
+saveRDS(leg, "Fructose_leg_pank4-1_leaves.RDS")
+# Fructose fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_29", "leaves", "Fructose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_29", "leaves", "Fructose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_29", "fruits", "Fructose nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_29", "fruits", "Fructose CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_29", "fruits", "Fructose", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Fructose_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+# Tyrosine fruits transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_112", "fruits", "Tyrosine nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+
+p2 <- make_box_dot_plot_cv("m_112", "fruits", "Tyrosine CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_112", "fruits", "Tyrosine", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Tyrosine_fruits_combined_transp1-1.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+p3 <- make_box_dot_plot_levene("m_112", "fruits", "Tyrosine levene transformed", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Tyrosine_fruits_box_dot_plot_levene_transp1-1.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+saveRDS(p1, "Tyrosine_nominal_pank4-1_fruits.RDS")
+saveRDS(p2, "Tyrosine_cv_pank4-1_fruits.RDS")
+saveRDS(p3, "Tyrosine_lt_pank4-1_fruits.RDS")
+saveRDS(leg, "Tyrosine_leg_pank4-1_fruits.RDS")
+# Tyrosine leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_112", "leaves", "Tyrosine nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_112", "leaves", "Tyrosine CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+leg <- get_legend(make_box_dot_plot("m_112", "leaves", "Tyrosine", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Tyrosine_leaves_combined.png",width = 16.5, height = 10, units = "cm", dpi = 300)
+
+
+p3 <- make_box_dot_plot_levene("m_112", "leaves", "Tyrosine", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+ggsave("Tyrosine_leaves_box_dot_plot_levene.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+saveRDS(p1, "Tyrosine_nominal_pank4-1_leaves.RDS")
+saveRDS(p2, "Tyrosine_cv_pank4-1_leaves.RDS")
+saveRDS(p3, "Tyrosine_lt_pank4-1_leaves.RDS")
+saveRDS(leg, "Tyrosine_leg_pank4-1_leaves.RDS")
+
+# Tyrosine fruits leaves transp1-1-------------------------------------------------------
+
+p1 <- make_box_dot_plot("m_112", "leaves", "Tyrosine nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p1
+
+p2 <- make_box_dot_plot_cv("m_112", "leaves", "Tyrosine CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p2
+
+p3 <- make_box_dot_plot("m_112", "fruits", "Tyrosine nominal", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p3
+
+p4 <- make_box_dot_plot_cv("m_112", "fruits", "Tyrosine CV", "none", c("MoneyMaker","*transp1-1*"), c(1,4))
+p4
+
+leg <- get_legend(make_box_dot_plot("m_112", "fruits", "Tyrosine", "bottom", c("MoneyMaker","*transp1-1*"), c(1,4)))
+leg
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Tyrosine_fruits_leaves_combined_transp1-1.png",width = 16.5, height = 20, units = "cm", dpi = 300)
+
+
+# Clustering k-means---------------------------------------------------------
+
+library(pheatmap)
+
+heat_base <- fc_1 %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker"),
+    tissue == "fruits")
+
+heat.GC <- heat_base %>% 
+  group_by(Compound_Name, met) %>% 
+  mutate(log_norm = log2(mean_fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm)),
+         z_score = (mean_fc - mean(mean_fc))/(max(mean_fc)-min(mean_fc))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(Compound_Name, Compound_Class, met),
+              names_from = group,
+              values_from = z_score) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+rownames(heat.GC) <- heat.GC$met
+
+mat.heat.GC <- heat.GC %>% 
+  select(contains("0.4"), contains("0.6"),
+         contains("0.8"),
+         contains("1"),
+         contains("leaves"), contains("fruits")) %>% as.matrix()
+
+set.seed(484)
+k_means <- kmeans(mat.heat.GC, centers = 6)
+
+clusters <- k_means$cluster
+
+clust_tidy <- tibble("clust" = clusters) %>% 
+  mutate(met = names(clusters))
+
+heat_base %>% 
+  left_join(clust_tidy) %>% 
+  left_join(classes_tidy) %>% 
+  group_by(Compound_Name, met) %>% 
+  mutate(log_norm = log2(mean_fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm)),
+         z_score = (mean_fc - mean(mean_fc))/(max(mean_fc)-min(mean_fc))) %>% 
+  ungroup() %>% 
+  group_by(clust, treatment) %>% 
+  mutate(n = n(),
+         ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other"))) %>% 
+  ungroup() %>% 
+  ggplot(aes(x = treatment, y = z_score, color = ChEBI)) +
+  geom_line(aes(group = met)) +
+  geom_label(aes(x = 0.7, y = -0.6, label = n), inherit.aes = F) +
+  facet_wrap(vars(clust), nrow = 2) +
+  scale_color_manual(values = cb_scale[5:8]) +
+  com_theme +
+  ylab("range-scaled level") +
+  theme(legend.position = "bottom",
+        strip.text = element_blank())
+
+ggsave("clusters_MoneyMaker.pdf",
+       width = 8.25,
+       height = 8.25,
+       units = "cm",
+       dpi = 300)
+
+saveRDS(last_plot(), "clusters_MoneyMaker.RDS")
+
+annotation_row <- heat.GC %>% 
+  select(Compound_Class)
+
+rownames(annotation_row) <- heat.GC$met
+
+annotation_col <- heat_base %>% 
+  distinct(tissue, treatment, genotype) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>%  
+  as.data.frame()
+
+rownames(annotation_col) <- annotation_col$group
+
+annotation_col <- annotation_col %>% 
+  select(-group, treatment, tissue, genotype)
+
+heat.GC_signif <- heat_base %>% 
+  distinct(tissue, treatment, alias, genotype, met) %>% 
+  #filter(genotype != "MoneyMaker") %>% 
+  left_join(sig_GC, by = c("met", "tissue", "treatment", "alias" = "group2")) %>% 
+  #left_join(sig_mets) %>% 
+  #filter(sig == T) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_")),
+         signif = if_else(p.value >= 0.05| is.na(p.value), "","X")) %>%
+  pivot_wider(id_cols = c(met),
+              names_from = group,
+              values_from = signif) %>% 
+  left_join(met_dat) %>% 
+  #left_join(GCid_classes) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+heat_cols <- colnames(mat.heat.GC)
+
+rownames(heat.GC_signif) <- heat.GC_signif$met
+
+mat.heat.GC_signif <- heat.GC_signif %>% 
+  select(all_of(heat_cols)) %>% as.matrix()
+
+ann_colors = list(
+  tissue = c(fruits = "red",leaves = "darkgreen"),
+  genotype = c(MoneyMaker = cb_scale[1], `*panK4-1*` = "brown", `log2-1` = "blue", `*transp1-1*` = cb_scale[4]),
+  treatment = c("0.4" = "red", "0.6" = "orange", "0.8" = "yellow", "1" = "green"))
+
+pheatmap.GC <- pheatmap(mat.heat.GC,
+                        color = colorRampPalette(c("blue", "white", "red"))(65),
+                        #cellwidth = 16,
+                        #cellheight = 4,
+                        breaks = seq(-3.25, 3.25, 0.1),
+                        clustering_distance_rows = dist((mat.heat.GC), method = "euclidean"),
+                        cluster_rows = T,
+                        cluster_cols = F,
+                        annotation_names_row = F,
+                        show_rownames = F,
+                        annotation_row = annotation_row,
+                        annotation_col = annotation_col,
+                        display_numbers = mat.heat.GC_signif,
+                        number_color = "black",
+                        fontsize_number = 6,
+                        # annotation_colors = ann_colors,
+                        #filename = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                        #                "cmQTL_val_1_heatmap_rel_tissue_wt.jpg",
+                        #                 sep = "_")
+)
+
+dev.off()
+
+
+# Correlation network -------------------------------------------------------------
+
+cor_theme <- theme(axis.text = element_blank(),
+                   axis.title = element_blank(),
+                   panel.background = element_rect(fill = "white"),
+                   strip.text = element_text(size = 10, family = "sans"),
+                   text = element_text(size = 6, family = "sans"),
+                   legend.position = "",
+                   legend.title = element_text(size = 8, family = "sans"),
+                   legend.text = element_text(size = 6, family = "sans"),
+                   plot.margin = unit(c(1,1,1,1), "pt"),
+                   legend.box.margin = margin(t = 0, r = 0, b = 0, l = 0 , unit = "mm"),
+                   legend.margin = margin(t = 0, r = 0, b = 0, l = 0 , unit = "mm"))
+
+library(viridis)
+library(igraph)
+library(ggnetwork)
+library(jmuOutlier)
+library(ggraph)
+
+#correlation matrices are preformed
+
+## Loop start --------------------------------------------------------------
+
+cor_base <- fc_1_ind %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker"),
+    tissue == "fruits")
+
+cor.GC <- cor_base %>% 
+  group_by(met) %>% 
+  mutate(log_norm = log2(fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(genotype, treatment, LIMS_ID),
+              names_from = met,
+              values_from = fc) %>% 
+  #arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+cor.GC_numeric <- cor.GC %>% 
+  select(starts_with("m_"))
+
+g_vec <- genotypes$genotype %>%
+  sort() %>%
+  as.character()
+
+g_vec_lab <- genotypes %>%
+  arrange(genotype) %>% 
+  select(genotype_label) %>% 
+  as_vector()
+
+setwd(current)
+latest <- str_sort(str_extract(list.files(pattern = "^\\d{6}_correlation$"),
+                               pattern = "^\\d{6}_correlation"),
+                   decreasing = T)[[1]]
+
+latest_Correlation <- str_c(current, "/", latest)
+
+setwd(latest_Correlation)
+
+## Start -------------------------------------------------------------------
+
+
+for(g in seq_along(genotypes$genotype)) {
+  print(str_c("starting with", g_vec_lab[[g]], sep = " "))
+
+latest_Correlation_date <- str_extract(latest, pattern = "^\\d{6}")
+
+corr_mat <- read_csv(list.files()[str_detect(list.files(),
+                                             str_c("corr_mat_",
+                                                   g_vec_lab[[g]],
+                                                   ".csv"))])
+
+corr_mat_comb <- corr_mat %>% 
+  filter(abs(cor_val) >= 0.75, p_adj <= 0.05) %>% 
+  mutate(Correlation = as_factor(if_else(pos_neg == "pos", "+", "-"))) %>% 
+  mutate(Correlation = fct_relevel(Correlation, c("+", "-")),
+         weight = abs(cor_val))
+
+#classes_tidy <- met_dat %>% 
+#  mutate(`ChEBI simplified` = if_else(str_detect(Compound_Class,"carbohydrate"),"carbohydrate or derivative",
+#                                      if_else(str_detect(Compound_Class,"amino_acid"), "amino acid or derivative",
+#                                              if_else(str_detect(Compound_Class,"carboxylic_acid"), "carboxylic acid",
+#                                                      "other"))))
+
+annotation_col_corr <- classes_tidy %>% 
+  select(ChEBI) %>% 
+  as.data.frame()
+
+rownames(annotation_col_corr) <- classes_tidy$met
+
+ann_colors_corr = list(
+  ChEBI= c("AA"= cb_scale[5],
+                        "CH" = cb_scale[6],
+                        "CA" = cb_scale[7],
+                        "other" = cb_scale[8])
+)
+
+
+
+ann_colors_graph = tibble(ChEBI= names(ann_colors_corr$ChEBI),
+                          color = ann_colors_corr$ChEBI)
+
+node_cols <- tibble(met = colnames(cor.GC_numeric)) %>% 
+  left_join(classes_tidy) %>% 
+  left_join(ann_colors_graph) %>% 
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other")))
+
+node_col_vec <- node_cols$color
+
+
+net_raw <- graph_from_data_frame(corr_mat_comb, vertices = node_cols, directed = F)
+
+set.seed(10000)
+
+if (g_vec_lab[[g]] == "MoneyMaker") {
+  layout<- layout_with_graphopt(net_raw , charge = 0.5,
+                                spring.length = 1,
+                                spring.constant = 100,
+                                mass = 100)
+  #layout <- layout_with_fr(net_raw)
+  
+  net_metric <- tibble(parameters = c("Number of nodes",
+                                      "Number of edges",
+                                      "Average node level centrality degree",
+                                      "Graph level centrality degree",
+                                      "Average node level centrality closeness",
+                                      "Average node level centrality betweenness",
+                                      "Graph level centrality betweenness",
+                                      "Graph diameter",
+                                      "Average distance"
+                                      ))
+  
+}
+
+node_cols <- tibble(met = colnames(cor.GC_numeric)) %>% 
+  left_join(classes_tidy) %>% 
+  left_join(ann_colors_graph)%>% 
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other")))
+
+node_col_vec <- node_cols$color
+
+
+net_raw <- graph_from_data_frame(corr_mat_comb, vertices = node_cols, directed = F)
+
+ggnetwork(net_raw, layout = layout) %>% 
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other"))) %>% 
+  ggplot(aes(x = x, y = y, xend = xend, yend = yend)) + 
+  geom_edges(aes(color = Correlation),curvature = 0.15) +
+  geom_nodes(aes(fill = ChEBI), color = "black",  shape = 21, size = 2) +
+  geom_nodetext(aes(label = Compound_Name), size = 2) +
+  #geom_nodetext(aes(label = if_else(Compound_Name == "malic_acid","Malic acid", ""))) +
+  theme_blank() +
+  scale_fill_manual(values = cb_scale[5:8], aesthetics = "fill") +
+  scale_color_manual(values = c(cb_scale[3], cb_scale[1]), aesthetics = "color") +
+  cor_theme +
+  theme(legend.position = c(0.5, 0.5))
+
+ggsave(str_c(g_vec_lab[[g]],
+             "_Correlation_network_wt_layout.pdf"),
+       width = 8.25,
+       height = 8.25,
+       units = "cm",
+       dpi = 300)
+
+ggnetwork(net_raw, layout = layout) %>% 
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other"))) %>% 
+  ggplot(aes(x = x, y = y, xend = xend, yend = yend)) + 
+  geom_edges(aes(color = Correlation),curvature = 0.15) +
+  geom_nodes(aes(fill = ChEBI), color = "black",  shape = 21, size = 2) +
+  theme_blank() +
+  scale_fill_manual(values = cb_scale[5:8], aesthetics = "fill") +
+  scale_color_manual(values = c(cb_scale[3], cb_scale[1]), aesthetics = "color") +
+  cor_theme +
+  theme(legend.position = "right") +
+  scale_x_continuous(expand = expansion(mult = 0.02)) +
+  scale_y_continuous(expand = expansion(mult = 0.02))
+
+ggsave(str_c(g_vec_lab[[g]],
+             "_Correlation_network_wt_layout_unlab.pdf"),
+       width = 8.25,
+       height = 8.25,
+       units = "cm",
+       dpi = 300)
+
+saveRDS(last_plot(),str_c(g_vec_lab[[g]],
+                          "_Correlation_network_wt_layout_unlab.RDS"))
+
+
+### Own layout and node filtered --------------------------------------------
+
+  node_cols <- tibble(met = colnames(cor.GC_numeric)) %>% 
+    left_join(classes_tidy) %>% 
+    left_join(ann_colors_graph) %>% 
+    filter(met %in% corr_mat_comb$met1 | met %in% corr_mat_comb$met2) %>%
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other")))
+  
+  net_raw <- graph_from_data_frame(corr_mat_comb, vertices = node_cols, directed = F)
+  
+  net_metric <- net_metric %>% add_column(.after = g,
+                                          "current" =
+                                            c(net_raw %>% length(),
+                                              edge.attributes(net_raw)$cor_val %>% length(),
+                                              degree(net_raw) %>% mean(),
+                                              centr_degree(net_raw)$centralization,
+                                              centr_clo(net_raw)$res %>% mean(na.rm = T), #returns NaN for some nodes
+                                              centr_betw(net_raw)$res %>% mean(),
+                                              centr_betw(net_raw)$centralization,
+                                              diameter(net_raw), # does not work with negative weight scores
+                                              mean_distance(net_raw) # does not work with negative weight scores
+                                            )
+  )
+  
+  colnames(net_metric)[g+1] <- g_vec_lab[[g]]
+  set.seed(10000)
+  layout_own <- layout_with_fr(net_raw)
+  
+  ggnetwork(net_raw, layout = layout_own) %>% 
+    mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other"))) %>% 
+    ggplot(aes(x = x, y = y, xend = xend, yend = yend)) + 
+    geom_edges(aes(color = Correlation),curvature = 0.15) +
+    geom_nodes(aes(fill = ChEBI), color = "black",  shape = 21, size = 2) +
+    geom_nodetext(aes(label = Compound_Name), size = 2) +
+    #geom_nodetext(aes(label = if_else(Compound_Name == "malic_acid","Malic acid", ""))) +
+    theme_blank() +
+    scale_fill_manual(values = cb_scale[5:8], aesthetics = "fill") +
+    scale_color_manual(values = c(cb_scale[3], cb_scale[1]), aesthetics = "color") +
+    cor_theme +
+    theme(legend.position = c(0.5, 0.5))
+  
+  ggsave(str_c(g_vec_lab[[g]],
+               "_Correlation_network_own.pdf"),
+         width = 8.25,
+         height = 8.25,
+         units = "cm",
+         dpi = 300)
+  
+  ggnetwork(net_raw, layout = layout_own) %>% 
+    mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other"))) %>% 
+    ggplot(aes(x = x, y = y, xend = xend, yend = yend)) + 
+    geom_edges(aes(color = Correlation),curvature = 0.15) +
+    geom_nodes(aes(fill = ChEBI), color = "black",  shape = 21, size = 2) +
+    #geom_nodetext(aes(label = Compound_Name), size = 2) +
+    #geom_nodetext_repel(aes(label = if_else(Compound_Name == "D-maltose","Malt", "")),inherit.aes = T) +
+    theme_blank() +
+    scale_fill_manual(values = cb_scale[5:8], aesthetics = "fill") +
+    scale_color_manual(values = c(cb_scale[3], cb_scale[1]), aesthetics = "color") +
+    cor_theme +
+    theme(legend.position = "right") +
+    scale_x_continuous(expand = expansion(mult = 0.02)) +
+    scale_y_continuous(expand = expansion(mult = 0.02))
+  
+  ggsave(str_c(g_vec_lab[[g]],
+               "_Correlation_network_own_unlab.pdf"),
+         width = 8.25,
+         height = 8.25,
+         units = "cm",
+         dpi = 300)
+  
+  saveRDS(last_plot(), str_c(g_vec_lab[[g]],
+               "_Correlation_network_own_unlab.RDS"))
+
+}
+
+## Loop end ----------------------------------------------------------------
+
+write_csv(net_metric, "Network_topology_metrics.csv")
+
+
+stop("current_end")
+
+
+# Test edgebundling -------------------------------------------------------
+
+library(edgebundle)
+test <- edge_bundle_force(net_raw, layout_own, compatibility_threshold = 0.2)
+
+ggplot(test) +
+  geom_path(aes(x = x, y = y, group = group)) +
+  geom_point(data = layout_own %>% as.data.frame(), aes(x = V1, y = V2), color = "red", size = 2)
+
+ggnetwork(net_raw, layout = test) %>% 
+  ggplot(aes(x = x, y = y, group = group,  xend = xend, yend = yend)) + 
+  geom_edges(aes(color = Correlation),curvature = 0.15) +
+  geom_nodes(aes(fill = ChEBI), color = "black",  shape = 21, size = 2) +
+  #geom_nodetext(aes(label = Compound_Name), size = 2) +
+  #geom_nodetext_repel(aes(label = if_else(Compound_Name == "D-maltose","Malt", "")),inherit.aes = T) +
+  theme_blank() +
+  scale_fill_manual(values = cb_scale[5:8], aesthetics = "fill") +
+  scale_color_manual(values = c(cb_scale[3], cb_scale[1]), aesthetics = "color") +
+  cor_theme +
+  theme(legend.position = "right") +
+  scale_x_continuous(expand = expansion(mult = 0.02)) +
+  scale_y_continuous(expand = expansion(mult = 0.02))
+
+# Test combine ------------------------------------------------------------
+
+#other methods
+
+set.seed(200)
+plot.igraph(net_raw, vertex.color = node_col_vec, vertex.label = "")
+
+ggraph(net_raw, layout = layout) +
+  geom_edge_arc0(aes(color = pos_neg), strength = 0.1) +
+  geom_node_point(aes(color = `ChEBI simplified`), size = 3)
+#  geom_node_text(aes(label = name))
+
+corr_mat_plot <- corr_mat_long %>% 
+  filter(abs(cor_val) >= 0.75) %>% 
+  select(met1, met2) %>% 
+  as.matrix()
+
+corr_mat_plot <- corr_mat
+corr_mat_plot[abs(corr_mat_plot)<0.5] <- 0
+
+net_raw <- graph_from_edgelist(corr_mat_plot, directed = F)
+net_raw <- graph_from_adjacency_matrix(corr_mat_plot,
+                                       weighted=T,
+                                       mode="undirected",
+                                       diag=F)
+
+stop <- T
+
+if (stop == T) {
+  stop(print("Automatically ended"))
+p1 <- make_box_dot_plot("m_81", "fruits", "Phenylalanine", "none", c("MoneyMaker","*log2-1*"))
+p1
+
+p2 <- make_box_dot_plot_levene("m_81", "fruits", "Phenylalanine", "none", c("MoneyMaker","*log2-1*"))
+p2
+
+p3 <- make_box_dot_plot_cv("m_81", "fruits", "Phenylalanine", "none", c("MoneyMaker","*log2-1*"))
+p3
+
+leg46 <- get_legend(make_box_dot_plot("m_81", "fruits", "Phenylalanine", "bottom", c("MoneyMaker","*log2-1*")))
+leg46
+
+main_plot <- plot_grid(p1,p2, nrow = 2, labels = "AUTO",rel_heights = c(1,1))
+main_plot
+
+main_plot_2 <- plot_grid(main_plot, p3, ncol = 2, labels = list("", "C"),rel_widths = c(3,1))
+main_plot_2
+
+comp_plot <- plot_grid(main_plot_2, leg46, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Phenylalanine_combined.png",width = 15.8, height = 16, units = "cm", dpi = 300)
+
+#sugars?
+
+p1 <- make_box_dot_plot("m_72", "fruits", "Maltose", "none", c("MoneyMaker","*transp1-1*"))
+p1
+
+p2 <- make_box_dot_plot_levene("m_72", "fruits", "Maltose", "none", c("MoneyMaker","*transp1-1*"))
+p2
+
+p3 <- make_box_dot_plot_cv("m_72", "fruits", "Maltose", "none", c("MoneyMaker","*transp1-1*"))
+p3
+
+leg46 <- get_legend(make_box_dot_plot("m_72", "fruits", "Maltose", "bottom", c("MoneyMaker","*transp1-1*")))
+leg46
+
+main_plot <- plot_grid(p1,p2, nrow = 2, labels = "AUTO",rel_heights = c(1,1))
+main_plot
+
+main_plot_2 <- plot_grid(main_plot, p3, ncol = 2, labels = list("", "C"),rel_widths = c(3,1))
+main_plot_2
+
+
+ggsave("Maltose_combined.png",width = 15.8, height = 16, units = "cm", dpi = 300)
+
+
+
+# Recycling ---------------------------------------------------------------
+# Correlation -------------------------------------------------------------
+
+library(viridis)
+library(igraph)
+library(ggnetwork)
+library(jmuOutlier)
+
+cor_base <- fc_1_ind %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker"),
+    tissue == "fruits")
+
+cor.GC <- cor_base %>% 
+  group_by(met) %>% 
+  mutate(log_norm = log2(fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(genotype, treatment, LIMS_ID),
+              names_from = met,
+              values_from = fc) %>% 
+  #arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+cor.GC_numeric <- cor.GC %>% 
+  select(starts_with("m_"))
+
+corr_mat <- cor(cor.GC_numeric, method = "spearman")
+
+for (i in seq_along(colnames(cor.GC_numeric))) {
+  
+}
+corr_p <- perm.cor.test(x = cor.GC_numeric, method = "spearman")
+
+
+classes_tidy <- met_dat %>% 
+  mutate(`ChEBI simplified` = if_else(str_detect(Compound_Class,"carbohydrate"),"carbohydrate or derivative",
+                                      if_else(str_detect(Compound_Class,"amino_acid"), "amino acid or derivative",
+                                              if_else(str_detect(Compound_Class,"carboxylic_acid"), "carboxylic acid",
+                                                      "other"))))
+
+annotation_row_corr <- classes_tidy %>% 
+  select(`ChEBI simplified`) %>% 
+  as.data.frame()
+
+rownames(annotation_row_corr) <- classes_tidy$met
+
+annotation_col_corr <- classes_tidy %>% 
+  select(`ChEBI simplified`) %>% 
+  as.data.frame()
+
+rownames(annotation_col_corr) <- classes_tidy$met
+
+
+ann_colors_corr = list(
+  `ChEBI simplified`= c("amino acid or derivative"= vir_scale[1],
+                        "carbohydrate or derivative" = vir_scale[2],
+                        "carboxylic acid" = vir_scale[3],
+                        "other" = vir_scale[4])
+)
+
+pheatmap <- pheatmap(corr_mat,
+                     color = colorRampPalette(c("blue","white", "red"))(7),
+                     cellwidth = 4,
+                     cellheight = 4,
+                     breaks = seq(-1.05, 1.05, 0.3),
+                     cluster_rows = T,
+                     cluster_cols = T,
+                     annotation_names_row = F,
+                     annotation_names_col = F,
+                     show_rownames = F,
+                     show_colnames = F,
+                     annotation_row = annotation_row_corr,
+                     annotation_col = annotation_col_corr,
+                     annotation_legend =T,
+                     legend = F,
+                     display_numbers = F,
+                     number_color = "black",
+                     fontsize_number = 6,
+                     annotation_colors = ann_colors_corr,
+                     #filename = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                     #                  "metabolite_correlation_heatmap_simple.jpg",
+                     #                  sep = "_")
+)
+
+library(igraph)
+
+ann_colors_graph = tibble(`ChEBI simplified`= names(ann_colors_corr$`ChEBI simplified`),
+                          color = ann_colors_corr$`ChEBI simplified`)
+
+node_cols <- tibble(met = colnames(cor.GC_numeric)) %>% 
+  left_join(classes_tidy) %>% 
+  left_join(ann_colors_graph)
+
+node_col_vec <- node_cols$color
+
+
+
+corr_mat_long <- corr_mat %>% 
+  as_tibble(rownames = "met1") %>% 
+  pivot_longer(cols = starts_with("m_"),
+               values_to = "cor_val",
+               names_to = "met2") %>% 
+  mutate(forward = str_c(met1, met2, sep = "_"),
+         dup_check = str_c(sort(str_extract_all(forward,
+                                                "(?<=m_)\\d+",
+                                                simplify = T)),
+                           collapse = "_")) %>% 
+  filter(abs(cor_val) >= 0.5, !met1 == met2)
+
+corr_mat_plot <- corr_mat_long %>% 
+  select(met1, met2) %>% 
+  as.matrix()
+
+corr_mat_plot[abs(corr_mat_plot)<0.5] <- 0
+
+net_raw <- graph_from_edgelist(corr_mat_plot, directed = F)
+
+net_raw <- graph_from_adjacency_matrix(corr_mat_plot,
+                                       weighted=T,
+                                       mode="undirected",
+                                       diag=F)
+set.seed(200)
+plot.igraph(net_raw, vertex.color = node_col_vec, vertex.label = "")
+
+edges <- E(net_raw) #%>% 
+names() %>% 
+  tibble()
+
+V(net_raw)$Compound_Class <- node_cols$`ChEBI simplified`
+E(net_raw)$cor <- corr_mat_plot
+
+test <- layout.fruchterman.reingold(net_raw)
+
+ggraph(net_raw, layout = test) +
+  geom_edge_link0(aes(color = cor)) +
+  geom_node_point(aes(color = Compound_Class), size = 3) 
+
+
+
+# Correlation network -------------------------------------------------------------
+
+cor_theme <- theme(axis.text = element_blank(),
+                   axis.title = element_blank(),
+                   panel.background = element_rect(fill = "white"),
+                   strip.text = element_text(size = 10, family = "sans"),
+                   text = element_text(size = 6, family = "sans"),
+                   legend.position = "",
+                   legend.title = element_text(size = 8, family = "sans"),
+                   legend.text = element_text(size = 6, family = "sans"),
+                   plot.margin = unit(c(1,1,1,1), "pt"),
+                   legend.box.margin = margin(t = 0, r = 0, b = 0, l = 0 , unit = "mm"),
+                   legend.margin = margin(t = 0, r = 0, b = 0, l = 0 , unit = "mm"))
+
+
+library(viridis)
+library(igraph)
+library(ggnetwork)
+library(jmuOutlier)
+library(ggraph)
+
+
+
+cor_base <- fc_1_ind %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker"),
+    tissue == "fruits")
+
+cor.GC <- cor_base %>% 
+  group_by(met) %>% 
+  mutate(log_norm = log2(fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(genotype, treatment, LIMS_ID),
+              names_from = met,
+              values_from = fc) %>% 
+  #arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+cor.GC_numeric <- cor.GC %>% 
+  select(starts_with("m_"))
+
+corr_mat <- cor(cor.GC_numeric, method = "spearman")
+
+x <- vector(mode = "list", length = length(colnames(cor.GC_numeric)))
+names(x) <- colnames(cor.GC_numeric)
+
+start <- Sys.time()
+for (i in seq_along(colnames(cor.GC_numeric))) {
+  #names(x[[i]]) <- colnames(cor.GC_numeric)
+  for (n in seq_along(colnames(cor.GC_numeric))) {
+    if(i > n) {
+      x[[i]][[n]] <- NA
+    } else {
+      x[[i]][[n]] <- perm.cor.test(cor.GC_numeric[[i]], cor.GC_numeric[[n]],
+                                   method = "spearman",
+                                   num.sim = 100
+      )$p.value
+    }
+  }
+}
+
+stop <- Sys.time()
+stop-start
+
+for (i in seq_along(colnames(cor.GC_numeric))) {
+  names(x[[i]]) <- colnames(cor.GC_numeric)
+}
+
+corr_p <- x %>% 
+  reduce(bind_rows)
+
+corr_p_long <- corr_p %>% 
+  mutate(met1 = colnames(cor.GC_numeric)) %>% 
+  pivot_longer(cols = starts_with("m_"),
+               values_to = "cor_p_val",
+               names_to = "met2") %>% 
+  mutate(#cor_num = row_number(),
+    met1_num = as.numeric(str_extract(met1,"(?<=m_)\\d+")),
+    met2_num = as.numeric(str_extract(met2,"(?<=m_)\\d+"))) %>% 
+  mutate(small = if_else(met1_num < met2_num, met1_num, met2_num),
+         big = if_else(met1_num > met2_num, met1_num, met2_num),
+         sorted = str_c(small, big, sep = "_")) %>% 
+  filter(!is.na(cor_p_val)) %>% 
+  #distinct(sorted, .keep_all = T) %>% 
+  mutate(p_adj = p.adjust(cor_p_val, method = "fdr")) %>% 
+  filter(!met1 == met2) %>% 
+  # group_by(cor_num) %>% 
+  mutate(cor_p_val_plot = if_else(p_adj > 0.05, 1, p_adj))
+
+corr_mat_long <- corr_mat %>% 
+  as_tibble(rownames = "met1") %>% 
+  pivot_longer(cols = starts_with("m_"),
+               values_to = "cor_val",
+               names_to = "met2") %>% 
+  mutate(#cor_num = row_number(),
+    met1_num = as.numeric(str_extract(met1,"(?<=m_)\\d+")),
+    met2_num = as.numeric(str_extract(met2,"(?<=m_)\\d+"))) %>% 
+  mutate(small = if_else(met1_num < met2_num, met1_num, met2_num),
+         big = if_else(met1_num > met2_num, met1_num, met2_num),
+         sorted = str_c(small, big, sep = "_")) %>% 
+  distinct(sorted, .keep_all = T) %>% 
+  filter(!met1 == met2) %>% 
+  mutate(pos_neg = as_factor(if_else(cor_val > 0, "pos", "neg")),
+         cor_val_plot = if_else(abs(cor_val)< 0.4, 0, cor_val),
+         weight = cor_val)
+
+corr_mat_comb <- corr_mat_long %>% 
+  left_join(corr_p_long) %>% 
+  filter(abs(cor_val_plot) >= 0.4 , cor_p_val_plot <= 0.05) %>% 
+  mutate(ChEBI = fct_relevel(ChEBI, c("AA", "CH", "CA", "other")))
+
+
+
+#
+# Clustering difference---------------------------------------------------------
+
+library(pheatmap)
+
+heat_base <- fc_1 %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker", "*panK4-1*"),
+    tissue == "fruits")
+
+heat.GC <- heat_base %>% 
+  group_by(Compound_Name, met) %>% 
+  mutate(log_norm = log2(mean_fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm)),
+         log_norm_level = log_norm_level + 1) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) 
+
+heat_GC_WT <- heat.GC %>% 
+  filter(genotype == "MoneyMaker") %>% 
+  select(met, treatment, log_norm_level_wt = log_norm_level)
+
+heat_GC_diff <- heat.GC %>% 
+  filter(genotype == "*panK4-1*") %>% 
+  left_join(heat_GC_WT) %>% 
+  mutate(log_norm_diff = log_norm_level_wt - log_norm_level)
+
+heat.GC <- heat_GC_diff %>% 
+  pivot_wider(id_cols = c(Compound_Name, Compound_Class, met),
+              names_from = group,
+              values_from = log_norm_diff) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+rownames(heat.GC) <- heat.GC$met
+
+mat.heat.GC <- heat.GC %>% 
+  select(contains("0.4"), contains("0.6"),
+         contains("0.8"),
+         contains("1"),
+         contains("leaves"), contains("fruits")) %>% as.matrix()
+
+plot(hclust(dist(mat.heat.GC)))
+
+all_clust <- hclust(dist(mat.heat.GC, method = "euclidean"))
+
+clusters <- cutree(all_clust, k = 4)
+
+clust_tidy <- tibble("clust" = clusters) %>% 
+  mutate(met = names(clusters))
+
+heat_GC_diff %>% 
+  left_join(clust_tidy) %>% 
+  group_by(Compound_Name, met) %>% 
+  # mutate(log_norm = log2(mean_fc),
+  #         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+  #         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm))) %>% 
+  ungroup() %>% 
+  ggplot(aes(x = treatment, y = log_norm_diff, color = Compound_Class)) +
+  geom_line(aes(group = met)) +
+  facet_wrap(vars(clust))
+
+annotation_row <- heat.GC %>% 
+  select(Compound_Class)
+
+rownames(annotation_row) <- heat.GC$met
+
+annotation_col <- heat_base %>% 
+  distinct(tissue, treatment, genotype) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>%  
+  as.data.frame()
+
+rownames(annotation_col) <- annotation_col$group
+
+annotation_col <- annotation_col %>% 
+  select(-group, treatment, tissue, genotype)
+
+heat.GC_signif <- heat_base %>% 
+  distinct(tissue, treatment, alias, genotype, met) %>% 
+  #filter(genotype != "MoneyMaker") %>% 
+  left_join(sig_GC, by = c("met", "tissue", "treatment", "alias" = "group2")) %>% 
+  #left_join(sig_mets) %>% 
+  #filter(sig == T) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_")),
+         signif = if_else(p.value >= 0.05| is.na(p.value), "","X")) %>%
+  pivot_wider(id_cols = c(met),
+              names_from = group,
+              values_from = signif) %>% 
+  left_join(met_dat) %>% 
+  #left_join(GCid_classes) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+heat_cols <- colnames(mat.heat.GC)
+
+rownames(heat.GC_signif) <- heat.GC_signif$met
+
+mat.heat.GC_signif <- heat.GC_signif %>% 
+  select(all_of(heat_cols)) %>% as.matrix()
+
+ann_colors = list(
+  tissue = c(fruits = "red",leaves = "darkgreen"),
+  genotype = c(MoneyMaker = cb_scale[1], `*panK4-1*` = "brown", `log2-1` = "blue", `*transp1-1*` = cb_scale[4]),
+  treatment = c("0.4" = "red", "0.6" = "orange", "0.8" = "yellow", "1" = "green"))
+
+pheatmap.GC <- pheatmap(mat.heat.GC,
+                        color = colorRampPalette(c("blue", "white", "red"))(65),
+                        #cellwidth = 16,
+                        #cellheight = 4,
+                        breaks = seq(-3.25, 3.25, 0.1),
+                        clustering_distance_rows = dist((mat.heat.GC), method = "euclidean"),
+                        cluster_rows = T,
+                        cluster_cols = F,
+                        annotation_names_row = F,
+                        show_rownames = F,
+                        annotation_row = annotation_row,
+                        annotation_col = annotation_col,
+                        display_numbers = mat.heat.GC_signif,
+                        number_color = "black",
+                        fontsize_number = 6,
+                        # annotation_colors = ann_colors,
+                        #filename = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                        #                "cmQTL_val_1_heatmap_rel_tissue_wt.jpg",
+                        #                 sep = "_")
+)
+
+dev.off()
+
+
+
+
+
+# Clustering ---------------------------------------------------------
+
+library(pheatmap)
+
+heat_base <- fc_1 %>% 
+  filter(#Compound_Class %in% c("Phospholipid", "Phosphatidylcholine"),
+    genotype %in% c("MoneyMaker"),
+    tissue == "fruits")
+
+heat.GC <- heat_base %>% 
+  group_by(Compound_Name, met) %>% 
+  mutate(log_norm = log2(mean_fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm)),
+         z_score = (mean_fc - mean(mean_fc))/(max(mean_fc)-min(mean_fc))) %>% 
+  ungroup() %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>% 
+  pivot_wider(id_cols = c(Compound_Name, Compound_Class, met),
+              names_from = group,
+              values_from = z_score) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+rownames(heat.GC) <- heat.GC$met
+
+mat.heat.GC <- heat.GC %>% 
+  select(contains("0.4"), contains("0.6"),
+         contains("0.8"),
+         contains("1"),
+         contains("leaves"), contains("fruits")) %>% as.matrix()
+
+plot(hclust(dist(mat.heat.GC, method = "maximum"), method = "ward.D2"))
+
+all_clust <- hclust(dist(mat.heat.GC, method = "maximum"))
+
+clusters <- cutree(all_clust, k = 4)
+
+clust_tidy <- tibble("clust" = clusters) %>% 
+  mutate(met = names(clusters))
+
+heat_base %>% 
+  left_join(clust_tidy) %>% 
+  group_by(Compound_Name, met) %>% 
+  mutate(log_norm = log2(mean_fc),
+         log_norm = if_else(is.infinite(log_norm), 0, log_norm),
+         log_norm_level = (log_norm - mean(log_norm))/(max(log_norm)-min(log_norm)),
+         z_score = (mean_fc - mean(mean_fc))/(sd(mean_fc))) %>% 
+  ungroup() %>% 
+  ggplot(aes(x = treatment, y = z_score, color = Compound_Class)) +
+  geom_line(aes(group = met)) +
+  facet_wrap(vars(clust))
+
+annotation_row <- heat.GC %>% 
+  select(Compound_Class)
+
+rownames(annotation_row) <- heat.GC$met
+
+annotation_col <- heat_base %>% 
+  distinct(tissue, treatment, genotype) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_"))) %>%  
+  as.data.frame()
+
+rownames(annotation_col) <- annotation_col$group
+
+annotation_col <- annotation_col %>% 
+  select(-group, treatment, tissue, genotype)
+
+heat.GC_signif <- heat_base %>% 
+  distinct(tissue, treatment, alias, genotype, met) %>% 
+  #filter(genotype != "MoneyMaker") %>% 
+  left_join(sig_GC, by = c("met", "tissue", "treatment", "alias" = "group2")) %>% 
+  #left_join(sig_mets) %>% 
+  #filter(sig == T) %>% 
+  mutate(group = as_factor(str_c(tissue, treatment, genotype, sep = "_")),
+         signif = if_else(p.value >= 0.05| is.na(p.value), "","X")) %>%
+  pivot_wider(id_cols = c(met),
+              names_from = group,
+              values_from = signif) %>% 
+  left_join(met_dat) %>% 
+  #left_join(GCid_classes) %>% 
+  arrange(Compound_Class, Compound_Name) %>% 
+  as.data.frame()
+
+heat_cols <- colnames(mat.heat.GC)
+
+rownames(heat.GC_signif) <- heat.GC_signif$met
+
+mat.heat.GC_signif <- heat.GC_signif %>% 
+  select(all_of(heat_cols)) %>% as.matrix()
+
+ann_colors = list(
+  tissue = c(fruits = "red",leaves = "darkgreen"),
+  genotype = c(MoneyMaker = cb_scale[1], `*panK4-1*` = "brown", `log2-1` = "blue", `*transp1-1*` = cb_scale[4]),
+  treatment = c("0.4" = "red", "0.6" = "orange", "0.8" = "yellow", "1" = "green"))
+
+pheatmap.GC <- pheatmap(mat.heat.GC,
+                        color = colorRampPalette(c("blue", "white", "red"))(65),
+                        #cellwidth = 16,
+                        #cellheight = 4,
+                        breaks = seq(-3.25, 3.25, 0.1),
+                        clustering_distance_rows = dist((mat.heat.GC), method = "euclidean"),
+                        cluster_rows = T,
+                        cluster_cols = F,
+                        annotation_names_row = F,
+                        show_rownames = F,
+                        annotation_row = annotation_row,
+                        annotation_col = annotation_col,
+                        display_numbers = mat.heat.GC_signif,
+                        number_color = "black",
+                        fontsize_number = 6,
+                        # annotation_colors = ann_colors,
+                        #filename = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                        #                "cmQTL_val_1_heatmap_rel_tissue_wt.jpg",
+                        #                 sep = "_")
+)
+
+dev.off()
+
+
+
+
+testtheme <- theme(axis.text.x = element_markdown(angle = 45, hjust = 1, margin = unit(c(1,0,0,0), "mm")),
+                   axis.title.x = element_blank(),
+                   panel.background = element_rect(fill = "white"),
+                   panel.border = element_rect(color = "black",fill = NA),
+                   text = element_text(size = 8, family = "Arial Narrow"),
+                   legend.title = element_blank(),
+                   legend.text = element_markdown(),
+                   legend.position = "right",
+                   legend.direction = "vertical",
+                   plot.margin = unit(c(1,0,1,2), "mm"),
+                   legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm")) 
+
+
+make_box_point_plot <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_GC %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  fc_1_ind_plot <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  plot_out <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = fc)) +
+    geom_boxplot(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_point(aes(fill = genotype, color = genotype),size = 2, shape = 21) +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale, aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+make_box_beeswarm_plot <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_GC %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  fc_1_ind_plot <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  plot_out <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = fc)) +
+    geom_boxplot(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_beeswarm(aes(fill = genotype, color = genotype),size = 2, shape = 21, cex = 2, priority = "density") +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale, aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+make_box_dot_line_plot <- function(plot_met, plot_tissue, plot_label, plot_legend, plot_genotypes){
+  
+  sig_bar <- sig_GC %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val,
+           treatment = as_factor(treatment)) %>% 
+    filter(met == plot_met, tissue == plot_tissue,
+           group1 %in% plot_genotypes, group2 %in% plot_genotypes)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  fc_1_plot <- fc_1%>% 
+    filter(met == plot_met, tissue == plot_tissue, genotype %in% plot_genotypes) %>% 
+    mutate(treatment = as_factor(treatment))
+  
+  plot_out <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue, genotype %in% plot_genotypes) %>% 
+    mutate(treatment = as_factor(treatment)) %>% 
+    ggplot(position = "dodge", aes(x = treatment, y = fc)) +
+    geom_boxplot(aes(fill = genotype), color = "black") +
+    geom_dotplot(aes(fill = genotype, color = genotype), position = position_dodge(0.75),
+                 binaxis = "y", stackdir = "center", binwidth = 0.1, dotsize = 1) +
+    geom_smooth(aes(x = treatment, y = fc, color = genotype, group = genotype),method = "lm", position = position_dodge(0.75)) +
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    #facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale, aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+p1 <- make_box_dot_line_plot("m_70", "fruits", "Malic acid", "none", c("MoneyMaker","*panK4-1*"))
+p1
+ggsave("Malic_acid_box_dot_line_plot_nominal.png",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+make_col_plot_cv <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_GC_cv %>%  
+    left_join(genotypes, by = c("alias1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("alias2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y_cv) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_cv %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = mean_fc)) +
+    geom_col(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_errorbar(aes(ymin = (mean_fc-se), ymax = (mean_fc + se)), position = position_dodge(0.9), width = 0.25, size = 0.75)+
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    #facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("CV fold-change")+
+    ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+  
+  plot_out
+  
+}
+
+make_col_plot <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_GC %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  fc_1_ind_plot <- fc_1_ind %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  plot_out <- fc_1 %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = mean_fc)) +
+    geom_col(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_jitter(aes(fill = genotype, color = genotype, y = fc), size = 2, shape = 21, data = fc_1_ind_plot) +
+    geom_errorbar(aes(ymin = (mean_fc-se), ymax = (mean_fc + se)), position = position_dodge(0.9), width = 0.25, size = 0.75)+
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_manual(values = cb_scale, aesthetics = "fill") +
+    scale_color_manual(values = bw_scale, aesthetics = "color")
+  
+  plot_out
+  
+}
+
+# Figure 46 ---------------------------------------------------------------
+
+p1 <- make_col_plot("m_70", "fruits", "Malic acid", "none")
+p1
+
+
+p2 <- make_col_plot_cv("m_119", "fruits", "Malic acid CV", "none")
+p2
+
+
+leg46 <- get_legend(make_col_plot("m_123", "fruits", "sucrose", "bottom"))
+leg46
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg46, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("figure_46.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+# Figure 47 ---------------------------------------------------------------
+
+p1 <- make_col_plot("m_74", "fruits", "Phenylalanine", "none")
+p1
+
+p2 <- make_col_plot_cv("m_74", "fruits", "Phe CV", "none")
+p2
+
+
+leg46 <- get_legend(make_col_plot("m_74", "fruits", "sucrose", "bottom"))
+leg46
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO",rel_widths = c(3,1))
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg46, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("figure_47.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+# Figure 48 ---------------------------------------------------------------
+
+p1 <- make_col_plot_cv("m_25", "fruits", "F6P CV", "none")
+p1
+
+p2 <- make_col_plot_cv("m_38", "fruits", "G6P CV", "none")
+p2
+
+p3 <- make_col_plot_cv("m_65", "fruits", "Maltose CV", "none")
+p3
+
+leg48 <- get_legend(make_col_plot("m_65", "fruits", "sucrose", "bottom"))
+leg48
+
+main_plot <- plot_grid(p1,p2,p3, ncol = 3, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg48, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("figure_48.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+}
+  
+  
\ No newline at end of file
diff --git a/workflows/Phenotype_analysis/210114_cmQTL_val_phenotype_analysis.R b/workflows/Phenotype_analysis/210114_cmQTL_val_phenotype_analysis.R
new file mode 100644
index 0000000..d6f45eb
--- /dev/null
+++ b/workflows/Phenotype_analysis/210114_cmQTL_val_phenotype_analysis.R
@@ -0,0 +1,826 @@
+rm(list = ls())
+
+
+# Libraries ---------------------------------------------------------------
+
+library(tidyverse)
+library(openxlsx)
+library(lubridate)
+library(broom)
+library(ggtext)
+library(glue)
+library(ggpubr)
+library(markdown)
+library(cowplot)
+library(RVAideMemoire)
+
+setwd(dirname(rstudioapi::getActiveDocumentContext()$path))
+getwd()
+
+pt <- readxl::read_xlsx("../200923_samplelist_WIJESI-030820-13_cmQTL_validation.xlsx")
+
+line_info <- readxl::read_xlsx("../WIJESI-030820-13- Culturereport-051220-7624.xlsx")
+
+genotypes <- readxl::read_xlsx("../Genotype_names.xlsx")
+
+pt_tidy <- pt %>% 
+  mutate(col_pot = as.integer(col_pot),
+         comment = if_else(is.na(comment), "NA", comment),
+         irrigation = as_factor(irrigation),
+         #LIMS_ID = as.double(str_replace(LIMS_ID, "(?<=244)1", "")),
+         fruit_num_dropped = if_else(is.na(fruit_num_dropped), 0, fruit_num_dropped),
+         weight_sartorius_dropped = if_else(is.na(weight_sartorius_dropped), 0, weight_sartorius_dropped),
+         total_fruit_num = fruit_num_dropped + fruit_num_harvest,
+         total_fruit_weight = weight_sartorius_dropped + weight_sartorius_harvest,
+         #total_fruit_weight = if_else(str_detect(comment, "beefsteak"), NaN, total_fruit_weight),
+         av_fruit_weight = total_fruit_weight/total_fruit_num,
+         biomass = total_fruit_weight + shoot_fresh_weight,
+         harvest_index = total_fruit_weight/biomass,
+         days_until_flower = sow_date %--% flower_date %/% days(1),
+         days_until_redripe = sow_date %--% redripe_date %/% days(1)) %>% 
+  left_join(line_info, by = c("LIMS_ID" = "aliquot")) %>% 
+  left_join(genotypes)  %>%
+  mutate(plantline = as_factor(plantline),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "panK4-1", "log2-1", "transp1-1")),
+         irrigation = fct_relevel(irrigation, c("0.4", "0.6", "0.8", "1")),
+         group = str_c(genotype, irrigation, sep = "_"))
+
+pt_tidy %>% 
+  mutate(genotype = as_factor(if_else(genotype == "MoneyMaker", glue("{genotype}"),glue("*{genotype}*"))),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*"))) %>% 
+  ggplot(aes(x = col_pot, y = irrigation, color = genotype)) +
+  geom_point( size = 8) +
+  xlab("Column") +
+  theme(text = element_text(size = 18),
+        legend.text = element_markdown())
+
+#ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+#             "cmQTL_val1_layout.jpg",
+#             sep = "_"),
+#       width = 27,
+#       height = 9,
+#       units = "cm",
+#       dpi = 300)
+
+pt_vars <- c(#"fruit_num_dropped", "weight_sartorius_dropped",
+             "total_fruit_num", "total_fruit_weight",
+             "av_fruit_weight",# "days_until_flower", 
+             "days_until_redripe","shoot_fresh_weight", "harvest_index",
+             "biomass", "shoot_dry_weight")
+
+pt_long <- pt_tidy %>% 
+  pivot_longer(cols = all_of(pt_vars),
+               names_to = "pt_var",
+               values_to = "value") %>% 
+  group_by(plantline, alias, genotype, irrigation, pt_var) %>% 
+  mutate(rep = row_number()) %>% 
+  filter(!is.na(value)) %>% 
+  arrange(pt_var) %>% 
+  ungroup()
+
+pt_long %>% 
+  group_by(plantline, alias, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, color = genotype)) +
+  geom_boxplot() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+pt_long %>% 
+  group_by(plantline, alias, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, fill = alias)) +
+  #stat_summary(aes(color = alias)) +
+  #geom_boxplot(position = "identity")+
+  #geom_dotplot(binaxis = "y", stackdir = "center") +
+  geom_smooth(aes(group = alias, color = alias), method = "lm", formula = y ~ poly(x,2), se = T) +
+  facet_wrap(facets = vars(pt_var),scales = "free", nrow = 2)
+
+WT <- pt_long %>% 
+  filter(plantline == "967514") %>% 
+  rename(WT_value = value) %>% 
+  select(irrigation, pt_var, WT_value, rep)
+
+# Summary statistics ------------------------------------------------------
+
+pt_vars_label <- c("Total fruit number per plant", "Total fruit weight per plant/ g",
+                   "Average fruit weight/ g", "Days until ripe fruit", "Shoot fresh weight/ g",
+                   "Harvest index", "Biomass", "Shoot dry weight/ g")
+
+pt_vars_plot <- cbind(pt_vars, pt_vars_label) %>% as_tibble()
+
+sum_stat <- pt_long %>% 
+  left_join(WT, by = c("irrigation", "pt_var", "rep")) %>% 
+  group_by(plantline, genotype, alias, irrigation, pt_var) %>% 
+  summarise(mean = mean(value),
+            median = median(value),
+            sd = sd(value),
+            n = n(),
+            se = sd/sqrt(n),
+            ttest = t.test(value, WT_value)$p.value,
+            iqr = IQR(value),
+            Q1 = quantile(value)["25%"],
+            Q3 = quantile(value)["75%"]) %>% 
+  ungroup() %>%
+  group_by(irrigation, pt_var) %>% 
+  mutate(p_adj = p.adjust(ttest, method = "fdr"),
+         pt_var = as_factor(pt_var),
+         alias = as_factor(alias),
+         irrigation = as_factor(irrigation),
+         genotype = as_factor(if_else(genotype == "MoneyMaker", glue("{genotype}"),glue("*{genotype}*"))),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*"))) %>% 
+  ungroup()
+
+
+
+#dry weight biomass still missing
+#AOV TukeyHSD?
+#test manova
+
+pt_tidy_numeric <- pt_tidy %>% 
+  select(all_of(pt_vars))
+
+pt_norm <- pt_long %>% 
+  group_by(irrigation, genotype, pt_var) %>% 
+  summarise(norm = shapiro.test(value)$p.value)
+
+pt_norm_wt <- pt_norm %>% 
+  filter(genotype == "MoneyMaker") %>% 
+  select(irrigation, comp = genotype, pt_var, wt_norm = norm)
+
+pt_norm_mut <- pt_norm %>% 
+  filter(genotype != "MoneyMaker") %>% 
+  select(irrigation, genotype, pt_var, norm)
+
+pt_norm_comb <- pt_norm_wt %>% 
+  left_join(pt_norm_mut)
+
+pt_variance <- map(.x = pt_tidy_numeric, .f = ~pairwise.var.test(.x, pt_tidy$group, p.method = "none"))
+
+pt_variance_tidy <- pt_variance %>% 
+  map(tidy) %>% 
+  map2(.y = names(pt_variance), .f = ~.x %>% mutate(pt_vars = .y)) %>% 
+  purrr::reduce(bind_rows) %>% 
+  separate(group1,
+           into = c("genotype1", "irrigation1"),
+           sep = "_") %>% 
+  separate(group2,
+           into = c("genotype2", "irrigation2"),
+           sep = "_") %>% 
+  filter(irrigation1 == irrigation2, genotype1 == "MoneyMaker"& genotype2 != "MoneyMaker"|
+         genotype1 != "MoneyMaker" & genotype2 == "MoneyMaker") %>% 
+  mutate(genotype = if_else(genotype1 == "MoneyMaker", genotype2, genotype1),
+         comp = "MoneyMaker") %>% 
+  select(genotype, comp, irrigation = irrigation1, pt_var = pt_vars, p.value)
+
+prereq <- pt_norm_comb %>% 
+  left_join(pt_variance_tidy) %>% 
+  mutate(fulfill = if_else(wt_norm >= 0.05, norm >= 0.05, p.value >= 0.05))
+
+prereq_sum <- prereq %>% 
+  group_by(fulfill) %>% 
+  summarise(n = n()) %>% 
+  ungroup() %>% 
+  mutate(percent = 100*n/sum(n))
+
+pt_aov <- map(.x = pt_tidy_numeric, .f = ~aov(.x ~ alias* irrigation, data = pt_tidy))
+
+pt_aov_tidy <- pt_aov %>% 
+  map(tidy) %>% 
+  map2(.y = names(pt_aov), .f = ~.x %>% mutate(pt_vars = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+pt_aov_table <- pt_aov_tidy %>% 
+  left_join(pt_vars_plot) %>% 
+  filter(term != "Residuals") %>% 
+  select(term, pt_vars_label, p.value) %>% 
+  pivot_wider(names_from = term, values_from = p.value)
+
+write.xlsx(pt_aov_table, str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                                       "anova_table.xlsx",
+                                       sep = "_"))
+
+pt_tuk <- map(.x = pt_aov, .f = ~TukeyHSD(.x)) %>% 
+  map(.f = tidy) %>% 
+  map2(.y = names(pt_aov), .f = ~.x %>% mutate(var = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+# General differences -----------------------------------------------------
+
+sig_pt_overall <- pt_tuk %>% 
+  filter(term == "alias") %>% 
+  separate(col = contrast, into = c("group1", "group2"), sep = "-")
+
+lines <- sum_stat %>% distinct(alias,plantline) %>% 
+  mutate(plantline = as.double(as.character(plantline))) %>% 
+  left_join(genotypes)
+
+sig_pt_overall_table <- sig_pt_overall %>%  
+  filter(group1 == "967514 MM WT" | group2 == "967514 MM WT") %>% 
+  ungroup() %>% 
+  left_join(lines, by = c("group1" = "alias")) %>% 
+  select(var, group1, group2, adj.p.value, genotype1 = genotype) %>%
+  left_join(lines, by = c("group2" = "alias")) %>% 
+  select(var,adj.p.value, genotype1, genotype2 = genotype) %>% 
+  left_join(pt_vars_plot, by = c("var" = "pt_vars")) %>% 
+    pivot_wider(names_from = genotype2,
+                values_from = adj.p.value)
+
+write.xlsx(sig_pt_overall_table, str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                                       "overall_significance.xlsx",
+                                       sep = "_"))
+
+# Pairwise significant changes --------------------------------------------
+
+
+sig_pt_groups <- pt_tuk %>% 
+  filter(term == "alias:irrigation") %>% 
+  separate(col = contrast, into = c("group1", "group2"), sep = "-")
+
+sig_pt <- sig_pt_groups %>%  
+  separate(group1, into = c("alias1", "irrigation1"), sep = ":") %>% 
+  separate(group2, into = c("alias2", "irrigation2"), sep = ":") %>% 
+  filter(irrigation1 == irrigation2) %>% 
+  filter(alias1 == "967514 MM WT" | alias2 == "967514 MM WT") %>% 
+  ungroup() %>% 
+  mutate(p.signif = if_else(adj.p.value <= 0.05, "*", "ns")) %>% 
+  left_join(sum_stat, by = c("var" = "pt_var", "irrigation1" = "irrigation", "alias1" = "alias")) %>% 
+  select(var, irrigation1, irrigation2, alias1, alias2, p.signif, mean1 = mean, se1 = se, genotype1 = genotype) %>%
+  left_join(sum_stat, by = c("var" = "pt_var", "irrigation2" = "irrigation", "alias2" = "alias")) %>% 
+  select(var, irrigation1, irrigation2, alias1, alias2, p.signif, mean1, mean2 = mean, se1, se2 = se, genotype1, genotype2 = genotype) %>% 
+  group_by(irrigation1, var) %>% 
+  mutate(tot_val1 = mean1 + se1,
+         tot_val2 = mean2 + se2,
+         y.position = 1.1*(max(tot_val1, tot_val2))) %>% 
+  rename(pt_var = var,
+         group1 = genotype1,
+         group2 = genotype2,
+         irrigation = irrigation1) %>% 
+  mutate(pt_var = as_factor(pt_var),
+         group1 = as_factor(group1),
+         group2 = as_factor(group2),
+         irrigation = as_factor(irrigation)) %>% 
+  ungroup()
+
+pt_vars
+
+plot_out <- vector("list", length = length(pt_vars))
+
+for(pt in seq_along(pt_vars)) {
+  sig_pt_temp <- sig_pt %>% 
+    filter(pt_var == pt_vars[[pt]])
+  
+plot_out[[pt]] <-   sum_stat %>% 
+    filter(pt_var == pt_vars[[pt]]) %>%
+    ggplot(aes(genotype, mean)) +
+    geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+    geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.75) +
+    facet_grid(cols = vars(irrigation), scales = "fixed")  +
+    stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.1,
+                       hide.ns = T) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 14),
+          legend.text = element_markdown(),
+          legend.position = "right") +
+    ggtitle(pt_vars_label[[pt]]) +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+}
+
+pdf(file = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),"cmQTL_val1_phenotypes.pdf"),
+    width = 9,
+    height = 4)
+
+for (pt in seq_along(pt_vars)) {
+  print(plot_out[[pt]])
+}
+
+dev.off()
+
+
+
+sum_stat %>% 
+  ggplot(aes(irrigation, mean, color = alias)) +
+  geom_point() +
+  geom_line(aes(group = plantline)) + 
+  geom_errorbar(aes(ymin = mean - sd, ymax = mean + sd)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+
+
+sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_pointrange(aes(ymin = mean - se, ymax = mean + se, shape = genotype, fill = genotype)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23))
+
+sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_point(aes(shape = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype, width = 0.25)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23))
+
+
+ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+             "cmQTL_val1_phenotypic.jpg",
+             sep = "_"),
+       width = 183,
+       height = 100,
+       units = "mm",
+       dpi = 300)
+
+
+# Overview for supplemental -----------------------------------------------
+
+pt_long %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  group_by(plantline, genotype, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, fill = genotype)) +
+  #stat_summary(aes(color = genotype)) +
+  #geom_violin(aes(color = genotype),fill = NA, position = "identity", trim = T, scale = "width", alpha = 1)+
+  geom_dotplot(binaxis = "y", stackdir = "center") +
+  geom_smooth(aes(group = genotype, color = genotype), method = "lm", formula = y ~ poly(x,2), se = F, size = 1.5) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23))
+
+
+
+sum_stat %>% 
+  ggplot(aes(irrigation, mean, color = alias)) +
+  geom_point() +
+  geom_line(aes(group = plantline)) +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+             "cmQTL_val1_phenotypic.jpg",
+             sep = "_"),
+       width = 183,
+       height = 100,
+       units = "mm",
+       dpi = 300)
+
+library(bootstrap)
+
+theta <- function(x){
+  sd(x)/mean(x)
+}
+
+CV <- sum_stat %>% 
+  ungroup() %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(grand_mean = mean(mean),
+            grand_sd = sd(mean)) %>% 
+  mutate(cv = grand_sd/grand_mean)
+
+cv_jack <- sum_stat %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(cv = jackknife(mean, theta)$jack.values) %>% 
+  mutate(jack_rep = row_number()) %>% 
+  ungroup() 
+
+cv_jack_wide <- cv_jack %>% 
+  pivot_wider(id_cols = c(plantline, genotype, alias, pt_var, jack_rep),
+              names_from = pt_var,
+              values_from = cv)
+
+cv_jack_numeric <- cv_jack_wide %>% 
+  select(-c(alias, genotype, plantline, jack_rep))
+
+cv_jack_sum_stat <- cv_jack %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(mean_cv = mean(cv),
+            sd = sd(cv),
+            n = n(),
+            se = sd/sqrt(n))
+
+
+pt_cv_jack <- map(.x = cv_jack_numeric, .f = ~aov(.x ~ alias, data = cv_jack_wide))
+
+pt_jack_t <- map(.x = cv_jack_numeric, .f = ~pairwise.t.test(x = .x , g = cv_jack_wide$alias)) %>% 
+  map(.f = tidy) #%>% 
+
+pt_jack_t_tidy <- pt_jack_t %>% 
+  map2(.y = names(pt_jack_t), .f = ~.x %>% mutate(var = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+sig_pt_cv_groups <- pt_jack_t_tidy %>% 
+  filter(group1 == "967514 MM WT" | group2 == "967514 MM WT") %>% 
+  group_by(var) %>% 
+  mutate(adj.p.value = p.adjust(p.value)) %>% 
+  ungroup() %>% 
+  mutate(p.signif = if_else(adj.p.value <= 0.05, "*", "ns")) %>% 
+  left_join(cv_jack_sum_stat, by = c("var" = "pt_var", "group1" = "alias")) %>% 
+  select(var, group1, group2, p.signif, mean_cv1 = mean_cv, se1 = se, genotype1 = genotype) %>%
+  left_join(cv_jack_sum_stat, by = c("var" = "pt_var",  "group2" = "alias")) %>% 
+  select(var, group1, group2, p.signif, mean_cv1, mean_cv2 = mean_cv, se1, se2 = se, genotype1, genotype2 = genotype) %>% 
+  group_by(var) %>% 
+  mutate(tot_val1 = mean_cv1 + se1,
+         tot_val2 = mean_cv2 + se2,
+         y.position = 1.1*(max(tot_val1, tot_val2))) %>% 
+  rename(pt_var = var) %>% 
+  mutate(pt_var = as_factor(pt_var),
+         group1 = genotype1,
+         group2 = genotype2) %>% 
+  ungroup()
+
+cv_jack %>% 
+  ggplot(aes(alias, cv, fill = alias)) +
+  geom_boxplot() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+
+
+  
+CV %>% 
+  ggplot(aes(alias, cv, fill = alias)) +
+  geom_col() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+x = rnorm(10)
+
+
+
+# Jackknife plots---------------------------------------------------------------
+
+
+
+
+pt_vars
+pt_vars_label <- c("Total fruit number per plant", "Total fruit weight per plant/ g",
+                   "Average fruit weight/ g", "Days until ripe fruit", "Shoot fresh weight/ g",
+                   "Harvest index", "Biomass", "Shoot dry weight/ g")
+
+plot_out <- vector("list", length = length(pt_vars))
+
+for(pt in seq_along(pt_vars)) {
+  sig_pt_temp <- sig_pt_cv_groups %>% 
+    filter(pt_var == pt_vars[[pt]])
+  
+  plot_out[[pt]] <-   cv_jack_sum_stat %>% 
+    filter(pt_var == pt_vars[[pt]]) %>%
+    ggplot(aes(genotype, mean_cv)) +
+    geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+    geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.75) +
+    stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.1,
+                       hide.ns = T) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 14),
+          legend.text = element_markdown(),
+          legend.position = "right") +
+    ggtitle(pt_vars_label[[pt]]) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+    ylab("mean CV")
+}
+
+plot_out[[1]]
+
+pdf(file = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),"cmQTL_val1_phenotypes_cv.pdf"),
+    width = 9,
+    height = 4)
+
+
+
+
+
+
+for (pt in seq_along(pt_vars)) {
+  print(plot_out[[pt]])
+}
+
+dev.off()
+
+
+
+plot(bootstrap::jackknife(x = x, theta = theta)$jack.values)
+
+# Figure 40 ---------------------------------------------------------------
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "total_fruit_weight")
+
+p1<-   sum_stat %>% 
+  filter(pt_var == "total_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(0,1150) +
+  ggtitle("Total fruit weight per plant/ g") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p1
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p2<-   sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(c(0,65))+
+  ggtitle("Average fruit weight/ g") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p2
+
+leg40 <- get_legend(sum_stat %>% 
+                      filter(pt_var == "av_fruit_weight") %>%
+                      ggplot(aes(genotype, mean)) +
+                      geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                      geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+                      facet_grid(cols = vars(irrigation), scales = "fixed")  +
+                      stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                         step.increase = 0.07,
+                                         hide.ns = T) +
+                      theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                            panel.background = element_rect(fill = "white"),
+                            panel.border = element_rect(color = "black",fill = NA),
+                            text = element_text(size = 14),
+                            legend.text = element_markdown(),
+                            legend.position = "right") +
+                      ggtitle("Average fruit weight/ g") +
+                      scale_fill_grey(start = 1, end = 0, aesthetics = "fill"))
+leg40
+
+main_plot <- plot_grid(p1,p2, ncol = 1, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg40, ncol = 2, rel_widths =  c(3,1))
+comp_plot
+
+ggsave("figure_40.wmf",width = 15.8, height = 12, units = "cm", dpi = 300)
+
+# Figure 41 ---------------------------------------------------------------
+
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p1 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,1,0), "mm")) +
+  ggtitle("Average fruit weight/ g") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p1
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "harvest_index")
+
+p2 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "harvest_index") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,1,0), "mm")) +
+  ggtitle("Harvest index") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p2
+
+leg41 <-   get_legend(cv_jack_sum_stat %>% 
+  filter(pt_var == "harvest_index") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "bottom") +
+  ggtitle("Harvest index") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV"))
+
+
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg41, ncol = 1, rel_heights =  c(10,1)) + theme(plot.margin = unit(c(0,0,5,0), "mm"))
+comp_plot
+
+ggsave("figure_41.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+
+# Figure means presentation -----------------------------------------------
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "total_fruit_weight")
+
+p1<-   sum_stat %>% 
+  filter(pt_var == "total_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(0,1150) +
+  ggtitle("Total fruit weight per plant/ g") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p1
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p2<-   sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(c(0,65))+
+  ggtitle("Average fruit weight/ g") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p2
+
+leg40 <- get_legend(sum_stat %>% 
+                      filter(pt_var == "av_fruit_weight") %>%
+                      ggplot(aes(genotype, mean)) +
+                      geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                      geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+                      facet_grid(cols = vars(irrigation), scales = "fixed")  +
+                      stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                         step.increase = 0.07,
+                                         hide.ns = T) +
+                      theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                            panel.background = element_rect(fill = "white"),
+                            panel.border = element_rect(color = "black",fill = NA),
+                            text = element_text(size = 14),
+                            legend.text = element_markdown(),
+                            legend.position = "right") +
+                      ggtitle("Average fruit weight/ g") +
+                      scale_fill_grey(start = 1, end = 0, aesthetics = "fill"))
+leg40
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg40, ncol = 2, rel_widths =  c(6,1))
+comp_plot
+
+ggsave("Av_tot_fruit_weight_mean.wmf",width = 33.5, height = 12, units = "cm", dpi = 300)
+
+# Figure CV average fruit weight ---------------------------------------------------------------
+
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p1 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,0,0), "mm")) +
+  ggtitle("Average fruit weight/ g") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p1
+
+
+
+leg41 <-   get_legend(cv_jack_sum_stat %>% 
+                        filter(pt_var == "av_fruit_weight") %>%
+                        ggplot(aes(genotype, mean_cv)) +
+                        geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                        geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+                        stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                           step.increase = 0.1,
+                                           hide.ns = T) +
+                        theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                              panel.background = element_rect(fill = "white"),
+                              panel.border = element_rect(color = "black",fill = NA),
+                              text = element_text(size = 14),
+                              legend.text = element_markdown(),
+                              legend.position = "right") +
+                        ggtitle("Harvest index") +
+                        scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+                        ylab("mean CV"))
+
+
+
+main_plot <- plot_grid(p1, ncol = 1, labels = "")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg41, ncol = 2, rel_widths =  c(3,2)) + theme(plot.margin = unit(c(0,0,0,0), "mm"))
+comp_plot
+
+ggsave("av_fruit_weight_cv_presentation.wmf",width = 10, height = 10, units = "cm", dpi = 300)
+
+
diff --git a/workflows/Phenotype_figures/Figure maker_phenotypes.R b/workflows/Phenotype_figures/Figure maker_phenotypes.R
new file mode 100644
index 0000000..ca17cf9
--- /dev/null
+++ b/workflows/Phenotype_figures/Figure maker_phenotypes.R	
@@ -0,0 +1,1448 @@
+
+# Library and Directory ---------------------------------------------------
+
+
+rm(list = ls())
+library(tidyverse)
+library(openxlsx)
+library(lubridate)
+library(broom)
+library(ggtext)
+library(glue)
+library(ggpubr)
+library(markdown)
+library(cowplot)
+library(ggbeeswarm)
+library(extrafont)
+library(devEMF)
+library(RVAideMemoire)
+
+setwd(dirname(rstudioapi::getActiveDocumentContext()$path))
+getwd()
+
+# Directory setting -------------------------------------------------------
+
+
+setwd(dirname(rstudioapi::getActiveDocumentContext()$path))
+getwd()
+
+current <- getwd()
+source <- str_c(current,"/..")
+
+cur_date <- str_c(str_replace_all(Sys.Date(),"^.{2}|-",""))
+
+out <- str_c(cur_date, "Figures", sep = "_")
+
+if (file.exists(out)) {
+  cat("The folder already exists")
+} else {
+  dir.create(out)
+}
+
+out_dir <- str_c(current, out, sep = "/")
+
+setwd(source)
+
+# Data loading ------------------------------------------------------------
+
+
+
+pt <- readxl::read_xlsx("200923_samplelist_WIJESI-030820-13_cmQTL_validation.xlsx")
+
+line_info <- readxl::read_xlsx("WIJESI-030820-13- Culturereport-051220-7624.xlsx")
+
+genotypes <- readxl::read_xlsx("Genotype_names.xlsx")
+
+setwd(out_dir)
+# Data combination --------------------------------------------------------
+
+com_theme <- theme(axis.text.x = element_markdown(angle = 45, hjust = 1, size = 8, family = "sans"),
+                   axis.text.y = element_text(size = 8, family = "sans"),
+                   axis.title.x = element_blank(),
+                   axis.title.y = element_text(size = 8, family = "sans"),
+                   panel.background = element_rect(fill = "white"),
+                   panel.border = element_rect(color = "black",fill = NA),
+                   strip.text = element_text(size = 8, family = "sans"),
+                   text = element_text(size = 8, family = "sans"),
+                   legend.title = element_blank(),
+                   legend.text = element_markdown(size = 8),
+                   plot.margin = unit(c(1,1,1,1), "mm"),
+                   legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm"))
+
+
+cb_scale <- c("#E69F00", "#56B4E9", "#009E73", "#F0E442",
+              "#0072B2", "#D55E00", "#CC79A7","#000000")
+bw_scale <- c("black", "black", "black", "black", "black", "black", "black")
+
+
+pt_tidy <- pt %>% 
+  mutate(col_pot = as.integer(col_pot),
+         comment = if_else(is.na(comment), "NA", comment),
+         irrigation = as_factor(irrigation),
+         #LIMS_ID = as.double(str_replace(LIMS_ID, "(?<=244)1", "")),
+         fruit_num_dropped = if_else(is.na(fruit_num_dropped), 0, fruit_num_dropped),
+         weight_sartorius_dropped = if_else(is.na(weight_sartorius_dropped), 0, weight_sartorius_dropped),
+         total_fruit_num = fruit_num_harvest, #fruit_num_dropped,
+         total_fruit_weight = weight_sartorius_harvest, #weight_sartorius_dropped,
+         #total_fruit_weight = if_else(str_detect(comment, "beefsteak"), NaN, total_fruit_weight),
+         av_fruit_weight = total_fruit_weight/total_fruit_num,
+         biomass = total_fruit_weight + shoot_fresh_weight,
+         harvest_index = total_fruit_weight/biomass,
+         days_until_flower = sow_date %--% flower_date %/% days(1),
+         days_until_redripe = sow_date %--% redripe_date %/% days(1)) %>% 
+  left_join(line_info, by = c("LIMS_ID" = "aliquot")) %>% 
+  left_join(genotypes)  %>%
+  mutate(plantline = as_factor(plantline),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "panK4-1", "log2-1", "transp1-1")),
+         irrigation = fct_relevel(irrigation, c("0.4", "0.6", "0.8", "1")),
+         group = str_c(genotype, irrigation, sep = "_"))
+
+pt_tidy %>% 
+  mutate(genotype = as_factor(if_else(genotype == "MoneyMaker", glue("{genotype}"),glue("*{genotype}*"))),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*"))) %>% 
+  ggplot(aes(x = col_pot, y = irrigation, color = genotype)) +
+  geom_point( size = 8) +
+  xlab("Column") +
+  theme(text = element_text(size = 18),
+        legend.text = element_markdown())
+
+ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+             "cmQTL_val1_layout.jpg",
+             sep = "_"),
+       width = 27,
+       height = 9,
+       units = "cm",
+       dpi = 300)
+
+pt_vars <- c(#"fruit_num_dropped", "weight_sartorius_dropped",
+  "total_fruit_num", "total_fruit_weight",
+  "av_fruit_weight",# "days_until_flower", 
+  "days_until_redripe","shoot_fresh_weight", "harvest_index",
+  "biomass", "shoot_dry_weight")
+
+pt_long <- pt_tidy %>% 
+  pivot_longer(cols = all_of(pt_vars),
+               names_to = "pt_var",
+               values_to = "value") %>% 
+  group_by(plantline, alias, genotype, irrigation, pt_var) %>% 
+  mutate(rep = row_number()) %>% 
+  filter(!is.na(value)) %>% 
+  arrange(pt_var) %>% 
+  ungroup()
+
+pt_long %>% 
+  group_by(plantline, alias, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, color = genotype)) +
+  geom_boxplot() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+pt_long %>% 
+  group_by(plantline, alias, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, fill = alias)) +
+  #stat_summary(aes(color = alias)) +
+  #geom_boxplot(position = "identity")+
+  #geom_dotplot(binaxis = "y", stackdir = "center") +
+  geom_smooth(aes(group = alias, color = alias), method = "lm", formula = y ~ poly(x,2), se = T) +
+  facet_wrap(facets = vars(pt_var),scales = "free", nrow = 2)
+
+WT <- pt_long %>% 
+  filter(plantline == "967514") %>% 
+  rename(WT_value = value) %>% 
+  select(irrigation, pt_var, WT_value, rep)
+
+# Summary statistics ------------------------------------------------------
+
+pt_vars_label <- c("Fruit number per plant", "Total fruit weight",
+                   "Average fruit weight", "Days until ripe fruit", "Shoot fresh weight",
+                   "Harvest index", "Biomass", "Shoot dry weight")
+
+pt_vars_plot <- cbind(pt_vars, pt_vars_label) %>% as_tibble()
+
+sum_stat <- pt_long %>% 
+  left_join(WT, by = c("irrigation", "pt_var", "rep")) %>% 
+  group_by(plantline, genotype, alias, irrigation, pt_var) %>% 
+  summarise(mean = mean(value),
+            median = median(value),
+            sd = sd(value),
+            n = n(),
+            se = sd/sqrt(n),
+            ttest = t.test(value, WT_value)$p.value,
+            iqr = IQR(value),
+            Q1 = quantile(value)["25%"],
+            Q3 = quantile(value)["75%"]) %>% 
+  ungroup() %>%
+  group_by(irrigation, pt_var) %>% 
+  mutate(p_adj = p.adjust(ttest, method = "fdr"),
+         pt_var = as_factor(pt_var),
+         alias = as_factor(alias),
+         irrigation = as_factor(irrigation),
+         genotype = as_factor(if_else(genotype == "MoneyMaker", glue("{genotype}"),glue("*{genotype}*"))),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*"))) %>% 
+  ungroup()
+
+
+# Estimation of normality and equal variances -----------------------------
+
+#dry weight biomass still missing
+#AOV TukeyHSD?
+#test manova
+
+pt_tidy_numeric <- pt_tidy %>% 
+  select(all_of(pt_vars))
+
+pt_norm <- pt_long %>% 
+  group_by(irrigation, genotype, pt_var) %>% 
+  summarise(norm = shapiro.test(value)$p.value)
+
+pt_norm_wt <- pt_norm %>% 
+  filter(genotype == "MoneyMaker") %>% 
+  select(irrigation, comp = genotype, pt_var, wt_norm = norm)
+
+pt_norm_mut <- pt_norm %>% 
+  filter(genotype != "MoneyMaker") %>% 
+  select(irrigation, genotype, pt_var, norm)
+
+pt_norm_comb <- pt_norm_wt %>% 
+  left_join(pt_norm_mut)
+
+pt_variance <- map(.x = pt_tidy_numeric, .f = ~pairwise.var.test(.x, pt_tidy$group, p.method = "none"))
+
+pt_variance_tidy <- pt_variance %>% 
+  map(tidy) %>% 
+  map2(.y = names(pt_variance), .f = ~.x %>% mutate(pt_vars = .y)) %>% 
+  purrr::reduce(bind_rows) %>% 
+  separate(group1,
+           into = c("genotype1", "irrigation1"),
+           sep = "_") %>% 
+  separate(group2,
+           into = c("genotype2", "irrigation2"),
+           sep = "_") %>% 
+  filter(irrigation1 == irrigation2, genotype1 == "MoneyMaker"& genotype2 != "MoneyMaker"|
+           genotype1 != "MoneyMaker" & genotype2 == "MoneyMaker") %>% 
+  mutate(genotype = if_else(genotype1 == "MoneyMaker", genotype2, genotype1),
+         comp = "MoneyMaker") %>% 
+  select(genotype, comp, irrigation = irrigation1, pt_var = pt_vars, p.value)
+
+prereq <- pt_norm_comb %>% 
+  left_join(pt_variance_tidy) %>% 
+  mutate(fulfill = if_else(wt_norm >= 0.05, norm >= 0.05, p.value >= 0.05))
+
+prereq_sum <- prereq %>% 
+  group_by(fulfill) %>% 
+  summarise(n = n()) %>% 
+  ungroup() %>% 
+  mutate(percent = 100*n/sum(n))
+
+pt_aov <- map(.x = pt_tidy_numeric, .f = ~aov(.x ~ alias* irrigation, data = pt_tidy))
+
+pt_aov_tidy <- pt_aov %>% 
+  map(tidy) %>% 
+  map2(.y = names(pt_aov), .f = ~.x %>% mutate(pt_vars = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+pt_aov_table <- pt_aov_tidy %>% 
+  left_join(pt_vars_plot) %>% 
+  filter(term != "Residuals") %>% 
+  select(term, pt_vars_label, p.value) %>% 
+  pivot_wider(names_from = term, values_from = p.value)
+
+write.xlsx(pt_aov_table, str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                               "anova_table.xlsx",
+                               sep = "_"))
+
+pt_tuk <- map(.x = pt_aov, .f = ~TukeyHSD(.x)) %>% 
+  map(.f = tidy) %>% 
+  map2(.y = names(pt_aov), .f = ~.x %>% mutate(var = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+# General differences -----------------------------------------------------
+
+sig_pt_overall <- pt_tuk %>% 
+  filter(term == "alias") %>% 
+  separate(col = contrast, into = c("group1", "group2"), sep = "-")
+
+lines <- sum_stat %>% distinct(alias,plantline) %>% 
+  mutate(plantline = as.double(as.character(plantline))) %>% 
+  left_join(genotypes)
+
+sig_pt_overall_table <- sig_pt_overall %>%  
+  filter(group1 == "967514 MM WT" | group2 == "967514 MM WT") %>% 
+  ungroup() %>% 
+  left_join(lines, by = c("group1" = "alias")) %>% 
+  select(var, group1, group2, adj.p.value, genotype1 = genotype) %>%
+  left_join(lines, by = c("group2" = "alias")) %>% 
+  select(var,adj.p.value, genotype1, genotype2 = genotype) %>% 
+  left_join(pt_vars_plot, by = c("var" = "pt_vars")) %>% 
+  pivot_wider(names_from = genotype2,
+              values_from = adj.p.value)
+
+write.xlsx(sig_pt_overall_table, str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+                                       "overall_significance.xlsx",
+                                       sep = "_"))
+
+# Pairwise significant changes --------------------------------------------
+
+per_comp_y <- pt_long %>% 
+  group_by(pt_var, irrigation) %>% 
+  summarise(max_y = max(value),
+            min_y = min(value))
+
+sig_pt_groups <- pt_tuk %>% 
+  filter(term == "alias:irrigation") %>% 
+  separate(col = contrast, into = c("group1", "group2"), sep = "-")
+
+sig_pt <- sig_pt_groups %>%  
+  separate(group1, into = c("alias1", "irrigation1"), sep = ":") %>% 
+  separate(group2, into = c("alias2", "irrigation2"), sep = ":") %>% 
+  filter(irrigation1 == irrigation2) %>% 
+  filter(alias1 == "967514 MM WT" | alias2 == "967514 MM WT") %>% 
+  ungroup() %>% 
+  mutate(p.signif = if_else(adj.p.value <= 0.05, "*", "ns")) %>% 
+  left_join(sum_stat, by = c("var" = "pt_var", "irrigation1" = "irrigation", "alias1" = "alias")) %>% 
+  select(var, irrigation1, irrigation2, alias1, alias2, p.signif, mean1 = mean, se1 = se, genotype1 = genotype, adj.p.value) %>%
+  left_join(sum_stat, by = c("var" = "pt_var", "irrigation2" = "irrigation", "alias2" = "alias")) %>% 
+  select(var, irrigation1, irrigation2, alias1, alias2, p.signif, mean1, mean2 = mean, se1, se2 = se, genotype1, genotype2 = genotype, adj.p.value) %>% 
+  rename(pt_var = var,
+         group1 = genotype1,
+         group2 = genotype2,
+         irrigation = irrigation1) %>% 
+  left_join(per_comp_y) %>% 
+  mutate(y.position = 1.1*(max_y)) %>% 
+  mutate(pt_var = as_factor(pt_var),
+         group1 = as_factor(group1),
+         group2 = as_factor(group2),
+         irrigation = as_factor(irrigation)) %>% 
+  ungroup()
+
+genotypes_label <- sum_stat %>% 
+  distinct(plantline, genotype)
+
+pt_vars
+
+plot_out <- vector("list", length = length(pt_vars))
+
+for(pt in seq_along(pt_vars)) {
+  
+  binwidth <- per_comp_y %>% 
+    filter(pt_var == pt_vars[[pt]]) %>% 
+    summarise(min_y_comp = min(min_y),
+              max_y_comp = max(max_y)) %>% 
+    ungroup() %>% 
+    mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+    select(binwidth) %>% 
+    as_vector() %>%
+    as.numeric()
+  
+  sig_pt_temp <- sig_pt %>% 
+    filter(pt_var == pt_vars[[pt]])
+  
+  plot_out[[pt]] <-  pt_long %>% 
+    select(-genotype) %>% 
+    left_join(genotypes_label) %>% 
+    left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+    filter(pt_var == pt_vars[[pt]]) %>%
+    ggplot(aes(genotype, value)) +
+    geom_boxplot(position = "dodge", color = "black", aes(fill = genotype)) + 
+    geom_dotplot(aes(fill = genotype), color = "black", binaxis = "y", stackdir = "center", binwidth = binwidth) +
+    facet_grid(cols = vars(irrigation), scales = "fixed")  +
+    stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.1,
+                       hide.ns = T) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1, margin = unit(c(1,0,0,0), "mm")),
+          axis.title.x = element_blank(),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 8, family = "Arial Narrow"),
+          legend.title = element_blank(),
+          legend.text = element_markdown(),
+          legend.position = "right",
+          legend.direction = "vertical",
+          plot.margin = unit(c(1,0,1,2), "mm"),
+          legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm")) +
+    scale_shape_manual(values = c(1,2,22,23)) +
+    scale_color_manual(values = cb_scale, aesthetics = "color")  +
+    scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+    xlab(label = "") +
+    ggtitle(pt_vars_label[[pt]])
+}
+
+plot_out[[pt]]
+pdf(file = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),"cmQTL_val1_phenotypes.pdf"),
+    width = 6.5,
+    height = 3)
+
+for (pt in seq_along(pt_vars)) {
+  print(plot_out[[pt]])
+}
+
+dev.off()
+
+if (!is.null(dev.list())) {
+  dev.off()
+}
+
+sum_stat %>% 
+  ggplot(aes(irrigation, mean, color = alias)) +
+  geom_point() +
+  geom_line(aes(group = plantline)) + 
+  geom_errorbar(aes(ymin = mean - sd, ymax = mean + sd)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+
+
+sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_pointrange(aes(ymin = mean - se, ymax = mean + se, shape = genotype, fill = genotype)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23))
+
+sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_point(aes(shape = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype, width = 0.25)) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23))
+
+
+ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+             "cmQTL_val1_phenotypic.jpg",
+             sep = "_"),
+       width = 183,
+       height = 100,
+       units = "mm",
+       dpi = 300)
+
+
+# Overview for supplemental -----------------------------------------------
+
+bins <- pt_long %>% 
+  group_by(pt_var) %>% 
+  summarise(binwidth = max(value)/60)
+
+cb_scale <- c("#E69F00", "#56B4E9", "#009E73", "#F0E442",
+              "#0072B2", "#D55E00","#000000")
+
+pt_long %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  group_by(plantline, genotype, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value, fill = genotype)) +
+  geom_dotplot(binaxis = "y", stackdir = "center") +
+  geom_smooth(aes(group = genotype, color = genotype), method = "lm", formula = y ~ poly(x,2), se = F, size = 1) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8, family = "Arial Narrow"),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  scale_color_manual(values = cb_scale, aesthetics = "color")
+
+sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype), size = 0.3) + 
+  geom_point(shape = 1, size = 2) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype, width = 0.25), size = 0.3) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 8, family = "Arial Narrow"),
+        legend.text = element_markdown(),
+        legend.position = "right") +
+  #scale_shape_manual(values = c(1,2,22,23)) +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  scale_color_manual(values = cb_scale, aesthetics = "color")
+
+ggsave("Overview_phenotype_dot_line.svg",width = 17.0, height = 8, units = "cm", dpi = 300)
+
+overview <- pt_long %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  mutate(genotype = as_factor(if_else(genotype == "MoneyMaker", glue("{genotype}"),glue("*{genotype}*"))),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*"))) %>% 
+  group_by(plantline, genotype, irrigation, pt_var, pt_vars_label) %>% 
+  summarise(mean = mean(value)) %>% 
+  ggplot(aes(irrigation, mean, fill = genotype)) +
+  #geom_dotplot(binaxis = "y", stackdir = "center") +
+  geom_line(aes(group = genotype, color = genotype), size = 0.5) +
+  geom_point(aes(group = genotype, color = genotype), size = 0.5) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1, size = 8, family = "sans"),
+        axis.title.x = element_text(size = 8, family = "sans"),
+        axis.title.y = element_text(size = 8, family = "sans"),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        strip.text = element_text(size = 6, family = "sans"),
+        text = element_text(size = 8, family = "sans"),
+        legend.title = element_text(size = 10, family = "sans"),
+        legend.text = element_markdown(size = 8),
+        legend.position = "right") +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  scale_color_manual(values = cb_scale, aesthetics = "color"); overview
+
+ggsave("Overview_phenotype_line_mean.png", width = 17.0, height = 8, units = "cm", dpi = 300, scale = 1)
+
+pt_long %>% 
+  #filter(genotype == "MoneyMaker") %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  group_by(plantline, genotype, irrigation, pt_var) %>% 
+  ggplot(aes(irrigation, value)) +
+  geom_boxplot(fill = "orange", size = 0.25) +
+  geom_smooth(aes(group = pt_var), method = "lm", formula = y ~ poly(x,2), se = F, size = 1) +
+  facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  com_theme +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  scale_color_manual(values = cb_scale, aesthetics = "color")
+
+ggsave("Overview_phenotype_box_line_all_g_aggregated.png",width = 17.0, height = 8, units = "cm", dpi = 300)
+saveRDS(last_plot(), "Overview_phenotype_box_line_all_g_aggregated.RDS")
+
+sum_stat_out <- sum_stat %>% 
+  mutate(genotype = str_remove_all(genotype, "\\*")) %>% 
+  left_join(pt_norm) %>% 
+  left_join(pt_variance_tidy) %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  left_join(sig_pt, by = c("irrigation", "pt_var", "alias" = "alias2")) %>% 
+  mutate(genotype = as_factor(genotype),
+         genotype = fct_relevel(genotype, c("MoneyMaker", "panK4-1", "log2-1", "transp1-1"))) %>% 
+  arrange(pt_vars_label, irrigation, genotype) %>% 
+  select(Genotype = genotype, Irrigation = irrigation, Trait = pt_vars_label,
+         Mean = mean, SD = sd, SE = se, N = n, Normality = norm, `Equal variance` = p.value,
+         `P-value` = adj.p.value)
+
+write.xlsx(sum_stat_out, "Summary_statistics.xlsx")
+
+pt_raw_out <- pt_long %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>%
+  arrange(pt_vars_label, irrigation, genotype) %>% 
+  select(Genotype = genotype, Irrigation = irrigation, Replicate = rep, Trait = pt_vars_label,
+         Value = value)
+
+write.xlsx(pt_raw_out, "Raw_data_phenotypes.xlsx")
+
+
+# Figure part panK4-1 total fruit number ----------------------------------
+
+A <- sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  filter(pt_var == "total_fruit_num", genotype == "MoneyMaker" | genotype == "*panK4-1*") %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_point(aes(color = genotype),
+             size = 1) +
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype),
+                width = 0.1) +
+  geom_text(aes(x = "0.8", y = 12, label = "TukeyHSD: 1.30*10^-3"), inherit.aes = F, size = 2) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  #facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  com_theme +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_color_manual(values = cb_scale, aesthetics = "color")
+
+A
+
+ggsave("Total_fruit_number_panK4.png",
+       width = 8.25,
+       height = 3,
+       units = "cm",
+       dpi = 300)
+
+saveRDS(A, "Total_fruit_number_panK4.RDS")
+
+# Figure part log2-1 average fruit weight ----------------------------------
+
+
+B <- sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  filter(pt_var == "av_fruit_weight", genotype == "MoneyMaker" | genotype == "*log2-1*") %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_point(aes(color = genotype),
+             size = 1) +
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype),
+                width = 0.1) +
+  geom_text(aes(x = "0.6", y = 17, label = "TukeyHSD: 6.69*10^-6"), inherit.aes = F, size = 2) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  #facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  com_theme +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_color_manual(values = cb_scale[c(1,3)], aesthetics = "color")
+
+B
+
+ggsave("Average_fruit_weight_log2-1.png",
+       width = 8.25,
+       height = 3,
+       units = "cm",
+       dpi = 300)
+
+saveRDS(B, "Average_fruit_weight_log2-1.RDS")
+
+# Figure part transp1-1 days until red ripe ----------------------------------
+
+
+C <- sum_stat %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  filter(pt_var == "days_until_redripe", genotype == "MoneyMaker" | genotype == "*transp1-1*") %>% 
+  ggplot(aes(irrigation, mean, color = genotype)) +
+  geom_line(aes(group = genotype)) + 
+  geom_point(aes(color = genotype),
+                  size = 1) +
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se, color = genotype),
+                width = 0.1) +
+  geom_text(aes(x = "0.6", y = 110, label = "TukeyHSD: 3.80*10^-3"), inherit.aes = F, size = 2) +
+  #geom_text(aes(label = is_sig), nudge_y = 10) +
+  #facet_wrap(nrow = 2, ncol = 4, facets = vars(pt_vars_label),scales = "free")+
+  com_theme +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_color_manual(values = cb_scale[c(1,4)], aesthetics = "color")
+
+C
+
+ggsave("Days_until_red_ripe_transp1-1.png",
+       width = 8.25,
+       height = 3,
+       units = "cm",
+       dpi = 300)
+
+saveRDS(C, "Days_until_red_ripe_transp1-1.RDS")
+# Combining plots ---------------------------------------------------------
+
+
+main_plot <- plot_grid(A,B,C, nrow = 1, labels = "AUTO",  label_size = 12)
+
+main_plot
+
+ggsave("Selected_phenotypes.png",
+       width = 17.0,
+       height = 6,
+       units = "cm",
+       dpi = 300)
+
+ 
+sum_stat %>% 
+  ggplot(aes(irrigation, mean, color = alias)) +
+  geom_point() +
+  geom_line(aes(group = plantline)) +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+ggsave(str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),
+             "cmQTL_val1_phenotypic.jpg",
+             sep = "_"),
+       width = 183,
+       height = 100,
+       units = "mm",
+       dpi = 300)
+
+# CV analysis -------------------------------------------------------------
+
+
+
+library(bootstrap)
+
+theta <- function(x){
+  sd(x)/mean(x)
+}
+
+CV <- sum_stat %>% 
+  ungroup() %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(grand_mean = mean(mean),
+            grand_sd = sd(mean)) %>% 
+  mutate(cv = grand_sd/grand_mean)
+
+cv_jack <- sum_stat %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(cv = jackknife(mean, theta)$jack.values) %>% 
+  mutate(jack_rep = row_number()) %>% 
+  ungroup() 
+
+cv_jack_wide <- cv_jack %>% 
+  pivot_wider(id_cols = c(plantline, genotype, alias, jack_rep),
+              names_from = pt_var,
+              values_from = cv)
+
+cv_jack_numeric <- cv_jack_wide %>% 
+  select(-c(alias, genotype, plantline, jack_rep))
+
+cv_jack_sum_stat <- cv_jack %>% 
+  group_by(plantline, genotype, alias, pt_var) %>% 
+  summarise(mean_cv = mean(cv),
+            sd = sd(cv),
+            n = n(),
+            se = sd/sqrt(n))
+
+
+pt_cv_jack <- map(.x = cv_jack_numeric, .f = ~aov(.x ~ alias, data = cv_jack_wide))
+
+pt_jack_t <- map(.x = cv_jack_numeric, .f = ~pairwise.t.test(x = .x , g = cv_jack_wide$alias)) %>% 
+  map(.f = tidy) #%>% 
+
+pt_jack_t_tidy <- pt_jack_t %>% 
+  map2(.y = names(pt_jack_t), .f = ~.x %>% mutate(var = .y)) %>% 
+  purrr::reduce(bind_rows)
+
+sig_pt_cv_groups <- pt_jack_t_tidy %>% 
+  filter(group1 == "967514 MM WT" | group2 == "967514 MM WT") %>% 
+  group_by(var) %>% 
+  mutate(adj.p.value = p.adjust(p.value)) %>% 
+  ungroup() %>% 
+  mutate(p.signif = if_else(adj.p.value <= 0.05, "*", "ns")) %>% 
+  left_join(cv_jack_sum_stat, by = c("var" = "pt_var", "group1" = "alias")) %>% 
+  select(var, group1, group2, p.signif, mean_cv1 = mean_cv, se1 = se, genotype1 = genotype) %>%
+  left_join(cv_jack_sum_stat, by = c("var" = "pt_var",  "group2" = "alias")) %>% 
+  select(var, group1, group2, p.signif, mean_cv1, mean_cv2 = mean_cv, se1, se2 = se, genotype1, genotype2 = genotype) %>% 
+  group_by(var) %>% 
+  mutate(tot_val1 = mean_cv1 + se1,
+         tot_val2 = mean_cv2 + se2,
+         y.position = 1.1*(max(tot_val1, tot_val2))) %>% 
+  rename(pt_var = var) %>% 
+  mutate(pt_var = as_factor(pt_var),
+         group1 = genotype1,
+         group2 = genotype2) %>% 
+  ungroup()
+
+cv_jack %>% 
+  ggplot(aes(alias, cv, fill = alias)) +
+  geom_boxplot() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+
+
+
+CV %>% 
+  ggplot(aes(alias, cv, fill = alias)) +
+  geom_col() +
+  facet_wrap(facets = vars(pt_var),scales = "free")
+
+x = rnorm(10)
+
+# Figure Average fruit weight ---------------------------------------------
+
+
+## Nominal all lines -----------------------------------------------------
+
+binwidth <- pt_long %>% 
+  filter(pt_var == "av_fruit_weight",
+         genotype %in% c("MoneyMaker", "*panK4-1*")) %>% 
+  group_by(pt_var) %>% 
+  summarise(min_y_comp = min(value),
+            max_y_comp = max(value)) %>% 
+  ungroup() %>% 
+  mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+  select(binwidth) %>% 
+  as_vector() %>%
+  as.numeric()
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "av_fruit_weight",
+         group1 %in% c("MoneyMaker", "*panK4-1*"),
+         group2 %in% c("MoneyMaker", "*panK4-1*"))
+
+A <-  pt_long %>% 
+  select(-genotype) %>% 
+  left_join(genotypes_label) %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  filter(pt_var == "av_fruit_weight", genotype %in% c("MoneyMaker", "*panK4-1*")) %>%
+  ggplot(aes(genotype, value)) +
+  geom_boxplot(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_dotplot(aes(fill = genotype), color = "black", binaxis = "y", stackdir = "center", binwidth = binwidth, dotsize = 1.5) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1, size = 8, family = "sans"),
+        axis.title.x = element_text(size = 8, family = "sans"),
+        axis.title.y = element_text(size = 8, family = "sans"),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        strip.text = element_text(size = 6, family = "sans"),
+        text = element_text(size = 8, family = "sans"),
+        legend.title = element_text(size = 10, family = "sans"),
+        legend.text = element_markdown(size = 8),
+        legend.position = "bottom",
+        legend.direction = "horizontal",
+        plot.margin = unit(c(1,0,1,2), "mm"),
+        legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm")) +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_color_manual(values = cb_scale, aesthetics = "color")  +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  xlab(label = "")
+
+A
+
+leg <- get_legend(A)
+
+A <- A + theme(legend.position = "")
+A
+
+## CV all lines ------------------------------------------------------------
+
+per_comp_y_cv <- cv_jack %>% 
+  group_by(pt_var) %>% 
+  summarise(max_y = max(cv),
+            min_y = min(cv))
+
+binwidth <- per_comp_y_cv %>% 
+  filter(pt_var == "av_fruit_weight") %>% 
+  summarise(min_y_comp = min(min_y),
+            max_y_comp = max(max_y)) %>% 
+  ungroup() %>% 
+  mutate(binwidth = (max_y_comp - min_y_comp)/50) %>% 
+  select(binwidth) %>% 
+  as_vector() %>%
+  as.numeric()
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "av_fruit_weight")
+
+B <-  cv_jack %>% 
+  select(-genotype) %>% 
+  left_join(genotypes_label) %>% 
+  left_join(pt_vars_plot, by = c("pt_var" = "pt_vars")) %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, cv)) +
+  geom_boxplot(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_dotplot(aes(fill = genotype), color = "black", binaxis = "y", stackdir = "center", binwidth = binwidth, dotsize = 1.5) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1, size = 8, family = "sans"),
+        axis.title.x = element_text(size = 8, family = "sans"),
+        axis.title.y = element_text(size = 8, family = "sans"),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        strip.text = element_text(size = 6, family = "sans"),
+        text = element_text(size = 8, family = "sans"),
+        legend.title = element_text(size = 10, family = "sans"),
+        legend.text = element_markdown(size = 8),
+        legend.position = "",
+        legend.direction = "vertical",
+        plot.margin = unit(c(1,1,1,2), "mm"),
+        legend.margin = margin(t = 0, r = 2, b = 0, l = 2 , unit = "mm")) +
+  scale_shape_manual(values = c(1,2,22,23)) +
+  scale_color_manual(values = cb_scale, aesthetics = "color")  +
+  scale_fill_manual(values = cb_scale, aesthetics = "fill") + 
+  xlab(label = "")
+
+B
+
+
+# Combining plots ---------------------------------------------------------
+
+
+main_plot <- plot_grid(A,B, nrow = 1, labels = "AUTO", rel_widths = c(2,1), label_size = 12)
+main_plot
+
+comp_plot <- plot_grid(main_plot, ncol = 1, leg, rel_heights = c(10,1))
+comp_plot
+
+ggsave("Average_fruit_weight_all_lines.png",
+       width = 17.0,
+       height = 8,
+       units = "cm",
+       dpi = 300)
+
+# Jackknife plots---------------------------------------------------------------
+
+
+
+
+pt_vars
+pt_vars_label <- c("Total fruit number per plant", "Total fruit weight per plant",
+                   "Average fruit weight", "Days until ripe fruit", "Shoot fresh weight",
+                   "Harvest index", "Biomass", "Shoot dry weight")
+
+plot_out <- vector("list", length = length(pt_vars))
+
+for(pt in seq_along(pt_vars)) {
+  sig_pt_temp <- sig_pt_cv_groups %>% 
+    filter(pt_var == pt_vars[[pt]])
+  
+  plot_out[[pt]] <-   cv_jack_sum_stat %>% 
+    filter(pt_var == pt_vars[[pt]]) %>%
+    ggplot(aes(genotype, mean_cv)) +
+    geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+    geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.75) +
+    stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.1,
+                       hide.ns = T) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 14),
+          legend.text = element_markdown(),
+          legend.position = "right") +
+    ggtitle(pt_vars_label[[pt]]) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+    ylab("mean CV")
+}
+
+plot_out[[1]]
+
+pdf(file = str_c(str_replace_all(Sys.Date(),"^.{2}|-",""),"cmQTL_val1_phenotypes_cv.pdf"),
+    width = 9,
+    height = 4)
+
+
+
+
+
+
+for (pt in seq_along(pt_vars)) {
+  print(plot_out[[pt]])
+}
+
+dev.off()
+
+
+
+plot(bootstrap::jackknife(x = x, theta = theta)$jack.values)
+
+stop(print("current stop"))
+
+# Figure 40 ---------------------------------------------------------------
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "total_fruit_weight")
+
+p1<-   sum_stat %>% 
+  filter(pt_var == "total_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(0,1150) +
+  ggtitle("Total fruit weight per plant") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p1
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p2<-   sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(c(0,65))+
+  ggtitle("Average fruit weight") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p2
+
+leg40 <- get_legend(sum_stat %>% 
+                      filter(pt_var == "av_fruit_weight") %>%
+                      ggplot(aes(genotype, mean)) +
+                      geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                      geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+                      facet_grid(cols = vars(irrigation), scales = "fixed")  +
+                      stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                         step.increase = 0.07,
+                                         hide.ns = T) +
+                      theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                            panel.background = element_rect(fill = "white"),
+                            panel.border = element_rect(color = "black",fill = NA),
+                            text = element_text(size = 14),
+                            legend.text = element_markdown(),
+                            legend.position = "right") +
+                      ggtitle("Average fruit weight") +
+                      scale_fill_grey(start = 1, end = 0, aesthetics = "fill"))
+leg40
+
+main_plot <- plot_grid(p1,p2, ncol = 1, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg40, ncol = 2, rel_widths =  c(3,1))
+comp_plot
+
+ggsave("figure_40.wmf",width = 15.8, height = 12, units = "cm", dpi = 300)
+
+# Figure 41 ---------------------------------------------------------------
+
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p1 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,1,0), "mm")) +
+  ggtitle("Average fruit weight") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p1
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "harvest_index")
+
+p2 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "harvest_index") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,1,0), "mm")) +
+  ggtitle("Harvest index") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p2
+
+leg41 <-   get_legend(cv_jack_sum_stat %>% 
+                        filter(pt_var == "harvest_index") %>%
+                        ggplot(aes(genotype, mean_cv)) +
+                        geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                        geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+                        stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                           step.increase = 0.1,
+                                           hide.ns = T) +
+                        theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                              panel.background = element_rect(fill = "white"),
+                              panel.border = element_rect(color = "black",fill = NA),
+                              text = element_text(size = 14),
+                              legend.text = element_markdown(),
+                              legend.position = "bottom") +
+                        ggtitle("Harvest index") +
+                        scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+                        ylab("mean CV"))
+
+
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg41, ncol = 1, rel_heights =  c(10,1)) + theme(plot.margin = unit(c(0,0,5,0), "mm"))
+comp_plot
+
+ggsave("figure_41.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
+
+
+# Figure means presentation -----------------------------------------------
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "total_fruit_weight")
+
+p1<-   sum_stat %>% 
+  filter(pt_var == "total_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(0,1150) +
+  ggtitle("Total fruit weight per plant") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p1
+
+sig_pt_temp <- sig_pt %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p2<-   sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+  facet_grid(cols = vars(irrigation), scales = "fixed")  +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.07,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.margin = unit(c(0,0,0,0), "cm")) +
+  xlab("") +
+  ylim(c(0,65))+
+  ggtitle("Average fruit weight") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+
+p2
+
+leg40 <- get_legend(sum_stat %>% 
+                      filter(pt_var == "av_fruit_weight") %>%
+                      ggplot(aes(genotype, mean)) +
+                      geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                      geom_errorbar(aes(ymin = mean - se, ymax = mean + se), position = position_dodge(width = 0.9), width = 0.25) +
+                      facet_grid(cols = vars(irrigation), scales = "fixed")  +
+                      stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                         step.increase = 0.07,
+                                         hide.ns = T) +
+                      theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                            panel.background = element_rect(fill = "white"),
+                            panel.border = element_rect(color = "black",fill = NA),
+                            text = element_text(size = 14),
+                            legend.text = element_markdown(),
+                            legend.position = "right") +
+                      ggtitle("Average fruit weight") +
+                      scale_fill_grey(start = 1, end = 0, aesthetics = "fill"))
+leg40
+
+main_plot <- plot_grid(p1,p2, ncol = 2, labels = "")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg40, ncol = 2, rel_widths =  c(6,1))
+comp_plot
+
+ggsave("Av_tot_fruit_weight_mean.wmf",width = 33.5, height = 12, units = "cm", dpi = 300)
+
+# Figure CV average fruit weight ---------------------------------------------------------------
+
+
+sig_pt_temp <- sig_pt_cv_groups %>% 
+  filter(pt_var == "av_fruit_weight")
+
+p1 <-   cv_jack_sum_stat %>% 
+  filter(pt_var == "av_fruit_weight") %>%
+  ggplot(aes(genotype, mean_cv)) +
+  geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+  geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+  stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                     step.increase = 0.1,
+                     hide.ns = T) +
+  theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+        panel.background = element_rect(fill = "white"),
+        panel.border = element_rect(color = "black",fill = NA),
+        text = element_text(size = 14),
+        legend.text = element_markdown(),
+        legend.position = "",
+        plot.title = element_text(size = 14),
+        plot.margin = unit(c(0,0,0,0), "mm")) +
+  ggtitle("Average fruit weight") +
+  xlab("") +
+  scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+  ylab("mean CV")
+p1
+
+
+
+leg41 <-   get_legend(cv_jack_sum_stat %>% 
+                        filter(pt_var == "av_fruit_weight") %>%
+                        ggplot(aes(genotype, mean_cv)) +
+                        geom_col(position = "dodge", color = "black", aes(fill = genotype)) + 
+                        geom_errorbar(aes(ymin = mean_cv - se, ymax = mean_cv + se), position = position_dodge(width = 0.9), width = 0.25) +
+                        stat_pvalue_manual(sig_pt_temp, label = "p.signif", y.position = "y.position",
+                                           step.increase = 0.1,
+                                           hide.ns = T) +
+                        theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+                              panel.background = element_rect(fill = "white"),
+                              panel.border = element_rect(color = "black",fill = NA),
+                              text = element_text(size = 14),
+                              legend.text = element_markdown(),
+                              legend.position = "right") +
+                        ggtitle("Harvest index") +
+                        scale_fill_grey(start = 1, end = 0, aesthetics = "fill") +
+                        ylab("mean CV"))
+
+
+
+main_plot <- plot_grid(p1, ncol = 1, labels = "")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg41, ncol = 2, rel_widths =  c(3,2)) + theme(plot.margin = unit(c(0,0,0,0), "mm"))
+comp_plot
+
+ggsave("av_fruit_weight_cv_presentation.wmf",width = 10, height = 10, units = "cm", dpi = 300)
+
+
+
+
+setwd(dirname(rstudioapi::getActiveDocumentContext()$path))
+getwd()
+
+library(tidyverse)
+library(ggpubr)
+library(glue)
+library(ggtext)
+library(cowplot)
+
+rm(list = ls())
+fc_1 <- read_csv("211012_fc_1.csv") %>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+sig_lip <- read_csv("211012_sig_lip.csv")
+
+fc_cv <- read.csv("211012_fc_cv.csv")%>% 
+  mutate(genotype = fct_relevel(genotype, c("MoneyMaker", "*panK4-1*", "*log2-1*", "*transp1-1*")))
+sig_lip_cv <- read_csv("211012_sig_lip_cv_groups.csv")
+
+met_dat <- fc_1 %>% 
+  distinct(met, Compound_Name, Compound_Class)
+
+genotypes <- fc_1 %>% 
+  distinct(alias, genotype)
+
+per_comp_y <- fc_1 %>%
+  group_by(tissue, treatment, met) %>% 
+  summarise(tot_val = max(mean_fc + se))
+
+make_col_plot <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_lip %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_1 %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = mean_fc)) +
+    geom_col(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_errorbar(aes(ymin = (mean_fc-se), ymax = (mean_fc + se)), position = position_dodge(0.9), width = 0.25, size = 0.75)+
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend,
+          plot.margin = unit(c(0,0,0,0), "cm")) +
+    ylab("Mean fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+  
+  plot_out
+  
+}
+
+per_comp_y_cv <- fc_cv %>%
+  group_by(tissue, met) %>% 
+  summarise(tot_val = max(mean_fc + se))
+
+make_col_plot_cv <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_lip_cv %>%  
+    left_join(genotypes, by = c("alias1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("alias2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y_cv) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  #ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_cv %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = mean_fc)) +
+    geom_col(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_errorbar(aes(ymin = (mean_fc-se), ymax = (mean_fc + se)), position = position_dodge(0.9), width = 0.25, size = 0.75)+
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    #facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_markdown(angle = 45, hjust = 1),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend) +
+    ylab("CV fold-change")+
+    #ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+  
+  plot_out
+  
+}
+
+#make_col_plot("m_1", "fruits", "test")
+
+make_col_plot_no_x <- function(plot_met, plot_tissue, plot_label, plot_legend){
+  
+  sig_bar <- sig_lip %>%  
+    left_join(genotypes, by = c("group1" = "alias")) %>%
+    rename(genotype1 = genotype) %>% 
+    left_join(genotypes, by = c("group2" = "alias")) %>% 
+    rename(genotype2 = genotype) %>% 
+    select(-group1, -group2) %>% 
+    #mutate(genotype1 = as_factor(if_else(genotype1 == "MoneyMaker", glue("{genotype1}"),glue("*{genotype1}*"))),
+    #       genotype2 = as_factor(if_else(genotype2 == "MoneyMaker", glue("{genotype2}"),glue("*{genotype2}*")))) %>% 
+    rename(group1 = genotype1, group2 = genotype2) %>% 
+    left_join(per_comp_y) %>% 
+    mutate(y.position = 1.1 * tot_val) %>% 
+    filter(met == plot_met, tissue == plot_tissue)
+  
+  ylim_top <- 1.1*max(sig_bar$y.position)
+  
+  plot_out <- fc_1 %>% 
+    filter(met == plot_met, tissue == plot_tissue) %>% 
+    ggplot(aes(x = genotype, y = mean_fc)) +
+    geom_col(position = "dodge", aes(fill = genotype), color = "black") +
+    geom_errorbar(aes(ymin = (mean_fc-se), ymax = (mean_fc + se)), position = position_dodge(0.9), width = 0.25, size = 0.75)+
+    stat_pvalue_manual(sig_bar, label = "p.signif", y.position = "y.position",
+                       step.increase = 0.07,
+                       hide.ns = T) +
+    facet_grid(cols = vars(treatment)) +
+    theme(axis.text.x = element_blank(),
+          panel.background = element_rect(fill = "white"),
+          panel.border = element_rect(color = "black",fill = NA),
+          text = element_text(size = 10),
+          legend.text = element_markdown(),
+          legend.position = plot_legend,
+          plot.margin = unit(c(0,0,0,0), "cm")) +
+    ylab("Mean fold-change")+
+    ylim(c(0, ylim_top))+
+    xlab("") +
+    ggtitle(label = plot_label) +
+    scale_fill_grey(start = 1, end = 0, aesthetics = "fill")
+  
+  plot_out
+  
+}
+
+# Figure 58 ---------------------------------------------------------------
+
+p1 <- make_col_plot_no_x("Cluster_08623", "leaves", "TAG 48:1", "none")
+p1
+
+p2 <- make_col_plot_no_x("Cluster_08498", "leaves", "TAG 48:3 (1)", "none")
+p2
+
+p3 <- make_col_plot_no_x("Cluster_08438", "leaves", "TAG 48:4", "none")
+p3
+
+p4 <- make_col_plot_no_x("Cluster_09298", "leaves", "TAG 50:3 (1)", "none")
+p4
+
+p5 <- make_col_plot("Cluster_09251", "leaves", "TAG 50:4 (1)", "none")
+p5
+
+p6 <- make_col_plot("Cluster_10916", "leaves", "TAG 54:5", "none")
+p6
+
+leg58 <- get_legend(make_col_plot("Cluster_08623", "leaves", "sucrose", "bottom"))
+leg58
+
+main_plot <- plot_grid(p1,p2,p3,p4,p5,p6, ncol = 2, rel_heights = c(1,1,1.35), labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg58, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("figure_58.wmf",width = 15.8, height = 16, units = "cm", dpi = 300)
+
+# Figure 61 ---------------------------------------------------------------
+
+p1 <- make_col_plot_cv("Cluster_09079", "leaves", "TAG 50:7 (2)", "none")
+p1
+
+p2 <- make_col_plot_cv("Cluster_10388", "leaves", "TAG 52:0", "none")
+p2
+
+p3 <- make_col_plot_cv("Cluster_09920", "leaves", "TAG 52:7 (2)", "none")
+p3
+
+p4 <- make_col_plot_cv("Cluster_11255", "leaves", "TAG 54:0", "none")
+p4
+
+leg61 <- get_legend(make_col_plot("Cluster_09079", "leaves", "sucrose", "bottom"))
+leg61
+
+main_plot <- plot_grid(p1,p2,p3,p4, ncol = 4, labels = "AUTO")
+main_plot
+
+comp_plot <- plot_grid(main_plot, leg61, ncol = 1, rel_heights = c(10,1))
+comp_plot
+
+ggsave("figure_61.wmf",width = 15.8, height = 8, units = "cm", dpi = 300)
-- 
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