Download Resources for the map-based cloning of tga1

Wang et al - Supplement, Page 1
Methods
Genetic mapping
The maize inbred line W22 was used as the recurrent parent into which a chromosome segment
carrying tga1-teosinte1 was introduced by backcross breeding to create the W22:tga1 isogenic
line (Dorweiler et al., 1993, Science 262:233-235). From the W22  W22:tga1 F2 population, 10
recombinant inbred lines with cross-overs between markers su1 and bt2, which flank tga1, were
recovered. These 10 lines were use to identify new marker loci within a ~3 cM interval
surrounding tga1. A large W22  W22:tga1 F2 fine-mapping population of 3106 plants, which
segregated for Tga1-maize vs. tga1-teosinte1, was generated by self-pollinating F1 hybrids of the
parental stocks.
DNAs were isolated using a simplified cTAB method similar to that described elsewhere
(Doebley and Stec, 1993, Genetics 134:559-570) except that only one CHCl3 extraction was
performed and the ethanol washes were eliminated. One cm2 leaf tissue samples were ground in
96 deep well microtiter plates using a Retsch MM300 Mixer Mill. For a complete list of the
marker loci, primer sequences, PCR conditions, gel conditions and restriction endonucleases for
CAPS markers see Supplementary Information.
Our mapping strategy involved first screening all 3106 F2 plants with markers b91.k20 and
umc1511. The 623 plants with cross-overs between these two markers were subsequently
screened with be25.a15. The 81 plants with cross-overs between b91.k20 and be25.a15 were
then screened with b331f21, bnlg252 and umc1390. The 15 plants with cross-overs between
bnlg252 and umc1390 were screened for bm22.7. Finally, the seven plants with cross-overs
within the ~6 kb segment between bnlg252 and bm22.7 were screened for a series of single
nucleotide polymorphisms and one indel. Phenotypes of F2 plants with cross-overs between
markers b91.k20 and be25.a15 were scored by visual inspection of mature ears. Although
Tga1-maize is essentially dominant to tga1-teosinte1, the heterozygous class is distinct and
intermediate between the two homozygous classes (Dorweiler and Doebley, 1997, Amer. J. Bot.
84:1313-1322), and thus we were able to fully classify the plants into three phenotypic classes.
The genotypic data when combined with the plant phenotypes were used to construct a genetic
map for the tga1 genic region, placing the element controlling the difference between the maize
and teosinte phenotypes into a 1042 bp segment.
EMS mutagenesis
Fresh W22 pollen was suspended in a 0.06% solution of ethyl methanesulfonate (EMS) in
paraffin oil (Fisher 0121-1) for 45 min with shaking at 5 min intervals [Neuffer, M. G. in The
Maize Handbook (eds. Freeling, M. & Walbot, V.) 212-219 (Springer-Verlag, New York,
1993)]. A ratio of ten parts EMS solution to one part pollen was used. The EMS treated pollen
was then applied to silks of the W22:tga1 stock with a camel hair brush. To prevent self
pollination, the W22:tga1 stock was detasseled. Approximately, 2,500 plants were screened.
Nucleic acid analyses
After genetic mapping demonstrated that tga1 lies within the BAC clone ZMMBBc126f15, we
shotgun sequenced this clone to 7X coverage. The BAC DNA was sheared by nebulization and
purified DNA fragments were cloned into pBluescript vector and sequenced from each end. The
Wang et al - Supplement, Page 2
sequence reads were assembled using PHRED and PHRAP software and the assemblies viewed
in CONSED (www.phrap.org). Clone-mates were used to verify and create the assembly with
the XGAP program (www-gap.dcs.st-and.ac.uk). A final remaining gap was closed by designing
PCR primers matching the contig-ends, amplifying across the gap, and sequencing the gap
fragment. This work produced a final assembly of ~169 kb in length (AY883559).
BLAST (www.ncbi.nlm.nih.gov) searches identified a match between the ~6 kb region
between bnlg252 and bm22.7 to which tga1 was mapped and the rice EST AK109469. We used
this rice EST and the corresponding maize genomic sequence as guides to predict the location of
the intron-exon boundaries of tga1. We confirmed that the predicted maize gene is expressed by
RT-PCR. One g of total RNA from young W22 ears was reverse-transcribed using Superscript
III (Invitrogen) and a tga1-specific primer (GCTCACCTCGCTGTCGTT). We then performed
PCR with primers (AGGGCTAGGACTAGCGGTTG, GCTCACCTCGCTGTCGTT) that
amplify a region from 31 bp 5' of the putative start codon to 99 bp 5' of the predicted stop codon.
The PCR conditions using QIAGEN PCR Taq Core Kit were 96 C for 2', then 35 cycles of 96 C
for 30", 57 C for 30", 72 C for 1.5', followed by a final extension of 72 C for 5'. The PCR
product was cloned into pCR2.1 TOPO vector (Invitrogen) and sequenced to verify gene
structure (Genbank AY883560).
We sequenced Tga1-maize, tga1-teosinte1 and tga1-ems1 (Genbank AY883560AY883568) as follows. PCR primers were designed to amplify and subsequently sequence exon
1 (ATAGATAAACGCCCGCGGAAT, AACGAACGAGATGAAGGTTTTTCA), exon 2
(ATCATGTGCCCACCCTACTACCTA, CGTCTTGTCGCAACTAATGTATGC), exon 3
(AATTGCCAGCAGATTGGCATA and TGCAGGAAAAAGAACAGAAATCAA), and two
overlapping portions of the 5' untranslated region (UTR) (region 1:
GCAGGGAGTTGCAGATAGAC, GCGTTTATCTATCGACAGAC; region 2:
TAGCCCCAATAATTGTAGCACAGG, TCGTTCGTTTCCTCCTGTAGCTAC). Sequences
were amplified from genomic DNA using QIAGEN PCR Taq Core Kit under the following
conditions: 96 C, 4' and then 35 cycles of 96 C, 1'; appropriate annealing temperature (exon 1, 64
C; exon 2, 59 C; exon 3, 60 C; 5' UTR-region-1, 60 C; 5' UTR-region-2, 63 C) 1'; 72 C, 1' per
kb, followed by a final extension step at 72 C for 5'. PCR products were purified (QIAquick
purification kit) and directly sequenced using ABI BigDye (Applied Biosystems).
Gene expression assays
Total RNA was isolated from immature ears (2-3 cm in length) and other tissues using TRI
reagent (Molecular Research Center). At 2 cm, immature ears include organ primordia ranging
from spikelet-pair primordia to floret primordia that lack differentiated silks. At 3 cm, immature
ears include organ primordia ranging from spikelet-pair primordia to floret primordia that have
differentiated silks. RNA was isolated from immature tassels that ranged in size from 1 to 5 cm.
At 1 cm, immature tassel have spikelet-pair and spikelet primordia. At 5 cm, immature tassels
have completely differentiated flowers. For Northern blots, ~12 µg total RNA of each sample
was subjected to electrophoresis in a 1.5% agarose gel in the presence of 0.5 M formaldehyde
and then transferred to Zeta-Probe GT blotting membrane (Bio-Rad) according to the
manufacturer's instructions. A cloned DNA fragment developed for in situ hybridization (below)
was labeled with 32P as previously described (Doebley and Stec, 1993, Genetics 134:559-570).
Filters were probed at 68 C in 0.5 M NaHPO4, pH 7.2, and 7% SDS for 16 h, followed with two
30-min washes at 68 C in 40 mM KHPO4, pH 7.2, and 5% SDS and one 30-min wash in 40 mM
Wang et al - Supplement, Page 3
NaHPO4, pH 7.2, and 1% SDS at 68 C. Washed filters were exposed to a phosphor screen
(Molecular Dynamics) and signal detected on a Storm PhosphorImager (Molecular Dynamics).
cDNA for real-time PCR was produced using 1 µg of total RNA isolated from 16
immature ears each of W22 and W22:tga1. Immature ears were developmentally staged by
measuring ear length to insure that both the W22 and W22:tga1 genotypes were represented by
developmentally equivalent samples, 27 mm ± 0.9 and 28 ± 0.9, respectively. RNA was treated
with amplification grade DNaseI (Invitrogen) and then reversed transcribed into cDNA with
random hexamers using Taqman Reverse Transcription Reagent Kit (Applied Biosystems).
Reactions were done in 50 µl for 10 min at 25 C, 90 min at 37 C and 5 min at 95 C. Taqman
primers and probe sets for real-time PCR were designed using Primer Express software (Applied
Biosystems) with attention paid to avoiding the conserved regions of genes. For tga1, the
forward primer, reverse primer and probe are CCATGGCTTCTGCTAGCTTTATC,
GGATGCGCAAATGGTGAGA and 6FAM-CAAGCCAGCAAGGCACGCGA-TAMRA.
Maize Beta-tublin2 (Genbank X52879) was selected as an endogenous control for PCR
quantification and its forward primer, reverse primer and probe are
CCTATAACGCCACGCTCTCTGT, CATTGTCCAGCACCATGCA and 6FAMCACCAGCTCGTGGAGAACGCCG-TAMRA. Real-time PCR reactions were performed on an
ABI Prism 7000 sequence detection system (Applied Biosystems) by using 1 µl of reverse
transcription product per sample in 25 µl containing 12.5 µl Taqman Universal PCR Master Mix
(Applied Biosystems), 900 nM of each primer and 250 nM probe. The PCR program was run for
2 min at 50 C, 10 min at 95 C and 40 cycles with 15 s at 95 C and 1 min at 60 C. One of the RT
samples was taken randomly to do a dilution series (0.25x, 0.5x, 1x, 2x) to construct standard
curves for both tga1 and beta-tublin2. Two replicates were analyzed for each of the 32 samples.
The standard curves were then used to calculate tga1 and beta-tublin2 transcript levels in all the
samples. tga1 expression levels were normalized relative to the endogenous reference betatublin2. The calculation procedures for standard Curve method followed manufacturer's
recommendations.
Methods for preparing tissue samples and in situ hybridization with digoxigenin-labeled
RNA probes followed procedures described elsewhere32. A tga1 891 bp RT-PCR fragment that
excluded the SBP-domain and included 80 bp of the 3' UTR was amplified using primers
CAGCTGCAGAAAGCGTCT and TAAAGCCAGTATCTCCCTACCAGAT and cloned into
pGEM-T vector (Promega). Two plasmids with different insert orientation were isolated and
linearized using SpeI, and then used as templates to generate anti-sense/sense probes. Probes
were synthesized by T7 RNA polymerase with the DIG RNA labeling Kit (Roche).
Protein assays
The 3' end of tga1 excluding the SBP-box was PCR amplified using primers
CACCATGGCTTCTGCTAGCT and CTACTGCCACGAGAAGGGCA from a tga1 RT-cDNA
clone. The PCR fragment was cloned into pET151/D-TOPO vector (Invitrogen) and transformed
into BL21 codonPlus (DE3)-RIPL cells (Stratagene). The Histag-TGA1 (amino acids 181-432)
fusion protein was purified using a His-bind purification kit (Novagen). After a further
purification by SDS-PAGE gel, the TGA1 fusion protein was used as an antigen to generate
rabbit polyclonal anti-TGA1 antibody (Invitrogen). The specificity of the antibody to TGA1 was
tested by Western blot analysis.
Protein from immature maize ears (9 mm to 22 mm in length) was extracted using a plant
total protein extraction kit (Sigma). Protein concentrations were determined by protein assay
Wang et al - Supplement, Page 4
using Quick start Bradford dye regent (Bio-Rad). For each ear sample, 25 µg of protein was
loaded into a 10% SDS-PAGE gel and transferred to immoblion-P membrane (Millipore) using a
mini trans-blot cell (Bio-Rad). The blot was probed with anti-TGA1 primary antibody and then
affinity purified HRP-conjugated goat anti-rabbit secondary antibody (Kirkegaard & Perry
Laboratory). Then, immune-Star HRP substrate (Bio-Rad) was added and the chemiluminescent
signal on the blots was detected with X-ray film. The blots were stripped in a buffer of 25mM
glycine-HCl, pH 2.0 and 1% SDS, and re-probed with an anti-ACTIN (sc-1616R) antibody
(Santa Cruz Biotechnology) as a loading control.
Molecular evolution
We sequenced the tga1 promoter and coding regions for a set of 16 diverse landraces of maize
and 12 teosinte individuals (Z. mays ssp. parviglumis) and the outgroup Zea diploperennis
(Genbank AY883436-AY883558) using the PCR primers and conditions listed above. PCR
products from Z. diploperennis were cloned into the TA vector (pCR 2.1-TOPO kit, Invitrogen)
and at least four clones were sequenced. Nucleotide diversity (π), and Tajima's D statistic
(Tajima, 1989, Genetics 123:585-595), and the Hudson-Kreitman-Aguadé (HKA) tests (Hudson
et al., 1987, Genetics 116:153-159) were calculated in DnaSP Version 4.0 (ref. 33). For the
HKA tests, Z. diploperennis was used as the outgroup and adh1, adh2, glb1, and te1 as control
loci (Tenaillon et al., 2004, Mol. Biol. Evol. 21:1214-1225; White and Doebley, 1999, Genetics
153:1455-1462). The overall HKA P-value was obtained by summing the individual 2 values
for the four control genes.
We used the method of Przeworski (2003, Genetics 164:1667-1676) to estimate the time
since fixation of the favored allele and the selection coefficient. For this analysis, we used
sequences from the promoter region upstream of the ORF (-1 to -1360). The population size of
maize was set to 100,000 and the position of the selected site was set to +18 (the position of the
site controlling the fixed amino acid difference between maize and teosinte). The population
mutation parameter (=0.02626) and the population recombination parameter (=0.0649) were
estimated from the teosinte data. The observed number of segregating sites (12), Tajima’s D
(-2.02428) and the number of haplotypes (10) were estimated from the maize sequence data. A
total of 10,000 simulations were performed. We also used the method of Kim and Stephan
(2002, Genetics 160:765-777) to test for a reduction in polymorphism using the combined
promoter and exon 1 sequences. For this analysis, the neutral distribution was calculated with
the following parameters estimated from the teosinte data: Rn=0.1205, =0.02626 for the
promoter region, and =0.01335 for exon 1. Gene conversion was not allowed by setting the
parameter Gn=0. A total of 10,000 simulations were performed using the program ssw and these
were then evaluated using the program clsw.
Material Availability
Novel materials directly described in this publication may be available for non-commercial
research purposes upon acceptance and signing of a material transfer agreement. In some cases,
such materials may contain or be derived from materials obtained from a third party. In such
cases, distribution of material will be subject to the requisite permission from any third-party
owners, licensors or controllers of all or parts of the material. Obtaining any permissions will be
the sole responsibility of the requestor.
Wang et al - Supplement, Page 5
Results
Genomic walk:
We developed a physical map and a set of molecular markers for the tga1 region. We employed
both the public maize BAC library made from maize inbred B73 (www.genome.arizona.edu),
and the DuPont maize BAC library made from inbred Mo17 (Fengler et al., 2003, An integrated
physical map in maize. Maize Genetics Conference Abstracts 45: 122). Below, we use the
contig numbers for the B73 and Mo17 BAC libraries that were in use at the time of our research.
Since these numbers change over time as the physical map for maize is improved, current contig
numbers can be found by searching the maize physical map for any of the BAC clones at the
Arizona Genomics Institute (www.genome.arizona.edu).
We screened the Mo17 BAC library with marker npi316, which was known to be tightly linked
to tga1 (Dorweiler et al., 1993, Science 262:233-235), and identified contig 47 as harboring
npi316 homologous sequences. Using BAC end sequences from contig 47, we developed two
markers, b91.k20 and b23.l24. We oriented these markers relative to tga1 by screening a set of
10 maize recombinant inbred lines (RILs) with cross-overs within a few centiMorgans (cM) of
tga1. These RILs were isolated from the W22  W22:tga1 F2 population previously described
(Dorweiler et al., 1993, Science 262:233-235) and a second F2 population of the same parentage.
All 10 RILs have a cross-over between markers su1 and bt2, which flank tga1. These analyses
revealed that two of the RILs possessed a cross-over between b91.k20 and b23.l24 within contig
47 (Table 1). Furthermore, three of the 10 RILs had cross-overs between b91.k20 and tga1,
while one RIL had a cross-over between b23.l24 and tga1. These data enabled us to orient
contig 47 relative to the genetic map based on the RILs (Figure S1).
b91.k20
npi316
b23.l24
tga1
b0166h14a
umc1390
be25.a15
umc201
bt2
umc1511
Line
W22
W22:tga1
99S-446
99S-448
99S-450
99S-462
99S-474-1
99S-482
99S-488
02S-57-5
su1
Table 1. Genotypes for the 10 RIL lines and their parents.
m
t
t
t
t
m
t
m
m
m
m
t
t
t
t
m
-
m
t
m
t
t
m
m
m
m
m
m
t
m
t
t
m
m
m
m
m
m
t
m
t
t
m
m
t
m
m
m
t
t
m
t
m
m
t
t
m
t
m
m
t
m
t
t
m
m
t
m
t
m
t
m
m
t
t
m
t
m
t
m
t
m
m
m
t
m
t
t
t
m
t
m
m
m
t
m
t
t
t
Wang et al - Supplement, Page 6
02S-58-1
m
02S-61-3
t
"-" indicates no assay performed.
t
m
t
m
t
m
t
m
t
m
t
m
t
t
m
t
m
t
m
Next, BAC end and overgo sequences from contig 47 were used in BLAST searches against the
rice genomic sequence (www.ncbi.nlm.nih.gov/blast/), identifying two rice chromosome 8 BACs
(AP004015 and AP003920) as sharing sequence homology with Mo17 contig 47. Because we
knew the orientation of contig 47 relative to the genetic map for the 10 RILs, we could infer that
the next rice BAC moving toward (or beyond) tga1 should be AP005529. The AP005529
sequence was then used to BLAST the BAC end and overgo sequences for the Mo17 BAC
library, identifying Mo17 contig 3208 as potentially adjacent to contig 47. Two markers
(umc1390 and be25.a15) from contig 3208 were mapped with the 10 RILs, confirming that
contig 47 and 3208 were closely linked on maize chromosome 4 (Table 1). We also observed
one cross-over within contig 3208 between umc1390 and be25.a15 among the 10 RILs. Marker
be25.a15 showed one recombinant with tga1, but umc1390 was perfectly associated with tga1
among the 10 RILs. Thus, we were able to orient contig 3208 with our genetic map and contig 47
(Figure S1). These data also confirmed that contigs 47 and 3208 flank tga1.
Figure S1. Map of the tga1 region showing the relationship between tga1 and molecular markers
in the Mo17 and B73 contigs as discerned from mapping with 10 RILs.
We then identified B73 contigs that correspond to the Mo17 contigs 47 and 3208 by selecting
B73 contigs that hybridized to the same set of overgo probes as the two Mo17 contigs. B73
contig 236 corresponds to Mo17 contig 47, and B73 contig 4068 corresponds to Mo17 contig
3208. To determine if the two B73 contigs overlap, we first examined the High-Information
Content-Fingerprint (HICF) Fingerprint-Contigs (FPC) at the Arizona Genomics Institute
(http://www.genome.arizona.edu/fpc_hicf/maize/) and identified BACs b0442p01 from contig
Wang et al - Supplement, Page 7
236 and b0331f21 from contig 4068 as being closest to the gap for their respective contigs. DNA
from these two BAC clones was digested with EcoRI-XhoI, cloned and 24 random clones from
each BAC sequenced. Low-copy sequences were identified and used to design PCR primers.
PCR reactions were then performed on multiple BAC clones from both contigs located near the
gap as inferred from the HICF-FPC map. Primer sequences derived from one b0442p01
subclone gave positive PCR results not only for itself (b0442p01 of ctg236), but also for
b0184d07, b0283h11, b0135g01 and b0472h04 (all from ctg4068). We also examined BAC end
sequences from clones near the gap and identified ZMMBBb0184D07.f (ctg4068) as a low-copy
sequence. Primer sequences from this BAC end gave positive PCR results on itself (b0184d07)
as well as b0283h11, b0135g01(ctg4068) and b0442p01 (ctg236). Another BAC end sequence,
ZMMBBb0135G01.r (ctg4068), which has an intermediate copy number, gives positive PCR on
b0283h11 (ctg4068), and b0442p01 and b0237i07 (ctg236). On the basis of these experiments,
we merged contigs 236 and 4068 into a single supercontig within which tga1 is located (Figures
S1 and S2).
To determine the approximate size of the supercontig and the approximate physical distances
between our markers, we selected a set of B73 BACs that form a tiling path across the
supercontig. These BACs were screened by PCR for our markers to determine the BACs on
which each marker is located. The distances between the markers were approximated by
counting the number of consensus bands (CB). The B73 HICF-FPC map was down-loaded from
the Arizona Genomics Institute. The CB map uses the number of CBs between two markers as
the map scale. By multiplying the number of CBs by the average CB size, which is 1.4 kb for
our contig (Will Nelson, University of Arizona, personal communication), an approximate
distance is obtained. These analyses enabled us to create a preliminary physical map for the tga1
region (Figure S2). We emphasize because of the limits of this approach, these estimated
physical distances are very rough.
Figure S2. Schematic physical map of the supercontig showing a tiling path of BAC clones and
the positions of our molecular markers. BACs in blue are from B73 contig 236 and BACs in red
are from B73 contig 4068. Orange boxes indicate the markers that gave a positive PCR
amplification with a specific BAC, e.g. marker be25.a15 was positive for BACs b0398l24 and
c0213e02. Approximate physical distances are shown.
Wang et al - Supplement, Page 8
We selected or developed additional markers from within the super contig for fine mapping of
tga1. To screen a large F2 population and identify more cross-overs, we needed two markers that
flank tga1 and are suitable for high-throughput PCR-based analysis. We chose marker b91.k20
which is distal to tga1 and at the distal end of the supercontig. Unfortunately, no markers on the
proximal end of the supercontig were amenable for high-throughput PCR-based genotyping in
our laboratory. Therefore, we selected umc1511, which resides some distance off the proximal
end of the supercontig, but shows an easily and reliably scored polymorphism between W22 and
W22:tga1. A list of our marker loci, primer sequences and scoring methods is given in Table 2.
Additionally, we used several markers described at the MaizeGDB (www.maizegdb.org)
including: bnlg252, bt2, npi316, su1, umc201, and umc1511.
Wang et al - Supplement, Page 9
Table 2. List of markers.
Marker
Type
b0166h14a
+/b0331f21a
CAPs
bnlg252
SSR
b91.k20
size
be25.a15
CAPs
bm22.7
+/umc1390
SNP
umc1511
SSR
b23.l24
SNP
be163.d18
+/-
Forward Primer
CCCTTGGATCTGTAGTCAAC
CTAAACATTGGCCCATGTAC
CGTTCTCCGTACAGCACAGACCAACGT
AATAAAATAGAGGAACGTCA
GTGTCCACCGTATGGCTTG
CCCTTCTCGTGGCAGTAGTC
CCTCGAAACAGATGCCTGAGTC
CAGACAGATCCATCCAGCACATAC
ACAAATATGCAACGTGTCTT
CAGGGCCTATAGCTCTAAGA
Reverse Primer
AAAATCACGAGGGTTCTTAT
GGTTAGCCATGCGGAAGTAG
CTCAGATGAACTCCTCAGCAGCTGTAGCCT
TGCTGCAAAGGATTACTGAT
TGCTTCGCCAGACCTGCG
CACAGTTAGCCGTGAACCAA
AAATGATCCCGAAGCCTGAGAC
GTTTGTAGGCTTCGTTTTCCTTCA
TTCAGCTAGAACGGAACAGA
TGATCCATCAGCCGTTAAAG
Marker
Program
Ta
PCR Mix
Maize (W22)
% Agarose
Enzyme
W22:tga1
b0166h14a
A
60
Supermix
+
1
N/A
b0331f21a
A
55
Core Kit
lower band
upper band
4
Bcl I
bnlg252
B
55
Supermix
upper band
lower band
4
N/A
b91.k20
A
50
Core Kit
upper band
lower band
2
N/A
be25.a15
A
57
Core Kit
upper band
lower band
2
Tai I
bm22.7
A
55
Core Kit
+
1
N/A
SSRintron1
B
55
Core Kit
upper band
lower band
4
N/A
umc1390
A
50
Supermix
C
G
N/A
N/A
umc1511
A
55
Core Kit
lower
upper band
4
N/A
sbp3130L/3629R
C
60
Core Kit
upper band
lower band
1
N/A
b23.l24
A
55
Supermix
C
A
N/A
N/A
be163.d18
A
60
Supermix
+
1
N/A
Marker types include: +/-, for which only one genotype gives a PCR product; CAPs, cleaved-amplified polymorphisms for
which the restriction enzyme is listed; SSR, simple sequence repeat; size, for which one genotype gives a larger PCR
product than the other due to insertion/deletion differences; and SNP, single nucleotide polymorphisms. Ta = annealing
temperature. PCR Mix, either PCR Supermix (Stratagene) or Taq Core Kit (Invitrogen). Program is the PCR program. A =
96C 2’, 34 times (95C 45”, Ta 1’, 72C 1’), 72C 10’; B = 95C 4’, 34 times (95C 1’, Ta 1’, 72C 2’), 72C 10’; C = 96C 4’,34 times
(96C 1’ Ta 1’, 72C 1’), 72C 5’. N/A = not applicable.
Table 3. SNP polymorphisms used to map tga1 within the supercontig. The maize W22 allele I
shown in red and the W22:tga1 allele in blue.
umc1390
CCTCGAAACAGATGCCTGAGTCATCACCAGCTCGACATCCTTGGCCTCGACACCAGTTTCGTCAAC
CTCCTCCTC[C/G]TCGTTGTCCTCCAGGCCCAGGCCAGATGTCTCAGGCTTCGGGATCATTT
b23.l24
GCAAGCAAAACCAGCATCTAAAGCCCGCTCGCAGCAGCCACCGCTGCATTTCGCAGCCTTTCTGT
CATACACTTGCTAAAATCTGCGCCCTCCACGGCCACCTCTAAGCATGTAATGTACCGTGCCAAGCT
GAATTCCGTGCT[C/A]TGAGAGTTTGTCAGGTCCTTGAGATGGGATCACTCAAGATGGCTGGAAAA
GCACCCGGTGCTGCTGCGCTGCCAGAGTCCTCATGTGCCTACTTGCTTCAAGAACTAAAGGTACAC
AAAGTTTTTTTTTCCAACAAAAGAATATGTATCTGTTCCGTTCTAGCTGAA
Once we had localized the causative site that differentiates Tga1-maize and tga1-teosinte1 to the
~6Kb segment described in the text, we further mapped the causative site to the 1042 kb segment
by DNA sequence analysis of the seven plants carrying cross-overs between bnlg252 and
bm22.7. Figure S3 shows the positions of the sequence polymorphisms relative to the putative
start site as described in the text.
Wang et al - Supplement, Page 10
Figure S3. DNA sequence of the tga1 genic region for the B73 BAC ZMMBBc126f15. Positions
of the polymorphisms used to map the causative site for the difference between the W22 maize and
tga1-teosinte1 alleles are highlighted in pink.
-2000
-1900
-1800
-1700
-1600
-1500
-1400
-1300
-1200
-1100
-1000
-900
-800
-700
-600
-500
-400
-300
-200
-100
1
101
201
301
401
501
601
701
801
901
1001
1101
1201
1301
1401
1501
1601
1701
1801
1901
2001
2101
2201
2301
2401
2501
2601
2701
2801
2901
3001
3101
3201
3301
3401
---------------------cacgtcactcaagggtcattttcctatttcctgttccccagctgagctatagctcccacagggacagtcccacccaaaa
cgtaccaatcgtgtgccatcgcgtctttttctcttccattttttttccttacaaaaatcgcttatattatattattggcacgaaaactactcagatgaac
tcctcagcagctgtagcctctctctctctctctctctctctctcacttttttctttaaaattatctgtacgtacgtagatataaatatagctatgttacc
actactagtgacacactagacgtgttggtctgtgctgtacggagaacgaaggtataaacaaatgcgttgcaaaagcgcagttcgacgacgatagatgtat
atgtatactccacaaaagcccgagctgtcttttgtttttggagaaaaaaataaataacgtgtggttcaggaacaaaagctagcggggttgctactaattg
ctactgttgttgataaacacgtcctgatcagacgccacttgcttggatctgttttaataataataataataataataataataatattattattattatt
attattattatttcctttgctttctttttttgaactgattcttttgtatgttgcaataataatgttaaaagacaaaagtaacggcagggagttgcagata
gacctaatatcaaaggctgtcagggcccttttggaacgcagggattttcacttcacaggtgcttttttttatgaaaatgaaccggttcttatgatttctt
tttttctatgtttcatcatacgagtggaactcgaacatccatcccaaatattagaagacaaaagtaacagtaggagagctgagaagcctgagatagaccc
aatatcaattaattaaagacttttatagcaaatccaaaaggatgcaaaaacacccccgtcaaaaaaataattattagggacaggttacatttttttggag
gcaaatatggtgcatacttcaacagttttattcaaaacaaaggctaaaagaccgtaaaccgagcctatattttttttaaaaaaactagatccatctgccc
cattccgcaaacgattggggagacactacgtcgctcaaatatgtgggggtgggggggggggataggttgaaagcgggttaccgtatttttccaacaatga
tggatccgtcgggctgatttggtgacaaggggatcccggaaggattgaagagaattgaggggaaaatgaactaatttccctctccattatctcgggatcc
agggtcaccaaatcaactctaaatctgtttttcgggttgttttaagtgcttttggagttactcttatacgaatttgagtcatttttttttaaaaaaaaga
taaatcaaaatatacattagtctgtacatagagtgactgctctgaaaaaaataaaaaaactgtagagacaccttagccccaataattgtagcacaggcaa
caggtgcacagcacaacatagccccattgcatgcatggctgcagtgtgacacatggcggtggggccctgcccactgttcctccttcagggacggaaaggt
tggttgcggccccaccatggcgccaagtaatatcgccgctgctctctctctctcttttcatgcacacgctcctctctccctccctcccaccattgctaca
gtcgcagctgtctgtgtctgcaaagtactgactgctcccactccactccacccccagttccgggccactcgggcatcgtttctgttgctgcaaatctttg
ttggctgctgctgccttgctatctatctatacaccgcccgcaccttccattcctcctcctccgaagcagcaggcagcagctgcatcgcacctcacacctc
tcgtgtccatcgatccagccgccgccgcagctgcagctctcacttcactgttgctgtgccacctcctcgtcgcctgtagtgtctgtcgatagataaacgc
ccgcggaatgagagggaaggagcggaagctgcagcgggcgcgcgcgtgcaagggctaggactagcggttgcaacgtcggcgcgcgcggcgtacgtcgggc
atggattgggatctcaacgcggcgggcgcgtgggacctcgcggagctggagcgggaccacgcggccgcggcgccgtcgtcggggggccacgccgccaatg
ctgccgcggcgggcacggggacggagagccgcccgccggcgcccggggcagcaggggcacccgccgagtgctccgtggacctgaagctgggcgggatggg
cgagtgcgagcccggcgcggcccgcagggagagggaggccgcggcgggggcggcgaagcggccgcgccccgccgggcccggcgggcagcagcagcagcag
cagtgcccgtcgtgcgcggtggacgggtgcagggcggacctgggcaagtgccgcgactaccaccggcggcacaaggtgtgcgaggcgcactccaagaccc
ccgtcgtcgtcgtcgccggccgcgagatgcgcttctgccagcagtgcagcaggtagtatccccgccttcttttcccatggggggctggtgtagtgtagtg
tagctcgtccctgtctcgtttcaaggatgcacaactttaccttttccggcttgcctttttttttttcgttatcttttttctctctctctttttcctgaaa
accaaagagatgaaaaaccttcatctcgttcgttcgtttcctcctgtagctacggtacctgaattattggcacgcctttttcctttctcccggcctcctc
ctgcgctcgctgctgctgctgcacactgctctcaggcaggcctagcgttcgtttccttcactttctctgacgccctgatgcgaattaacatctgctgctc
cccaatcgtctgctcaagattcagcccgctgccacggacacgtgagctcctgcgcttgcttttccgagcggtttcttgctttcacgccctttggcggcga
cgggacgggatgcccccccccccccccccccggtactctgccattggctcctcctctagtcggcgctgcttgcttcccggcgacagttcgccaccgccgc
agatgaagcgccgccacggtttggcgtgccgcgttgagcgagacaggggtgtactcccagctttggctaccatcaatcatttgatgtttaggtgccgcgt
tgtgagctgtactgctacagtatctctgcagaatgtattctgtagtagtatgaaaaggtaaaggcggcgtactgctatagtatctctgcagaatgttcgg
aggagtacaccaactgaaagaggtttaggaaaattgcatcgtggatctagaattctagatgcagtagtgtagcctacagtagccctgtacacacataaag
gcatttttcttataaaattgtctcgcaaaatgggatttttttgtgctaattatagggtgctttagcgccacctgggccgtataggatgctttagcgcaac
atttctgaacaaccctatgcagcttccatagaccaccacaggcattcccgcatgcagtttgctctaaatgcctccttttcatttttactatccctgaacg
acgacccatccatttctttttccgtctcagggaagtagccattaaatgctagcagtctttttttaaagtatcgcttggtttgagatgttattaggcccgg
tttggttggaaaaaccgctccattttaatctcctttagtttgtaaattacagtactaaactgttttagtctttagtatctcgaggagtgactaaaagaga
ctaaaccatataaatttcaccttttatctcccatttatttcagttacactaatgacgggagaatgctaaagtgtattttagtcatcttataattgattta
gtgtgttttaaatacttccttagtctataaaattaaacaggatataggctaaactttagttggactaaaccatataaatttcgtctggtgaagtttacgg
cagtacagcgctgtgttggcgcccagttttgtcttgctcatatggacagcagagacaggtcccgctctcctgtggtggacctaaacctaaacagcgtagg
ttcagatcgttcctggagcgatcgacgctgtggcaagcgatcctaatgcgatgctctggtgacatgtcctgtttctgtttacgccggaacatcttgctcg
gtggggacaattctcaatcattgatcactttgtcttctgctttaaattgcagactttgtaggttaatagtaagttgtcagccagtgagccaacagattct
cagctttctgtttaccgtctgtgtggtccaggcttgacaggtcttatagtattttcctgtctctaataagcatggacaaggaacattggtccctggtgtg
atgtgtattaactataacaagcatgtatgtgcgccacagtacattaactattctgacaataattacgttgctgtttcttcagtgaggaccatcttagcat
accgttggatcatgtgcccaccctactacctaggaccatctattcttctaaaaaaaactgagtatcttatacttgtttagaattatcctaattcaaagaa
tcctgtggtcctaatcgattatgccatcattcattggtcagcatattacactgcaactttctcgtttgcccgtactcgaattcagttgtctgatcaagag
ttttttttttgttttttgtttatgtaggttccatctacttgcggagttcgacgccgacaagcgcagctgcagaaagcgtctggacgggcacaatcgccgc
cgcaggaagccacagccagacaccatggcttctgctagctttatcgcaagccagcaaggtcattttcgtactcaccaattttgctttggttttttttctc
cccaagtctgtacttggttcgaatacatctacattgtttccatcttgctggtgctggacaaagaaaaggtaaaacgtcagaacagatctgtgcacacata
aaccatgagaagtaaaagactgctctgttaaacttgtagagcttgcgtagcatttcagcatgtttcagaactagaattcggttcataagataagatgcta
ctctagcatccctgcatacattagttgcgacaagacgaagattacattgttgacgcatcaagttattatgaacctcttctgtttctgtcaattgtgtcct
catatttagtgcaaggtcttgctaatgcctttaagttaaaaaagattagtatactgtatgtttctttgcctttcagatgtgttctagtacaaactacttg
tatgcttcaccacttgtagattggcatgtatatgagtctcaaacttccaacacaattgccagcagattggcatatagaactgcaataaaaattctgattc
acattgtatgaaacggcgcacagaatcaatttatcagttatgtcacctgatcgacatttcttcccaattgatcaggcacgcgattctcaccatttgcgca
tccaagactggaggcgagctggccgccgggggtgatgaaaaccgaggagagtccatatcacatcactcacc
The polymorphisms are given by position relative to the start site (maize allele | teosinte allele).
-1453 (a|g), -1024 (a|t), +18 (c|g), +462 (insertion of ccttgtttaatttgtt between the two C’s | no
insertion), +2335 (a|g), +2368 (c|t), +2851 (c|g), +3442 (a|c)
-2001
-1901
-1801
-1701
-1601
-1501
-1401
-1301
-1201
-1101
-1001
-901
-801
-701
-601
-501
-401
-301
-201
-101
-1
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
3100
3200
3300
3400
Wang et al - Supplement, Page 11
EMS mutagenesis:
We confirmed that the SBP-domain gene just described is tga1 by ethyl methanesulfonate (EMS)
mutagensis. We performed a directed EMS mutagenesis in which EMS treated pollen of maize
inbred W22 was applied to silks of a version of W22 (W22:tga1) that carries a teosinte allele at
tga1. Since the teosinte allele is essentially recessive to the maize allele, progeny of this cross
will exhibit the teosinte phenotype (Figure 1g) only if the pollen carried an EMS induced
recessive allele of tga1. We recovered a single EMS mutant allele, tga1-ems1. Plants
homozygous for tga1-ems1 (Figure 1h) match the phenotype of the teosinte allele, although
phenotypic expression of tga1-ems1 seems more environmentally labile than tga1-teosinte1.
DNA sequence analysis of the tga1-ems1 revealed that it differs from its parental (W22) allele by
a non-conservative amino acid substitution of a phenylalanine for a leucine at position 5 (Figure
2b). The tga1-ems1 allele confirms our conclusion from the positional cloning that tga1 is the
SBP gene, and it demonstrates that a single amino acid substitution is sufficient to confer the
difference between the maize and teosinte phenotypes.