Developing molecular tools and insights into the Penstemon genome using genomic reduction and next-generation sequencing

Abstract

Penstemon's unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain specific insights into the Penstemon genome.
We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers produced simple banding patterns in 90% of the species x marker interactions (~84% were polymorphic). Twelve of the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between any two apparent homologous sequences from the four source species. We selected 192 homologous sequences, meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest genome surveyed (P. dissectus).
We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa. Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa), DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon phylogenetic studies and breeding efforts.

Full text

Developing molecular tools and insights into the
Penstemon genome using genomic reduction and
next-generation sequencing
Dockter et al.
Dockter et al. BMC Genetics 2013, 14:66
http://www.biomedcentral.com/1471-2156/14/66

RES E AR C H A R T I C L E Open Access
Developing molecular tools and insights into the
Penstemon genome using genomic reduction and
next-generation sequencing
Rhyan B Dockter
1
, David B Elzinga
1
, Brad Geary
1
, P Jeff Maughan
1
, Leigh A Johnson
2
, Danika Tumbleson
1
,
JanaLynn Franke
1
, Keri Dockter
1
and Mikel R Stevens
1*
Abstract
Background: Penstemon’s unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for
the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought
tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives
were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain
specific insights into the Penstemon genome.
Results: We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify
homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing
three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique
interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers
produced simple banding patterns in 90% of the species × marker interactions (~84% were polymorphic). Twelve of
the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible
marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between
any two apparent homologous sequences from the four source species. We selected 192 homologous sequences,
meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker
functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were
approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest
genome surveyed (P. dissectus).
Conclusions: We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa.
Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa),
DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in
next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously
sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our
understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study
identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon
phylogenetic studies and breeding efforts.
Keywords: Breeding domesticated Penstemon, Genome reduction, Homologous sequences, LTR retroelements,
Plantaginaceae, Pyrosequencing, Repetitive elements
* Correspondence: mikel_stevens@byu.edu
1
Plant and Wildlife Sciences Department, Brigham Young University, Provo,
UT 84602, USA
Full list of author informa tion is available at the end of the article
© 2013 Dockter et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Dockter et al. BMC Genetics 2013, 14:66
http://www.biomedcentral.com/1471-2156/14/66

Background
Interest is increasing in drought tolerant landscape
plants due to wa ter shortages experienced by many
municipalities, especially in the Southwestern US [1,2].
However, the increased use of drought tolerant species also
carries concerns regarding the introduction of non-native
and potentially invasive species [3,4]. One way to address
both issues is to landscape with native xeric flora [3].
Penstemon Mitchell (Plantaginaceae) has excellent potential
for xeric landscapes and some Penstemon cultivars, adapted
to mild climates, are already used throughout Europe as
landscape plants [5-10]. Despite its potential, few
Penstemon cultivars are used in xeric landscapes and
there has been little to no drought or cold tolerant
cultivar development for such landscapes [6-8,10-12].
Penstemon, with over 270 species, is one of the largest
and most diverse plant genera of those that are strictly
indigenous to North and Central America. This genus
features a deep diversity in morphology, including a
broad assortment of colors , flowers , and leaf struc-
tures. Penstemon’s putative cent er of origin is the arid
Intermountain West of the United States [13,14] and
has frequently been discussed as an untapped resource for
xeric landscape cultivar development [5-7,9-11,15-17].
Because domestication and cultivar development, of any
species , is slow, costly, and time consuming, few in
the landscape industry have invested in native species
breeding. However, given the recent and dramatic decrease
in costs and relative ease of genotyping, we anticipate the
wider utilization of marker assisted selection to accelerate
breeding programs of native species, including d rought
tolerant Penstemon [18-20].
PCR-based markers are now essential tools to facilitate
plant domestication, plant breeding, germplasm con-
ser vation, phylogenetics, and genetic mapping studies
[19-22]. Not surprisingly, little molecular or traditional
genetic work has been reported for Penstemon [23]. To
achieve broad resolution of the genome with three of the
most efficient markers, SSRs (simple sequence repeats or
microsatellites), INDELs (insertions/deletions), and SNPs
(single nucleotide polymorphisms), vast amounts of
DNA sequence are needed, particularly for SNPs where
sufficient read depth is needed to distinguish true
polymorphisms from sequence noise [24-26]. With
the development of next-generation sequencing (e.g., Roche
454-pyrosequencing) the cost of high-throughput marker
discovery has been dramatically reduced [18]. Additionally,
Maughan et al. [25] described a simple genome reduction
method, known as GR-RSC ( genome reduction using
restrict ion site conservation), which reduces the gen-
ome by > 90% thereby, making it feasible to redundantly
sequence the remaining genome with next-generation
sequencing technologies. This process is repeated across
multiple cultivars or species, with comparisons identifying
many inter- and intraspecific homologous loci. Genomic
reduction techniques consistently identify homologous
loci between related species [20,27], and GR-RSC has
enabled the identification and development of interspecific
homologous SNPs [20].
We utilized GR-RSC to identify homologous sequences
in four diploid (2n =2x =16)Penstemon species chosen to
represent a range of taxonomic and genome size diversity
[5,14]. Included in our analysis are two closely related
species from the subgenus Dasanthera (P. davidsonii
Greene and P. fruticosus (Pursh) Greene var. fruticosus),
one from the subgenus Habroanthus (P. cyananthus
Hook. var. cyananthus), and one (P. dissectus Elliot) from
the monophyletic subgenus Dissecti, which is phenotypically
divergent from all other Penstemon species. This experi-
mental design allowed us to make broad inter- and intra-
subgenera comparisons in Penstemon. The objectives of our
study were three-fold: First, identify homologous SSR and
INDEL markers from the four diverse species and test their
conservation across 93, mostly xerophilic, Penstemon taxa.
Second, identify conserved homologous sequences for SNPs
for use in future interspecific studies. Third, assess observed
variation in the GR-RSC sequences to gain insights into the
Penstemon genome and possible reasons for the large size
variation previously identified among the diploid taxa [5].
Methods
Plant material and DNA extraction
DNA from P. cyananthus, P. davidsonii, P. dissectus,andP.
fruticosus leaf tissue was extracted using the CT AB purifica-
tion method [28] with modifications [29] for the GR-RSC
technique. The source localities and identification of these
plants have been reported previously [5]. A single sample
from each spe cies with the highest quality and DNA
concentration, as determined using a ND-1000 spectropho-
tometer (NanoDrop Technologies Inc., Montchanin, DE),
was selected to provide the 500 ng of DNA necessary for
the genome reduction protocol.
For the molecular marker experiments, we used 93
Penstemon taxa. Leaf tissue was colle cted mostly from
wild populations in the United States Intermountain
West (Table 1). Each field-collected sample was identi-
fied to species and (or) variety using taxonomic keys
specific to the area [30,31]. We extracted DNA using
Qiagen DNeasy Plant Mini Kit (Qiagen Inc., Valencia ,
CA), and concentrations were diluted to 25–35 ng/μL.
Genome reduction, barcode addition and 454
pyrosequencing
Genome reduction followed Maughan et al. [25]. Briefly,
for each sample, EcoRI and BfaI were used for the initial
restriction digest, after which a biotin-labeled adapter was
ligated to the EcoRI restriction site and a non-labeled
adapter was ligated independently to the BfaIrestriction
Dockter et al. BMC Genetics 2013, 14:66 Page 2 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 1 Penstemon taxa (with collection counties) utilized in the 12 marker analysis with respective marker sizes
Species County
1
Marker sizes in bp
PS004 PS011 PS012 PS014 PS017 PS032 PS034 PS035 PS048 PS052 PS053 PS075
Subgenus Dasanthera
P. davidsonii Purchased
2
460 500 360 370 700 370 320, 950 520 440 220 320 140
P. fruticosus v. fruticosus Purchased 460 500 360 410 700 340 360 520 420 220 320 140
P. montanus v. montanus Utah 480 430 390 370 450 310 340, 310 470 430 200 390 115
Subgenus Dissecti
P. dissectus Purchased 440 860 370 380 750 370 320 920 380 220 320, 450 140
Subgenus Habroanthus
P. ammophilus Kane 480 800 400 430 470 300 1250, 340 470 420 230 360 125
P. barbatus v. torreyi Garfield 420 800 400 490 500 320 340 500 410 200 360 110
P. barbatus v. trichander San Juan 650, 480 850 420 500, 490 520 310 310 500 450, 410 200 370 130
P. comarrhenus Garfield 650, 480 850 420 490 470 330, 310 310 500 430 200 360 125
P. compactus Cache 440 850 400 500 490 300 300 480 410 210 390, 360 125
P. cyananthus v. cyananthus Wasatch 420 860 400 410 750 370 310, 340 630 420 220 160, 320 160
P. cyananthus v. subglaber Box Elder 440, 420 850 400 490, 470 500, 450 340, 310 310, 280 520 410 210 360 120
P. cyanocaulis Emery 440 310 420 490, 470 520 330, 320 320 480 420 210 350 120
P. eatonii v. eatonii Utah 420 800 420 490 450 320 300 500 NM
3
210 350 135, 125
P. eatonii v. undosus Washington 420 850 420 470 420 320 290 650, 500 410 210 340 125
P. fremontii Uintah 480 850 400 430 490 320, 310 340 500 420 220 370 130
P. gibbensii Daggett 480, 440 850 420 490 420 320 300 480 430 220 360 130
P. idahoensis Box Elder 440 800 400 410 470 310 340 500 430 250 340 130
P. laevis Kane 440 850 400 470 470 310 350, 320 500 420 220 360 125
P. leiophyllus v. leiophyllus Iron 480 850, 490 420 430 450 310 340 480 430 220 350 120
P. longiflorus Beaver 440 800 420, 400 470 470, 450 330, 310 310 500 450 230 350, 220 125
P. navajoa San Juan 480 800 400 490 550 330, 300 360, 340 500 450 230 410 135, 130
P. parvus Garfield 480 850 450 500, 490 490 320 300 500 430 210 380, 360 130
P. pseudoputus Garfield 480 800 420, 400 430 490, 420 320 340 480 450 230, 220 350 130
P. scariosus v. albifluvis Uintah 440 850 400 490 490 310 320 480 410 210 370 115
P. scariosus v. cyanomontanus Uintah 440 850 400 490 490 330 310 500 420 210 360 115
P. scariosus v. garrettii Duchesne 490, 480 850 420 430 490 320 420, 340 520 430 230 360 125
P. scariosus v. scariosus Sevier 480 1500, 1300 400 470 520 340, 310 310 500 430 210 360 130
P. speciosus Box Elder 440 800 420 500, 490 490 320 340, 310 520 310 210 360 120
Dockter et al. BMC Genetics 2013, 14:66 Page 3 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 1 Penstemon taxa (with collection counties) utilized in the 12 marker analysis with respective marker sizes (Continued)
P. strictiformis San Juan 480 850 400 470 500, 470 370, 310 350 500 410 220 370 125
P. strictus Wasatch 480 850 400 410 450 310 350 520, 500 430 230 340 110
P. subglaber Sevier 480 850 420, 400 470 490 310 350 500 430 220 350 115
P. tidestromii Juab 390 850 420 470 490 310 300 480 400 190 360 140, 120
P. uintahensis Duchesne 480 850 420 490 450 340 300 520 410 220 380 120
P. wardii Sevier 480 800 420 430 490, 450 310 310 520 420 220 340 135, 120
Subgenus Penstemon
P. abietinus Sevier 440 AD
4
390 400 520 320 340 500 430 230 350 125
P. acaulis Daggett 570 490 420 430 470 320 350 480 420 220 340 120
P. ambiguus v. laevissimus Washington 520 850 390 490 470 320 1250, 340 500 400 220 AD 120
P. angustifolius v. dulcis Millard 440 850, 600 400 490 520 370, 150 310 520 420 220 360 125
P. angustifolius v. venosus San Juan 480 310 390 470 470 320, 150 340 550 450 220 360 135
P. angustifolius v. vernalensis Daggett 480 800 390 430 470 370, 150 350 500 420 220 380 125
P. atwoodii Kane 440 800 420 490, 390 470 300 310 480 400 180 360, 280 115
P. bracteatus Garfield 440 850 400 500 AD 330, 310 320 520 420 230 380 125
P. breviculus San Juan 650, 480 190 400 AD 470 500, 220 320 480 430 210 350 125
P. caespitosus v. caespitosus Uintah 440 850 390 390 490 320 NM 190 NM 210 390, 370 115
P. caespitosus v. desertipicti Washington 440 230 390 470, 370 470, 360 330 350 1000, 300 430 210 400, 380 130
P. caespitosus v. perbrevis Wasatch 420 490 390 430, 400 470 320 350 520 380 220 340 120
P. carnosus Emery 440 850 420 490 490 330, 300 310 500 430 220 350 130, 120
P. concinnus Beaver 440 800 420 430, 400 500 480 350 480 420 190 700, 360 120
P. confusus Washington 480 850 420 490 520 300 320 480 450 220 350 125
P. crandallii v. atratus San Juan 420 490 390 500 450 370 320 280 400 190 350 120
P. crandallii v. crandallii San Juan 420 340, 190 390 500 450 370, 340 310 280 380 190 350 115
P. deustus v. pedicellatus Teton 420 850 420 430 550 340 320 550 340 230 370 130
P. dolius v. dolius Millard NM 710 400 400 490, 320 530, 300 320 480 450, 420 180 340 105
P. dolius v. duchesnensis Duchesne 420 AD 420 400 500 340 320 480 410 180 360, 340 140, 120
P. eriantherus v. cleburnei Daggett 420 850 420 410 450 480 300 500 490, 420 190 390, 360 140, 130
P. flowersii Uintah 480 AD 420, 400 490, 430 470 300 350 520 420 220 360 125
P. franklinii Iron 480 800 400 430 470 320, 300 350 520 420 240 380 125
P. goodrichii Uintah 420 650 390 400 490 480 310 480 400 200 370, 350 135
P. grahamii Uintah 420 850 400 390 470 530, 320 350 500 420 230 500, 370 120
P. humilis v. brevifolius Cache 390 850 370 500 450 340, 320 320 480 500 220 280 115
Dockter et al. BMC Genetics 2013, 14:66 Page 4 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 1 Penstemon taxa (with collection counties) utilized in the 12 marker analysis with respective marker sizes (Continued)
P. humilis v. humilis Box Elder 420 850 390 410 520 330, 310 360 500 500, 470 220 350 120
P. humilis v. obtusifolius Washington 420 800 390 520, 490 AD 330 340 480 470 200 350 120
P. immanifestus Millard 480 710 420 490 380 300 320 480 410, 380 220 400, 360 120
P. lentus v. albiflorus San Juan NM 430 390 430 450 320 300 500 400 210 470, 370 140
P. lentus v. lentus San Juan 480 850 400 430 470 300 310 500 410 210 400, 370 145
P. linarioides v. sileri Washington 420 850 370 490, 390 470 330, 310 350 470 400 210 370 125
P. marcusii Emery 390 800 450 370 490 310 340, 320 500 NM 200 390, 360 120
P. moffatii Grand 390 800 420 390 490 330 340 480 430 290, 200 380, 350 140
P. nanus Millard 480 800 420 390 470 280 320 470 NM 180 360 120
P. ophianthus Sevier 520 850 420 370 900, 750 330, 310 310 480 420 190 AD 115
P. pachyphyllus v. congestus Kane 480 850 400 430 470, 380 320 310 520 410 250 370 170
P. pachyphyllus v. mucronatus Daggett 440 800 390, 370 430 500 320 300, 280 520, 500 430 220 350 120
P. pachyphyllus v. pachyphyllus Duchesne 480 850 390 410 490 370, 330 340, 240 400, 190 500, 430 290, 230 380, 220 125
P. palmeri v. palmeri Washington 440 850 400 500, 490 520, 490 330 310 500 430 210 380 125
P. petiolatus Washington 420 1000 400 500, 490 500 330 300 480 420 210 380 145
P. pinorum Washington 480 800 420 610 500 480 310 480 470 200 390 125
P. procerus v. aberrans Garfield 440 1000, 850 450, 370 520 520 330 360 480 410 220 370 115
P. procerus v. procerus Iron 420 850, 550 370 490 470 340, 310 360 470 470 220 340 120
P. radicosus Daggett 420 AD 420 490 470 330, 310 310 500 450 200 360 125
P. rydbergii v. aggregatus Box Elder 420 850 400 520 500 340 360 520 470 210 380 115
P. rydbergii v. rydbergii Rich 420 710 400 520 500 370 320 500 470, 430 AD 390 115
P. thompsoniae Kane 420 AD 370 500 450 340, 320 340 500 410 220 390, 370 130
P. tusharensis Beaver 420 1300, 230 370 430 450 320 320 500, 300 410 230 340 120
P. utahensis San Juan 480 410 420 430 490 300 310 500 410 220 370 125
P. watsonii Sevier 420 AD 370 490 470 320 350 480 490 220 350 120
P. whippleanus Iron 420 800 400 370 450 310 350 500 430 210 370 105
P. yampaensis Daggett 570 710 400 430, 390 490 500, 320 310 480 410 260, 230 340 120
Subgenus Saccanthera
P. leonardii v. higginsii Washington 390 1300 420, 400 490, 430 550, 520 320 310 480 470 250 AD 125
P. leonardii v. leonardii Utah 440 800 420 430 490 320 340, 320 480 500 240 370 120
P. leonardii v. patricus Tooele 440 850 370 470 AD 370 310 550, 520 470 230 380 115
P. platyphyllus Salt Lake 420 800 400 430 470 330 310 520 430 240 AD 135
P. rostriflorus Washington 420 1100, 430 400 410 420 320, 300 290 500 490, 430 470 370 120
P. sepalulus Utah 420 800 400 470 450 330 310 500 430 230 390 130
Dockter et al. BMC Genetics 2013, 14:66 Page 5 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 1 Penstemon taxa (with collection counties) utilized in the 12 marker analysis with respective marker sizes (Continued)
Total unique molecular weight bands 9 18 6 10 14 12 12 13 12 11 17 11
Total pairs of dual molecular weight bands 6 7 7 16 11 28 14 7 8 4 20 7
Total monomorphic markers 85 80 86 76 80 65 78 86 81 88 69 86
Total NM 2 0 0 0 0 0 1 0 4 0 0 0
Total AD 0 6 0 1 2 0 0 0 0 1 4 0
1
All counties are in Utah except Teton, Co. which is in Wyoming.
2
Purchased = P. davidsonii and P. fruticosus were purchased from nurseries in Utah Co., Utah while P. dissectus was purchased from a nursery in Aiken Co., South Carolina.
3
NM = no marker.
4
AD = ambiguous data (usually multiple bands and or smearing).
Dockter et al. BMC Genetics 2013, 14:66 Page 6 of 33
http://www.biomedcentral.com/1471-2156/14/66

site. Next, a non-labeled size exclusion step using Chroma
Spin + TE-400 columns (Clontech Laboratories, Inc.,
Mountain View, CA) and magnetic biotin-streptavidin
separation (Dynabeads M-280 Streptavidin, Invitrogen Life
Science Corporation, Carlsbad, CA) was performed. Unique
multiplex identifiers (MID) barcodes were added independ-
ently to each species using primers complementary to the
adapter and cut sites (Table 2). Preliminary amplification
was performed using 95°C for 1 min., 22 cycles of 95°C for
15 s , 65°C for 30 s, and 68°C for 2 min. P CR prod-
ucts were loaded into a 1.2% agarose Fla shgel DNA
Cassette (Lonza Co rporation; Rockland, ME) to verify
smearing and adequate amplification in preparation
for pyrosequencing.
After the initial PCR, concentrations of each of the
four species samples were determined fluorometrically
using PicoGreen® dye (Invitro gen, Carl sbad, CA).
Samples were then pooled using approximately equal
molar concentrations o f each species except for P.
cyananthus (genome size = 1C = 893 Mbp), where the
molar concentration was doubled to maintain a similar
genomic representation compared to the other three
species with smaller genome sizes (P. dissectus, 1C = 462
Mbp; P. davidsonii,1C=483Mbp; and P. fruticosus,
1C = 476 Mbp; [5]). DNA fragments between 500–600 bp
were selected following Maughan et al. [25]. Sequencing
was performed by the Brigham Young University
DNA Sequencing Center (Provo, UT) using a half
454-pyrosequencing plate, Roche-454 GS GLX instrument,
and Titanium reagents (Brandord, CT).
Sequence assembly
Sequence data were sorted by species using their unique
MID species barcode (Table 2) by means of the software
package CLC Bio Workbench (v. 2.6.1; Katrinebjerg ,
Aarhus N, Denmark). Following sorting (Table 2), assem-
blies were performed using Roche’s de novo assembler,
Newbler (v. 2.6), which yields consensus sequences (contigs)
of all i ndividual reads, from each independent species ,
for use in subse quent analyses.
A full assembly (all individual reads of all four species
pooled together) wa s performed by Newbler with
“complex genome” parameter set and a trim file with
MID barcodes specified; all other parameters were left
to their defaults. For all subsequent species a ssemblies
(all individual reads of one species), these same parameters
were used with a few added conservative options selected:
an expected depth of ‘10’ (20 default), a minimum overlap
length of ‘50’ (40 default), and a minimum overlap identity
of 95% (90% default).
Repeat element identif ication
Assembled sequences from all four species were masked
for possible genome wide repetitive elements using a
combination of RepeatModeler and RepeatMasker [32].
RepeatModeler is a de novo repeat element family identifi-
cation and modeling algorithm that implements RECON
[33] and RepeatScout [34]. RepeatModeler scanned all
contigs from the four Penstemon species assemblies and
produced a predicted repeat element library of predictive
models to find repeat elements. Using this reference
library, RepeatMasker then scanned the four species to
filter out repetitive elements. Singletons were omitted
from the analysis. To assess possible repetitive element
biases with RepeatMasker when implementing a denovo
library from RepeatModeler, we analyzed the GR-RSC data
from Arabidopsis RIL’s (recombinant inbred lines) Ler-O
and Col-4 from Maughan et al’s. [35] study, compared to
the Arabidopsis non-reduced genome downloaded from
TAIR (The Arabidopsis Information Resource) [36].
Marker development, verification, and use
To identify SSR s, INDELs, and SNPs, we used soft-
ware MIS A and SNP_Finder_Plus (custom Perl-script),
respectively [25,37,38]. RepeatMasker was used to identify
and mask transposable elements. MISA parameters were
set as follows: di-nucleotide motifs had a minimum of
eight repeats, tri-nucleotide motifs had a minimum of six
repeats , tetra-nucleotide motifs had a minimum of
five repeat s , and 100 bp was set as the interruption
Table 2 The four multiplex identifiers (MID) barcodes (adapter) primers used for the genomes of Penstemon
cyananthus, P. dissectus, P. davidsonii, and P. fruticosus
Species MID ID # EcoR1 MID primer
1
Bfa1 MID primer
2
P. cyananthus MID 1 5′- ACGAGTGCGTGACTGCGTACCAATTC 5′- ACGAGTGCGTGATGAGTCCTGAGTA
P. dissectus MID 2 5′- ACGCTCGACAGACTGCGTACCAATTC 5′- ACGCTCGACAGATGAGTCCTGAGTA
P. davidsonii MID 3 5′- AGACGCACTCGACTGCGTACCAATTC 5′- AGACGCACTCGATGAGTCCTGAGTA
P. fruticosus MID 4 5′- AGCACTGTAGGACTGCGTACCAATTC 5′- AGCACTGTAGGATGAGTCCTGAGTA
1
The “AATTC” at the 3′ end the primer was where adapters complement the enzyme EcoR1 cut site and the preceding “C” is where the base was changed to
avoid further enzymatic cleavage of the fragment.
2
The “TA” at the 3′ end of the primer was where adapters complement the enzyme Bfa1 cut site and the preceding “G” is where the base was changed to avoid
further enzymatic cleavage of the fragment.
Dockter et al. BMC Genetics 2013, 14:66 Page 7 of 33
http://www.biomedcentral.com/1471-2156/14/66

(max difference between two purported SSR alleles). For
the comparison of SSR frequency and repeat motifs
across species , “unmasked” assembly files were used to
remove bia s caused by masking low complexity reads.
The following parameters were used to define the heur-
istic thresholds for SNP_Finder_Plus: 8× minimum read
depth f or the SNP, 30% proportion of the reads
representing the minor allele and 90% identity (an indi-
cation of homozygosity within a single spe cies used in a
dual-species assembly) required for each SNP locus.
These parameters also helped c ompensate for sequen-
cing and assembly errors, which allow greater confi-
dence in calling base pair discrepancies as actual SNPs
in the dual-species assemblies and the confident identi-
fication of heterozygosity in the individual a ssemblies.
For both individual assemblies and dual species assem-
blies SNPs reported are those conforming to the afore-
mentioned parameters.
All genomic sequences matching the abov e criteria
were used for marker development. Primer3 v2.0 [39]
was used to identify primers for amplifying these
markers, with the following parameters: optimal primer
size = 20 (range = 18–27); product size range = 100–500
base bp; Tm range = 50–60°C with 55°C optimum; and
maximum polynucleotid e = 3. Allowing PCR products
greater than 200 bp greatly increased the possibility of
INDELs in the PCR products.
The PCR (SSR/INDEL) mark ers were validated using
the original four species as template DNA. Each 10 μl
PCR reaction had ~ 30 ng genomic DNA, 0.05 mM
dNTPs, 0.1 mM cresol red, 1.0 μl of 10X PCR buffer
(Sigma-Aldrich, St. Louis, MO), 0.5 units of JumpStart ™
Taq DNA Polymerase (Sigma-Aldrich, St. Louis, MO) and
0.5 μM (each) of the forward and reverse primers. The
thermal cycler (Mastercycler® Pro; Eppendorf International;
Hamburg, Germany) was set as follows: 94°C for 30 s,
45 cycles of 92°C for 20 s, (primer specific annealing
temperature)°C for 1 min. 30 s, 72°C for 2 min., and 72°C
for 7 min. (final extension). Following PCR reactions,
DNA wa s loaded into 3% Metaphor® agarose (Lonza
Corporation; Rockland, ME) gels and run us ing a gel
electrophoresis box at 100 V for 2 h. Optimal
annealing temperatures for each SSR/INDEL marker
were sele cted ba sed on clarity of bands produced over
varying annealing temperatures. Only SSR/INDEL
markers with one or two reproducible bands are
reported in the marker studies (Tables 1 and 3). The same
conditions used for marker validation were used in the
SSR/INDEL marker studies, except gel electrophoresis
times were increased to 4 h at 100 V.
The gels were evaluated and scored as: 1 = marker
present; 0 = marker absent based upon molecular weight.
The results were then analyzed to assess the strength
of hierarchical signal in these data using 10,000
replications of fast bootstrapping as implemented in
PAUP* v. 4.0b10 [40].
Our interspecific SNP genotyping was accomplished
using Fluidigm (Fluidigm Corp., South San Francisco, CA)
nanofluidic Dynamic Array Integrated Fluidic Circuit
(IFC) Chips [40] on the EP-1TM System (Fluidigm Corp.,
South San Francisco, CA) and competitive allele-specific
PCR KASPar chemistry (KBioscience Ltd., Hoddesdon,
UK). A 5 μL sample mix, consisting of 2.25 μLgenomic
DNA (20 ng μL
-1
), 2.5 μL of 2x KBiosciences Allele Spe-
cific PCR (KASP) reagent Mix (KBioscience Ltd.), and
0.25 μL of 20x GT sample loading reagent (Fluidigm
Corp., South San Francisco, CA) was prepared for each
DNA sample. Similarly, a 4 μL 10x KASP Assay,
containing 0.56 μL of the KASP assay primer mix (allele
specific primers at 12 μM and the common reverse primer
at 30 μM), 2 μL of 2x Assay Loading Reagent (Fluidigm
Corp., South San Francisco, CA), and 1.44 μL DNase-free
water was prepared for each SNP assay.
The two assay mixes were added to the dynamic array
chip, mixed, and then thermal cycled using an integrated
fluidic circuit Controller HX and FC1 thermal cycler
(Fluidigm Corp., South San Francisco, CA). The thermo
cycler was set as follows: 70°C for 30 min; 25°C for 10 min
for thermo mixing of components followed by hot-start
Taq polymerase activation at 94°C 15 min then a touch-
down amplification protocol consisting of 10 cycles for
94°C for 20 sec, 65°C for 1 min (decreasing 0.8°C per
cycle), 26 cycles of 94°C for 20 sec, 57°C for 1 min, and
then hold at 20°C for 30 sec. Five end-point fluorescent
images of the chip were acquired using the EP-1TM
imager (Fluidigm Corp., South San Francisco, CA), once
after the initial touchdown cycles were complete and then
after each additional run on “addition al touc hdown
cycles.” The extra cycles were run four times , with an
analysis of the chip after each run.
The determination of each SNP allele was based on a
minimum of at least two of three SNP genotyping experi-
ments. The primers were then analyzed for functionality
using the results from each of the five stops for each chip,
which were compared to determine the most accurate call.
Functionality was determined by number of calls verses
no calls, and consistency.
Cross species sequencing verification
To evaluate the DNA sequence homology and polymorph-
ism type (SSR or INDEL) at specific marker amplicons
(Table 1) across the Penstemon genus, DNA samples from
each of five species (P. cyananthus, P. davidsonii, P.
dissectus, P. fruticosus,andP. pachyphyllus) were amplified
and Sanger sequenced. We accomplished the PCR
amplification using Qiagen HotStarTaq Plus Master
Mix (Valencia, CA, USA) according to the manufacturer’s
recommendations. The amplification protocol consisted
Dockter et al. BMC Genetics 2013, 14:66 Page 8 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 3 Summary of marker characteristics including the primary SSR motif identified in the original GR-RSC
(genome reduction using restriction site conservation) sequence, primer sequences, EFL (expected fragment length), total bands, and fragment sizes
Marker
name
1
Primary
motif
Forward primer (5′-3′) GenBank
accession ID
EFL Total unique
bands
Fragment size
Reverse primer (5′-3′) P. cyananthus P. davidsonii P. dissectus P. fruticosus
PS003
(di,f)
(AT)
8
TGCCTCTGTCTTTACATTCCAA JQ966997 217 3 360 260 250 260
CATGAAGCACTGCAAATCCA
PS004
(da,f)
(ATT)
6
TGTTTCAATTGCTGTCCACAT JQ951613 476 3 420 460 440 460
TTGTCTGTCCAAACGGTAGGT
PS005
(c,di,f)
(GAA)
6
GCCCAACTTCCGTAATTGAA JQ966998 303 3 260, 300 260 280 280
AACTGCTTGCCACTCGACTC
PS009
(c,da,f)
(TGA)
6
ACCTCGAACTTGACGGTCC JQ966999 466 4 370, 650 540 650 600
TTCTGAGGAGAAACCAAGGG
PS011
(da,f)
(GA)
8
AAGTGCGACACTGGATGTCTT JQ951614 435 2 860 500 860 500
GCAGCTTCAGCTCCAGAAAT
PS012
(c)
(TA)
8
TCCATATTGTAACCAACAATGACTG JQ951615 402 3 400 360 370 360
TGAATGGCAAACCGTAATCA
PS013
(f)
(TA)
8
GAAGAATTGATTTAAACAAGATGCAA JQ967000 399 2 400 650 650 400
TCAGTACGTGAGAAACTTGATCAATAA
PS014
(c)
(TGA)
6
CGATTTGGTATAGTTGGATTACGA JQ951616 409 3 410 370 380 410
CCTTCATCACCCGGTACTTG
PS015
(di)
(TCG)
6
GCCGAGTTTCAAGAAAGCAA JQ967001 409 2 490 500 490 490
AATTACGACCTGCCACGC
PS016
(c,di)
(CT)
8
CATGGCCCTTTCTTCACACT JQ967002 447 3 NM
2
1,100 1,060 1,030
GACGCGGTTGGCTATACAGT
PS017
(da,di)
(AG)
9
GAAGGCTTAGCATAAATCCTCAAA JQ951617 455 2 750 700 750 700
ATTAGGCTCCCACGAACAAA
PS019
(c,di)
(AG)
8
AATCCCACAGCCCATACAAA JQ967003 473 1 380 380 380 380
TGAATTGAGTCCTATACCCTATTTCAA
PS021
(f)
(CT)
8
CTTTAGCTTAGCTGGAATACACGTT JQ967004 386 3 350 450 450 420
AGATTCTTGCATCACAGTTCAATTA
PS023
(da)
(AG)
8
GCTGGAGAATAACATGGCG JQ967005 469 4 310 480 120, 740 480
CCATCTTGCAAGTCCATACG
PS024
(da,f)
(CTG)
6
CTTCTTGCCCTGTGCCTCT JQ967006 403 2 430 430 400 430
CCACCACCAACAACAACAAC
PS025
(c,di)
(TC)
9
GCACATGAATGAAGGAATGC JQ967007 440 3 440 410 440 400
ACGATCTGTGAAGGAACCCA
Dockter et al. BMC Genetics 2013, 14:66 Page 9 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 3 Summary of marker characteristics including the primary SSR motif identified in the original GR-RSC
(genome reduction using restriction site conservation) sequence, primer sequences, EFL (expected fragment length), total bands, and fragment sizes
(Continued)
PS026
(c,da,di,f)
(CTT)
6
ACTTAATAATGCCTCCTTGTGTCA JQ967008 465 1 460 NM 460 460
TTCCGCAACGTTGTATTTGA
PS028
(di)
(AC)
9
GGGAGGCAGGTAACAACAAA JQ967009 316 4 950 400, 460 320 400, 460
TACCTCTGCCGAACTGGATT
PS029
(di)
(TA)
8
ACCAAGTTGTTGGATGTTTGG JQ967010 440 3 840 500 500 420
GGTTTGGAATGAGACTTAGAAGGA
PS032
(c,di)
(GT)
9
ACAAAGTCTCCTCAATCGCC JQ951618 328 2 370 370 370 340
GCATGTACCGTGCACACACT
PS034
(c)
(AC)
9
CCAAACAAATCAAACAGCACTC JQ951619 322 5 310, 340 320, 950 320 360
CATGCGAATCAGTGTTGCTAA
PS035
(da,f)
(TC)
9
TTGCACAGCTACTTTGGCAT JQ951620 486 3 630 520 920 520
ATCTGTCCAAGGCATGGAAT
PS036
(c,di)
(TA)
8
TTCCTAATTTGGTAGCTGCAATC JQ967011 405 3 770 770, 820 590 770
TCCGAGGAACTATTGCCATT
PS038
(c,da)
(TA)
8
GTAATTACTTCGGCAGTTTGTTAATTT JQ967012 100 1 NM 100 100 NM
GGTGCGACCTAATTACGTTTCTAT
PS040
(da)
(CA)
9
TAAAGAGGCTTAAGCGCGG JQ967013 399 3 380 390 410 390
ACCTGAAGAGCTGCGGAGTA
PS041
(c,da,di,f)
(AT)
8
TTTCCGCAAGAGAAGAGCAT JQ967014 249 3 270 670 270 240
CTTGTGCACGATTCCATTGT
PS045
(c,da)
(CT)
8
GCCACATACATGAAACGTGAA JQ967015 366 4 460 NM 440 120, 400
CGAACTCTCTTGTGTTTCTCCC
PS047
(c,di,f)
(AC)
8
ACACGACATCGTTTCAGCAA JQ967016 428 3 470, 510 440 470 470
GCGTATGGAGAGATTTGGGA
PS048
(c,di)
(CA)
9
GCATTAGATGCCGAAATATCTACAA JQ951621 436 3 420 440 380 420
TGCCTGTAGGTTGATTTCCTTT
PS049
(c,da,di,f)
(AG)
8
CCCATCAATAAAGAAAGAAAGAAAGA JQ967017 436 2 460 460 1,000 460
GGTGAAACCCTGTCCTAAACC
PS050
(c,di)
(AT)
9
GTGTAACCTCTGAACAAGTTTACTGAA JQ967018 434 2 480 460 480 460
TGCAGTGAGCCATGCTATTC
PS051
(c,di,f)
(TG)
8
TGTAACACGACAATTTAACTCTTTCA JQ967019 352 1 280 NM 280 280
CGAGAACTCTTTCCGAGAACC
Dockter et al. BMC Genetics 2013, 14:66 Page 10 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 3 Summary of marker characteristics including the primary SSR motif identified in the original GR-RSC
(genome reduction using restriction site conservation) sequence, primer sequences, EFL (expected fragment length), total bands, and fragment sizes
(Continued)
PS052
(c,da,di,f)
(AC)
9
CGCGGTCAATCTTGAAATCT JQ951622 206 1 220 220 220 220
TGACTTCCTCTCTCTCTCTCACAC
PS053
(di)
(AC)
8
AATCATAGTCTCGAGCGCGT JQ951623 410 3 160, 320 320 320, 450 320
GAGATAAATTAGATCAGCGCATCA
PS054
(c,da,f)
(GA)
8
TCGTTAAGCAATCTCGGAGC JQ967020 192 3 200 200 180 190
TCGACTGGAGAGCAAAGCA
PS055
(f)
(AG)
8
TGTGGTCCGGTTCCATAAAC JQ967021 412 4 960 500 1,040 470
TTTGTCTCCCTAATATGTGTGATGAT
PS056
(da,di)
(TG)
8
CATGTTTCAGGATTGGGCTT JQ967022 319 4 690 450 230 340
CGGTTACACACAGGTTGTTGA
PS057
(da,f)
(AT)
8
TGCCTAATGGACCTGATCCT JQ967023 402 2 570 440 570 440
CCCAATTGTTTGAAGAAAGAACA
PS058
(da)
(AT)
9
GTGCAACCAATGCAACTAATTC JQ967024 469 1 NM 720 NM 720
TCTCTCATTTCCAATGATTTCTCA
PS059
(di)
(CT)
8
CATCAATTGACACACAAGCAGA JQ967025 312 2 930 340 340 340
TCGAATCTTAAAGAAACACATCCA
PS060
(c,di)
(AC)
9
CCATGAGAAGTAGATGACTGGGA JQ967026 484 2 560 560 560 540
TTGTAATTATGATTAACTTCCCTCGTT
PS061
(da,f)
(TA)
8
CGACCAATCATCAACCAACA JQ967027 453 3 480 480, 530 450, 480 NM
GACGGGCAGAATAATTGGAA
PS062
(c,di)
(TA)
9
TGGAGAGGGTACGAAAGTGC JQ967028 320 2 350 290 350 290
CAACGATCGATTATTAGCACCA
PS064
(c,da,f)
(AG)
8
ATGGATGCCCTATGGGTACA JQ967029 437 4 490 500, 680 470 470
TGAAATGGAGGGAGTAATATAAACAA
PS066
(di)
(GA)
9
CAAGGATGCAGGCTCTCATT JQ967030 434 2 250 480 250, 480 480
CTCTGCTCGTCGTAGTGCAA
PS068
(c,da,di,f)
(GA)
8
TTTGGGATGCATTTCTCCAC JQ967031 463 2 500 500 480 480
TCAAAGTGACATCTTCCAACAAA
PS069
(di)
(GT)
8
CATTGGGTCAGATTTGGCTT JQ967032 309 4 220 210 390 350
GCTTTCAGTTTGTATATTTGTGCC
PS071
(c,di)
(AT)
8
AAGATGGCCCTGATCTGTTG JQ967033 446 1 NM NM 490 NM
TTCGTGGGAGTTGCAAATTA
Dockter et al. BMC Genetics 2013, 14:66 Page 11 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 3 Summary of marker characteristics including the primary SSR motif identified in the original GR-RSC
(genome reduction using restriction site conservation) sequence, primer sequences, EFL (expected fragment length), total bands, and fragment sizes
(Continued)
PS074
(c,da,di,f)
(AAG)
6
AGAAATCTCGCTCTCCACGA JQ967034 168 1 170 170 170 170
CGACAACCTTAGTGATCGCTTT
PS075
(c,da,di,f)
(TA)
8
CACCACTTTCGCAGCATTTA JQ951624 120 2 160 140 140 140
CAAATTACATTATTGTATGGAAACACG
PS076
(c,di)
(GTG)
6
CTGACAGCAACATGAACATGAA JQ967035 161 1 170 170 170 170
CAATCTTTGCCAATTTCCCA
1
Parentheses indicates the species possessing sequence from which primers were designed (c = P. cyananthus,da=P. davidsonii,di=P. dissectus, f = P. fruticosus).
2
NM = no marker.
Dockter et al. BMC Genetics 2013, 14:66 Page 12 of 33
http://www.biomedcentral.com/1471-2156/14/66

of an initial denaturation step of 5 min at 95°C, followed
by 40 cycles of amplification consisting of 30 sec denatur-
ation at 94°C, 30 sec for primer annealing at 55°C and
1 min of extension at 72°C. PCR products were separated
on 1% aga rose gels run in 0.5X TBE and visualized
by ethidium bromide staining and UV transillumination.
PCR products were purified using a standard ExoSAP
(Exonuclease I/Shrimp Alkaline Phosphatase) protocol
and sequenced directly as PCR products. DNA sequencing
was performed at the Brigham Young University DNA Se-
quencing Center (Provo, UT, USA) using s tandard
ABI Prism Taq dye-terminator cycle- sequencing
methodology. DNA sequences were analyzed, assembled
and aligned using Geneious software (Biomatters ,
Auckland, New Zealand).
Gene ontology
We used BLASTX [41] on assembled sequences of all
four species to compare with the GenBank refseq-protein
database [42] with a threshold of < 1.0e
-15
. Blast2GO
(v2.4.2) was used to map the blast hits and annotate them
to putative cellular components, biological processes, and
molecular functions found in the blast database [43]. For
species comparisons, the GO level 3 was used for cellular
components and level 2 wa s used for both biological
processes and mole cular functions.
Assembled sequences of all four species were also
compared to all available Antirrhinum and Mimulus
(genera more or less related to Penstemon) genes on
GenBank (downloaded 23 June 2011). Comparisons were
made using BLA STN [41] with an e-value threshold
of <1.0e
-13
.
Results and discussion
Genome reduction, pyrosequencing and species
assemblies
Given that a full 454 pyrosequencing plate using Titanium
reagents is capable of producing 1.3 million reads
averaging ~400 bp each [25], we expected a half plate to
produce approximately 250 Mbp from 650,000 reads. Our
reaction produced 287 Mbp from 733,413 reads, 20%
more than expected, with an average read length of
392 bp. In total, 93.8, 46.4, 48.8, and 53.3 Mbp were
sequenced from P. cyananthus, P. dissectus, P. davidsonii
and P. fruticosus, respectively, closely resembling the
2:1:1:1 ratio of DNA pooled from each species for sequen-
cing (Table 4). Likewise, from our de novo assemblies, we
identified nearly twice as many contigs, 9,714 in P.
cyananthus than the 4,777 found in P. fruticosus,for
example, which was expected because we sequenced
approximately twice as much DNA from P. cyananthus
than the other three species. There was 0.6% of P.
cyananthus genome represented compared to 0.5% average
coverage of the other three species (Table 4); thus,
essentially an equal genome representation from each spe-
cies was realized using the GR-RSC technique by pooling
approximately equal genome molar concentrations in the
sequencing reaction. The contigs of this study have been
deposited at DDBJ/EMBL/GenBank as a Whole Genome
Shotgun project under the accessions AKKG00000000 (P.
cyananthus), AKKH00000000 (P. dissectus), AKKI00000000
(P. davidsonii), and AKKJ00000000 (P. fruticosus). The
version described in this paper is the first version for each
accession, XXXX01000000.
DNA sequences produced by the GR-RSC technique
represent a broad sample of the genome. With this sample,
we can begin to estimate genome-wide characteristics, such
as GC content, frequency of repeat elements, and so forth.
From the genome reduction, GC content was measured to
be 36.4%, 34.5%, 35.3%, and 35.15% for P. cyananthus,
P. dissectus, P. davidsonii and P. fruticosus, respectively
(Table 4), matching the average 35% GC content reported
for dicots [44]. Using the dicot average GC content a
priori, we estimated a theoretical frequency of the BfaI
and EcoRI recognition sites. The theoretical GC content
in combination with estimated genome sizes of the four
species [5] suggested the GR-RSC should have rendered a
104 fold reduction of the genome of each species. With a
reduced genome of these species, the 650,000 reads that
were sequenc e suggest an average of 11× coverage;
however the observed read depth wa s 8.5×, 22.7% less
than expected (Table 4). This lighter coverage is partly due
to the lower than expected specificity of reads. An average
of 48.2% of the reads were matched to contigs with the
other 51.8% either too short or lacking in homology to
successfully match to a contig (Table 4).
The full assembly of all four Penstemon, using the
Newbler de novo assembler, produced a total of 44,966
contigs, representing 16.4 Mbp, or 5.7% of our total
sequence. In the individual species assemblies of P.
cyananthus, P. dissectus, P. davidsonii,andP. fruticosus,a
total of 9,714, 5,364, 4,882, and 4,777 contigs were created
representing 4.6, 2.6, 2.4, and 2.3 Mbp of assembled bases
respectively. These contigs represent, on average, 0.5% of
the total genomes being sequenced (Table 4).
Marker analysis
We utilized assembly contigs from genomic sequence of
all four species with “masked” multiple repeats, such as
transposons, to identify SSRs. Penstemon cyananthus, P.
dissectus, P. davidsonii,andP. fruticosus had 97, 113, 49,
and 58 SSRs identified respectively (Table 5). There were
more SSRs identified in P. dissectus than P. cyananthus,
which has a 1.9 times larger genome and a higher repre-
sentation of sequence than P. dissectus (Table 5). This
inverse relationship between genome size and SSR s
content agrees with observations in other plant genomes
[45]. Some SSRs were found as putative homologs in
Dockter et al. BMC Genetics 2013, 14:66 Page 13 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 4 Summary data from 454-pyrosequencing and Newbler de-novo assembly (v.2.0.01) of Penstemon cyananthus, P. dissectus, P. davidsonii, and P.
fruticosus
Assembly Genome size
(Mbp)
1
GC content Reads Bases
2
% Reads
assembled
% Bases
assembled
Contigs
created
Bases in
assembly
% Genome
represented
Average
coverage
Bases shared
between assemblies
P. cyananthus 893 36.4% 199,329 87,753,792 53.1% 50.0% 9,714 4,623,755 0.5% 7.7X
P. dissectus 462 34.5% 98,868 43,304,550 52.8% 50.9% 5,364 2,629,819 0.6% 8.2X
P. davidsonii 483 35.3% 103,963 45,599,742 45.8% 43.5% 4,882 2,376,141 0.5% 9.1X
P. fruticosus 476 35.2% 113,146 49,786,980 41.0% 38.8% 4,777 2,322,606 0.5% 8.9X
P. cyananthus × P. dissectus 298,197 131,058,342 53.0% 50.1% 14,523 6,915,079 338,495
P. cyananthus × P. davidsonii 303,292 133,353,534 49.9% 46.9% 14,254 6,757,023 242,873
P. cyananthus × P. fruticosus 312,475 137,540,772 47.8% 44.9% 14,134 6,705,536 240,825
P. dissectus × P. davidsonii 202,831 88,904,292 48.3% 46.1% 10,053 4,855,491 150,469
P. dissectus × P. fruticosus 212,014 93,091,530 45.7% 43.5% 9,873 4,774,539 177,886
P. davidsonii × P. fruticosus 217,109 95,386,722 44.0% 41.7% 9,184 4,442,194 256,553
Full Penstemon Assembly 730,215 265,987,500 47.9% 46.4% 44,966 16,363,589
1
The diploid (2n = 2x = 16) genome size as reported by Broderick et al. [5].
2
Bases denotes the total number of bases used to create the assembly and not the total number of bases sequenced.
Dockter et al. BMC Genetics 2013, 14:66 Page 14 of 33
http://www.biomedcentral.com/1471-2156/14/66

multiple species; after eliminating redundancies, we tallied
133 unique SSRs (Table 3). We generated primer pairs sur-
rounding 77 of these SSRs large enough to potentially cap-
ture INDELs, of these, 51 produced 1 or 2 reproducible
bands with no or few faint superfluous bands. From those
51, there was an overall success rate of 94% with 42 (82%)
being polymorphic between the four species (Table 3).
To assess the possibility of utilizing these markers in
interspecific plant improvement studies, 12 of the 51
SSR/INDEL markers (Table 3) were tested on 93 mostly
xeric Penstemon taxa (72 species [Table 1]) representing
five of six subgenera recognized in the genus [14]. The
overall success rate of the markers was 98% with 100%
being polymorphic across the 93 taxa. Without sequencing
each band and/or doing inheritance studies on each marker
it is not possible to clearly determine if a polymorphism of
a given marker is a variant of an allele or a new locus.
Howe ver, we did amplify and sequenc e the amplicon
produced at 11 of these markers in five Penstemon species
(P. cyananthus, P. davidsonii, P. dissectus, P. fruticosus,and
P. pachyphyllus). P. pachyphyllus var. pachyphyllus repre-
sents the largest subgenus (Penstemon)inthegenus.These
five species represented four of the presently classified six
Penstemon subgenera. Of the 55 attempted sequences, 60%
produced high quality sequences results which could be
compared to the original 454 contigs containing the
microsatellites. Using BLASTN (v2.2.25+) [41] we found
that 33 sequences matched the respective microsatellite-
containing contigs from which the SSR/INDEL markers
were derived with an e-value of no more than 1.0e
-36
.An
example of the types of polymorphism (SSRs and INDEL)
found at these loci across the various species is represented
graphically for the marker PS035 (Figure 1). For 22
(40%) of the 55 attempted sequences , we were unable
to obtain high quality sequence information. In the
majority of the se c a ses (94%) the lack of high q uality
data was clearly due to the amplification of multiple
amplicons (seen as multiple bands in gel electrophoresis)
Table 5 Data obtained from MISA (SSR), Blast2GO (GO) and RepeatMasker (RM)
Penstemon species
P. cyananthus P. dissectus P. davidsonii P. fruticosus
SSR Total SSRs
1
97 113 49 58
SSRs/Assembly Length 2.1E-05 (~1/48000) 4.3E-05 (~1/23000) 2.1E-05 (~1/48000) 2.5E-05 (~1/40000)
Repeat Type di- 44.3% 40.7% 46.9% 48.3%
tri- 45.4% 43.4% 44.9% 41.4%
tetra- 10.3% 15.9% 8.2% 10.3%
GO Contigs Analyzed 9,714 5,364 4,882 4,777
Blast Hits Found
2
1,899 1,125 1,121 1,091
Annotated Hits 1,430 844 388 826
% Blast Hits 19.5% 21.0% 23.0% 22.8%
% Annotated 14.7% 15.7% 7.9% 17.3%
RM Masked Repeat Elements 28.5% 16.8% 17.4% 16.1%
Retroelements (LTR) 7.8% 3.0% 4.9% 4.6%
DNA Transposons 0.3% 0.9% 1.0% 1.0%
Other Repeats
3
20.4% 12.9% 11.6% 10.5%
1
For MISA, “unmasked” individual species assemblies were used.
2
Sequence compared to the GenBank refseq-protein database e-value threshold of <1.0e
-15
.
3
Other Repeats includes: lines, unclassified repeats, satellites, simple repeats, and low complexity sequence.
Figure 1 An example of SSR and INDEL found in the comparisons of four Penstemon species in the sequences of marker Pen035.
Dockter et al. BMC Genetics 2013, 14:66 Page 15 of 33
http://www.biomedcentral.com/1471-2156/14/66

which impeded the seque ncing of the PCR reaction.
The source of the multiple amplicons may be from
heterozygousity at t he locus or from the amplification
of paralogous loci.
Both the sequence data (Figure 1) as well as the
marker size data (Tables 1 and 3) are clear evidence of
sequence conservation, and probable homologous loci,
in many of the SSR/INDEL markers. Marker PS012,
the apparent most conser ve d marker, had six unique
molecular weight bands and was present in all 93 taxa.
The marker with the most diversity in its molecular
weights was PS011 which had 18 variants and was not
readable in seven of the 93 taxa. Of the 1,116 possible
marker × ta xa inte ractions , 22 (2.0%) did not p roduce
reliable data. Seven of those 22 (0.5%) were absent of
any product with the remaining 15 producing multiple
bands (reported as ambiguous data). Clearly readable
double bands were found in 135 of the 1,116 (12.1%)
marker × taxa interactions (Table 1).
Our data suggest a high degree of sequence conservation
across the genus, favoring the present hypothesis of a
recent and rather rapid evolutionary radiation of the genus
[13,14]. Furthermore, our data agree with Morgante et al.
[45] who suggest that SSR presence in non-coding
sequence are highly conserved and predate recent genome
expansions of many plants. Some of our markers differed in
length by as much as 570 bp (Tables 1 and 3) suggesting
thepresenceofINDELsandpossiblyadditionalSSRs
(Table 3). We confirmed the presence of INDELs in the
sample of 11 markers which we sequenced (Figure 1). In
some instances, these large fragment length variances
may be amplifying a different locus, which is a recognized
concern when using SSR based markers above the species
level [46,47]. INDELs are useful as PCR based markers
since they, like SSRs, are codominant and abundant in the
genome and are commonly used in genetic mapping [26].
By combining the SSRs we identified in the source
sequence for each of these markers with potential INDELs,
alleles will be easily and inexpensively identified by gel
electrophoresis.
To assess the possibility of phylogenetic (i.e., hierarchical)
structure of the variation within these SSR/INDEL data at
thebroadtaxonomicscaleofoursurvey,weanalyzedthe
12 marker data set (Table 1) with P AUP*. Fast bootstrap-
ping recovered a largely unresolved topology suggesting
rampant homoplasy. Or one or more of these markers
represent more than one locus. These results are similar to
what others have reported about SSR type markers. SSRs
have demonstrated utility for population and intraspecific
relationships, such as cultivar differentiation; however, they
can be problematic when used to reconstruct relationships
above the species level where length differences are
expected to poorly reflect homology [47,48]. Nonetheless,
with over 96% of these SSR/INDEL regions being
conserved across Penstemon, these markers have potential
for studies of interspecific hybridization and cultivar
development.
Interspecific Penstemon breeding is complex [7,11,15,49];
thus, having a set of inexpensive and easily used SSR/
INDEL markers, which amplify across the genus, will
have utility in understanding the results of some wide
crosses. Empirical studies of various Penstemon interspe-
cific crosses have ranged from a clearly recognizable
intermediate phenotype of the two parents, to the F
1
es-
sentially mimicking one of the two parents, usually
mirroring the female parent. Furthermore, in some in-
stances the F
2
’s and additional generations continue to
mimic the female parent to the point that Viehmeyer
[49] began to question if apomixis was involved. An ex-
ample of this phenomenon was a ‘Flathead Lake’ × P.
cobaea interspecific cross. It was not until the hybrid
progeny of this cross was crossed with other interspecific
hybrids when the progeny gave a much wider range of
phenotypes [49]. A probable reason for this phenomenon
is “unequal segregation” which has been described in
other w ide crosses [50,51]. Thus through the use of
these SSR/INDEL markers, regions of the genome can
be identified which are unusual genotypic combina-
tions, for that specific cross , and sele ctions made ac-
cordingly [51-54]. Thus increasing the number of
unique genotype /phenotype plants to be grown out to
maturity from thousands of seedlings. Since many
Penstemon require two years before their first anthe-
sis, using markers to identify the greatest number of
genotypic diverse plants is potentially very useful in
the breeding of this crop.
Beyond amplification ability, we also assessed the com-
position and trends of all SSRs identified. On average,
adenine and thymine rich repea t motifs were the most
common repeat type in the di-, tri-, and tetra-nucleotide
repeat motifs (Figure 2). In general, AT motifs are the
most common motifs in noncoding regions of most
plant genomes [45]. More variation was observed in the
repeat motifs in the tetra-nucleotide repeats across the
four species. Even closely related P. fruticosus and P.
dav idsonii had completely distinct tetra-nucleotide re-
peat motifs (Figure 2). This is likely due, in part, to the
rarity of the motifs and high number of possible nucleo-
tide combinations. Several studies have found that the
hypothetical origins of some SSRs are retrotransposition
events [48,55,56] and, as such, may be useful in develop-
ing part of a unique “fingerprint” for a given species.
SNP analysis
Using our SNP discovery parameters of an 8× minimum
coverage, and 30% representation of the minor allele, we
identified an average of one SNP per 2,890 bp across the
four species ranging from P. cyananthus (1/1,855 bp) to
Dockter et al. BMC Genetics 2013, 14:66 Page 16 of 33
http://www.biomedcentral.com/1471-2156/14/66

P. fruticosus (1/3,777 bp). The three species with similar
genome sizes all had similar SNP frequencies (Table 6).
As reported in other plant species [57,58], we found that
the frequencies of bp transitions (A↔GorC↔T) were
more common compared to transversions (A↔T, A↔C,
G↔C, G↔T) in Penstemon by an average factor of 1.5
(Table 6). This is close to the 1.4 factor in Arabidopsis
[35]. In the dual species assemblies, using the same
parameters and a 90% SNP identity, the average transition
to transversion mutation rate was lower at 1.2 (Table 6).
In the dual species assembly , we found an average
of 1 SNP/97 bp between homologous sequence assemblies
Table 6 SNP type and distributions along with SNP comparisons of sequences found within and between species
(homologous sequence comparisons) using SNP_Finder_Plus (8X min. coverage, 30% min. minor allele, 90% min. identity)
Species assembly SNP Average
coverage
SNPs/assembly
length
1
SNP distribution
A↔CA↔GA↔TC↔GC↔TG↔T
P. cyananthus 2,493 16.4 0.000539 (~1/1855 bp) 10.7% 29.5% 13.9% 4.3% 30.2% 9.5%
P. dissectus 737 14.3 0.000280 (1/3568 bp) 9.8% 30.7% 15.6% 4.6% 27.4% 9.8%
P. davidsonii 713 14.4 0.000300 (~1/3333 bp) 11.9% 26.4% 15.2% 3.9% 28.3% 11.8%
P. fruticosus 615 12.4 0.000265 (~1/3777 bp) 11.7% 27.2% 17.9% 4.2% 25.4% 12.0%
Homologous sequence comparisons
P. cyananthus × P. dissectus 3,253 10.6 0.009610 (~1/104 bp) 11.7% 27.5% 16.0% 7.1% 27.1% 10.6%
P. cyananthus × P. davidsonii 1,958 10.7 0.008062 (~1/124 bp) 11.1% 27.6% 15.8% 7.1% 28.5% 9.9%
P. cyananthus × P. fruticosus 2,015 10.6 0.008367 (~1/119 bp) 10.6% 27.2% 16.7% 6.8% 28.7% 10.1%
P. dissectus × P. davidsonii 2,348 10.8 0.015605 (~1/64 bp) 12.6% 26.7% 15.5% 7.5% 27.3% 10.4%
P. dissectus × P. fruticosus 2,133 10.0 0.011991 (~1/83 bp) 12.0% 26.4% 16.5% 7.6% 27.2% 10.4%
P. davidsonii × P. fruticosus 2,156 10.1 0.008404 (~1/119 bp) 12.8% 28.2% 14.5% 7.2% 27.2% 10.1%
1
Assembly length is bases shared between assemblies (see Table 4).
25
29
9
13
6
14
7
7
12
3
7
8
18
25
5
11
16
12
9
3
2
4
4
6
1
3
3
2
4
2
2
2
1
1
1
1
1
2
6
1
2
5
1
2
1
1
3
3
1
1
1
1
2
1
1
1
1
1
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
P. cyananthus
P. dissectus
P. davidsonii
P. fruticosus
P. cyananthus
P. dissectus
P. davidsonii
P. fruticosus
P. cyananthus
P. dissectus
P. davidsonii
P. fruticosus
Di-nucleotide Repeats Tri-nucleotide Repeats Tetra-nucleotide Repeats
Percent Repeats
SSR Motifs
AGTC/TCAG
AGGG/TCCC
AGAT/TCTA
AAGT/TTCA
AAGG/TTCC
AGGC/TCC
G
AATT/TTAA
AATC/TTAG
AAAC/TTTG
ACAT/TGTA
AAAG/TTTC
AAAT/TTTA
AGC/TCG
ACG/TGC
AGG/TCC
AAC/TTG
AGT/CTA
ACT/GTA
ACC/TGG
AAG/TTC
AAT/TTA
AC/TG
AG/TC
AT/TA
Figure 2 Simple sequence repeat (SSR) motif distributions identified in each of the four Penstemon (P. cyananthus, P. dissectus,
P. davidsonii, and P. fruticosus) sequences using the program MISA.
Dockter et al. BMC Genetics 2013, 14:66 Page 17 of 33
http://www.biomedcentral.com/1471-2156/14/66

of any two of the four species. The frequency of SNPs be-
tween homologous sequences of P. dissectus and P.
davidsonii was the highest at 1/64 bp, with the lowest being
between P. cyananthus and P. davidsonii at 1/119 bp.
These results are in line with previous molecular based
studies [5,14]. Penstemon davidsonii and P. fruticosus both
belong to subgenus Dasanth e r a, while P. cyananthus and
either P. davidsonii or P. fruticosus homologous sequences
had fewer SNPs at 1/124 and 1/119, respe ctively. All
homologous sequence comparison involving P. dissectus
had the highest density of SNPs ( Table 6) suggesting
that P. dissectus is the most evolutionary distant of the
four species.
It is important, for a high degree of c onfidence in
the results, when the “SNP identity” par ameter in
SNP_Finder_Plus to have tw o or more independent
samples from the same species. This requirement wa s
not met for each of the species assemblies, thus, introdu-
cing a weakness in our interspecific SNP comparisons.
Although with the parameters of a minimum 8× coverage
and minor allele frequency set at least 30%, a putative
SNP must be present in at least three of the eight contig
reads, thus providing some protection from mislabeling a
sequencing and/or assembly error as a SNP. Furthermore,
when doing across species comparisons the average
SNP coverage was actually 14.4× ( Table 6). Therefore,
on average, five identical putative SNPs represented
the minor allele.
To understand the viability of our interspe cific SNP
as markers, we utilized the 1 ,958 P. davidsonii × P.
cyananthus and 2,348 P. davidsonii × P. dissectus SNPs
identified in the 14,254 and 10,053 respective homologous
contig parings (Tables 4 and 6). After removing contigs
absent of identifiable SNPs, putative repetitive elements,
and nonnuclear plastid DNA, 431 remained. Of these
contigs, 99 were homologous across all three spe cies
(P. cyananthus, P. davidsonii and P. dissectus)another
164 were only in the P. davidsonii × P. cyananthus
comparisons while the remaining 168 were in the
P. davidsonii × P. dissectus contigs. Of those 431
contigs, we selected the first 192 for SNP marker de-
velopment, 86 from each of the spe c ies comparisons.
These contigs were utilized for competitive allele-
specific PCR SNP primer design using PrimerPicker
(KBioscience Ltd., Hoddesdon, UK).
Of the 192 SNP markers tested, using KASPar genotyping
chemistry, 75 (39%) of produced consistent result s for
P. cyananthus, P. davidsonii, P. dissectus,andP. fruticosus
(Table 7). All 75 SNP markers indicated polymorphisms
between P. cyananthus, P. davidsonii,andP. dissectus,
where only 16 (21% of the 75) produced results in
P.
fruticosus (Table 7). These results suggest that it is possible
to develop intrageneric SNPs for Penstemon. However, it
is unclear as to how viable these markers will be for use
across all the species of the genome since only 21%
worked on all the species used in this GR-RSC study.
Repetitive elements
We identified 28.5%, 16.8%, 17.4% and 16.1% of the
respective sequence from P. cyananthus, P. dissectus, P.
dav idsonii, and P. frutic osus as repeat elements using
RepeatModeler and RepeatMasker. Of these elements,
3.0-7.8% were identified as LTR (long terminal repeat)
retroelements, 0.3-1.0% transposons and the remainder
were unclassified (Table 5). Since RepeatModeler utilizes
RECON and RepeatScout to create a de novo model in
RepeatMasker in place of the Arabidopsis model, details
about the subcategories of LTRs and transposons which
are included in the model could not be addressed.
Maughan et al. [35] utilized GR-RSC on the Arabidopsis
lines Ler-0 and Col-4. Utilizing RepeatModeler, then
RepeatMasker on their sequence data from these lines, we
found an average of 6.2% were identified as repetitive ele-
ments, of which 4.4% were identified as LTR retroelements
and 0.4% were transposons. By way of comparison,
the downloaded full “ non-genome reduced” sequence
of Arabidopsis line TAIR10 had a similar 7.4% of the
sequence identified a s repeat elements of which 3.0%
were LTR retroelements and 0.2% were transposons
(Table 5 and Figures 3 and 4). These data suggest that the
GR-RSC method reflects, at least for repetitive elements,
similar proportions as to that found in the full seque nce
of Arabidopsis.
Broderick et al. [5] hypothesized that the broad range
found in Penstemon genome sizes, of the same ploidy,
may be explained by retrotransposons. Lynch [60]
detailed a relationship betw een genome size and repeat
elements suggesting a linear relationship between the
number of elements and genomes size [60-62]. The four
Penstemon species used in this study provide insufficient
evidence to establish a linear relationship between
genome size and repeat elements in Penstemon. However,
the three smaller, similar sized, Penstemon genomes
possess comparable quantities of repetitive elements
whereas P. cyananthus (the largest genome) has nearly
double the number of repeat elements compared to the
other three species (Figure 3).
Not only do repetitive elements largely influence genome
size, but they are also likely to evolve more rapidly than do
low-copy sequence [62,63]. Thus, repetitive elements of a
species take on unique “fingerprints” which become
valuable in phylogenetic relationship studies [64,65].
Thus, our limited four Penstemon species genomic data
set suggest agreement with the two hypotheses that firstly,
repetitive elements are a major component of the genome
size variation identified by Broderick et al. [5]. Secondly,
these elements are variable between the species we
tested suggesting the possibility of identifying species
Dockter et al. BMC Genetics 2013, 14:66 Page 18 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays
Name Contig
Source
1
SNP Allele
GenBank
Accession #
2
SNP
Type
Allele Specific A1
Forward (5′→3′)
3
Common Allele
Specific Reverse
5′→3′)
P.
davidsonii
P.
cyananthus
P.
dissectus
P.
fruticosus
P.
pachyphyllus
P. cyananthus +
P. davidsonii
P. dissectus +
P. davidsonii
Allele Specific A2
Forward (5′→3′)
3
PenSNP00001 00336CD JX649978 A/G AAGATTGCA
TGGAGAGGA
AATGGATT
CGATCCAAA
TGGCAGATC
CGAGAAA
X
4
YX X Y Y
AGATTGCAT
GGAGAGGAA
ATGGATC
PenSNP00002 00405CD JX649979 C/T ACGCGAGTA
ATAAGTTGG
TTTTCTTC
CCAACACTT
CCGCAGAAG
CTCTTAA
YX YXY H H
GACGCGAGT
AATAAGTTG
GTTTTCTTT
PenSNP00003 02625CD JX649980 A/T AAAAGCTCC
CAAACATGA
CTATGAACT
AATTCTTCGA
CACTTGAAGA
GAGCGTAA
YX Y Y H H
AAAAGCTCC
CAAACATGA
CTATGAACA
PenSNP00004 02857CD JX649981 A/C ATCAAATGA
ACTTGTCTC
ATGAGCCT
GCAACAAGGT
GCAAAAAATT
GTAGCGTAA
XY X X H H
CAAATGAAC
TTGTCTCATG
AGCCG
PenSNP00005 03943CD JX649982 A/G ACTACCAAA
ACTACCCTT
CCCTTA
GGGGTACAGA
GTTGAGAAGA
AGGAA
XY X X H H
ACTACCAAA
ACTACCCTTC
CCTTG
PenSNP00006 04420CD JX649983 A/C TGTCTCTAA
ATCGATATG
ATGAGGCT
GTGGTTCTTC
CCCTTTAGA
GGACTT
YX YXY H H
GTCTCTAAA
TCGATATGAT
GAGGCG
Dockter et al. BMC Genetics 2013, 14:66 Page 19 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00007 08446CD JX649984 A/T GGCAACATC
CTCAGCAGA
GACA
CCGACTCCCT
TAGCAATCTT
AGCAT
YX X X H X
GGCAACAT
CCTCAGCA
GAGACT
PenSNP00008 11303CD JX649985 C/G GGGTGGTA
TTGGTTAC
TTTTATGGG
CGGTATAAGA
GCAACTAAGC
TAAATGACTT
YX Y Y H H
GGGTGGTAT
TGGTTACTT
TTATGGC
PenSNP00009 11357CD JX649986 C/T ACAATATTTG
ATAATTCATT
CTCAAGTGCG
AAGCATGCAG
TGAGACAAAA
GCTAAGAT
XY XYX H H
CACAATATTT
GATAATTCAT
TCTCAAGTGCA
PenSNP00010 11935CD JX649987 A/C AGCCTGATTA
TCCCTTAAAC
CCAATT
GAATCACGG
CGGGGGAG
CAAAT
XY X X Y
GCCTGATTAT
CCCTTAAAC
CCAATG
PenSNP00011 12047CD JX649988 C/T TTTGGCACT
GCAGTGAC
CATC
TGCTCCAGT
CCGAAGGA
AGTTGAAT
XY Y X Y Y
CTTTTGGCAC
TGCAGTGAC
CATT
PenSNP00012 12119CD JX649989 A/G AAGATAGAC
GTGGTATTTC
TTCAGCA
GCAATTAG
TCACAGAC
CATAGTGG
XY X X H H
AGATAGACG
TGGTATTTCT
TCAGCG
PenSNP00013 12398CD JX649990 A/T TATTTTCCTT
TCTGCAATC
TCAACATTGA
GTTGAGTGTG
ATTTTAGAGT
GCATTTAGTT
XY X X Y Y
ATTTTCCTTT
CTGCAATCT
CAACATTGT
Dockter et al. BMC Genetics 2013, 14:66 Page 20 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00014 13398CD JX649991 A/C AGGCCTGTGG
CTGACTTGTCA
GGCATATCT
TTGCCCGTT
TCCACAA
XY XXX H H
GGCCTGTGG
CTGACTTGTCC
PenSNP00015 13752CD JX649992 A/C AAATGCTC
CCTCATTTTG
ACCATATGA
GTCAACGG
ATTTGTGGA
AGTCGGTA
YX Y Y H H
ATGCTCCCT
CATTTTGAC
CATATGC
PenSNP00016 14394CD JX649993 C/G TGAAAATTTC
AGATTTAATG
AACAAACAGTC
AGACTTGTAA
CAAATTCCTT
GGGTCCAAA
XY X Y H
GAAAATTTCA
GATTTAATGAA
CAAACAGTG
PenSNP00017 14661CD JX649994 A/G TGACCAAGGA
ATCTGTTCAAG
AACTT
CTTCTACTGTG
GCTGTTTCACC
TCTA
YX Y H H
GACCAAGGA
ATCTGTTCAA
GAACTC
PenSNP00018 15226CD JX649995 G/T TACCTCCAAT
TGTGATGCA
ACATTAG
CTAAGTGA
GAAGCACA
AGGA
XY X X H H
CTTACCTCCA
ATTGTGATGC
AACATTAT
PenSNP00019 17421CD JX649996 G/T ATCCTCCTC
CTTTGCATC
AAAGC
GAGCCAA
CCTCGACT
GCTTCTATTT
YX Y Y X H
CATCCTCC
TCCTTTGC
ATCAAAGA
PenSNP00020 17816CD JX649997 A/G AAGGACTG
AGTACCAA
GACAGATCT
GCCAGGGTA
CTGAACCTG
TCTTTTA
XY X X H H
GGACTGAG
TACCAAGA
CAGATCC
Dockter et al. BMC Genetics 2013, 14:66 Page 21 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00021 18745CD JX649998 C/T AGCATATTG
AAAAGATC
AGTCGCATAG
CAGCTGCTCC
TATCCAATC
TTCGAA
YX Y Y H H
AAAGCATAT
TGAAAAGAT
CAGTCGCATAA
PenSNP00022 19267CD JX649999 A/G AAATACCT
GAGCTTCT
GCCTCTTGT
GATGCTCGT
CATCTTGCT
CAACGAT
XY XYX H H
ACCTGAG
CTTCTGCC
TCTTGC
PenSNP00023 21409CD JX650000 C/G ACCATTCAG
GTAATATTT
CCAAAGGC
AGCGGTTCT
AGAACCGT
CAATGCTT
YX YYY X H
ACCATTCAG
GTAATATTT
CCAAAGGG
PenSNP00024 22934CD JX650001 A/G GTACAATTGT
CAAGTGTGTA
TTTTCTTACATA
GCACTGCAC
CATTCATGC
CCTAAAA
YX Y Y H H
ACAATTGTCA
AGTGTGTATT
TTCTTACATG
PenSNP00025 22942CD JX650002 A/T ATCCGATTCT
TCGTCTACTA
TGCCA
AGAAAAGCA
CAAGCTGAA
ATCAGGGAA
XY X Y H H
ATCCGATTCT
TCGTCTACTA
TGCCT
PenSNP00026 27992CD JX650003 A/G TCCTCCTCG
TCTCTTCCT
CTT
CTTGGACCGT
CCAAAGAAG
GAAAGAA
YX Y Y H H
CCTCCTCG
TCTCTTCC
TCTC
PenSNP00027 01179DD JX650004 A/G TCGACCC
CAACCTG
TCACA
CTTGCTTGGTT
TCGGAAAGAG
YX Y Y H H
CTTCGACC
CCAACCTG
TCACG
Dockter et al. BMC Genetics 2013, 14:66 Page 22 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00028 01235DD JX650005 C/T TGTGATCTT
TGGTTTGAA
CTTTGTC
CTACCAAAC
TCACTCTAAC
ATCCGGAT
XY X X H H
CACTTGTGAT
CTTTGGTTTG
AACTTTGTT
PenSNP00029 01600DD JX650006 A/G TGGTCTTGTT
CTTTACCATT
ACGCAT
GAAGTAGCTG
CCATGGAAA
AGGAAGTT
XX YXX X H
GGTCTTGTT
CTTTACCAT
TACGCAC
PenSNP00030 04630DD JX650007 A/G AGTAGTACA
GAATACTTAA
AACTATCACCA
GTTGGGGGA
GTTGCCTTCT
TGAAAT
XX Y X X H
GTAGTACAGA
ATACTTAAAA
CTATCACCG
PenSNP00031 05304DD JX650008 C/T AGTTTTCCTTT
TGTCCTTATG
TGCAG
AAGGCTTAGC
TTGGATGATA
TCCTACAA
YY X Y Y Y
CAGTTTTCCT
TTTGTCCTTA
TGTGCAA
PenSNP00032 05884DD JX650009 A/T GTCACCGCC
TCCGATTGA
GATT
CGGCTTTTGA
CGCCGCCGT
AAA
XY X X H H
GTCACCGCC
TCCGATTGA
GATA
PenSNP00033 06956DD JX650010 G/T GTTGATTCTA
CAGATCTTAA
TTCTTGATTG
TACTACAAA
GGGTAAAAAG
TGCAATTCATA
XY X X H H
AGTTGATTCTA
CAGATCTTAA
TTCTTGATTT
PenSNP00034 08307DD JX650011 C/G ACATTAAGG
GTCCACCAA
AAATCCG
GCGCAATTAA
AATCTCTTAAA
TCACCTGGT
YY X Y H
ACATTAAGG
GTCCACCAA
AAATCCC
Dockter et al. BMC Genetics 2013, 14:66 Page 23 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00035 08352DD JX650012 A/T AGTACAAGGA
AAAACCTTTTA
TTAGTAAGTATA
CTGACACAA
ACCCATTCTA
ATATGACCAA
XY X X H H
AGTACAAGGA
AAAACCTTTTAT
TAGTAAGTATT
PenSNP00036 08488DD JX650013 A/T GTGTTGGAG
AGCCAGGT
GCGA
GTATTGAGGAT
CATTCTGACAA
AAAACATA
YX Y Y H H
GTGTTGGAG
AGCCAGGTG
CGT
PenSNP00037 08608DD JX650014 C/T GTAGATAAG
TTGATTGCGA
GAGGC
CCAAACAAAT
GCACCACATT
CTCCTT
XY XYX Y H
GGTAGATAA
GTTGATTGC
GAGAGGT
PenSNP00038 08831DD JX650015 A/T TTTGAACTGC
CATGTAAAGT
TGTTTTAGA
ATTTTGAACCA
AGGAGCTATC
AGAGG
XY X X Y H
TTGAACTGCC
ATGTAAAGTT
GTTTTAGT
PenSNP00039 08947DD JX650016 A/T GGGATCGTAA
AACTCAGGAA
AAATGA
TCAGATACTC
GTGGGGTCTT
CGATT
XY XHX Y H
GGGATCGTAA
AACTCAGGAA
AAATGT
PenSNP00040 08959DD JX650017 A/G AGAGAATGAAG
AAGGAGAAGGA
AGAAA
CTCCTACGG
TTGCATTATC
GGTAGTA
YX Y Y Y Y
GAGAATGAAGA
AGGAGAAGGA
AGAAG
PenSNP00041 09272DD JX650018 A/T TTCTACAAAAC
AATCAGCAGTC
ATCATT
TCGACACCTT
TTGCCTTATC
TTGAA
XY X X Y H
TCTACAAAAC
AATCAGCAGT
CATCATA
Dockter et al. BMC Genetics 2013, 14:66 Page 24 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00042 09369DD JX650019 C/T GTTTTTATACG
CATCCATATAC
ATAATAATAG
GGTTCACTCT
CCAGAAATAA
AATCTTATAT
YX Y Y H X
GTTTTTATACGC
ATCCATATACAT
AATAATAA
PenSNP00043 09764DD JX650020 A/G AATTCAACGTC
AAATTGCAAG
GTTGCA
TTCACTATAC
CGGCTGAGT
TGGCAT
YX Y Y H H
CAACGTCAAATT
GCAAGGTTGCG
PenSNP00044 10765DD JX650021 A/G TTTTTTAATAAAT
ATCCTGGTGGAT
AATTTAT
AAATTGAGT
GGATGGCTA
GGAAGACTAA
XY XYX H H
TTTTTAATAAAT
ATCCTGGTGGA
TAATTTAC
PenSNP00045 10870DD JX650022 A/T AGATCTGGAG
ACTAAAT
CGAAGAGTT
TGGGTGGGC
GGAT
XY X X Y Y
AGATCTGGAG
ACTAAAA
PenSNP00046 11107DD JX650023 C/T GTCCGACGTG
ACAATGCAGC
CGCCGTCAA
AGAGACTTT
GTTGGAT
YX Y Y H H
CTGTCCGACGT
GACAATGCAGT
PenSNP00047 11531DD JX650024 C/T AGAAGATTCTT
CGGCTGGGAGC
TCTTCACATG
ATTACGACAA
TGGCTGAAT
XY X X H H
AAGAAGATTCTT
CGGCTGGGAGT
PenSNP00048 11655DD JX650025 A/G ACGTCCATGGA
GGACCATAAA
GCTGTCTTCC
TGCAAGGAA
CTTCTT
XY X X H H
CTACGTCCATGG
AGGACCATAAG
PenSNP00049 11974DD JX650026 G/T AAAATGCATGTA
GTTTGGTTTACG
CACACCCCC
AAAGGAAG
AATAGCAT
YX Y Y H H
AAAATGCATGTA
GTTTGGTTTACT
Dockter et al. BMC Genetics 2013, 14:66 Page 25 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00050 13159DD JX650027 C/T TGAATGTACTTT
TCATTGATAGA
GAACG
AACAATAGT
ACAACACAAC
TAAAGCAGAGA
YY X Y Y H
GTTGAATGTAC
TTTTCATTGATA
GAGAACA
PenSNP00051 13463DD JX650028 G/T GCCTTTGACG
GCGAAGGAT
TTC
GCAAGCACGG
CACTAAGCCCTT
XY X X H H
CGCCTTTGA
CGGCGAAG
GATTTA
PenSNP00052 14334DD JX650029 A/G AGAAACAAC
AAATACGAA
TAAATCACCCA
TTCGAAAATTG
TGCTTGAATCA
CGCAGT
XX YHX X Y
GAAACAACA
AATACGAATA
AATCACCCG
PenSNP00053 00290DD03373CD JX650030 C/T TGCCTTTGCG
TCGCCACAATC
AGCTAAGAGA
TGGGCAGACT
TTACAAAAT
YX Y Y H H
CTTGCCTTTG
CGTCGCCAC
AATT
PenSNP00054 00354DD04637CD JX650031 A/G GCAAAAGG
GAACCCTCA
TTTCGTT
TACTTGTCTGG
GACTTTTCCTT
TCTCTTT
XX Y X X H
CAAAAGGGA
ACCCTCATTT
CGTC
PenSNP00055 01161DD11697CD JX650032 A/G ACTGGTAAA
TACACTACG
TTCACAGT
GAAACACAGCA
GCCCAACGACA
TAT
YY XXY Y H
CTGGTAAA
TACACTAC
GTTCACAGC
PenSNP00056 01323DD15501CD JX650033 A/G ACCTGAAGA
ATTTGTTCAC
TACTTCGT
GGATCGGGTGGA
ACGATTTGTGTT
XY X X H H
CCTGAAGAA
TTTGTTCACT
ACTTCGC
Dockter et al. BMC Genetics 2013, 14:66 Page 26 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00057 01541DD02481CD JX650034 A/G AATTAGAAC
CACATCCAC
TGATTCCAA
GGAGCCCAAA
CCTTTTACATT
CTTTTCTA
YX Y Y H H
AGAACCACA
TCCACTGAT
TCCAG
PenSNP00058 02019DD03127CD JX650035 A/G GTGATTGTTA
AATCTGAATA
TATAATTTCTTTT
GTACGAGGCT
TCGAAAAAGA
CCAGAT
XY X X H H
GTGATTGTTA
AATCTGAATA
TATAATTTCTTTC
PenSNP00059 02851DD17191CD JX650036 A/G AAGAGGTTGA
TCCTAAGTTA
TCGAGA
GAAGAAAATC
ATTGTCCAC
ATCTCGTGTA
XY X X Y H
AGAGGTTGAT
CCTAAGTTAT
CGAGG
PenSNP00060 03089DD14703CD JX650037 C/T TTTCAGAGTC
ACTAATGTTC
TCACG
GCATTTCTTG
TCCATCTCTT
CAAGATGTA
XX YYX X H
GTTTTCAGAG
TCACTAATGT
TCTCACA
PenSNP00061 03423DD25897CD JX650038 A/C AATTCTTCTA
CGTCCATTTG
ATCGGAT
TATTCTTAGA
CATGGACAT
GGAAATTGAGA
YX Y Y H H
CTTCTACGT
CCATTTGAT
CGGAG
PenSNP00062 04632DD19186CD JX650039 A/T AAATGGGT
CAGCTGAA
ATTTCCGCA
CTCTTCTTTAC
TCTGTTTTTTCT
TCTTTTT
YY X Y H
AAATGGGT
CAGCTGAA
ATTTCCGCT
PenSNP00063 05160DD08243CD JX650040 C/G TCGATCGTTG
AAATGATAAT
TGATACAAG
GATCCCATA
GACTTCTTTT
AAGGATTCTAA
YX YYY H H
CGATCGTTG
AAATGATAA
TTGATACAAC
Dockter et al. BMC Genetics 2013, 14:66 Page 27 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00064 06332DD03627CD JX650041 A/C ATCAAATGCC
ATAGATCCTG
CAGATTT
ACATTTCCTAC
ACCAACTTCTT
CCACTA
XX Y X X H
CAAATGCCA
TAGATCCTG
CAGATTG
PenSNP00065 08748DD13630CD JX650042 C/G AGCTGTTC
AGGAGGT
TCATGAATG
CACCATGTGA
ACCAACACT
ATTGTCATTT
XY X X H H
AGCTGTTCA
GGAGGTTCA
TGAATC
PenSNP00066 09773DD14323CD JX650043 A/G TCATGCCCA
TTCCCCA
CCTGGTATGA
ACATGGGGA
GGTTAT
YY X Y Y
CATGCCCA
TTCCCCG
PenSNP00067 10248DD06150CD JX650044 C/G TGTGTCATT
GAAATCAA
TCCGC
GTTTCATATCT
CCCTTTGAGC
TTCTTGAA
YY XYY Y H
GCTTGTGTC
ATTGAAAT
CAATCCGG
PenSNP00068 10624DD11358CD JX650045 A/G GTGGCAGT
GTGAAACT
GCATCA
GTTTTTCCCT
GGGTGCTA
AGGTTCAT
YX Y Y H H
GTGGCAGT
GTGAAACT
GCATCG
PenSNP00069 11267DD06273CD JX650046 A/C ACCAAATA
CTTATTAGC
TCCAGTCGAA
GACTGAAG
GATGTTGC
GAGAGGC
YY X Y Y H
CCAAATAC
TTATTAGCT
CCAGTCGAC
PenSNP00070 11564DD17128CD JX650047 C/T TGGACTTG
GCATTGAA
ACAAAAGATC
ATATGAAA
CTCCCCAC
AAGAAA
YX Y X H H
AATTGGAC
TTGGCATTGA
AACAAAAGATT
Dockter et al. BMC Genetics 2013, 14:66 Page 28 of 33
http://www.biomedcentral.com/1471-2156/14/66

Table 7 Penstemon SNP marker name, GenBank dbSNP accession ID, polymorphism type, KASPar™ primer sequences (A1, A2 and common allele specific reverse) for
all 75 functional SNP assays (Continued)
PenSNP00071 11647DD17264CD JX650048 C/G GCACGAGCC
AAAATCCT
GAGC
ATTGGCATG
TGTATCCT
GTGTGGGA
XY XYX H H
GCACGAGCC
AAAATCCT
GAGG
PenSNP00072 11671DD17144CD JX650049 C/T GTGCAGCA
ACCCCTATT
CATGAC
CCTGTCCAA
AACATATGAT
CTTCATTGGAA
XY X H H
ATGTGCAG
CAACCCCT
ATTCATGAT
PenSNP00073 12915DD17470CD JX650050 A/G AAGAAAAG
GGTGGACAA
ATTAAACCGT
CAGAACAAC
ATCATACTTG
ATAAATCTCTT
XX Y X X H
GAAAAGGGT
GGACAAATT
AAACCGC
PenSNP00074 13828DD14937CD JX650051 C/T GTAAGATAT
GCTGCCAGA
TGG
CTCTGAAGAA
GTTTTTGTCCT
TGATAGCTA
YY X Y Y H
GTAAGATAT
GCTGCCAG
ATGA
PenSNP00075 14286DD18608CD JX650052 G/T GTATTGAG
AGCCACT
ACCGG
CCACTTGAAT
TGTTTGAAGA
GTTTGGGAA
YX Y Y X H
CTGTATTGA
GAGCCAC
TACCGT
1
These contigs have been deposited at DDBJ/EMBL/GenBank as a Whole Genome Shotgun project under the accessions AKKG00000000 (P. cyananthus), AKKH00000000 (P. dissectus), AKKI00000000 (P. davidsonii), and
AKKJ00000000 (P. fruticosus). The version described in this paper is the first version for each accession, XXXX01000000.
2
The GenBank accession identification for the full sequence for each allele with the specific SNP bp identified.
3
KASPar™ primers: A1 and A2 primers are SNP allele specific. All A1 Forward primers had the follow universal primer GAAGGTGACCAAGTTCATGCT added to the 5′ end of the allele specific sequence. All A2 Forward
primers had the follow universal primer GAAGGTCGGAGTCAACGGATT added to end of the 5′ allele specific sequence.
4
H = heterozygous compared to either homozygous condition for either “ X” or “Y”.
Dockter et al. BMC Genetics 2013, 14:66 Page 29 of 33
http://www.biomedcentral.com/1471-2156/14/66

specific repetitive elements. However, without further
comparisons we were unable to identify specific repetitive
elements associated with the four Penstemon species used
in this study.
Gene ontology
Using BLA STX we identified an average of 21.5% of
the contigs across t he four species a s putative genes
with an average of 13.9% annotated by Bla st2GO
(Table 5). These pu tative genes were compared and
contrasted in a more detailed study by Dockter [23].
Furthermore, he compared the Penstemon sequences
to known genes from the related genera Antirrhinum
and Mimulus, and identified nine putative Penstemon genes
from Antirrhinu m and 14 from Mimulus with an e-value
below 1.0e
-13
. Three genes (NADH dehydrogenase from M.
aurantia cus, ribosomal protein L10 from M. guttatus,
and ribosomal protein subunit 2 from M. aurantia cus,
M. szechuanensis,andM. tenellus var. tenellus)were
perfect hit s (e-value = 0.0).
Conclusions
Penstemon are recognized for their phenotypic vari-
ation and their adaptation to multiple environments
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
0200400600 8001000
Percent Repeats Masked
Genome Size (Mbp)
Percent Repeats Masked/Genome Size
% Repeats Masked LTR Elements Masked
Linear (% Re
p
eats Masked) Linear (LTR Elements Masked)
Figure 4 Relationship between genome size and repeat elements in Penstemon including the relationship of both LTRs and total
repeat elements to genome size for both genome reduced Penstemon and genome reduced/non-genome reduced Arabidopsis
1
(yellow).
1
Genome reduced A. thaliana sequence from Maughan et al. [35]; A. thaliana RILs Ler-0 and Col-4; Non-genome reduced A. thaliana
sequence downloaded from TAIR (The Arabidopsis Information Resource) as whole chromosomes; Genome size as reported by Broderick et al.
and Schmuths et al. [5,59].
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
P. cyananthus P. dissectus P. davidsonii P. fruticosus A. thaliana
(Ler-0)
A. thaliana
(Col-4)
A. thaliana
(TAIR10)
Percentage of Sequence Masked
Repeat Masker Results
Retroelements (LTR) DNA Trans
p
osons Other Re
p
eats
Figure 3 Percentage of retroelements, DNA transposons and other unclassified repeats in Penstemon cyananthus, P. dissectus,
P. davidsonii, P. fruticosus, and both genome reduced and non-genome reduced Arabidopsis
1
.
1
Genome reduced A. t hali ana sequence
from Maughan et al. [35]; A. thaliana RILs Ler-0 and Col-4; Non-genome reduced A. thaliana sequence downloaded from TAIR
(The Arabidopsis Information Resource) as whole chromosomes; the diploid (2n = 2x = 16) genome size as reported by Broderick et al.
and Schmuths et al. [5,59].
Dockter et al. BMC Genetics 2013, 14:66 Page 30 of 33
http://www.biomedcentral.com/1471-2156/14/66

[6-8,13,14,17,30,31]. Brode rick et al. [5] found that
this diversity is reflected by a wide range in their genome
sizes. Nevertheless, even with this demonstrated plasticity
we have identified evidence that there is a high level of
sequence conservation across the genus. This apparent
sequence conservation is in harmony with the hypothesis
that Penstemon ha s rapidly irradiated to its variety of
species rather recently in evolutionary time [13,14].
Furthermore, our study identified evidence that the
genome size variation in Penstemon is rooted in the
amount of repetitive element s in each species.
Despite the large differences in Penstemon’s genome
size, the finding that the genus has a great deal of
sequence conservation is invaluable for the development
of interspecific markers. The further development and
mapping of a number of conserved markers will facilitate
the domestication of xeric Penstemon cultivars via
interspecific hybridization which are largely unexploited
largely due to crossing barriers [6-8,10-12,15]. Viehmeyer
[16] hypothesized that it might be possible to develop
Penstemon breeding lines that would facilitate the
indirect interspecific hybridiz ation of any two spe cies
within the genus. He and others have used traditional
breeding techniques to develop a number of interspecific
hybrids [7,1 1,15,17,66]. Clarifying the phylog enetic
relationships within the genus should facilitate these
objectives [ 67]. In the largest Penstemon phylogenetic
study conducted to date, W olfe et al. [14] sequenced
the ITS and two chloroplas t genes in 163 species.
They concluded that many spe cies are polyphyletic in
their origins thus making them difficult to discriminate
between one another; thus, requiring additional molecular
studies to more accurately define taxonomic relationships.
We tested 51 SSR/INDEL ba sed markers (Table 3),
and identified several thousand inter- and intraspecific
SNPs (Table 6), all of which have potential as both
inter- and intra specific markers. Of the 51 SSR s/
INDELs we sele cted 12 to test across 93 Penstemon
taxa. The resulting data was used to more clearly
definethephylogeneticrelationshipsofthosetaxabut
our results were incoherent. It is possible that some
of these markers may represent more than one locus
in the Penstemon genome. This situation has been
identified by others as a potential weakness in using
SSR based markers in interspecific phylogenetic studies
[46,47]. A major reason for the vagary in Penstemon’s
phylogeny is that it appears to have quite recently evolved
and rapidly radiated leaving weak species boundaries
[13,14]. Furthermore, thereareanumberofreports
of spec iation via natural interspecific hybridization
found within the genus [14,68-73]. Therefore, like
Wolfe et al. [14], we concluded that better marker
data sets will be required to reduce present phylogenetic
ambiguity.
To gain clearer insights into the relationships of
Penstemon it will take carefully designed large sc ale
sequencing studies. There are methods which are
showing promise to do su ch studies e conomica lly.
One exa mple would be to utilize GR-RSC o r similar
methods which will sample large quantities of homolo-
gous sequence of a genome at ever decreasing costs
[18,20,74]. Since our SSR/INDEL, sequenc e, and SNP
data have demonstrated broad applicability across
Penstemon it becomes e vident that further studies
utilizing this same GR-RSC protocol and downstream
analysis on additional species would allow broader
comparisons o f putative genes, repeat elements, SNPs
and SSRs , facilitating a much better understanding of
the genus. Furthermore, using this technique on carefully
selected parent s and their segregating progeny would
allow Penstemon genetic mapping studies w hich
would greatly enhance the ability to do breeding and
domestication studies within the genus. Historically,
studies of this na ture would have been unthinkable;
however, mass homologous loci sequence studies are
rapidly becoming feasible [18,20,74]. In the interim it
is possible to take the data we report here and further test
the75SNPswehavereportedherealongwithothers
not yet de veloped and for around US$0.05/data point
[18,20] do a much broader study. Studies on homolo-
gous SNPs across many Penstemon taxa, similar to
the Amaranthus study of Maughan et al. [20], should
assist in developing improved insights into Penstemon
phylogenetic relationships and produce high quality
genetic maps from carefully designed segregating
Penstemon populations.
Competing interests
The authors declare no competing interests.
Authors’ contributions
Rhyan B Dockter, David B Elzinga, Brad Geary, P Jeff Maughan, Leigh A
Johnson, Danika Tumbleson, JanaLynn Franke, Keri Dockter, and Mikel R
Stevens. RBD preformed the GR-RSC technique and either carried out or
oversaw the all other steps of the study and participated in all planning and
design of all experiments as well as their analysis and did the initial drafting
of the manuscript. DBE did or assisted in all bioinfomatics performed in this
study. BG participated in the design of all aspects of the study as well as
advised RBD and was involved in the editing and revising of the manuscript.
PJM advised and assisted in the GR-RSC technique as well as advised RBD in
relevant issues of the bioinfomatics of the study and was involved in the
editing and revising of the manuscript. LAJ advised and assisted RBD and
MRS in the taxonomy related issues of the study and was involved in the
editing and revising of the manuscript. DT, JF, and KD carried out all aspects,
including basic analysis, of the marker studies reported. MRS was the senior
advisor of RBD and was intricately involved in all aspects of the study and
the manuscript. All authors both read and approved the final manuscript.
Acknowledgements
We acknowledge Shaun Broderick, a graduate student, Tiffany Austin, and
Aaron King, undergraduates, for their laboratory assistance and Robert Byers
a graduate student and Scott Yourstone, an undergraduate, for their
bioinformatic assistance, all from Brigham Young University. This research
was funded in part by an Annaley Naegle Redd Assistantship from the
Brigham Young University Charles Redd Center for Western Studies and a
Dockter et al. BMC Genetics 2013, 14:66 Page 31 of 33
http://www.biomedcentral.com/1471-2156/14/66

Year-End Funding Grant from the Department of Plant and Wildlife Sciences,
Brigham Young University.
Author details
1
Plant and Wildlife Sciences Department, Brigham Young University, Provo,
UT 84602, USA.
2
Biology Department, Brigham Young University, Provo, UT
84602, USA.
Received: 15 September 2012 Accepted: 1 August 2013
Published: 8 August 2013
References
1. St Hilaire R, Arnold MA, Wilkerson DC, Devitt DA, Hurd BH, Lesikar BJ, Lohr
VI, Martin CA, McDonald GV, Morris RL, Pittenger DR, Shaw DA, Zoldoske DF:
Efficient water use in residential urban landscapes. HortScience 2008,
43:2081–2092.
2. Martin CA: Landscape water use in Phoenix, Arizona. Desert Plants 2001,
17:26–31.
3. Bradley BA, Blumenthal DM, Early R, Grosholz ED, Lawler JJ, Miller LP, Sorte
CJB, D’Antonio CM, Diez JM, Dukes JS, Ibanez I, Olden JD: Global change,
global trade, and the next wave of plant invasions. Front Ecol Environ
2012, 10:20–28.
4. Burt JW, Muir AA, Piovia-Scott J, Veblen KE, Chang AL, Grossman JD, Weiskel
HW: Preventing horticultural introductions of invasive plants: potential
efficacy of voluntary initiatives. Biol Invasions 2007, 9:909–923.
5. Broderick SR, Stevens MR, Geary B, Love SL, Jellen EN, Dockter RB, Daley SL,
Lindgren DT: AsurveyofPenstemon’s genome size. Genome 2011, 54:160–173.
6. Lindgren D, Wilde E: Growing Penstemons: Species, Cultivars and Hybrids.
Haverford, PA: Infinity Publishing Com; 2003.
7. Lindgren DT: Breeding Penstemon.InBreeding Ornamental Plants. Edited by
Callaway DJ, Callaway MB. Portland, Oregon: Timber Press; 2000:196–212.
8. Nold R: Penstemons. Portland, Oregon: Timber Press; 1999.
9. Viehmeyer G: Let’s breed better Penstemon. Bul Amer Penstemon Soc 1955,
14:275–288.
10. Way D, James P: The Gardener’s Guide to Growing Penstemon. Portland, OR:
Timber Press; 1998.
11. Lindgren DT, Schaaf DM: Penstemon : a summary of interspecific crosses.
HortScience 2007, 42:494–498.
12. Lindgren D: List and Description of Named Cultivars in the Genus Penstemon
(2006). Lincoln, Nebraska: University of Nebraska-Lincoln Extension; EC1255; 2006.
13. Straw RM: A redefinition of Penstemon
(Scrophulariaceae). Brittonia 1966,
18:80–95.
14. Wolfe AD, Randle CP, Datwyler SL, Morawetz JJ, Arguedas N, Diaz J:
Phylogeny, taxonomic affinities, and biogeography of Penstemon
(Plantaginaceae) based on ITS and cpDNA sequence data. Amer J Bot
2006, 93:1699–1713.
15. Uhlinger RD, Viehmeyer G: Penstemon in your Garden. Lincoln, Nebraska:
University of Nebraska College of Agriculture The Agricultural Experiment
Station; 1971. Station Circular 105.
16. Viehmeyer G: Reversal of evolution in the genus Penstemon. Am Nat 1958,
92:129–137.
17. Viehmeyer G: Advances in Penstemon breeding. Bul Amer Penstemon Soc
1973, 32:16–21.
18. Cronn R, Knaus BJ, Liston A, Maughan PJ, Parks M, Syring JV, Udall J:
Targeted enrichment strategies for next-generation plant biology.
Amer J Bot 2012, 99:291–311.
19. Heslop-Harrison JS: Exploiting novel germplasm. Aust J Agric Res 2002,
53:873–879.
20. Maughan PJ, Smith SM, Fairbanks DJ, Jellen EN: Development,
characterization, and linkage mapping of single nucleotide polymorphisms
in the grain amaranths (Amaranthus sp.). Plant Gen 2011, 4:1–10.
21. Bernardo R: Molecular markers and selection for complex traits in plants:
learning from the last 20 years. Crop Sci 2008, 48:1649–1664.
22. Tanksley SD, McCouch SR: Seed banks and molecular maps: unlocking
genetic potential from the wild. Science 1997, 277:1063–1066.
23. Dockter RB: Genome snapshot and molecular marker development in
Penstemon (Plantaginaceae). M.S. Thesis. Brigham Young University,
Department of Plant and Wildlife Sciences; 2011.
24. Santana QC, Coetzee MPA, Steenkamp ET, Mlonyeni OX, Hammond GNA,
Wingfield MJ, Wingfield BD: Microsatellite discovery by deep sequencing
of enriched genomic libraries. Biotechniques 2009, 46:217–
223.
25. Maughan PJ, Yourstone SM, Jellen EN, Udall JA: SNP discovery via genomic
reduction, barcoding and 454-pyrosequencing in amaranth. Plant Gen
2009, 2:260–270.
26. Păcurar DI, Păcurar ML, Street N, Bussell JD, Pop TI, Gutierrez L, Bellini C: A
collection of INDEL markers for map-based cloning in seven Arabidopsis
accessions. J Exp Bot 2012, 63:2491–2501.
27. Althoff DM, Gitzendanner MA, Segraves KA: The utility of amplified
fragment length polymorphisms in phylogenetics: a comparison of
homology within and between genomes. Syst Biol 2007, 56:477– 484.
28. Sambrook J, Fritcsh EF, Maniatis T: Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor, N.Y: Cold Spring Harbor Lab; 1989.
29. Todd JJ, Vodkin LO: Duplications that suppress and deletions that restore
expression from a chalcone synthase multigene family. Plant Cell 1996,
8:687–699.
30. Holmgren NH: Penstemon.InIntermountain Flora: Vascular Plants of the
Intermountain West. Volume 4. Edited by Cronquist A, Holmgren AH,
Holmgren NH, Reveal JL, Holmgren PK. Bronx, New York, USA: New York
Botanical Garden; 1984:370–457.
31. Welsh SL, Atwood ND, Goodrich S, Higgins LC: A Utah Flora. 4th edition.
Provo, Utah: Brigham Young University; 2008.
32. RepeatMasker. [http://www.repeatmasker.org].
33. Bao Z, Eddy SR: Automated de novo identification of repeat sequence
families in sequenced genomes. Genome Res 2002, 12:1269–1276.
34. Price AL, Jones NC, Pevzner PA: De novo identification of repeat families
in large genomes. Bioinformatics 2005, 21(Suppl 1):I351–I358.
35. Maughan PJ, Yourstone SM, Byers RL, Smith SM, Udall JA: Single-nucleotide
polymorphism genotyping in mapping populations via genomic
reduction and next-generation sequencing: proof-of-concept. Plant Gen
2010, 3:1–13.
36. Rhee SY, Beavis W, Berardini TZ, Chen GH, Dixon D, Doyle A, Garcia-Hernandez
M, Huala E, Lander G, Montoya M, Miller N, Mueller LA, Mundodi S, Reiser L,
Tacklind J, Weems DC, Wu YH, Xu I, Yoo D, Yoon J, Zhang PF: The Arabidopsis
Information Resource (TAIR): a model organism database providing a
centralized, curated gateway to Arabidopsis biology, research materials and
community. Nucleic Acids Res 2003, 31:
224–228.
37. Thiel T, Michalek W, Varshney RK, Graner A: Exploiting EST databases for
the development and characterization of gene-derived SSR-markers in
barley (Hordeum vulgare L.). Theor Appl Genet 2003, 106:411–422.
38. Stajich JE, Block D, Boulez K, Brenner SE, Chervitz SA, Dagdigian C, Fuellen
G, Gilbert JGR, Korf I, Lapp H, Lehväslaiho H, Matsalla C, Mungall CJ,
Osborne BI, Pocock MR, Schattner P, Senger M, Stein LD, Stupka E, Wilkinson
MD, Birney E: The Bioperl toolkit: Perl modules for the life sciences.
Genome Res 2002, 12:1611–1618.
39. Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for
biologist programmers.InBioinformatics Methods and Protocols: Methods in
Molecular Biology. Edited by Krawetz S, Misener S. Totowa, NJ: Humana
Press; 2000:365–386.
40. PAUP* Phylogenetic analysis using parsimony (*and other methods).
[http://paup.csit.fsu.edu/].
41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment
search tool. J Mol Biol 1990, 215:403–410.
42. GenBank. [http://www.ncbi.nlm.nih.gov/genbank/].
43. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M: Blast2GO: a
universal tool for annotation, visualization and analysis in functional
genomics research. Bioinformatics 2005, 21:3674–3676.
44. Kawabe A, Miyashita NT: Patterns of codon usage bias in three dicot and
four monocot plant species. Genes Genet Syst 2003, 78:343–352.
45. Morgante M, Hanafey M, Powell W: Microsatellites are preferentially
associated with nonrepetitive DNA in plant genomes. Nat Genet 2002,
30:194–200.
46. Robinson JP, Harris SA: Amplified fragment length polymorphisms and
microsatellites: a phylogenetic perspective.InEU-Compendium: Which
DNA Marker for Which Purpose?. Edited by Gillet EM. Göttingen, Germany:
Institut für Forstgenetik und Forstpflanzenzüchtung, Universität Göttingen;
1999:95–121.
47. Ochieng JW, Steane DA, Ladiges PY, Baverstock PR, Henry RJ, Shepherd M:
Microsatellites retain phylogenetic signals across genera in eucalypts
(Myrtaceae). Genet Mol Biol 2007, 30:1125–1134.
48. Nadir E, Margalit H, Gallily T, Ben-Sasson SA: Microsatellite spreading in the
human genome: evolutionary mechanisms and structural implications.
Proc Natl Acad Sci USA 1996,
93:6470–6475.
Dockter et al. BMC Genetics 2013, 14:66 Page 32 of 33
http://www.biomedcentral.com/1471-2156/14/66

49. Viehmeyer G: Reports dealing in large part with hybridization and
selection. Bul Amer Penstemon Soc 1965, 24:95–100.
50. Zamir D, Tadmor Y: Unequal segregation of nuclear genes in plants.
Bot Gaz 1986, 147:355–358.
51. Eshed Y, Zamir D: A genomic library of Lycopersicon pennellii in L.
esculentum: A tool for fine mapping of genes. Euphytica 1994, 79:175–179.
52. Robbins MD, Masud MAT, Panthee DR, Gardner RG, Francis DM, Stevens MR:
Marker assisted selection for coupling phase resistance to Tomato
spotted wilt virus and Phytophthora infestans (late blight) in tomato.
HortScience 2010, 45:1424–1428.
53. Canady MA, Meglic V, Chetelat RT: A library of Solanum lycopersicoides
introgression lines in cultivated tomato. Genome 2005, 48:685–697.
54. Canady MA, Ji YF, Chetelat RT: Homeologous recombination in Solanum
lycopersicoides introgression lines of cultivated tomato. Genetics 2006,
174:1775–1788.
55. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, McCouch S:
Computational and experimental analysis of microsatellites in rice
(Oryza sativa L.): frequency, length variation, transposon associations,
and genetic marker potential. Genome Res 2001, 11:1441–1452.
56. Parida SK, Kalia SK, Kaul S, Dalal V, Hemaprabha G, Selvi A, Pandit A, Singh A,
Gaikwad K, Sharma TR, Srivastava PS, Singh NK, Mohapatra T: Informative
genomic microsatellite markers for efficient genotyping applications in
sugarcane. Theor Appl Genet 2009, 118:327–338.
57. Zhang FK, Zhao ZM: The influence of neighboring-nucleotide
composition on single nucleotide polymorphisms (SNPs) in the mouse
genome and its comparison with human SNPs. Genomics 2004,
84:785–795.
58. Morton BR, Bi IV, McMullen MD, Gaut BS: Variation in mutation dynamics
across the maize genome as a function of regional and flanking base
composition. Genetics 2006, 172:569–577.
59. Schmuths H, Meister A, Horres R, Bachmann K: Genome size variation
among accessions of Arabidopsis thaliana. Ann Bot 2004, 93:317–321.
60. Lynch M: The Origins of Genome Architecture. Sunderland, MA: Sinauer
Associates, Inc; 2007.
61. Lynch M, Conery JS: The origins of genome complexity. Science 2003,
302:1401–1404.
62. Kidwell MG: Transposable elements and the evolution of genome size in
eukaryotes. Genetica 2002, 115:49–63.
63. Raskina O, Barber JC, Nevo E, Belyayev A: Repetitive DNA and
chromosomal rearrangements: speciation-related events in plant
genomes. Cytogenet Genome Res 2008, 120:351–357.
64. Kolano B, Gardunia BW, Michalska M, Bonifacio A, Fairbanks D, Maughan PJ,
Coleman CE, Stevens MR, Jellen EN, Maluszynska J: Chromosomal
localization of two novel repetitive sequences isolated from the
Chenopodium quinoa Willd. genome. Genome 2011, 54:710–717.
65. Kubis S, Schmidt T, Heslop-Harrison JS: Repetitive DNA elements as a
major component of plant genomes. Ann Bot 1998, 82(Suppl A):45–55.
66. Meyers B: A summary of Bruce Meyers’ Penstemon hybridizations.
Bul Amer Penstemon Soc 1998, 57:2–11.
67. Friedt W, Snowdon RJ, Ordon F, Ahlemeyer J: Plant breeding: assessment
of genetic diversity in crop plants and its exploitation in breeding.
Prog Bot 2007, 68:151–178.
68. Wolfe AD, Elisens WJ: Diploid hybrid speciation in Penstemon
(Scrophulariaceae) revisited. Amer J Bot 1993, 80:1082–1094.
69. Wolfe AD, Elisens WJ: Nuclear ribosomal DNA restriction site variation in
Penstemon section Peltanthera (Scrophulariaceae): an evaluation of
diploid hybrid speciation and evidence for introgression. Amer J Bot
1994, 81:
1627–1635.
70. Wolfe AD, Elisens WJ: Evidence of chloroplast capture and pollen-mediated
gene flow in Penstemon sect. Peltanthera (Scrophulariaceae). Syst Bot 1995,
20:395–412.
71. Datwyler SL, Wolfe AD: Phylogenetic relationships and morphological
evolution in Penstemon subg. Dasanthera (Veronicaceae). Syst Bot 2004,
29:165–176.
72. Wolfe AD, Xiang Q-Y, Kephart SR: Assessing hybridization in natural
populations of Penstemon (Scrophulariaceae) using hypervariable
intersimple sequence repeat (ISSR) bands. Mol Ecol 1998, 7:1107–1125.
73. Wolfe AD, Xiang Q-Y, Kephart SR: Diploid hybrid speciation in Penstemon
(Scrophulariaceae). Proc Natl Acad Sci USA 1998, 95:5112–5115.
74. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell
SE: A robust, simple genotyping-by-sequencing (GBS) approach for high
diversity species. PLoS One 2011, 6:e19379.
doi:10.1186/1471-2156-14-66
Cite this article as: Dockter et al.: Developing molecular tools and
insights into the Penstemon genome using genomic reduction and
next-generation sequencing. BMC Genetics 2013 14:66.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Dockter et al. BMC Genetics 2013, 14:66 Page 33 of 33
http://www.biomedcentral.com/1471-2156/14/66