Genome-wide association study dissects the genetic architecture of

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1 Supplementary Information for Genome-wide association study dissects the genetic architecture of oil biosynthesis in maize kernels Hui Li 1,6, Zhiyu Peng 2,6, Xiaohong Yang 1,6, Weidong Wang 1,6, Junjie Fu 3,6, Jianhua Wang 1,6, Yingjia Han 1, Yuchao Chai 1, Tingting Guo 1, Ning Yang 4, Jie Liu 4, Marilyn L. Warburton 5, Yanbing Cheng 2, Xiaomin Hao 1, Pan Zhang 1, Jinyang Zhao 2, Yunjun Liu 3, Guoying Wang 3, Jiansheng Li 1, Jianbing Yan 4 1 National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing , China. 2 BGI-Shenzhen, Shenzhen, , China 3 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing , China 4 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan , China. 5 United States Department of Agriculture-Agricultural Research Service: Corn Host Plant Resistance Research Unit, Box 9555, Mississippi State, MS, 39762, USA 6 These authors contributed equally to this work. Correspondence should be addressed to J.Y. (yjianbing@mail.hzau.edu.cn), J.L. (lijiansheng@cau.edu.cn) and G.W. (gywang@caas.net.cn). This file includes: Supplementary Figures 1-13 Supplementary Tables 1-11

2 Supplementary Figure 1 Percentage of 10 kernel oil components detected by gas chromatography and measured in 508 diverse inbred lines. Supplementary Figure 2 Distributions of maize kernel oil concentration and composition in 508 inbred lines. Supplementary Figure 3 Manhattan plots of each chromosome resulting from the GWAS results for oil concentration in maize kernels. Supplementary Figure 4 Manhattan and quantile-quantile plots resulting from the GWAS results for oil compositional traits in maize kernels. Supplementary Figure 5 The simplified lipid metabolic pathway in maize, modified from Arabidopsis and other plants. Supplementary Figure 6 Manhattan plots for the expression GWAS of proposed candidate genes with eqtl. Supplementary Figure 7 The co-expression network of the 54 most highly connected genes among 67 genes with expression data. Supplementary Figure 8 The validation of FAD2 via re-sequencing in an independent panel. Supplementary Figure 9 The validation of ACP via re-sequencing in an independent panel. Supplementary Figure 10 The validation of LACS via re-sequencing in an independent panel. Supplementary Figure 11 The validation of WRI1a via re-sequencing in an independent panel. Supplementary Figure 12 The co-expression network of WRI1a. Supplementary Figure 13 The validation of COPII via re-sequencing in an independent panel. Supplementary Table 1 Phenotypic analysis, correlations and heritability of all measured traits Supplementary Table 2 Correlations among all the measured traits Supplementary Table 3 SNPs and candidate genes significantly associated with oil compositional traits in two association panels of 368 and 508 maize inbred lines Supplementary Table 4 SNPs significantly associated with traits after conditioning upon lead SNPs of all oil traits from primary GWAS analysis Supplementary Table 5 List of possible additional candidate genes within 100 kb flanking region of the 74 lead SNPs Supplementary Table 6 Comparison of the associations of oil concentration between regular and high-oil lines Supplementary Table 7 Comparison of the associations of oil compositional traits between regular and high-oil lines Supplementary Table 8 Confirmation of significant candidate genes in three linkage populations Supplementary Table 9 Correlation analysis between the phenotype and the expression levels of proposed candidate genes with eqtls Supplementary Table 10 The full list of genes in the co-expression network of WRI1a Supplementary Table 11 List of the primers used in this study

3 Supplementary Figure 1 Percentage of 10 kernel oil components detected by gas chromatography and measured in 508 diverse inbred lines. Oil concentration was calculated as the sum of all the compositions. C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C20:0, arachidic acid; C20:1, gadoleic acid; C22:0, behenic acid; C24:0, lignoceric acid.

4 Supplementary Figure 2 Distributions of maize kernel oil concentration and composition in 508 inbred lines.the left plots are the frequency distributions and the right are the density distributions.

5 Supplementary Figure 3 Manhattan plots of each chromosome resulting from the GWAS results for oil concentration in maize kernels. The dashed horizontal line depicts the Bonferroni-adjusted significance threshold ( ). For each chromosome, the significant unique SNPs are indicated with red dots. The manhanttan plot of chromosome 9 is not shown as there were no significant loci detected on this chromosome. The full gene names are listed in Table 1.

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10 Supplementary Figure 4 Manhattan and quantile-quantile plots resulting from the GWAS results for oil compositional traits in maize kernels. The dashed horizontal line in Manhattan plot depicts the Bonferroni-adjusted significance threshold ( ). The lead sites significantly associated with corresponding traits are indicated by red dots on the Manhattan plots. SFA: saturated fatty acid; USFA: Unsaturated fatty acid. Jianbing Yan 11/13/12 7:33 PM Deleted:

11 Supplementary Figure 5 The simplified lipid metabolic pathway in maize, modified from Arabidopsis and other plants 30. The candidate genes corresponding to significant loci detected by GWAS are in orange. The plasmid, endoplasmic reticulum (ER), oil body and peroxisome, are marked with light green, light blue, light orange and light purple, respectively. Abbreviations: PDHC, pyruvate dehydrogenase complex; ACC, acetyl-coa carboxylase; MCMT, malonyl-coa:acp malonyltransferase; FAS, fatty acid synthase; KAS, ketoacyl-acp synthase; SAD, stearoyl-acp desaturase; ACP, acyl carrier protein; FAT, acyl-acp thioesterase; FFA, free fatty acid; LACS, long chain acyl-coa synthase; FAD2, ER oleate desaturase; FAD3, ER linoleate desaturase; PC, phosphatidylcholine; G3P, glycerol 3-phosphate;GPAT, glycerol phosphate acyltransferase; LPAAT, lysophosphatidic acid (LPA) acyltransferase; PP, phosphatidic acid (PA) phosphatase; DGAT, acyl-coa: diacylglycerol (DAG) acyltransferase; FAE2, fatty acid elongase; KCS, ketoacyl-coa synthase; VLCFA, very long chain fatty acid; TAGL, triacylglycerol (TAG) lipase; MAGL: monoacylglycerol (MAG) lipase; CTS, COMATOSE (an ABC transporter); ACT, acyl-coa thioesterase; ACX, acyl-coa oxidase.

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15 Supplementary Figure 6 Manhattan plots for the expression GWAS of proposed candidate genes with eqtl. A P) the top Manhattan plots of each panel are the GWAS results for oil compositional traits (A L) and the GWAS results after conditioning upon 63 lead SNPs (M P). The red dots are the proposed candidate genes with eqtls. The small Manhattan plots are the expression GWAS results for the candidate genes, and the red diamonds are the most significant sites associated with the expression level of each candidate gene.

16 Supplementary Figure 7 The co-expression network of the 54 most highly connected genes among 67 genes with expression data.

17 Supplementary Figure 8 The validation of FAD2 via re-sequencing in an independent panel. (a) Association between polymorphisms within the gene and the phenotypic variation in oleic acid levels in three environments. The x axis is the distance in base pairs along the gene; the first base of the start codon is defined as position 1. The y axis is the probability expressed as log 10 P from a mixed linear model. (b) The structure and functional domain of FAD2. The filled gray boxes represent the exons, open boxes indicate the UTRs, the black dashed lines mark the region re-sequenced in this study, and the orange ellipse represents the location of the conserved functional domain in gene. The red stars represent the lead polymorphisms (PZE ) detected by original GWAS and the proposed functional polymorphisms (S230A) detected by re-sequencing. R1, R2, and R3 are amplicons of

18 sequencing reaction; the primer sequences are given in Supplementary Table 11. (c) A representation of the pair-wise r 2 value(a measure of LD) among the significantly associated sites, where the color of each box corresponds to the r 2 value according to the legend. The purple star represents the proposed functional polymorphic locus. (d) Plots of the results from the correlation analysis. The x axis represents the normalized expression level of FAD2 in kernels collected at 15 DAP. The y axis represents the levels of oleic acid and linoleic acid, and the ratio of the two components. The r value is a Pearson correlation coefficient. (e) Box plot of normalized FAD2 expression for each allele of S230A site. The P value is based on a two-tailed t-test. (f) Linkage mapping result for chromosome 4 in a RIL population derived from By804 and B73. The x axis is the genetic map position, and the y axis is the LOD value. The purple line is the empirical significance threshold (LOD = 2.5). QTLs for C18:1 from two environments in Beijing 2005 (B 05) and Beijing 2006 (B 06) were identified; FAD2 is located within these QTLs. Jianbing Yan 11/13/12 7:34 PM Deleted: BY804

19 Supplementary Figure 9 The validation of ACP via re sequencing in an independent panel. (a) Association between polymorphisms within the gene and the phenotypic variation in oil concentration in three environments. The x axis is the distance in base pairs along the gene; the first base of the start codon is defined as position 1. The y axis is the probability expressed as log 10 P from a mixed linear model. (b) The structure and functional domain of ACP. Filled gray boxes represent exons, open boxes indicate the UTRs, the black dashed lines mark the region re-sequenced in this study and the orange ellipse represents the location of the conserved functional domain in gene. The red star represent the lead polymorphisms (M1c ) detected by original GWAS. The red triangle represents the proposed functional polymorphisms (InDel_8) by re-sequencing. The marker, InDel_8, represents the allele with 8-bp or without insertions. R1, R2, and R3 are amplicons of sequencing reaction; the primer sequences are given in Supplementary Table 11. (c) A representation of the pair-wise r 2 value (a measure of LD) among the significantly associated sites, where the color of each box corresponds to the r 2 value according to the legend. The purple star represents the proposed functional polymorphic locus. (d) Plots of the results from the correlation analysis. The x axis represents the normalized expression level of ACP in kernels collected at 15 DAP. The y axis represents the total oil concentration. The r value is a Pearson correlation coefficient. (e) Box plot of normalized ACP expression for each allele of InDel_8 site. The P value is based on a two-tailed t-test. The phenotypic variation explained (R 2 ) from ANOVA analysis.

20 Supplementary Figure 10 The validation of LACS via re-sequencing in an independent panel. (a) Association between polymorphisms within the gene and the phenotypic variation in oil concentration in three environments. The x axis is the distance in base pairs along the gene; the first base of the start codon is defined as position 1. The y axis is the probability expressed as log 10P from a mixed linear model. (b) The structure and functional domain of LACS. Filled gray boxes represent exons, open boxes indicate the UTRs, the black dashed lines mark the region re-sequenced in this study and the location of the conserved functional domain in gene. The red star represent the lead polymorphisms (M2c ) detected by original GWAS. The red triangle represents the proposed functional polymorphisms (InDel_146/472) by re-sequencing. The marker, InDel_146/472, represents the allele with a 146 bp or 472 bp insertion. R1, R2, and R3 are amplicons of sequencing reaction; the primer sequences are given in Supplementary Table 11. (c) A representation of the pair wise r 2 value (a measure of LD) among the significantly associated sites, where the color of each box corresponds to the r 2 value according to the legend. The purple star represents the proposed functional polymorphic locus. (d) Plots of the results from the correlation analysis. The x axis represents the normalized expression of LACS in kernels collected at 15 DAP. The y axis represents the total oil concentration. The r value is a Pearson correlation coefficient. (e) Box plot of normalized LACS expression for each allele of InDel_146/472 site. The P value is based on a two-tailed t-test. The phenotypic variation explained (R 2 ) from ANOVA analysis.

21 Supplementary Figure 11 The validation of WRI1a via re sequencing in an independent panel. (a) Association between polymorphisms within the gene and the phenotypic variation in oil concentration in three environments. The x axis is the distance in base pairs along the gene; the first base of the start codon is defined as position 1. The y axis is the probability expressed as log 10P from a mixed linear model. (b) The structure and functional domain of WRI1a. Filled gray boxes represent exons, open boxes indicate the UTRs, the black dashed lines mark the region re-sequenced in this study and the orange ellipse represents the location of the conserved functional domains in gene. The red star represent the lead polymorphisms (M2c ) detected by original GWAS. The red triangle represents the proposed functional polymorphisms (InDel_2000) by re-sequencing. InDel_2000 represents the allele with 2000-bp or without insertions. R1, R2, and R3 are amplicons of sequencing reaction; the primer sequences are given in Supplementary Table 11. (c) A representation of the pair wise r 2 value (a measure of LD) among the significantly associated sites, where the color of each box corresponds to the r 2 value according to the legend. The purple star represents the proposed functional polymorphic locus. (d) Plots of the results from the correlation analysis. The x axis represents the normalized expression of WRI1a in kernels collected at 15 DAP. The y axis represents the total oil concentration. The r value is a Pearson correlation coefficient. (e) Box plot of normalized WRI1a expression for each allele of InDel_2000 site. The P value is based on a two tailed t-test. The phenotypic variation explained (R 2 ) from ANOVA analysis.

22 Supplementary Figure 12 The co expression network of WRI1a. Genes shown in light blue are annotated as transcription factors. Genes shown in yellow were previously reported as the putative target genes of WRI1a 33. Genes shown in orange are involved in late glycolysis and fatty acid biosynthesis in plastids. Genes shown in light green are mainly related to carbohydrate metabolism, amino acid metabolism and transmembrane transport. Genes shown in gray have an unknown function. The full list of genes can be found in Supplementary Table 10. Abbreviations: PDHE1, pyruvate dehydrogenase E1; PDHE2, pyruvate dehydrogenase E2; LT, lipoyltransferase; OAT, oleoyl ACP, thioesterase; KHT, ketopantoate hydroxymethyltransferase; PK, pyruvate kinase; ACAT2, acetoacetyl CoA thiolase 2; ACP, acyl carrier protein; OLE, oleosin; PPT, phosphoenolpyruvate/phosphate translocator; KAS II, ketoacyl ACP synthase II; OAS III, oxoacyl ACP synthase III; SAD, stearoyl ACP desaturase; FAD2, fatty acid desaturase 2; GH, glycosyl hydrolases; GP, ADP glucose pyrophosphorylase; PK, protein kinase; S/TPK, serine/ threonine kinase protein; P450, cytochrome P450; ZFP, zinc finger protein; GRF, growth regulating factor; UF, unknown function.

23 Supplementary Figure 13 The validation of COPII via re-sequencing in an independent panel. (a) Association between polymorphisms within the gene and the phenotypic variation in oil concentration in three environments. The x axis is the distance in base pairs along the gene; the first base of the start codon is defined as position 1. The y axis is the probability expressed as log 10P from a mixed linear model. (b) The structure and functional domain of COPII. Filled gray boxes represent exons, open boxes indicate the UTRs, the black dashed lines mark the region re-sequenced in this study and the orange ellipse represents the location of the conserved functional domain in gene. The red star represent the lead polymorphisms (M8c ) detected by original GWAS. The red triangle represents the proposed functional polymorphisms (InDel_20) by re-sequencing. InDel_20 represents the allele with 20-bp or without insertions. R1, R2, R3, and R4 are amplicons of sequencing reaction; the primer sequences are given in Supplementary Table 11. (c) A representation of the pair wise r 2 value (a measure of LD) among the significantly associated sites, where the color of each box corresponds to the r 2 value according to the legend. The purple star represents the proposed functional polymorphic locus. (d) Plots of the results from the correlation analysis. The x axis represents the normalized expression of COPII in kernels collected at 15 DAP. The y axis represents the total oil concentration. The r value is a Pearson correlation coefficient. (e) Box plot of normalized COPII expression for each allele of InDel_20 site. The P value is based on a two-tailed t-test. (f) Linkage mapping result for chromosome 8 in the F 2:3 population derived from Dan340 and K22. The x axis is the genetic map position, and the y axis is the LOD value. The gray line is the empirical significance threshold (LOD = 2.5). A QTL for total oil was identified; COPII is located within this QTL interval.

24 ARY TABLES 1 11 able 1 Phenotypic analysis, correlations and heritability of all measured traits of variation DF c Oil C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:0 ment ** ** 0.09** 1.10** ** 79.23** 1.13** 0.56** 0.22** 0.16** 0.12** pe ** 9.96** 0.01** 0.98** ** ** 0.29** 0.03** 0.01** 0.01** 0.02** ** lity for H b s.d ± ± ± ± ± ± ± ± ± ± ± 0.09 of variation DF C16:0/C16:1 C16:0/C18:0 C18:0/C18:1 C18:1/C18:2 C18:2/C18:3 C18:0/C20:0 C20:0/C20:1 C20:0/C22:0 C22:0/C24:0 SFA/USFA ment ** 22.92** 0.01** 0.27** ** 22.46** 6.39** 15.66** 1.14** 0.05** otype ** 12.26** 0.00** 0.15** ** 2.56** 0.33** 0.77** 0.09** 0.00** r itability CI for H b ge n ± s.d ± ± ± ± ± ± ± ± ± ± 0.03 ce, showing the mean square and degrees of freedom. b 95% confidence interval (CI) for broad sense heritability. **, P < 0.01; *, P < 0.05; s.d., standard of freedom.

25 able 2 Correlations among all the measured traits C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:0 C16:0/C16:1 C16:0/C18:0 C18:0/C18:1 C18:1/C18:2 C18:2/C18:3 C18:0/C20:0 C20:0/C20:1 C20:0/C22:0 C22:0/C24:0 SFA/USFA 0.35** 0.19** 0.48** 0.33** 0.69** ** 0.60** 0.10* 0.36** 0.20** 0.40** 0.71** 0.23** ** 0.29** 0.43** 0.36** 0.12** 0.34** ** 0.16** ** 0.35** 0.20** 0.55** 0.13** 0.25** 0.41** 0.28** 0.16** 0.33** 0.13** 0.95** 0.13** ** ** 0.19** 0.30** 0.80** 0.23** ** 0.13** 0.15** 0.28** 0.17** 0.32** 0.08** 0.21** 0.27** 0.21** 0.47** 0.18** 0.09* 0.17** 0.10* 0.79** 0.65** 0.22** 0.09* 0.64** 0.62** 0.61** 0.12** 0.19** ** 0.95** 0.54** 0.21** 0.32** 0.19** 0.34** 0.16** 0.33** 0.56** 0.98** 0.12** ** 0.23** 0.27** 0.09** 0.27** 0.94** 0.43** 0.34** 0.34** ** 0.11* 0.24** 0.48** 0.96** ** 0.21** ** 0.19** 0.48** 0.39** 0.10* 0.13** 0.34** 0.48** ** 0.26** 0.47** 0.82** 0.14** ** 0.24** 0.28** ** 0.05* 0.47** 0.15** 0.31** 0.09** 0.41** 0.68** 0.37** 0.10* 0.29** 0.20** 0.23** 0.10* 0.35** 0.62** 0.13** 0.31** 0.38** ** 0.16** 0.16** 0.22** 0.27** 0.10** 0.40** 0.42** 0.28** 0.14** 0.19** 0.41** 0.30** ** 0.44** ** 0.01 ** 0.11** ** 0.07** 0.27** 0.67** 0.49** 0.72** ** ** 0.38** 0.67** 0.30** 0.60** 0.22** 0.37** ** 0.26** 0.12** 0.33** 0.19** 0.46** 0.41** 0.32** 0.69** 0.10* 0.30** 0.10* 0.29** 0.45** 0.49** 0.13** 0.61** 0.47** 0.37** ** 0.81** 0.08** 0.16** 0.11** 0.11** 0.09** 0.20** 0.07** 0.09** ** 0.15** 0.13** ** 0.10* 0.22** ** 0.16** 0.79** 0.32** 0.24** 0.29** 0.29** 0.21** 0.19** 0.25** 0.07** 0.44** 0.30** 0.26** 0.64** 0.45** 0.62** 0.15** 0.30** ** ** 0.54** 0.44** 0.20** 0.27** 0.31** 0.11** 0.13** 0.17** 0.46** 0.51** ** 0.56** 0.10* ** 5** ** 0.98** 0.96** 0.42** 0.18** 0.21** 0.16** 0.28** 0.16** 0.29** 0.48** ** 0.21** 0.17** 2** 0.32** 0.06* ** 0.81** 0.10** 0.05* 0.30** 0.41** 0.16** 0.20** ** ** 0.17** 0.39** 0** 0.12** 0.63** 0.08** ** 0.35** 0.52** 0.61** 0.46** ** 0.48** 0.07** 0.16** 0.14** 0.53** 0.13** 0.14** ** 0.20** 0.60** 0.06* 0.06* ** 0.46** 0.21** 0.11** 0.10** 0.45** 0.55** * 0.12** 0.20** 0.15** 0.37** 6** 0.12** 0.56** 0.46** 0.42** 0.50** 0.13** 0.19** 0.61** 0.52** ** 0.12** 0.43** 0.38** 0.46** 0.29** 0.11** 0.17** 8** 0.20** 0.09** 0.19** 0.18** 0.25** 0.27** 0.18** 0.28** 0.45** 0.16** 0.12** 0.05* 0.18** 0.18** 0.12** 0.09** * ** 0.29** 0.25** 0.27** 0.05* 0.21** 0.46** 0.11** 0.41** 0.38** 0.21** 0.23** 0.38** 0.17** 0.31** 0.15** 0.36** 0.11** 0.04 above and below the diagonal are the genetic and phenotypic correlation coefficients, respectively. **, P < 0.01; *, P < 0.05.

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27 able 3 SNPs and candidate genes significantly associated with oil compositional traits in two association panels of 368 and 508 Chr. Position b SNPs Lead trait c Other trait d Alleles e MAF f P value g QTL h j QTL eqtl Annotation k direction i PZE C20:1 A/G N # Ketoacyl CoA Synthase, KCS S1,S2,S3,S M1c C16:1 G/C Y + (D/C) Haem peroxidase M1c C16:1 A/C Y + (D/C) Unknown M1c C20:0 G/A N # Unknown M2c C18:3 A/C N # N.S* A cytochrome b5 containing fusion protein, CYTOCHROMB5 30,S5,S6,S7,S8,S M2c C18:0 C/T N # N.S* Unknown M3c C18:0/C18:1 T/A Phosphatidylinositol/phosphat idylcholine transfer protein, PITP S10,S M3c C16:1 C/G N # N.S* Unknown PZE C18:1 C18:2,C18:0/C A/C Y + (C) N.S* Oleate desaturase, FAD2 18:1, (refs.22,30,71,72,73,74) C18:1/C18: M4c C180/C181 C/A N # Sequence specific DNA binding transcription factor activity M4c C18:0 C/G N # N.S* Unknown M4c C16:0 SFA/USFA T/C N # Nutrient reservoir activity PUT 163a C18:0 C/A N # Zinc finger Jianbing Yan Formatted: S

28 Chr. Position b SNPs Lead trait c Other trait d Alleles e MAF f P value g QTL h j QTL eqtl Annotation k direction i PZA C16:0 A/G Y + (D) N.S* COPII coated vesicles, COPII 30, M5c C20:0/C22:0 A/G N # N.A* Unknown M6c C18:0/C20:0 T/C Y + (D/C) N.S* WD 40 repeats M6c C20:0 A/G Y + (B) Acyl ACP thioesterases, FATB.a $ 24,30,S12,S13,S M6c C22:0 C20:0,C24:0 T/A N # Unknown M6c C24:0 G/T Y + (B) Protein binding M6c C20:1 G/A Y + (B/C) Vegetative storage protein M6c C22:0 C/A N # Structural constituent of ribosome M6c C20:0 C/T Y + (B) N.S* Zinc finger PZE C20:0 A/G N # Unknown M6c C18:0/C18:1 G/T N # N.S* Unknown M6c C18:2 G/A N # N.S* Unknown M6c C18:0 G/C N # Fasciclin like arabinogalactan protein M6c C20:1 T/C N # N.S* Unknown M8c C18:1 G/C Y + (C) N.S* Unknown M8c C20:0 A/G Y + (C) Unknown M8c C18:0 T/C Y + (C) Unknown M9c C16:0 A/G Y + (D/C) Unknown M9c SFA/USFA G/A Y + (B/D/C) Acyl transferase M9c C16:0 C16:0/C16:1,C G/T Y + (D/C) Acyl ACP thioesterases, 16:0/C18:0,SF FATB.b $ 24,30,S12,S13,S14 Jianbing Yan Formatted: S

29 Chr. Position b SNPs Lead trait c Other trait d Alleles e MAF f P value g QTL h j QTL eqtl Annotation k direction i A/USFA,C18:0 /C18: PZB C18:0/C20:0 A/G N # N.S* Fatty acid elongase, FAE2 30,S15,S16,S PZE C18:2/C18:3 C18:3 C/A Y + (B) Unknown M10c C18:1/C18:2 C18:1 C/G N # Hydrolase activity M10c C18:0 T/C Y + (C) N.S* RNA polymerase II candidate gene in the locus or the nearest annotated gene to the lead SNP. b Position in base pairs for the lead SNP according to B73 reference sequence.60, c The oil trait associated with the highest P value among oil compositional traits. d Additional oil trait with a high probability of 10 6 ). e Major allele (first) and minor allele. The underlined base is the favorable allele. f Minor allele frequency (MAF). g P values of the lead trait only. h The ated in one of the QTL intervals reported previously (see supplementary references indicated in brackets) or in the BY804/B73 RIL population (B) or in one populations K22/Dan340 (D) and CI7/K22 (C). # The candidate was not located in any QTL interval. + The candidate gene was located in one or more QTL nd C populations. i The allele effect direction in B, D and C populations. High parent. Low parent. Not segregated. j SNP located within 200 kb of the ene, that was most significantly associated with the expression level of this gene. N.S., not significant (P > ); N.A., not available (no expression e gene). k Each candidate gene is annotated according to Interproscan ( The supplementary references for these genes are t the end of the annotation. $ There are two copies of this gene in the maize genome.

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31 able 4 SNPs significantly associated with traits after conditioning upon lead SNPs of all oil traits from primary GWAS analysis Chr. Position b SNP Lead trait c Other trait d e Alleles MAF f P value g QTL h QTL direction i j eqtl Annotation k M1c C22:0/C24:0 C/T Y + (D) N.A. Acyl CoA oxidase, M1c C22:0/C24:0 A/G Y + (B) ACX S18,S19,S Acetyl CoA synthetase 30,S SYN27357 C18:1/C18:2 C/A N # Unknown M2c C22:0/C24:0 C/A N # N.S.* Protein tyrosine phosphatase M4c C22:0/C24:0 T/C N # Nutrient reservoir activity M4c C18:3 C/G N # Unknown M4c C16:0/C16:1 C/T N # Protein phosphatase 2C, PP2C M5c C20:0/C20:1 G/T N # N.A.* 4A nuclear receptor M6c C22:0/C24:0 C/A N # N.A.* Epoxide hydrolase, EH 51, M8c C18:1/C18:2 C18:1 C/G Y + (C) Translation elongation factor, EF1B M8c C22:0/C24:0 T/C N # Unknown ical candidate gene either in the locus or the nearest annotated gene to the lead SNP. b Position in base pairs for the lead SNP according to version 5b.60 of e sequence ( c The lipid traits associated with the highest P value among all traits. d Additional lipid traits with P < e Major or allele. The underlined base indicates the favorable allele. f Minor allele frequency (MAF). g P values of the lead trait only. h The candidate gene is located in ervals reported previously (see supplementary references indicated in brackets) or in the BY804/B73 RIL population (B) or in one or both of the F 2:3/F 2:4 an340 (D) and CI7/K22 (C). # The candidate was not located in any QTL interval. + The candidate gene was located in one or more QTL intervals in the B, D. i The allele effect direction in B, D and C populations. Not segregated. j SNP located within 200 kb of the candidate gene, that was most significantly e expression of this gene. N.S., not significant (P > ); V. A., not available. k Each candidate gene is annotated according to InterProScan k/interpro). The supplementary references for these genes are included in brackets at the end of the annotation.

32 Table 5 List of possible additional candidate genes within a 100 kb flanking region of the 74 lead SNPs Chr. Position Gene list b GRMZM2G031718,GRMZM2G172448,GRMZM2G172410,GRMZM2G GRMZM5G855347,GRMZM2G064663,GRMZM2G064732,GRMZM2G GRMZM2G496991,GRMZM2G153393,GRMZM5G815358,GRMZM2G GRMZM2G144995,GRMZM2G GRMZM2G None GRMZM2G005887,GRMZM2G009530,GRMZM2G GRMZM5G839017,GRMZM2G059064,GRMZM2G GRMZM2G110328,GRMZM2G110304,GRMZM2G110294,GRMZM2G GRMZM2G062394,GRMZM2G062377,GRMZM2G062289,GRMZM2G047299,GRMZM2G047223,GRMZM2G349062,GRMZM2G046231,GR MZM2G046267,GRMZM2G GRMZM2G051675,GRMZM2G None GRMZM2G GRMZM2G455491,GRMZM2G GRMZM2G358619,GRMZM2G GRMZM5G881605,GRMZM2G082974,GRMZM2G083094,GRMZM2G GRMZM2G471529,GRMZM2G GRMZM2G176570,GRMZM2G GRMZM2G044055,GRMZM2G044092,GRMZM2G118362,GRMZM2G GRMZM2G003682,GRMZM2G GRMZM2G148532,GRMZM2G GRMZM2G088273,GRMZM2G088365,GRMZM2G088441,GRMZM2G388461,GRMZM2G045387,GRMZM2G346895,GRMZM2G346897,GR MZM2G044104,GRMZM2G044152,GRMZM2G GRMZM2G054651,GRMZM2G133756,GRMZM2G GRMZM5G828945,GRMZM2G055520,GRMZM2G055499,GRMZM5G890820,GRMZM2G152573

33 Chr. Position Gene list b GRMZM2G059021,GRMZM2G GRMZM2G064960,GRMZM2G365298,GRMZM2G065030,GRMZM2G GRMZM2G122846,GRMZM2G122843,GRMZM2G122811,GRMZM2G GRMZM2G GRMZM2G166713,GRMZM2G125378,GRMZM2G125387,GRMZM2G125455,GRMZM2G125512,GRMZM2G GRMZM2G008839,GRMZM2G008819,GRMZM2G008691,GRMZM2G GRMZM2G441541,GRMZM2G GRMZM2G173903,GRMZM2G AC _FG002,GRMZM2G345055,GRMZM2G GRMZM2G GRMZM2G339018,GRMZM2G041310,GRMZM2G041159,GRMZM2G580389,GRMZM2G GRMZM2G147809,GRMZM2G147814,GRMZM2G147862,GRMZM2G GRMZM2G007810,AC _FG003,GRMZM2G GRMZM2G064852,GRMZM2G GRMZM2G363437,GRMZM2G GRMZM2G GRMZM2G149452,GRMZM2G GRMZM2G GRMZM2G139796,GRMZM2G079080,GRMZM2G GRMZM2G335618,GRMZM2G567373,GRMZM2G GRMZM2G055209,GRMZM2G GRMZM2G GRMZM2G GRMZM2G367631,GRMZM2G331283,GRMZM2G GRMZM2G030583,GRMZM2G116986,GRMZM2G GRMZM2G442215,GRMZM2G442244

34 Chr. Position Gene list b GRMZM5G826194,GRMZM5G GRMZM2G None GRMZM2G366778,GRMZM2G065757,GRMZM2G GRMZM2G142068,GRMZM2G167786,GRMZM2G167860,GRMZM2G010953,GRMZM2G GRMZM2G GRMZM2G527017,GRMZM2G GRMZM2G097141,GRMZM2G097103,GRMZM2G003103,GRMZM5G GRMZM2G052926,GRMZM2G GRMZM2G GRMZM2G None GRMZM2G043737,GRMZM5G800211,GRMZM5G847358,GRMZM2G None GRMZM2G098721,GRMZM2G098750,GRMZM2G GRMZM2G103179,GRMZM2G005461,GRMZM2G005339,GRMZM2G485304,GRMZM2G104269,GRMZM2G GRMZM2G391473,GRMZM5G899300,GRMZM2G GRMZM2G GRMZM2G None GRMZM2G028307,GRMZM2G091819,AC _FG None None GRMZM2G043737,GRMZM5G800211,GRMZM5G847358,GRMZM2G according to chromosome and physical position, which are listed in Table 1, Supplementary Tables 3 and 4. b Other possible candidate genes besides

35 able 6 Comparison of the associations of oil concentration between regular and high-oil lines P value* Alleles* MAF* P value (2) a Alleles (2) b MAF (2) c P value (3) a Alleles (3) b MAF(3) c T/C T/C T/C T/A T/A T/A C/A C/A C/A T/C T/C C/T A/G A/G A/G T/G T/G T/G C/G C/G C/G C/T C/T C/T G/C G/C G/C A/G A/G A/G A/G A/G A/G G/C G/C G/C G/A G/A G/A C/T C/T C/T G/C G/C G/C A/G A/G A/G G/C G/C G/C C/T C/T C/T C/T C/T C/T C/A C/A C/A G/T G/T G/T G/T G/T G/T G/A G/A G/A A/C A/C A/C T/C T/C T/C 0.13

36 P value* Alleles* MAF* P value (2) a Alleles (2) b MAF (2) c P value (3) a Alleles (3) b MAF(3) c G/A G/A G/A 0.22 ed in Table1. (2) Associations of oil concentration in regular lines excluding high-oil lines. (3) Associations of oil concentration in high-oil lines excluding of association. b Major allele (first), minor allele. The underlined bases are the favorable alleles. c Minor allele frequency (MAF).

37 able 7 Comparison of the associations of oil compositional traits between regular and high-oil lines P value * Alleles * MAF * P value (2) a Alleles (2) b MAF (2) b P value (3) a Alleles (3) b MAF (3) c A/G A/G A # G/C G/C G # A/C A/C A # G/A G/A A/G A/C A/C A # C/T C/T T/C T/A T/A A/T C/G C/G G/C A/C A/C C/A C/A C/A C/A C/G C/G G/C T/C T/C C/T C/A C/A C # A/G A/G G/A A/G A/G A/G T/C T/C C/T A/G A/G A # T/A T/A T # G/T G/T G* G/A G/A A/G C/A C/A C # C/T C/T T/C A/G A/G G/A G/T G/T T/G 0.17

38 P value * Alleles * MAF * P value (2) a Alleles (2) b MAF (2) b P value (3) a Alleles (3) b MAF (3) c G/A G/A A/G G/C G/C G # T/C T/C T # G/C G/C G # A/G A/G G/A T/C T/C T # A/G A/G A/G G/A G/A A/G G/T G/T T/G A/G A/G G/A C/A C/A A/G C/G C/G C # T/C T/C C/T 0.09 listed in Supplementary Table 3. (2) Associations of oil concentration in regular lines excluding high-oil lines. (3) Associations of oil concentration in high-oil ular lines. a P value of association. b Major allele (first), minor allele. The underlined bases are the favorable alleles. C Minor allele frequency (MAF). # Not

39 able 8 Confirmation of significant candidate genes in three linkage populations Chr. Confidence Interval (Mb) a Population b Trait A c D d R 2 (%) e 1 B C22:0/C24: D (BJ $ ) C 1 B C16:1 D / C(BJ, WH*) 0.01/ / / / B (05BJ, 06BJ # ) C18:0/C18:1 0.01/ / /20.30 D C 1 B C16: D (BJ $ ) C (WH*) B (06BJ # ) C22:0/C24: D C 3 B Oil D (BJ $ ) / C(BJ, WH*) 0.20/ / / / B (05BJ, 06BJ # ) C18:1 2.35/ / /10.29 D / C (BJ, WH*) 0.94/ / / B C16: D (BJ $ ) C 6 B C18:0/C20: D (BJ $ ) C / B (05BJ, 06BJ # ) C20:0 0.05/ / /30.36 D C / B(05BJ, 06BJ # ) C24:0 0.02/ / /24.65 D C / B (05BJ, 06BJ # ) C20:1 0.03/ / /21.18 D C (HN*) / B (05BJ, 06BJ # ) C18:1/C18:2 0.17/ / /39.48 D C / B (05BJ/06BJ # ) C20:0 0.05/ / /29.37 D C

40 Chr. Confidence Interval (Mb) a Population b Trait A c D d R 2 (%) e 8 B Oil / D (BJ, WH $ ) 0.13/ / / C (BJ*) B Oil / D (WH $ ) C 8 B Oil / D(BJ, WH $ ) 0.03/ / /17.06 C 8 B C18:1/C18:2 D / / C (BJ, HN, WH*) 0.03/0.02/ /0.00/ /6.16/ B C18:1 D / / C (BJ, HN, WH*) 1.34/0.94/ /0.15/ /7.54/ B C20:0 D C(WH*) B C18:0 D / / C (BJ, HN, WH*) 0.19/0.12/ /0.11/ /9.94/ / B (05BJ, 06BJ # ) C16:0 1.28/ / / / D (BJ, HN $ ) 1.88/ / / / C (BJ, HN*) 1.56/ / / / B (05BJ, 06BJ # ) SFA/USFA 0.02/ / / / D (BJ,WH $ ) 0.02/ / / / / C (BJ, HN, WH*) 0.02/0.02/ /0.00/ /28.76/ / B (05BJ, 06BJ # ) C16:0 1.28/ / / / / D (BJ, HN, WH $ ) 1.88/0.99/ / 0.08/ /6.41/ / / C (BJ, HN, WH*) 1.56/1.33/ / 0.06/ /31.78/ / B (05BJ, 06BJ # ) C18:2/C18:3 4.60/ / /17.49 D C / B (05BJ, 06BJ # ) Oil 0.31/ / /10.53 D C 10 B C18:0 D C (HN*) ion of the candidate gene refers to version 5b.60 of the maize reference sequence, flanked by the confidence interval of the QTL to the left and right of the ulations B, D and C correspond to the By804 B73 RIL, K22 D340 and CI7 K22 populations, respectively. # Phenotypic data were collected in year 2005 (05) ijing (BJ) for QTL analysis in B population. $ F 2:3 families of the D population were harvested for phenotyping in Beijing 2009, and F 2:4 families were obtained

41 s, Hainan in 2009 and Wuhan in 2010 (HN and WH, respectively). *F2:3 families of the C population were harvested for phenotyping in Beijing, and F2:4 d from Hainan and Wuhan. c Additive effect. d Dominant effect. e The phenotypic variation explained by this QTL.

42 able 9 Correlation analysis between the phenotype and the expression of proposed candidate genes with eqtls Lead trait Oil C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:0 C16:0/C16:1 C16:0/C18:0 C18:0/C18:1 C18:1/C18:2 C18:2/C18:3 C18:0/C20:0 C20:0/C20:1 C20:0/C22:0 C22:0/C24:0 SFA/USFA C20:1 r p < Oil r p C16:1 r p C16:1 r p < < < C20:0 r p Oil r p C18:3 r p < < C18:0/C18:1 r p < < < < < < Oil r p Oil r p C18:0/C18:1 r p Oil r p C16:0 r p C18:0 r p Oil r p < < Oil r p Oil r p Oil r p Oil r p C22:0 r p < < < < < C24:0 r p < < C20:1 r p < < < < < < < C22:0 r p < <

43 Lead trait Oil C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:0 C16:0/C16:1 C16:0/C18:0 C18:0/C18:1 C18:1/C18:2 C18:2/C18:3 C18:0/C20:0 C20:0/C20:1 C20:0/C22:0 C22:0/C24:0 SFA/USFA C20:0 r p C18:0 r p < C20:1 r p Oil r p C20:0 r p C18:0 r p < < C16:0 r p SFA/USFA r p C16:0 r p C18:1/C18:2 r p Oil r p C22:0/C24:0 r p C18:1/C18:2 r p C22:0/C24:0 r p C18:3 r p C16:0/C16:1 r p C18:1/C18:2 r p C22:0/C24:0 r p xpression of candidate gene correlates with target trait at P < 0.01; blue highlighting, expression of candidate gene does not correlate with target trait but with other traits at P <, expression of candidate does not correlate with any trait at P < 0.01.