Supporting Information
Table S1. Repeat motif, primer sequences, annealing temperature (Ta), size of the
sequenced alleles and GenBank accession numbers of the microsatellite loci of N.
attenuata
Locus
Primer sequence (5’-3’)
Repeat
motif
Ta
Allel
Accession
(°C)
e size
No.
(bp)
NA023
(TC)5
F:[6FAM]TCACTCCCTGTCTCCTTTGC
62
173
JF327433
62
88
JF327434
60
144
JF430155
60
95
JF327435
60
93
JF327436
62
241
JF327437
62
197
JF327438
62
218
JF327439
R:AATTCGAGCACCTTCAGCAG
NA137
NA149
(TTC)1
F:[6FAM]ACTTTCCCCCATCTTCACCT
9
R:ACCAGGGGCTACCTGTCTTT
(AAG)3
F:[6FAM]TCCAGGTCAACAAAATCAAG
C
R:GATGTCATTGTGCTGTCACG
NA341
NA441
(TC)20
F:[HEX]AAGTCTCGTGTGGTTGCTTT
(GT)9
R:AAAGGGCAATGTGTCTAGCTC
(ACA)5
F:[HEX]TGGCCTCCATATGTAACCTAC
C
R:TCCAGACACCACTTGTGGAA
NA537
(GAA)3
F:[HEX]GGTCACCGTCTTCTTCTCCA
R:CCCAAATTTATCACGCAACC
NA539
NA541
(AG)12
F:[HEX]GCGTGAAGCAACTAGAGAGA
(CAG)3
GA
(CAA)4
R: CCATCCATTGCTGCTGATAC
(AAC)4
F:[HEX]CCATGTTCACTGCCTTGTTG
.(ACA)
R:TCCACTTTGCATTTGCACTC
3
(TAA)5
(CTT)3
Ta, annealing temperature of the primer pairs during PCR amplification reaction.
1
Table S2. Multiplex grouping and description of 16 polymorphic microsatellite markers for
20 N. attenuata native accessions.
Groups
Group 1
Group 2
Group 3
Marker
DyeSet
Repeat motif
Na149a
Na149b
Na341a
Na541a
Na541b
Na541c
Na023a
Na023b
Na023c
Na441a
Na537a
Na537b
Na537c
Na137a
Na539a
Na539b
FAM
FAM
HEX
HEX
HEX
HEX
FAM
FAM
FAM
HEX
HEX
HEX
HEX
FAM
HEX
HEX
AAG
AAG
TC & GT
AAC&ACA&TAA&CTT
AAC&ACA&TAA&CTT
AAC&ACA&TAA&CTT
TC
TC
TC
ACA
GAA
GAA
GAA
TTC
AG&CAG&CAA
AG&CAG&CAA
Allele size
(bp)
102-132
138-192
95-121
127-154
167-176
215-242
105-139
150-184
214-238
88-94
95-131
172-175
239-251
87-141
84-94
201-240
Na
Ho
He
I
5
6
6
8
4
5
8
6
5
3
9
2
6
8
3
5
0.300
0.400
0.450
0.450
0.600
0.500
0.550
0.700
0.400
0.500
0.500
0.350
0.350
0.650
0.300
0.350
0.685
0.684
0.759
0.821
0.635
0.734
0.820
0.730
0.509
0.595
0.810
0.469
0.573
0.733
0.646
0.709
1.376
1.415
1.603
1.879
1.142
1.430
1.869
1.501
1.022
0.997
1.866
0.662
1.209
1.615
1.067
1.387
Na= Number of allele, Ho= Observed heterozygosity, He= Expected heterozygosity, I=
Noof Heterozygotes
Shannon-Weaver Information index. H o 
, where N is the number of
N
alleles; He  1   pi2 , where pi is the frequency of ith allele; I   pi ln pi , where ln is the
natural logarithm.
2
Figure S1. Reproducibility of nectar nicotine concentrations. Mean (±SD) nectar nicotine
concentration per flower from flowers of five genotypes (Fig.1) from which nectar was
collected in November 2009 (white bar; data from Fig. 1) or in October 2011 (black bar)
from glasshouse grown plants. Utah wild type (UT) is fully comparable to EV (Schwachtje et
al. 2008)
3
Figure S2. Allele size ranges for the 16 markers used for the genotyping of N. attenuata.
Markers with non-overlapping allele size ranges amplified either by FAM labeled primers
(A) or by HEX labeled primers (B) were selected for 3 different multiplex groups (shaded in
different colors).
4
Figure S3. Consistent resolution of uniplex and multiplex genotyping for paternity analysis
of seeds in mixed pollination. Percentage of seeds sired by 11 different pollen genotypes
from mixed hand-pollinations of emasculated flowers with equal numbers of viable pollen
grains in two inbred accessions of N. attenuata, UT-WT (A, C) and AZ-WT (B, D). Pollen
genotypes most successful in siring seeds in UT-WT and AZ-WT are presented in exploded
pie diagrams. Paternity of randomly selected 96 seeds from single capsules of UT-WT and
AZ-WT plants were determined by genotyping with 16 microsatellite markers amplified
separately (uniplex PCR reaction, A, B). Paternity of the same 96 samples from UT-WT and
AZ-WT were determined by multiplex method of genotyping (C, D).
5
Figure S4. Consistency of microsatellite genotyping in different uniplex and multiplex PCR
reactions and among biological replicates. Correlation of Shannon-Weaver Information index
(I) of comparable resolution of the 16 polymorphic microsatellite markers by uniplex and
multiplex genotyping technique for UT-WT (A) and AZ-WT (B) maternal genotypes. r2,
correlation coefficient.
6
Figure S5. Seed paternity in an open-pollinated capsule from a single plant in a native
population. Percentage of different natural paternal genotypes, detected by microsatellite
genotyping, in 30 randomly selected seeds from a single, open-pollinated capsule collected
from a single plant growing in a large native population in Utah. The analysis revealed that
more than half of the seeds resulted from outcrossing.
References
Schwachtje, J., Kutschbach, S. and Baldwin, I.T. (2008) Reverse genetics in ecological
research. . PLoS ONE, 3, e1543.
7