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1 Supporting Information for Metabolomic Assessments of Key Maize Resources: GC-MS and NMR Profiling of Grain from B73 Hybrids of the Nested Association Mapping (NAM) Founders and of Geographically Diverse Landraces Tyamagondlu V. Venkatesh 1,*, Alexander W. Chassy 2, Oliver Fiehn 2,3, Sherry Flint-Garcia 4,5,*, Qin Zeng 1, Kirsten Skogerson 1, George G. Harrigan 1 1 Monsanto Company, 800 North Lindbergh Boulevard; St. Louis, Missouri, 63167, USA Corresponding author: tvvenkatesh@gmail.com, Phone: , Fax: Genome Center - Metabolomics, University of California at Davis, Davis, California, 95616, USA 3 King Abudalaziz University, Biochemistry Department, Jeddah, Saudi-Arabia 4 USDA-ARS, Columbia, Missouri, 65211, USA 5 Division of Plant Sciences, University of Missouri, Columbia, Missouri, 65211, USA. * Corresponding author: Sherry.Flint-Garcia@ARS.USDA.GOV, Phone: , Fax:
2 ASSOCITED CONTENT AC_File_1_GC_MS_ALL_NAM.xlxs. This file contains data for all 504 metabolites measured for the NAM-derived hybrids. It also includes worksheets showing PCA and PLS scores. AC_File_2_GC_MS_KNOWN_NAM.xlxs This file contains data for the 174 identified metabolites (identified to level 1 of the Metabolomics Standards Initiative 18b ) measured for the NAM-derived hybrids. It also includes worksheets showing PCA and PLS scores. AC_File_3_GC_MS_ALL_LANDRACE.xlxs This file contains data for all 504 metabolites measured for the landrace-derived hybrids. It also includes worksheets showing PCA and PLS scores. AC_File_4_GC_MS_KNOWN_LANDRACE.xlxs This file contains data for the 174 identified metabolites (identified to level 1 of the Metabolomics Standards Initiative 18b ) measured for the landrace-derived hybrids. It also includes worksheets showing PCA and PLS scores. AC_File_5_GC_MS tables-formatted.xlxs This file contains data for all identified metabolites categorized according to chemical class. It also highlights those excluded from multivariate analysis. Metabolites were removed (red text font) if the log of their average peak area was less than 2.5 (peak area of <316) or if the average relative standard deviation (of log peak area) of replicate analyses was greater than 10% (average RSD > 150%). AC_File_6_GC_MS_CORRELATIONS.xlxs This shows results from analysis of correlations between GC-MS data and agronomic/phenotypic and grain composition data for each of the two hybrid sets. AC_File_7_GC_MS_PCA_PLS.pptx Results of PCA, PLS and HCA on GC-MS data of NAM and landrace hybrids AC_File_8_NMR_Metabolites_Quantitation.xlxs- This file contains data for all 33 quantified metabolites (identified based on Chenomx assignments) as well as calculates means, minimum, and maximum values for metabolites within a breeding group. AC_File_9_NMR_CORRELATIONS.xlxs This file shows results from analysis of correlations between NMR data and agronomic/phenotypic and grain composition data for each of the two hybrid sets file 2
3 SUPPORTING TABLES Supporting Table 1: Grouping information for the NAM founder inbreds for PLS-DA NAM FOUNDER + INBRED GEOGRAPHIC ORIGIN BROAD GROUP BREEDING GROUP HP301 Indiana northern flint popcorn Il14H Illinois northern flint sweet corn P39 Indiana northern flint sweet corn B97 Iowa temperate non-stiff stalk Ky21 Kentucky temperate non-stiff stalk M162W South Africa temperate non-stiff stalk Mo17 Missouri temperate non-stiff stalk MS71 Michigan temperate non-stiff stalk Oh43 Ohio temperate non-stiff stalk Oh7B Ohio temperate non-stiff stalk M37W South Africa mixed* mixed Mo18W Missouri mixed* mixed Tx303 Texas mixed* mixed CML103 Mexico tropical tropical/subtropical CML228 Mexico tropical tropical/subtropical CML247 Mexico tropical tropical/subtropical CML277 Mexico tropical tropical/subtropical CML322 Mexico tropical tropical/subtropical CML333 Mexico tropical tropical/subtropical CML52 Mexico tropical tropical/subtropical CML69 Mexico tropical tropical/subtropical Ki11 Thailand tropical tropical/subtropical Ki3 Thailand tropical tropical/subtropical NC350 North Carolina tropical tropical/subtropical NC358 North Carolina tropical tropical/subtropical Tzi8 Nigeria tropical tropical/subtropical *In the metabolomic univariate analyses the broad group combined both the mixed and temperate lines. + The inbreds were classified by population structure using genetic marker data as described in Flint-Garcia, et al. 12 Brief descriptions of these groups are also provided in Venkatesh, et al. 13 Three biological replicates were available for all hybrids except CML277, M37W, Oh43, and Oh7B (two replicates). 3
4 Supporting Table 2: Grouping information for the landrace inbreds for PLS-DA LANDRACE INBRED + GEOGRAPHIC ORIGIN BROAD GROUP MR01 (Araguito) Venezuela tropical/subtropical MR02 (Assiniboine) North Dakota northern flint MR03 (Bolita) Mexico tropical/subtropical MR04 (Canilla) Venezuela tropical/subtropical MR05 (Cateto) Bolivia tropical/subtropical MR06 (Chapalote) Mexico intermediate* MR07 (Comiteco) Mexico tropical/subtropical MR08 (Costeno) Venezuela tropical/subtropical MR09 (Cravo Riogranense) Brazil tropical/subtropical MR10 (Cristalino Norteno) Chile tropical/subtropical MR11 (Cuban Flint) Cuba tropical/subtropical MR12 (Havasupai) Arizona, northern flint MR13 (Hickory King) US temperate* MR14 (Longfellow Flint) US northern flint MR15 (Palomero de Jalisco) Mexico Intermediate* MR16 (Pepetilla) Mexico tropical/subtropical MR18 (Reventador) Mexico tropical/subtropical MR19 (Santo Domingo) New Mexico northern flint MR20 (Shoe Peg) Missouri temperate* MR21 (Tabloncillo) Mexico tropical/subtropical MR22 (Tuxpeno) Mexico tropical/subtropical MR23 (Zapalote Chico) Mexico tropical/subtropical MR25 (Poropo) Bolivia tropical/subtropical MR26 (Pollo) Colombia tropical/subtropical *In the metabolomic univariate analyses the broad group combined both the intermediate and temperate lines. + The geographical origins of landrace inbreds are described in Hufford, et al. 14a Brief descriptions of these groups are also provided in Venkatesh, et al. 13 Three biological replicates were available for all hybrids except Bolita, Chapalote, Costeno, Cuban Flint, and Tabloncillo (two replicates), and Canilla and Havasupai (one replicate). Some assays were further sample-limited. Thus, for the GC-MS and NMR experiments only two replicates of Araguito, Chapalote, Peptilla, and Pollo were available. 4
5 Supporting Table 3. Correlation Of Canonical Scores And NMR-Based metabolite Data Group Metabolite Canon[1] Canon[2] LR Hybrids 4.Aminobutyrate LR Hybrids Acetate LR Hybrids Alanine LR Hybrids Arginine LR Hybrids Asparagine LR Hybrids Aspartate LR Hybrids Choline LR Hybrids Dimethylamine LR Hybrids Formate LR Hybrids Fructose LR Hybrids Fumarate LR Hybrids Galactose LR Hybrids Glucose LR Hybrids Glutamate LR Hybrids Glutamine LR Hybrids Histidine LR Hybrids Isoleucine LR Hybrids Lactate LR Hybrids Leucine LR Hybrids Lysine LR Hybrids Malate LR Hybrids Maltose LR Hybrids Methionine LR Hybrids Methylamine LR Hybrids Phenylalanine LR Hybrids Proline LR Hybrids Succinate LR Hybrids Sucrose LR Hybrids Threonine LR Hybrids Trigonelline LR Hybrids Tyrosine LR Hybrids Valine NAM Hybrids 4.Aminobutyrate NAM Hybrids Acetate NAM Hybrids Alanine NAM Hybrids Arginine NAM Hybrids Asparagine NAM Hybrids Aspartate NAM Hybrids Choline NAM Hybrids Dimethylamine NAM Hybrids Formate
6 NAM Hybrids Fructose NAM Hybrids Fumarate NAM Hybrids Galactose NAM Hybrids Glucose NAM Hybrids Glutamate NAM Hybrids Glutamine NAM Hybrids Histidine NAM Hybrids Isoleucine NAM Hybrids Lactate NAM Hybrids Leucine NAM Hybrids Lysine NAM Hybrids Malate NAM Hybrids Maltose NAM Hybrids Methionine NAM Hybrids Methylamine NAM Hybrids Phenylalanine NAM Hybrids Proline NAM Hybrids Succinate NAM Hybrids Sucrose NAM Hybrids Threonine NAM Hybrids Trigonelline NAM Hybrids Tyrosine NAM Hybrids Valine
7 SUPPORTING FIGURES Supporting Figure 1. Prior to multivariate analyses, metabolite peaks were removed if the log of their average peak area was less than 2.5 (peak area of <316) or if the average relative standard deviation (of log peak area) of replicate analyses was greater than 10% (average RSD > 150%). This resulted in the removal of 171 (about 25%) metabolites leaving 504 quality metabolite peaks included in multivariate analyses 7
8 Supporting Figure 2. PLS of NAM Broad Group (includes 174 known metabolites (identified to level 1 of the Metabolomics Standards Initiative 18b ) key metabolites that contributed to observed pattern of the sub groups are listed in colored rectangles corresponding to each group. 8
9 Supporting Figure 3. PLS of landraces Broad group (includes 174 known metabolites, (known metabolites, identified to level 1 of the Metabolomics Standards Initiative 18b ) - key metabolites that contributed to observed pattern of the sub groups are listed in colored rectangles corresponding to each group. 9
10 Supporting Figure 4. Box plots for NMR Metabolite Values (identified based on Chenomx assignments) for Hybrids Derived from the NAM Founders and B73 10
11 Supporting Figure 5. Box plots for NMR Metabolite Values (identified based on Chenomx assignments) for Hybrids Derived from Landrace lines and B73 11
12 12
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