Supplemental Data
Fig. S1: Markers developed for the candidate genes in the MK-pathway and
genotyping of an interspecific F2 population. (A) Representative gel image of ACP1
PCR products separated on a 1% (w/v) agarose gel. Image includes products from the
control DNA of the wild species (PI), cultivated line (M82), and portion of the F2
population. Upper single band (200 bp) represents homozygous for the wild allele.
Lower single band (150 bp) represents homozygous for the cultivated allele. Triple
band represents heterozygous (the third band could be a chimeric product of the two
alleles). (B) Representative gel image of screening the F2 population with a CAPS
marker for the KAS1 locus (PCR products digested with Taq1). Upper single band
(1200 bp) represents homozygous for the wild allele, lower single band (1000 bp)
represents homozygous for the cultivated allele (200 bp band is not in the picture) and
double band represents heterozygous. (C-H) Example of genotyping the F2
population with HRM markers at the following loci: (C) Acetyl-CoA carboxylase
(ACC); (D) Malonyl-CoA:ACP transacylase (MaCoA-ACP trans); (E) 3-ketoacylACP synthase III (KAS3); (F) 2,3-trans-enoyl-ACP reductase; (G) Acyl carrier protein
2 (ACPII); (H) Methylketone synthase 1 (MKS1). Arrows point to the three genotypes
in each locus: (1) homozygous for the wild allele, (2) heterozygous, (3) homozygous
for the cultivated allele.
Fig. S2: Genetic mapping using Solanum pennellii introgression lines (ILs) with the
HRM technology. (A) Screening the ILs using an HRM marker for MKS1.
Introgression line 1-4 is the only line that shares the same pattern with S. pennellii,
thereby localizing it to bin 1-I (B). Chromosomal location of loci ACC and MaCoAACP trans is shown as well.
Fig.S3 . Homology model of tomato MKS2 templated on the structure of a putative
thioesterase from Thermus thermophilus (PDB ID: 1Z54). The hotdog-fold
absolutely conserved homodimeric interface is represented both in the foreground
(blue and gold monomers) and background (green and rose). The less conserved
tetrameric assembly also depicted here is found in both 1Z54 and 4HBT (PDB ID:
1LO9). External binding of phosphopantetheinylated cofactors at the edges of the
conserved homodimeric interface delivers thioester-activated substrates to one of four
identical internal active sites, illustrated here by two stick molecules of coenzyme A
borrowed from another related 4HBT-subfamily crystal structure (PDB ID: 2CYE).
Table S.I: Primers and PCR conditions:
Gene
Forward Primer
Reverse Primer
Tm°C
ACP1
TCGCCATTTGTTAAGAAGCACTTTG
TCAGACCCCTCGATCTCTTTCAC
58
KAS I
TCGCCATTTGTTAAGAAGCACTTTG
TCAGACCCCTCGATCTCTTTCAC
55
Table S.I: Primers and PCR conditions. Approximately 50-100 ng DNA was used as
a template for a 25-μL reaction containing 0.4 μM forward and reverse primers, 0.625
units of Taq DNA polymerase (Peqlab Sawady, Erlangen, Germany), 2.5 μL of 10X
PCR buffer S, and 17 μL DDW. The following reaction profile was used: 60 s at
94°C, 35 cycles of 20 s at 94°C, 20 s at Tm°C, 30 s at 68°C, and a final extension for
10 min at 68°C.
Table S.II: Primers and HRM conditions:
Gene
Forward Primer
Reverse Primer
Tm°C
Acetyl-CoA carboxylase
CAATGCCAATGCTTAATTATTCTTC
TCAAGTTCCAATGAGAGTAATGTTC
55
65-85
Malonyl-CoA:ACP transacylase
ATCCGCGCTCATTATGCTAC
TGAAAGCTGGGCAGAGAAAT
60
72-85
3-Ketoacyl-ACP synthase III
TGCTGTGAAGTTTGGGTCTG
TGAGGCTTTGAGAGGTTTCTTC
60
68-84
Enoyl-ACP reductase
GAGCACTATGAGTTTCAATTTTGG
GAAGCTATGGATTGGCTTCG
60
73-80
ACP2
AGGCACCTAACCGTGTATCG
TGGCTGGATTCACTCTGATG
60
65-85
MKS1
TAAGCGAGTGTTCATTGTTG
CGATCTCTTTCACTTCATCA
56
65-85
MKS2
TGGAGGCAAGAGGAATAGCA
CAAATGTGGTTAGACATTACAAGCA
60
70-84
Table S.II: Primers and HRM conditions. Approximately 250-500 ng DNA were used
as template for a 25-μl reaction containing 0.4 μM forward and reverse primers, 1 unit
of Taq DNA polymerase, 2.5 μL of 10X PCR buffer S, 1.5 μM syto9 (Invitrogen) and
14 μL DDW. The reaction profile was: 60 s at 94°C, 35 cycles of 20 s at 94°C, 20 s at
Tm, 30 s at 68°C. Temperature was raised by increments of 0.1°C.
HRM°C