Supplementary Table 1. Threeprimer sets for obtaining theDGAT1-2 sequence and four functional markers developed for this gene.

Primer set or marker / Primer sequence (5'–3') / Amplified regiona / Size of PCR fragments (bp)
DGAT seq1 / Forward: AGAGCACGATGAGTCTGGAGT / –912 to 411 / 1,323
Reverse: GGCAGAGTTGACTGGAAGC
DGAT seq2 / Forward: CCGTATTTATTGGTTGCTCTGG / 1,421 to 2,691 / 1,271
Reverse: GCTTCATGTTAGGAAAGGGGTT
DGAT seq3 / Forward: TGCCACTACCTAACCATCATCT / 6,256 to 7,258 / 1,002
Reverse: GGATCGAGTCCAGGATCTC
HO06 / Forward: TGCCACTACCTAACCATC / 6,256 to 6,340 / 87 of 84
Reverse: ACATCGGCTGTCCGACTA
DAGT04 / Forward: TACTATCTGGATTTGTGC / 3,036 to 3,764 / 1,002 or 728
Reverse: TGGTAACAAAGTGTTGGG

a Amplified region was from the genomic DNA sequence of DGAT1-2 in the B73 inbred line; theadenine nucleotide of the start codon ATG was considered asposition [1.]

Supplementary Table 2.Maize inbredlinesused in the expression analysis.

Type / ID / Name / Heterotic groupa
High-oil inbred lines (N= 8)
EA01 / By807 / BHO
EA02 / By4944 / BHO
EA03 / By4960 / BHO
EA04 / Gy923 / AIHO
EA05 / Gy462 / AIHO
EA06 / Ry713 / RYD
EA07 / Ry732 / RYD
EA08 / Sy999 / Syn.D.O.
Normal inbred lines (N= 12)
EA09 / Ye478 / Reid
EA10 / Ye107 / Reid
EA11 / Shen5003 / Reid
EA12 / HuangC / Reid
EA13 / B73 / Reid
EA14 / Mo17 / Lancaster
EA15 / M0113 / Lancaster
EA16 / Hai014 / Tang SPT
EA17 / Xi502 / Tang SPT
EA18 / Zong3 / Zi330
EA19 / 8701 / Tem-tropic I
EA20 / Qi205 / Others

aInformation from Teng et al. (2004) and Yang et al. (2010b).

Supplementary Table 3. qHO6 genotype and seed oleic acid concentrationin maize landraces and early inbred lines.

Line / Group / Oleic acid (%)a / qHO6b / Accession #
CANADA YELLOW FLINT / Northern Flint / 40.1 ± 4.3 / +/ / AMES 2755
GRAY'S RHODE ISLAND FLINT / Northern Flint / 42.7 ± 4.7 / + / AMES 2751
PALMER RHODE ISLAND FLINT / Northern Flint / 40.1 ± 1.9 / + / AMES 2749
QUICKS RHODE ISLAND FLINT / Northern Flint / 37.5 ± 2.9 / + / AMES 2752
WAKEFIELDS RHODE ISLAND FLINT / Northern Flint / 47.8 ± 6.5 / + / AMES 2750
ASSINIBOINE / Northern Flint / 47.7 ± 6.1 / +/ / PI 213793
CONCHO BROWN / Northern Flint / 35.1 ± 3.6 / +/ / PI 213748
GASPEFLINT / Northern Flint / 47.5 ± 4.1 / + / PI 213803
GASPEFLINT / Northern Flint / 39.4 ± 6.3 / +/ / PI 401757
GEHU / Northern Flint / 47.7 ± 4.5 / +/ / PI 267161
GEHU YELLOW / Northern Flint / 39.8 ± 2.3 / +/ / PI 213762
KING PHILIP / Northern Flint / 47.4 ± 5.6 / + / PI 217460
MANDAN BUE FLOUR / Northern Flint / 40.3 ± 4.6 / +/ / PI 213763
MANDAN YELLOW FLINT / Northern Flint / 45.3 ± 3.9 / + / PI 213800
SIOUX TRIBE / Northern Flint / 49.2 ± 4.0 / +/ / PI 401755
WINNEBAGO MIXED / Northern Flint / 30.3 ± 5.4 /  / PI 213774
GOURDESEED DENT / Southern Dent and Southwestern Population / 32.5 ± 4.3 /  / PI 213715
HICKORY KING / Southern Dent and Southwestern Population / 41.1 ± 5.1 / + / PI 311237
HICKORY KING / Southern Dent and Southwestern Population / 35.3 ± 4.1 / + / PI 221831
LAGUNA PUEBLO / Southern Dent and Southwestern Population / 36.9 ± 3.1 / +/ / PI 218169
MEXICAN JUNE / Southern Dent and Southwestern Population / 41.4 ± 5.3 / + / PI 311243
PAPAGO FLOUR CORN / Southern Dent and Southwestern Population / 40.7 ± 3.8 / +/– / PI 217410
PAPAGO FLOUR CORN / Southern Dent and Southwestern Population / 35.3 ± 4.0 / +/– / PI 218179
SANTO DOMINGOPUEBLO / Southern Dent and Southwestern Population / 39.6 ± 4.1 / +/– / PI 218143
SHOEPEG / Southern Dent and Southwestern Population / 37.8 ± 4.7 / + / PI 269743
TESUQUE PUEBLO / Southern Dent and Southwestern Population / 40.1 ± 4.5 / +/– / PI 218137
WHITE DENT / Southern Dent and Southwestern Population / 39.9 ± 4.8 / + / PI 311232
KRUG YELLOW DENT / Corn Belt Dent / 22.4 ± 2.7 / – / PI 213699
LANCASTER SURE CROP / Corn Belt Dent / 37.2 ± 4.4 / – / PI 213697
LEAMING / Corn Belt Dent / 33.7 ± 5.0 / +/– / PI 221878
MIDLAND YELLOW DENT / Corn Belt Dent / 39.6 ± 3.6 / + / PI 213712
MILLER YELLOW DENT / Corn Belt Dent / 21.5 ± 2.5 / – / 213726
MINNESOTA 13 / Corn Belt Dent / 29.0 ± 7.6 / +/– / AMES 23518
OSTERLAND REIDS YELLOW DENT / Corn Belt Dent / 29.1 ± 6.0 / – / PI 213706
REID YELLOW DENT / Corn Belt Dent / 25.8 ± 4.7 / – / PI 222613
I205 / Inbred / 24.8 ± 3.2 / –
B37 / Inbred / 23.4 ± 1.7 / –
A632 / Inbred / 19.4 ± 1.4 / –
B84 / Inbred / 24.5 ± 2.5 / –
CO109 / Inbred / 26.2 ± 3.4 / –
C103 / Inbred / 42.2 ± 2.1 / +
Mo17 / Inbred / 19.2 ± 0.6 / –
Oh43 / Inbred / 19.7 ± 1.2 / –
A619 / Inbred / 19.6 ± 0.6 / –
N28 / Inbred / 21.5 ± 1.5 / –

aAverage oleic acid concentration(%) ± SD in 10 plants from each population.

bqHO6 genotype was determined by re-sequencing the DGAT1-2 region responsible for oil and oleic acid differences.

Supplementary Table 4.Haplotypes constructed based on the four significant polymorphisms in DGAT1-2 in 120 normal lines of the association panel.

Haplotype / Frequency / Oil (%)a / Oleic acid (%)a
G/T/0/3 / 42 / 8.48 ± 2.69 / 35.9% ± 4.9%
G/C/3/3 and T/C/3/3 / 11 / 5.67 ± 1.92 / 34.0% ± 2.6%
G/C/9/0 and G/C/3/0 / 9 / 4.11 ± 0.28 / 24.9% ± 3.0%
T/C/3/0 / 79 / 4.50 ± 0.78 / 27.0% ± 5.7%
R2b / 55% / 42%
P valuec / 4.38 × 10–22 / 2.01 × 10–14

a The mean and SD of kernel oil content and oleic acid concentration.

bR2 values from ANOVA of blup data showing the percentage of phenotypic variation explained.

cP value from ANOVA analysis of blup data.

The frequency of the T/C/3/3haplotype was very small and hence counted with the G/C/3/3 haplotype, and G/C/3/0 was counted with G/C/9/0 for the same reason.

Supplementary Table 5. Maize inbred linesused for marker testing.

ID / Inbred lines / Oil content (%)
1 / By804 / 10.91
2 / By809 / 11.68
3 / Gy237 / 12.79
4 / Gy246 / 12.57
5 / Ry684 / 9.65
6 / Ry713 / 7.58
7 / Sy1052 / 8.00
8 / Sy998 / 7.47
9 / B73 / 4.06
10 / Ye478 / 4.41
11 / Ye107 / 2.99
12 / K10 / 4.08
13 / HuangC / 4.59
14 / Dong237 / 3.69
15 / Ji846 / 4.41
16 / ZaC546 / 3.90
17 / Yu374 / 3.64
18 / 4F1 / 4.27
19 / HTH-17 / 3.74
20 / Mo17 / 3.90
21 / Zi330 / 3.68
22 / Zong31 / 3.35
23 / LK11 / 3.75
24 / Hai268 / 3.78
25 / Zong3 / 3.35
26 / Dan340 / 4.21
27 / Chang7-2 / 3.92
28 / HZS / 4.42
29 / Xi502 / 3.69
30 / BT1 / 5.04
31 / SW1611 / 4.28

Supplementary Fig. 1Polymorphism test of 31 maize inbred lineswith two functional markers (details are shown in Supplementary Table 5).

aThe PCR products of the DGAT04 primer pair detected on a 1% (w/v) agarose gelstained with ethidium bromide. M, Trans2K Plus DNA Marker (TransGen Biotech, Inc.,Beijing,China).

b The PCR products of the HO06 primer pair detected on a PAGE gel using the DNA silver staining method.

Supplementary Fig.1