DOI: 10.5061/dryad.3ffbg79sv
title: Moisture-responsive root-branching pathways identified in diverse maize breeding germplasm

description: Plants grow complex root systems to extract unevenly distributed resources
 from soils. Spatial differences in soil moisture are perceived by root
 tips leading to the patterning of new root branches towards available
 water, a process called hydropatterning. Little is known about
 hydropatterning behavior and its genetic basis in crops plants. Here, we
 develop an assay to measure hydropatterning in maize and reveal
 substantial differences between tropical/subtropical and temperate maize
 breeding germplasm that likely resulted from divergent selection. Genetic
 dissection of hydropatterning confirmed the regulatory role of auxin and
 revealed that the gaseous hormone ethylene locally inhibits root branching
 from air-exposed tissues. Our results demonstrate how distinct signaling
 pathways translate spatial patterns of water availability to developmental
 programs that determine root architecture.
description: All methods can be found in the supplementary materials of the
 related publication.
description: # Moisture-responsive root-branching pathways identified in diverse maize
 breeding germplasm The data we submitted was collected for a project with
 the aim to characterize hydropatterning and elucidate genetic control of
 hydropatterning in maize (*Zea mays*). Validation of maize candidate genes
 for hydropatterning was carried out in Arabidopsis (*Arabidopsis
 thaliana*) using mutants of gene orthologs. We included the following: (1)
 Laser cutter files to re-create acrylic pieces needed to construct the
 hydropatterning assay for maize (LaserCutterFiles.zip). (2) Raw data from
 the maize portion of the project (MaizeHydropatterning.zip). The folder
 contains all data needed to execute the related R scripts. (3) Raw data
 from the Arabidopsis portion of the project
 (ArabidopsisHydropatterning.zip). The folder contains all data needed to
 execute the related R scripts. (4) A copy of the supplementary data of the
 manuscript (Supplementary_data_S1_to_S16.xlsx). (5) Scripts to run the
 Genome and Transcriptome Wide Association Studies (GWAS_TWAS.zip). (6)
 Scripts to perform image analysis related to the hydropatterning assay in
 ImageJ (ImageJ.zip). (7) A Markdown file that contains the Qst-Fst
 analysis portion of the project (Qst-Fst.zip). (8) R scripts for the
 analysis of maize and Arabidopsis raw data (R_code.zip). ## Description of
 the data and file structure ### Maize hydropatterning assay construction
 The maize hydropatterning assay was constructed using two different shapes
 of acrylic sheets that were cut using a laser cutter (Universal Laser
 Systems VLS 4.60). #### File structure: ``` LaserCutterFiles.zip |-
 Rack.svg |- Sheet.svg ``` #### File description: ##### Rack.svg Scalable
 Vector Graphics file that contains the traces to cut the acrylic panels of
 the Rhizo-sheet rack. The file can be opened and edited with programs such
 as Adobe Illustrator or Inkscape. ##### Sheet.svg Scalable Vector Graphics
 file that contains the traces to cut the Acrylic sheet for the Rhizo-sheet
 assembly. The file can be opened and edited with programs such as Adobe
 Illustrator or Inkscape. ### Maize hydropatterning analysis ZIP archive of
 data files that contain raw data and additional information needed for the
 analysis of hydropatterning in maize. The folder "data/" should
 be placed into the folder "MaizeHydropatterning/" (contained in
 the ZIP archive R_code.zip). That way all scripts in
 "MaizeHydropatterning/" can be executed. #### File structure:
 ``` MaizeHydropatterning.zip |- data/ | |- Pheno/ | | |-
 MaizeDiversityPanel_Info.csv | | |- Rep1/ | | | |- GWAS1_LRcount.csv | | |
 |- GWAS1_Positions.csv | | |- Rep2/ | | | |- GWAS2_LRcount.csv | | | |-
 GWAS2_Positions.csv | | |- Rep3/ | | | |- GWAS3_LRcount.csv | | | |-
 GWAS3_Positions.csv | | |- pheno.BLUP.csv | | |- all_data.BLUP.csv | | |-
 EXP023/ | | | |- EXP023_LRcount.csv | | | |- EXP023_Positions.csv | | | |-
 summary_data.csv | |- GWAS/ | | |- FarmCPUpp_output/ | | | |-
 AIRdensity.GWAS_noMAF.csv | | | |- AIRdensity.GWAS_0.022MAF.csv | | | |-
 AIRdensity.GWAS_0.044MAF.csv | | |- input/ | | | |-
 AIRdensity_sigSNP_LD.ld | | | |- gwas_sigSNP_AIRdensity_posB73v3.bed | | |
 |- gwas_sigSNP_AIRdensity_posB73v4.bed | | | |-
 gwas_sigSNP_AIRdensity_posB73v5.bed | | |- output/ | | | |-
 gwas_sigSNP_AIRdensity_posB73v2.bed | |- TWAS/ | | |-
 AIRdensity_run41000.txt | | |- expression.rpm.txt | | |-
 SRR_genotype_key.csv | | |- eQTL/ | | |- |- GRMZM2G002765.GWAS.csv | | |-
 |- eQTL_sigSNP_posB73v2.bed | | |- |- eQTL_sigSNP_posB73v3.bed | | |- |-
 eQTL_sigSNP_posB73v4.bed | | |- |- eQTL_sigSNP_posB73v5.bed | |-
 GeneModels/ | | |- Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3 | |-
 OtherData/ | | |- MuralEtAl2022GigaSci/ | | | |-
 SupplementalDataFileS2_GenotypesAndTraitValues_subset.csv | | | |-
 SupplementalDataFileS3_PhenotypeMetaData.csv | | |- PopStructure/ | | | |-
 ADMIXTURE_withOutgroups.csv | | | |- maize_pca.csv ``` #### File
 descriptions: ##### data/Pheno/MaizeDiversityPanel\_Info.csv Scalable
 Vector Graphics file that contains the traces to cut the acrylic panels of
 the Rhizo-sheet rack. * *SortNo*: Unique internal number to identify each
 maize inbred line. * *PlantID*: Unique ID assigned to each maize seed
 stock by the USDA National Plant Germplasm System. * *PlantName*: Common
 name of maize inbred line. * *genotype*: Internally used genotype name of
 maize inbred line. * *Subpopulation_FlintGarcia*: Subpopulation assigned
 by Flint-Garcia et.al. Plant J. 2005 Dec;44(6):1054-64. doi:
 10.1111/j.1365-313X.2005.02591.x. Assignments: [ss] stiff stalk, [nss]
 non-stiff stalk, [ts] tropical/subtropical, [mixed] less than 80%
 subpopulation identity of ss/nss/ts, [popcorn] popcorn outgroup, [sweet]
 sweet corn outgroup. * *Subpopulation*: Subpopulation assignments done in
 this study. Assignments: [ss] stiff stalk, [nss] non-stiff stalk, [ts]
 tropical/subtropical, [mixed] less than 80% subpopulation identity of
 ss/nss/ts, [popcorn] popcorn outgroup, [sweet] sweet corn outgroup. #####
 data/Pheno/Rep\[1/2/3]/GWAS\[1/2/3]\_LRcount.csv Data of lateral root
 counts from the hydropatterning assay. Three rounds of phenotyping were
 conducted (Rep1/Rep2/Rep3). * *Position*: ID of physical position in the
 hydropatterning assay. This position is translated to a genotype in the
 GWAS[1/2/3]_Position.csv files. * *X*: X-coordinate of lateral root in the
 picture that the lateral roots were counted on. * *Y*: Y-coordinate of
 lateral root in the picture that the lateral roots were counted on. *
 *Slice*: Lateral root emerged on the contact-side [2] or air-side [1].
 ##### data/Pheno/Rep\[1/2/3]/GWAS\[1/2/3]\_Position.csv File that
 translated the Position information into a specific genotype (SortNo). *
 *Box*: Box ID for the hydropatterning assay. We used boxes 1-15. Each box
 was able to hold 20 plants. * *Position*: ID of physical position in the
 hydropatterning assay. * SortNo: Unique internal number to identify each
 maize inbred line. * *SeedNo*: Five seeds were germinated for each inbred
 line. This number specifies which seed was transplanted into the
 hydropatterning assay. ##### data/Pheno/pheno.BLUP.csv Best Linear
 Unbiased Predictions of phenotypes for each maize inbred line grown in the
 hydropatterning assay and used in the GWAS/TWAS analysis. * *genotype*:
 Internally used genotype name of maize inbred line. * *AIRdensity*:
 Air-side lateral root density. * *pAIR*: Percent air-side lateral root. *
 *CONTdensity*: Contact-side lateral root density. * *LRBD*: Total lateral
 root density. * *Diameter*: Average diameter of primary root. *
 *AnthocyaninValue*: Anthocyanin coloration value on primary root. #####
 data/Pheno/all\_data.BLUP.csv Best Linear Unbiased Predictions of
 phenotypes for all maize inbred line grown in the hydropatterning assay. *
 *genotype*: Internally used genotype name of maize inbred line. *
 *AIRdensity*: Air-side lateral root density. * *pAIR*: Percent air-side
 lateral roots. * *CONTdensity*: Contact-side lateral root density. *
 *LRBD*: Total lateral root density. * *Diameter*: Average diameter of
 primary root. * *AnthocyaninValue*: Anthocyanin coloration value on
 primary root. ##### data/Pheno/EXP023/EXP023\_LRcount.csv Data of lateral
 root counts from the hydropatterning assay for the comparison of
 hydropatterning on primary and crown roots. * *Position*: ID of physical
 position in the hydropatterning assay. This position is translated to a
 genotype in the EXP023_Position.csv file. * *X*: X-coordinate of lateral
 root in the picture that the lateral roots were counted on. * *Y*:
 Y-coordinate of lateral root in the picture that the lateral roots were
 counted on. * *Slice*: Lateral root emerged on the contact-side [2] or
 air-side [1]. ##### data/Pheno/EXP023/EXP023\_Positions.csv File that
 translated the Position information into a specific genotype (SortNo). *
 *Box*: Box ID for the hydropatterning assay. We used boxes 1-15. Each box
 was able to hold 20 plants. * *Position*: ID of physical position in the
 hydropatterning assay. * genotype: Internally used genotype name of maize
 inbred line. * *RootType*: Screening of hydropatterning on primary or
 crown root. * *Date*: Date of seedling transfer to the hydropatterning
 assay. * *Time*: Time of seedling transfer to the hydropatterning assay;
 [AM] morning, [PM] afternoon. ##### data/Pheno/EXP023/summary\_data.csv
 Summarized data from the comparison of hydropatterning on primary and
 crown roots. * *genotype*: Internally used genotype name of maize inbred
 line. * *RootType*: Screening of hydropatterning on primary or crown root.
 * *N_replicates*: How many biological replicates were summarized. *
 *LRBD_mean*: Mean value of total lateral root density [lateral roots/cm].
 * *LRBD_median*: Median value of total lateral root density [lateral
 roots/cm]. * *LRBD_sd*: Standard deviation value of total lateral root
 density [lateral roots/cm]. * *CONTdensity_mean*: Mean value of
 contact-side lateral root density [lateral roots/cm]. *
 *CONTdensity_median*: Median value of contact-side lateral root density
 [lateral roots/cm]. * *CONTdensity_sd*: Standard deviation value of
 contact-side lateral root density [lateral roots/cm]. * *AIRdensity_mean*:
 Mean value of air-side lateral root density [lateral roots/cm]. *
 *AIRdensity_median*: Median value of air-side lateral root density
 [lateral roots/cm]. * *AIRdensity_sd*: Standard deviation value of
 air-side lateral root density [lateral roots/cm]. * *pAIR_mean*: Mean
 value of percent air-side lateral roots [%]. * *pAIR_median*: Median value
 of percent air-side lateral roots [%]. * *pAIR_sd*: Standard deviation
 value of percent air-side lateral roots [%]. #####
 data/GWAS/FarmCPUpp\_output/AIRdensity.GWAS\_\[noMAF/0.022MAF/0.044MAF].csv Output from Genome Wide Association Study using FarmCPUpp with AIRdensity (data/Pheno/pheno.BLUP.csv) as input phenotype. Output for three minor allele frequency levels: noMAF = 0.044, 0.022MAF = 0.022, 0.044MAF = 0.044. * *SNP*: SNP ID in the form of [Chromosome]_[Position] in B73 RefGen_v2. * *Chromosome*: Chromosome that the SNP is on in B73 RefGen_v2. * *Position*: SNP position in B73 RefGen_v2. * *p.value*: Significance value. * *estimate*: Effect estimate of SNP. * *stderr*: Standard deviation of estimate. * *tvalue*: FDR corrected significance value. ##### data/GWAS/output/gwas\_sigSNP\_AIRdensity\_posB73v2.bed BED file as input to translate SNP positions from GWAS (B73 RefGen_v2) to newer versions of the B73 genome. * *Column1*: Chromosome in B73 RefGen_v2. * *Column2*: Position in B73 RefGen_v2. * *Column3*: Position in B73 RefGen_v2. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/GWAS/input/AIRdensity\_sigSNP\_LD.ld Linkage analysis of significant SNPs from GWAS using PLINK. * *CHR_A*: Chromosome ID of SNP A in B73 RefGen_v2. * *BP_A*: Position on chromosome of SNP A in B73 RefGen_v2. * *SNP_A*: SNP ID of SNP A in B73 RefGen_v2. * *CHR_B*: Chromosome ID of SNP B in B73 RefGen_v2. * *BP_B*: Position on chromosome of SNP B in B73 RefGen_v2. * *SNP_B*: SNP ID of SNP B in B73 RefGen_v2. * *R2*: R2 value of linkage between SNP A and B. ##### data/GWAS/input/gwas\_sigSNP\_AIRdensity\_posB73v3.bed Translation of SNP positions from GWAS (B73 RefGen_v2) to B73 RefGen_v3. * *Column1*: Chromosome in B73 RefGen_v3. * *Column2*: Position in B73 RefGen_v3. * *Column3*: Position in B73 RefGen_v3. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/GWAS/input/gwas\_sigSNP\_AIRdensity\_posB73v4.bed Translation of SNP positions from GWAS (B73 RefGen_v2) to Zm-B73-REFERENCE-GRAMENE-4.0. * *Column1*: Chromosome in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column2*: Position in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column3*: Position in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/GWAS/input/gwas\_sigSNP\_AIRdensity\_posB73v5.bed Translation of SNP positions from GWAS (B73 RefGen_v2) to Zm-B73-REFERENCE-NAM-5.0. * *Column1*: Chromosome in Zm-B73-REFERENCE-NAM-5.0. * *Column2*: Position in Zm-B73-REFERENCE-NAM-5.0. * *Column3*: Position in Zm-B73-REFERENCE-NAM-5.0. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/TWAS/AIRdensity\_run41000.txt Output from the Transcriptome Wide Association Study with AIRdensity (data/Pheno/pheno.BLUP.csv) as input phenotype. * *Genotype*: Internally used genotype name of maize inbred line. * *Effect*: Effect size related to air-side LR density (BLUP). * *EffectVar*: Effect variance related to air-side LR density (BLUP). * *ModelFreq*: Model frequency (arbitrary unit). * *map-posn*: Map position for gene in the form of [Chromosome]_[Position]. ##### data/TWAS/expression.rpm.txt Root gene expression data for maize inbred lines from Kremling et.al. Nature. 2018 Mar 22;555(7697):520-523. doi: 10.1038/nature25966. Data was used as input for TWAS. * *genotype*: SRR identifier of genotype. * \[Gene IDs 1...n]: Column names are gene IDs (B73 RefGen_v2). Values are normalized expression by genotype in roots. ##### data/TWAS/SRR\_genotype\_key.csv Key to relate SRR identifier to genotype for TWAS analysis in file "data/TWAS/expression.rpm.txt". * *genotype*: Internally used genotype name of maize inbred line. * *SRR*: SRR identifier of genotype. ##### data/TWAS/eQTL/GRMZM2G002765.GWAS.csv Output from eQTL mapping of ZmAXR1 using FarmCPUpp with ZmAXR1 root gene expression as an input. * *SNP*: SNP ID in the form of [Chromosome]_[Position] in B73 RefGen_v2. * *Chromosome*: Chromosome that the SNP is on in B73 RefGen_v2. * *Position*: SNP position in B73 RefGen_v2. * *p.value*: Significance value. * *estimate*: Effect estimate of SNP. * *stderr*: Standard deviation of estimate. * *tvalue*: FDR corrected significance value. ##### data/TWAS/eQTL/eQTL\_sigSNP\_posB73v2.bed BED file as input to translate SNP positions from eQTL mapping (B73 RefGen_v2) to newer versions of the B73 genome. * *Column1*: Chromosome in B73 RefGen_v2. * *Column2*: Position in B73 RefGen_v2. * *Column3*: Position in B73 RefGen_v2. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/TWAS/eQTL/eQTL\_sigSNP\_posB73v3.bed Translation of SNP positions from eQTL mapping (B73 RefGen_v2) to B73 RefGen_v3. * *Column1*: Chromosome in B73 RefGen_v3. * *Column2*: Position in B73 RefGen_v3. * *Column3*: Position in B73 RefGen_v3. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/TWAS/eQTL/eQTL\_sigSNP\_posB73v4.bed Translation of SNP positions from eQTL mapping (B73 RefGen_v2) to Zm-B73-REFERENCE-GRAMENE-4.0. * *Column1*: Chromosome in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column2*: Position in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column3*: Position in Zm-B73-REFERENCE-GRAMENE-4.0. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/TWAS/eQTL/eQTL\_sigSNP\_posB73v5.bed Translation of SNP positions from eQTL mapping (B73 RefGen_v2) to Zm-B73-REFERENCE-NAM-5.0. * *Column1*: Chromosome in Zm-B73-REFERENCE-NAM-5.0. * *Column2*: Position in Zm-B73-REFERENCE-NAM-5.0. * *Column3*: Position in Zm-B73-REFERENCE-NAM-5.0. * *Column4*: SNP ID in B73 RefGen_v2. ##### data/GeneModels/Zm-B73-REFERENCE-NAM-5.0\_Zm00001eb.1.gff3 Gene model files used to associate genes to significant SNPs for GWAS and eQTL mapping. The file was downloaded from: [https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/](https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/) ##### data/OtherData/MuralEtAl2022GigaSci/SupplementalDataFileS2\_GenotypesAndTraitValues\_subset.csv Supplementary data file 2 from Mural et.al. *GigaScience*, Volume 11, 2022, giac080, [https://doi.org/10.1093/gigascience/giac080](https://doi.org/10.1093/gigascience/giac080). Data were used for correlation analysis between hydropatterning traits and other below and above group plant traits. ##### data/OtherData/MuralEtAl2022GigaSci/SupplementalDataFileS3\_PhenotypeMetaData.csv Supplementary data file 3 from Mural et.al. *GigaScience*, Volume 11, 2022, giac080, [https://doi.org/10.1093/gigascience/giac080](https://doi.org/10.1093/gigascience/giac080). Data were used for correlation analysis between hydropatterning traits and other below and above group plant traits. ##### data/OtherData/PopStructure/ADMIXTURE\_withOutgroups.csv Population structure analysis output from ADMIXTURE v1.3.0 (k=3). * *Sample*: Taxon ID of genotypes according to Zea mays HapMap v3.2.1. * *genotype*: Internally used genotype name of maize inbred line. * *Subpopulation_FlintGarcia*: Subpopulation assigned by Flint-Garcia et.al. Plant J. 2005 Dec;44(6):1054-64. doi: 10.1111/j.1365-313X.2005.02591.x. Assignments: [ss] stiff stalk, [nss] non-stiff stalk, [ts] tropical/subtropical, [mixed] less than 80% subpopulation identity of ss/nss/ts, [popcorn] popcorn outgroup, [sweet] sweet corn outgroup. * *Prop_ts*: Tropical/subtropical ancestry component. * *Prop_ss*: Stiff stalk ancestry component. * *Prop_nss*: Non-stiff stalk ancestry component. * *Prop_pop*: A priory assignment of popcorn ancestry. * *Prop_sweet*: A priory assignment of sweet corn ancestry. * *Subpopulation*: Subpopulation assignments done in this study. Assignments: [ss] stiff stalk, [nss] non-stiff stalk, [ts] tropical/subtropical, [mixed] less than 80% subpopulation identity of ss/nss/ts, [popcorn] popcorn outgroup, [sweet] sweet corn outgroup. ##### data/OtherData/PopStructure/maize\_pca.csv Principal component analysis of population structure. * *ind*: Taxon ID of genotypes according to Zea mays HapMap v3.2.1. * *PC*[1-20]: Principal components 1-20. * *pop*: Subpopulation assignments done in this study. Assignments: [ss] stiff stalk, [nss] non-stiff stalk, [ts] tropical/subtropical, [mixed] less than 80% subpopulation identity of ss/nss/ts, [popcorn] popcorn outgroup, [sweet] sweet corn outgroup. ### Arabidopsis hydropatterning analysis ZIP archive of data files that contain raw data and additional information needed for the analysis of hydropatterning in Arabidopsis. The folder "data/" should be placed into the folder "ArabidopsisHydropatterning/" (contained in the ZIP archive R_code.zip). That way all scripts in "ArabidopsisHydropatterning/" can be executed. #### File structure: ``` ArabidopsisHydropatterning.zip |- data/ | |- MutantScreen/ | | |- HA_[YYYY_MM_DD].csv | | |- HA_MutantInfo.csv | | |- stats_output.csv | |- EthylenePathway/ | | |- HA_[YYYY_MM_DD].csv | | |- HA_MutantInfo.csv | |- Pharma/ | | |- HA_[YYYY_MM_DD].csv ``` #### File descriptions: ##### data/MutantScreen/HA\_\[YYYY\_MM\_DD].csv Raw data files collected for hydropatterning assays conducted with Arabidopsis plants comparing Col-0 wild-type and mutant plants for Arabidopsis gene orthologs to maize candidate genes from GWAS and TWAS. * *Date*: Date when the data was collected. * *Line*: Name of the Arabidopsis line/accession/mutant. * *Plate*: Number assigned to the plate replicate. * *Plant*: Replicate plant on a plate. * *Group*: Grouping variable constructed from date and name of mutant tested. * *MediaSide*: Number of lateral roots counted that grew towards the media (contact-side lateral roots/seedling). * *AirSide*: Number of lateral roots counted that grew towards the air (air-side lateral roots/seedling). * *Notes*: Notes on plants that did not germinate, or were small, or grew into their roots into the media. * *RootLength*: Total primary root length of the plant from hypocotyl to primary root tip (cm). * *RootLength_TtoB*: Primary root length between the first and last lateral root counted (cm). ##### data/MutantScreen/HA\_MutantInfo.csv Info on Arabidopsis mutants related to maize candidate genes screened in the hydropatterning assay. * *Group*: Grouping variable linked to the "Group" in the HA_[YYYY_MM_DD].csv files * *Method*: GWAS or TWAS. * *Order*: Internal ordering number. * *Suborder*: Internal ordering number. * *Locus_B73v*5: If method is "GWAS", SNP linked to the candidate gene in maize. * *MaizeGene_B73v*5: Gene ID of maize candidate gene related to Arabidopsis ortholog. * *Gene*: Arabidopsis ortholog gene to maize candidate gene. * *Description*: Arabidopsis gene description * *Allele*: Name of mutant allele. If no specific name: a/b * *Germplasm*: Germplasm ID of Arabidopsis mutant. * *Stock*: Stock ID name of Arabidopsis mutant. ##### data/MutantScreen/stats\_output.csv Summary output and statistical comparisons between Col-0 wild-type and mutant for all Arabidopsis mutants of ortholog genes to maize candidate genes that were screened for their hydropatterning behavior in this study. * *Group*: Grouping variable linked to the "Group" in the HA_[YYYY_MM_DD].csv files * *Gene*: Arabidopsis ortholog gene to maize candidate gene. * *Description*: Arabidopsis gene description * *Allele*: Name of mutant allele. If no specific name: a/b * *Germplasm*: Germplasm ID of Arabidopsis mutant. * *N_WT*: Total number of Col-0 wild-type plants across all plates per group. * *N_Mutant*: Total number of mutant plants across all plates per group. * *RootLength_WT*: Average seedling primary root length for Col-0 wild-type plants (cm). * *RootLength_Mutant*: Average seedling primary root length for mutant plants (cm). * *RootLength_DiffEst*: Estimated difference in seedling primary root length between WT and mutant (cm). * *RootLength_*p: *p*-value for primary root length difference using a paired (by plate) ttest. * *AirDensity_WT*: Average air-side lateral root density for Col-0 wild-type plants (air-side lateral roots/cm). * *AirDensity_Mutant*: Average air-side lateral root density for mutant plants (air-side lateral roots/cm). * *AirDensity_DiffEst*: Estimated difference for air-side lateral root density between WT and mutant (air-side lateral roots/cm). * *AirDensity_p*: *p*-value for air-side lateral root density difference using a paired (by plate) ttest. * *MediaDensity_TtoB_WT*: Average contact-side lateral root density for Col-0 wild-type plants (contact-side lateral roots/cm). * *MediaDensity_TtoB_Mutant*: Average contact-side lateral root density for mutant plants (contact-side lateral roots/cm). * *MediaDensity_TtoB_DiffEst*: Estimated difference for contact-side lateral root density between WT and mutant (contact-side lateral roots/cm). * *MediaDensity_p*: *p*-value for contact-side lateral root density difference using a paired (by plate) ttest. * *pAIR_WT*: Average percent air-side lateral roots for Col-0 wild-type plants (%). * *pAIR_Mutant*: Average percent air-side lateral roots for mutant plants (%). * *pAIR_DiffEst*: Estimated difference for percent air-side lateral roots between WT and mutant (%). * *pAir_p*: *p*-value for percent air-side lateral roots difference using a paired (by plate) ttest. ##### data/EthylenePathway/HA\_\[YYYY\_MM\_DD].csv Raw data files collected for hydropatterning assays conducted with Arabidopsis plants comparing Col-0 wild-type and mutant plants for Arabidopsis genes in the ethylene pathway. * *Date*: Date when the data was collected. * *Line*: Name of the Arabidopsis line/accession/mutant. * *Plate*: Number assigned to the plate replicate. * *Plant*: Replicate plant on a plate. * *Group*: Grouping variable constructed from date and name of mutant tested. * *MediaSide*: Number of lateral roots counted that grew towards the media (contact-side lateral roots/seedling). * *AirSide*: Number of lateral roots counted that grew towards the air (air-side lateral roots/seedling). * *Notes*: Notes on plants that did not germinate, or were small, or grew into their roots into the media. * *RootLength*: Total primary root length of the plant from hypocotyl to primary root tip (cm). * *RootLength_TtoB*: Primary root length between the first and last lateral root counted (cm). ##### data/EthylenePathway/HA\_MutantInfo.csv Info on Arabidopsis mutants related to the ethylene pathway screened in the hydropatterning assay. * *Group*: Grouping variable linked to the "Group" in the HA_[YYYY_MM_DD].csv files * *Gene*: Arabidopsis gene ID. * *Description*: Arabidopsis gene description * *Allele*: Name of mutant allele. If no specific name: a/b * *Germplasm*: Germplasm ID of Arabidopsis mutant. * *Stock*: Stock ID name of Arabidopsis mutant. ##### data/Pharma/HA\_\[YYYY\_MM\_DD].csv Raw data files collected for hydropatterning assays conducted with Arabidopsis plants where a chemical treatment was applied. * *Date*: Date when the data was collected. * *Line*: Name of the Arabidopsis line/accession/mutant. * *Treatment*: Chemical treatment that was applied. * *TestType*: Compare treatment between [Lines] or across [Treatment] levels. * *Plate*: Number assigned to the plate replicate. * *Plant*: Replicate plant on a plate. * *Group*: Grouping variable constructed from date and name of mutant tested. * *MediaSide*: Number of lateral roots counted that grew towards the media (contact-side lateral roots/seedling). * *AirSide*: Number of lateral roots counted that grew towards the air (air-side lateral roots/seedling). * *Notes*: Notes on plants that did not germinate, or were small, or grew into their roots into the media. * *RootLength*: Total primary root length of the plant from hypocotyl to primary root tip (cm). * *RootLength_TtoB*: Primary root length between the first and last lateral root counted (cm). ### Supplementary materials Supplementary data file that was submitted with the manuscript. #### File descriptions: ##### data S1: Data S1 provides an overview on the maize inbred lines that were obtained from the USDA National Plant Germplasm System to characterize hydropatterning. * *Accession (GRIN)*: Unique ID assigned to each maize seed stock by the USDA National Plant Germplasm System. * *Inbred Name*: Name of maize inbred line according to the USDA National Plant Germplasm System. * *Taxonomy*: Taxonomy name. * *SRA accessions (SRR)*: Sequence Read Archive ID for each inbred line. * *Phenotyped replicates*: Number of biological replicate seedlings in characterized using the hydropatterning assay. * *Phenotyped subset*: Indicates whether hydropatterning was characterized for the inbred line. * *GWAS subset*: Indicates whether inbred line was included in Genome Wide Association Study. * *TWAS subset*: Indicates whether inbred line was included in Transcriptome Wide Association Study. ##### data S2: Summary data of hydropatterning behavior for primary roots of all maize inbred lines phenotyped in this study. * *Inbred Name*: Name of maize inbred line according to the USDA National Plant Germplasm System. * *Phenotyped replicates*: Number of biological replicate seedlings in characterized using the hydropatterning assay. * *Contact-side LR density - Median (cm^-1*): Median contact-side lateral root density (contact-side lateral roots/cm). * *Contact-side LR density - SD (cm^-1)*: Standard deviation of contact-side lateral root density (contact-side lateral roots/cm). * *Air-side LR density - Median (cm^-1)*: Median air-side lateral root density (air-side lateral roots/cm). * *Air-side LR density - SD (cm^-1*): Standard deviation of air-side lateral root density (air-side lateral roots/cm). * *Percent air-side LRs - Median (%)*: Median percent air-side lateral roots (%). * *Percent air-side LRs - SD (%)*: Standard deviation of percent air-side lateral roots (%). ##### data S3: Summary data on comparison of hydropatterning on primary and crown roots for a subset of 21 maize inbred lines. * *Inbred Name*: Name of maize inbred line according to the USDA National Plant Germplasm System. * *Root type*: Specifies whether hydropatterning was characterized in primary or crown roots. * *Phenotyped replicates*: Number of biological replicate seedlings in characterized using the hydropatterning assay. * *Contact-side LR density - Median (cm^-1*): Median contact-side lateral root density (contact-side lateral roots/cm). * *Contact-side LR density - SD (cm^-1)*: Standard deviation of contact-side lateral root density (contact-side lateral roots/cm). * *Air-side LR density - Median (cm^-1)*: Median air-side lateral root density (air-side lateral roots/cm). * *Air-side LR density - SD (cm^-1*): Standard deviation of air-side lateral root density (air-side lateral roots/cm). * *Percent air-side LRs - Median (%)*: Median percent air-side lateral roots (%). * *Percent air-side LRs - SD (%)*: Standard deviation of percent air-side lateral roots (%). ##### data S4: Summary statistic of the partial correlation analysis to study links between hydropatterning traits and traits of field-grown maize plants. * *Field-grown maize traits*: Traits collected by Mural et.al. *GigaScience*, Volume 11, 2022, giac080, [https://doi.org/10.1093/gigascience/giac080](https://doi.org/10.1093/gigascience/giac080). * *Hydropatterning traits*: Hydropatterning trait used in the the analysis. * *Partial pearson's correlation coeff*.: Partial pearson's correlation coefficient calculated between the field trait and hydropatterning trait. * *p-value*: Significance value. * *FDR-adjusted p-value*: FDR-adjusted significance value to account for multiple testing. ##### data S5: Output of the ancestry analysis (k=3) using ADMIXTURE. Sweet corn and popcorn subpopulations were defined a priori according to Flint-Garcia et.al. Plant J. 2005 Dec;44(6):1054-64. doi: 10.1111/j.1365-313X.2005.02591.x. * *PlantName*: Name of maize inbred line according to the USDA National Plant Germplasm System. * *Proportion_ts*: Tropical/subtropical ancestry component. * *Proportion_ss*: Stiff stalk ancestry component. * *Proportion_nss*: Non-stiff stalk ancestry component. * *Subpopulation*: Subpopulation assignments done in this study. Assignments: [ss] stiff stalk, [nss] non-stiff stalk, [ts] tropical/subtropical, [mixed] less than 80% subpopulation identity of ss/nss/ts, [popcorn] popcorn outgroup, [sweet] sweet corn outgroup. ##### data S6: Summary overview of the Qst-Fst analysis for hydropatterning traits. * *Comparison*: Specifies whether this Qst-Fst comparison was done on a global level (all subpopulations) or pair-side (between two subpopulations). * *Trait*: Hydropatterning trait that was used for the Qst calculations. * *Qst*: Qst estimate related to the trait in the "Trait" column. * *CI.lower*: Lower limit of the confidence interval of Qst. * *CI.upper*: Upper limit of the confidence interval of Qst. * *Fst.mean*: Mean Fst value. * *Qst_Fst.mean*: Mean difference between Qst and Fst. * *CI_Fst.mean*: Difference between Mean Fst value and lower limit of the Qst confidence interval. * *Difference.mean*: Indicates whether there is a difference according to "*CI_Fst.mean*". * *Fst.weighted*: Weighted Fst values based on resampling. * *Qst_Fst.weighted*: Differences between Qst and weighted Fst. * *CI_Fst.weighted*: Difference between weighted Fst value and lower limit of the Qst confidence interval. * *Difference.weighted*: Indicates whether there is a difference according to "*CI_Fst.weighted*". * *Overlap.raw*: Significance value for Qst-Fst differences. * *Significance.raw*: Asterisk significance denotation for Qst-Fst differences. * *Overlap.fdr*: FDR-adjusted significance value for Qst-Fst differences. * *Significance.fdr*: Asterisk significance denotation for FDR-adjusted Qst-Fst differences. * *pop1*: If pair-side comparison, then this specifies the first subpopulation. * *pop2*: If pair-side comparison, then this specifies the second subpopulation. ##### data S7: Summary statistic of Transcriptome Wide Association Study (TWAS) for air-side lateral root density at model frequency &gt; 0.05. * *TWAS-gene*: Gene ID (Zm-B73-REFERENCE-NAM-5.0). * *Chromosome*: Chromosome number. * *Effect*: Effect size for air-side lateral root density (Best Linear Unbiased Prediction). * *EffectVar*: Variance of effect size. * *Model frequency*: Model frequency. ##### data S8: Summary statistics of expression Quantitative Trait Loci (eQTL) for AUXIN RESISTANT 1 (ZmAXR1; Zm00001eb211770) at FDR &lt; 0.05. * *e-SNP*: ID of the SNP that was found to be significantly associated with the expression of ZmAXR1 in roots. * *Chromosome*: Chromosome number of the SNP. * *Position (B73v5)*: SNP position according to Zm-B73-REFERENCE-NAM-5.0. * *estimate*: Effect estimate. * *stderr*: Standard deviation. * *p-value*: Significance value. * *q-value*: FDR-corrected significance value. ##### data S9: Expression Associated SNPs (e-SNP) for AUXIN RESISTANT 1 (ZmAXR1; Zm00001eb211770) and co-located SNP-genes within a 20 kb window. * *e-SNP*: ID of the SNP that was found to be significantly associated with the expression of ZmAXR1 in roots. * *SNP-gene*: e-SNP associated gene ID (Zm-B73-REFERENCE-NAM-5.0). * *Notes*: Note on which gene ID refers to ZmAXR1. ##### data S10: Combined summary statistics of Genome Wide Association Study (GWAS) for air-side lateral root density at FDR &lt; 0.05 for three MAF cutoffs (≥ 0.004, ≥ 0.022, ≥ 0.044). * *TAS*: Trait Associated SNP. * *Chromosome*: Chromosome number of the TAS. * *Position (B73v5)*: SNP position according to Zm-B73-REFERENCE-NAM-5.0. * *minor allele effect estimate*: Effect estimate. * *stderr*: Standard deviation. * *p-value*: Significance value. * *q-value*: FDR-corrected significance value. * *MAF cutoff*: Minor allele frequency cutoff value at which the TAS was detected. ##### data S11: Maize Trait Associated SNPs (TAS) for air-side lateral root density and co-located SNP-genes within a 20 kb window. * *TAS*: Trait Associated SNP from GWAS. * *SNP-gene*: TAS associated gene ID (Zm-B73-REFERENCE-NAM-5.0). * *Notes*: Indicates which TAS is associated with ZmFLA4 and if the gene is located more than 10 kb up- or downstream of the TAS (in cases where not gene was detected within the 20 kb window). ##### data S12: * Arabidopsis mutant alleles used for orthologous validation of maize candidate genes from TWAS and GWAS. * *Method*: Specifies whether the Arabidopsis mutant is an ortholog to a maize candidate gene from the TWAS or GWAS analysis. * *Maize gene*: Associated maize gene ID (Zm-B73-REFERENCE-NAM-5.0). * *At gene*: Arabidopsis gene ID. * *Description*: Description of the Arabidopsis gene. * *Allele*: Specifies any published names for the mutant or if non available a/b. * *Germplasm name*: Germplasm ID of Arabidopsis mutant. * *Stock number*: Stock ID name of Arabidopsis mutant. * *Allele mutagen*: Describes how the mutant was created. * *Left primer (LP)*: Left primer sequence for genotyping. * *Right primer (RP)*: Right primer sequence for genotyping. * *Border primer (BP)*: Border primer sequence specific to T-DNA. * *Primer combination for T-DNA detection*: Specifies which primers are used together to detect T-DNA insertion. ##### data S13: Statistical summary of mutant analysis in Arabidopsis. * *Gene*: Arabidopsis gene ID. * *Description*: Description of the Arabidopsis gene. * *Allele*: Specifies any published names for the mutant or if non available a/b. * *Germplasm*: Germplasm ID of Arabidopsis mutant. * *N.plates_WT*: Number of plates with Col-0 wild-type plants that were phenotyped. * *N.plates_Mutant*: Number of plates with mutant plants that were phenotyped. * *RootLength_WT_cm*: Median primary root length of Col-0 wild-type plants (cm). * *RootLength_Mutant_cm*: Median primary root length of mutant plants (cm). * *RootLength_DiffEst_cm*: Estimated difference in primary root length between Col-0 wild-type and mutant plants (cm). * *RootLength_p-value*: Statistical significance calculated using a paired t-test to compare difference in primary root length between Col-0 wild-type and mutant plants grown on the same plate. * *AirsideLRdensity_WT_LRs/cm*: Median air-side lateral root density of Col-0 wild-type plants (air-side lateral roots/cm). * *AirsideLRdensity_Mutant_LRs/cm*: Median air-side lateral root density of mutant plants (air-side lateral roots/cm). * *AirsideLRdensity_DiffEst_LRs/cm*: Estimated difference in air-side lateral root density between Col-0 wild-type and mutant plants (air-side lateral roots/cm). * *AirsideLRdensity_p-value*: Statistical significance calculated using a paired t-test to compare difference in air-side lateral root density between Col-0 wild-type and mutant plants grown on the same plate. * *ContactsideLRdensity_WT_LRs/cm*: Median contact-side lateral root density of Col-0 wild-type plants (contact-side lateral roots/cm). * *ContactsideLRdensity_Mutant_LRs/cm*: Median contact-side lateral root density of mutant plants (contact-side lateral roots/cm). * *ContactsideLRdensity_DiffEst_LRs/cm*: Estimated difference in contact-side lateral root density between Col-0 wild-type and mutant plants (contact-side lateral roots/cm). * *ContactsideLRdensity_p-value*: Statistical significance calculated using a paired t-test to compare difference in contact-side lateral root density between Col-0 wild-type and mutant plants grown on the same plate. * *PercentairsideLRs_WT_%*: Median percentage of air-side lateral roots in Col-0 wild-type plants (%). * *PercentairsideLRs_Mutant_%*: Median percentage of air-side lateral roots in mutant plants (%). * *PercentairsideLRs_DiffEst_%*: Estimated difference in percentage of air-side lateral roots between Col-0 wild-type and mutant plants (%). * *PercentairsideLRs_p-value*: Statistical significance calculated using a paired t-test to compare difference in percentage of air-side lateral roots between Col-0 wild-type and mutant plants grown on the same plate. ##### data S14: Arabidopsis mutant alleles used for the investigation of the ethylene pathway. * *At gene(s)*: Arabidopsis gene ID(s) associated with the mutant(s). * *Mutant allele*: Specifies any published name(s) for the mutant(s). * *Source*: How where the mutant(s) obtained. * *Germplasm name*: Germplasm ID of Arabidopsis mutant. * *Stock number*: Stock ID of Arabidopsis mutant. * *Allele mutagen*: Describes how the mutant was created. ##### data S15: Best Linear Unbiased Predictions (BLUPs) of hydropatterning traits. * *Inbred Name*: Name of the maize inbred line. * *Contact-side LR density (BLUP)*: Best Linear Unbiased Prediction of contact-side lateral root density. * *Air-side LR density (BLUP)*: Linear Unbiased Prediction of sir-side lateral root density. * *Percent air-side LRs (BLUP)*: Linear Unbiased Prediction of percent air-side lateral roots. ##### data S16: Hybridization Chain Reaction probes for maize inbred B73. Individual probes for each target were combined. * *Target*: Maize gene ID (Zm-B73-REFERENCE-NAM-5.0) for the gene targeted in the Hybridization Chain Reaction experiment. * *Probe #*: Numerical probe ID. * *Probe-binding Sequences*: Sequence of Hybridization Chain Reaction probes. ## Code/Software ZIP archive files with code that was used in this study. ### Genome and Transcriptome Wide Association Studies Code that was used to run the Genome and Transcriptome Wide Association Studies. #### File structure: ``` GWAS_TWAS.zip |- MaizeHydropatterning_FarmCPUpp.GWAS.R |- MaizeHydropatterning_TWASrun41000.inp ``` #### Code description: ##### MaizeHydropatterning\_FarmCPUpp.GWAS.R R code needed to execute the FarmCPUpp analysis for maize air-side lateral root density. ##### MaizeHydropatterning\_TWASrun41000.inp Code used to execute the TWAS analysis for maize air-side lateral root density. ### ImageJ macros Custom Fiji/ImageJ macros to process the images from the maize and Arabidopsis hydropatterning assays. #### File structure: ``` ImageJ.zip |- MaizeHydropatterningAssay/ | |- 00_ImageSeparation.ijm | |- 01_RegisteredStacks.ijm | |- 02_SegmentTrace.ijm | |- Register.py |- ArabidopsisHydropatterningAssay/ | |- Arabidopsis_RootTrace.ijm ``` #### Code description: ##### MaizeHydropatterningAssay/00\_ImageSeparation.ijm Fiji/ImageJ macro code to separate individual roots from root scans for the maize hydropatterning assay. ##### MaizeHydropatterningAssay/01\_RegisteredStacks.ijm Fiji/ImageJ macro code to align contact- and air-side root images from the maize hydropatterning assay. ##### MaizeHydropatterningAssay/02\_SegmentTrace.ijm Fiji/ImageJ macro code to trace and straighten primary roots from the maize hydropatterning assay. ##### MaizeHydropatterningAssay/Register.py Additional python code needed for the macro script "MaizeHydropatterningAssay/01_RegisteredStacks.ijm". ##### ArabidopsisHydropatterningAssay/Arabidopsis\_RootTrace.ijm Fiji/ImageJ macro code to measure root length from scanned plates of Arabidopsis. ### Evolutionary analyses Code used for the evolutionary analysis. #### File structure: ``` Qst-Fst.zip |- Qst-Fst_06-12.html ``` #### Code description: ##### Qst-Fst\_06-12.html Markdown of the R code and outputs from the Qst-Fst analysis. ### R scripts The R scripts are related to two RStudio project files (MaizeHydropatterning.Rproj and ArabidopsisHydropatterning.Rproj). To run the scripts within those projects add the data files contained MaizeHydropatterning.zip ArabidopsisHydropatterning.zip accordingly. #### File structure: ``` R_code.zip |- MaizeHydropatterning/ | |- MaizeHydropatterning.Rproj | |- MaizeHydropatterning_PhenoAnalysis.Rmd | |- MaizeHydropatterning_PartialCor.Rmd | |- MaizeHydropatterning_gwasAnalysis.Rmd | |- MaizeHydropatterning_twasAnalysis.Rmd | |- MaizeHydropatterning_eqtlAnalysis.Rmd |- ArabidopsisHydropatterning/ | |- ArabidopsisHydropatterning.Rproj | |- ArabidopsisHydropatterning_MutantScreen.Rmd | |- ArabidopsisHydropatterning_EthylenePathway.Rmd | |- ArabidopsisHydropatterning_Pharma.Rmd ``` #### Code description: ##### MaizeHydropatterning/MaizeHydropatterning.Rproj Rstudio project file for the maize hydropatterning analysis. ##### MaizeHydropatterning/MaizeHydropatterning\_PhenoAnalysis.Rmd R script to analyze and raw data collected from the maize hydropatterning assays. ##### MaizeHydropatterning/MaizeHydropatterning\_PartialCor.Rmd R script to calculate the partical correlation statistics between hydropatterning and above and below ground maize phenotypic data from Mural et.al. *GigaScience*, Volume 11, 2022, giac080, [https://doi.org/10.1093/gigascience/giac080](https://doi.org/10.1093/gigascience/giac080). ##### MaizeHydropatterning/MaizeHydropatterning\_gwasAnalysis.Rmd R script to analyze GWAS output data and extract maize candidate genes. ##### MaizeHydropatterning/MaizeHydropatterning\_twasAnalysis.Rmd R script to analyze TWAS output data. ##### MaizeHydropatterning/MaizeHydropatterning\_eqtlAnalysis.Rmd R script to analyze eQTL output data for ZmAXR1 and extract maize candidate genes. ##### ArabidopsisHydropatterning/ArabidopsisHydropatterning.Rproj Rstudio project file for the Arabidopsis hydropatterning analysis. ##### ArabidopsisHydropatterning/ArabidopsisHydropatterning\_MutantScreen.Rmd R script to analyze Arabidopsis hydropatterning assay raw data for mutants of Arabidopsis ortholog genes of maize candidate genes. ##### ArabidopsisHydropatterning/ArabidopsisHydropatterning\_EthylenePathway.Rmd R script to analyze Arabidopsis hydropatterning assay raw data for mutants in the ethylene pathway. ##### ArabidopsisHydropatterning/ArabidopsisHydropatterning\_Pharma.Rmd R script to analyze Arabidopsis hydropatterning assay raw data from experiments with chemical treatment.