inv4m Analysis Pipeline

Modulation of Flowering Time, Plant Height, and Growth Regulation Gene Networks

Comprehensive analysis of the maize chromosomal inversion Inv4m: phenotypic effects on flowering and growth, gene-by-environment interactions, JMJ candidate genes, and WGCNA network perturbation

2 Research Papers
8 Main Figures
7 Supplementary Figures
35+ Analysis Scripts
1

Inversion Paper

Complete (8 main figures, 6 supplementary)

Genomic Analysis (Figure 1)

Figure 1 Assembly

Multi-panel composition of the breeding scheme, repeat annotation, AnchorWave dotplots, breakpoint self-similarity, and population genotypes

Figure 1 (assembly)

Synteny & Repeat Annotation

Repeat tracks, breakpoint segments, and AnchorWave/jcvi dotplots for B73, Mi21, PT, TIL18

Figure 1 panels B–D

Genotype & SNP Distribution

Inv4m-correlated SNP panel and chromosome distribution; produces the Figure 1 E–F panels and the Figure S1 SNP distribution plot

Figure 1 panels E–F, Figure S1, Table 1

Breakpoint Tables

Inv4m delimitation across four genomes (B73, Mi21, PT, TIL18) and knob-repeat composition at the breakpoints

Tables S1, S2

Phenotypic Effects

Phenotype Analysis

Effect of Inv4m on plant height, flowering time (DTA, DTS), and harvest index

Figure 2

SAM Morphology

DIC microscopy analysis of shoot apical meristem dimensions

Figure 2 (SAM panels)

Internode Analysis

Internode length profiles, node count, and height validation

Figure S4

GxE Analysis

Genotype × Environment interactions across 3 field environments (PA2022, PA2025, NC2025)

Figure S2, Figure S3, Table S4

Transcriptomics

Figure 3 Assembly

8-panel composite figure showing global and local transcriptomic effects

Figure 3 (complete assembly)

Differential Expression

Limma analysis of Inv4m effects on gene expression with plant-level blocking

Figure 3 (MDS panel), Table S3

Volcano Plot

Visualization of DEGs with labeled candidate genes (Figure 3 panel C)

Figure 3 panel C

Manhattan Plots

Genome-wide visualization of expression effects by chromosome

Figure 3 panels D, E, G, H

Phenotype Association Filter

Overlap of Inv4m DEGs with published flowering time and plant height candidate gene lists (GWAS Atlas, tibbs-cortes, wang2021, li2022)

Table 2

Sequence Divergence vs DE

CDS sequence divergence between Mi21 and B73 vs differential expression of Inv4m DEGs

Supplementary Figure (divergence)

Network Analysis

Trans Coexpression Network

MaizeNetome-validated trans-regulatory network of Inv4m DEGs (reference / dataset-specific edge split)

Figure 5 (network data)

Network GO Enrichment

Gene Ontology enrichment of trans-regulated gene networks; produces the Figure 5 annotation overlay

Figure 5 (GO panel)

WGCNA Module Perturbation (Figure 6)

Consensus network analysis showing module disruption in Inv4m: dendrogram, kWithin permutation Z-score density, and hub-gene connectivity

Figure 6, Table S5 (preservation)

WGCNA Bootstrap Support

Bootstrap support comparison between the Genotype Response and Leaf Gradient reference networks

Figure S5

WGCNA Field Perturbation Pipeline

7-step consensus WGCNA pipeline (data prep → consensus networks → bootstrap support → preservation → module annotation). Shell-orchestrated with checkpoint support.

Figure 6 panels, Figure S6 (GO), Table S5 (preservation)

JMJ Cluster Analysis (Figure 7)

JMJ Cluster Expression

Gene-level (V5 annotation) total cluster expression; 5:1 copy-number contrast t-test of B73 vs Inv4m across PA2022 field and Crow 2020 growth-chamber tissues. Also covers the cell-proliferation companion genes (pcna2, smo4).

Figure 7 panel A

Pink Module Characterization

30 DEGs co-expressed with jmj2/jmj4 in the pink WGCNA module (growth network)

Table S7

5-Genome Microsynteny

jcvi pipeline for JMJ cluster synteny across B73, PT, TIL18, CML457, CML459

Figure 7 panel B (bash script)

JMJ 5-Paralog Split (planned supplementary)

Transcript-level expression of all 5 JMJ paralogs (jmj9, psi, jmj6, jmj2, jmj4) using a kallisto-corrected cDNA reference where each paralog is a distinct entry. Read-distribution diagnostic.

Planned supplementary figure (manuscript TODO)

Crow 2020 Reanalysis

Reanalysis of growth chamber dataset for JMJ expression validation

Figure 7 panel A (Crow data)
2

Phosphorus Paper

Complete (12 scripts)

Foundation

Spatial Correction

Spatial correction for field phenotypes using SpATS models

Differential Expression

RNA-seq analysis identifying DEGs under leaf × phosphorus treatment interaction

Differential Lipid Analysis

Lipidomics analysis of membrane remodeling under phosphorus stress

Phenotypes

Growth Curves

Longitudinal growth analysis comparing Inv4m and control genotypes

Ionome Profiling

Mineral nutrient analysis (P, K, Ca, Mg, Fe, Zn, etc.) across genotypes

Phenotype Marginal Means

Estimated marginal means for phenotypic traits with statistical comparisons

Enrichment

GO Enrichment

Gene Ontology enrichment analysis of differentially expressed genes

KEGG Enrichment

KEGG pathway analysis linking DEGs to metabolic pathways

LION Enrichment

Lipid ontology enrichment for differentially abundant lipids

Synthesis

Transcription Indices

Gene expression indices for photosynthesis, senescence, and stress response

Volcano Plots

Visualization of differential expression results with annotated genes